Journal: J Mol Cell Cardiol

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Abstract

Intrafibrillar and perinuclear mitochondrial heterogeneity in adult cardiac myocytes.

Lu X, Thai PN, Lu S, Pu J, Bers DM

Mitochondria are involved in multiple cellular functions, in addition to their core role in energy metabolism. Mitochondria localized in different cellular locations may have different morphology, Ca handling and biochemical properties and may interact differently with other intracellular structures, causing functional specificity. However, most prior studies have utilized isolated mitochondria, removed from their intracellular environment. Mitochondria in cardiac ventricular myocytes are highly organized, with a majority squeezed between the myofilaments in longitudinal chains (intrafibrillar mitochondria, IFM). There is another population of perinuclear mitochondria (PNM) around and between the two nuclei typical in myocytes. Here, we take advantage of live myocyte imaging to test for quantitative morphological and functional differences between IFM and PNM with respect to calcium fluxes, membrane potential, sensitivity to oxidative stress, shape and dynamics. Our findings show higher mitochondrial Ca uptake and oxidative stress sensitivity for IFM vs. PNM, which may relate to higher local energy demand supporting the contractile machinery. In contrast to IFM which are remarkably static, PNM are relatively mobile, appear to participate readily in fission/fusion dynamics and appear to play a central role in mitochondrial genesis and turnover. We conclude that while IFM may be physiologically tuned to support local myofilament energy demands, PNM may be more critical in mitochondrial turnover and regulation of nuclear function and import/export. Thus, important functional differences are present in intrafibrillar vs. perinuclear mitochondrial subpopulations.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Oct 2019; 136:72-84
Lu X, Thai PN, Lu S, Pu J, Bers DM
J Mol Cell Cardiol: 30 Oct 2019; 136:72-84 | PMID: 31491377
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Abstract

The axis of local cardiac endogenous Klotho-TGF-β1-Wnt signaling mediates cardiac fibrosis in human.

Liu Q, Zhu LJ, Waaga-Gasser AM, Ding Y, ... Charytan DM, Hsiao LL
Background
Cardiovascular fibrosis is a major contributor to cardiovascular disease, the primary cause of death in patients with chronic kidney disease (CKD). We previously reported expression of endogenous Klotho in human arteries, and that CKD is a state of Klotho deficiency, resulting in vascular calcification, but myocardial expression of Klotho is poorly understood. This study aimed to further clarify endogenous Klotho\'s functional roles in cardiac fibrosis in patients with underlying CKD.
Methods and results
Human atrial appendage specimens were collected during cardiac surgery from individuals with or without CKD. Cardiac fibrosis was quantified using trichrome staining. For endogenous Klotho functional studies, primary human cardiomyocytes (HCMs) were treated with uremic serum from CKD patients or recombinant human TGF-β1. The effects of endogenous Klotho in HCMs were studied using Klotho-siRNA and Klotho-plasmid transfection. Both gene and protein expression of endogenous Klotho are found in human heart, but decreased Klotho expression is clearly associated with the degree of cardiac fibrosis in CKD patients. Moreover, we show that endogenous Klotho is expressed by HCMs and cardiac fibroblasts (HCFs) but that HCM expression is suppressed by uremic serum or TGF-β1. Klotho knockdown or overexpression aggravates or mitigates TGF-β1-induced fibrosis and canonical Wnt signaling in HCMs, respectively. Furthermore, co-culture of HCMs with HCFs increases TGF-β1-induced fibrogenic proteins in HCFs, but overexpression of endogenous Klotho in HCMs mitigates this effect, suggesting functional crosstalk between HCMs and HCFs.
Conclusions
Our data from analysis of human hearts as well as functional in vitro studies strongly suggests that the loss of cardiac endogenous Klotho in CKD patients, specifically in cardiomyocytes, facilitates intensified TGF-β1 signaling which enables more vigorous cardiac fibrosis through upregulated Wnt signaling. Upregulation of endogenous Klotho inhibits pathogenic Wnt/β-catenin signaling and may offer a novel strategy for prevention and treatment of cardiac fibrosis in CKD patients.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:113-124
Liu Q, Zhu LJ, Waaga-Gasser AM, Ding Y, ... Charytan DM, Hsiao LL
J Mol Cell Cardiol: 30 Oct 2019; 136:113-124 | PMID: 31520610
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Abstract

Acetaldehyde dehydrogenase 2 deficiency exacerbates cardiac fibrosis by promoting mobilization and homing of bone marrow fibroblast progenitor cells.

Li X, Weng X, Shi H, Gao R, ... Sun A, Ge J

Cardiac fibrosis is a common feature of various cardiovascular diseases. Previous studies showed that acetaldehyde dehydrogenase 2 (ALDH2) deficiency exacerbated pressure overload-induced heart failure. However, the role and mechanisms of cardiac fibrosis in this process remain largely unknown. This study aimed to investigate the effect of ALDH2 deficiency on cardiac fibrosis in transverse aortic constriction (TAC) induced pressure overload model in mice. Echocardiography and histological analysis revealed cardiac dysfunction and enhanced cardiac fibrosis in TAC-operated animals; ALDH2 deficiency further aggravated these changes. ALDH2 chimeric mice were generated by bone marrow (BM) transplantation of WT mice into the lethally irradiated ALDH2KO mice. The proportion of circulating fibroblast progenitor cells (FPCs) and ROS level in BM after TAC were significantly higher in ALDH2KO mice than in ALDH2 chimeric mice. Furthermore, FPCs were isolated and cultured for in vitro mechanistic studies. The results showed that the stem cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR4) axis played a major role in the recruitment of FPCs. In conclusion, our research reveals that increased bone marrow FPCs mobilization and myocardial homing contribute to the enhanced cardiac fibrosis and dysfunction induced by TAC in ALDH2 KO mice via exacerbating accumulation of ROS in BM and myocardial SDF-1 expression.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 23 Oct 2019; epub ahead of print
Li X, Weng X, Shi H, Gao R, ... Sun A, Ge J
J Mol Cell Cardiol: 23 Oct 2019; epub ahead of print | PMID: 31668970
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Abstract

Deficiency of NONO is associated with impaired cardiac function and fibrosis in mice.

Xu X, Jiang H, Lu Y, Zhang M, ... Ni M, Zhang Y

Non-POU-domain-containing octamer-binding protein (NONO), a component of multifunctional Drosophila behavior/human splicing (DBHS) family, plays an important role in regulating glucose and fat metabolism, circadian cycles, cell division, collagen formation and fibrosis. Dysfunctional variants of NONO have been described as the cause of congenital heart defects in males. However, the effects of NONO deficiency on the ventricular function and cardiac fibrosis as well as the related mechanisms are not clear. In the present study, we aimed to reveal the overall phenotypes, cardiac function and fibroblasts in NONO knockout (NONO KO) mice compared with the wild-type (WT) male littermates. The results showed that the birth rate of NONO mice was much lower than their WT male littermates at the time of weaning. The body weight of NONO mice was 19% lower than that of WT male littermates (27.2 ± 1.49 g vs. 22.01 ± 1.20 g, P < .001). NONO KO mice exhibited continuous higher mortality from birth to a year later (P < .05). Compared with those in the WT mice, the heart weight was lower(142.0 ± 8.7 mg vs. 179.0 ± 10.4 mg, P < .001), the heart weight to body weight ratio (HW/BW) was similar, the E/A ratio was higher (1.80 ± 0.47 vs. 1.44 ± 0.26, P < .05), and the left ventricular end diastolic diameter (LVEDd) was significantly lower (2.72 ± 0.51 mm vs.3.54 ± 0.43 mm, P < .001) in the NONO KO mice. We also found excessive matrix deposition in vivo. In vitro, NONO deficiency led to fibroblasts hyperproliferation, while migration was inhibited, which would induce collagen maturation and deposition. Conversely, overexpression of NONO inhibited fibroblasts proliferation and increased migration which reduced collagen deposition. RNA-seq of cardiac fibroblasts further indicated that NONO deficiency upregulated the cell cycle regulators, which included cyclin B2, the origin recognition complex 1 (ORC1) and cell division cycle 6 (CDC6), while downregulated the migration regulators, which included myosins, integrin and coagulation factor II. Overexpression of NONO further verified the effects of these indicators. In conclusion, our study demonstrated that NONO deficiency was associated with developing heart defects in mice. Hyperproliferation of cardiac fibroblasts with dramatically excessive collagen secretion might be the cause of heart defects of NONO KO mice.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 17 Oct 2019; 137:46-58
Xu X, Jiang H, Lu Y, Zhang M, ... Ni M, Zhang Y
J Mol Cell Cardiol: 17 Oct 2019; 137:46-58 | PMID: 31634484
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Abstract

Molecular machinery and interplay of apoptosis and autophagy in coronary heart disease.

Dong Y, Chen H, Gao J, Liu Y, Li J, Wang J

Coronary heart disease (CHD) is a common heart disease and the leading cause of cardiovascular death. Apoptosis and autophagy are two forms of programmed cell deaths which participate in the pathogenesis, development and prognosis of CHD. They are activated by several different pathways respectively and can interact with each other through the Beclin 1-Bcl-2/Bcl-xL complex, mTOR, TRAIL, TNF-α, ER stress and nucleus p53 pathways. Excessive apoptosis can promote myocardial ischemia, ischemia/reperfusion (I/R) injury, post-ischemia cardiac remodeling and coronary atherosclerosis except for VSMC-induced atherosclerosis progress. In contrast, activated autophagy protects heart from myocardial ischemia injury and post-ischemia cardiac remodeling, but can exert controversial effects on I/R injury and coronary atherosclerosis. Therefore, considering the pathological significance and mechanisms of apoptosis and autophagy underlying CHD, therapeutic implication of targeting apoptosis and autophagy is obvious. Fortunately, some therapeutic drugs and pharmacologic compounds involving mTOR inhibitor and AMPK activator have been reported to regulate apoptosis and autophagy. Although recent studies are limited and insufficient, they have pointed out the complex interplay between apoptosis and autophagy and further provided treatment concept for CHD by balancing the switch between the two responses.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:27-41
Dong Y, Chen H, Gao J, Liu Y, Li J, Wang J
J Mol Cell Cardiol: 30 Oct 2019; 136:27-41 | PMID: 31505198
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Abstract

TAK1/Map3k7 enhances differentiation of cardiogenic endoderm from mouse embryonic stem cells.

Hunter A, Dai Y, Brown KJ, Muise-Helmericks R, Foley AC

Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFβ-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFβ target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 23 Oct 2019; epub ahead of print
Hunter A, Dai Y, Brown KJ, Muise-Helmericks R, Foley AC
J Mol Cell Cardiol: 23 Oct 2019; epub ahead of print | PMID: 31668971
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Abstract

Genetic ablation and pharmacological inhibition of immunosubunit β5i attenuates cardiac remodeling in deoxycorticosterone-acetate (DOCA)-salt hypertensive mice.

Cao HJ, Fang J, Zhang YL, Zou LX, ... Guo SB, Li HH

Hypertensive cardiac remodeling is a major cause of heart failure. The immunoproteasome is an inducible form of the proteasome and its catalytic subunit β5i (also named LMP7) is involved in angiotensin II-induced atrial fibrillation; however, its role in deoxycorticosterone-acetate (DOCA)-salt-induced cardiac remodeling remains unclear. C57BL/6 J wild-type (WT) and β5i knockout (β5i KO) mice were subjected to uninephrectomy (sham) and DOCA-salt treatment for three weeks. Cardiac function, fibrosis, and inflammation were evaluated by echocardiography and histological analysis. Protein and gene expression levels were analyzed by quantitative real-time PCR and immunoblotting. Our results showed that after 21 days of DOCA-salt treatment, β5i expression and chymotrypsin-like activity were the most significantly increased factors in the heart compared with the sham control. Moreover, DOCA-salt-induced elevation of blood pressure, adverse cardiac function, chamber and myocyte hypertrophy, interstitial fibrosis, oxidative stress, and inflammation were markedly attenuated in β5i KO mice. These findings were verified in β5i inhibitor PR-957-treated mice. Moreover, blocking of PTEN (the gene of phosphate and tensin homolog deleted on chromosome ten) markedly attenuated the inhibitory effect of β5i knockout on DOCA-salt-induced cardiac remodeling. Mechanistically, DOCA-salt stress upregulated the expression of β5i, which promoted the degradation of PTEN and the activation of downstream signals (AKT/mTOR, TGF-β1/Smad2/3, NOX, and NF-κB), which ultimately led to cardiac hypertrophic remodeling. This study provides new evidence of the critical role of β5i in DOCA-salt-induced cardiac remodeling through the regulation of PTEN stability, and indicates that the inhibition of β5i may be a promising therapeutic target for the treatment of hypertensive heart diseases.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Oct 2019; 137:34-45
Cao HJ, Fang J, Zhang YL, Zou LX, ... Guo SB, Li HH
J Mol Cell Cardiol: 16 Oct 2019; 137:34-45 | PMID: 31629736
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Abstract

Effects of the combination of tanshinone IIA and puerarin on cardiac function and inflammatory response in myocardial ischemia mice.

Gao S, Li L, Li L, Ni J, ... Mao J, Fan G
Background
Ventricular remodeling is a major pathological process of normal heart failure. With the aging of society, poor diet control, social, psychological and other risk factors in our country, the incidence of myocardial infarction and hypertension is reported to increase yearly. Many treatment methods have effectively delayed the occurrence of ventricular remodeling. However, in order to prevent and delay the occurrence and development of ventricular remodeling, the new treatment strategy cannot be ignored.
Methods
In this study, we used male C57BL/6 mice (8 weeks old), weight 23 g-27 g, SPF grade. According to the established methods of the research group, the left anterior descending branch of the coronary artery (LAD) was used to make the model of myocardial ischemia, and which was evaluated by the change of EF value in mice. The experiment included seven groups: sham operation group, model group, metoprolol group, puerarin group, tanshinone IIA group, tanshinone IIA: puerarin =1:1 group, tanshinone IIA: puerarin =1:2 group. The changes of cardiac function in each group were observed by echocardiography and hemodynamics after the drug delivery cycle was 3d, 7d, 14d and 28d. Detection of 3d serum enzyme indexes LDH, CK and CK-MB by automatic biochemical analyzer. The expression of CD11b, F4/80, Ly6C in cardiac tissues were detected by flow cytometry at 3d and 7d. The expression of IL-1β and TNF- α in serum were detected by ELISA. IL-1β, IL-6, IL-10, iNOS and other related genes were detected by RT-PCR method. HE, Masson staining and immunohistochemical staining were used to observe the changes of myocardial histomorphology in mice. We also examined the effects of different drug treatments on the proliferation and function of Raw264.7 cells, H9C2 cells and HUVECs. Western blot examined the effects of different drug treatments on the expression of inflammatory pathway related proteins TLR4 and C/EBP-β.
Results
1. Echocardiographic results showed that with the prolongation of ischemic time, the ejection fraction of the model group, the shortening rate of the short axis of the left ventricle, the flow rate of the outflow tract were significantly decreased, and the structure of the ventricle was significantly changed. Hemodynamic tests showed that the maximum and maximum rate of decline in the post-ischemia model group were significantly reduced, with increased systolic and diastolic volume, and a decrease in pressure difference. After treatment with drugs, all groups improved, but tanshinone IIA: puerarin = 1:1 group can significantly improve the above indicators after 28d of administration, which can effectively relieve the deterioration of cardiac function caused by acute myocardial infarction. 2. After administration for 3 and 7 days, the inflammatory cell CD11b monocytes and the F4/80 phenotype macrophages in heart tissue were detected by flow cytometry, and it was found that tanshinone IIA: puerarin = 1:1 can inhibit the release of inflammatory cells. The results of RT-PCR showed that the tanshinone IIA: puerarin = 1:1 group significantly improved the expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, and iNOS. In the immunohistochemical analysis of iNOS and Arg-1, the tanshinone IIA and puerarin 1:1 treatment group was able to inhibit the expression of M1 macrophages in the early stage of inflammation and promote the expression of M2 macrophages. 3. The cardiac index increased significantly and the serum TGF-β increased after 28d. The combination of tanshinone IIA and puerarin could significantly reduce these indexes. HE, Masson, Sirius red and immunohistochemical staining were found in the combination of tanshinone IIA and puerarin can significantly reduce the structure of acute ischemic myocardial cell damage and interstitial edema, reduce collagen synthesis, and fibroblasts release, thereby inhibiting myocardial fibrosis and heart remodeling. 4. MTT assay showed a significantly greater proliferation of above two cells types treated with tanshinone IIA: puerarin =1:1 and more nodes and meshes were found in tanshinone IIA: puerarin =1:1 group compared with other groups. 5. The combination of tanshinone IIA and puerarin could regulate inflammation through inhibiting the expression of TLR4 protein, but up-regulating the expression of C/EBP-β protein.
Conclusion
The combination of tanshinone IIA and puerarin inhibits the immersion of inflammatory cells. Improving hemodynamics by improving cardiac function, reducing the destruction of cardiac myocytes, reducing collagen synthesis, inhibiting myocardial fibrosis and ventricular remodeling. Through the whole experiment, tanshinone IIA: puerarin = 1:1 is the best.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Oct 2019; 137:59-70
Gao S, Li L, Li L, Ni J, ... Mao J, Fan G
J Mol Cell Cardiol: 16 Oct 2019; 137:59-70 | PMID: 31629735
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Abstract

A knock-in mutation at cysteine 144 of TRIM72 is cardioprotective and reduces myocardial TRIM72 release.

Fillmore N, Casin KM, Sinha P, Sun J, ... Kohr MJ, Murphy E

TRIM72 is a membrane repair protein that protects against ischemia reperfusion (I/R) injury. We previously identified Cys (C144) on TRIM72 as a site of S-nitrosylation. To study the importance of C144, we generated a knock-in mouse with C144 mutated to a serine (TRIM72 C144S). We subjected ex vivo perfused mouse hearts to 20 min of ischemia followed by 90 min of reperfusion and observed less injury in TRIM72 C144S compared to WT hearts. Infarct size was smaller (54 vs 27% infarct size) and cardiac functional recovery (37 vs 62% RPP) was higher for the TRIM72 C144S mouse hearts. We also demonstrated that TRIM72 C144S hearts were protected against I/R injury using an in vivo LAD occlusion model. As TRIM72 has been reported to be released from muscle we tested whether C144 is involved in TRIM72 release. After I/R there was significantly less TRIM72 in the perfusate normalized to total released protein from the TRIM72 C144S compared to WT hearts, suggesting that C144 of TRIM72 regulates myocardial TRIM72 release during I/R injury. In addition to TRIM72\'s protective role in I/R injury, TRIM72 has also been implicated in cardiac hypertrophy and insulin resistance, and secreted TRIM72 has recently been shown to impair insulin sensitivity. However, insulin sensitivity (measured by glucose and insulin tolerance) of TRIM72 C144S mice was not impaired. Further, whole body metabolism, as measured using metabolic cages, was not different in WT vs TRIM72 C144S mice and we did not observe enhanced cardiac hypertrophy in the TRIM72 C144S mice. In agreement, protein levels of the TRIM72 ubiquitination targets insulin receptor β, IRS1, and focal adhesion kinase were similar between WT and TRIM72 C144S hearts. Overall, these data indicate that mutation of TRIM72 C144 is protective during I/R and reduces myocardial TRIM72 release without impairing insulin sensitivity or enhancing the development of hypertrophy.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:95-101
Fillmore N, Casin KM, Sinha P, Sun J, ... Kohr MJ, Murphy E
J Mol Cell Cardiol: 30 Oct 2019; 136:95-101 | PMID: 31536744
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Abstract

Comparative study on isolation and mitochondrial function of adult mouse and rat cardiomyocytes.

Liu B, Li A, Qin Y, Tian X, ... Jiang W, Gong G
Background
Cultured adult mouse and rat cardiomyocytes are the best and low-cost cell model for cardiac cellular physiology, pathology, drug toxicity screening, and intervention. The functions of mouse cardiomyocytes decline faster than rat cardiomyocytes in culture conditions. However, little is known about the difference of mitochondrial function between cultured mouse and rat myocytes.
Methods and results
A large number of adult mouse and rat cardiomyocytes were comparative isolated using a simple perfusion system. Cardiomyocytes mitochondrial functions were measured after 2 h, 1 day, 2 days, 3 days, and 4 days culture by monitoring mitoflashes. We found that the mitochondrial function of mouse myocytes was remarkedly declined on the third day. Then, we focused on the third day cultured mouse and rat myocytes, comparatively analyzing the respiration function and superoxide generation stimulated by pyruvate/malate/ADP and the mitochondrial permeability transition pore (mPTP) opening induction. Mouse myocytes showed lower respiration and mitoflash activity, but without the change of maximum uncoupled respiration when compared with rat myocytes. Although the response to superoxide production stimulated by respiration substrates was slower than rat myocytes, the basal superoxide generation is faster than the rat. The faster mitochondrial reactive oxygen species (ROS) generation of mouse myocytes upon laser stimulation triggered the faster mPTP opening compared with the rat. Finally, antioxidant MitoTEMPO pretreatment preserved the mitochondrial function of mouse myocytes on the third day.
Conclusions
The mitochondrial function and stability are different between cultured mouse and rat cardiac myocytes beyond 3 days even though they both belong to Muridae. Mitochondrial ROS impairs the mitochondrial functions of mouse cardiomyocytes on the third day. Suppressing superoxide maintained the mitochondrial function of mouse myocytes on the third day.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:64-71
Liu B, Li A, Qin Y, Tian X, ... Jiang W, Gong G
J Mol Cell Cardiol: 30 Oct 2019; 136:64-71 | PMID: 31521710
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Abstract

Enhancement of human iPSC-derived cardiomyocyte maturation by chemical conditioning in a 3D environment.

Huang CY, Maia-Joca RPM, Ong CS, Wilson I, ... Tomaselli GF, Reich DH

Recent advances in the understanding and use of pluripotent stem cells have produced major changes in approaches to the diagnosis and treatment of human disease. An obstacle to the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and drug discovery is their immature state relative to adult myocardium. We show the effects of a combination of biochemical factors, thyroid hormone, dexamethasone, and insulin-like growth factor-1 (TDI) on the maturation of hiPSC-CMs in 3D cardiac microtissues (CMTs) that recapitulate aspects of the native myocardium. Based on a comparison of the gene expression profiles and the structural, ultrastructural, and electrophysiological properties of hiPSC-CMs in monolayers and CMTs, and measurements of the mechanical and pharmacological properties of CMTs, we find that TDI treatment in a 3D tissue context yields a higher fidelity adult cardiac phenotype, including sarcoplasmic reticulum function and contractile properties consistent with promotion of the maturation of hiPSC derived cardiomyocytes.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 22 Oct 2019; epub ahead of print
Huang CY, Maia-Joca RPM, Ong CS, Wilson I, ... Tomaselli GF, Reich DH
J Mol Cell Cardiol: 22 Oct 2019; epub ahead of print | PMID: 31655038
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Abstract

Death receptor 5 contributes to cardiomyocyte hypertrophy through epidermal growth factor receptor transactivation.

Tanner MA, Thomas TP, Grisanti LA

Cardiomyocyte survival and death contributes to many cardiac diseases. A common mechanism of cardiomyocyte death is through apoptosis however, numerous death receptors (DR) have been virtually unstudied in the context of cardiovascular disease. Previous studies have identified TNF-related apoptosis inducing ligand (TRAIL) and its receptor, DR5, as being altered in a chronic catecholamine administration model of heart failure, and suggest a role of non-canonical signaling in cardiomyocytes. Furthermore, multiple clinical studies have identified TRAIL or DR5 as biomarkers in the prediction of severity and mortality following myocardial infarction and in heart failure development risk suggesting a role of DR5 signaling in the heart. While TRAIL/DR5 have been extensively studied as a potential cancer therapeutic due to their ability to selectively activate apoptosis in cancer cells, TRAIL and DR5 are highly expressed in the heart where their function is uncharacterized. However, many non-transformed cell types are resistant to TRAIL-induced apoptosis suggesting non-canonical functions in non-cancerous cell types. Our goal was to determine the role of DR5 in the heart with the hypothesis that DR5 does not induce cardiomyocyte apoptosis but initiates non-canonical signaling to promote cardiomyocyte growth and survival. Histological analysis of hearts from mice treated with a DR5 agonists showed increased hypertrophy with no differences in cardiomyocyte death, fibrosis or function. Mechanistic studies in the heart and isolated cardiomyocytes identified ERK1/2 activation with DR5 agonist treatment which contributed to hypertrophy. Furthermore, epidermal growth factor receptor (EGFR) was activated following DR5 agonist treatment through activation of MMP and HB-EGFR cleavage and specific inhibitors of MMP and EGFR prevented DR5-mediated ERK1/2 signaling and hypertrophy. Taken together, these studies identify a previously unidentified role for DR5 in the heart, which does not promote apoptosis but acts through non-canonical MMP-EGFR-ERK1/2 signaling mechanisms to contribute to cardiomyocyte hypertrophy.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:1-14
Tanner MA, Thomas TP, Grisanti LA
J Mol Cell Cardiol: 30 Oct 2019; 136:1-14 | PMID: 31473246
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Abstract

Cardiotoxicity screening of illicit drugs and new psychoactive substances (NPS) in human iPSC-derived cardiomyocytes using microelectrode array (MEA) recordings.

Zwartsen A, de Korte T, Nacken P, de Lange DW, Westerink RHS, Hondebrink L

The use of recreational drugs, including new psychoactive substances (NPS), is paralleled by emergency department visits of drug users with severe cardiotoxicity. Drug-induced cardiotoxicity can be the (secondary) result of increased norepinephrine blood concentrations, but data on potential drug-induced direct effects on cardiomyocyte function are scarce. The presence of hundreds of NPS therefore calls for efficient screening models to assess direct cardiotoxicity. We investigated effects of four reference compounds (3-30 nM dofetilide, nifedipine and isoproterenol, and 1-10 μM mexiletine) and six recreational drugs (0.01-100 μM cocaine, 0.01-1000 μM amphetamine, MDMA, 4-fluoroamphetamine, α-PVP and MDPV) on cardiomyocyte function (beat rate, spike amplitude and field potential duration (FPD ≈ QT interval in ECGs)), using Pluricyte® human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes cultured on ready-to-use CardioPlate™ multi-well microelectrode arrays (mwMEAs). Moreover, the effects of exposure to recreational drugs on cell viability were assessed. Effects of reference compounds were in accordance with the literature, indicating the presence of hERG potassium (dofetilide), sodium (mexiletine) and calcium (nifedipine) channels and α-adrenergic receptors (isoproterenol). All recreational drugs decreased the spike amplitude at 10-100 μM. All amphetamine-type stimulants and α-PVP decreased the beat rate at 300 μM, while cocaine and MDPV did so at 10 μM and 30 μM, respectively. All drugs increased the FPD, however at varying concentrations. MDMA, MDPV and amphetamine affected cardiomyocyte function at concentrations relevant for human exposure, while other drugs affected cardiomyocyte function only at higher concentrations (≥ 10 μM). Cell viability was only mildly affected at concentrations well above the lowest concentrations affecting cardiomyocyte function. We demonstrate that MEA recordings of hiPSC-derived cardiomyocytes enable screening for acute, direct effects on cardiomyocyte function. Our data further indicate that tachycardia in patients exposed to recreational drugs is likely due to indirect drug effects, while prolonged repolarization periods (prolonged QT interval) could (partly) result from direct drug effects on cardiomyocyte function.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:102-112
Zwartsen A, de Korte T, Nacken P, de Lange DW, Westerink RHS, Hondebrink L
J Mol Cell Cardiol: 30 Oct 2019; 136:102-112 | PMID: 31526813
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Abstract

Deficiency of nuclear receptor interaction protein leads to cardiomyopathy by disrupting sarcomere structure and mitochondrial respiration.

Yang KC, Chuang KW, Yen WS, Lin SY, ... Chen WP, Chen SL
Background
Cardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms.
Methods and results
NRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6 hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6 mice.
Conclusions
NRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Oct 2019; 137:9-24
Yang KC, Chuang KW, Yen WS, Lin SY, ... Chen WP, Chen SL
J Mol Cell Cardiol: 16 Oct 2019; 137:9-24 | PMID: 31629737
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Abstract

The evolutionarily conserved C-terminal peptide of troponin I is an independently configured regulatory structure to function as a myofilament Ca-desensitizer.

Wong S, Feng HZ, Jin JP

The C-terminal end segment of troponin subunit I (TnI) is a structure highly conserved among the three muscle type-specific isoforms and across vertebrate species. Partial deletion or point mutation in this segment impairs cardiac muscle relaxation. In the present study, we characterized the C-terminal 27 amino acid peptide of human cardiac TnI (HcTnI-C27) for its role in modulating muscle contractility. Biologically or chemically synthesized HcTnI-C27 peptide retains an epitope structure in physiological solutions similarly to that in intact TnI as recognized by an anti-TnI C-terminus monoclonal antibody (mAb TnI-1). Protein binding studies found that HcTnI-C27 retains the binding affinity for tropomyosin as previously shown with intact cardiac TnI. A restrictive cardiomyopathy mutation R192H in this segment abolishes the bindings to mAb TnI-1 and tropomyosin, demonstrating a pathogenic loss of function. Contractility studies using skinned muscle preparations demonstrated that addition of HcTnI-C27 peptide reduces the Ca-sensitivity of myofibrils without decreasing maximum force production. The results indicate that the C-terminal end segment of TnI is a regulatory element of troponin, which retains the native configuration in the form of free peptide to confer an effect on myofilament Ca-desensitization. Without negative inotropic impact, this short peptide may be developed into a novel reagent to selectively facilitate cardiac muscle relaxation at the activated state as a potential treatment for heart failure.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:42-52
Wong S, Feng HZ, Jin JP
J Mol Cell Cardiol: 30 Oct 2019; 136:42-52 | PMID: 31505197
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Abstract

Atorvastatin protects cardiomyocyte from doxorubicin toxicity by modulating survivin expression through FOXO1 inhibition.

Oh J, Lee BS, Lim G, Lim H, ... Chung JH, Kang SM
Background
Survivin has an anti-apoptotic effect against anthracycline-induced cardiotoxicity. Clinically, statin use is associated with a lower risk for heart failure in breast cancer patients with anthracycline chemotherapy. So, the purpose of our study was to investigate whether survivin mediates the protective effect of statin against anthracycline-induced cardiotoxicity.
Methods
Mice were treated once a week with 5 mg/kg doxorubicin for 4 weeks with or without atorvastatin 20 mg/kg every day then heart tissues were analyzed. Molecular and cellular biology analyses were performed with H9c2 cell lysates.
Results
Doxorubicin suppressed survivin expression via activation of FOXO1 in H9c2 cardiomyocytes. Whereas, atorvastatin inhibited FOXO1 by increasing phosphorylation and inhibiting nuclear localization. Doxorubicin induced FOXO1 binding to STAT3 and prevented STAT3 from interacting with Sp1. However, atorvastatin inhibited these interactions and stabilized STAT3/Sp1 transcription complex. Chromatin immunoprecipitation analysis demonstrated that doxorubicin decreased STAT3/Sp1 complex binding to survivin promoter, whereas atorvastatin stabilized this binding. In mouse model, atorvastatin rescued doxorubicin-induced reduction of survivin expression and of heart function measured by cardiac magnetic resonance imaging.
Conclusions
Our study suggested a new pathophysiologic mechanism that survivin mediated protective effect of atorvastatin against doxorubicin-induced cardiotoxicity via FOXO1/STAT3/Sp1 transcriptional network.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 18 Dec 2019; epub ahead of print
Oh J, Lee BS, Lim G, Lim H, ... Chung JH, Kang SM
J Mol Cell Cardiol: 18 Dec 2019; epub ahead of print | PMID: 31866378
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Abstract

Diastolic dysfunction is initiated by cardiomyocyte impairment ahead of endothelial dysfunction due to increased oxidative stress and inflammation in an experimental prediabetes model.

Waddingham MT, Sonobe T, Tsuchimochi H, Edgley AJ, ... Hamdani N, Pearson JT

Coronary microvessel endothelial dysfunction and nitric oxide (NO) depletion contribute to elevated passive tension of cardiomyocytes, diastolic dysfunction and predispose the heart to heart failure with preserved ejection fraction. We examined if diastolic dysfunction at the level of the cardiomyocytes precedes coronary endothelial dysfunction in prediabetes. Further, we determined if myofilaments other than titin contribute to impairment. Utilizing synchrotron microangiography we found young prediabetic male rats showed preserved dilator responses to acetylcholine in microvessels. Utilizing synchrotron X-ray diffraction we show that cardiac relaxation and cross-bridge dynamics are impaired by myosin head displacement from actin filaments particularly in the inner myocardium. We reveal that increased PKC activity and mitochondrial oxidative stress in cardiomyocytes contributes to rho-kinase mediated impairment of myosin head extension to actin filaments, depression of soluble guanylyl cyclase /PKG activity and consequently stiffening of titin in prediabetes ahead of coronary endothelial dysfunction.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 25 Oct 2019; epub ahead of print
Waddingham MT, Sonobe T, Tsuchimochi H, Edgley AJ, ... Hamdani N, Pearson JT
J Mol Cell Cardiol: 25 Oct 2019; epub ahead of print | PMID: 31669609
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Abstract

Conversion of human cardiac progenitor cells into cardiac pacemaker-like cells.

Raghunathan S, Islas JF, Mistretta B, Iyer D, ... Schwartz RJ, McConnell BK

We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Oct 2019; epub ahead of print
Raghunathan S, Islas JF, Mistretta B, Iyer D, ... Schwartz RJ, McConnell BK
J Mol Cell Cardiol: 30 Oct 2019; epub ahead of print | PMID: 31678351
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Abstract

Carbonic anhydrase II/sodium-proton exchanger 1 metabolon complex in cardiomyopathy of ob type 2 diabetic mice.

Jaquenod De Giusti C, Blanco PG, Lamas PA, Carrizo Velasquez F, ... Portiansky EL, Alvarez BV

Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na/H exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob) and control heterozygous littermates (ob). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob mice at 9 and 16 weeks. ob mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of obvs. ob mice, and augmented Ser phosphorylation on NHE1 in ob hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob mice. NHE1-dependent rate of intracellular pH (pH) normalization after transient acid loading of isolated cardiomyocytes was higher in ob mice vs. ob. NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob mice, which may lead to perturbation of pH and [Na] and [Ca] handling in these diseased hearts.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:53-63
Jaquenod De Giusti C, Blanco PG, Lamas PA, Carrizo Velasquez F, ... Portiansky EL, Alvarez BV
J Mol Cell Cardiol: 30 Oct 2019; 136:53-63 | PMID: 31518570
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Abstract

Doxorubicin induces cardiomyocyte pyroptosis via the TINCR-mediated posttranscriptional stabilization of NLR family pyrin domain containing 3.

Meng L, Lin H, Zhang J, Lin N, ... Chi J, Guo H
Aims
Doxorubicin (DOX), a widely used powerful chemotherapeutic component for cancer treatment, can give rise to severe cardiotoxicity that limits its clinical use. Pyroptosis is characterized by proinflammation and has been defined as a new type of programmed cell death in recent years. However, whether the DOX-induced cardiotoxicity is related to pyroptosis, and if so, which genes are involved in this process is largely unknown. In this study, we sought to identify the effect of DOX on cardiomyocyte pyroptosis and further reveal the underlying regulatory mechanism.
Methods and results
In vitro and in vivo experiments showed that DOX treatment induced cardiomyocyte pyroptosis as evidenced by increased cell death and upregulated expression levels of NLR family pyrin domain containing 3 (NLRP3), caspase-3, IL-1β, IL-18 and GMDSD-N. Inhibition of NLRP3 rescued the DOX-induced pyroptosis. qRT-PCR showed that TINCR lncRNA was upregulated by DOX treatment and knockdown of TINCR reversed the DOX-induced pyroptosis both in vitro and in vivo. Mechanistic investigations revealed that TINCR increased NLRP3 level via recruiting IGF2BP1 to enhance NLRP3 mRNA. And the effect of TINCR on cardiomyocyte pyroptosis was attenuated by the inhibition of NLRP3 or IGF2BP1. Finally, TINCR was not involved in DOX-induced pyroptosis in cancer cells.
Conclusion
TINCR mediates the DOX-induced cardiotoxicity and pyroptosis in an IGF2BP1-dependent manner. Therefore, TINCR may serve as a promising therapeutic target to overcome the cardiotoxicity of chemotherapy for cancer therapy.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:15-26
Meng L, Lin H, Zhang J, Lin N, ... Chi J, Guo H
J Mol Cell Cardiol: 30 Oct 2019; 136:15-26 | PMID: 31445005
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Abstract

MicroRNA-125a-3p affects smooth muscle cell function in vascular stenosis.

Hu W, Chang G, Zhang M, Li Y, ... Wen D, Wang S
Aims
Many studies have indicated that microRNAs are closely related to the process of peripheral arterial disease (PAD). Previously, we found that microRNA-125a-3p (miR-125a-3p) in restenotic arteries after interventional therapy of lower extremity vessels was notably decreased compared with that of normal control arteries. However, its role in the development of vascular stenosis is not yet clearly understood. The purpose of this study was to investigate the expression, regulatory mechanism and function of miR-125a-3p in the process of vascular stenosis.
Methods and results
Quantitative reverse-transcription polymerase chain reaction assays indicated that miR-125a-3p in restenotic arteries after interventional therapy was significantly lower than that in normal control arteries. Immunofluorescence and in situ hybridization co-staining assays in arterial sections demonstrated that miR-125a-3p was mainly expressed in the medial smooth muscle layer. Transfection of miR-125a-3p mimics into cultured vascular smooth muscle cells (VSMCs) effectively inhibited cell proliferation and migration. Then, western blot and luciferase activity assays showed that recombinant human mitogen-activated protein kinase 1 (MAPK1) was a functional target of miR-125a-3p and was involved in miR-125a-3p-mediated cell effects. Finally, the lentiviral infection of miR-125a-3p in balloon-injured rat carotid vascular walls showed that miR-125a-3p overexpression significantly reduced the probability of neointimal membrane production.
Conclusions
miR-125a-3p can effectively inhibit the function of VSMCs and the occurrence of vascular stenosis by targeting MAPK1. This study introduces a new molecular mechanism of PAD. We show that regulation of the miR-125a-3p level has the potential to provide a new treatment for PAD and other proliferative vascular diseases.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Oct 2019; 136:85-94
Hu W, Chang G, Zhang M, Li Y, ... Wen D, Wang S
J Mol Cell Cardiol: 30 Oct 2019; 136:85-94 | PMID: 31499051
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Abstract

Shp2 in myocytes is essential for cardiovascular and neointima development.

Gong H, Ni J, Xu Z, Huang J, ... Cheng H, Ke Y

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Shp2, cause Noonan syndrome and LEOPARD syndrome, inherited multifaceted diseases including cardiac and vascular defects. However, the function of Shp2 in blood vessels, especially in vascular smooth muscle cells (VSMCs), remains largely unknown. We generated mice in which Shp2 was specifically deleted in VSMCs and embryonic cardiomyocytes using the SM22α-Cre transgenic mouse line. Conditional Shp2 knockout resulted in massive hemorrhage, cardiovascular defects and embryonic lethality at the late embryonic developmental stage (embryonic date 16.5). The thinning of artery walls in Shp2-knockout embryos was due to decreased VSMC number and reduced extracellular matrix deposition. Myocyte proliferation was decreased in Shp2-knockout arteries and hearts. Importantly, cardiomyocyte-specific Shp2-knockout did not cause similar vascular defects. Shp2 was required for TGFβ1-induced expression of ECM components, including collagens in VSMCs. In addition, collagens were sufficient to promote Shp2-inefficient VSMC proliferation. Finally, Shp2 was deleted in adult mouse VSMCs by using SMMHC-CreER and tamoxifen induction. Shp2 deletion dramatically inhibited the expression of ECM components, proliferation of VSMCs and neointima formation in a carotid artery ligation model. Therefore, Shp2 is required for myocyte proliferation in cardiovascular development and vascular remodeling through TGFβ1-regulated collagen synthesis.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 17 Oct 2019; 137:71-81
Gong H, Ni J, Xu Z, Huang J, ... Cheng H, Ke Y
J Mol Cell Cardiol: 17 Oct 2019; 137:71-81 | PMID: 31634485
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Abstract

Identification and characterization of self-association domains on small ankyrin 1 isoforms.

Subramaniam J, Yang P, McCarthy MJ, Cunha SR

In striated muscles, the large scaffolding protein obscurin and a small SR-integral membrane protein sAnk1.5 control the retention of longitudinal SR across the sarcomere. How a complex of these proteins facilitates localization of longitudinal SR has yet to be resolved, but we hypothesize that obscurin interacts with a complex of sAnk1.5 proteins. To begin to address this hypothesis, we demonstrate that sAnk1.5 interacts with itself and identify two domains mediating self-association. Specifically, we show by co-precipitation and FLIM-FRET analysis that sAnk1.5 and another small AnkR isoform (sAnk1.6) interact with themselves and each other. We demonstrate that obscurin interacts with a complex of sAnk1.5 proteins and that this complex formation is enhanced by obscurin-binding. Using FLIM-FRET analysis, we show that obscurin interacts with sAnk1.5 alone and with sAnk1.6 in the presence of sAnk1.5. We find that sAnk1.5 self-association is disrupted by mutagenesis of residues Arg64-Arg69, residues previously associated with obscurin-binding. Molecular modeling of two interacting sAnk1.5 monomers facilitated the identification of Gly31-Val36 as an additional site of interaction, which was subsequently corroborated by co-precipitation and FLIM-FRET analysis. In closing, these results support a model in which sAnk1.5 forms large oligomers that interact with obscurin to facilitate the retention of longitudinal SR throughout skeletal and cardiac myocytes.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 04 Feb 2020; epub ahead of print
Subramaniam J, Yang P, McCarthy MJ, Cunha SR
J Mol Cell Cardiol: 04 Feb 2020; epub ahead of print | PMID: 32035138
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Abstract

Pseudouridine and N-formylmethionine associate with left ventricular mass index: Metabolome-wide association analysis of cardiac remodeling.

Razavi AC, Bazzano LAA, He J, Li S, ... Kinchen J, Kelly TN
Background
Heart failure (HF) is the fastest growing form of cardiovascular disease both nationally and globally, underlining a need to phenotype subclinical HF intermediaries to improve primary prevention.
Objectives
We aimed to identify novel metabolite associations with left ventricular (LV) remodeling, one upstream HF intermediary, among a community-based cohort of individuals.
Methods
We examined 1052 Bogalusa Heart Study participants (34.98% African American, 57.41% female, aged 33.6-57.5 years). Measures of LV mass and relative wall thickness (RWT) were obtained using two-dimensional-guided echocardiographic measurements via validated eqs. LV mass was indexed to height to calculate left ventricular mass index (LVMI). Untargeted metabolomic analysis of fasting serum samples was conducted. In combined and ethnicity-stratified analyses, multivariable linear and multinomial logistic regression models tested the associations of metabolites with the continuous LVMI and RWT and categorical LV geometry phenotypes, respectively, after adjusting for demographic and traditional cardiovascular disease risk factors.
Results
Pseudouridine (B = 1.38; p = 3.20 × 10) and N-formylmethionine (B = 1.65; 3.30 × 10) were significantly associated with LVMI in the overall sample as well significant in Caucasians, with consistent effect direction and nominal significance (p < .05) in African Americans. Upon exclusion of individuals with self-report myocardial infarction or congestive HF, we similarly observed a 1.33 g/m and 1.52 g/m higher LVMI for each standard deviation increase in pseudouridine and N-formylmethionine, respectively. No significant associations were observed for metabolites with RWT or categorical LV remodeling outcomes.
Conclusions
The current analysis identified novel associations of pseudouridine and N-formylmethionine with LVMI, suggesting that mitochondrial-derived metabolites may serve as early biomarkers for LV remodeling and subclinical HF.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 10 Feb 2020; epub ahead of print
Razavi AC, Bazzano LAA, He J, Li S, ... Kinchen J, Kelly TN
J Mol Cell Cardiol: 10 Feb 2020; epub ahead of print | PMID: 32057737
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Abstract

Salvianolic acid B improves myocardial function in diabetic cardiomyopathy by suppressing IGFBP3.

Li CL, Liu B, Wang ZY, Xie F, ... Zhang MX, Liu DS
Background
Salvianolic acid B (Sal B) is the representative component of phenolic acids derived from the dried root and rhizome of Salvia miltiorrhiza Bge. (Labiatae), which has been widely used for the treatment of cardiovascular and cerebrovascular diseases. However, the effect of Sal B diabetic cardiomyopathy (DCM) is still unclear.
Methods
Type 1 diabetes mellitus was induced in C57BL/6 J mice by streptozotocin (STZ) treatment, whereas meanwhile Salvianolic Acid B (Sal B (15 or 30 mg/kg/d) was intraperitoneally injected for 16 weeks. At the end of this period, cardiac function was assessed by echocardiography, and total collagen deposition was evaluated by Masson\'s trichrome and Picrosirius Red staining. Human umbilical vein endothelial cells exposed to hypoxia were used to investigate the effect of different doses of Sal B on angiogenesis and tube formation in vitro. Transcriptome sequencing was performed to identify potential targets of Sal B.
Results
Sal B ameliorated left ventricular dysfunction and remodeling, and decreased collagen deposition in the heart of diabetic mice. Administration of Sal B increased the expression of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and VEGFA in a dose-dependent manner and promoted angiogenesis both in vivo and in vitro. Furthermore, Sal B reduced HG-induced insulin-like growth factor-binding protein 3 (IGFBP3) expression, induced the phosphorylation of extracellular signal-regulated protein kinase and protein kinase B (AKT) activities, enhanced cell proliferation, and activated VEGFR2/VEGFA signaling in endothelial cells. The underlying mechanisms involve SalB that enhances IGFBP3 promoter DNA methylation and induce nuclear translocation of IGFBP3 in HUVECs under hypoxia.
Conclusions
Sal B promoted angiogenesis and alleviated cardiac fibrosis and cardiac remodeling in DCM by suppressing IGFBP3.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 22 Jan 2020; epub ahead of print
Li CL, Liu B, Wang ZY, Xie F, ... Zhang MX, Liu DS
J Mol Cell Cardiol: 22 Jan 2020; epub ahead of print | PMID: 31982427
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Abstract

Metabolic regulation of Kv channels and cardiac repolarization by Kvβ2 subunits.

Kilfoil PJ, Chapalamadugu KC, Hu X, Zhang D, ... Tipparaju SM, Nystoriak MA

Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvβ subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvβ subunits regulate myocardial K currents and repolarization. Here, we tested the hypothesis that Kvβ2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvβ2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvβ2) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced I, I, and I densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvβ2 mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P) accelerated Kv inactivation in wild type, but not Kvβ2 myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH] and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvβ2, hearts. Taken together, these results suggest that Kvβ2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate I and I, and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 18 Oct 2019; 137:93-106
Kilfoil PJ, Chapalamadugu KC, Hu X, Zhang D, ... Tipparaju SM, Nystoriak MA
J Mol Cell Cardiol: 18 Oct 2019; 137:93-106 | PMID: 31639389
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Abstract

Transcriptome signature of ventricular arrhythmia in dilated cardiomyopathy reveals increased fibrosis and activated TP53.

Haywood ME, Cocciolo A, Porter KF, Dobrinskikh E, ... Mestroni L, Taylor MRG
Aims
One-third of DCM patients experience ventricular tachycardia (VT), but a clear biological basis for this has not been established. The purpose of this study was to identify transcriptome signatures and enriched pathways in the hearts of dilated cardiomyopathy (DCM) patients with VT.
Methods and results
We used RNA-sequencing in explanted heart tissue from 49 samples: 19 DCM patients with VT, 16 DCM patients without VT, and 14 non-failing controls. We compared each DCM cohort to the controls and identified the genes that were differentially expressed in DCM patients with VT but not without VT. Differentially expressed genes were evaluated using pathway analysis, and pathways of interest were investigated by qRT-PCR validation, Western blot, and microscopy. There were 590 genes differentially expressed in DCM patients with VT that are not differentially expressed in patients without VT. These genes were enriched for genes in the TGFß1 and TP53 signaling pathways. Increased fibrosis and activated TP53 signaling was demonstrated in heart tissue of DCM patients with VT.
Conclusions
Our study supports that distinct biological mechanisms distinguish ventricular arrhythmia in DCM patients.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Jan 2020; epub ahead of print
Haywood ME, Cocciolo A, Porter KF, Dobrinskikh E, ... Mestroni L, Taylor MRG
J Mol Cell Cardiol: 16 Jan 2020; epub ahead of print | PMID: 31958463
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Impact:
Abstract

Progranulin deficiency leads to enhanced age-related cardiac hypertrophy through complement C1q-induced β-catenin activation.

Zhu Y, Ohama T, Kawase R, Chang J, ... Sakata Y, Yamashita S
Aims
Age-related cardiac hypertrophy and subsequent heart failure are predicted to become increasingly serious problems in aging populations. Progranulin (PGRN) deficiency is known to be associated with accelerated aging in the brain. We aimed to evaluate the effects of PGRN deficiency on cardiac aging, including left ventricular hypertrophy.
Methods and results
Echocardiography was performed on wild-type (WT) and PGRN-knockout (KO) mice every 3 months from 3 to 18 months of age. Compared to that of WT mice, PGRN KO mice exhibited age-dependent cardiac hypertrophy and cardiac dysfunction at 18 months. Morphological analyses showed that the heart weight to tibia length ratio and cross-sectional area of cardiomyocytes at 18 months were significantly increased in PGRN KO mice relative to those in WT mice. Furthermore, accumulation of lipofuscin and increases in senescence markers were observed in the hearts of PGRN KO mice, suggesting that PGRN deficiency led to enhanced aging of the heart. Enhanced complement C1q (C1q) and activated β-catenin protein expression levels were also observed in the hearts of aged PGRN KO mice. Treatment of PGRN-deficient cardiomyocytes with C1q caused β-catenin activation and cardiac hypertrophy. Blocking C1q-induced β-catenin activation in PGRN-depleted cardiomyocytes attenuated hypertrophic changes. Finally, we showed that C1 inhibitor treatment reduced cardiac hypertrophy and dysfunction in old KO mice, possibly by reducing β-catenin activation. These results suggest that C1q is a crucial regulator of cardiac hypertrophy induced by PGRN ablation.
Conclusion
The present study demonstrates that PGRN deficiency enhances age-related cardiac hypertrophy via C1q-induced β-catenin activation. PGRN is a potential therapeutic target to prevent cardiac hypertrophy and dysfunction.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 18 Dec 2019; epub ahead of print
Zhu Y, Ohama T, Kawase R, Chang J, ... Sakata Y, Yamashita S
J Mol Cell Cardiol: 18 Dec 2019; epub ahead of print | PMID: 31866375
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Impact:
Abstract

High hydrostatic pressure induces atrial electrical remodeling through angiotensin upregulation mediating FAK/Src pathway activation.

Li X, Deng CY, Xue YM, Yang H, ... Wu SL, Rao F

Hypertension is an independent risk factor for atrial fibrillation (AF), although its specific mechanisms remain unclear. Previous research has been focused on cyclic stretch, ignoring the role of high hydrostatic pressure. The present study aimed to explore the effect of high hydrostatic pressure stimulation on electrical remodeling in atrial myocytes and its potential signaling pathways. Experiments were performed on left atrial appendages from patients with chronic AF or sinus rhythm, spontaneously hypertensive rats (SHRs) treated with or without valsartan (10 mg/kg/day) and HL-1 cells were exposed to high hydrostatic pressure using a self-developed device. Whole-cell patch-clamp recordings and western blots demonstrated that the amplitudes of I, I, and I were reduced in AF patients with corresponding changes in protein expression. Angiotensin protein levels increased and Ang1-7 decreased, while focal adhesion kinase (FAK) and Src kinase were enhanced in atrial tissue from AF patients and SHRs. After rapid atrial pacing, AF inducibility in SHR was significantly higher, accompanied by a decrease in I, upregulation of I and I, and a shortened action potential duration. Angiotensin upregulation and FAK/Src activation in SHR were inhibited by angiotensin type 1 receptor inhibitor valsartan, thus, preventing electrical remodeling and reducing AF susceptibility. These results were verified in HL-1 cells treated with high hydrostatic pressure, and demonstrated that electrical remodeling regulated by the FAK-Src pathway could be modulated by valsartan. The present study indicated that high hydrostatic pressure stimulation increases AF susceptibility by activating the renin-angiotensin system and FAK-Src pathway in atrial myocytes.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 28 Jan 2020; epub ahead of print
Li X, Deng CY, Xue YM, Yang H, ... Wu SL, Rao F
J Mol Cell Cardiol: 28 Jan 2020; epub ahead of print | PMID: 32006532
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Impact:
Abstract

Age- and sex-dependent differences in extracellular matrix metabolism associate with cardiac functional and structural changes.

Grilo GA, Shaver PR, Stoffel HJ, Morrow CA, ... Iyer RP, de Castro Brás LE

Age-related remodeling of the heart causes structural and functional changes in the left ventricle (LV) that are associated with a high index of morbidities and mortality worldwide. Some cardiac pathologies in the elderly population vary between genders revealing that cardiac remodeling during aging may be sex-dependent. Herein, we analyzed the effects of cardiac aging in male and female C57Bl/6 mice in four age groups, 3, 6, 12, and 18 month old (n = 6-12 animals/sex/age), to elucidate which age-related characteristics of LV remodeling are sex-specific. We focused particularly in parameters associated with age-dependent remodeling of the LV extracellular matrix (ECM) that are involved in collagen metabolism. LV function and anatomical structure were assessed both by conventional echocardiography and strain analysis. We then measured ECM proteins that directly affect LV contractility and remodeling. All data were analyzed across ages and between sexes and were directly linked to LV functional changes. Echocardiography confirmed an age-dependent decrease in chamber volumes and LV internal diameters, indicative of concentric remodeling. As in humans, animals displayed preserved ejection fraction with age. Notably, changes to chamber dimensions and volumes were temporally distinct between sexes. Complementary to the traditional echocardiography, speckle tracking echocardiography (STE) revealed that circumferential strain rate declined in 18 month old females, compared to younger animals, but not in males, suggesting STE as an earlier indicator for changes in cardiac function between sexes. Age-dependent collagen deposition and expression in the endocardium did not differ between sexes; however, other factors involved in collagen metabolism were sex-specific. Specifically, while decorin, osteopontin, Cthrc1, and Ddr1 expression were age-dependent but sex-independent, periostin, lysyl oxidase, and Mrc2 displayed age-dependent and sex-specific differences. Moreover, our data also suggest that with age males and females have distinct TGFβ signaling pathways. Overall, our results give evidence of sex-specific molecular changes during physiological cardiac remodeling that associate with age-dependent structural and functional dysfunction. These data highlight the importance of including sex-differences analysis when studying cardiac aging.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 20 Jan 2020; epub ahead of print
Grilo GA, Shaver PR, Stoffel HJ, Morrow CA, ... Iyer RP, de Castro Brás LE
J Mol Cell Cardiol: 20 Jan 2020; epub ahead of print | PMID: 31978395
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Impact:
Abstract

Exosomal non-coding RNAs (Exo-ncRNAs) in cardiovascular health.

De Giusti CJ, Santalla M, Das S

Extracellular vesicles (EVs) play a role in the pathophysiological processes and in different diseases, including cardiovascular disease. Out of several categories of EVs, exosomes (smallest - 30 to 150 nm) are gaining most of the focus as the next generation of biomarkers and in therapeutic strategies. This is because exosomes can be differentiated from other types of EVs based on the expression of tetraspanin molecules on the surface. More importantly, exosomes can be traced back to the cell of origin by identifying the unique cellular marker(s) on the exosomal surface. Recently, several researchs have demonstrated an important and underappreciated mechanism of paracrine cell-cell communication involving exosomal transfer, and its subsequent functional impact on recipient cells. Exosomes are enriched in proteins, mRNAs, miRNAs, and other non-coding RNAs, which can potentially alter myocardial function. Additionally, different stages of tissue damage can also be identified by measuring these bioactive molecules in the circulation. There are several aspects of this new concept still unknown. Therefore, in this review, we have summarized the knowledge we have so far and highlighted the potential of this novel concept of next generation biomarkers and therapeutic intervention.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 24 Oct 2019; epub ahead of print
De Giusti CJ, Santalla M, Das S
J Mol Cell Cardiol: 24 Oct 2019; epub ahead of print | PMID: 31669445
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Abstract

Late sodium current in human, canine and guinea pig ventricular myocardium.

Horváth B, Hézső T, Szentandrássy N, Kistamás K, ... Varró A, Nánási PP

Although late sodium current (I) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of I in canine and guinea pig ventricular cells and compare them to I recorded in undiseased human hearts. I was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human I monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine I could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo I profile in guinea pig was due to the slower decay of I in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of I. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of I. These results should be taken into account when pharmacological studies with I are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with I inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of I in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Jan 2020; epub ahead of print
Horváth B, Hézső T, Szentandrássy N, Kistamás K, ... Varró A, Nánási PP
J Mol Cell Cardiol: 16 Jan 2020; epub ahead of print | PMID: 31958464
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Abstract

Programmed death-ligand 1 triggers PASMCs pyroptosis and pulmonary vascular fibrosis in pulmonary hypertension.

Zhang M, Xin W, Yu Y, Yang X, ... Wang X, Zhu D

Pyroptosis is a pro-inflammatory form of programmed cell death, whose genesis directly depended on caspase-1 activation. Pulmonary hypertension (PH) is a disease characterized, in part, by vascular fibrosis. Up to now, there is no report on the relationship between pyroptosis and vascular fibrosis in PH. Here, we confirmed that pyroptosis had occurred in the media of pulmonary arteries in two PH rat models and hypoxic human pulmonary arterial smooth muscle cells (hPASMCs). Caspase-1 inhibition attenuated the pathogenesis of PH, as assessed by vascular remodeling, right ventricular systolic pressure, right ventricle hypertrophy and hemodynamic parameters of pulmonary vasculature. Moreover, caspase-1 inhibition suppressed pulmonary vascular fibrosis as demonstrated by Masson staining, as well as immunohistochemistry and Western blot analysis of fibrillar collagen. In addition, Programmed death-ligand 1 (PD-L1) was markedly increased in PH, which was regulated by the transcription factor STAT1. Furthermore, PD-L1 knockdown in hPASMCs repressed the onset of hypoxia-induced pyroptosis and fibrosis. Overall, these data identify a critical STAT1-dependent posttranscriptional modification that promotes PD-L1 expression in the pyroptosis of PASMCs to modulate pulmonary vascular fibrosis and accelerate the progression of PH.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 12 Nov 2019; epub ahead of print
Zhang M, Xin W, Yu Y, Yang X, ... Wang X, Zhu D
J Mol Cell Cardiol: 12 Nov 2019; epub ahead of print | PMID: 31733200
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Abstract

Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs.

Stroik DR, Ceholski DK, Bidwell PA, Mleczko J, ... Cornea RL, Thomas DD

There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLB) that were engineered to compete with the binding of inhibitory wild-type PLB (PLB). Our therapeutic strategy is to relieve PLB inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLB (L31A/I40A) that effectively competes with PLB binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLB enhances SERCA Ca-ATPase activity by increasing enzyme Ca affinity (1/K) in PLB-inhibited HEK cell homogenates. For an initial assessment of PLB physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLB enhances the amplitude of SR Ca release and the rate of SR Ca re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLB rescues arrhythmic Ca transients and alleviates decreased Ca transport. Thus, we propose that PLB transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca transport and thus contractility in underlying systolic heart failure.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 17 Nov 2019; epub ahead of print
Stroik DR, Ceholski DK, Bidwell PA, Mleczko J, ... Cornea RL, Thomas DD
J Mol Cell Cardiol: 17 Nov 2019; epub ahead of print | PMID: 31751570
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Abstract

Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study.

Fan C, Fast VG, Tang Y, Zhao M, ... Zhu W, Zhang J
Background
Cardiomyocytes that have been differentiated from CCND2-overexpressing human induced-pluripotent stem cells (hiPSC-CCND2 CMs) can proliferate when transplanted into mouse hearts after myocardial infarction (MI). However, it is unknown whether remuscularization can replace the thin LV scar and if the large muscle graft can electrophysiologically synchronize to the recipient myocardium. Our objectives are to evaluate the structural and functional potential of hiPSC-CCND2 CMs in replacing the LV thin scar.
Methods
NOD/SCID mice were treated with hiPSC-CCND2 CMs (i.e., the CCND2 group), hiPSC-CCND2 CMs (the CCND2 group), or an equal volume of PBS immediately after experimentally-induced myocardial infarction. The treatments were administered to one site in the infarcted zone (IZ), two sites in the border zone (BZ), and a fourth group of animals underwent Sham surgery.
Results
Six months later, engrafted cells occupied >50% of the scarred region in CCND2 animals, and exceeded the number of engrafted cells in CCND2 animals by ~8-fold. Engrafted cells were also more common in the IZ than in the BZ for both cell-treatment groups. Measurements of cardiac function, infarct size, wall thickness, and cardiomyocyte hypertrophy were significantly improved in CCND2 animals compared to animals from the CCND2 or PBS-treatment groups. Measurements in the CCND2 and PBS groups were similar, and markers for cell cycle activation and proliferation were significantly higher in hiPSC-CCND2 CMs than in hiPSC-CCND2 CMs. Optical mapping of action potential propagation indicated that the engrafted hiPSC-CCND2 CMs were electrically coupled to each other and to the cells of the native myocardium. No evidence of tumor formation was observed in any animals.
Conclusions
Six months after the transplantation, CCND2-overexpressing hiPSC-CMs proliferated and replaced >50% of the myocardial scar tissue. The large graft hiPSC-CCND2 CMs also electrically integrated with the host myocardium, which was accompanied by a significant improvement in LV function.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Oct 2019; 137:25-33
Fan C, Fast VG, Tang Y, Zhao M, ... Zhu W, Zhang J
J Mol Cell Cardiol: 16 Oct 2019; 137:25-33 | PMID: 31629738
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Abstract

Diaphragm weakness and proteomics (global and redox) modifications in heart failure with reduced ejection fraction in rats.

Kelley RC, McDonagh B, Brumback B, Walter GA, Vohra R, Ferreira LF

Inspiratory dysfunction occurs in patients with heart failure with reduced ejection fraction (HFrEF) in a manner that depends on disease severity and by mechanisms that are not fully understood. In the current study, we tested whether HFrEF effects on diaphragm (inspiratory muscle) depend on disease severity and examined putative mechanisms for diaphragm abnormalities via global and redox proteomics. We allocated male rats into Sham, moderate (mHFrEF), or severe HFrEF (sHFrEF) induced by myocardial infarction and examined the diaphragm muscle. Both mHFrEF and sHFrEF caused atrophy in type IIa and IIb/x fibers. Maximal and twitch specific forces (N/cm) were decreased by 19 ± 10% and 28 ± 13%, respectively, in sHFrEF (p < .05), but not in mHFrEF. Global proteomics revealed upregulation of sarcomeric proteins and downregulation of ribosomal and glucose metabolism proteins in sHFrEF. Redox proteomics showed that sHFrEF increased reversibly oxidized cysteine in cytoskeletal and thin filament proteins and methionine in skeletal muscle α-actin (range 0.5 to 3.3-fold; p < .05). In conclusion, fiber atrophy plus contractile dysfunction caused diaphragm weakness in HFrEF. Decreased ribosomal proteins and heighted reversible oxidation of protein thiols are candidate mechanisms for atrophy or anabolic resistance as well as loss of specific force in sHFrEF.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 04 Feb 2020; epub ahead of print
Kelley RC, McDonagh B, Brumback B, Walter GA, Vohra R, Ferreira LF
J Mol Cell Cardiol: 04 Feb 2020; epub ahead of print | PMID: 32035137
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Abstract

Large scale, unbiased analysis of elementary calcium signaling events in cardiac myocytes.

Tian Q, Schröder L, Schwarz Y, Flockerzi A, ... Bruns D, Lipp P

The identification of spatiotemporally restricted Ca signals, Ca sparks, was instrumental for our understanding of cardiac Ca homeostasis. High-speed 2D confocal imaging enables acquisition of such Ca sparks with high-content information but their full appreciation is constrained by the lack of unbiased and easy-to-use analysis tools. We developed a software toolset for unbiased and automatic Ca spark analysis for huge data sets of subcellular Ca signals. iSpark was developed to be scanner and detector independent. In myocytes from hearts subjected to various degrees of hypertrophy we acquired >5.000.000 Ca sparks from 14 mice. The iSpark-enabled analysis of this large Ca spark data set showed that the highly organized distribution of Ca sparks present in healthy cells disarrayed concomitant with the development of aberrant transverse tubules and disease severity. Thus, iSpark represents a versatile and universal tool for analyzing local Ca signaling in healthy as well as diseased, aberrant local Ca signal transduction. The results from the unbiased analysis of large data sets provide a deeper insight into possible mechanisms contributing to the onset and progression of cardiac diseases such as hypertrophy.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:79-89
Tian Q, Schröder L, Schwarz Y, Flockerzi A, ... Bruns D, Lipp P
J Mol Cell Cardiol: 29 Sep 2019; 135:79-89 | PMID: 31419438
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Abstract

Homocysteine accelerates atherosclerosis by inhibiting scavenger receptor class B member1 via DNMT3b/SP1 pathway.

Guo W, Zhang H, Yang A, Ma P, ... Nie L, Jiang Y

Homocysteine (Hcy) is an independent risk factor for atherosclerosis, which is characterized by lipid accumulation in the atherosclerotic plaque. Increasing evidence supports that as the main receptor of high-density lipoprotein, scavenger receptor class B member 1 (SCARB1) is protective against atherosclerosis. However, the underlying mechanism regarding it in Hcy-mediated atherosclerosis remains unclear. Here, we found the remarkable inhibition of SCARB1 expression in atherosclerotic plaque and Hcy-treated foam cells, whereas overexpression of SCARB1 can suppress lipid accumulation in foam cells following Hcy treatment. Analysis of SCARB1 promoter showed that no significant change of methylation level was observed both in vivo and in vitro under Hcy treatment. Moreover, it was found that the negative regulation of DNMT3b on SCARB1 was due to the decreased recruitment of SP1 to SCARB1 promoter. Thus, we concluded that inhibition of SCARB1 expression induced by DNMT3b at least partly accelerated Hcy-mediated atherosclerosis through promoting lipid accumulation in foam cells, which was attributed to the decreased binding of SP1 to SCARB1 promoter. In our point, these findings will provide novel insight into an epigenetic mechanism for atherosclerosis.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 12 Nov 2019; epub ahead of print
Guo W, Zhang H, Yang A, Ma P, ... Nie L, Jiang Y
J Mol Cell Cardiol: 12 Nov 2019; epub ahead of print | PMID: 31733201
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Abstract

Sam68 impedes the recovery of arterial injury by augmenting inflammatory response.

Han S, Xu S, Zhou J, Qiao A, ... Zhang J, Qin G
Objective
The role of Src-associated-in-mitosis-68-kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 promotes TNF-α-induced NF-κB activation in fibroblasts. Here we sought to dissect the molecular mechanism by which Sam68 regulates NF-κB signaling and its functional significance in vascular injury.
Approach and results
The endothelial denudation injury was induced in the carotid artery of Sam68-null (Sam68) and WT mice. Sam68 mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines in the injured vessels. Remarkably, the ameliorated vascular remodeling was recapitulated in WT mice after receiving transplantation of bone marrow (BM) from Sam68 mice, suggesting the effect was attributable to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α-induced expression of TNF-α, IL-1β, and IL-6 and in the level of nuclear phospho-p65, indicating attenuated NF-κB activation; and these results were confirmed in peritoneal and BM-derived macrophages of Sam68 vs. WT mice. Furthermore, co-immunoprecipitation and mass-spectrometry identified Filamin A (FLNA) as a novel Sam68-interacting protein upon TNF-α treatment. Loss- and gain-of-function experiments suggest that Sam68 and FLNA are mutually dependent for NF-κB activation and pro-inflammatory cytokine expression, and that the N-terminus of Sam68 is required for TRAF2-FLNA interaction.
Conclusions
Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery by interacting with FLNA to stabilize TRAF2 on the cytoskeleton and consequently potentiate NF-κB signaling.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 18 Oct 2019; 137:82-92
Han S, Xu S, Zhou J, Qiao A, ... Zhang J, Qin G
J Mol Cell Cardiol: 18 Oct 2019; 137:82-92 | PMID: 31639388
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Abstract

Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes.

Ashwood C, Waas M, Weerasekera R, Gundry RL

Cell surface glycoproteins play critical roles in maintaining cardiac structure and function in health and disease and the glycan-moiety attached to the protein is critical for proper protein folding, stability and signaling (Ajit Varki [1]). However, despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. In the current study, we used porous graphitized carbon liquid chromatography interfaced with mass spectrometry (PGC-LC-MS) to generate glycan structure libraries for primary human heart tissue homogenate, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM). Altogether, we established the first reference structure libraries of the cardiac glycome containing 265 N- and O-glycans. Comparing the N-glycome of CM enriched from primary heart tissue to that of heart tissue homogenate, the same pool of N-glycan structures was detected in each sample type but the relative signal of 21 structures significantly differed between samples, with the high mannose class increased in enriched CM. Moreover, by comparing primary CM to hiPSC-CM collected during 20-100 days of differentiation, dynamic changes in the glycan profile throughout in vitro differentiation were observed and differences between primary and hiPSC-CM were revealed. Namely, >30% of the N-glycome significantly changed across these time-points of differentiation and only 23% of the N-glycan structures were shared between hiPSC-CM and primary CM. These observations are an important complement to current genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. Finally, these data are expected to support future regenerative medicine efforts by informing targets for evaluating the immunogenic potential of hiPSC-CM and harnessing differences between immature, proliferative hiPSC-CM and adult primary CM.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 19 Jan 2020; epub ahead of print
Ashwood C, Waas M, Weerasekera R, Gundry RL
J Mol Cell Cardiol: 19 Jan 2020; epub ahead of print | PMID: 31972267
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Abstract

Effects of sympatho-vagal interaction on ventricular electrophysiology and their modulation during beta-blockade.

Chin SH, Allen E, Brack KE, Ng GA
Aims
The effects of sympatho-vagal interaction on heart rate (HR) changes are characterized by vagal dominance resulting in accentuated antagonism. Complex autonomic modulation of ventricular electrophysiology may exert prognostic arrhythmic impact. We examined the effects of concurrent sympathetic (SNS) and vagus (VNS) nerve stimulation on ventricular fibrillation threshold (VFT) and standard restitution (RT) in an isolated rabbit heart preparation with intact dual autonomic innervation, with and without beta-blockade.
Methods and results
Monophasic action potentials were recorded from left ventricular epicardial surface of dual-innervated isolated heart preparations from New Zealand white rabbits (n = 18). HR, VFT and RT were measured during different stimulation protocols (Protocol 1: VNS-SNS; Protocol 2: SNS-VNS) involving low- and high-frequency stimulations. A sub-study of Protocol 2 was performed in the presence of metoprolol tartrate. In both protocols, HR changes were characterized by vagal-dominant bradycardic component, affirming accentuated antagonism. During concurrent high-frequency VNS (HV), SNS prevails in lowering VFT in a frequency-sensitive manner during low (LS) or high (HS)-frequency stimulations (HV-LS: -2.8 ± 0.8 mA; HV-HS: -4.0 ± 0.9 mA, p < .05 vs. HV), with accompanying steepening of relative RT slope gradients (HV-LS: 223.54 ± 37.41%; HV-HS: 295.20 ± 60.86%, p < .05 vs. HV). In protocol 2, low (LV) and high (HV) vagal stimulations during concurrent HS raised VFT (HS-LV: 1.0 ± 0.4 mA; HS-HV: 3.0 ± 0.6 mA, p < .05 vs HS) with associated flattening of RT slopes (HS-LV: 32.40 ± 4.97%;HS-HV: 38.07 ± 6.37%; p < .05 vs HS). Metoprolol abolished accentuated antagonism in HR changes, reduced VFT and flattened RT globally during SNS-VNS.
Conclusions
Accentuated antagonism is absent in ventricular electrophysiological changes during sympatho-vagal interaction with sympathetic effect prevailing, suggesting a different mechanism at the ventricular level from heart rate effects. Metoprolol nullified accentuated antagonism with additional anti-fibrillatory effect beyond adrenergic blockade during sympatho-vagal stimulations.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 27 Jan 2020; epub ahead of print
Chin SH, Allen E, Brack KE, Ng GA
J Mol Cell Cardiol: 27 Jan 2020; epub ahead of print | PMID: 32004506
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Abstract

Defining decreased protein succinylation of failing human cardiac myofibrils in ischemic cardiomyopathy.

Ali HR, Michel CR, Lin YH, McKinsey TA, ... Woulfe KC, Fritz KS

Succinylation is a post-translational modification of protein lysine residues with succinyl groups derived from succinyl CoA. Succinylation is considered a significant post-translational modification with the potential to impact protein function which is highly conserved across numerous species. The role of succinylation in the heart, especially in heart failure and myofibril mechanics, remains largely unexplored. Mechanical parameters were measured in myofibrils isolated from failing hearts of ischemic cardiomyopathy patients and non-failing donor controls. We employed mass spectrometry to quantify differential protein expression in myofibrils from failing ischemic cardiomyopathy hearts compared to non-failing hearts. In addition, we combined peptide enrichment by immunoprecipitation with liquid chromatography tandem mass spectrometry to quantitatively analyze succinylated lysine residues in these myofibrils. Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ischemic cardiomyopathy hearts, including lower resting tension and a faster rate of activation. Of the 100 differentially expressed proteins, 46 showed increased expression in ischemic heart failure, while 54 demonstrated decreased expression in ischemic heart failure. Our quantitative succinylome analysis identified a total of 572 unique succinylated lysine sites located on 181 proteins, with 307 significantly changed succinylation events. We found that 297 succinyl-Lys demonstrated decreased succinylation on 104 proteins, while 10 residues demonstrated increased succinylation on 4 proteins. Investigating succinyl CoA generation, enzyme activity assays demonstrated that α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were significantly decreased in ischemic heart failure. An activity assay for succinyl CoA synthetase demonstrated a significant increase in ischemic heart failure. Taken together, our findings support the hypothesis that succinyl CoA production is decreased and succinyl CoA turnover is increased in ischemic heart failure, potentially resulting in an overall decrease in the mitochondrial succinyl CoA pool, which may contribute to decreased myofibril protein succinylation in heart failure.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 09 Dec 2019; epub ahead of print
Ali HR, Michel CR, Lin YH, McKinsey TA, ... Woulfe KC, Fritz KS
J Mol Cell Cardiol: 09 Dec 2019; epub ahead of print | PMID: 31836543
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Abstract

EZH2 as a novel therapeutic target for atrial fibrosis and atrial fibrillation.

Song S, Zhang R, Mo B, Chen L, ... Yu Y, Wang Q

Angiotensin II (Ang-II)-induced fibroblast differentiation plays an important role in the development of atrial fibrosis and atrial fibrillation (AF). Here, we show that the expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is increased in atrial muscle and atrial fibroblasts in patients with AF, accompanied by significant atrial fibrosis and atrial fibroblast differentiation. In addition, EZH2 is induced in murine models of atrial fibrosis. Furthermore, either pharmacological GSK126 inhibition or molecular silencing of EZH2 can inhibit the differentiation of atrial fibroblasts and the ability to produce ECM induced by Ang-II. Simultaneously, inhibition of EZH2 can block the Ang-II-induced migration of atrial fibroblasts. We found that EZH2 promotes fibroblast differentiation mainly through the Smad signaling pathway and can form a transcription complex with Smad2 to bind to the promoter region of the ACTA2 gene. Finally, our in vivo experiments demonstrated that the EZH2 inhibitor GSK126 significantly inhibited Ang-II-induced atrial enlargement and fibrosis and reduced AF vulnerability. Our results demonstrate that targeting EZH2 or EZH2-regulated genes might present therapeutic potential in AF.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:119-133
Song S, Zhang R, Mo B, Chen L, ... Yu Y, Wang Q
J Mol Cell Cardiol: 29 Sep 2019; 135:119-133 | PMID: 31408621
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Abstract

Cardioprotective effects of idebenone do not involve ROS scavenging: Evidence for mitochondrial complex I bypass in ischemia/reperfusion injury.

Perry JB, Davis GN, Allen ME, Makrecka-Kuka M, ... Shaikh SR, Brown DA

Novel therapeutic strategies to treat mitochondrial deficiencies in acute coronary syndromes are needed. Complex I of the mitochondrial electron transport system is damaged following ischemia/reperfusion (I/R) injury. This disruption contributes to aberrant electron transport, diminished bioenergetics, an altered redox environment, and mitochondrial damage involved in tissue injury. In this study, we determined the cardiac and mitochondrial effects of idebenone, a benzoquinone currently in several clinical trials with purported \'antioxidant\' effects. We employed complimentary models of ischemia/reperfusion injury in perfused hearts, permeabilized cardiac fibers, isolated mitochondria, and in cells to elucidate idebenone\'s cardioprotective mechanism(s). In ex vivo whole hearts, infarct size was markedly reduced with post-ischemic idebenone treatment (25 ± 5% area at risk, AAR) compared to controls (56 ± 6% AAR, P < .05). Several parameters of hemodynamic function were also significantly improved after idebenone treatment. Parallel studies of anoxia/reoxygenation were conducted using isolated mitochondria and permeabilized ventricular fibers. In isolated mitochondria, we simultaneously monitored respiration and ROS emission. Idebenone treatment modestly elevated succinate-derived HO production when compared to vehicle control (1.34 ± 0.05 vs 1.21 ± 0.05%, HO/O respectively, P < .05). Isolated mitochondria subjected to anoxia/reoxygenation demonstrated higher rates of respiration with idebenone treatment (2360 ± 69 pmol/s*mg) versus vehicle control (1995 ± 101 pmol/s*mg). Both mitochondria and permeabilized cardiac fibers produced high rates of HO after anoxia/reoxygenation, with idebenone showing no discernable attenuation on HO production. These insights were further investigated with studies in mitochondria isolated from reperfused ventricle. The profound decrease in complex-I dependent respiration after ischemia/reperfusion (701 ± 59 pmolO/s*mg compared to 1816 ± 105 pmol O/s*mg in normoxic mitochondria) was attenuated with idebenone treatment (994 ± 76 vs pmol O/s*mg, P < .05). Finally, the effects of idebenone were determined using permeabilized cell models with chemical inhibition of complex I. ADP-dependent oxidative phosphorylation capacity was significantly higher in complex-I inhibited cells treated acutely with idebenone (89.0 ± 4.2 pmol/s*million cells versus 70.1 ± 8.2 pmol/s*million cells in untreated cells). Taken together, these data indicate that the cardioprotective effects of idebenone treatment do not involve ROS-scavenging but appear to involve augmentation of the quinone pool, thus providing reducing equivalents downstream of complex I. As this compound is already in clinical trials for other indications, it may provide a safe and useful approach to mitigate ischemia/reperfusion injury in patients.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:160-171
Perry JB, Davis GN, Allen ME, Makrecka-Kuka M, ... Shaikh SR, Brown DA
J Mol Cell Cardiol: 29 Sep 2019; 135:160-171 | PMID: 31445917
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Abstract

Saraf-dependent activation of mTORC1 regulates cardiac growth.

Sanlialp A, Schumacher D, Kiper L, Varma E, ... Freichel M, Völkers M

Pathological cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signaling pathways regulating intracellular Ca homeostasis that control adaptive and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the store-operated calcium entry-associated regulatory factor (Saraf), encoded by the Tmem66 gene, on cardiac growth control in vitro and in vivo. Saraf is a single-pass membrane protein located at the sarco/endoplasmic reticulum and regulates intracellular calcium homeostasis. We found that Saraf expression was upregulated in the hypertrophied myocardium and was sufficient for cell growth in response to neurohumoral stimulation. Increased Saraf expression caused cell growth, which was associated with dysregulation of calcium-dependent signaling and sarcoplasmic reticulum calcium content. In vivo, Saraf augmented cardiac myocyte growth in response to angiotensin II and resulted in increased cardiac remodeling together with worsened cardiac function. Mechanistically, Saraf activated mTORC1 (mechanistic target of rapamycin complex 1) and increased protein synthesis, while mTORC1 inhibition blunted Saraf-dependent cell growth. In contrast, the hearts of Saraf knockout mice and Saraf-deficient myocytes did not show any morphological or functional alterations after neurohumoral stimulation, but Saraf depletion resulted in worsened cardiac function after acute pressure overload. SARAF knockout blunted transverse aortic constriction cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for SARAF in compensatory myocyte growth. Collectively, these results reveal a novel link between sarcoplasmic reticulum calcium homeostasis and mTORC1 activation that is regulated by Saraf.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 11 Mar 2020; epub ahead of print
Sanlialp A, Schumacher D, Kiper L, Varma E, ... Freichel M, Völkers M
J Mol Cell Cardiol: 11 Mar 2020; epub ahead of print | PMID: 32173353
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Abstract

Alteration of purinergic signaling in diabetes: Focus on vascular function.

Zhou R, Dang X, Sprague RS, Mustafa SJ, Zhou Z

Diabetes is an important risk factor for the development of cardiovascular disease including atherosclerosis and ischemic heart disease. Vascular complications including macro- and micro-vascular dysfunction are the leading causes of morbidity and mortality in diabetes. Disease mechanisms at present are unclear and no ideal therapies are available, which urgently calls for the identification of novel therapeutic targets/agents. An altered nucleotide- and nucleoside-mediated purinergic signaling has been implicated to cause diabetes-associated vascular dysfunction in major organs. Alteration of both purinergic P1 and P2 receptor sensitivity rather than the changes in receptor expression accounts for vascular dysfunction in diabetes. Activation of P2X receptors plays a crucial role in diabetes-induced retinal microvascular dysfunction. Recent findings have revealed that both ecto-nucleotidase CD39, a key enzyme hydrolyzing ATP, and CD73, an enzyme regulating adenosine turnover, are involved in the renal vascular injury in diabetes. Interestingly, erythrocyte dysfunction in diabetes by decreasing ATP release in response to physiological stimuli may serve as an important trigger to induce vascular dysfunction. Nucleot(s)ide-mediated purinergic activation also exerts long-term actions including inflammatory and atherogenic effects in hyperglycemic and diabetic conditions. This review highlights the current knowledge regarding the altered nucleot(s)ide-mediated purinergic signaling as an important disease mechanism for the diabetes-associated vascular complications. Better understanding the role of key receptor-mediated signaling in diabetes will provide more insights into their potential as targets for the treatment.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 10 Feb 2020; epub ahead of print
Zhou R, Dang X, Sprague RS, Mustafa SJ, Zhou Z
J Mol Cell Cardiol: 10 Feb 2020; epub ahead of print | PMID: 32057736
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Abstract

MiR322 mediates cardioprotection against ischemia/reperfusion injury via FBXW7/notch pathway.

Chen Z, Su X, Shen Y, Jin Y, ... Weintraub NL, Tang Y

Myocardial ischemia/reperfusion (MI/R) causes loss of cardiomyocytes via oxidative stress-induced cardiomyocyte apoptosis. miR322, orthologous to human miR-424, was identified as an ischemia-induced angiogenic miRNA, but its cellular source and function in the setting of acute MI/R remains largely unknown. Using LacZ-tagged miR322 cluster reporter mice, we observed that vascular endothelial cells are the major cellular source of the miR322 cluster in adult hearts. Moreover, miR322 levels were significantly reduced in the heart at 24 h after MI/R injury. Intramyocardial injection of mimic-miR322 significantly diminished cardiac apoptosis (as determined by expression levels of active caspase 3 by Western blot analysis and immunostaining for TUNEL) and reduced infarct size by about 40%, in association with reduced FBXW7 and increased active Notch 1 levels in the ischemic hearts. FBXW7, which is an ubiquitin ligase that is crucial for activated Notch1 turnover, was identified as a direct target of miR322 via FBXW7 3\'UTR reporter assay. Co-injection of FBXW7 plasmid with mimic-miR322 in ischemic hearts abolished the effect of mimic-miR322 to reduce apoptosis and infarct size in MI/R hearts. These data identify FBXW7 as a direct target of miR322 and suggest that miR322 could have potential therapeutic application for cardioprotection against ischemia/reperfusion-induced injury.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2019; 133:67-74
Chen Z, Su X, Shen Y, Jin Y, ... Weintraub NL, Tang Y
J Mol Cell Cardiol: 30 Jul 2019; 133:67-74 | PMID: 31150734
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Abstract

Mechanical strain induces a pro-fibrotic phenotype in human mitral valvular interstitial cells through RhoC/ROCK/MRTF-A and Erk1/2 signaling pathways.

Blomme B, Deroanne C, Hulin A, Lambert C, ... Radermecker M, Colige A

The mitral valve is a complex multilayered structure populated by fibroblast-like cells, valvular interstitial cells (VIC) which are embedded in an extracellular matrix (ECM) scaffold and are submitted to the mechanical deformations affecting valve at each heartbeat, for an average of 40 million times per year. Myxomatous mitral valve (MMV) is the most frequent heart valve disease characterized by disruption of several valvular structures due to alterations of their ECM preventing the complete closure of the valve resulting in symptoms of prolapse and regurgitation. VIC and their ECM exhibit reciprocal dynamic processes between the mechanical signals issued from the ECM and the modulation of VIC phenotype responsible for ECM homeostasis of the valve. Abnormal perception and responsiveness of VIC to mechanical stress may induce an inappropriate adaptative remodeling of the valve progressively leading to MMV. To investigate the response of human VIC to mechanical strain and identify the molecular mechanisms of mechano-transduction in these cells, a cyclic equibiaxial elongation of 14% at the cardiac frequency of 1.16 Hz was applied to VIC by using a Flexercell-4000 T™ apparatus for increasing time (from 1 h to 8 h). We showed that cyclic stretch induces an early (1 h) and transient over-expression of TGFβ2 and αSMA. CTGF, a profibrotic growth factor promoting the synthesis of ECM components, was strongly induced after 1 and 2 h of stretching and still upregulated at 8 h. The mechanical stress-induced CTGF up-regulation was dependent on RhoC, but not RhoA, as demonstrated by siRNA-mediated silencing approaches, and further supported by evidencing RhoC activation upon cell stretching and suppression of cell response by pharmacological inhibition of the effector ROCK1/2. It was also dependent on the MEK/Erk1/2 pathway which was activated by mechanical stress independently of RhoC and ROCK. Finally, mechanical stretching induced the nuclear translocation of myocardin related transcription factor-A (MRTF-A) which forms a transcriptional complex with SRF to promote the expression of target genes, notably CTGF. Treatment of stretched cultures with inhibitors of the identified pathways (ROCK1/2, MEK/Erk1/2, MRTF-A translocation) blocked CTGF overexpression and abrogated the increased MRTF-A nuclear translocation. CTGF is up-regulated in many pathological processes involving mechanically challenged organs, promotes ECM accumulation and is considered as a hallmark of fibrotic diseases. Pharmacological targeting of MRTF-A by newly developed inhibitors may represent a relevant therapy for MMV.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:149-159
Blomme B, Deroanne C, Hulin A, Lambert C, ... Radermecker M, Colige A
J Mol Cell Cardiol: 29 Sep 2019; 135:149-159 | PMID: 31442470
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Abstract

Hypoplastic left heart syndrome: From bedside to bench and back.

Saraf A, Book WM, Nelson TJ, Xu C

Hypoplastic Left Heart Syndrome (HLHS) is a complex Congenital Heart Disease (CHD) that was almost universally fatal until the advent of the Norwood operation in 1981. Children with HLHS who largely succumbed to the disease within the first year of life, are now surviving to adulthood. However, this survival is associated with multiple comorbidities and HLHS infants have a higher mortality rate as compared to other non-HLHS single ventricle patients. In this review we (a) discuss current clinical challenges associated in the care of HLHS patients, (b) explore the use of systems biology in understanding the molecular framework of this disease, (c) evaluate induced pluripotent stem cells as a translational model to understand molecular mechanisms and manipulate them to improve outcomes, and (d) investigate cell therapy, gene therapy, and tissue engineering as a potential tool to regenerate hypoplastic cardiac structures and improve outcomes.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:109-118
Saraf A, Book WM, Nelson TJ, Xu C
J Mol Cell Cardiol: 29 Sep 2019; 135:109-118 | PMID: 31419439
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Impact:
Abstract

Cardioprotection of (±)-sodium 5-bromo-2-(α-hydroxypentyl) benzoate (BZP) on mouse myocardium I/R injury through inhibiting 12/15-LOX-2 activity.

Xiao Y, Song C, Lin Q, Shi X, ... Zhao W, Chang J

(±)-Sodium5-bromo-2-(α-hydroxypentyl) benzoate (brand name: brozopine, BZP, 1a), derived from L-3-n-butylphthalide (L-NBP), has been reported to protect the brain from stoke and has been approved by CFDA in Phase I-II clinical trials. However, it remains to be investigated whether 1a may exhibit any cardioprotective effect on ischemia-reperfusion (I/R) injury. In the current study, C57BL/6 and ICR mice were pretreated with 1a, and myocardium I/R were then performed. We found that 1a not only significantly reduced the infarct size and improved cardiac contractile function after acute MI/R in both species, but also protected hearts from chronic MI-related cardiac injury. Mechanically, we found that 1a physically binds to 12/15-LOX-2 using molecular docking. The shRNA-mediated 12/15-LOX-2 knockdown almost completely blocked the protective effect of 1a. Our findings, for the first time, strongly indicate that 1a may serve as a potent and promising cardioprotective agent in treatment of I/R related injury, at least partially through targeting 12/15-LOX-2.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:52-66
Xiao Y, Song C, Lin Q, Shi X, ... Zhao W, Chang J
J Mol Cell Cardiol: 29 Sep 2019; 135:52-66 | PMID: 31362020
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Abstract

The cardiac CaMKII-Na1.5 relationship: From physiology to pathology.

Takla M, Huang CCL, Jeevaratnam K

The SCN5A gene encodes Na1.5, which, as the cardiac voltage-gated Na channel\'s pore-forming α subunit, is crucial for the initiation and propagation of atrial and ventricular action potentials. The arrhythmogenic propensity of inherited SCN5A mutations implicates the Na channel in determining cardiomyocyte excitability under normal conditions. Cytosolic kinases have long been known to alter the kinetic profile of Na1.5 inactivation via phosphorylation of specific residues. Recent substantiation for both the role of calmodulin-dependent kinase II (CaMKII) in modulating the properties of the Na1.5 inactivation gate and the significant rise in oxidation-dependent autonomous CaMKII activity in structural heart disease has raised the possibility of a novel pathway for acquired arrhythmias - the CaMKII-Na1.5 relationship. The aim of this review is to: (1) outline the relationship\'s transition from physiological adaptation to pathological vicious circle; and (2) discuss the relative merits of each of its components as pharmacological targets.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Jan 2020; epub ahead of print
Takla M, Huang CCL, Jeevaratnam K
J Mol Cell Cardiol: 16 Jan 2020; epub ahead of print | PMID: 31958466
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Abstract

Cellular cross-talks in the diseased and aging heart.

Wagner JUG, Dimmeler S

Communication between cells is an important, evolutionarily conserved mechanism which enables the coordinated function of multicellular organisms. Heterogeneity within cell populations drive a remarkable network of cellular cross-talk that allows the heart to function as an integrated unit with distinct tasks allocated to sub-specialized cells. During diseases and aging, cells acquire an overt disordered state that significantly contributes to an altered cellular cross-talk and hence drive cardiac remodeling processes and cardiovascular diseases. However, adaptive mechanisms, and phenotypic changes in subpopulations of cells (e.g. reparative macrophages or fibroblasts) can also contribute to repair and regeneration. In this article, we review the cellular cross-talks between immune cells, endothelial cells, fibroblasts and cardiomyocytes that control heart failure by contributing to cardiac dysfunction and aging, or by mediating repair and regeneration of the heart after injury.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 25 Nov 2019; epub ahead of print
Wagner JUG, Dimmeler S
J Mol Cell Cardiol: 25 Nov 2019; epub ahead of print | PMID: 31783034
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Abstract

Remodeling of substrate consumption in the murine sTAC model of heart failure.

Turer A, Altamirano F, Schiattarella GG, May H, ... Malloy CR, Merritt ME
Background
Energy metabolism and substrate selection are key aspects of correct myocardial mechanical function. Myocardial preference for oxidizable substrates changes in both hypertrophy and in overt failure. Previous work has shown that glucose oxidation is upregulated in overpressure hypertrophy, but its fate in overt failure is less clear. Anaplerotic flux of pyruvate into the tricarboxylic acid cycle (TCA) has been posited as a secondary fate of glycolysis, aside from pyruvate oxidation or lactate production.
Methods and results
A model of heart failure that emulates both valvular and hypertensive heart disease, the severe transaortic constriction (sTAC) mouse, was assayed for changes in substrate preference using metabolomic and carbon-13 flux measurements. Quantitative measures of O consumption in the Langendorff perfused mouse heart were paired with C isotopomer analysis to assess TCA cycle turnover. Since the heart accommodates oxidation of all physiological energy sources, the utilization of carbohydrates, fatty acids, and ketones were measured simultaneously using a triple-tracer NMR method. The fractional contribution of glucose to acetyl-CoA production was upregulated in heart failure, while other sources were not significantly different. A model that includes both pyruvate carboxylation and anaplerosis through succinyl-CoA produced superior fits to the data compared to a model using only pyruvate carboxylation. In the sTAC heart, anaplerosis through succinyl-CoA is elevated, while pyruvate carboxylation was not. Metabolomic data showed depleted TCA cycle intermediate pool sizes versus the control, in agreement with previous results.
Conclusion
In the sTAC heart failure model, the glucose contribution to acetyl-CoA production was significantly higher, with compensatory changes in fatty acid and ketone oxidation not reaching a significant level. Anaplerosis through succinyl-CoA is also upregulated, and is likely used to preserve TCA cycle intermediate pool sizes. The triple tracer method used here is new, and can be used to assess sources of acetyl-CoA production in any oxidative tissue.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:144-153
Turer A, Altamirano F, Schiattarella GG, May H, ... Malloy CR, Merritt ME
J Mol Cell Cardiol: 30 Aug 2019; 134:144-153 | PMID: 31340162
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Abstract

MicroRNA-1976 regulates degeneration of the sinoatrial node by targeting Ca1.2 and Ca1.3 ion channels.

Zhang J, Wei F, Ding L, Wang L, ... Yang B, Fan J

Sick sinus syndrome (SSS) is primarily a disease of the elderly, and age-dependent decrease in Ca1.2 and Ca1.3 Ca channels within the sinus node has been shown to play an important role in sinoatrial node (SAN) degeneration; however, posttranscriptional mechanisms regulating decrease in Ca1.2 and Ca1.3 Ca channels remain unclear. Some studies have reported that microRNAs (miRNAs) are involved in age-related cardiovascular diseases. Nevertheless, little is known about the roles of miRNAs in age-related SSS. This study investigated whether miR-1976 was involved in the regulation of SAN degeneration by targeting Ca1.2 and Ca1.3 Ca channels. First, using microarray-based miRNA expression profiling and qRT-PCR, we confirmed that miR-1976 was upregulated in the plasma of patients with age-related SSS relative to healthy controls. By employing target gene prediction software, luciferase assay and western blotting, we further confirmed Ca1.2 and Ca1.3 as direct targets of miR-1976. Furthermore, miR-1976 levels in rabbit SAN tissues were negatively correlated with Ca1.2 and Ca1.3 expression and intrinsic heart rates but positively correlated with corrected sinus node recovery time (CSNRT). Additionally, miR-1976 transgenic mice displayed attenuated Ca1.2 and Ca1.3 protein expression, which led to sinus node dysfunction. These results suggest that miR-1976 plays an important role in the SAN aging process by targeting Ca1.2 and Ca1.3. Thus, miR-1976 could have great potential as a noninvasive diagnostic tool and therapeutic target for SSS. These findings may reveal important insights into the pathogenesis of SSS.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Aug 2019; 134:74-85
Zhang J, Wei F, Ding L, Wang L, ... Yang B, Fan J
J Mol Cell Cardiol: 30 Aug 2019; 134:74-85 | PMID: 31302118
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Abstract

Narciclasine inhibits angiogenic processes by activation of Rho kinase and by downregulation of the VEGF receptor 2.

Bräutigam J, Bischoff I, Schürmann C, Buchmann G, ... Brandes RP, Fürst R

The process of angiogenesis is involved in several pathological conditions, such as tumor growth or age-related macular degeneration. Although the available anti-angiogenic drugs have improved the therapy of these diseases, major drawbacks, such as unwanted side effects and resistances, still exist. Consequently, the search for new anti-angiogenic substances is still ongoing. Narciclasine, a plant alkaloid from different members of the Amaryllidaceae family, has extensively been characterized as anti-tumor compound. Beyond the field of cancer, the compound has recently been shown to possess anti-inflammatory properties. Surprisingly, potential actions of narciclasine on endothelial cells in the context of angiogenesis have been neglected so far. Thus, we aimed to analyze the effects of narciclasine on angiogenic processes in vitro and in vivo and to elucidate the underlying mechanism. Narciclasine (100-300 nM) effectively inhibited the proliferation, undirected and directed migration, network formation and angiogenic sprouting of human primary endothelial cells. Moreover, narciclasine (1 mg/kg/day) strongly reduced the VEGF-triggered angiogenesis in vivo (Matrigel plug assay in mice). Narciclasine mediated its anti-angiogenic effects in part by a RhoA-independent activation of the Rho kinase ROCK. Most importantly, however, the compound reduced the de novo protein synthesis in endothelial cells by approx. 50% without exhibiting considerable cytotoxic effects. As a consequence, narciclasine diminished the presence of proteins with a short half-life, such as the VEGF receptor 2, which is the basis for its anti-angiogenic effects. Taken together, our study highlights narciclasine as an interesting anti-angiogenic compound that is worth to be further evaluated in preclinical studies.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:97-108
Bräutigam J, Bischoff I, Schürmann C, Buchmann G, ... Brandes RP, Fürst R
J Mol Cell Cardiol: 29 Sep 2019; 135:97-108 | PMID: 31381906
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Abstract

Cardiac expression of the microsomal triglyceride transport protein protects the heart function during ischemia.

Klevstig M, Arif M, Mannila M, Svedlund S, ... Ehrenborg E, Borén J
Aims
The microsomal triglyceride transport protein (MTTP) is critical for assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins and is most abundant in the liver and intestine. Surprisingly, MTTP is also expressed in the heart. Here we tested the functional relevance of cardiac MTTP expression.
Materials and methods
We combined clinical studies, advanced expression analysis of human heart biopsies and analyses in genetically modified mice lacking cardiac expression of the MTTP-A isoform of MTTP.
Results
Our results indicate that lower cardiac MTTP expression in humans is associated with structural and perfusion abnormalities in patients with ischemic heart disease. MTTP-A deficiency in mice heart does not affect total MTTP expression, activity or lipid concentration in the heart. Despite this, MTTP-A deficient mice displayed impaired cardiac function after a myocardial infarction. Expression analysis of MTTP indicates that MTTP expression is linked to cardiac function and responses in the heart.
Conclusions
Our results indicate that MTTP may play an important role for the heart function in conjunction to ischemic events.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 14 Sep 2019; 137:1-8
Klevstig M, Arif M, Mannila M, Svedlund S, ... Ehrenborg E, Borén J
J Mol Cell Cardiol: 14 Sep 2019; 137:1-8 | PMID: 31533023
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Abstract

Metabolic reprogramming orchestrates CD4 T-cell immunological status and restores cardiac dysfunction in autoimmune induced-dilated cardiomyopathy mice.

Wu J, Sun P, Chen Q, Sun Y, ... Zhang M, Yu B

Cellular autoimmune responses, especially those mediated by T-cells, play vital roles in the immunopathogenesis of dilated cardiomyopathy (DCM). Metabolic reprogramming directly controls T-cell function, imprinting distinct functional fates. However, its contribution to T-cell dysfunction and the immunopathogenesis of DCM is unknown. Here, we found that in DCM patients, CD4 T-cells exhibited immune dysfunction and glycolytic metabolic reprogramming based on extracellular acidification and oxygen consumption rates. Similar results were observed in splenic and cardiac CD4 T-cells from autoimmune-induced DCM mice. In vitro, the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) reversed T-cell dysfunction; thus, heightened metabolic activity directly controls CD4 T-cell immunological status. Adoptive transfer of CD4 T-cells from DCM mice to normal recipients induced cardiac remodeling and cardiac T-cell dysfunction. Strikingly, these effects were abolished by preconditioning cells with 2-DG, indicating that CD4 T-cell dysfunction partially induced by metabolic reprogramming contributes to cardiac remodeling. Moreover, the microRNA let-7i modulated the metabolism and function of T-cells from DCM mice by directly targeting Myc. Collectively, our results show that metabolic reprogramming occurs in T-cells of autoimmune-induced DCM mice and patients. Further, our findings highlight that glycolytic metabolism is a critical contributor to T-cell dysfunction and DCM immunopathogenesis. Our data position the modulation of the metabolism as a central integrator for T-cell function, representing a promising strategy against autoimmune-mediated DCM progression.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:134-148
Wu J, Sun P, Chen Q, Sun Y, ... Zhang M, Yu B
J Mol Cell Cardiol: 29 Sep 2019; 135:134-148 | PMID: 31398346
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Abstract

Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression.

Chen B, Zhao Y, Han D, Zhao B, ... Xu MJ, Zhao G
Aims
Wnt signaling plays a critical role in vascular calcification (VC). Wnt factors induce different physiological and pathological effects on cardiovascular functions. Wnt1, a ligand of Wnt/β-catenin signaling, promotes pro-angiogenesis and reduces myocardial infarction. The role of Wnt1 on VC in chronic kidney disease (CKD) is not fully understood.
Methods and results
We used human vascular smooth muscle cells (VSMCs) and a rat model of chronic renal failure (CRF), and observed a native protective mechanism by which VC is reduced via the activation of Wnt1 and its transcriptional target ANKH inorganic pyrophosphate transport regulator (ANKH) gene. ANKH is an essential calcification inhibitor that effluxes inorganic pyrophosphate (PPi) from VSMCs to play an inhibitory role in VC. Vascular ANKH and plasma PPi were significantly downregulated in the rat model of CRF. The knockdown or inhibition of ANKH reversed the effect of Wnt1 on VC in VSMCs. Clinical analysis revealed low plasma levels of Wnt1 and PPi were associated with CKD in patients. Applying a Wnt/β-catenin signaling agonist can alleviate the progression of VC.
Conclusion
This work reveals the ANKH regulation of Wnt1 in VSMCs is essential for blocking VC. Our findings may contribute to the development of medications that target Wnt signaling and/or ANKH to inhibit VC.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:10-21
Chen B, Zhao Y, Han D, Zhao B, ... Xu MJ, Zhao G
J Mol Cell Cardiol: 29 Sep 2019; 135:10-21 | PMID: 31356809
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Abstract

CHIR99021 and fibroblast growth factor 1 enhance the regenerative potency of human cardiac muscle patch after myocardial infarction in mice.

Fan C, Tang Y, Zhao M, Lou X, ... Zhu W, Zhang J
Background
We have shown that genetic overexpression of cell cycle proteins can increase the proliferation of transplanted cardiomyocytes derived from human induced-pluripotent stem cells (hiPSC-CMs) in animal models of myocardial infarction (MI). Here, we introduce a new, non-genetic approach to promote hiPSC-CM cell cycle activity and proliferation in transplanted human cardiomyocyte patches (hCMPs).
Methods
Mice were randomly distributed into 5 experimental groups (n = 10 per group). One group underwent Sham surgery, and the other 4 groups underwent MI induction surgery followed by treatment with hCMPs composed of hiPSC-CMs and nanoparticles that contained CHIR99021 and FGF1 (the NP-hCMP group), with hCMPs composed of hiPSC-CMs and empty nanoparticles (the NP-hCMP group); with patches containing the CHIR99021/FGF-loaded nanoparticles but lacking hiPSC-CMs (the NP-Patch group), or patches lacking both the nanoparticles and cells (the E-Patch group). Cell cycle activity was evaluated via Ki67 and Aurora B expression, bromodeoxyuridine incorporation, and phosphorylated histone 3 levels (immunofluorescence); engraftment via human cardiac troponin T or human nuclear antigen expression (immunofluorescence) and bioluminescence imaging; cardiac function via echocardiography; infarct size and wall thickness via histology; angiogenesis via isolectin B4 expression (immunofluorescence); and apoptosis via TUNEL and caspace 3 expression (immunofluorescence).
Results
Combined CHIR99021- and FGF1-treatment significantly increased hiPSC-CM cell cycle activity both in cultured cells (by 4- to 6-fold) and in transplanted hCMPs, and compared to treatment with NP-hCMPs, NP-hCMP transplantation increased hiPSC-CM engraftment by ~4-fold and was associated with significantly better measurements of cardiac function, infarct size, wall thickness, angiogenesis, and hiPSC-CM apoptosis four weeks after MI induction.
Conclusions
Nanoparticle-mediated CHIR99021 and FGF1 delivery promotes hiPSC-CM cell cycle activity and proliferation, as well as the engraftment and regenerative potency of transplanted hCMPs, in a mouse MI model.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 09 Mar 2020; epub ahead of print
Fan C, Tang Y, Zhao M, Lou X, ... Zhu W, Zhang J
J Mol Cell Cardiol: 09 Mar 2020; epub ahead of print | PMID: 32169551
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Impact:
Abstract

Gut microbe-derived metabolite trimethylamine N-oxide accelerates fibroblast-myofibroblast differentiation and induces cardiac fibrosis.

Yang W, Zhang S, Zhu J, Jiang H, ... Sun A, Ge J
Background
Trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, has been associated with poor prognosis in coronary heart disease. However, the role and underlying mechanisms of TMAO in the cardiac fibrosis after myocardial infarction (MI) remains unclear.
Methods
We used mouse MI models and primary cardiac fibroblasts cultures to study the role of TMAO in the heart and in cardiac fibroblasts. C57BL/6 mice were fed a control diet, high choline (1.2%) or/and DMB diet or a diet containing TMAO (0.12%) starting 3 weeks before MI. DMB, a structural analogue of choline, inhibited microbial TMA lyases and reduced the level of TMAO in mice. Cardiac function was measured 7 days after MI using echocardiography. One week post MI, myocardial tissues were collected to evaluate cardiac fibrosis, and blood samples were evaluated for TMAO levels. The expression of TGF-β receptor, P-Smad2, α-SMA or collagen I in myocardial tissues and fibroblasts were analyzed by western blot or immunocytochemistry.
Results
We demonstrated that cardiac function and cardiac fibrosis were significantly deteriorated in mice fed either TMAO or high choline diets compared with the control diet, and DMB reversed the cardiac function damage of high choline diet (p < .05). Cardiomyocyte necrosis, apoptosis and macrophage infiltration after MI was significantly increased after treatment with TMAO or high choline diets. The size and migration of fibroblasts were increased after TMAO treatment compared with non-treated fibroblasts in vitro. Furthermore, TMAO increased TGF-β receptor I expression, which promoted the phosphorylation of Smad2 and up-regulated the expression of α-SMA and collagen I. The ubiquitination of TGF-βRI was decreased in neonatal mouse fibroblasts after TMAO treatment. TMAO also inhibited the expression of smurf2. Inhibition of TGF-β1 receptor with the small molecule inhibitor SB431542 decreased TGF-β receptor I expression, reduced the phosphorylation of Smad2, down-regulated TMAO-induced α-SMA and collagen I expression in cardiac fibroblasts.
Conclusions
Cardiac function and cardiac fibrosis were significantly exacerbated in mice fed diets supplemented with either choline or TMAO, probably through accelerating the transformation of fibroblasts into myofibroblasts, indicating activation of TGF-βRI/Smad2 pathway.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Aug 2019; 134:119-130
Yang W, Zhang S, Zhu J, Jiang H, ... Sun A, Ge J
J Mol Cell Cardiol: 30 Aug 2019; 134:119-130 | PMID: 31299216
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Impact:
Abstract

MIR148A family regulates cardiomyocyte differentiation of human embryonic stem cells by inhibiting the DLL1-mediated NOTCH signaling pathway.

Fang X, Miao S, Yu Y, Ding F, ... Hu S, Lei W

MicroRNAs (miRNAs), as a class of naturally occurring RNAs, play important roles in cardiac physiology and pathology. There are many miRNAs that show multifarious expression patterns during cardiomyocyte genesis. Here, we focused on the MIR148A family, which is composed of MIR148A, MIR148B and MIR152, and shares the same seed sequences. The expression levels of all MIR148A family members progressively increased during the differentiation of human embryonic stem cells (hESCs) into cardiomyocytes. The deletion of MIR148A family (MIR148A-TKO) resulted in a decreased proportion of cardiomyocytes after cardiac induction, which was restored by the ectopic expression of MIR148A family members. Transcriptome analyses indicated that the MIR148A family could partially repress paraxial mesodermal differentiation from primitive streak cells. In turn, these miRNAs promoted lateral mesoderm and cardiomyocyte differentiation. Furthermore, the NOTCH ligand Delta-like 1 (DLL1) was validated as the target gene of MIR148A family, and knockdown of DLL1 could promote the cardiomyocyte differentiation of MIR148A-TKO hESCs. Thus, our results demonstrate MIR148A family could promote cardiomyocyte differentiation by inhibiting undesired paraxial mesoderm lineage commitment, which improves our understanding on cardiomyocyte differentiation from hESCs.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:1-12
Fang X, Miao S, Yu Y, Ding F, ... Hu S, Lei W
J Mol Cell Cardiol: 30 Aug 2019; 134:1-12 | PMID: 31233755
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Impact:
Abstract

High-fat diet improves tolerance to myocardial ischemia by delaying normalization of intracellular PH at reperfusion.

Inserte J, Aluja D, Barba I, Ruiz-Meana M, ... Castellano J, Garcia-Dorado D

Reports on the effect of obesity on the myocardial tolerance to ischemia are contradictory. We have described that obesity induced by high-fat diet (HFD) reduces infarct size in B6D2F1 mice submitted to transient coronary occlusion. In this study, we analysed the mechanism by which dietary obesity modifies the susceptibility to myocardial ischemia and the robustness of this effect. B6D2F1 (BDF), C57BL6/J (6J), C57BL6/N (6N) male mice and BDF female mice were fed with a HFD or control diet for 16 weeks. In all three strains, HFD induced obesity with hyperinsulinemia and hypercholesterolemia and without hyperglycemia, hypertension, ventricular remodelling or cardiac dysfunction. In obese mice from all three strains PDK4 was overexpressed and HSQC NMR spectroscopy showed reduced C-glutamate and increased C-lactate and C-alanine, indicating uncoupling of glycolysis from glucose oxidation. In addition, HFD induced mild respiratory uncoupling in mitochondria from BDF and 6N mice in correlation with UCP3 overexpression. In studies performed in isolated perfused hearts submitted to transient ischemia these changes were associated with reduced ATP content and myocardial PCr/ATP ratio at baseline, and delayed pHi recovery (PNMR) and attenuated hypercontracture at the onset of reperfusion. Finally, in mice subjected to 45 min of coronary occlusion and 24 h of reperfusion, HFD significantly reduced infarct size respect to their respective control diet groups in male BDF (39.4 ± 6.1% vs. 19.9 ± 3.2%, P = 0.018) and 6N mice (38.0 ± 4.1 vs. 24.5 ± 2.6%, P = 0.017), and in female BDF mice (35.3 ± 4.4% vs. 22.3 ± 2.5%, P = 0.029), but not in male 6J mice (40.2 ± 3.4% vs. 34.1 ± 3.8%, P = 0.175). Our results indicate that the protective effect of HFD-induced obesity against myocardial ischemia/reperfusion injury is influenced by genetic background and appears to critically depend on inhibition of glucose oxidation and mild respiratory mitochondrial uncoupling resulting in prolongation of acidosis at the onset of reperfusion.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2019; 133:164-173
Inserte J, Aluja D, Barba I, Ruiz-Meana M, ... Castellano J, Garcia-Dorado D
J Mol Cell Cardiol: 30 Jul 2019; 133:164-173 | PMID: 31194987
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Abstract

Distinct roles of myofibroblast-specific Smad2 and Smad3 signaling in repair and remodeling of the infarcted heart.

Huang S, Chen B, Su Y, Alex L, ... Conway SJ, Frangogiannis NG

TGF-βs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-β1, -β2 and -β3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2019; 132:84-97
Huang S, Chen B, Su Y, Alex L, ... Conway SJ, Frangogiannis NG
J Mol Cell Cardiol: 29 Jun 2019; 132:84-97 | PMID: 31085202
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Abstract

A small-molecule LF3 abrogates β-catenin/TCF4-mediated suppression of Na1.5 expression in HL-1 cardiomyocytes.

Zhao L, Sun L, Lu Y, Li F, Xu H

Increased nuclear β-catenin interacting with T-cell factor 4 (TCF4) affects the expression of target genes including SCN5A in ischemic heart disease, which is characterized by frequent ventricular tachycardia/fibrillation. A complex of β-catenin and TCF4 inhibits cardiac Na channel activity by reducing Na1.5 expression through suppressing SCN5A promoter activity in HL-1 cardiomyocytes. LF3, a 4-thioureido-benzenesulfonamide derivative and an inhibitor of β-catenin/TCF4 interaction, has been shown to block the self-renewal capacity of cancer stem cells. We performed studies to determine if LF3 can reverse suppressive effects of β-catenin/TCF4 signaling on the expression of Na1.5 in HL-1 cardiomyocytes. Western blotting and real-time qRT-PCR analyses showed that 10 μM LF3 significantly increased the expression of Na1.5 but it did not alter β-catenin and TCF4 expression. Subcellular fractionation analysis demonstrated that LF3 significantly increased the levels of Na1.5 in both membrane and cytoplasm. Whole-cell patch-clamp recordings revealed that Na currents were significantly increased with no changes in the steady-state parameters, activation and inactivation time constants and recovery from inactivation of Na channel in HL-1 cells treated with LF3. Immunoprecipitation exhibited that LF3 blocked the interaction of β-catenin and TCF4. Luciferase reporter assays performed in HEK 293 cells and HL-1 revealed that LF3 increased the SCN5A promoter activity in HL-1 cells and prevented β-catenin suppressive effect on SCN5A promoter activity in HEK 293 cells. Taken together, we conclude that LF3, an inhibitor of β-catenin/TCF4 interaction, elevates Na1.5 expression, leading to increase Na channel activity in HL-1 cardiomyocytes.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:90-96
Zhao L, Sun L, Lu Y, Li F, Xu H
J Mol Cell Cardiol: 29 Sep 2019; 135:90-96 | PMID: 31419437
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Abstract

Patterns of ascending aortic dilatation and predictors of surgical replacement of the aorta: A comparison of bicuspid and tricuspid aortic valve patients over eight years of follow-up.

Agnese V, Pasta S, Michelena HI, Minà C, ... Pilato M, Bellavia D
Background
Predictors of thoracic aorta growth and early cardiac surgery in patients with bicuspid aortic valve are undefined. Our aim was to identify predictors of ascending aorta dilatation and cardiac surgery in patients with bicuspid aortic valve (BAV).
Methods
Forty-one patients with BAV were compared with 165 patients with tricuspid aortic valve (TAV). All patients had LV EF > 50%, normal LV dimensions, and similar degree of aortic root or ascending aorta dilatation at enrollment. Patients with more than mild aortic stenosis or regurgitation were excluded. A CT-scan was available on 76% of the population, and an echocardiogram was repeated every year for a median time of 4 years (range: 2 to 8 years). Patterns of aortic expansion in BAV and TAV groups were analyzed by a mixed-effects longitudinal linear model. In the time-to-event analysis, the primary end point was elective or emergent surgery for aorta replacement.
Results
BAV patients were younger, while the TAV group had greater LV wall thickness, arterial hypertension, and dyslipidemia than BAV patients. Growth rate was 0.46 ± 0.04 mm/year, similar in BAV and TAV groups (p = 0.70). Predictors of cardiac surgery were aorta dimensions at baseline (HR 1.23, p = 0.01), severe aortic regurgitation developed during follow-up (HR 3.49, p 0.04), family history of aortic aneurysm (HR 4.16, p 1.73), and history of STEMI (HR 3.64, p < 0.001).
Conclusions
Classic baseline risk factors were more commonly observed in TAV aortopathy compared with BAV aortopathy. However, it is reassuring that, though diagnosed with aneurysm on average 10 years earlier and in the absence of arterial hypertension, BAV patients had a relatively low growth rate, similar to patients with a tricuspid valve. Irrespective of aortic valve morphology, patients with a family history of aortic aneurysm, history of coronary artery disease, and those who developed severe aortic regurgitation at follow-up, had the highest chances of being referred for surgery.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:31-39
Agnese V, Pasta S, Michelena HI, Minà C, ... Pilato M, Bellavia D
J Mol Cell Cardiol: 29 Sep 2019; 135:31-39 | PMID: 31348923
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Abstract

MicroRNAs fingerprint of bicuspid aortic valve.

Sabatino J, Wicik Z, De Rosa S, Eyileten C, ... Postula M, Indolfi C

Aortic valve tissue is largely exposed to high blood flow. Cells belonging to aortic valve tissues are able to detect and respond to flow conditions changes. Bicuspid aortic valve (BAV) presents altered morphology, with only two abnormal cusps instead of three. This results in an alteration of blood flow dynamics on valve cusps and aortic wall, which may, in turn, increase the risk to develop aortic stenosis and/or regurgitation, endocarditis, aortopathy and/or aortic dissection. MicroRNAs (miRNAs) are short RNA strands regulating gene expression mainly through the inhibition of their target mRNAs. They are largely involved in cardiovascular pathophysiology and heart disease. More recently, it has been observed that the expression of specific miRNAs can be modulated in response to changes in hemodynamic conditions. Using a bioinformatic approach, this article analyses available scientific evidence about the differential expression of miRNAs in the bicuspid aortic valve, with a focus on the differential modulation compared to the calcific-degenerative tricuspid aortic valve.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:98-106
Sabatino J, Wicik Z, De Rosa S, Eyileten C, ... Postula M, Indolfi C
J Mol Cell Cardiol: 30 Aug 2019; 134:98-106 | PMID: 31278905
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Abstract

Phenotyping an adult zebrafish lamp2 cardiomyopathy model identifies mTOR inhibition as a candidate therapy.

Dvornikov AV, Wang M, Yang J, Zhu P, ... Cao H, Xu X

Adult zebrafish is an emerging vertebrate model for studying genetic basis of cardiomyopathies; but whether the simple fish heart can model essential features of hypertrophic cardiomyopathy (HCM) remained unknown. Here, we report a comprehensive phenotyping of a lamp2 knockout (KO) mutant. LAMP2 encodes a lysosomal protein and is a causative gene of Danon disease that is characterized by HCM and massive autophagic vacuoles accumulation in the tissues. There is no effective therapy yet to treat this most lethal cardiomyopathy in the young. First, we did find the autophagic vacuoles accumulation in cardiac tissues from lamp2 KO. Next, through employing a set of emerging phenotyping tools, we revealed heart failure phenotypes in the lamp2 KO mutants, including decreased ventricular ejection fraction, reduced physical exercise capacity, blunted β-adrenergic contractile response, and enlarged atrium. We also noted changes of the following indices suggesting cardiac hypertrophic remodeling in lamp2 KO: a rounded heart shape, increased end-systolic ventricular volume and density of ventricular myocardium, elevated actomyosin activation kinetics together with increased maximal isometric tension at the level of cardiac myofibrils. Lastly, we assessed the function of lysosomal-localized mTOR on the lamp2-associated Danon disease. We found that haploinsufficiency of mtor was able to normalize some characteristics of the lamp2 KO, including ejection fraction, β-adrenergic response, and the actomyosin activation kinetics. In summary, we demonstrate the feasibility of modeling the inherited HCM in the adult zebrafish, which can be used to develop potential therapies.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2019; 133:199-208
Dvornikov AV, Wang M, Yang J, Zhu P, ... Cao H, Xu X
J Mol Cell Cardiol: 30 Jul 2019; 133:199-208 | PMID: 31228518
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Abstract

Global knockout of ROMK potassium channel worsens cardiac ischemia-reperfusion injury but cardiomyocyte-specific knockout does not: Implications for the identity of mitoKATP.

Papanicolaou KN, Ashok D, Liu T, Bauer TM, ... Foster DB, O\'Rourke B

The renal-outer-medullary‑potassium (ROMK) channel, mutated in Bartter\'s syndrome, regulates ion exchange in kidney, but its extra-renal functions remain unknown. Additionally, ROMK was postulated to be the pore-forming subunit of the mitochondrial ATP-sensitive K channel (mitoK), a mediator of cardioprotection. Using global and cardiomyocyte-specific knockout mice (ROMK-GKO and ROMK-CKO respectively), we characterize the effects of ROMK knockout on mitochondrial ion handling, the response to pharmacological K channel modulators, and ischemia/reperfusion (I/R) injury. Mitochondria from ROMK-GKO hearts exhibited a lower threshold for Ca-triggered permeability transition pore (mPTP) opening but normal matrix volume changes during oxidative phosphorylation. Isolated perfused ROMK-GKO hearts exhibited impaired functional recovery and increased infarct size after I/R injury, particularly when I/R was preceded by an ischemic preconditioning (IPC) protocol. Because ROMK-GKO mice exhibited severe renal defects and cardiac remodeling, we further characterized ROMK-CKO hearts to avoid confounding systemic effects. Mitochondria from ROMK-CKO hearts had unchanged matrix volume responses during oxidative phosphorylation and still swelled upon addition of a mitoK opener, but exhibited a lower threshold for mPTP opening, similar to GKO mitochondrial. Nevertheless, I/R induced damage was not exacerbated in ROMK-CKO hearts, either ex vivo or in vivo. Lastly, we examined the response of ROMK-CKO hearts to ex vivo I/R injury with or without IPC and found that IPC still protected these hearts, suggesting that cardiomyocyte ROMK does not participate significantly in the cardioprotective pathway elicited by IPC. Collectively, our findings from these novel strains of mice suggest that cardiomyocyte ROMK is not a central mediator of mitoK function, although it can affect mPTP activation threshold.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 27 Jan 2020; epub ahead of print
Papanicolaou KN, Ashok D, Liu T, Bauer TM, ... Foster DB, O'Rourke B
J Mol Cell Cardiol: 27 Jan 2020; epub ahead of print | PMID: 32004507
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Abstract

Piezo1 and outflow tract and aortic valve development.

Faucherre A, Maati HMO, Nasr N, Pinard A, ... Zaffran S, Jopling C
Aims
During embryogenesis, the onset of circulatory blood flow generates a variety of hemodynamic forces which reciprocally induce changes in cardiovascular development and performance. It has been known for some time that these forces can be detected by as yet unknown mechanosensory systems which in turn promote cardiogenic events such as outflow tract and aortic valve development. PIEZO1 is a mechanosensitive ion channel present in endothelial cells where it serves to detect hemodynamic forces making it an ideal candidate to play a role during cardiac development. We sought to determine whether PIEZO1 is required for outflow tract and aortic valve development.
Methods and results
By analysing heart development in zebrafish we have determined that piezo1 is expressed in the developing outflow tract where it serves to detect hemodynamic forces. Consequently, disrupting Piezo1 signalling leads to defective outflow tract and aortic valve development and indicates this gene may be involved in the etiology of congenital heart diseases. Based on these findings, we analysed genomic data generated from patients who suffer from left ventricular outflow tract obstructions (LVOTO) and identified 3 probands who each harboured potentially pathogenic variants in PIEZO1. Subsequent in vitro and in vivo assays indicates that these variants behave as dominant negatives leading to an inhibition of normal PIEZO1 mechanosensory activity. Expressing these dominant negative PIEZO1 variants in zebrafish endothelium leads to defective aortic valve development.
Conclusion
These data indicate that the mechanosensitive ion channel piezo1 is required for OFT and aortic valve development.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 02 Apr 2020; epub ahead of print
Faucherre A, Maati HMO, Nasr N, Pinard A, ... Zaffran S, Jopling C
J Mol Cell Cardiol: 02 Apr 2020; epub ahead of print | PMID: 32251670
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Abstract

Roles and mechanisms of SUMOylation on key proteins in myocardial ischemia/reperfusion injury.

Chen J, Luo Y, Wang S, Zhu H, Li D

Myocardial ischemia/reperfusion (MI/R) injury has a great influence on the prognosis of patients with acute coronary occlusion. The underlying mechanisms of MI/R injury are complex. While the incidence of MI/R injury is increasing every year, the existing therapies are not satisfactory. Recently, small ubiquitin-related modifier (SUMO), which is a post-translational modification and involved in many cell processes, was found to play remarkable roles in MI/R injury. Several proteins that can be SUMOylated were found to interfere with different mechanisms of MI/R injury. Sarcoplasmic reticulum Ca ATPase pump SUMOylation alleviated calcium overload. Among the histone deacetylase (HDAC) members, SUMOylation of HDAC4 reduced reactive oxygen species generation, whereas Sirt1 played protective roles in the SUMOylated form. Dynamic-related protein 1 modified by different SUMO proteins exerted opposite effects on the function of mitochondria. SUMOylation of hypoxia-inducible factors was fundamental in oxygen homeostasis, while eukaryotic elongation factor 2 SUMOylation induced cardiomyocyte apoptosis. The impact of other SUMOylation substrates in MI/R injury remains unclear. Here we reviewed how these SUMOylated proteins alleviated or exacerbated myocardial impairments by effecting the MI/R injury mechanisms. This may suggest methods for relieving MI/R injury in clinical practice and provide a reference for further study of SUMOylation in MI/R injury.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:154-164
Chen J, Luo Y, Wang S, Zhu H, Li D
J Mol Cell Cardiol: 30 Aug 2019; 134:154-164 | PMID: 31344368
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Abstract

Potent hERG channel inhibition by sarizotan, an investigative treatment for Rett Syndrome.

Cheng H, Du C, Zhang Y, James AF, ... Abdala AP, Hancox JC

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder associated with respiratory abnormalities and, in up to ~40% of patients, with prolongation of the cardiac QT interval. QT prolongation calls for cautious use of drugs with a propensity to inhibit hERG channels. The STARS trial has been undertaken to investigate the efficacy of sarizotan, a 5-HT receptor agonist, at correcting RTT respiratory abnormalities. The present study investigated whether sarizotan inhibits hERG potassium channels and prolongs ventricular repolarization. Whole-cell patch-clamp measurements were made at 37 °C from hERG-expressing HEK293 cells. Docking analysis was conducted using a recent cryo-EM structure of hERG. Sarizotan was a potent inhibitor of hERG current (I; IC of 183 nM) and of native ventricular I from guinea-pig ventricular myocytes. 100 nM and 1 μM sarizotan prolonged ventricular action potential (AP) duration (APD) by 14.1 ± 3.3% (n = 6) and 29.8 ± 3.1% (n = 5) respectively and promoted AP triangulation. High affinity I inhibition by sarizotan was contingent upon channel gating and intact inactivation. Mutagenesis experiments and docking analysis implicated F557, S624 and Y652 residues in sarizotan binding, with weaker contribution from F656. In conclusion, sarizotan inhibits I/I, accessing key binding residues on channel gating. This action and consequent ventricular AP prolongation occur at concentrations relevant to those proposed to treat breathing dysrhythmia in RTT. Sarizotan should only be used in RTT patients with careful evaluation of risk factors for QT prolongation.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:22-30
Cheng H, Du C, Zhang Y, James AF, ... Abdala AP, Hancox JC
J Mol Cell Cardiol: 29 Sep 2019; 135:22-30 | PMID: 31362019
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Abstract

Smooth muscle-specific TMEM16A expression protects against angiotensin II-induced cerebrovascular remodeling via suppressing extracellular matrix deposition.

Zeng XL, Sun L, Zheng HQ, Wang GL, ... Ma MM, Guan YY

Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca-activated Cl channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-β1/Smad3, and non-canonical TGF-β1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl-sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-β1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:131-143
Zeng XL, Sun L, Zheng HQ, Wang GL, ... Ma MM, Guan YY
J Mol Cell Cardiol: 30 Aug 2019; 134:131-143 | PMID: 31301303
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Abstract

Electrophysiologic and molecular mechanisms of a frameshift NPPA mutation linked with familial atrial fibrillation.

Menon A, Hong L, Savio-Galimberti E, Sridhar A, ... Kupershmidt S, Darbar D

A frameshift (fs) mutation in the natriuretic peptide precursor A (NPPA) gene, encoding a mutant atrial natriuretic peptide (Mut-ANP), has been linked with familial atrial fibrillation (AF) but the underlying mechanisms by which the mutation causes AF remain unclear. We engineered 2 transgenic (TG) mouse lines expressing the wild-type (WT)-NPPA gene (H-WT-NPPA) and the human fs-Mut-NPPA gene (H-fsMut-NPPA) to test the hypothesis that mice overexpressing the human NPPA mutation are more susceptible to AF and elucidate the underlying electrophysiologic and molecular mechanisms. Transthoracic echocardiography and surface electrocardiography (ECG) were performed in H-fsMut-NPPA, H-WT-NPPA, and Non-TG mice. Invasive electrophysiology, immunohistochemistry, Western blotting and patch clamping of membrane potentials were performed. To examine the role of the Mut-ANP in ion channel remodeling, we measured plasma cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity in the 3 groups of mice. In H-fsMut-NPPA mice mean arterial pressure (MAP) was reduced when compared to H-WT-NPPA and Non-TG mice. Furthermore, injection of synthetic fs-Mut-ANP lowered the MAP in H-WT-NPPA and Non-TG mice while synthetic WT-ANP had no effect on MAP in the 3 groups of mice. ECG characterization revealed significantly prolonged QRS duration in H-fsMut-NPPA mice when compared to the other two groups. Trans-Esophageal (TE) atrial pacing of H-fsMut-NPPA mice showed increased AF burden and AF episodes when compared with H-WT-NPPA or Non-TG mice. The cardiac Na (NaV1.5) and Ca (CaV1.2/CaV1.3) channel expression and currents (I, I) and action potential durations (APD/APD/APD) were significantly reduced in H-fsMut-NPPA mice while the rectifier K channel current (I) was markedly increased when compared to the other 2 groups of mice. In addition, plasma cGMP levels were only increased in H-fsMut-NPPA mice with a corresponding reduction in plasma cAMP levels and PKA activity. In summary, we showed that mice overexpressing an AF-linked NPPA mutation are more prone to develop AF and this risk is mediated in part by remodeling of the cardiac Na, Ca and K channels creating an electrophysiologic substrate for reentrant AF.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2019; 132:24-35
Menon A, Hong L, Savio-Galimberti E, Sridhar A, ... Kupershmidt S, Darbar D
J Mol Cell Cardiol: 29 Jun 2019; 132:24-35 | PMID: 31077706
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Abstract

Dynamic palmitoylation regulates trafficking of K channel interacting protein 2 (KChIP2) across multiple subcellular compartments in cardiac myocytes.

Murthy A, Workman SW, Jiang M, Hu J, ... Deschenes I, Tseng GN
Background
K channel interacting protein 2 (KChIP2), initially cloned as Kv4 channel modulator, is a multi-tasking protein. In addition to modulating several cardiac ion channels at the plasma membrane, it can also modulate microRNA transcription inside nuclei, and interact with presenilins to modulate Ca release through RyR2 in the cytoplasm. However, the mechanism regulating its subcellular distribution is not clear.
Objective
We tested whether palmitoylation drives KChIP2 trafficking and distribution in cells, and whether the distribution pattern of KChIP2 in cardiac myocytes is sensitive to cellular milieu.
Method
We conducted imaging and biochemical experiments on palmitoylatable and unpalmitoylatable KChIP2 variants expressed in COS-7 cells and in cardiomyocytes, and on native KChIP2 in myocytes.
Results
In COS-7 cells, palmitoylatable KChIP2 clustered to plasma membrane, while unpalmitoylatable KChIP2 exhibited higher cytoplasmic mobility and faster nuclear entry. The same differences in distribution and mobility were observed when these KChIP2 variants were expressed in cardiac myocytes, indicating that the palmitoylation-dependent distribution and trafficking are intrinsic properties of KChIP2. Importantly, acute stress in a rat model of cardiac arrest/resuscitation induced changes in native KChIP2 resembling those of KChIP2 depalmitoylation, promoting KChIP2 nuclear entry.
Conclusion
The palmitoylation status of KChIP2 determines its subcellular distribution in cardiac myocytes. Stress promotes nuclear entry of KChIP2, diverting it from ion channel modulation at the plasma membrane to other functions in the nuclear compartment.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:1-9
Murthy A, Workman SW, Jiang M, Hu J, ... Deschenes I, Tseng GN
J Mol Cell Cardiol: 29 Sep 2019; 135:1-9 | PMID: 31362018
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Impact:
Abstract

Disruption of the CCL1-CCR8 axis inhibits vascular Treg recruitment and function and promotes atherosclerosis in mice.

Vila-Caballer M, González-Granado JM, Zorita V, Abu Nabah YN, ... Mallat Z, Andrés V

The CC chemokine 1 (CCL1, also called I-309 or TCA3) is a potent chemoattractant for leukocytes that plays an important role in inflammatory processes and diseases through binding to its receptor CCR8. Here, we investigated the role of the CCL1-CCR8 axis in atherosclerosis. We found increased expression of CCL1 in the aortas of atherosclerosis-prone fat-fed apolipoprotein E (Apoe)-null mice; moreover, in vitro flow chamber assays and in vivo intravital microscopy demonstrated an essential role for CCL1 in leukocyte recruitment. Mice doubly deficient for CCL1 and Apoe exhibited enhanced atherosclerosis in aorta, which was associated with reduced plasma levels of the anti-inflammatory interleukin 10, an increased splenocyte Th1/Th2 ratio, and a reduced regulatory T cell (Treg) content in aorta and spleen. Reduced Treg recruitment and aggravated atherosclerosis were also detected in the aortas of fat-fed low-density lipoprotein receptor-null mice treated with CCR8 blocking antibodies. These findings demonstrate that disruption of the CCL1-CCR8 axis promotes atherosclerosis by inhibiting interleukin 10 production and Treg recruitment and function.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2019; 132:154-163
Vila-Caballer M, González-Granado JM, Zorita V, Abu Nabah YN, ... Mallat Z, Andrés V
J Mol Cell Cardiol: 29 Jun 2019; 132:154-163 | PMID: 31121182
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Impact:
Abstract

Tachycardia-induced CD44/NOX4 signaling is involved in the development of atrial remodeling.

Chen WJ, Chang SH, Chan YH, Lee JL, ... Tsai FC, Yeh YH

Atrial fibrillation (AF) is associated with oxidative stress and Ca-handling abnormalities in atrial myocytes. Our prior study has demonstrated the involvement of CD44, a membrane receptor for hyaluronan (HA), in the pathogenesis of AF. This study further evaluated whether CD44 and its related signaling mediate atrial tachycardia-induced oxidative stress and Ca-handling abnormalities. Tachypacing in atrium-derived myocytes (HL-1 cell line) induced the activation of CD44-related signaling, including HA and HA synthase (HAS) expression. Blocking HAS/HA/CD44 signaling attenuated tachypacing-induced oxidative stress (NADPH oxidase [NOX] 2/4 expression) and Ca-handling abnormalities (oxidized Ca/calmodulin-dependent protein kinase II [ox-CaMKII] and phospho-ryanodine receptor type 2 [p-RyR2] expression) in HL-1 myocytes. Furthermore, a direct association between CD44 and NOX4 was documented in tachy-paced HL-1 myocytes and atrial tissues from AF patients. In vitro, Ca spark frequencies in atrial myocytes isolated from CD44 mice were lower than those from wild-type mice. Furthermore, administration of an anti-CD44 blocking antibody in atrial myocytes isolated from wild-type mice diminished the frequency of Ca spark. Ex vivo tachypacing models of CD44 mice exhibited a lower degree of oxidative stress and expression of ox-CaMKII/p-RyR2 in their atria than those of wild-type mice. In vivo, burst atrial pacing stimulated a less inducibility of AF in CD44mice than in wild-type mice. In conclusion, atrial tachypacing-induced Ca-handling abnormalities are mediated via CD44/NOX4 signaling, which provides a possible explanation for the development of AF.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 29 Sep 2019; 135:67-78
Chen WJ, Chang SH, Chan YH, Lee JL, ... Tsai FC, Yeh YH
J Mol Cell Cardiol: 29 Sep 2019; 135:67-78 | PMID: 31419440
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Impact:
Abstract

Targeting HIF-1α in combination with PPARα activation and postnatal factors promotes the metabolic maturation of human induced pluripotent stem cell-derived cardiomyocytes.

Gentillon C, Li D, Duan M, Yu WM, ... Brown LA, Xu C

Immature phenotypes of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) limit the utility of these cells in clinical application and basic research. During cardiac development, postnatal cardiomyocytes experience high oxygen tension along with a concomitant downregulation of hypoxia-inducible factor 1α (HIF-1α), leading to increased fatty acid oxidation (FAO). We hypothesized that targeting HIF-1α alone or in combination with other metabolic regulators could promote the metabolic maturation of hiPSC-CMs. We examined the effect of HIF-1α inhibition on the maturation of hiPSC-CMs and investigated a multipronged approach to promote hiPSC-CM maturation by combining HIF-1α inhibition with molecules that target key pathways involved in the energy metabolism. Cardiac spheres of highly-enriched hiPSC-CMs were treated with a HIF-1α inhibitor alone or in combination with an agonist of peroxisome proliferator activated receptor α (PPARα) and three postnatal factors (triiodothyronine hormone T3, insulin-like growth factor-1 and dexamethasone). HIF-1α inhibition significantly increased FAO and basal and maximal respiration of hiPSC-CMs. Combining HIF-1α inhibition with PPARα activation and the postnatal factors further increased FAO and improved mitochondrial maturation in hiPSC-CMs. Compared with mock-treated cultures, the cultures treated with the five factors had increased mitochondrial content and contained more cells with mitochondrial distribution throughout the cells, which are features of more mature cardiomyocytes. Consistent with these observations, a number of transcriptional regulators of mitochondrial metabolic processes were upregulated in hiPSC-CMs treated with the five factors. Furthermore, these cells had significantly increased Ca transient kinetics and contraction and relaxation velocities, which are functional features for more mature cardiomyocytes. Therefore, targeting HIF-1α in combination with other metabolic regulators significantly improves the metabolic maturation of hiPSC-CMs.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2019; 132:120-135
Gentillon C, Li D, Duan M, Yu WM, ... Brown LA, Xu C
J Mol Cell Cardiol: 29 Jun 2019; 132:120-135 | PMID: 31082397
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Impact:
Abstract

Inactivation of cysteine 674 in the SERCA2 accelerates experimental aortic aneurysm.

Que Y, Shu X, Wang L, Hu P, ... Chen H, Tong X

Sarcoplasmic/endoplasmic reticulum Ca ATPase 2 (SERCA2) is vital to maintain intracellular calcium homeostasis. SERCA2 cysteine 674 (C674) is highly conservative and its irreversible oxidation is upregulated in human and mouse aortic aneurysms, especially in smooth muscle cells (SMCs). The contribution of SERCA2 and its redox C674 in the development of aortic aneurysm remains enigmatic. Objective: Our goal was to investigate the contribution of inactivation of C674 to the development of aortic aneurysm and the mechanisms involved. Approach and results: Using SERCA2 C674S knock-in (SKI) mouse line, in which half of C674 was substituted by serine 674 (S674) to represent partial irreversible oxidation of C674 in aortic aneurysm, we found that in aortic SMCs the replacement of C674 by S674 resulted in SMC phenotypic modulation. In SKI SMCs, the increased intracellular calcium activated calcium-dependent calcineurin, which promoted the nuclear translocation of nuclear factor of activated T-lymphocytes (NFAT) and nuclear factor kappa-B (NFκB), while inhibition of calcineurin blocked SMC phenotypic modulation. Besides, the replacement of C674 by S674 accelerated angiotensin II-induced aortic aneurysm. Conclusions: Our results indicate that the inactivation of C674 by causing the accumulation of intracellular calcium to activate calcineurin-mediated NFAT/NFκB pathways, resulted in SMC phenotypic modulation to accelerate aortic aneurysm, which highlights the importance of C674 redox state in the development of aortic aneurysms.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 04 Feb 2020; epub ahead of print
Que Y, Shu X, Wang L, Hu P, ... Chen H, Tong X
J Mol Cell Cardiol: 04 Feb 2020; epub ahead of print | PMID: 32035136
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Abstract

Pseudo-bipolar spindle formation and cell division in postnatal binucleated cardiomyocytes.

Leone M, Engel FB
Background
The majority of adult human, mouse and rat cardiomyocytes is not diploid mononucleated. Nevertheless, the current literature on heart regeneration based on cardiomyocyte proliferation focuses mainly on the proliferation capacity of diploid mononucleated cardiomyocytes, instead of the more abundant mononucleated polyploid or binucleated cardiomyocytes. Here, we aimed at a better understanding of the process of mitosis and cell division in postnatal binucleated cardiomyocytes.
Methods and results
Postnatal rat binucleated cardiomyocytes were stimulated to re-enter the cell cycle either by fetal bovine serum or a combination of fibroblast growth factor 1 and p38 MAP kinase inhibitor. Phase-contrast videos revealed that binucleated cardiomyocytes form one metaphase plate and preferentially undergo afterwards cytokinesis failure. The maximum rate of cell division of video-recorded binucleated cardiomyocytes was around 6%. Immunofluorescence analyses of centriole number in mitotic binucleated cardiomyocytes revealed that these cells contain more than four centrioles, which can be paired as well as unpaired. In agreement with multiple and/or unpaired centrioles, multipolar spindle formation was observed in mitotic binucleated cardiomyocytes using fluorescence live imaging of tubulin-GFP. Multipoles were transient and resolved into pseudo-bipolar spindles both in case of cell division and cytokinesis failure. Notably, centrioles were in most cases unevenly distributed among daughter cells.
Conclusions
Our results indicate that postnatal binucleated cardiomyocytes upon stimulation can enter mitosis, cope with their multiple and/or unpaired centrioles by forming pseudo-bipolar spindles, and divide.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:69-73
Leone M, Engel FB
J Mol Cell Cardiol: 30 Aug 2019; 134:69-73 | PMID: 31301302
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Abstract

Intracardiac administration of neutrophil protease cathepsin G activates noncanonical inflammasome pathway and promotes inflammation and pathological remodeling in non-injured heart.

Miller SA, Kolpakov MA, Guo X, Du B, ... Dell\'Italia LJ, Sabri A
Background
Inflammatory serine proteases (ISPs) play an important role in cardiac repair after injury through hydrolysis of dead cells and extracellular matrix (ECM) debris. Evidence also suggests an important role of ISPs in the coordination of the inflammatory response. However, the effect of ISPs on inflammation is obfuscated by the confounding factors associated with cell death and inflammatory cell infiltration induced after cardiac injury. This study investigated whether neutrophil-derived cathepsin G (Cat.G) influences inflammation and remodeling in the absence of prior cardiac injury and cell death.
Methods and results
Intracardiac catheter delivery of Cat.G (1 mg/kg) in rats induced significant left ventricular (LV) dilatation and cardiac contractile dysfunction at day 5, but not at day 2, post-delivery compared to vehicle-treated animals. Cat.G delivery also significantly increased matrix metalloprotease activity and collagen and fibronectin degradation at day 5 compared to vehicle-treated rats and these changes were associated with increased death signaling pathways and myocyte apoptosis. Mechanistic analysis shows that Cat.G-treatment induced potent chemotactic activity in hearts at day 2 and 5 post-delivery, characterized by processing and activation of interleukin (IL)-1β and IL-18, stimulation of inflammatory signaling pathways and accumulation of myeloid cells when compared to vehicle-treated rats. Cat.G-induced processing of IL-1β and IL-18 was independent of the canonical NLRP-3 inflammasome pathway and treatment of isolated cardiomyocytes with inhibitors of NLRP-3 or caspase-1 failed to reduce Cat.G-induced cardiomyocyte death. Notably, rats treated with IL-1 receptor antagonist (IL-1Ra) show reduced inflammation and improved cardiac remodeling and function following Cat.G delivery.
Conclusions
Cat.G exerts potent chemoattractant and pro-inflammatory effects in non-stressed or injured heart in part through processing and activation of IL-1 family cytokines, subsequently leading to adverse cardiac remodeling and function. Thus, targeting ISPs could be a novel therapeutic strategy to reduce cardiac inflammation and improve cardiac remodeling and function after injury or stress.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Aug 2019; 134:29-39
Miller SA, Kolpakov MA, Guo X, Du B, ... Dell'Italia LJ, Sabri A
J Mol Cell Cardiol: 30 Aug 2019; 134:29-39 | PMID: 31252040
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Abstract

LncRNA PCFL promotes cardiac fibrosis via miR-378/GRB2 pathway following myocardial infarction.

Sun F, Zhuang Y, Zhu H, Wu H, ... Pan Z, Lu Y

Long noncoding RNAs (lncRNAs) are a class of novel molecular regulators in cardiac development and diseases. However, the role of specific lncRNAs in cardiac fibrosis remains to be fully explored. The aim of the present study was to investigate the effects and underlying mechanisms of lncRNA PCFL (pro-cardiac fibrotic lncRNA) on cardiac fibrosis after myocardial infarction (MI). Cardiac fibroblasts (CFs) with gain and loss of function of PCFL and mice with global knockout or overexpression of PCFL were used to explore the effects of PCFL on cardiac fibrosis. The data showed that PCFL was significantly increased in hearts of mice subjected to MI and CFs treated with transforming growth factor-β1 (TGF-β1). Overexpression of PCFL promoted collagen production and CF proliferation, while silencing PCFL exhibited the opposite effects. Compared with wild type MI mice, heterozygous knockout of PCFL (PCFL) in mice significantly improved heart function and reduced cardiac fibrosis after MI. While overexpression of PCFL impaired cardiac function and aggravated MI-induced cardiac fibrosis. The mechanistic data demonstrated that PCFL functioned as a sponge of miR-378. Luciferase reporter assay confirmed the interaction of PCFL with miR-378. MiR-378 inhibited collagen production by suppressing its target gene, GRB2 (growth factor receptor bound protein 2). Knockdown of PCFL led to an increase of miR-378. Silencing of miR-378 reserved the inhibitory effects of PCFL knockdown on collagen production, cell proliferation and GRB2 expression. In conclusion, the study identifies a novel pro-fibrotic lncRNA, PCFL, and the mechanism involves the direct interaction of PCFL with miR-378, which in turn relieves the inhibition effect of miR-378 on GRB2 and promotes cardiac fibrosis.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2019; 133:188-198
Sun F, Zhuang Y, Zhu H, Wu H, ... Pan Z, Lu Y
J Mol Cell Cardiol: 30 Jul 2019; 133:188-198 | PMID: 31220469
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Abstract

Impaired SIRT3 activity mediates cardiac dysfunction in endotoxemia by calpain-dependent disruption of ATP synthesis.

Koentges C, Cimolai MC, Pfeil K, Wolf D, ... Zirlik A, Bugger H
Background
Sepsis-induced cardiomyopathy contributes to the high mortality of septic shock in critically ill patients. Since the underlying mechanisms are incompletely understood, we hypothesized that sepsis-induced impairment of sirtuin 3 (SIRT3) activity contributes to the development of septic cardiomyopathy.
Methods and results
Treatment of mice with lipopolysaccharide (LPS) for 6 h resulted in myocardial NAD depletion and increased mitochondrial protein acetylation, indicating impaired myocardial SIRT3 activity due to NAD depletion. LPS treatment also resulted in impaired cardiac output in isolated working hearts, indicating endotoxemia-induced cardiomyopathy. Maintaining normal myocardial NAD levels in LPS-treated mice by Poly(ADP-ribose)polymerase 1 (PARP1) deletion prevented cardiac dysfunction, whereas additional SIRT3 deficiency blunted this beneficial effect, indicating that impaired SIRT3 activity contributes to cardiac dysfunction in endotoxemia. Measurements of mitochondrial ATP synthesis suggest that LPS-induced contractile dysfunction may result from cardiac energy depletion due to impaired SIRT3 activity. Pharmacological inhibition of mitochondrial calpains using MDL28170 normalized LPS-induced cleavage of the ATP5A1 subunit of ATP synthase and normalized contractile dysfunction, suggesting that cardiac energy depletion may result from calpain-mediated cleavage of ATP5A1. These beneficial effects were completely blunted by SIRT3 deficiency. Finally, a gene set enrichment analysis of hearts of patients with septic, ischemic or dilated cardiomyopathy revealed a sepsis-specific suppression of SIRT3 deacetylation targets, including ATP5A1, indicating a functional relevance of SIRT3-dependent pathways in human sepsis.
Conclusions
Impaired SIRT3 activity may mediate cardiac dysfunction in endotoxemia by facilitating calpain-mediated disruption of ATP synthesis, suggesting SIRT3 activation as a potential therapeutic strategy to treat septic cardiomyopathy.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2019; 133:138-147
Koentges C, Cimolai MC, Pfeil K, Wolf D, ... Zirlik A, Bugger H
J Mol Cell Cardiol: 30 Jul 2019; 133:138-147 | PMID: 31201798
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Abstract

Mechanism of 17β-estradiol stimulated integration of human mesenchymal stem cells in heart tissue.

Mihai MC, Popa MA, Suica VI, Antohe F, ... Simionescu M, Dubey RK

Scarcity of gender specific donor hearts highlights the importance of mesenchymal stem cells (MSCs) as a therapeutic tool for heart repair. However, inefficient incorporation, retention, and activity of MSCs in cardiac tissue remain an obstacle. Since surges in follicular estradiol (E2; μmolar-range) trigger tissue remodeling (e.g. ovulation) and E2 exerts beneficial actions on the cardiovascular system, we hypothesized that E2 may promote/improve MSC-mediated cardiac repair processes. Using Wharton\'s jelly (WJ)-derived MSCs we assessed the effects of E2 on MSC proliferation, directed migration, and engraftment in murine heart slices (using xCELLigence real-time cell-impedance system, DNA quantification, and microscopy) and on MSC-induced angiogenesis in vivo (matrigel plug assay). Protein expression was assessed by Western blotting, ELISA/Luminex, and proteomic analysis; whereas mRNA expression was assessed by qRT-PCR. MSCs expressed estrogen receptors (ERs) -alpha and -beta. E2 promoted MSC proliferation and up-regulated mRNA and protein expression of ER-alpha, ER-beta, extracellular matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase (MMP) -9, yet down-regulated MMP-2 expression. Moreover, E2 up-regulated expression of vascular endothelial growth factor (VEGF)-A, VEGFR-2, vascular cell adhesion protein-1 (VCAM-1), and angiogenin (ANG) and stimulated nitric oxide (NO) production via ER. Proteomic analysis of MSCs showed that E2 up-regulated 47 proteins, down-regulated 7 proteins, and increased the expression of key biochemical components/pathways involved in tissue repair. In MSCs co-cultured with murine heart-slices, E2 significantly induced MSC migration in an ER-alpha-dependent fashion and significantly increased the secretion of MMP-2, MMP-9, ANG, and VEGF. In an in vivo matrigel assay, E2-treated MSCs increased angiogenesis and hemoglobin content. In conclusion, E2-treatment increases the incorporation of MSCs in heart slices and promotes MSC-induced angiogenesis. These beneficial effects are mediated via increases in molecules/pathways involved in tissue remodeling and angiogenesis. We speculate that E2 may enhance MSC ability to repair/regenerate cardiac tissue.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Jul 2019; 133:115-124
Mihai MC, Popa MA, Suica VI, Antohe F, ... Simionescu M, Dubey RK
J Mol Cell Cardiol: 30 Jul 2019; 133:115-124 | PMID: 31201797
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Abstract

Inhibition of Rho-associated kinases suppresses cardiac myofibroblast function in engineered connective and heart muscle tissues.

Santos GL, Hartmann S, Zimmermann WH, Ridley A, Lutz S

Cardiac fibrosis is a hallmark of heart failure for which there is no effective pharmacological therapy. By genetic modification and in vivo inhibitor approaches it was suggested that the Rho-associated kinases (ROCK1 and ROCK2) are involved in pro-fibrotic signalling in cardiac fibroblasts and that they may serve as targets for anti-fibrotic therapies. We demonstrate that simultaneous inhibition of ROCK1 and ROCK2 strongly interfered with tissue formation and their biomechanical properties in a model of engineered connective tissue (ECT), comprised of cardiac fibroblasts and collagen. These effects were observed with both rat and human ECT. Inhibitors of different chemistries, including the isoquinoline inhibitors Fasudil and H1152P as well as the pyrazol-phenyl inhibitor SR-3677, showed comparable effects. By combined treatment of ECT with TGF-β and H1152P, we could identify ROCK as a mediator of TGF-β-dependent tissue stiffening. Moreover, expression analyses suggested that lysyl oxidase (LOX) is a downstream target of the ROCK-actin-MRTF/SRF pathway and inhibition of this pathway by Latrunculin A and CCG-203971 showed similar anti-fibrotic effects in the ECT model as ROCK inhibitors. In line with the collagen crosslinking function of LOX, its inhibition by β-aminopropionitrile resulted in reduced ECT stiffness, but let tissue compaction unaffected. Finally, we show that ROCK inhibition also reduced the compaction and stiffness of engineered heart muscle tissues. Our results indicate that pharmacological inhibition of ROCK has a strong anti-fibrotic potential which is in part due to a decrease in the expression of the collagen crosslinking enzyme lysyl oxidase.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:13-28
Santos GL, Hartmann S, Zimmermann WH, Ridley A, Lutz S
J Mol Cell Cardiol: 30 Aug 2019; 134:13-28 | PMID: 31233754
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Abstract

A Notch more: Molecular players in bicuspid aortic valve disease.

Lee A, Wei S, Schwertani A

The prevalence of calcification of human aortic valve in populations 65 years old and greater is estimated to be 2-3%. Bicuspid aortic valve disease (BAVD) is a common etiology of aortic stenosis in populations aged 60-75 years of age; 30-50% of operated cases of aortic stenosis were due to calcified BAVD. Dysregulation of the Notch and the canonical Wnt pathway has been well documented to be associated with calcification of the aortic valve. However, recent studies have increased this scope to include the non-canonical pathway where Wnt5a, Wnt5b and Wnt 11 levels were significantly greater in calcified human aortic valves than normal valves, and with Wnt5b specifically being implicated in BAVD pathogenesis. More recently, Lipoprotein(a) [Lp(a)] has been implicated as a key player in the pathogenesis of calcific aortic valve disease. It has been shown that the osteogenic effect of Lp(a) is mediated through the oxidized phospholipid pathway as well as oxidized phospholipid independent pathways involving mitogen-activated protein kinases (MAPK), glycogen synthase kinase (GSK) and Wnt. Moving forward, further work needs to be conducted in order to elucidate the crosstalk between the different signaling cascades, specifically with regard to BAVD.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:62-68
Lee A, Wei S, Schwertani A
J Mol Cell Cardiol: 30 Aug 2019; 134:62-68 | PMID: 31150732
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Abstract

Exosomal CagA derived from Helicobacter pylori-infected gastric epithelial cells induces macrophage foam cell formation and promotes atherosclerosis.

Yang S, Xia YP, Luo XY, Chen SL, ... Li YN, Hu B
Background
Seroepidemiological studies have highlighted a positive relation between CagA-positive Helicobacter pylori (H. pylori), atherosclerosis and related clinic events. However, this link has not been well validated. The present study was designed to explore the role of H. pylori PMSS1 (a CagA-positive strain that can translocate CagA into host cells) and exosomal CagA in the progression of atherosclerosis.
Methods
To evaluate whether H. pylori accelerates or even induces atherosclerosis, H. pylori-infected C57/BL6 mice and ApoE mice were maintained under different dietary conditions. To identify the role of H. pylori-infected gastric epithelial cells-derived exosomes (Hp-GES-EVs) and exosomal CagA in atherosclerosis, ApoE mice were given intravenous or intraperitoneal injections of saline, GES-EVs, Hp-GES-EVs, and recombinant CagA protein (rCagA).
Findings
CagA-positive H. pylori PMSS1 infection does not induce but promotes macrophage-derived foam cell formation and augments atherosclerotic plaque growth and instability in two animal models. Meanwhile, circulating Hp-GES-EVs are taken up in aortic plaque, and CagA is secreted in Hp-GES-EVs. Furthermore, the CagA-containing EVs and rCagA exacerbates macrophage-derived foam cell formation and lesion development in vitro and in vivo, recapitulating the pro-atherogenic effects of CagA-positive H. pylori. Mechanistically, CagA suppresses the transcription of cholesterol efflux transporters by downregulating the expression of transcriptional factors PPARγ and LXRα and thus enhances foam cell formation.
Interpretation
These results may provide new insights into the role of exosomal CagA in the pathogenesis of CagA-positive H. pylori infection-related atherosclerosis. It is suggested that preventing and eradicating CagA-positive H. pylori infection could reduce the incidence of atherosclerosis and related events.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Sep 2019; 135:40-51
Yang S, Xia YP, Luo XY, Chen SL, ... Li YN, Hu B
J Mol Cell Cardiol: 29 Sep 2019; 135:40-51 | PMID: 31352044
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Abstract

Involvement of circulating inflammatory factors in prognosis and risk of cardiovascular disease.

Haybar H, Shokuhian M, Bagheri M, Davari N, Saki N

Cardiovascular disease (CVD) is an inflammatory disease that different factors play a crucial role in the development of clinical outcome of this disease. Inflammation could have effects on initiation, progression, and clinical complications of CVD. Previous studies have indicated that delineating the underlying mechanisms of inflammatory factors involved in this disease should be considerably beneficial both as predictive markers and targets for advancement of appropriate therapeutic approaches in offsetting development and progression of cardiovascular complications. Mechanisms of inflammatory factors involved in CVD combined with the development of atherosclerosis, reperfusion injury, and myocardial infarction caused by changes in processes such as endothelial cells function and hemostasis can contribute to the development of clinical outcome in CVD. Therefore, it can be stated that recognition of inflammatory mechanisms involved in this disease can be a promising tool for evaluation of prognosis in CVD patients. In this article, our goal is to evaluate the possible role of changes in the expressions of inflammatory factors in CVD as well as their relationship with prognosis of this disease.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2019; 132:110-119
Haybar H, Shokuhian M, Bagheri M, Davari N, Saki N
J Mol Cell Cardiol: 29 Jun 2019; 132:110-119 | PMID: 31102585
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Abstract

The lymphocyte adapter protein: A negative regulator of myocardial ischemia/reperfusion injury.

Huang J, Sun Y, Chen L, Ma G

Myocardial ischemia/reperfusion (I/R) injury is the major limitation for the cardioprotective action of revascularization after myocardial infarction. Lymphocyte adapter protein (Lnk), an adapter protein, has a regulatory role in multiple signaling pathways by functioning as a scaffold for different substrates. However, the involvement of Lnk in myocardial I/R injury remains to be established. In this study, increased expression of Lnk was detected upon the development of myocardial I/R injury. Mice were genetically engineered to investigate the role of Lnk in this pathological process. Upon I/R, myocardial infarction, cardiac dysfunction, inflammation and apoptosis were increased in Lnk-deficient hearts. However, cardiomyocyte-specific overexpression of Lnk protected the hearts against myocardial I/R injury. Mechanistically, we observed that the activation of Akt, but neither ERK1/2 nor STAT3, was influenced by the expression of Lnk upon myocardial I/R injury. Furthermore, the requirement of PI3K-Akt activation for the cardioprotective effect of Lnk was confirmed in rescue experiments using the PI3K inhibitor LY294002. Taken together, our data provide a potential diagnostic and therapeutic strategy for myocardial I/R injury.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:107-118
Huang J, Sun Y, Chen L, Ma G
J Mol Cell Cardiol: 30 Aug 2019; 134:107-118 | PMID: 31301301
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Abstract

The metalloproteinase ADAM15 is upregulated by shear stress and promotes survival of endothelial cells.

Babendreyer A, Molls L, Simons IM, Dreymueller D, ... Schnakenberg U, Ludwig A

Reduced shear stress resulting from disturbed blood flow can impair endothelial integrity and drive the development of vascular inflammatory lesions. Metalloproteinases of the ADAM family have been implicated in the regulation of cell survival and inflammatory responses. Here we investigate the mechanism and function of ADAM15 upregulation in primary flow cultured endothelial cells. Transcriptomic analysis indicated that within the ADAM family ADAM15 mRNA is most prominently upregulated (4-fold) when endothelial cells are exposed to physiologic shear stress. This induction was confirmed in venous, arterial and microvascular endothelial cells and is associated with increased presence of ADAM15 protein in the cell lysates (5.6-fold) and on the surface (3.1-fold). The ADAM15 promoter contains several consensus sites for the transcription factor KLF2 which is also upregulated by shear stress. Induction of endothelial KLF2 by simvastatin treatment is associated with ADAM15 upregulation (1.8-fold) which is suppressed by counteracting simvastatin with geranylgeranyl pyrophosphate. KLF2 overexpression promotes ADAM15 expression (2.1-fold) under static conditions whereas KLF2 siRNA knockdown prevents ADAM15 induction by shear stress. Functionally, ADAM15 promotes survival of endothelial cells challenged by growth factor depletion or TNF stimulation as shown by ADAM15 shRNA knockdown (1.6-fold). Exposure to shear stress increases endothelial survival while additional knockdown of ADAM15 reduces survival (6.7-fold) under flow conditions. Thus, physiologic shear stress resulting from laminar flow promotes KLF2 induced ADAM15 expression which contributes to endothelial survival. The absence of ADAM15 at low shear stress or static conditions may therefore lead to increased endothelial damage and promote vascular inflammation.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2019; 134:51-61
Babendreyer A, Molls L, Simons IM, Dreymueller D, ... Schnakenberg U, Ludwig A
J Mol Cell Cardiol: 30 Aug 2019; 134:51-61 | PMID: 31271758
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Abstract

Myocardial maladaptation to pressure overload in CB2 receptor-deficient mice.

Duerr GD, Heinemann JC, Kley J, Eichhorn L, ... Zimmer A, Dewald O
Background
Adaptation to aortic valve stenosis leads to myocardial hypertrophy, which has been associated with inflammation, fibrosis and activation of the endocannabinoid system. Since the endocannabinoid system and the CB2 receptor provide cardioprotection and modulate immune response in experimental ischemia, we investigated the role of CB2 in a mouse model of cardiac pressure overload.
Methods
Transverse aortic constriction was performed in CB2 receptor-deficient (Cnr2) mice and their wild-type littermates (Cnr2). After echocardiography and Millar left heart catheter hemodynamic evaluation hearts were processed for histological, cellular and molecular analyses.
Results
The endocannabinoid system showed significantly higher anandamide production and CB2 receptor expression in Cnr2 mice. Histology showed non-confluent, interstitial fibrosis with rare small areas of cardiomyocyte loss in Cnr2 mice. In contrast, extensive cardiomyocyte loss and confluent scar formation were found in Cnr2 mice accompanied by significantly increased apoptosis and left ventricular dysfunction when compared with Cnr2 mice. The underlying cardiac maladaptation in Cnr2 mice was associated with significantly reduced expression of myosin heavy chain isoform beta and less production of heme oxygenase-1. Cnr2 hearts presented after 7 days with stronger proinflammatory response including significantly higher TNF-alpha expression and macrophage density, but lower density of CD4+ and B220+ cells. At the same time, we found increased apoptosis of macrophages and adaptive immune cells. Higher myofibroblast accumulation and imbalance in MMP/TIMP-regulation indicated adverse remodeling in Cnr2 mice.
Conclusions
Our study provides mechanistic evidence for the role of the endocannabinoid system in myocardial adaptation to pressure overload in mice. The underlying mechanisms include production of anandamide, adaptation of contractile elements and antioxidative enzymes, and selective modulation of immune cells action and apoptosis in order to prevent the loss of cardiomyocytes.

Copyright © 2019 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2019; 133:86-98
Duerr GD, Heinemann JC, Kley J, Eichhorn L, ... Zimmer A, Dewald O
J Mol Cell Cardiol: 30 Jul 2019; 133:86-98 | PMID: 31181227
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Impact:

This program is still in alpha version.