Journal: J Mol Cell Cardiol

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Abstract

Can polarization of macrophage metabolism enhance cardiac regeneration?

Lantz C, Becker A, Thorp EB
While largely appreciated for their antimicrobial and repair functions, macrophages have emerged as indispensable for the development, homeostasis, and regeneration of tissue, including regeneration of the neonatal heart. Upon activation, mammalian neonatal macrophages express and secrete factors that coordinate angiogenesis, resolution of inflammation, and ultimately cardiomyocyte proliferation. This is contrary to adult macrophages in the adult heart, which are incapable of inducing significant levels of cardiac regeneration. The underlying mechanisms by which pro-regenerative macrophages are activated and regulated remain vague. A timely hypothesis is that macrophage metabolism contributes to this proliferative and regenerative potential. This is because we now appreciate the significant contributions of metabolites to immune cell programming and function, beyond solely bioenergetics. After birth, the metabolic milieu of the neonate is subject to significant alterations in oxygenation and nutrient supply, which will affect how metabolic substrates are catabolized. In this context, we discuss potential roles for select macrophage metabolic pathways during cardiac regeneration.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 18 Jul 2021; epub ahead of print
Lantz C, Becker A, Thorp EB
J Mol Cell Cardiol: 18 Jul 2021; epub ahead of print | PMID: 34293342
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Abstract

Early adaptive chromatin remodeling events precede pathologic phenotypes and are reinforced in the failing heart.

Chapski DJ, Cabaj M, Morselli M, Mason RJ, ... Vondriska TM, Rosa-Garrido M
The temporal nature of chromatin structural changes underpinning pathologic transcription are poorly understood. We measured chromatin accessibility and DNA methylation to study the contribution of chromatin remodeling at different stages of cardiac hypertrophy and failure. ATAC-seq and reduced representation bisulfite sequencing were performed in cardiac myocytes after transverse aortic constriction (TAC) or depletion of the chromatin structural protein CTCF. Early compensation to pressure overload showed changes in chromatin accessibility and DNA methylation preferentially localized to intergenic and intronic regions. Most methylation and accessibility changes observed in enhancers and promoters at the late phase (3 weeks after TAC) were established at an earlier time point (3 days after TAC), before heart failure manifests. Enhancers were paired with genes based on chromatin conformation capture data: while enhancer accessibility generally correlated with changes in gene expression, this feature, nor DNA methylation, was alone sufficient to predict transcription of all enhancer interacting genes. Enrichment of transcription factors and active histone marks at these regions suggests that enhancer activity coordinates with other epigenetic factors to determine gene transcription. In support of this hypothesis, ChIP-qPCR demonstrated increased enhancer and promoter occupancy of GATA4 and NKX2-5 at Itga9 and Nppa, respectively, concomitant with increased transcription of these genes in the diseased heart. Lastly, we demonstrate that accessibility and DNA methylation are imperfect predictors of chromatin structure at the scale of A/B compartmentalization-rather, accessibility, DNA methylation, transcription factors and other histone marks work within these domains to determine gene expression. These studies establish that chromatin reorganization during early compensation after pathologic stimuli is maintained into the later decompensatory phases of heart failure. The findings reveal the rules for how local chromatin features govern gene expression in the context of global genomic structure and identify chromatin remodeling events for therapeutic targeting in disease.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 13 Jul 2021; epub ahead of print
Chapski DJ, Cabaj M, Morselli M, Mason RJ, ... Vondriska TM, Rosa-Garrido M
J Mol Cell Cardiol: 13 Jul 2021; epub ahead of print | PMID: 34273410
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Abstract

FNDC5/Irisin attenuates diabetic cardiomyopathy in a type 2 diabetes mouse model by activation of integrin αV/β5-AKT signaling and reduction of oxidative/nitrosative stress.

Lin C, Guo Y, Xia Y, Li C, ... Yan W, Tao L
Irisin, the cleaved form of the fibronectin type III domain containing 5 (FNDC5) protein, is involved in metabolism and inflammation. Recent findings indicated that irisin participated in cardiovascular physiology and pathology. In this study, we investigated the effects of FNDC5/irisin on diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice. Downregulation of myocardial FNDC5/irisin protein expression and plasma irisin levels was observed in db/db mice compared to db/+ controls. Moreover, echocardiography revealed that db/db mice exhibited normal cardiac systolic function and impaired diastolic function. Adverse structural remodeling, including cardiomyocyte apoptosis, myocardial fibrosis, and cardiac hypertrophy were observed in the hearts of db/db mice. Sixteen-week-old db/db mice were intramyocardially injected with adenovirus encoding FNDC5 or treated with recombinant human irisin via a peritoneal implant osmotic pump for 4 weeks. Both overexpression of myocardial FNDC5 and exogenous irisin administration attenuated diastolic dysfunction and cardiac structural remodeling in db/db mice. Results from in vitro studies revealed that FNDC5/irisin protein expression was decreased in high glucose (HG)/high fat (HF)-treated cardiomyocytes. Increased levels of inducible nitric oxide synthase (iNOS), NADPH oxidase 2 (NOX2), 3-nitrotyrosine (3-NT), reactive oxygen species (ROS), and peroxynitrite (ONOO-) in HG/HF-treated H9C2 cells provided evidence of oxidative/nitrosative stress, which was alleviated by treatment with FNDC5/irisin. Moreover, the mitochondria membrane potential (ΔΨm) was decreased and cytochrome C was released from mitochondria with increased levels of cleaved caspase-3 in HG/HF-treated H9C2 cells, indicating the presence of mitochondria-dependent apoptosis, which was partially reversed by FNDC5/irisin treatment. Mechanistic studies showed that activation of integrin αVβ5-AKT signaling and attenuation of oxidative/nitrosative stress were responsible for the cardioprotective effects of FNDC5/irisin. Therefore, FNDC5/irisin mediates cardioprotection in DCM by inhibiting myocardial apoptosis, myocardial fibrosis, and cardiac hypertrophy. These findings implicate that FNDC5/irisin as a potential therapeutic intervention for DCM, especially in type 2 diabetes mellitus (T2DM).

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 02 Jul 2021; 160:27-41
Lin C, Guo Y, Xia Y, Li C, ... Yan W, Tao L
J Mol Cell Cardiol: 02 Jul 2021; 160:27-41 | PMID: 34224725
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Abstract

YAP1/TEAD1 upregulate platelet-derived growth factor receptor beta to promote vascular smooth muscle cell proliferation and neointima formation.

Osman I, Dong K, Kang X, Yu L, ... Zhang W, Zhou J
We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRβ expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRβ expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRβ expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRβ, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:20-32
Osman I, Dong K, Kang X, Yu L, ... Zhang W, Zhou J
J Mol Cell Cardiol: 29 Jun 2021; 156:20-32 | PMID: 33753119
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Abstract

Diverse functional responses to high glucose by primary and permanent hybrid endothelial cells in vitro.

Uruski P, Mikuła-Pietrasik J, Drzewiecki M, Budkiewicz S, ... Tykarski A, Książek K
Various types of human endothelial cells, including human umbilical vein endothelial cells (HUVECs) and the established hybrid EAhy926 cells, are used in experimental research. Here, we compared the biological properties of HUVECs and EAhy926 cells under normal (5 mM) and high glucose (30 mM; HG) conditions. The results showed that HG induced cellular senescence and a stronger DNA damage response in HUVECs than in EAhy926 cells. The magnitude of oxidative stress elicited in HUVECs by HG was also greater than that elicited in their established counterparts. Both endothelial cell types promoted the progression of breast (MCF7), ovarian (OVCAR-3), and lung (A549) cancer cells; however, the effects elicited by HG-treated HUVECs on adhesion (MCF7, OVCAR-3), proliferation (OVCAR-3), and migration (OVCAR-3) were more pronounced. Finally, HG stimulated the production of a higher number of proangiogenic agents in HUVECs than in EAhy926 cells. Collectively, our study shows that the functional properties of primary and established endothelial cells exposed to HG differ substantially, which seems to result from the higher sensitivity of the former to this stressor. The interchangeability of both types of endothelial cells in biomedical research should be considered with great care to avoid losing some biological effects due to the choice of cells with higher stress tolerance.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:1-6
Uruski P, Mikuła-Pietrasik J, Drzewiecki M, Budkiewicz S, ... Tykarski A, Książek K
J Mol Cell Cardiol: 29 Jun 2021; 156:1-6 | PMID: 33731316
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Abstract

Application of genetic cell-lineage tracing technology to study cardiovascular diseases.

Sun X, Lyu L, Zhong X, Ni Z, Xu Q
Cardiovascular diseases are leading causes that threaten people\'s life. To investigate cells that are involved in disease development and tissue repair, various technologies have been introduced. Among these technologies, lineage tracing is a powerful tool to track the fate of cells in vivo, providing deep insights into cellular behavior and plasticity. In cardiac diseases, newly formed cardiomyocytes and endothelial cells are found from proliferation of local cells, while fibroblasts and macrophages are originated from diverse cell sources. Similarly, in response to vascular injury, various sources of cells including media smooth muscle cells, endothelium, resident progenitors and bone marrow cells are involved in lesion formation and/or vessel regeneration. In summary, current review summarizes the development of lineage tracing techniques and their utilizations in investigating roles of different cell types in cardiovascular diseases.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 29 Jun 2021; 156:57-68
Sun X, Lyu L, Zhong X, Ni Z, Xu Q
J Mol Cell Cardiol: 29 Jun 2021; 156:57-68 | PMID: 33745891
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Abstract

New calcification model for intact murine aortic valves.

Kruithof BPT, van de Pol V, Los T, Lodder K, ... Goumans MJ, Ajmone Marsan N
Calcific aortic valve disease (CAVD) is a common progressive disease of the aortic valves, for which no medical treatment exists and surgery represents currently the only therapeutic solution. The development of novel pharmacological treatments for CAVD has been hampered by the lack of suitable test-systems, which require the preservation of the complex valve structure in a mechanically and biochemical controllable system. Therefore, we aimed at establishing a model which allows the study of calcification in intact mouse aortic valves by using the Miniature Tissue Culture System (MTCS), an ex vivo flow model for whole mouse hearts. Aortic valves of wild-type mice were cultured in the MTCS and exposed to osteogenic medium (OSM, containing ascorbic acid, β-glycerophosphate and dexamethasone) or inorganic phosphates (PI). Osteogenic calcification occurred in the aortic valve leaflets that were cultured ex vivo in the presence of PI, but not of OSM. In vitro cultured mouse and human valvular interstitial cells calcified in both OSM and PI conditions, revealing in vitro-ex vivo differences. Furthermore, endochondral differentiation occurred in the aortic root of ex vivo cultured mouse hearts near the hinge of the aortic valve in both PI and OSM conditions. Dexamethasone was found to induce endochondral differentiation in the aortic root, but to inhibit calcification and the expression of osteogenic markers in the aortic leaflet, partly explaining the absence of calcification in the aortic valve cultured with OSM. The osteogenic calcifications in the aortic leaflet and the endochondral differentiation in the aortic root resemble calcifications found in human CAVD. In conclusion, we have established an ex vivo calcification model for intact wild-type murine aortic valves in which the initiation and progression of aortic valve calcification can be studied. The in vitro-ex vivo differences found in our studies underline the importance of ex vivo models to facilitate pre-clinical translational studies.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:95-104
Kruithof BPT, van de Pol V, Los T, Lodder K, ... Goumans MJ, Ajmone Marsan N
J Mol Cell Cardiol: 29 Jun 2021; 156:95-104 | PMID: 33744308
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Abstract

Impact of etiology on force and kinetics of left ventricular end-stage failing human myocardium.

Mashali MA, Saad NS, Canan BD, Elnakish MT, ... Mohler PJ, Janssen PML
Background
Heart failure (HF) is associated with highly significant morbidity, mortality, and health care costs. Despite the significant advances in therapies and prevention, HF remains associated with poor clinical outcomes. Understanding the contractile force and kinetic changes at the level of cardiac muscle during end-stage HF in consideration of underlying etiology would be beneficial in developing targeted therapies that can help improve cardiac performance.
Objective
Investigate the impact of the primary etiology of HF (ischemic or non-ischemic) on left ventricular (LV) human myocardium force and kinetics of contraction and relaxation under near-physiological conditions.
Methods and results
Contractile and kinetic parameters were assessed in LV intact trabeculae isolated from control non-failing (NF; n = 58) and end-stage failing ischemic (FI; n = 16) and non-ischemic (FNI; n = 38) human myocardium under baseline conditions, length-dependent activation, frequency-dependent activation, and response to the β-adrenergic stimulation. At baseline, there were no significant differences in contractile force between the three groups; however, kinetics were impaired in failing myocardium with significant slowing down of relaxation kinetics in FNI compared to NF myocardium. Length-dependent activation was preserved and virtually identical in all groups. Frequency-dependent activation was clearly seen in NF myocardium (positive force frequency relationship [FFR]), while significantly impaired in both FI and FNI myocardium (negative FFR). Likewise, β-adrenergic regulation of contraction was significantly impaired in both HF groups.
Conclusions
End-stage failing myocardium exhibited impaired kinetics under baseline conditions as well as with the three contractile regulatory mechanisms. The pattern of these kinetic impairments in relation to NF myocardium was mainly impacted by etiology with a marked slowing down of kinetics in FNI myocardium. These findings suggest that not only force development, but also kinetics should be considered as a therapeutic target for improving cardiac performance and thus treatment of HF.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:7-19
Mashali MA, Saad NS, Canan BD, Elnakish MT, ... Mohler PJ, Janssen PML
J Mol Cell Cardiol: 29 Jun 2021; 156:7-19 | PMID: 33766524
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Abstract

Creld1 regulates myocardial development and function.

Beckert V, Rassmann S, Kayvanjoo AH, Klausen C, ... Mass E, Wachten D
CRELD1 (Cysteine-Rich with EGF-Like Domains 1) is a risk gene for non-syndromic atrioventricular septal defects in human patients. In a mouse model, Creld1 has been shown to be essential for heart development, particularly in septum and valve formation. However, due to the embryonic lethality of global Creld1 knockout (KO) mice, its cell type-specific function during peri- and postnatal stages remains unknown. Here, we generated conditional Creld1 KO mice lacking Creld1 either in the endocardium (KOTie2) or the myocardium (KOMyHC). Using a combination of cardiac phenotyping, histology, immunohistochemistry, RNA-sequencing, and flow cytometry, we demonstrate that Creld1 function in the endocardium is dispensable for heart development. Lack of myocardial Creld1 causes extracellular matrix remodeling and trabeculation defects by modulation of the Notch1 signaling pathway. Hence, KOMyHC mice die early postnatally due to myocardial hypoplasia. Our results reveal that Creld1 not only controls the formation of septa and valves at an early stage during heart development, but also cardiac maturation and function at a later stage. These findings underline the central role of Creld1 in mammalian heart development and function.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:45-56
Beckert V, Rassmann S, Kayvanjoo AH, Klausen C, ... Mass E, Wachten D
J Mol Cell Cardiol: 29 Jun 2021; 156:45-56 | PMID: 33773996
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Abstract

Role of myeloid-derived chemokine CCL5/RANTES at an early stage of atherosclerosis.

Jongstra-Bilen J, Tai K, Althagafi MG, Siu A, ... Hyduk SJ, Cybulsky MI
One of the hallmarks of atherosclerosis is ongoing accumulation of macrophages in the artery intima beginning at disease onset. Monocyte recruitment contributes to increasing macrophage abundance at early stages of atherosclerosis. Although the chemokine CCL5 (RANTES) has been studied in atherosclerosis, its role in the recruitment of monocytes to early lesions has not been elucidated. We show that expression of Ccl5 mRNA, as well as other ligands of the CCR5 receptor (Ccl3 and Ccl4), is induced in the aortic intima of Ldlr-/- mice 3 weeks after the initiation of cholesterol-rich diet (CRD)-induced hypercholesterolemia. En face immunostaining revealed that CCL5 protein expression is also upregulated at 3 weeks of CRD. Blockade of CCR5 significantly reduced monocyte recruitment to 3-week lesions, suggesting that chemokine signaling through CCR5 is critical. However, we observed that Ccl5-deficiency had no effect on early lesion formation and CCL5-blockade did not affect monocyte recruitment in Ldlr-/- mice. Immunostaining of the lesions in Ldlr-/- mice and reciprocal bone marrow transplantation (BMT) of Ccl5+/+ and Ccl5-/- mice revealed that CCL5 is expressed by both myeloid and endothelial cells. BMT experiments were carried out to determine if CCL5 produced by distinct cells has functions that may be concealed in Ccl5-/-Ldlr-/- mice. We found that hematopoietic cell-derived CCL5 regulates monocyte recruitment and the abundance of intimal macrophages in 3-week lesions of Ldlr-/- mice but plays a minor role in 6-week lesions. Our findings suggest that there is a short window in early lesion formation during which myeloid cell-derived CCL5 has a critical role in monocyte recruitment and macrophage abundance.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:69-78
Jongstra-Bilen J, Tai K, Althagafi MG, Siu A, ... Hyduk SJ, Cybulsky MI
J Mol Cell Cardiol: 29 Jun 2021; 156:69-78 | PMID: 33781821
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Abstract

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Lynch TL, Kumar M, McNamara JW, Kuster DWD, ... Warshaw DM, Sadayappan S
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein\'s amino terminal (N\')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N\'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N\'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:33-44
Lynch TL, Kumar M, McNamara JW, Kuster DWD, ... Warshaw DM, Sadayappan S
J Mol Cell Cardiol: 29 Jun 2021; 156:33-44 | PMID: 33781820
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Abstract

LITAF acts as a novel regulator for pathological cardiac hypertrophy.

Xiang M, Yang F, Zhou Y, Li W, ... Wang PX, Chen M
Pathological hypertrophy generally progresses to heart failure. Exploring effective and promising therapeutic targets might lead to progress in preventing its detrimental outcomes. Our current knowledge about lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) is mainly limited to regulate inflammation. However, the role of LITAF in other settings that are not that relevant to inflammation, such as cardiac remodeling and heart failure, remains largely unknown. In the present study, we found that the expression of LITAF decreased in hypertrophic hearts and cardiomyocytes. Meanwhile, LITAF protected cultured neonatal rat cardiomyocytes against phenylephrine-induced hypertrophy. Moreover, using LITAF knockout mice, we demonstrated that LITAF deficiency exacerbated cardiac hypertrophy and fibrosis compared with wild-type mice. Mechanistically, LITAF directly binds to the N-terminal of ASK1, thus disrupting the dimerization of ASK1 and blocking ASK1 activation, ultimately inhibiting ASK1-JNK/p38 signaling over-activation and protecting against cardiac hypertrophy. Furthermore, AAV9-mediated LITAF overexpression attenuated cardiac hypertrophy in vivo. Conclusions: Our findings uncover the novel role of LITAF as a negative regulator of cardiac remodeling. Targeting the interaction between LITAF and ASK1 could be a promising therapeutic strategy for pathological cardiac remodeling.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:82-94
Xiang M, Yang F, Zhou Y, Li W, ... Wang PX, Chen M
J Mol Cell Cardiol: 29 Jun 2021; 156:82-94 | PMID: 33823186
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Abstract

Sarcoplasmic reticulum-mitochondria communication; implications for cardiac arrhythmia.

Hamilton S, Terentyeva R, Clements RT, Belevych AE, Terentyev D
Sudden cardiac death due to ventricular tachyarrhythmias remains the major cause of mortality in the world. Heart failure, diabetic cardiomyopathy, old age-related cardiac dysfunction and inherited disorders are associated with enhanced propensity to malignant cardiac arrhythmias. Both defective mitochondrial function and abnormal intracellular Ca2+ homeostasis have been established as the key contributing factors in the pathophysiology and arrhythmogenesis in these conditions. This article reviews current advances in understanding of bidirectional control of ryanodine receptor-mediated sarcoplasmic reticulum Ca2+ release and mitochondrial function, and how defects in crosstalk between these two organelles increase arrhythmic risk in cardiac disease.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 29 Jun 2021; 156:105-113
Hamilton S, Terentyeva R, Clements RT, Belevych AE, Terentyev D
J Mol Cell Cardiol: 29 Jun 2021; 156:105-113 | PMID: 33857485
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Abstract

An organ-on-a-chip model for pre-clinical drug evaluation in progressive non-genetic cardiomyopathy.

Wang EY, Kuzmanov U, Smith JB, Dou W, ... Gramolini A, Radisic M
Angiotensin II (Ang II) presents a critical mediator in various pathological conditions such as non-genetic cardiomyopathy. Osmotic pump infusion in rodents is a commonly used approach to model cardiomyopathy associated with Ang II. However, profound differences in electrophysiology and pharmacokinetics between rodent and human cardiomyocytes may limit predictability of animal-based experiments. This study investigates the application of an Organ-on-a-chip (OOC) system in modeling Ang II-induced progressive cardiomyopathy. The disease model is constructed to recapitulate myocardial response to Ang II in a temporal manner. The long-term tissue cultivation and non-invasive functional readouts enable monitoring of both acute and chronic cardiac responses to Ang II stimulation. Along with mapping of cytokine secretion and proteomic profiles, this model presents an opportunity to quantitatively measure the dynamic pathological changes that could not be otherwise identified in animals. Further, we present this model as a testbed to evaluate compounds that target Ang II-induced cardiac remodeling. Through assessing the effects of losartan, relaxin, and saracatinib, the drug screening data implicated multifaceted cardioprotective effects of relaxin in restoring contractile function and reducing fibrotic remodeling. Overall, this study provides a controllable platform where cardiac activities can be explicitly observed and tested over the pathological process. The facile and high-content screening can facilitate the evaluation of potential drug candidates in the pre-clinical stage.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 29 Jun 2021; epub ahead of print
Wang EY, Kuzmanov U, Smith JB, Dou W, ... Gramolini A, Radisic M
J Mol Cell Cardiol: 29 Jun 2021; epub ahead of print | PMID: 34216608
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Abstract

Triiodothyronine maintains cardiac transverse-tubule structure and function.

Gilani N, Wang K, Muncan A, Peter J, ... Stout RF, Ojamaa K
Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca2+ mobilization and contractility, and clustering of dyadic proteins. Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 μg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca2+ channel (LTCC, Cav1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing. The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Cav1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 23 Jun 2021; 160:1-14
Gilani N, Wang K, Muncan A, Peter J, ... Stout RF, Ojamaa K
J Mol Cell Cardiol: 23 Jun 2021; 160:1-14 | PMID: 34175303
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Abstract

Function of histone methylation and acetylation modifiers in cardiac hypertrophy.

Qin J, Guo N, Tong J, Wang Z
Cardiac hypertrophy is an adaptive response of the heart to increased workload induced by various physiological or pathological stimuli. It is a common pathological process in multiple cardiovascular diseases, and it ultimately leads to heart failure. The development of cardiac hypertrophy is accompanied by gene expression reprogramming, a process that is largely dependent on epigenetic regulation. Histone modifications such as methylation and acetylation are dynamically regulated under cardiac stress. These consequently contribute to the pathogenesis of cardiac hypertrophy via compensatory or maladaptive transcriptome reprogramming. Histone methylation and acetylation modifiers play crucial roles in epigenetic remodeling during the pathogenesis of cardiac hypertrophy. Regulation of histone methylation and acetylation modifiers serves as a bridge between signal transduction and downstream gene reprogramming. Exploring the role of histone modifiers in cardiac hypertrophy provides novel therapeutic strategies to treat cardiac hypertrophy and heart failure. In this review, we summarize the recent advancements in functional histone methylation and acetylation modifiers in cardiac hypertrophy, with an emphasis on the underlying mechanisms and the therapeutic potential.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 23 Jun 2021; 159:120-129
Qin J, Guo N, Tong J, Wang Z
J Mol Cell Cardiol: 23 Jun 2021; 159:120-129 | PMID: 34175302
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Abstract

Matters of the heart: Cellular sex differences.

Walker CJ, Schroeder ME, Aguado BA, Anseth KS, Leinwand LA
Nearly all cardiovascular diseases show sexual dimorphisms in prevalence, presentation, and outcomes. Until recently, most clinical trials were carried out in males, and many animal studies either failed to identify the sex of the animals or combined data obtained from males and females. Cellular sex in the heart is relatively understudied and many studies fail to report the sex of the cells used for in vitro experiments. Moreover, in the small number of studies in which sex is reported, most of those studies use male cells. The observation that cells from males and females are inherently different is becoming increasingly clear - either due to acquired differences from hormones and other factors or due to intrinsic differences in genotype (XX or XY). Because of the likely contribution of cellular sex differences in cardiac health and disease, here, we explore differences in mammalian male and female cells in the heart, including the less-studied non-myocyte cell populations. We discuss how the heart\'s microenvironment impacts male and female cellular phenotypes and vice versa, including how secretory profiles are dependent on cellular sex, and how hormones contribute to sexually dimorphic phenotypes and cellular functions. Intracellular mechanisms that contribute to sex differences, including gene expression and epigenetic remodeling, are also described. Recent single-cell sequencing studies have revealed unexpected sex differences in the composition of cell types in the heart which we discuss. Finally, future recommendations for the design and consideration of cellular sex differences in the heart are provided.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 20 Jun 2021; epub ahead of print
Walker CJ, Schroeder ME, Aguado BA, Anseth KS, Leinwand LA
J Mol Cell Cardiol: 20 Jun 2021; epub ahead of print | PMID: 34166708
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Abstract

Prostaglandin E1 attenuates AngII-induced cardiac hypertrophy via EP3 receptor activation and Netrin-1upregulation.

Shen Y, Wang X, Yuan R, Pan X, ... He B, Shen L
Aims
Pathological cardiac hypertrophy induced by activation of the renin-angiotensin-aldosterone system (RAAS) is one of the leading causes of heart failure. However, in current clinical practice, the strategy for targeting the RAAS is not sufficient to reverse hypertrophy. Here, we investigated the effect of prostaglandin E1 (PGE1) on angiotensin II (AngII)-induced cardiac hypertrophy and potential molecular mechanisms underlying the effect.
Methods and results
Adult male C57 mice were continuously infused with AngII or saline and treated daily with PGE1 or vehicle for two weeks. Neonatal rat cardiomyocytes were cultured to detect AngII-induced hypertrophic responses. We found that PGE1 ameliorated AngII-induced cardiac hypertrophy both in vivo and in vitro. The RNA sequencing (RNA-seq) and expression pattern analysis results suggest that Netrin-1 (Ntn1) is the specific target gene of PGE1. The protective effect of PGE1 was eliminated after knockdown of Ntn1. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the PGE1-mediated signaling pathway changes are associated with the mitogen-activated protein kinase (MAPK) pathway. PGE1 suppressed AngII-induced activation of the MAPK signaling pathway, and such an effect was attenuated by Ntn1 knockdown. Blockade of MAPK signaling rescued the phenotype of cardiomyocytes caused by Ntn1 knockdown, indicating that MAPK signaling may act as the downstream effector of Ntn1. Furthermore, inhibition of the E-prostanoid (EP) 3 receptor, as opposed to the EP1, EP2, or EP4 receptor, in cardiomyocytes reversed the effect of PGE1, and activation of EP3 by sulprostone, a specific agonist, mimicked the effect of PGE1.
Conclusion
In conclusion, PGE1 ameliorates AngII-induced cardiac hypertrophy through activation of the EP3 receptor and upregulation of Ntn1, which inhibits the downstream MAPK signaling pathway. Thus, targeting EP3, as well as the Ntn1-MAPK axis, may represent a novel approach for treating pathological cardiac hypertrophy.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 17 Jun 2021; 159:91-104
Shen Y, Wang X, Yuan R, Pan X, ... He B, Shen L
J Mol Cell Cardiol: 17 Jun 2021; 159:91-104 | PMID: 34147480
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Impact:
Abstract

Dimethyl sulfoxide (DMSO) enhances direct cardiac reprogramming by inhibiting the bromodomain of coactivators CBP/p300.

Lim CK, Efthymios M, Tan W, Autio MI, ... Li PY, Foo RSY
Aims
Direct cardiac reprogramming represents an attractive way to reversing heart damage caused by myocardial infarction because it removes fibroblasts, while also generating new functional cardiomyocytes. Yet, the main hurdle for bringing this technique to the clinic is the lack of efficacy with current reprogramming protocols. Here, we describe our unexpected discovery that DMSO is capable of significantly augmenting direct cardiac reprogramming in vitro.
Methods and results
Upon induction with cardiac transcription factors- Gata4, Hand2, Mef2c and Tbx5 (GHMT), the treatment of mouse embryonic fibroblasts (MEFs) with 1% DMSO induced ~5 fold increase in Myh6-mCherry+ cells, and significantly upregulated global expression of cardiac genes, including Myh6, Ttn, Nppa, Myh7 and Ryr2. RNA-seq confirmed upregulation of cardiac gene programmes and downregulation of extracellular matrix-related genes. Treatment of TGF-β1, DMSO, or SB431542, and the combination thereof, revealed that DMSO most likely targets a separate but parallel pathway other than TGF-β signalling. Subsequent experiments using small molecule screening revealed that DMSO enhances direct cardiac reprogramming through inhibition of the CBP/p300 bromodomain, and not its acetyltransferase property.
Conclusion
In conclusion, our work points to a direct molecular target of DMSO, which can be used for augmenting GHMT-induced direct cardiac reprogramming and possibly other cell fate conversion processes.

Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 16 Jun 2021; 160:15-26
Lim CK, Efthymios M, Tan W, Autio MI, ... Li PY, Foo RSY
J Mol Cell Cardiol: 16 Jun 2021; 160:15-26 | PMID: 34146546
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Impact:
Abstract

Inhibition of Interleukin-21 prolongs the survival through the promotion of wound healing after myocardial infarction.

Kubota A, Suto A, Suga K, Iwata A, ... Kobayashi Y, Nakajima H
Ly6Clow macrophages promote scar formation and prevent early infarct expansion after myocardial infarction (MI). Although CD4+ T cells influence the regulation of Ly6Clow macrophages after MI, the mechanism remains largely unknown. Based on the hypothesis that some molecule(s) secreted by CD4+ T cells act on Ly6Clow macrophages, we searched for candidate molecules by focusing on cytokine receptors expressed on Ly6Clow macrophages. Comparing the transcriptome between Ly6Chigh macrophages and Ly6Clow macrophages harvested from the infarcted heart, we found that Ly6Clow macrophages highly expressed the receptor for interleukin (IL)-21, a pleiotropic cytokine which is produced by several types of CD4+ T cells, compared with Ly6Chigh macrophages. Indeed, CD4+ T cells harvested from the infarcted heart produce IL-21 upon stimulation. Importantly, the survival rate and cardiac function after MI were significantly improved in IL-21-deficient (il21-/-) mice compared with those in wild-type (WT) mice. Transcriptome analysis of infarcted heart tissue from WT mice and il21-/- mice at 5 days after MI demonstrated that inflammation is persistent in WT mice compared with il21-/- mice. Consistent with the transcriptome analysis, the number of neutrophils and matrix metalloproteinase (MMP)-9 expression were significantly decreased, whereas the number of Ly6Clow macrophages and MMP-12 expression were significantly increased in il21-/- mice. In addition, collagen deposition and the number of myofibroblasts in the infarcted area were significantly increased in il21-/- mice. Consistently, IL-21 enhanced the apoptosis of Ly6Clow macrophages. Finally, administration of neutralizing IL-21 receptor Fc protein increased the number of Ly6Clow macrophages in the infarcted heart and improved the survival and cardiac function after MI. Thus, IL-21 decreases the survival after MI, possibly through the delay of wound healing by inducing the apoptosis of Ly6Clow macrophages.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 15 Jun 2021; 159:48-61
Kubota A, Suto A, Suga K, Iwata A, ... Kobayashi Y, Nakajima H
J Mol Cell Cardiol: 15 Jun 2021; 159:48-61 | PMID: 34144051
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Abstract

The mechanistic target of rapamycin complex 1 critically regulates the function of mononuclear phagocytes and promotes cardiac remodeling in acute ischemia.

Chen G, Phan V, Luo X, Cao DJ
Monocytes and macrophages are cellular forces that drive and resolve inflammation triggered by acute myocardial ischemia. One of the most important but least understood regulatory mechanisms is how these cells sense cues from the micro-milieu and integrate environmental signals with their response that eventually determines the outcome of myocardial repair. In the current study, we investigated if the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) plays this role. We present evidence that support a robustly activated mTORC1 pathway in monocytes and macrophages in the infarcting myocardium.. Specific mTORC1 inhibition transformed the landscape of cardiac monocytes and macrophages into reparative cells that promoted myocardial healing. As the result, mTORC1 inhibition diminished remodeling and reduced mortality from acute ischemia by 80%. In conclusion, our data suggest a critical role of mTORC1 in regulating the functions of cardiac monocytes and macrophages, and specific mTORC1 inhibition protects the heart from inflammatory injury in acute ischemia. As mTOR/mTORC1 is a master regulator that integrates external signals with cellular responses, the study sheds light on how the cardiac monocytes and macrophages sense and respond to the ischemic environment..

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 14 Jun 2021; 159:62-79
Chen G, Phan V, Luo X, Cao DJ
J Mol Cell Cardiol: 14 Jun 2021; 159:62-79 | PMID: 34139235
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Impact:
Abstract

Serum response factor deletion 5 regulates phospholamban phosphorylation and calcium uptake.

Woulfe KC, Jeffrey DA, Da Silva JP, Wilson CE, ... Miyamoto SD, Sucharov CC
Aims
Pediatric dilated cardiomyopathy (pDCM) is characterized by unique age-dependent molecular mechanisms that include myocellular responses to therapy. We previously showed that pDCM, but not adult DCM patients respond to phosphodiesterase 3 inhibitors (PDE3i) by increasing levels of the second messenger cAMP and consequent phosphorylation of phospholamban (PLN). However, the molecular mechanisms involved in the differential pediatric and adult response to PDE3i are not clear.
Methods and results
Quantification of serum response factor (SRF) isoforms from the left ventricle of explanted hearts showed that PDE3i treatment affects expression of SRF isoforms in pDCM hearts. An SRF isoform lacking exon 5 (SRFdel5) was highly expressed in the hearts of pediatric, but not adult DCM patients treated with PDE3i. To determine the functional consequence of expression of SRFdel5, we overexpressed full length SRF or SRFdel5 in cultured cardiomyocytes with and without adrenergic stimulation. Compared to a control adenovirus, expression of SRFdel5 increased phosphorylation of PLN, negatively affected expression of the phosphatase that promotes dephosphorylation of PLN (PP2Cε), and promoted faster calcium reuptake, whereas expression of full length SRF attenuated calcium reuptake through blunted phosphorylation of PLN.
Conclusions
Taken together, these data indicate that expression of SRFdel5 in pDCM hearts in response to PDE3i contributes to improved function through regulating PLN phosphorylation and thereby calcium reuptake.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 14 Jun 2021; 159:28-37
Woulfe KC, Jeffrey DA, Da Silva JP, Wilson CE, ... Miyamoto SD, Sucharov CC
J Mol Cell Cardiol: 14 Jun 2021; 159:28-37 | PMID: 34139234
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Abstract

Systemic iron deficiency does not affect the cardiac iron content and progression of heart failure.

Paterek A, Oknińska M, Chajduk E, Polkowska-Motrenko H, Mączewski M, Mackiewicz U
Chronic heart failure (HF) is often accompanied by systemic iron deficiency (ID). However, effects of ID on cardiac iron status and progression of HF are unknown. To investigate these effects rats underwent LAD ligation to induce post-myocardial infarction HF or sham operation. After 3 weeks the animals from both groups were randomized into three subgroups: control, moderate ID and severe ID+anemia (IDA) by a combination of phlebotomy and low iron diet for 5 weeks. Serum and hepatic iron content were reduced by 55% and 70% (ID) and by 80% and 77% (IDA), respectively, while cardiac iron content was unchanged in HF rats. Changes in expression of all cardiomyocyte iron handling proteins indicating preserved cardiomyocytes iron status in HF and ID/IDA. Contractile function of LV cardiomyocytes, Ca2+ transient amplitude, sarcoplasmic reticulum Ca2+ release and SERCA2a function was augmented by ID and IDA and it was accompanied by an increase in serum catecholamines. Neither ID nor IDA affected left ventricular (LV) systolic or diastolic function or dimensions. To sum up, systemic ID does not result in cardiac ID and does not affect progression of HF and even improves contractile function and Ca2+ handling of isolated LV cardiomyocytes, however, at the cost of increased catecholamine level. This suggests that intravenous iron therapy should be considered as an additional therapeutic option in HF, preventing the increase of catecholaminergic drive with its well-known long-term adverse effects.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 14 Jun 2021; 159:16-27
Paterek A, Oknińska M, Chajduk E, Polkowska-Motrenko H, Mączewski M, Mackiewicz U
J Mol Cell Cardiol: 14 Jun 2021; 159:16-27 | PMID: 34139233
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Abstract

Exogenous extracellular matrix proteins decrease cardiac fibroblast activation in stiffening microenvironment through CAPG.

Wang X, Pierre V, Liu C, Senapati S, Park PS, Senyo SE
Controlling fibrosis is an essential part of regenerating the post-ischemic heart. In the post-ischemic heart, fibroblasts differentiate to myofibroblasts that produce collagen-rich matrix to physically stabilize the infarct area. Infarct models in adult mice result in permanent scarring unlike newborn animals which fully regenerate. Decellularized extracellular matrix (dECM) hydrogels derived from early-aged hearts have been shown to be a transplantable therapy that preserves heart function and stimulates cardiomyocyte proliferation and vascularization. In this study, we investigate the anti-fibrotic effects of injectable dECM hydrogels in a cardiac explant model in the context of age-associated tissue compliance. Treatments with adult and fetal dECM hydrogels were tested for molecular effects on cardiac fibroblast activation and fibrosis. Altered sensitivity of fibroblasts to the mechanosignaling of the remodeling microenvironment was evaluated by manipulating the native extracellular matrix in explants and also with elastomeric substrates in the presence of dECM hydrogels. The injectable fetal dECM hydrogel treatment decreases fibroblast activation and contractility and lowers the stiffness-mediated increases in fibroblast activation observed in stiffened explants. The anti-fibrotic effect of dECM hydrogel is most observable at highest stiffness. Experiments with primary cells on elastomeric substrates with dECM treatment support this phenomenon. Transcriptome analysis indicated that dECM hydrogels affect cytoskeleton related signaling including Macrophage capping protein (CAPG) and Leupaxin (LPXN). CAPG was down-regulated by the fetal dECM hydrogel. LPXN expression was decreased by stiffening the explants; however, this effect was reversed by dECM hydrogel treatment. Pharmacological disruption of cytoskeleton polymerization lowered fibroblast activation and CAPG levels. Knocking down CAPG expression with siRNA inhibited fibroblast activation and collagen deposition. Collectively, fibroblast activation is dependent on cooperative action of extracellular molecular signals and mechanosignaling by cytoskeletal integrity.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 09 Jun 2021; 159:105-119
Wang X, Pierre V, Liu C, Senapati S, Park PS, Senyo SE
J Mol Cell Cardiol: 09 Jun 2021; 159:105-119 | PMID: 34118218
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Abstract

Epigenetic alterations of TGFβ and its main canonical signaling mediators in the context of cardiac fibrosis.

Algeciras L, Palanca A, Maestro D, RuizdelRio J, Villar AV
Cardiac fibrosis is a pathological process that presents a continuous overproduction of extracellular matrix (ECM) components in the myocardium, which negatively influences the progression of many cardiac diseases. Transforming growth factor β (TGFβ) is the main ligand that triggers the production of pro-fibrotic ECM proteins. In the cardiac fibrotic process, TGFβ and its canonical signaling mediators are tightly regulated at different levels as well as epigenetically. Cardiac fibroblasts are one of the most important TGFβ target cells activated after cardiac injury. TGFβ-driven fibroblast activation is subject to epigenetic modulation and contributes to the progression of cardiac fibrosis, mainly through the expression of pro-fibrotic molecules implicated in the disease. In this review, we describe epigenetic regulation related to canonical TGFβ signaling in cardiac fibroblasts.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 09 Jun 2021; 159:38-47
Algeciras L, Palanca A, Maestro D, RuizdelRio J, Villar AV
J Mol Cell Cardiol: 09 Jun 2021; 159:38-47 | PMID: 34119506
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Impact:
Abstract

Organ-on-a-chip systems for vascular biology.

Mandrycky CJ, Howard CC, Rayner SG, Shin YJ, Zheng Y
Organ-on-a-chip (OOC) platforms involve the miniaturization of cell culture systems and enable a variety of novel experimental approaches. These range from modeling the independent effects of biophysical forces on cells to screening novel drugs in multi-organ microphysiological systems, all within microscale devices. As in living systems, the incorporation of vascular structure is a key feature common to almost all organ-on-a-chip systems. In this review we highlight recent advances in organ-on-a-chip technologies with a focus on the vasculature. We first present the developmental process of the blood vessels through which vascular cells assemble into networks and remodel to form complex vascular beds under flow. We then review self-assembled vascular models and flow systems for the study of vascular development and biology as well as pre-patterned vascular models for the generation of perfusable microvessels for modeling vascular and tissue function. We finally conclude with a perspective on developing future OOC approaches for studying different aspects of vascular biology. We highlight the fit for purpose selection of OOC models towards either simple but powerful testbeds for therapeutic development, or complex vasculature to accurately replicate human physiology for specific disease modeling and tissue regeneration.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 08 Jun 2021; 159:1-13
Mandrycky CJ, Howard CC, Rayner SG, Shin YJ, Zheng Y
J Mol Cell Cardiol: 08 Jun 2021; 159:1-13 | PMID: 34118217
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Impact:
Abstract

Circular RNA PVT1 silencing prevents ischemia-reperfusion injury in rat by targeting microRNA-125b and microRNA-200a.

Luo C, Ling GX, Lei BF, Feng X, ... Cai XW, Zheng BS
Circular RNAs (circRNAs) are essential regulators associated with many cardiac conditions, including myocardial infarction (MI). This study aimed to explore circRNA expression during MI development in an animal model and in hypoxia/reoxygenation (H/R)-treated cardiomyocytes. Microarray and real-time quantitative PCR showed that the circRNA PVT1 (circPVT1) was expressed at high levels in MI tissues and H/R-triggered cardiomyocytes. Loss-of-function assays were utilized for examining the influence of circPVT1 on cardiac function and cardiomyocyte properties. Cardiac function was measured by echocardiography at 7 d after MI. Reduced circPVT1 expression significantly decreased MI-triggered myocardial infarct size by 60% and prevented MI-triggered reductions in fractional shortening (%FS) and ejection fraction (EF%). Results of LDH, CCK-8, EdU staining, colony formation assays, and flow cytometry showed that circPVT1 silencing restored cell viability and proliferation while decreased apoptosis. Mechanistic experiments indicated that microRNAs (miR)-125b and miR-200a associated with circPVT1. We demonstrated that circPVT1 functioned as a competitive endogenous RNA (ceRNA) to sponge both miR-125b and miR-200a. Gain-of-function assays showed that miR-125b and miR-200a upregulation partially eliminated the effects of circPVT1 on cardiomyocyte properties. In addition, we found that the previously reported p53/TRAF6, SIRT7, Keap1/Nrf2, and PDCD4 pathways were regulated by the circPVT1/miR-125b/miR-200a axis. In conclusion, our study suggests that circPVT1 protects the myocardium from MI and H/R injury by preventing miR-125b- and miR-200a-mediated apoptotic signaling.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 04 Jun 2021; 159:80-90
Luo C, Ling GX, Lei BF, Feng X, ... Cai XW, Zheng BS
J Mol Cell Cardiol: 04 Jun 2021; 159:80-90 | PMID: 34097926
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Abstract

Ion current profiles in canine ventricular myocytes obtained by the \"onion peeling\" technique.

Horváth B, Kiss D, Dienes C, Hézső T, ... Bányász T, Nánási PP
The profiles of ion currents during the cardiac action potential can be visualized by the action potential voltage clamp technique. To obtain multiple ion current data from the same cell, the \"onion peeling\" technique, based on sequential pharmacological dissection of ion currents, has to be applied. Combination of the two methods allows recording of several ion current profiles from the same myocyte under largely physiological conditions. Using this approach, we have studied the densities and integrals of the major cardiac inward (ICa, INCX, INa-late) and outward (IKr, IKs, IK1) currents in canine ventricular cells and studied the correlation between them. For this purpose, canine ventricular cardiomyocytes were chosen because their electrophysiological properties are similar to those of human ones. Significant positive correlation was observed between the density and integral of ICa and IKr, and positive correlation was found also between the integral of ICa and INCX. No further correlations were detected. The Ca2+-sensitivity of K+ currents was studied by comparing their parameters in the case of normal calcium homeostasis and following blockade of ICa. Out of the three K+ currents studied, only IKs was Ca2+-sensitive. The density and integral of IKs was significantly greater, while its time-to-peak value was shorter at normal Ca2+ cycling than following ICa blockade. No differences were detected for IKr or IK1 in this regard. Present results indicate that the positive correlation between ICa and IKr prominently contribute to the balance between inward and outward fluxes during the action potential plateau in canine myocytes. The results also suggest that the profiles of cardiac ion currents have to be studied under physiological conditions, since their behavior may strongly be influenced by the intracellular Ca2+ homeostasis and the applied membrane potential protocol.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 02 Jun 2021; 158:153-162
Horváth B, Kiss D, Dienes C, Hézső T, ... Bányász T, Nánási PP
J Mol Cell Cardiol: 02 Jun 2021; 158:153-162 | PMID: 34089737
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Abstract

Metabolic remodeling precedes mTORC1-mediated cardiac hypertrophy.

Davogustto GE, Salazar RL, Vasquez HG, Karlstaedt A, ... Eckel-Mahan K, Taegtmeyer H
Rationale
The nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown.
Objective
To examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart.
Methods and results
We developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1iSA) through deletion of Tsc2. Prior to hypertrophy, rates of glucose uptake and oxidation, as well as protein and enzymatic activity of glucose 6-phosphate isomerase (GPI) were decreased, while intracellular levels of glucose 6-phosphate (G6P) were increased. Subsequently, hypertrophy developed. Transcript levels of the fetal gene program and pathways of exercise-induced hypertrophy increased, while hypertrophy did not progress to heart failure. We therefore examined the hearts of wild-type mice subjected to voluntary physical activity and observed early changes in GPI, followed by hypertrophy. Rapamycin prevented these changes in both models.
Conclusion
Activation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 31 May 2021; 158:115-127
Davogustto GE, Salazar RL, Vasquez HG, Karlstaedt A, ... Eckel-Mahan K, Taegtmeyer H
J Mol Cell Cardiol: 31 May 2021; 158:115-127 | PMID: 34081952
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Impact:
Abstract

5-Methoxytryptophan attenuates postinfarct cardiac injury by controlling oxidative stress and immune activation.

Hsu WT, Tseng YH, Jui HY, Kuo CC, Wu KK, Lee CM
Aims
Myocardial infarction (MI) remains a major cause of heart failure. 5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of L-tryptophan, exerts anti-inflammatory and antifibrotic effects, but MI impairs the biosynthesis of cardiac 5-MTP. Therefore, we evaluated the effect of exogenous 5-MTP administration on rescuing post-MI cardiac injury.
Methods and results
After a detailed pharmacokinetic analysis of 5-MTP, Sprague Dawley rats that had undergone left anterior descending coronary artery ligation received intraperitoneal administration of either 17 mg/kg 5-MTP or saline at 0.5 and 24 h after MI. Cardiac systolic function, infarction size, and fibrosis were evaluated using echocardiography, triphenyltetrazolium chloride staining, and Masson trichrome staining, respectively. Myocardial apoptosis was analyzed by staining for caspase-3 and cardiac troponin I. 5-MTP treatment decreased the infarct area and myocardial apoptosis; attenuated systolic dysfunction and left ventricular dilatation; and reduced cardiomyocyte hypertrophy, myocardial fibrosis, and infarct expansion. Crucially, 5-MTP alleviated oxidative stress by preserving mitochondrial antioxidant enzymes and downregulating reactive oxygen species-generating NADPH oxidase isoforms and endothelin-1. Consequently, 5-MTP-treated MI rat hearts exhibited lower levels of chemokines and cytokines, namely interleukin (IL)-1β, IL-18, IL-6, C-C motif chemokine ligand (CCL)-2, and CCL5, accompanied by reduced infiltration of CD11b+ cells and CD4+ T cells. Notably, 5-MTP protected against H2O2-induced damage in HL-1 cardiomyocytes and human umbilical vein endothelial cells in vitro.
Conclusion
5-MTP prevented post-MI cardiac injury by promoting mitochondrial stabilization and controlling redox imbalance. This cytoprotective effect ameliorated macrophage and T-cell infiltration, thus reducing the infarct size, attenuating fibrosis, and restoring myocardial function.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 31 May 2021; 158:101-114
Hsu WT, Tseng YH, Jui HY, Kuo CC, Wu KK, Lee CM
J Mol Cell Cardiol: 31 May 2021; 158:101-114 | PMID: 34087195
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Impact:
Abstract

Interleukin-1α dependent survival of cardiac fibroblasts is associated with StAR/STARD1 expression and improved cardiac remodeling and function after myocardial infarction.

Razin T, Melamed-Book N, Argaman J, Galin I, ... Leor J, Orly J
Aims
One unaddressed aspect of healing after myocardial infarction (MI) is how non-myocyte cells that survived the ischemic injury, keep withstanding additional cellular damage by stress forms typically arising during the post-infarction inflammation. Here we aimed to determine if cell survival is conferred by expression of a mitochondrial protein novel to the cardiac proteome, known as steroidogenic acute regulatory protein, (StAR/STARD1). Further studies aimed to unravel the regulation and role of the non-steroidogenic cardiac StAR after MI.
Methods and results
Following permanent ligation of the left anterior descending coronary artery in mouse heart, timeline western blot analyses showed that StAR expression corresponds to the inflammatory response to MI. Following the identification of StAR in mitochondria of cardiac fibroblasts in culture, confocal microscopy immunohistochemistry (IHC) identified StAR expression in left ventricular (LV) activated interstitial fibroblasts, adventitial fibroblasts and endothelial cells. Further work with the primary fibroblasts model revealed that interleukin-1α (IL-1α) signaling via NF-κB and p38 MAPK pathways efficiently upregulates the expression of the Star gene products. At the functional level, IL-1α primed fibroblasts were protected against apoptosis when exposed to cisplatin mimicry of in vivo apoptotic stress; yet, the protective impact of IL-1α was lost upon siRNA mediated StAR downregulation. At the physiological level, StAR expression was nullified during post-MI inflammation in a mouse model with global IL-1α deficiency, concomitantly resulting in a 4-fold elevation of apoptotic fibroblasts. Serial echocardiography and IHC studies of mice examined 24 days after MI revealed aggravation of LV dysfunction, LV dilatation, anterior wall thinning and adverse tissue remodeling when compared with loxP control hearts.
Conclusions
This study calls attention to overlooked aspects of cellular responses evolved under the stress conditions associated with the default inflammatory response to MI. Our observations suggest that LV IL-1α is cardioprotective, and at least one mechanism of this action is mediated by induction of StAR expression in border zone fibroblasts, which renders them apoptosis resistant. This acquired survival feature also has long-term ramifications on the heart recovery by diminishing adverse remodeling and improving the heart function after MI.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:125-137
Razin T, Melamed-Book N, Argaman J, Galin I, ... Leor J, Orly J
J Mol Cell Cardiol: 30 May 2021; 155:125-137 | PMID: 33130150
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Impact:
Abstract

Spen deficiency interferes with Connexin 43 expression and leads to heart failure in zebrafish.

Rattka M, Westphal S, Gahr BM, Just S, Rottbauer W
Genome-wide association studies identified Spen as a putative modifier of cardiac function, however, the precise function of Spen in the cardiovascular system is not known yet. Here, we analyzed for the first time the in vivo role of Spen in zebrafish and found that targeted Spen inactivation led to progressive impairment of cardiac function in the zebrafish embryo. In addition to diminished cardiac contractile force, Spen-deficient zebrafish embryos developed bradycardia, atrioventricular block and heart chamber fibrillation. Assessment of cardiac-specific transcriptional profiles identified Connexin 43 (Cx43), a cardiac gap junction protein and crucial regulator of cardiomyocyte-to-cardiomyocyte communication, to be significantly diminished in Spen-deficient zebrafish embryos. Similar to the situation in Spen-deficient embryos, Morpholino-mediated knockdown of cx43 in zebrafish resulted in cardiac contractile dysfunction, bradycardia, atrioventricular block and fibrillation of the cardiac chambers. Furthermore, ectopic overexpression of cx43 in Spen deficient embryos led to the reconstitution of cardiac contractile function and suppression of cardiac arrhythmia. Additionally, sensitizing experiments by simultaneously injecting sub-phenotypic concentrations of spen- and cx43-Morpholinos into zebrafish embryos resulted in pathological supra-additive effects. In summary, our findings highlight a crucial role of Spen in controlling cx43 expression and demonstrate the Spen-Cx43 axis to be a vital regulatory cascade that is indispensable for proper heart function in vivo.

Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:25-35
Rattka M, Westphal S, Gahr BM, Just S, Rottbauer W
J Mol Cell Cardiol: 30 May 2021; 155:25-35 | PMID: 33549680
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Impact:
Abstract

Evidence that the acetyltransferase Tip60 induces the DNA damage response and cell-cycle arrest in neonatal cardiomyocytes.

Wang X, Lupton C, Lauth A, Wan TC, ... Auchampach JA, Lough JW
Tip60, a pan-acetyltransferase encoded by the Kat5 gene, is enriched in the myocardium; however, its function in the heart is unknown. In cancer cells, Tip60 acetylates Atm (Ataxia-telangiectasia mutated), enabling its auto-phosphorylation (pAtm), which activates the DNA damage response (DDR). It was recently reported that activation of pAtm at the time of birth induces the DDR in cardiomyocytes (CMs), resulting in proliferative senescence. We therefore hypothesized that Tip60 initiates this process, and that depletion of Tip60 accordingly diminishes the DDR while extending the duration of CM cell-cycle activation. To test this hypothesis, an experimental model was used wherein a Myh6-driven Cre-recombinase transgene was activated on postnatal day 0 (P0) to recombine floxed Kat5 alleles and induce Tip60 depletion in neonatal CMs, without causing pathogenesis. Depletion of Tip60 resulted in reduced numbers of pAtm-positive CMs during the neonatal period, which correlated with reduced numbers of pH2A.X-positive CMs and decreased expression of genes encoding markers of the DDR as well as inflammation. This was accompanied by decreased expression of the cell-cycle inhibitors Meis1 and p27, activation of the cell-cycle in CMs, reduced CM size, and increased numbers of mononuclear/diploid CMs. Increased expression of fetal markers suggested that Tip60 depletion promotes a fetal-like proliferative state. Finally, infarction of Tip60-depleted hearts at P7 revealed improved cardiac function at P39 accompanied by reduced fibrosis, increased CM cell-cycle activation, and reduced apoptosis in the remote zone. These findings indicate that, among its pleiotropic functions, Tip60 induces the DDR in CMs, contributing to proliferative senescence.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:88-98
Wang X, Lupton C, Lauth A, Wan TC, ... Auchampach JA, Lough JW
J Mol Cell Cardiol: 30 May 2021; 155:88-98 | PMID: 33609538
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Impact:
Abstract

Mapping genetic changes in the cAMP-signaling cascade in human atria.

Garnier A, Bork NI, Jacquet E, Zipfel S, ... Fischmeister R, Molina CE
Aim
To obtain a quantitative expression profile of the main genes involved in the cAMP-signaling cascade in human control atria and in different cardiac pathologies.
Methods and results
Expression of 48 target genes playing a relevant role in the cAMP-signaling cascade was assessed by RT-qPCR. 113 samples were obtained from right atrial appendages (RAA) of patients in sinus rhythm (SR) with or without atrium dilation, paroxysmal atrial fibrillation (AF), persistent AF or heart failure (HF); and left atrial appendages (LAA) from patients in SR or with AF. Our results show that right and left atrial appendages in donor hearts or from SR patients have similar expression values except for AC7 and PDE2A. Despite the enormous chamber-dependent variability in the gene-expression changes between pathologies, several distinguishable patterns could be identified. PDE8A, PI3Kγ and EPAC2 were upregulated in AF. Different phosphodiesterase (PDE) families showed specific pathology-dependent changes.
Conclusion
By comparing mRNA-expression patterns of the cAMP-signaling cascade related genes in right and left atrial appendages of human hearts and across different pathologies, we show that 1) gene expression is not significantly affected by cardioplegic solution content, 2) it is appropriate to use SR atrial samples as controls, and 3) many genes in the cAMP-signaling cascade are affected in AF and HF but only few of them appear to be chamber (right or left) specific.
Topic
Genetic changes in human diseased atria.
Translational perspective
The cyclic AMP signaling pathway is important for atrial function. However, expression patterns of the genes involved in the atria of healthy and diseased hearts are still unclear. We give here a general overview of how different pathologies affect the expression of key genes in the cAMP signaling pathway in human right and left atria appendages. Our study may help identifying new genes of interest as potential therapeutic targets or clinical biomarkers for these pathologies and could serve as a guide in future gene therapy studies.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:10-20
Garnier A, Bork NI, Jacquet E, Zipfel S, ... Fischmeister R, Molina CE
J Mol Cell Cardiol: 30 May 2021; 155:10-20 | PMID: 33631188
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Abstract

Inhibiting microRNA-155 attenuates atrial fibrillation by targeting CACNA1C.

Wang J, Ye Q, Bai S, Chen P, ... Yao Y, Ma Y
Background
Reduction in L-type Ca2+ current (ICa,L) density is a hallmark of the electrical remodeling in atrial fibrillation (AF). The expression of miR-155, whose predicted target gene is the α1c subunit of the calcium channel (CACNA1C), was upregulated in atrial cardiomyocytes (aCMs) from patients with paroxysmal AF.The study is to determine miR-155 could target the gene expression of ICa,L and contribute to electrical remodeling in AF.
Methods
The expression of miR-155 and CACNA1C was assessed in aCMs from patients with paroxysmal AF and healthy control. ICa,L properties were observed after miR-155 transfection in human induced pluripotent stem cell derived atrial cardiomyocytes (hiPSC-aCMs). Furthermore, an miR-155 transgene (Tg) and knock-out (KO) mouse model was generated to determine whether miR-155 was involved in ICa,L-related electrical remodeling in AF by targeting CACNA1C.
Results
The expression level of miR-155 was increased, while the expression level of CACNA1C reduced in the aCMs of patients with AF. miR-155 transfection in hiPSC-aCMs produced changes in ICa,L properties qualitatively similar to those produced by AF. miR-155/Tg mice developed a shortened action potential duration and increased vulnerability to AF, which was associated with decreased ICa,L and attenuated by an miR-155 inhibitor. Finally, the genetic inhibition of miR-155 prevented AF induction in miR-155/KO mice with no changes in ICa,L properties.
Conclusions
The increased miR-155 expression in aCMs was sufficient for the reduction in the density of ICa,L and the underlying electronic remodeling. The inhibition of miR-155 prevented ICa,L-related electric remodeling in AF and might constitute a novel anti-AF approach targeting electrical remodeling.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:58-65
Wang J, Ye Q, Bai S, Chen P, ... Yao Y, Ma Y
J Mol Cell Cardiol: 30 May 2021; 155:58-65 | PMID: 33636223
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Abstract

The effect of variable troponin C mutation thin filament incorporation on cardiac muscle twitch contractions.

Mijailovich SM, Prodanovic M, Poggesi C, Powers JD, ... Geeves MA, Regnier M
One of the complexities of understanding the pathology of familial forms of cardiac diseases is the level of mutation incorporation in sarcomeres. Computational models of the sarcomere that are spatially explicit offer an approach to study aspects of mutational incorporation into myofilaments that are more challenging to get at experimentally. We studied two well characterized mutations of cardiac TnC, L48Q and I61Q, that decrease or increase the release rate of Ca2+ from cTnC, k-Ca, resulting in HCM and DCM respectively [1]. Expression of these mutations in transgenic mice was used to provide experimental data for incorporation of 30 and 50% (respectively) into sarcomeres. Here we demonstrate that fixed length twitch contractions of trabeculae from mice containing mutant differ from WT; L48Q trabeculae have slower relaxation while I61Q trabeculae have markedly reduced peak tension. Using our multiscale modelling approach [2] we were able to describe the tension transients of WT mouse myocardium. Tension transients for the mutant cTnCs were simulated with changes in k-Ca, measured experimentally for each cTnC mutant in whole troponin complex, a change in the affinity of cTnC for cTnI, and a reduction in the number of detached crossbridges available for binding. A major advantage of the multiscale explicit 3-D model is that it predicts the effects of variable mutation incorporation, and the effects of variations in mutation distribution within thin filaments in sarcomeres. Such effects are currently impossible to explore experimentally. We explored random and clustered distributions of mutant cTnCs in thin filaments, as well as distributions of individual thin filaments with only WT or mutant cTnCs present. The effects of variable amounts of incorporation and non-random distribution of mutant cTnCs are more marked for I61Q than L48Q cTnC. We conclude that this approach can be effective for study on mutations in multiple proteins of the sarcomere. SUMMARY: A challenge in experimental studies of diseases is accounting for the effect of variable mutation incorporation into myofilaments. Here we use a spatially explicit computational approach, informed by experimental data from transgenic mice expressing one of two mutations in cardiac Troponin C that increase or decrease calcium sensitivity. We demonstrate that the model can accurately describe twitch contractions for the data and go on to explore the effect of variable mutant incorporation and localization on simulated cardiac muscle twitches.

Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:112-124
Mijailovich SM, Prodanovic M, Poggesi C, Powers JD, ... Geeves MA, Regnier M
J Mol Cell Cardiol: 30 May 2021; 155:112-124 | PMID: 33636222
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Abstract

Global identification of S-palmitoylated proteins and detection of palmitoylating (DHHC) enzymes in heart.

Miles MR, Seo J, Jiang M, Wilson ZT, ... Ueberheide B, Tseng GN
High-throughput experiments suggest that almost 20% of human proteins may be S-palmitoylatable, a post-translational modification (PTM) whereby fatty acyl chains, most commonly palmitoyl chain, are linked to cysteine thiol groups that impact on protein trafficking, distribution and function. In human, protein S-palmitoylation is mediated by a group of 23 palmitoylating \'Asp-His-His-Cys\' domain-containing (DHHC) enzymes. There is no information on the scope of protein S-palmitoylation, or the pattern of DHHC enzyme expression, in the heart. We used resin-assisted capture to pull down S-palmitoylated proteins from human, dog, and rat hearts, followed by proteomic search to identify proteins in the pulldowns. We identified 454 proteins present in at least 2 species-specific pulldowns. These proteins are operationally called \'cardiac palmitoylome\'. Enrichment analysis based on Gene Ontology terms \'cellular component\' indicated that cardiac palmitoylome is involved in cell-cell and cell-substrate junctions, plasma membrane microdomain organization, vesicular trafficking, and mitochondrial enzyme organization. Importantly, cardiac palmitoylome is uniquely enriched in proteins participating in the organization and function of t-tubules, costameres and intercalated discs, three microdomains critical for excitation-contraction coupling and intercellular communication of cardiomyocytes. We validated antibodies targeting DHHC enzymes, and detected eleven of them expressed in hearts across species. In conclusion, we provide resources useful for investigators interested in studying protein S-palmitoylation and its regulation by DHHC enzymes in the heart. We also discuss challenges in these efforts, and suggest methods and tools that should be developed to overcome these challenges.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:1-9
Miles MR, Seo J, Jiang M, Wilson ZT, ... Ueberheide B, Tseng GN
J Mol Cell Cardiol: 30 May 2021; 155:1-9 | PMID: 33636221
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Abstract

Potential impacts of the cardiac troponin I mobile domain on myofilament activation and relaxation.

Creso JG, Campbell SG
The cardiac thin filament is regulated in a Ca2+-dependent manner through conformational changes of troponin and tropomyosin (Tm). It has been generally understood that under conditions of low Ca2+ the inhibitory peptide domain (IP) of troponin I (TnI) binds to actin and holds Tm over the myosin binding sites on actin to prevent crossbridge formation. More recently, evidence that the C-terminal mobile domain (MD) of TnI also binds actin has made for a more complex scenario. This study uses a computational model to investigate the consequences of assuming that TnI regulates Tm movement via two actin-binding domains rather than one. First, a 16-state model of the cardiac thin filament regulatory unit was created with TnI-IP as the sole regulatory domain. Expansion of this to include TnI-MD formed a 24-state model. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than would be required for IP acting alone. Indeed, setting actin affinities of the IP and MD to 25% of that assumed for the IP in the single-site model was sufficient to achieve precisely the same degree of Ca2+ regulation. We also tested the 24-state model\'s ability to represent steady-state experimental data in the case of disruption of either the IP or MD. We were able to capture qualitative changes in several properties that matched what was seen in the experimental data. Lastly, simulations were run to examine the effect of disruption of the IP or MD on twitch dynamics. Our results suggest that both domains are required to keep diastolic cross-bridge activity to a minimum and accelerate myofilament relaxation. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to complete Ca2+-dependent regulation of the thin filament.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:50-57
Creso JG, Campbell SG
J Mol Cell Cardiol: 30 May 2021; 155:50-57 | PMID: 33647310
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Abstract

Adaptation to exercise-induced stress is not dependent on cardiomyocyte α-adrenergic receptors.

Kaidonis X, Niu W, Chan AY, Kesteven S, ... Feneley M, Graham RM
The \'fight or flight\' response to physiological stress involves sympathetic nervous system activation, catecholamine release and adrenergic receptor stimulation. In the heart, this induces positive inotropy, previously attributed to the β1-adrenergic receptor subtype. However, the role of the α1A-adrenergic receptor, which has been suggested to be protective in cardiac pathology, has not been investigated in the setting of physiological stress. To explore this, we developed a tamoxifen-inducible, cardiomyocyte-specific α1A-adrenergic receptor knock-down mouse model, challenged mice to four weeks of endurance swim training and assessed cardiac outcomes. With 4-OH tamoxifen treatment, expression of the α1A-adrenergic receptor was knocked down by 80-89%, without any compensatory changes in the expression of other adrenergic receptors, or changes to baseline cardiac structure and function. Swim training caused eccentric hypertrophy, regardless of genotype, demonstrated by an increase in heart weight/tibia length ratio (30% and 22% in vehicle- and tamoxifen-treated animals, respectively) and an increase in left ventricular end diastolic volume (30% and 24% in vehicle- and tamoxifen-treated animals, respectively) without any change in the wall thickness/chamber radius ratio. Consistent with physiological hypertrophy, there was no increase in fetal gene program (Myh7, Nppa, Nppb or Acta1) expression. In response to exercise-induced volume overload, stroke volume (39% and 30% in vehicle- and tamoxifen-treated animals, respectively), cardiac output/tibia length ratio (41% in vehicle-treated animals) and stroke work (61% and 33% in vehicle- and tamoxifen-treated animals, respectively) increased, regardless of genotype. These findings demonstrate that cardiomyocyte α1A-adrenergic receptors are not necessary for cardiac adaptation to endurance exercise stress and their acute ablation is not deleterious.

Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:78-87
Kaidonis X, Niu W, Chan AY, Kesteven S, ... Feneley M, Graham RM
J Mol Cell Cardiol: 30 May 2021; 155:78-87 | PMID: 33647309
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Abstract

Txnip C247S mutation protects the heart against acute myocardial infarction.

Nakayama Y, Mukai N, Wang BF, Yang K, ... Kitsis RN, Yoshioka J
Rationale
Thioredoxin-interacting protein (Txnip) is a novel molecular target with translational potential in diverse human diseases. Txnip has several established cellular actions including binding to thioredoxin, a scavenger of reactive oxygen species (ROS). It has been long recognized from in vitro evidence that Txnip forms a disulfide bridge through cysteine 247 (C247) with reduced thioredoxin to inhibit the anti-oxidative properties of thioredoxin. However, the physiological significance of the Txnip-thioredoxin interaction remains largely undefined in vivo.
Objective
A single mutation of Txnip, C247S, abolishes the binding of Txnip with thioredoxin. Using a conditional and inducible approach with a mouse model of a mutant Txnip that does not bind thioredoxin, we tested whether the interaction of thioredoxin with Txnip is required for Txnip\'s pro-oxidative or cytotoxic effects in the heart.
Methods and results
Overexpression of Txnip C247S in cells resulted in a reduction in ROS, due to an inability to inhibit thioredoxin. Hypoxia (1% O2, 24 h)-induced killing effects of Txnip were decreased by lower levels of cellular ROS in Txnip C247S-expressing cells compared with wild-type Txnip-expressing cells. Then, myocardial ischemic injuries were assessed in the animal model. Cardiomyocyte-specific Txnip C247S knock-in mice had better survival with smaller infarct size following myocardial infarction (MI) compared to control animals. The absence of Txnip\'s inhibition of thioredoxin promoted mitochondrial anti-oxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage induced by MI. Furthermore, an unbiased RNA sequencing screen identified that hypoxia-inducible factor 1 signaling pathway was involved in Txnip C247S-mediated cardioprotective mechanisms.
Conclusion
Txnip is a cysteine-containing redox protein that robustly regulates the thioredoxin system via a disulfide bond-switching mechanism in adult cardiomyocytes. Our results provide the direct in vivo evidence that regulation of redox state by Txnip is a crucial component for myocardial homeostasis under ischemic stress.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:36-49
Nakayama Y, Mukai N, Wang BF, Yang K, ... Kitsis RN, Yoshioka J
J Mol Cell Cardiol: 30 May 2021; 155:36-49 | PMID: 33652022
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Abstract

Computational model of cardiomyocyte apoptosis identifies mechanisms of tyrosine kinase inhibitor-induced cardiotoxicity.

Grabowska ME, Chun B, Moya R, Saucerman JJ
Despite clinical observations of cardiotoxicity among cancer patients treated with tyrosine kinase inhibitors (TKIs), the molecular mechanisms by which these drugs affect the heart remain largely unknown. Mechanistic understanding of TKI-induced cardiotoxicity has been limited in part due to the complexity of tyrosine kinase signaling pathways and the multi-targeted nature of many of these drugs. TKI treatment has been associated with reactive oxygen species generation, mitochondrial dysfunction, and apoptosis in cardiomyocytes. To gain insight into the mechanisms mediating TKI-induced cardiotoxicity, this study constructs and validates a computational model of cardiomyocyte apoptosis, integrating intrinsic apoptotic and tyrosine kinase signaling pathways. The model predicts high levels of apoptosis in response to sorafenib, sunitinib, ponatinib, trastuzumab, and gefitinib, and lower levels of apoptosis in response to nilotinib and erlotinib, with the highest level of apoptosis induced by sorafenib. Knockdown simulations identified AP1, ASK1, JNK, MEK47, p53, and ROS as positive functional regulators of sorafenib-induced apoptosis of cardiomyocytes. Overexpression simulations identified Akt, IGF1, PDK1, and PI3K among the negative functional regulators of sorafenib-induced cardiomyocyte apoptosis. A combinatorial screen of the positive and negative regulators of sorafenib-induced apoptosis revealed ROS knockdown coupled with overexpression of FLT3, FGFR, PDGFR, VEGFR, or KIT as a particularly potent combination in reducing sorafenib-induced apoptosis. Network simulations of combinatorial treatment with sorafenib and the antioxidant N-acetyl cysteine (NAC) suggest that NAC may protect cardiomyocytes from sorafenib-induced apoptosis.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 155:66-77
Grabowska ME, Chun B, Moya R, Saucerman JJ
J Mol Cell Cardiol: 30 May 2021; 155:66-77 | PMID: 33667419
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Abstract

An oral absorbent, AST-120, restores vascular growth and blood flow in ischemic muscles in diabetic mice via modulation of macrophage transition.

Huang HL, Kuo CS, Chang TY, Chou RH, ... Wu CC, Huang PH

Background:
Diabetes has a pronounced effect on the peripheral vasculature. The accumulation of advanced glycation end products (AGEs) is regarded as the crucial mechanism responsible for vascular damage in diabetes, but it is not easy to be avoided from food. In this study, we aimed to investigate the effects of an oral absorbent, AST-120, on the accumulation of AGEs and changes in blood flow recovery in diabetic mice. Methods The mice were divided into four groups, wild-type (WT) mice without treatment, WT mice treated with 5% AST-120 mixed into pulverized chow, streptozotocin-induced diabetes mellitus (DM) mice, and DM mice treated with 5% AST-120. Six weeks after hind-limb ischemia surgery, blood flow reperfusion, histology, plasma AGE, and cytokine were examined. Bone marrow cells were cultured and derived into macrophages to evaluate the effects of AGEs on macrophage polarization. Results Plasma AGEs were significantly increased in diabetic mice. AST-120 could bind to AGEs and reduced their plasma concentrations. Histological analysis revealed fewer collateral vessels with corresponding impairment of blood flow recovery in diabetic mice. In these mice, AGE-positive and AGE receptor-positive macrophages were numerous in ischemic limbs compared with non- diabetic mice. In diabetic mice, macrophages in ischemic tissues demonstrated greater M1 polarization than M2 polarization; this pattern was reversed in the AST-120 treatment group. The change in macrophage polarization was associated with the corresponding expression of pro-inflammatory cytokines in the ischemic tissues. In cell cultures, AGEs triggered the transformation of bone marrow-derived macrophages into the M1 phenotype. The alterations in the polarization of macrophages were reversed after treatment with AST-120.
Conclusions:
Oral administration of AST-120 decreased the serum levels of AGEs in diabetic mice and improved neovascularization of ischemic limbs. This benefit may be due to, at least partially, the alterations in macrophage polarization and the associated changes in inflammatory cytokines.


Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 May 2021; 155:99-110
Huang HL, Kuo CS, Chang TY, Chou RH, ... Wu CC, Huang PH
J Mol Cell Cardiol: 30 May 2021; 155:99-110 | PMID: 33713645
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Abstract

Molecular remodeling of Cx43, but not structural remodeling, promotes arrhythmias in an arrhythmogenic canine model of nonischemic heart failure.

Yan J, Killingsworth C, Walcott G, Zhu Y, ... Ai X, Pogwizd SM
Background
Both gap junctional remodeling and interstitial fibrosis have been linked to impaired electrical conduction velocity (CV) and fatal ventricular arrhythmias in nonischemic heart failure (HF). However, the arrhythmogenic role of the ventricular gap junctional Cx43 in nonischemic HF remains in debate. Here, we assessed this in a newly developed arrhythmogenic canine model of nonischemic HF.
Methods and results
Nonischemic HF was induced in canines by combined aortic valve insufficiency and aortic constriction. Left ventricular (LV) myocardium from HF dogs showed similar pathological changes to that of humans. HF dogs had reduced LV function, widened QRS complexes, and spontaneous nonsustained ventricular tachycardia. CV was measured in intact LV epicardium with high-density grid mapping. Total (Cx43-T) and nonphosphorylated Cx43 (Cx43-NP) and histological interstitial fibrosis were assessed from these mapped LV tissues. Longitudinal CV, which was slowed in HF (49 ± 1 vs. 65 ± 2 cm/s in Ctl), was positively correlated with reduced total junctional Cx43 and negatively correlated with markedly increased junctional Cx43-NP (2-fold) in HF. Cx43 dephosphorylation in HF was associated with enhanced colocalization of PP2A at the level of Cx43. Unchanged action potential upstroke and transverse CV were associated with unaltered Cx43 lateralization and interstitial fibrosis in the nonischemic HF canine LV.
Conclusion
Our unique arrhythmogenic canine model of HF resembles human nonischemic HF (prior to the end stage). Cx43 remodeling occurs prior to the structural remodeling (with lack of fibrosis) in HF and it is crucial in slowed CV and ventricular arrhythmia development. Our findings suggest that altered Cx43 alone is arrhythmogenic and modulation of Cx43 has the anti-arrhythmic therapeutic potential for HF patients.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 May 2021; 158:72-81
Yan J, Killingsworth C, Walcott G, Zhu Y, ... Ai X, Pogwizd SM
J Mol Cell Cardiol: 30 May 2021; 158:72-81 | PMID: 34048725
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Abstract

The role of demethylases in cardiac development and disease.

Davis K, Azarcon P, Hickenlooper S, Bia R, ... Szulik MW, Franklin S
Heart failure is a worldwide health condition that currently has limited noninvasive treatments. Heart disease includes both structural and molecular remodeling of the heart which is driven by alterations in gene expression in the cardiomyocyte. Therefore, understanding the regulatory mechanisms which instigate these changes in gene expression and constitute the foundation for pathological remodeling may be beneficial for developing new treatments for heart disease. These gene expression changes are largely preceded by epigenetic alterations to chromatin, including the post-translational modification of histones such as methylation, which alters chromatin to be more or less accessible for transcription factors or regulatory proteins to bind and modify gene expression. Methylation was once thought to be a permanent mark placed on histone or non-histone targets by methyltransferases, but is now understood to be a reversible process after the discovery of the first demethylase, KDM1A/LSD1. Since this time, it has been shown that demethylases play key roles in embryonic development, in maintaining cellular homeostasis and disease progression. However, the role of demethylases in the fetal and adult heart remains largely unknown. In this review, we have compiled data on the 33 mammalian demethylases that have been identified to date and evaluate their expression in the embryonic and adult heart as well as changes in expression in the failing myocardium using publicly available RNA-sequencing and proteomic datasets. Our analysis detected expression of 14 demethylases in the normal fetal heart, and 5 demethylases in the normal adult heart. Moreover, 8 demethylases displayed differential expression in the diseased human heart compared to healthy hearts. We then examined the literature regarding these demethylases and provide phenotypic information of 13 demethylases that have been functionally interrogated in some way in the heart. Lastly, we describe the 6 arginine and lysine residues on histones which have been shown to be methylated but have no corresponding demethylase identified which removes these methyl marks. Overall, this review highlights our current knowledge on the role of demethylases, their importance in cardiac development and pathophysiology and provides evidence for the use of pharmacological inhibitors to combat disease.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 May 2021; 158:89-100
Davis K, Azarcon P, Hickenlooper S, Bia R, ... Szulik MW, Franklin S
J Mol Cell Cardiol: 30 May 2021; 158:89-100 | PMID: 34081951
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Abstract

An evolutionarily-conserved promoter allele governs HMG-CoA reductase expression in spontaneously hypertensive rat.

Khan AA, Sundar P, Natarajan B, Gupta V, ... Barthwal MK, Mahapatra NR
3-Hydroxy-3-methyl glutaryl-coenzyme A reductase (Hmgcr) encodes the rate-limiting enzyme in the cholesterol biosynthesis pathway. The regulation of Hmgcr in rat models of genetic hypertension (viz. Spontaneously Hypertensive Rat [SHR] and its normotensive control Wistar/Kyoto [WKY] strain) is unclear. Interestingly, Hmgcr transcript and protein levels are diminished in liver tissues of SHR as compared to WKY. This observation is consistent with the diminished plasma cholesterol level in SHR animals. However, the molecular basis of these apparently counter-intuitive findings remains completely unknown. Sequencing of the Hmgcr promoter in SHR and WKY strains reveals three variations: A-405G, C-62T and a 11 bp insertion (-398_-388insTGCGGTCCTCC) in SHR. Among these variations, A-405G occurs at an evolutionarily-conserved site among many mammals. Moreover, SHR-Hmgcr promoter displays lower activity than WKY-Hmgcr promoter in various cell lines. Transient transfections of Hmgcr-promoter mutants and in silico analysis suggest altered binding of Runx3 and Srebf1 across A-405G site. On the other hand, C-62T and -398_-388insTGCGGTCCTCC variations do not appear to contribute to the reduced Hmgcr promoter activity in SHR as compared to WKY. Indeed, chromatin immunoprecipitation assays confirm differential binding of Runx3 and Srebf1 to Hmgcr promoter leading to reduced expression of Hmgcr in SHR as compared to WKY under basal as well as cholesterol-modulated conditions. Taken together, this study provides, for the first time, molecular basis for diminished Hmgcr expression in SHR animals, which may account for the reduced circulating cholesterol level in this widely-studied model for cardiovascular diseases.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 May 2021; 158:140-152
Khan AA, Sundar P, Natarajan B, Gupta V, ... Barthwal MK, Mahapatra NR
J Mol Cell Cardiol: 30 May 2021; 158:140-152 | PMID: 34081950
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Impact:
Abstract

Molecular determinants of pro-arrhythmia proclivity of d- and l-sotalol via a multi-scale modeling pipeline.

DeMarco KR, Yang PC, Singh V, Furutani K, ... Clancy CE, Vorobyov I
Drug isomers may differ in their proarrhythmia risk. An interesting example is the drug sotalol, an antiarrhythmic drug comprising d- and l- enantiomers that both block the hERG cardiac potassium channel and confer differing degrees of proarrhythmic risk. We developed a multi-scale in silico pipeline focusing on hERG channel - drug interactions and used it to probe and predict the mechanisms of pro-arrhythmia risks of the two enantiomers of sotalol. Molecular dynamics (MD) simulations predicted comparable hERG channel binding affinities for d- and l-sotalol, which were validated with electrophysiology experiments. MD derived thermodynamic and kinetic parameters were used to build multi-scale functional computational models of cardiac electrophysiology at the cell and tissue scales. Functional models were used to predict inactivated state binding affinities to recapitulate electrocardiogram (ECG) QT interval prolongation observed in clinical data. Our study demonstrates how modeling and simulation can be applied to predict drug effects from the atom to the rhythm for dl-sotalol and also increased proarrhythmia proclivity of d- vs. l-sotalol when accounting for stereospecific beta-adrenergic receptor blocking.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 28 May 2021; 158:163-177
DeMarco KR, Yang PC, Singh V, Furutani K, ... Clancy CE, Vorobyov I
J Mol Cell Cardiol: 28 May 2021; 158:163-177 | PMID: 34062207
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Impact:
Abstract

CircHelz activates NLRP3 inflammasome to promote myocardial injury by sponging miR-133a-3p in mouse ischemic heart.

Bian Y, Pang P, Li X, Yu S, ... Du W, Yang B
Myocardial infarction (MI)-induced the activation of NLRP3 inflammasome has been well known to aggravate myocardial injury and cardiac dysfunction by causing inflammation and pyroptosis in the heart. Circular RNAs (circRNAs) have been demonstrated to play critical roles in cardiovascular diseases. However, the functions and mechanisms of circRNAs in modulating cardiac inflammatory response and cardiomyocyte pyroptosis remain largely unknown. We revealed that circHelz, a novel circRNA transcribed from the helicase with zinc finger (Helz) gene, was significantly upregulated in both the ischemic myocardium of MI mouse and neonatal mouse ventricular cardiomyocytes (NMVCs) exposed to hypoxia. Overexpression of circHelz caused cardiomyocyte injury in NMVCs by activating the NLRP3 inflammasome and inducing pyroptosis, while circHelz silencing reduced these effects induced by hypoxia. Furthermore, knockdown of circHelz remarkably attenuated NLRP3 expression, decreased myocardial infarct size, pyroptosis, inflammation, and increased cardiac function in vivo after MI. Overexpression of miR-133a-3p in cardiomyocytes greatly prevented pyroptosis in the presence of hypoxia or circHelz by targeting NLRP3 in NMVCs. Mechanistically, circHelz functioned as an endogenous sponge for miR-133a-3p via suppressing its activity. Overall, our results demonstrate that circHelz causes myocardial injury by triggering the NLRP3 inflammasome-mediated pro-inflammatory response and subsequent pyroptosis in cardiomyocytes by inhibiting miR-133a-3p function. Therefore, interfering with circHelz/miR-133a-3p/NLRP3 axis might be a promising therapeutic approach for ischemic cardiac diseases.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 24 May 2021; 158:128-139
Bian Y, Pang P, Li X, Yu S, ... Du W, Yang B
J Mol Cell Cardiol: 24 May 2021; 158:128-139 | PMID: 34043986
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Impact:
Abstract

Amino acid differences between diabetic older adults and non-diabetic older adults and their associations with cardiovascular function.

Kovalik JP, Zhao X, Gao F, Leng S, ... Zhong L, Koh AS
Background
Ageing and insulin resistant states such as diabetes mellitus frequently coexist and increase the risk of cardiovascular disease development among older adults. Here we investigate metabolic differences in amino acid profiles between ageing and diabetes mellitus, and their associations with cardiovascular function.
Methods
In a group of community older adults we performed echocardiography, cardiac magnetic resonance imaging as well as cross sectional and longitudinal metabolomics profiling based on current and archived sera obtained fifteen years prior to examination.
Results
We studied a total of 515 participants (women 50%, n = 255) with a mean age 73 (SD = 4.3) years. Diabetics had higher alanine (562 vs 448, p < 0.0001), higher glutamate (107 vs 95, p = 0.016), higher proline (264 vs 231, p = 0.008) and lower arginine (107 vs 117, p = 0.043), lower citrulline (30 vs 38, p = 0.006) levels (μM) compared to non-diabetics. Over time, changes in amino acid profiles differentiated diabetic older adults from non-diabetic older adults, with greater accumulation of alanine (p = 0.002), proline (p = 0.008) and (non-significant) trend towards greater accumulation of glycine (p = 0.057) among the older diabetics compared to the older non-diabetics. However, independent of diabetes status, amino acids were associated with cardiovascular functions in ageing, [archived valine (p = 0.011), leucine (p = 0.011), archived isoleucine (p = 0.0006), archived serine (p = 0.008), archived glycine (p = 0.006) methionine (p = 0.003)] which were associated with impairments in E/A ratio.
Conclusion
Markers of branched chain amino acids and one ‑carbon metabolism pathways were associated with changes in cardiovascular function in older adults regardless of diabetes status. However, nitrogen handling pathways were specifically altered among older adults with diabetes. These findings broaden our understanding into specific amino acid pathways that may be altered between diabetic and non-diabetic older adults, and their relevance to cardiovascular function in ageing.
Trial registration
ClinicalTrials.gov Identifier: NCT02791139.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 23 May 2021; 158:63-71
Kovalik JP, Zhao X, Gao F, Leng S, ... Zhong L, Koh AS
J Mol Cell Cardiol: 23 May 2021; 158:63-71 | PMID: 34033835
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Abstract

Dimethyl fumarate preserves left ventricular infarct integrity following myocardial infarction via modulation of cardiac macrophage and fibroblast oxidative metabolism.

Mouton AJ, Flynn ER, Moak SP, Aitken NM, ... do Carmo JM, Hall JE
Myocardial infarction (MI) is one of the leading causes of mortality and cardiovascular disease worldwide. MI is characterized by a substantial inflammatory response in the infarcted left ventricle (LV), followed by transition of quiescent fibroblasts to active myofibroblasts, which deposit collagen to form the reparative scar. Metabolic shifting between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is an important mechanism by which these cell types transition towards reparative phenotypes. Thus, we hypothesized that dimethyl fumarate (DMF), a clinically approved anti-inflammatory agent with metabolic actions, would improve post-MI remodeling via modulation of macrophage and fibroblast metabolism. Adult male C57BL/6J mice were treated with DMF (10 mg/kg) for 3-7 days after MI. DMF attenuated LV infarct and non-infarct wall thinning at 3 and 7 days post-MI, and decreased LV dilation and pulmonary congestion at day 7. DMF improved LV infarct collagen deposition, myofibroblast activation, and angiogenesis at day 7. DMF also decreased pro-inflammatory cytokine expression (Tnf) 3 days after MI, and decreased inflammatory markers in macrophages isolated from the infarcted heart (Hif1a, Il1b). In fibroblasts extracted from the infarcted heart at day 3, RNA-Seq analysis demonstrated that DMF promoted an anti-inflammatory/pro-reparative phenotype. By Seahorse analysis, DMF did not affect glycolysis in either macrophages or fibroblasts at day 3, but enhanced macrophage OXPHOS while impairing fibroblast OXPHOS. Our results indicate that DMF differentially affects macrophage and fibroblast metabolism, and promotes anti-inflammatory/pro-reparative actions. In conclusion, targeting cellular metabolism in the infarcted heart may be a promising therapeutic strategy.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 20 May 2021; 158:38-48
Mouton AJ, Flynn ER, Moak SP, Aitken NM, ... do Carmo JM, Hall JE
J Mol Cell Cardiol: 20 May 2021; 158:38-48 | PMID: 34023353
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Abstract

Mechanisms of flecainide induced negative inotropy: An in silico study.

Yang PC, Giles WR, Belardinelli L, Clancy CE
It is imperative to develop better approaches to predict how antiarrhythmic drugs with multiple interactions and targets may alter the overall electrical and/or mechanical function of the heart. Safety Pharmacology studies have provided new insights into the multi-target effects of many different classes of drugs and have been aided by the addition of robust new in vitro and in silico technology. The primary focus of Safety Pharmacology studies has been to determine the risk profile of drugs and drug candidates by assessing their effects on repolarization of the cardiac action potential. However, for decades experimental and clinical studies have described substantial and potentially detrimental effects of Na+ channel blockers in addition to their well-known conduction slowing effects. One such side effect, associated with administration of some Na+ channel blocking drugs is negative inotropy. This reduces the pumping function of the heart, thereby resulting in hypotension. Flecainide is a well-known example of a Na+ channel blocking drug, that exhibits strong rate-dependent block of INa and may cause negative cardiac inotropy. While the phenomenon of Na+ channel suppression and resulting negative inotropy is well described, the mechanism(s) underlying this effect are not. Here, we set out to use a modeling and simulation approach to reveal plausible mechanisms that could explain the negative inotropic effect of flecainide. We utilized the Grandi-Bers model [1] of the cardiac ventricular myocyte because of its robust descriptions of ion homeostasis in order to characterize and resolve the relative effects of QRS widening, flecainide off-target effects and changes in intracellular Ca2+ and Na+ homeostasis. The results of our investigations and predictions reconcile multiple data sets and illustrate how multiple mechanisms may play a contributing role in the flecainide induced negative cardiac inotropic effect.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 14 May 2021; 158:26-37
Yang PC, Giles WR, Belardinelli L, Clancy CE
J Mol Cell Cardiol: 14 May 2021; 158:26-37 | PMID: 34004185
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Abstract

Evidence for synergy between sarcomeres and fibroblasts in an in vitro model of myocardial reverse remodeling.

Shen S, Sewanan LR, Campbell SG
We have created a novel in-vitro platform to study reverse remodeling of engineered heart tissue (EHT) after mechanical unloading. EHTs were created by seeding decellularized porcine myocardial sections with a mixture of primary neonatal rat ventricular myocytes and cardiac fibroblasts. Each end of the ribbon-like constructs was fixed to a plastic clip, allowing the tissues to be statically stretched or slackened. Inelastic deformation was introduced by stretching tissues by 20% of their original length. EHTs were subsequently unloaded by returning tissues to their original, shorter length. Mechanical characterization of EHTs immediately after unloading and at subsequent time points confirmed the presence of a reverse-remodeling process, through which stress-free tissue length was increased after chronic stretch but gradually decreased back to its original value within 9 days. When a cardiac myosin inhibitor was applied to tissues after unloading, EHTs failed to completely recover their passive and active mechanical properties, suggesting a role for actomyosin contraction in reverse remodeling. Selectively inhibiting cardiomyocyte contraction or fibroblast activity after mechanical unloading showed that contractile activity of both cell types was required to achieve full remodeling. Similar tests with EHTs formed from human induced pluripotent stem cell-derived cardiomyocytes also showed reverse remodeling that was enhanced when treated with omecamtiv mecarbil, a myosin activator. These experiments suggest essential roles for active sarcomeric contraction and fibroblast activity in reverse remodeling of myocardium after mechanical unloading. Our findings provide a mechanistic rationale for designing potential therapies to encourage reverse remodeling in patient hearts.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 13 May 2021; 158:11-25
Shen S, Sewanan LR, Campbell SG
J Mol Cell Cardiol: 13 May 2021; 158:11-25 | PMID: 33992697
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Abstract

Increasing fatty acid oxidation elicits a sex-dependent response in failing mouse hearts.

Ritterhoff J, McMillen TS, Villet O, Young S, ... Caudal A, Tian R
Background
Reduced fatty acid oxidation (FAO) is a hallmark of metabolic remodeling in heart failure. Enhancing mitochondrial long-chain fatty acid uptake by Acetyl-CoA carboxylase 2 (ACC2) deletion increases FAO and prevents cardiac dysfunction during chronic stresses, but therapeutic efficacy of this approach has not been determined.
Methods
Male and female ACC2 f/f-MCM (ACC2KO) and their respective littermate controls were subjected to chronic pressure overload by TAC surgery. Tamoxifen injection 3 weeks after TAC induced ACC2 deletion and increased FAO in ACC2KO mice with pathological hypertrophy.
Results
ACC2 deletion in mice with pre-existing cardiac pathology promoted FAO in female and male hearts, but improved cardiac function only in female mice. In males, pressure overload caused a downregulation in the mitochondrial oxidative function. Stimulating FAO by ACC2 deletion caused unproductive acyl-carnitine accumulation, which failed to improve cardiac energetics. In contrast, mitochondrial oxidative capacity was sustained in female pressure overloaded hearts and ACC2 deletion improved myocardial energetics. Mechanistically, we revealed a sex-dependent regulation of PPARα signaling pathway in heart failure, which accounted for the differential response to ACC2 deletion.
Conclusion
Metabolic remodeling in the failing heart is sex-dependent which could determine the response to metabolic intervention. The findings suggest that both mitochondrial oxidative capacity and substrate preference should be considered for metabolic therapy of heart failure.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 11 May 2021; 158:1-10
Ritterhoff J, McMillen TS, Villet O, Young S, ... Caudal A, Tian R
J Mol Cell Cardiol: 11 May 2021; 158:1-10 | PMID: 33989657
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Abstract

Relevance of N6-methyladenosine regulators for transcriptome: Implications for development and the cardiovascular system.

Sweaad WK, Stefanizzi FM, Chamorro-Jorganes A, Devaux Y, Emanueli C, EU-CardioRNA COST Action CA17129
N6-methyladenosine (m6A) is the most abundant and well-studied internal modification of messenger RNAs among the various RNA modifications in eukaryotic cells. Moreover, it is increasingly recognized to regulate non-coding RNAs. The dynamic and reversible nature of m6A is ensured by the precise and coordinated activity of specific proteins able to insert (\"write\"), bind (\"read\") or remove (\"erase\") the m6A modification from coding and non-coding RNA molecules. Mounting evidence suggests a pivotal role for m6A in prenatal and postnatal development and cardiovascular pathophysiology. In the present review we summarise and discuss the major functions played by m6A RNA methylation and its components particularly referring to the cardiovascular system. We present the methods used to study m6A and the most abundantly methylated RNA molecules. Finally, we highlight the possible involvement of the m6A mark in cardiovascular disease as well as the need for further studies to better describe the mechanisms of action and the potential therapeutic role of this RNA modification.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 11 May 2021; epub ahead of print
Sweaad WK, Stefanizzi FM, Chamorro-Jorganes A, Devaux Y, Emanueli C, EU-CardioRNA COST Action CA17129
J Mol Cell Cardiol: 11 May 2021; epub ahead of print | PMID: 33991529
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Abstract

B cells modulate the expression of MHC-II on cardiac CCR2 macrophages.

Rocha-Resende C, Pani F, Adamo L
The uninjured murine heart contains a heterogeneous population of macrophages with disparate ontogenies and functions. These macrophages are often associated with blood vessels and can be subclassified based on the expression of CC chemokine receptor 2 (CCR2) and major histocompatibility complex class II (MHC-II). The biological cues that modulate these macrophage pool subpopulations have not been completely identified. It has been recently shown that a sub-population of circulating naïve B cells adheres to the myocardial microvasculature. We hypothesized that B cells might modulate the phenotype of myocardial macrophages. To test this hypothesis, we analyzed both the relative location of B cells and macrophages in myocardial histological section and the prevalence of myocardial macrophage subsets in hearts from B cell-deficient mice (μMT) and mice depleted of B cells through administration of an anti-CD20 antibody. We found that B cells pause in the microvasculature in proximity of macrophages and modulate the number of myocardial CCR2-MHC-IIhigh cells. Through in vitro studies we found that this is likely the result of a paracrine effect of B cells on the expression of MHC-II in CCR2- cells. These results reveal an unexpected relationship between B cells and resident macrophages and, highlighting a direct intramyocardial effect of circulating B cells, challenge the currently held belief that naïve recirculating B lymphocytes merely shuttle between lymphoid stations.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 07 May 2021; 157:98-103
Rocha-Resende C, Pani F, Adamo L
J Mol Cell Cardiol: 07 May 2021; 157:98-103 | PMID: 33971183
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Abstract

Piezo1 and BK channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation.

Jakob D, Klesen A, Allegrini B, Darkow E, ... Ravens U, Peyronnet R
Aims
Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF.
Methods and results
Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent \'big potassium channels\' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected.
Conclusions
Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 07 May 2021; 158:49-62
Jakob D, Klesen A, Allegrini B, Darkow E, ... Ravens U, Peyronnet R
J Mol Cell Cardiol: 07 May 2021; 158:49-62 | PMID: 33974928
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Abstract

Dual role of miR-1 in the development and function of sinoatrial cells.

Benzoni P, Nava L, Giannetti F, Guerini G, ... Baruscotti M, Barbuti A
miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating If currents; miR1OE SAN-like cells instead have only fast-activating If with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and If alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 05 May 2021; 157:104-112
Benzoni P, Nava L, Giannetti F, Guerini G, ... Baruscotti M, Barbuti A
J Mol Cell Cardiol: 05 May 2021; 157:104-112 | PMID: 33964276
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Abstract

Translational investigation of electrophysiology in hypertrophic cardiomyopathy.

Flenner F, Jungen C, Küpker N, Ibel A, ... Christ T, Carrier L
Hypertrophic cardiomyopathy (HCM) patients are at increased risk of ventricular arrhythmias and sudden cardiac death, which can occur even in the absence of structural changes of the heart. HCM mouse models suggest mutations in myofilament components to affect Ca2+ homeostasis and thereby favor arrhythmia development. Additionally, some of them show indications of pro-arrhythmic changes in cardiac electrophysiology. In this study, we explored arrhythmia mechanisms in mice carrying a HCM mutation in Mybpc3 (Mybpc3-KI) and tested the translatability of our findings in human engineered heart tissues (EHTs) derived from CRISPR/Cas9-generated homozygous MYBPC3 mutant (MYBPC3hom) in induced pluripotent stem cells (iPSC) and to left ventricular septum samples obtained from HCM patients. We observed higher arrhythmia susceptibility in contractility measurements of field-stimulated intact cardiomyocytes and ventricular muscle strips as well as in electromyogram recordings of Langendorff-perfused hearts from adult Mybpc3-KI mice than in wild-type (WT) controls. The latter only occurred in homozygous (Hom-KI) but not in heterozygous (Het-KI) mouse hearts. Both Het- and Hom-KI are known to display pro-arrhythmic increased Ca2+ myofilament sensitivity as a direct consequence of the mutation. In the electrophysiological characterization of the model, we observed smaller repolarizing K+ currents in single cell patch clamp, longer ventricular action potentials in sharp microelectrode recordings and longer ventricular refractory periods in Langendorff-perfused hearts in Hom-KI, but not Het-KI. Interestingly, reduced K+ channel subunit transcript levels and prolonged action potentials were already detectable in newborn, pre-hypertrophic Hom-KI mice. Human iPSC-derived MYBPC3hom EHTs, which genetically mimicked the Hom-KI mice, did exhibit lower mutant mRNA and protein levels, lower force, beating frequency and relaxation time, but no significant alteration of the force-Ca2+ relation in skinned EHTs. Furthermore, MYBPC3hom EHTs did show higher spontaneous arrhythmic behavior, whereas action potentials measured by sharp microelectrode did not differ to isogenic controls. Action potentials measured in septal myectomy samples did not differ between patients with HCM and patients with aortic stenosis, except for the only sample with a MYBPC3 mutation. The data demonstrate that increased myofilament Ca2+ sensitivity is not sufficient to induce arrhythmias in the Mybpc3-KI mouse model and suggest that reduced K+ currents can be a pro-arrhythmic trigger in Hom-KI mice, probably already in early disease stages. However, neither data from EHTs nor from left ventricular samples indicate relevant reduction of K+ currents in human HCM. Therefore, our study highlights the species difference between mouse and human and emphasizes the importance of research in human samples and human-like models.

Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 02 May 2021; 157:77-89
Flenner F, Jungen C, Küpker N, Ibel A, ... Christ T, Carrier L
J Mol Cell Cardiol: 02 May 2021; 157:77-89 | PMID: 33957110
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Abstract

Blocking endothelial TRPV4-Nox2 interaction helps reduce ROS production and inflammation, and improves vascular function in obese mice.

Gao M, Han J, Zhu Y, Tang C, ... Xiao W, Ma X
Obesity induces inflammation and oxidative stress, and ultimately leads to vasodilatory dysfunction in which Transient receptor potential vanilloid type 4 (TRPV4) and Nicotinamide Adenine Dinucleotide Phosphate Oxidase (Nox2) have been reported to be involved. However, little attention has been paid to the role of the TRPV4-Nox2 complex in these problems. The purpose of this study was to figure out the role of the TRPV4-Nox2 complex in obesity-induced inflammation, oxidative stress, and vasodilatory dysfunction. Using fluorescence resonance energy transfer and immunoprecipitation assays, we found enhanced TRPV4 and Nox2 interactions in obese mice. Using q-PCR, fluorescent dye dihydroethidium staining, and myotonic techniques, we found that obesity caused inflammation, oxidative stress, and vasodilatory dysfunction. Using adeno-associated viruses, we found that enhancement or attenuation of TRPV4-Nox2 interaction altered the vaso-function. Based on these findings, we found a small-molecule drug, M12, that interrupted the TRPV4-Nox2 interaction, thereby reducing inflammatory factors and reactive oxygen species production and helping to restore the vasodilatory function. In summary, our results revealed a new mechanism by which obesity-induced inflammation, oxidative stress, and vasodilatory dysfunction is caused by enhanced TRPV4-Nox2 interactions. Using M12 to interrupt the TRPV4-Nox2 interaction may have anti-inflammatory and anti-oxidative stress effects and help restore vasodilatory function and thus provide a new therapeutic approach to obesity.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Apr 2021; 157:66-76
Gao M, Han J, Zhu Y, Tang C, ... Xiao W, Ma X
J Mol Cell Cardiol: 27 Apr 2021; 157:66-76 | PMID: 33932464
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Abstract

Protein acetylation in cardiac aging.

Francois A, Canella A, Marcho LM, Stratton MS
Biological aging is attributed to progressive dysfunction in systems governing genetic and metabolic integrity. At the cellular level, aging is evident by accumulated DNA damage and mutation, reactive oxygen species, alternate lipid and protein modifications, alternate gene expression programs, and mitochondrial dysfunction. These effects sum to drive altered tissue morphology and organ dysfunction. Protein-acylation has emerged as a critical mediator of age-dependent changes in these processes. Despite decades of research focus from academia and industry, heart failure remains a leading cause of death in the United States while the 5 year mortality rate for heart failure remains over 40%. Over 90% of heart failure deaths occur in patients over the age of 65 and heart failure is the leading cause of hospitalization in Medicare beneficiaries. In 1931, Cole and Koch discovered age-dependent accumulation of phosphates in skeletal muscle. These and similar findings provided supporting evidence for, now well accepted, theories linking metabolism and aging. Nearly two decades later, age-associated alterations in biochemical molecules were described in the heart. From these small beginnings, the field has grown substantially in recent years. This growing research focus on cardiac aging has, in part, been driven by advances on multiple public health fronts that allow population level clinical presentation of aging related disorders. It is estimated that by 2030, 25% of the worldwide population will be over the age of 65. This review provides an overview of acetylation-dependent regulation of biological processes related to cardiac aging and introduces emerging non-acetyl, acyl-lysine modifications in cardiac function and aging.

Copyright © 2021. Published by Elsevier Ltd.

J Mol Cell Cardiol: 26 Apr 2021; 157:90-97
Francois A, Canella A, Marcho LM, Stratton MS
J Mol Cell Cardiol: 26 Apr 2021; 157:90-97 | PMID: 33915138
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This program is still in alpha version.