Journal: J Mol Cell Cardiol

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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. Published by Elsevier Ltd.

J Mol Cell Cardiol: 11 Apr 2021; epub ahead of print
Hamilton S, Terentyeva R, Clements RT, Belevych AE, Terentyev D
J Mol Cell Cardiol: 11 Apr 2021; epub ahead of print | PMID: 33857485
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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: 02 Apr 2021; 156:82-94
Xiang M, Yang F, Zhou Y, Li W, ... Wang PX, Chen M
J Mol Cell Cardiol: 02 Apr 2021; 156:82-94 | PMID: 33823186
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Abstract

The role of ATG-7 contributes to pulmonary hypertension by impacting vascular remodeling.

Yang X, Zhang L, Ye JQ, Wu XH, ... Chen LW, Li YM
Aim
Pulmonary hypertension (PH) is a pathophysiological syndrome with functional abnormalities of the pulmonary artery and heart, eventually becoming life threatening to the patients. Autophagy-related gene 7 (ATG)-7 is involved in many cardiovascular diseases, but little is known about the specific role of ATG-7 in the development of PH. We aimed to examine the expression of ATG-7 in PH patients and PH mice, specifically investigate pulmonary physiological responses in a mouse model with conditional deletion of ATG-7 in smooth muscle cells (SMCs) and further clarify the mechanism of PH caused by ATG-7 deficiency.
Methods and results
SMC-ATG-7-/- mice underwent echocardiography and subsequent pulmonary arterial pressure (PAP) checks. The PAP was lower in wild-type (WT) mice (22.6 ± 2.0 mmHg) than knockout (KO) mice (34.0 ± 2.5 mmHg; P < 0.001). Pulmonary artery resistance was increased in KO (17.61 ± 2.03 mm2·s-1) versus WT mice (8.91 ± 1.62 mm2·s-1; P < 0.005). Combined with these statistics, SMC-ATG7-/- mice were diagnosed with PH. The increase of ATG-7 expression in vessels from PH patients and PH mice were assessed and the effects of ATG-7 on vascular remodeling were investigated in SMCs using relevant methods. We also identified silencing ATG-7 in SMCs induced the increased level of Ca2+ and abnormal proliferation through PP2A/ 4EBP-1/ elf-4E pathway.
Conclusions
ATG-7 affects vascular remodeling and exerts a protective function during the pathogenesis of PH. Our study revealed a novel mechanism ATG-7 deficiency promotes cell proliferation via the interaction between PP2A, 4EBP1 and elf-4E.

Copyright © 2018. Published by Elsevier Ltd.

J Mol Cell Cardiol: 01 Apr 2021; epub ahead of print
Yang X, Zhang L, Ye JQ, Wu XH, ... Chen LW, Li YM
J Mol Cell Cardiol: 01 Apr 2021; epub ahead of print | PMID: 33819456
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Abstract

Ubiquitin-like protein FAT10 suppresses SIRT1-mediated autophagy to protect against ischemic myocardial injury.

Wan R, Yuan P, Guo L, Shao J, ... Jiang X, Hong K
Autophagy plays a deleterious role in ischemic myocardial injury. The deacetylase SIRT1 is a well-established regulator of autophagy that can be modified by the ubiquitin-like protein SUMO1. Our previous work demonstrated that another ubiquitin-like protein, FAT10, exerts cardioprotective effects against myocardial ischemia by stabilizing the caveolin-3 protein; however, the effects of FAT10 on autophagy through SIRT1 are unclear. Here, we constructed a Fat10-knockout rat model to evaluate the role of FAT10 in autophagy. In vivo and in vitro assays confirmed that FAT10 suppressed autophagy to protect the heart from ischemic myocardial injury. Mechanistically, FAT10 was mainly involved in the regulation of the autophagosome formation process. FAT10 affected autophagy through modulating SIRT1 degradation, which resulted in reduced SIRT1 nuclear translocation and inhibited SIRT1 activity via its C-terminal glycine residues. Notably, FAT10 competed with SUMO1 at the K734 modification site of SIRT1, which further reduced LC3 deacetylation and suppressed autophagy. Our findings suggest that FAT10 inhibits autophagy by antagonizing SIRT1 SUMOylation to protect the heart from ischemic myocardial injury. This is a novel mechanism through which FAT10 regulates autophagy as a cardiac protector.

Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:1-13
Wan R, Yuan P, Guo L, Shao J, ... Jiang X, Hong K
J Mol Cell Cardiol: 30 Mar 2021; 153:1-13 | PMID: 33307094
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Abstract

The role of β-adrenergic system remodeling in human heart failure: A mechanistic investigation.

Mora MT, Gong JQX, Sobie EA, Trenor B
β-adrenergic receptor antagonists (β-blockers) are extensively used to improve cardiac performance in heart failure (HF), but the electrical improvements with these clinical treatments are not fully understood. The aim of this study was to analyze the electrophysiological effects of β-adrenergic system remodeling in heart failure with reduced ejection fraction and the underlying mechanisms. We used a combined mathematical model that integrated β-adrenergic signaling with electrophysiology and calcium cycling in human ventricular myocytes. HF remodeling, both in the electrophysiological and signaling systems, was introduced to quantitatively analyze changes in electrophysiological properties due to the stimulation of β-adrenergic receptors in failing myocytes. We found that the inotropic effect of β-adrenergic stimulation was reduced in HF due to the altered Ca2+ dynamics resulting from the combination of structural, electrophysiological and signaling remodeling. Isolated cells showed proarrhythmic risk after sympathetic stimulation because early afterdepolarizations appeared, and the vulnerability was greater in failing myocytes. When analyzing coupled cells, β-adrenergic stimulation reduced transmural repolarization gradients between endocardium and epicardium in normal tissue, but was less effective at reducing these gradients after HF remodeling. The comparison of the selective activation of β-adrenergic isoforms revealed that the response to β2-adrenergic receptors stimulation was blunted in HF while β1-adrenergic receptors downstream effectors regulated most of the changes observed after sympathetic stimulation. In conclusion, this study was able to reproduce an altered β-adrenergic activity on failing myocytes and to explain the mechanisms involved. The derived predictions could help in the treatment of HF and guide in the design of future experiments.

Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:14-25
Mora MT, Gong JQX, Sobie EA, Trenor B
J Mol Cell Cardiol: 30 Mar 2021; 153:14-25 | PMID: 33326834
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Abstract

Elevated EZH2 in ischemic heart disease epigenetically mediates suppression of Na1.5 expression.

Zhao L, You T, Lu Y, Lin S, Li F, Xu H
Suppression of the cardiac sodium channel NaV1.5 leads to fatal arrhythmias in ischemic heart disease (IHD). However, the transcriptional regulation of NaV1.5 in cardiac ischemia is still unclear. Our studies are aimed to investigate the expression of enhancer of zeste homolog 2 (EZH2) in IHD and regulation of cardiac NaV1.5 expression by EZH2. Human heart tissue was obtained from IHD and non-failing heart (NFH) patients; mouse heart tissue was obtained from the peri-infarct zone of hearts with myocardial infarction (MI) and hearts with a sham procedure. Protein and mRNA expression were measured by immunoblotting, immunostaining, and qRT-PCR. Protein-DNA binding and promoter activity were analyzed by ChIP-qPCR and luciferase assays, respectively. Na+ channel activity was assessed by whole-cell patch clamp recordings. EZH2 and H3K27me3 were increased while NaV1.5 expression was reduced in IHD hearts and in mouse MI hearts compared to the controls. Reduced NaV1.5 and increased EZH2 mRNA levels were observed in mouse MI hearts. A selective EZH2 inhibitor, GSK126 decreased H3K27me3 and elevated NaV1.5 in HL-1 cells. Silencing of EZH2 expression decreased H3K27me3 and increased NaV1.5 in these cells. EZH2 and H3K27me3 were enriched in the promoter regions of Scn5a and were decreased by treatment with EZH2 siRNA. GSK126 inhibited the enrichment of H3K27me3 in the Scn5a promoter and enhanced Scn5a transcriptional activity. GSK126 significantly increased Na+ channel activity. Taken together, EZH2 is increased in ischemic hearts and epigenetically suppresses Scn5a transcription by H3K27me3, leading to decreased NaV1.5 expression and Na+ channel activity underlying the pathogenesis of arrhythmias.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:95-103
Zhao L, You T, Lu Y, Lin S, Li F, Xu H
J Mol Cell Cardiol: 30 Mar 2021; 153:95-103 | PMID: 33370552
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Abstract

Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis.

Riching AS, Danis E, Zhao Y, Cao Y, ... Buttrick PM, Song K
Direct reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression of GATA4, HAND2, MEF2C, TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-β) inhibition efficiently promote reprogramming. However, the mechanisms by which TGF-β blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-β signaling regulates the interaction between GATA4 and JMJD3. TGF-β activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation in GATA4 (V267M) that is associated with congenital heart disease exhibits reduced binding to JMJD3 and impairs cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-β pathway activation impairs cardiac gene programming, in part by interfering with GATA4-JMJD3 interactions.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:44-59
Riching AS, Danis E, Zhao Y, Cao Y, ... Buttrick PM, Song K
J Mol Cell Cardiol: 30 Mar 2021; 153:44-59 | PMID: 33359755
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Abstract

Considerations for using isolated cell systems to understand cardiac metabolism and biology.

McNally LA, Altamimi TR, Fulghum K, Hill BG
Changes in myocardial metabolic activity are fundamentally linked to cardiac health and remodeling. Primary cardiomyocytes, induced pluripotent stem cell-derived cardiomyocytes, and transformed cardiomyocyte cell lines are common models used to understand how (patho)physiological conditions or stimuli contribute to changes in cardiac metabolism. These cell models are helpful also for defining metabolic mechanisms of cardiac dysfunction and remodeling. Although technical advances have improved our capacity to measure cardiomyocyte metabolism, there is often heterogeneity in metabolic assay protocols and cell models, which could hinder data interpretation and discernment of the mechanisms of cardiac (patho)physiology. In this review, we discuss considerations for integrating cardiomyocyte cell models with techniques that have become relatively common in the field, such as respirometry and extracellular flux analysis. Furthermore, we provide overviews of metabolic assays that complement XF analyses and that provide information on not only catabolic pathway activity, but biosynthetic pathway activity and redox status as well. Cultivating a more widespread understanding of the advantages and limitations of metabolic measurements in cardiomyocyte cell models will continue to be essential for the development of coherent metabolic mechanisms of cardiac health and pathophysiology.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:26-41
McNally LA, Altamimi TR, Fulghum K, Hill BG
J Mol Cell Cardiol: 30 Mar 2021; 153:26-41 | PMID: 33359038
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Abstract

Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography.

Rog-Zielinska EA, Moss R, Kaltenbacher W, Greiner J, ... Kohl P, Cannell MB
Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:86-92
Rog-Zielinska EA, Moss R, Kaltenbacher W, Greiner J, ... Kohl P, Cannell MB
J Mol Cell Cardiol: 30 Mar 2021; 153:86-92 | PMID: 33359037
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Abstract

Blockade of sodium‑calcium exchanger via ORM-10962 attenuates cardiac alternans.

Szlovák J, Tomek J, Zhou X, Tóth N, ... Rodriguez B, Nagy N
Repolarization alternans, a periodic oscillation of long-short action potential duration, is an important source of arrhythmogenic substrate, although the mechanisms driving it are insufficiently understood. Despite its relevance as an arrhythmia precursor, there are no successful therapies able to target it specifically. We hypothesized that blockade of the sodium‑calcium exchanger (NCX) could inhibit alternans. The effects of the selective NCX blocker ORM-10962 were evaluated on action potentials measured with microelectrodes from canine papillary muscle preparations, and calcium transients measured using Fluo4-AM from isolated ventricular myocytes paced to evoke alternans. Computer simulations were used to obtain insight into the drug\'s mechanisms of action. ORM-10962 attenuated cardiac alternans, both in action potential duration and calcium transient amplitude. Three morphological types of alternans were observed, with differential response to ORM-10962 with regards to APD alternans attenuation. Analysis of APD restitution indicates that calcium oscillations underlie alternans formation. Furthermore, ORM-10962 did not markedly alter APD restitution, but increased post-repolarization refractoriness, which may be mediated by indirectly reduced L-type calcium current. Computer simulations reproduced alternans attenuation via ORM-10962, suggesting that it is acts by reducing sarcoplasmic reticulum release refractoriness. This results from the ORM-10962-induced sodium‑calcium exchanger block accompanied by an indirect reduction in L-type calcium current. Using a computer model of a heart failure cell, we furthermore demonstrate that the anti-alternans effect holds also for this disease, in which the risk of alternans is elevated. Targeting NCX may therefore be a useful anti-arrhythmic strategy to specifically prevent calcium driven alternans.

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

J Mol Cell Cardiol: 30 Mar 2021; 153:111-122
Szlovák J, Tomek J, Zhou X, Tóth N, ... Rodriguez B, Nagy N
J Mol Cell Cardiol: 30 Mar 2021; 153:111-122 | PMID: 33383036
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Abstract

Mechanisms underlying age-associated manifestation of cardiac sodium channel gain-of-function.

Nowak MB, Poelzing S, Weinberg SH
Cardiac action potentials are initiated by sodium ion (Na+) influx through voltage-gated Na+ channels. Na+ channel gain-of-function (GOF) can arise in inherited conditions due to mutations in the gene encoding the cardiac Na+ channel, such as Long QT syndrome type 3 (LQT3). LQT3 can be a \"concealed\" disease, as patients with LQT3-associated mutations can remain asymptomatic until later in life; however, arrhythmias can also arise early in life in LQT3 patients, demonstrating a complex age-associated manifestation. We and others recently demonstrated that cardiac Na+ channels preferentially localize at the intercalated disc (ID) in adult cardiac tissue, which facilitates ephaptic coupling and formation of intercellular Na+ nanodomains that regulate pro-arrhythmic early afterdepolarization (EAD) formation in tissue with Na+ channel GOF. Several properties related to ephaptic coupling vary with age, such as cell size and Na+ channel and gap junction (GJ) expression and distribution: neonatal cells have immature IDs, with Na+ channels and GJs primarily diffusively distributed, while adult myocytes have mature IDs with preferentially localized Na+ channels and GJs. Here, we perform an in silico study varying critical age-dependent parameters to investigate mechanisms underlying age-associated manifestation of Na+ channel GOF in a model of guinea pig cardiac tissue. Simulations predict that total Na+ current conductance is a critical factor in action potential duration (APD) prolongation. We find a complex cell size/ Na+ channel expression relationship: increases in cell size (without concurrent increases in Na+ channel expression) suppress EAD formation, while increases in Na+ channel expression (without concurrent increases in cell size) promotes EAD formation. Finally, simulations with neonatal and early age-associated parameters predict normal APD with minimal dependence on intercellular cleft width; however, variability in cellular properties can lead to EADs presenting in early developmental stages. In contrast, for adult-associated parameters, EAD formation is highly dependent on cleft width, consistent with a mechanism underlying the age-associated manifestation of the Na+ channel GOF.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Mar 2021; 153:60-71
Nowak MB, Poelzing S, Weinberg SH
J Mol Cell Cardiol: 30 Mar 2021; 153:60-71 | PMID: 33373643
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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: 25 Mar 2021; 156:69-78
Jongstra-Bilen J, Tai K, Althagafi MG, Siu A, ... Hyduk SJ, Cybulsky MI
J Mol Cell Cardiol: 25 Mar 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: 25 Mar 2021; 156:33-44
Lynch TL, Kumar M, McNamara JW, Kuster DWD, ... Warshaw DM, Sadayappan S
J Mol Cell Cardiol: 25 Mar 2021; 156:33-44 | PMID: 33781820
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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: 23 Mar 2021; 156:45-56
Beckert V, Rassmann S, Kayvanjoo AH, Klausen C, ... Mass E, Wachten D
J Mol Cell Cardiol: 23 Mar 2021; 156:45-56 | PMID: 33773996
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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: 21 Mar 2021; 156:7-19
Mashali MA, Saad NS, Canan BD, Elnakish MT, ... Mohler PJ, Janssen PML
J Mol Cell Cardiol: 21 Mar 2021; 156:7-19 | PMID: 33766524
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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: 18 Mar 2021; 156:20-32
Osman I, Dong K, Kang X, Yu L, ... Zhang W, Zhou J
J Mol Cell Cardiol: 18 Mar 2021; 156:20-32 | PMID: 33753119
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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: 18 Mar 2021; 156:57-68
Sun X, Lyu L, Zhong X, Ni Z, Xu Q
J Mol Cell Cardiol: 18 Mar 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: 17 Mar 2021; 156:95-104
Kruithof BPT, van de Pol V, Los T, Lodder K, ... Goumans MJ, Ajmone Marsan N
J Mol Cell Cardiol: 17 Mar 2021; 156:95-104 | PMID: 33744308
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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: 13 Mar 2021; 156:1-6
Uruski P, Mikuła-Pietrasik J, Drzewiecki M, Budkiewicz S, ... Tykarski A, Książek K
J Mol Cell Cardiol: 13 Mar 2021; 156:1-6 | PMID: 33731316
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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: 09 Mar 2021; 155:99-110
Huang HL, Kuo CS, Chang TY, Chou RH, ... Wu CC, Huang PH
J Mol Cell Cardiol: 09 Mar 2021; 155:99-110 | PMID: 33713645
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Impact:
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: 02 Mar 2021; 155:66-77
Grabowska ME, Chun B, Moya R, Saucerman JJ
J Mol Cell Cardiol: 02 Mar 2021; 155:66-77 | PMID: 33667419
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Impact:
Abstract

Precision medicine for heart failure based on molecular mechanisms: The 2019 ISHR Research Achievement Award Lecture.

Nomura S, Komuro I
Heart failure is a leading cause of death, and the number of patients with heart failure continues to increase worldwide. To realize precision medicine for heart failure, its underlying molecular mechanisms must be elucidated. In this review summarizing the \"The Research Achievement Award Lecture\" of the 2019 XXIII ISHR World Congress held in Beijing, China, we would like to introduce our approaches for investigating the molecular mechanisms of cardiac hypertrophy, development, and failure, as well as discuss future perspectives.

Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 27 Feb 2021; 152:29-39
Nomura S, Komuro I
J Mol Cell Cardiol: 27 Feb 2021; 152:29-39 | PMID: 33275937
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Abstract

The cardiac methylome: A hidden layer of RNA modifications to regulate gene expression.

Rajan KS, Ramasamy S, Garikipati VNS, Suvekbala V
Post-transcriptional RNA modification has been observed in all kingdoms of life and more than a hundred different types of RNA modifications decorate the chemical and topological properties of these ribose nucleotides. These RNA modifications can potentially alter the RNA structure and also affect the binding affinity of proteins, thus regulating the mRNA stability as well as translation. Emerging evidence suggest that these modifications are not static, but are dynamic; vary upon different cues and are cell-type or tissue-specific. The cardiac transcriptome is not exceptional to such RNA modifications and is enriched with the abundant base methylation such as N6-methyladenosine (m6A) and also 2\'-O-Methylation (Nm). In this review we will focus on the technologies available to map these modifications and as well as the contribution of these post-transcriptional modifications during various pathological conditions of the heart.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Feb 2021; 152:40-51
Rajan KS, Ramasamy S, Garikipati VNS, Suvekbala V
J Mol Cell Cardiol: 27 Feb 2021; 152:40-51 | PMID: 33279505
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Abstract

Protective role of ErbB3 signaling in myeloid cells during adaptation to cardiac pressure overload.

Yin H, Favreau-Lessard AJ, deKay JT, Herrmann YR, ... Sawyer DB, Ryzhov S
Background
Myeloid cells play an important role in a wide variety of cardiovascular disorders, including both ischemic and non-ischemic cardiomyopathies. Neuregulin-1 (NRG-1)/ErbB signaling has recently emerged as an important factor contributing to the control of inflammatory activation of myeloid cells after an ischemic injury. However, the role of ErbB signaling in myeloid cells in non-ischemic cardiomyopathy is not fully understood. This study investigated the role of ErbB3 receptors in the regulation of early adaptive response using a mouse model of transverse aortic constriction (TAC) for non-ischemic cardiomyopathy.
Methods and results
TAC surgery was performed in groups of age- and sex-matched myeloid cell-specific ErbB3-deficient mice (ErbB3MyeKO) and control animals (ErbB3MyeWT). The number of cardiac CD45 immune cells, CD11b myeloid cells, Ly6G neutrophils, and Ly6C monocytes was determined using flow cytometric analysis. Five days after TAC, survival was dramatically reduced in male but not female ErbB3MyeKO mice or control animals. The examination of lung weight to body weight ratio suggested that acute pulmonary edema was present in ErbB3MyeKO male mice after TAC. To determine the cellular and molecular mechanisms involved in the increased mortality in ErbB3MyeKO male mice, cardiac cell populations were examined at day 3 post-TAC using flow cytometry. Myeloid cells accumulated in control but not in ErbB3MyeKO male mouse hearts. This was accompanied by increased proliferation of Sca-1 positive non-immune cells (endothelial cells and fibroblasts) in control but not ErbB3MyeKO male mice. No significant differences in intramyocardial accumulation of myeloid cells or proliferation of Sca-1 cells were found between the groups of ErbB3MyeKO and ErbB3MyeWT female mice. An antibody-based protein array analysis revealed that IGF-1 expression was significantly downregulated only in ErbB3MyeKO mice hearts compared to control animals after TAC.
Conclusion
Our data demonstrate the crucial role of myeloid cell-specific ErbB3 signaling in the cardiac accumulation of myeloid cells, which contributes to the activation of cardiac endothelial cells and fibroblasts and development of an early adaptive response to cardiac pressure overload in male mice.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Feb 2021; 152:1-16
Yin H, Favreau-Lessard AJ, deKay JT, Herrmann YR, ... Sawyer DB, Ryzhov S
J Mol Cell Cardiol: 27 Feb 2021; 152:1-16 | PMID: 33259856
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Abstract

What we know about cardiomyocyte dedifferentiation.

Zhu Y, Do VD, Richards AM, Foo R
Cardiomyocytes (CMs) lost during cardiac injury and heart failure (HF) cannot be replaced due to their limited proliferative capacity. Regenerating the failing heart by promoting CM cell-cycle re-entry is an ambitious solution, currently vigorously pursued. Some genes have been proven to promote endogenous CM proliferation, believed to be preceded by CM dedifferentiation, wherein terminally differentiated CMs are initially reversed back to the less mature state which precedes cell division. However, very little else is known about CM dedifferentiation which remains poorly defined. We lack robust molecular markers and proper understanding of the mechanisms driving dedifferentiation. Even the term dedifferentiation is debated because there is no objective evidence of pluripotency, and could rather reflect CM plasticity instead. Nonetheless, the significance of CM transition states on cardiac function, and whether they necessarily lead to CM proliferation, remains unclear. This review summarises the current state of knowledge of both natural and experimentally induced CM dedifferentiation in non-mammalian vertebrates (primarily the zebrafish) and mammals, as well as the phenotypes and molecular mechanisms involved. The significance and potential challenges of studying CM dedifferentiation are also discussed. In summary, CM dedifferentiation, essential for CM plasticity, may have an important role in heart regeneration, thereby contributing to the prevention and treatment of heart disease. More attention is needed in this field to overcome the technical limitations and knowledge gaps.

Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 27 Feb 2021; 152:80-91
Zhu Y, Do VD, Richards AM, Foo R
J Mol Cell Cardiol: 27 Feb 2021; 152:80-91 | PMID: 33275936
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Impact:
Abstract

Inhibition of endoplasmic reticulum stress mediates the ameliorative effect of apelin on vascular calcification.

Li Y, Li Y, Li Y, Yang Z, ... Wang X, Teng X
Aims
Apelin is the endogenous ligand of G protein-coupled receptor APJ and play an important role in the regulation of cardiovascular homeostasis. We aimed to investigate whether apelin ameliorates vascular calcification (VC) by inhibition of endoplasmic reticulum stress (ERS).
Methods and results
VC model in rats was induced by nicotine plus vitamin D, while calcification of vascular smooth muscle cell (VSMC) was induced by beta-glycerophosphate. Alizarin Red S staining showed dramatic calcium deposition in the aorta of rats with VC, while calcium contents and ALP activity also increased in calcified aorta. Protein levels of apelin and APJ were decreased in the calcified aorta. In rats with VC, apelin treatment significantly ameliorated aortic calcification, compliance and stimulation of ERS. The ameliorative effect of apelin on VC and ERS was also observed in calcified VSMCs. ERS stimulator (tunicamycin or DTT) blocked the beneficial effect of apelin. Apelin treatment activated the PI3K/Akt signaling, blockage of which by wortmannin or inhibitor IV prevented the ameliorative effect of apelin, while ERS inhibitor 4-PBA rescued the blockade effect of wortmannin. Akt-induced GSK inhibition prevented the phosphorylation of PERK and IRE1, and the activation of these two major ERS branches. F13A blocked the ameliorative effect of apelin on VC and ERS, which was reversed by treatment with 4-PBA or Akt activator SC79
Conclusions:
Apelin ameliorated VC by binding to APJ and then prevented ERS activation by stimulating Akt signaling. These results might provide new target for therapy and prevention of VC.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Feb 2021; 152:17-28
Li Y, Li Y, Li Y, Yang Z, ... Wang X, Teng X
J Mol Cell Cardiol: 27 Feb 2021; 152:17-28 | PMID: 33279504
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Abstract

Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes.

Hesse M, Bednarz R, Carls E, Becker C, ... Fleischmann BK, Gilsbach R
The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization. Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy. In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 27 Feb 2021; 152:95-104
Hesse M, Bednarz R, Carls E, Becker C, ... Fleischmann BK, Gilsbach R
J Mol Cell Cardiol: 27 Feb 2021; 152:95-104 | PMID: 33290769
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Impact:
Abstract

Downregulation of the zinc transporter SLC39A13 (ZIP13) is responsible for the activation of CaMKII at reperfusion and leads to myocardial ischemia/reperfusion injury in mouse hearts.

Wang J, Cheng X, Zhao H, Yang Q, Xu Z
While Zn2+ dyshomeostasis is known to contribute to ischemia/reperfusion (I/R) injury, the roles of zinc transporters that are responsible for Zn2+ homeostasis in the pathogenesis of I/R injury remain to be addressed. This study reports that ZIP13 (SLC39A13), a zinc transporter, plays a role in myocardial I/R injury by modulating the Ca2+ signaling pathway rather than by regulating Zn2+ transport. ZIP13 is downregulated upon reperfusion in mouse hearts or in H9c2 cells at reoxygenation. Ca2+ but not Zn2+ was responsible for ZIP13 downregulation, implying that ZIP13 may play a role in I/R injury through the Ca2+ signaling pathway. In line with our assumption, knockout of ZIP13 resulted in phosphorylation (Thr287) of Ca2+-calmodulin-dependent protein kinase (CaMKII), indicating that downregulation of ZIP13 leads to CaMKII activation. Further studies showed that the heart-specific knockout of ZIP13 enhanced I/R-induced CaMKII phosphorylation in mouse hearts. In contrast, overexpression of ZIP13 suppressed I/R-induced CaMKII phosphorylation. Moreover, the heart-specific knockout of ZIP13 exacerbated myocardial infarction in mouse hearts subjected to I/R, whereas overexpression of ZIP13 reduced infarct size. In addition, knockout of ZIP13 induced increases of mitochondrial Ca2+, ROS, mitochondrial swelling, decrease in the mitochondrial respiration control rate (RCR), and dissipation of mitochondrial membrane potential (ΔΨm) in a CaMKII-dependent manner. These data suggest that downregulation of ZIP13 at reperfusion contributes to myocardial I/R injury through activation of CaMKII and the mitochondrial death pathway.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 27 Feb 2021; 152:69-79
Wang J, Cheng X, Zhao H, Yang Q, Xu Z
J Mol Cell Cardiol: 27 Feb 2021; 152:69-79 | PMID: 33307093
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Impact:
Abstract

Gut microbiome - A potential mediator of pathogenesis in heart failure and its comorbidities: State-of-the-art review.

Mamic P, Chaikijurajai T, Tang WHW
Gut microbiome (GMB) has been increasingly recognized as a contributor to development and progression of heart failure (HF), immune-mediated subtypes of cardiomyopathy (myocarditis and anthracycline-induced cardiotoxicity), response to certain cardiovascular drugs, and HF-related comorbidities, such as chronic kidney disease, cardiorenal syndrome, insulin resistance, malnutrition, and cardiac cachexia. Gut microbiome is also responsible for the \"gut hypothesis\" of HF, which explains the adverse effects of gut barrier dysfunction and translocation of GMB on the progression of HF. Furthermore, accumulating evidence has suggested that gut microbial metabolites, including short chain fatty acids, trimethylamine N-oxide (TMAO), amino acid metabolites, and bile acids, are mechanistically linked to pathogenesis of HF, and could, therefore, serve as potential therapeutic targets for HF. Even though there are a variety of proposed therapeutic approaches, such as dietary modifications, prebiotics, probiotics, TMAO synthesis inhibitors, and fecal microbial transplant, targeting GMB in HF is still in its infancy and, indeed, requires further preclinical and clinical evidence. In this review, we aim to highlight the role gut microbiome plays in HF pathophysiology and its potential as a novel therapeutic target in HF.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Feb 2021; 152:105-117
Mamic P, Chaikijurajai T, Tang WHW
J Mol Cell Cardiol: 27 Feb 2021; 152:105-117 | PMID: 33307092
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Impact:
Abstract

Mep1a contributes to Ang II-induced cardiac remodeling by promoting cardiac hypertrophy, fibrosis and inflammation.

Ge W, Hou C, Zhang W, Guo X, ... Zhao H, Wang J
Pathological cardiac remodeling, characterized by excessive deposition of extracellular matrix proteins and cardiac hypertrophy, leads to the development of heart failure. Meprin α (Mep1a), a zinc metalloprotease, previously reported to participate in the regulation of inflammatory response and fibrosis, may also contribute to cardiac remodeling, although whether and how it participates in this process remains unknown. Here, in this work, we investigated the role of Mep1a in pathological cardiac remodeling, as well as the effects of the Mep1a inhibitor actinonin on cardiac remodeling-associated phenotypes. We found that Mep1a deficiency or chemical inhibition both significantly alleviated TAC- and Ang II-induced cardiac remodeling and dysfunction. Mep1a deletion and blocking both attenuated TAC- and Ang II-induced heart enlargement and increases in the thickness of the left ventricle anterior and posterior walls, and reduced expression of pro-hypertrophic markers, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and myosin heavy chain beta (β-MHC). In addition, Mep1a deletion and blocking significantly inhibited TAC- and Ang II-induced cardiac fibroblast activation and production of extracellular matrix (ECM). Moreover, in Mep1a-/- mice and treatment with actinonin significantly reduced Ang II-induced infiltration of macrophages and proinflammatory cytokines. Notably, we found that in vitro, Mep1a is expressed in cardiac myocytes and fibroblasts and that Mep1a deletion or chemical inhibition both markedly suppressed Ang II-induced hypertrophy of rat or mouse cardiac myocytes and activation of rat or mouse cardiac fibroblasts. In addition, blocking Mep1a in macrophages reduced Ang II-induced expression of interleukin (IL)-6 and IL-1β, strongly suggesting that Mep1a participates in cardiac remodeling processes through regulation of inflammatory cytokine expression. Mechanism studies revealed that Mep1a mediated ERK1/2 activation in cardiac myocytes, fibroblasts and macrophages and contributed to cardiac remodeling. In light of our findings that blocking Mep1a can ameliorate cardiac remodeling via inhibition of cardiac hypertrophy, fibrosis, and inflammation, Mep1a may therefore serve as a strong potential candidate for therapeutic targeting to prevent cardiac remodeling.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Feb 2021; 152:52-68
Ge W, Hou C, Zhang W, Guo X, ... Zhao H, Wang J
J Mol Cell Cardiol: 27 Feb 2021; 152:52-68 | PMID: 33301800
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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: 26 Feb 2021; 155:36-49
Nakayama Y, Mukai N, Wang BF, Yang K, ... Kitsis RN, Yoshioka J
J Mol Cell Cardiol: 26 Feb 2021; 155:36-49 | PMID: 33652022
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Impact:
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: 25 Feb 2021; 155:50-57
Creso JG, Campbell SG
J Mol Cell Cardiol: 25 Feb 2021; 155:50-57 | PMID: 33647310
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Impact:
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: 25 Feb 2021; 155:78-87
Kaidonis X, Niu W, Chan AY, Kesteven S, ... Feneley M, Graham RM
J Mol Cell Cardiol: 25 Feb 2021; 155:78-87 | PMID: 33647309
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Impact:
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: 23 Feb 2021; 155:58-65
Wang J, Ye Q, Bai S, Chen P, ... Yao Y, Ma Y
J Mol Cell Cardiol: 23 Feb 2021; 155:58-65 | PMID: 33636223
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Impact:
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 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 which showed 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. Published by Elsevier Ltd.

J Mol Cell Cardiol: 22 Feb 2021; epub ahead of print
Mijailovich SM, Prodanovic M, Poggesi C, Powers JD, ... Geeves MA, Regnier M
J Mol Cell Cardiol: 22 Feb 2021; epub ahead of print | PMID: 33636222
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Impact:
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: 22 Feb 2021; 155:1-9
Miles MR, Seo J, Jiang M, Wilson ZT, ... Ueberheide B, Tseng GN
J Mol Cell Cardiol: 22 Feb 2021; 155:1-9 | PMID: 33636221
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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: 21 Feb 2021; 155:10-20
Garnier A, Bork NI, Jacquet E, Zipfel S, ... Fischmeister R, Molina CE
J Mol Cell Cardiol: 21 Feb 2021; 155:10-20 | PMID: 33631188
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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: 17 Feb 2021; 155:88-98
Wang X, Lupton C, Lauth A, Wan TC, ... Auchampach JA, Lough JW
J Mol Cell Cardiol: 17 Feb 2021; 155:88-98 | PMID: 33609538
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Impact:
Abstract

Hypertrophic signaling compensates for contractile and metabolic consequences of DNA methyltransferase 3A loss in human cardiomyocytes.

Madsen A, Krause J, Höppner G, Hirt MN, ... Eschenhagen T, Stenzig J
The role of DNA methylation in cardiomyocyte physiology and cardiac disease remains a matter of controversy. We have recently provided evidence for an important role of DNMT3A in human cardiomyocyte cell homeostasis and metabolism, using engineered heart tissue (EHT) generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a knockout of the de novo DNA methyltransferase DNMT3A. Unlike isogenic control EHT, knockout EHT displayed morphological abnormalities such as lipid accumulations inside cardiomyocytes associated with impaired mitochondrial metabolism, as well as functional defects and impaired glucose metabolism. Here, we analyzed the role of DNMT3A in the setting of cardiac hypertrophy. We induced hypertrophic signaling by treatment with 50 nM endothelin-1 and 20 μM phenylephrine for one week and assessed EHT contractility, morphology, DNA methylation, and gene expression. While both knockout EHTs and isogenic controls showed the expected activation of the hypertrophic gene program, knockout EHTs were protected from hypertrophy-related functional impairment. Conversely, hypertrophic treatment prevented the metabolic consequences of a loss of DNMT3A, i.e. abolished lipid accumulation in cardiomyocytes likely by partial normalization of mitochondrial metabolism and restored glucose metabolism and metabolism-related gene expression of knockout EHT. Together, these data suggest an important role of DNA methylation not only for cardiomyocyte physiology, but also in the setting of cardiac disease.

Copyright © 2021 University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. Published by Elsevier Ltd.. All rights reserved.

J Mol Cell Cardiol: 11 Feb 2021; 154:115-123
Madsen A, Krause J, Höppner G, Hirt MN, ... Eschenhagen T, Stenzig J
J Mol Cell Cardiol: 11 Feb 2021; 154:115-123 | PMID: 33582159
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Abstract

Periostin-expressing Schwann cells and endoneurial cardiac fibroblasts contribute to sympathetic nerve fasciculation after birth.

Hortells L, Meyer EC, Thomas ZM, Yutzey KE
Background
The intracardiac nervous system (ICNS) is composed of neurons, in association with Schwann cells (SC) and endoneurial cardiac fibroblasts (ECF). Besides heart rhythm control, recent studies have implicated cardiac nerves in postnatal cardiac regeneration and cardiomyocyte size regulation, but cardiac SC and ECF remain understudied. During the postnatal period, the ICNS undergoes intense remodeling with nerve fasciculation and elongation throughout the myocardium, partially guided by the extracellular matrix (ECM). Here we report the origins, heterogeneity, and functions of SC and ECF that develop in proximity to neurons during postnatal ICNS maturation.
Methods and results
Periostin lineage (Postn+) cells include cardiac Remak SC and ECF during the postnatal period in mice. The developmental origins of cardiac SC and ECF were examined using Rosa26eGFP reporter mice bred with Wnt1Cre, expressed in Neural crest (NC)-derived lineages, or tamoxifen-inducible Tcf21MerCreMer, expressed predominantly in epicardial-derived fibroblast lineages. ICNS components are NC-derived with the exceptions of the myelinating Plp1+ SC and the Tcf21+ lineage-derived intramural ventricular ECF. In addition, Postn+ lineage GFAP- Remak SC and ECF are present around the fasciculating cardiac nerves. Whole mount studies of the NC-derived cells confirmed postnatal maturation of the complex ICNS network from P0 to P30. Sympathetic, parasympathetic, and sensory neurons fasciculate from P0 to P7 indicated by co-staining with PSA-NCAM. Ablation of Postn+ cells from P0 to P6 or loss of Periostin leads to reduced fasciculation of cardiac sympathetic nerves. In addition, collagen remodeling surrounding maturing nerves of the postnatal heart is reduced in Postn-null mice.
Conclusions
Postn+ cells include cardiac SC and ECF during postnatal nerve maturation, and these cells have different embryonic origins. At P7, the Postn+ cells associated with cardiac nerves are mainly Remak SC and ECF. Ablation of the Postn+ cells from P0 to P6 and also loss of Postn in Postn-null mice leads to reduced fasciculation of cardiac nerves at P7.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 10 Feb 2021; 154:124-136
Hortells L, Meyer EC, Thomas ZM, Yutzey KE
J Mol Cell Cardiol: 10 Feb 2021; 154:124-136 | PMID: 33582160
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Impact:
Abstract

β-adrenergic receptor N-terminal cleavage by ADAM17; the mechanism for redox-dependent downregulation of cardiomyocyte β-adrenergic receptors.

Zhu J, Steinberg SF
β1-adrenergic receptors (β1ARs) are the principle mediators of catecholamine action in cardiomyocytes. We previously showed that the β1AR extracellular N-terminus is a target for post-translational modifications that impact on signaling responses. Specifically, we showed that the β1AR N-terminus carries O-glycan modifications at Ser37/Ser41, that O-glycosylation prevents β1AR N-terminal cleavage, and that N-terminal truncation influences β1AR signaling to downstream effectors. However, the site(s) and mechanism for β1AR N-terminal cleavage in cells was not identified. This study shows that β1ARs are expressed in cardiomyocytes and other cells types as both full-length and N-terminally truncated species and that the truncated β1AR species is formed as a result of an O-glycan regulated N-terminal cleavage by ADAM17 at R31↓L32. We identify Ser41 as the major O-glycosylation site on the β1AR N-terminus and show that an O-glycan modification at Ser41 prevents ADAM17-dependent cleavage of the β1-AR N-terminus at S41↓L42, a second N-terminal cleavage site adjacent to this O-glycan modification (and it attenuates β1-AR N-terminal cleavage at R31↓L32). We previously reported that oxidative stress leads to a decrease in β1AR expression and catecholamine responsiveness in cardiomyocytes. This study shows that redox-inactivation of cardiomyocyte β1ARs is via a mechanism involving N-terminal truncation at R31↓L32 by ADAM17. In keeping with the previous observation that N-terminally truncated β1ARs constitutively activate an AKT pathway that affords protection against doxorubicin-dependent apoptosis, overexpression of a cleavage resistant β1AR mutant exacerbates doxorubicin-dependent apoptosis. These studies identify the β1AR N-terminus as a structural determinant of β1AR responses that can be targeted for therapeutic advantage.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 05 Feb 2021; 154:70-79
Zhu J, Steinberg SF
J Mol Cell Cardiol: 05 Feb 2021; 154:70-79 | PMID: 33556394
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Impact:
Abstract

Rad-GTPase contributes to heart rate via L-type calcium channel regulation.

Levitan BM, Ahern BM, Aloysius A, Brown L, ... Andres DA, Satin J
Sinoatrial node cardiomyocytes (SANcm) possess automatic, rhythmic electrical activity. SAN rate is influenced by autonomic nervous system input, including sympathetic nerve increases of heart rate (HR) via activation of β-adrenergic receptor signaling cascade (β-AR). L-type calcium channel (LTCC) activity contributes to membrane depolarization and is a central target of β-AR signaling. Recent studies revealed that the small G-protein Rad plays a central role in β-adrenergic receptor directed modulation of LTCC. These studies have identified a conserved mechanism in which β-AR stimulation results in PKA-dependent Rad phosphorylation: depletion of Rad from the LTCC complex, which is proposed to relieve the constitutive inhibition of CaV1.2 imposed by Rad association. Here, using a transgenic mouse model permitting conditional cardiomyocyte selective Rad ablation, we examine the contribution of Rad to the control of SANcm LTCC current (ICa,L) and sinus rhythm. Single cell analysis from a recent published database indicates that Rad is expressed in SANcm, and we show that SANcm ICa,L was significantly increased in dispersed SANcm following Rad silencing compared to those from CTRL hearts. Moreover, cRadKO SANcm ICa,L was not further increased with β-AR agonists. We also evaluated heart rhythm in vivo using radiotelemetered ECG recordings in ambulating mice. In vivo, intrinsic HR is significantly elevated in cRadKO. During the sleep phase cRadKO also show elevated HR, and during the active phase there is no significant difference. Rad-deletion had no significant effect on heart rate variability. These results are consistent with Rad governing LTCC function under relatively low sympathetic drive conditions to contribute to slower HR during the diurnal sleep phase HR. In the absence of Rad, the tonic modulated SANcm ICa,L promotes elevated sinus HR. Future novel therapeutics for bradycardia targeting Rad - LTCC can thus elevate HR while retaining βAR responsiveness.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 05 Feb 2021; 154:60-69
Levitan BM, Ahern BM, Aloysius A, Brown L, ... Andres DA, Satin J
J Mol Cell Cardiol: 05 Feb 2021; 154:60-69 | PMID: 33556393
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Impact:
Abstract

Clinical epigenomics for cardiovascular disease: Diagnostics and therapies.

Fischer MA, Vondriska TM
The study of epigenomics has advanced in recent years to span the regulation of a single genetic locus to the structure and orientation of entire chromosomes within the nucleus. In this review, we focus on the challenges and opportunities of clinical epigenomics in cardiovascular disease. As an integrator of genetic and environmental inputs, and because of advances in measurement techniques that are highly reproducible and provide sequence information, the epigenome is a rich source of potential biosignatures of cardiovascular health and disease. Most of the studies to date have focused on the latter, and herein we discuss observations on epigenomic changes in human cardiovascular disease, examining the role of protein modifiers of chromatin, noncoding RNAs and DNA modification. We provide an overview of cardiovascular epigenomics, discussing the challenges of data sovereignty, data analysis, doctor-patient ethics and innovations necessary to implement precision health.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 05 Feb 2021; 154:97-105
Fischer MA, Vondriska TM
J Mol Cell Cardiol: 05 Feb 2021; 154:97-105 | PMID: 33561434
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Impact:
Abstract

Phosphoproteomics of the developing heart identifies PERM1 - An outer mitochondrial membrane protein.

Aravamudhan S, Türk C, Bock T, Keufgens L, ... Braun T, Krüger M
Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1-/- mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.

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

J Mol Cell Cardiol: 04 Feb 2021; 154:41-59
Aravamudhan S, Türk C, Bock T, Keufgens L, ... Braun T, Krüger M
J Mol Cell Cardiol: 04 Feb 2021; 154:41-59 | PMID: 33549681
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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: 04 Feb 2021; 155:25-35
Rattka M, Westphal S, Gahr BM, Just S, Rottbauer W
J Mol Cell Cardiol: 04 Feb 2021; 155:25-35 | PMID: 33549680
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Impact:
Abstract

Computation-assisted targeted proteomics of alternative splicing protein isoforms in the human heart.

Han Y, Wood SD, Wright JM, Dostal V, Lau E, Lam MPY
Alternative splicing is prevalent in the heart and implicated in many cardiovascular diseases, but not every alternative transcript is translated and detecting non-canonical isoforms at the protein level remains challenging. Here we show the use of a computation-assisted targeted proteomics workflow to detect protein alternative isoforms in the human heart. We build on a recent strategy to integrate deep RNA-seq and large-scale mass spectrometry data to identify candidate translated isoform peptides. A machine learning approach is then applied to predict their fragmentation patterns and design protein isoform-specific parallel reaction monitoring detection (PRM) assays. As proof-of-principle, we built PRM assays for 29 non-canonical isoform peptides and detected 22 peptides in a human heart lysate. The predictions-aided PRM assays closely mirrored synthetic peptide standards for non-canonical sequences. This approach may be useful for validating non-canonical protein identification and discovering functionally relevant isoforms in the heart.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 04 Feb 2021; 154:92-96
Han Y, Wood SD, Wright JM, Dostal V, Lau E, Lam MPY
J Mol Cell Cardiol: 04 Feb 2021; 154:92-96 | PMID: 33549679
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Impact:
Abstract

MicroRNA-21 regulates right ventricular remodeling secondary to pulmonary arterial pressure overload.

Chang WT, Fisch S, Dangwal S, Mohebali J, ... Chen FY, Liao R
Right ventricular (RV) function is a critical determinant of survival in patients with pulmonary arterial hypertension (PAH). While miR-21 is known to associate with vascular remodeling in small animal models of PAH, its role in RV remodeling in large animal models has not been characterized. Herein, we investigated the role of miR-21 in RV dysfunction using a sheep model of PAH secondary to pulmonary arterial constriction (PAC). RV structural and functional remodeling were examined using ultrasound imaging. Our results showed that post PAC, RV strain significantly decreased at the basal region compared with t the control. Moreover, such dysfunction was accompanied by increases in miR-21 levels. To determine the role of miR-21 in RV remodeling secondary to PAC, we investigated the molecular alteration secondary to phenylephrine induced hypertrophy and miR21 overexpression in vitro using neonatal rat ventricular myocytes (NRVMs). We found that overexpression of miR-21 in the setting of hypertrophic stimulation augmented only the expression of proteins critical for mitosis but not cytokinesis. Strikingly, this molecular alteration was associated with an eccentric cellular hypertrophic phenotype similar to what we observed in vivo PAC animal model in sheep. Importantly, this hypertrophic change was diminished upon suppressing miR-21 in NRVMs. Collectively, our in vitro and in vivo data demonstrate that miR-21 is a critical contributor in the development of RV dysfunction and could represent a novel therapeutic target for PAH associated RV dysfunction.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 03 Feb 2021; 154:106-114
Chang WT, Fisch S, Dangwal S, Mohebali J, ... Chen FY, Liao R
J Mol Cell Cardiol: 03 Feb 2021; 154:106-114 | PMID: 33548242
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Impact:
Abstract

A peptide of the amino-terminus of GRK2 induces hypertrophy and yet elicits cardioprotection after pressure overload.

Bledzka KM, Manaserh IH, Grondolsky J, Pfleger J, ... Koch WJ, Schumacher SM
G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 \"interactome\" that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (βARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, βARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, βARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify βARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and βARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in βARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male βARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the βARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.

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

J Mol Cell Cardiol: 03 Feb 2021; 154:137-153
Bledzka KM, Manaserh IH, Grondolsky J, Pfleger J, ... Koch WJ, Schumacher SM
J Mol Cell Cardiol: 03 Feb 2021; 154:137-153 | PMID: 33548241
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Impact:
Abstract

Xanthine oxidoreductase-mediated injury is amplified by upregulated AMP deaminase in type 2 diabetic rat hearts under the condition of pressure overload.

Igaki Y, Tanno M, Sato T, Kouzu H, ... Nakamura T, Miura T
Background
We previously reported that upregulated AMP deaminase (AMPD) contributes to diastolic ventricular dysfunction via depletion of the adenine nucleotide pool in a rat model of type 2 diabetes (T2DM), Otsuka Long-Evans-Tokushima Fatty rats (OLETF). Meanwhile, AMPD promotes the formation of substrates of xanthine oxidoreductase (XOR), which produces ROS as a byproduct. Here, we tested the hypothesis that a functional link between upregulated AMPD and XOR is involved in ventricular dysfunction in T2DM rats.
Methods and results
Pressure-volume loop analysis revealed that pressure overloading by phenylephrine infusion induced severer left ventricular diastolic dysfunction (tau: 14.7 ± 0.8 vs 12.5 ± 0.7 msec, left ventricular end-diastolic pressure: 18.3 ± 1.5 vs 12.2 ± 1.3 mmHg, p < 0.05) and ventricular-arterial uncoupling in OLETF than in LETO, non-diabetic rats, though the baseline parameters were comparable in the two groups. While the pressure overload did not affect AMPD activity, it increased XOR activity both in OLETF and LETO, with OLETF showing significantly higher XOR activity than that in LETO (347.2 ± 17.9 vs 243.2 ± 6.1 μg/min/mg). Under the condition of pressure overload, myocardial ATP level was lower, and levels of xanthine and uric acid were higher in OLETF than in LETO. Addition of exogenous inosine, a product of AMP deamination, to the heart homogenates augmented XOR activity. OLETF showed 68% higher tissue ROS levels and 47% reduction in mitochondrial state 3 respiration compared with those in LETO. Overexpression of AMPD3 in H9c2 cells elevated levels of hypoxanthine and ROS and reduced the level of ATP. Inhibition of XOR suppressed the production of tissue ROS and mitochondrial dysfunction and improved ventricular function under the condition of pressure overload in OLETF.
Conclusions
The results suggest that increases in the activity of XOR and the formation of XOR substrates by upregulated AMPD contribute to ROS-mediated diastolic ventricular dysfunction at the time of increased cardiac workload in diabetic hearts.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 03 Feb 2021; 154:21-31
Igaki Y, Tanno M, Sato T, Kouzu H, ... Nakamura T, Miura T
J Mol Cell Cardiol: 03 Feb 2021; 154:21-31 | PMID: 33548240
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Impact:
Abstract

Computational modeling approaches to cAMP/PKA signaling in cardiomyocytes.

McCabe KJ, Rangamani P
The cAMP/PKA pathway is a fundamental regulator of excitation-contraction coupling in cardiomyocytes. Activation of cAMP has a variety of downstream effects on cardiac function including enhanced contraction, accelerated relaxation, adaptive stress response, mitochondrial regulation, and gene transcription. Experimental advances have shed light on the compartmentation of cAMP and PKA, which allow for control over the varied targets of these second messengers and is disrupted in heart failure conditions. Computational modeling is an important tool for understanding the spatial and temporal complexities of this system. In this review article, we outline the advances in computational modeling that have allowed for deeper understanding of cAMP/PKA dynamics in the cardiomyocyte in health and disease, and explore new modeling frameworks that may bring us closer to a more complete understanding of this system. We outline various compartmental and spatial signaling models that have been used to understand how β-adrenergic signaling pathways function in a variety of simulation conditions. We also discuss newer subcellular models of cardiovascular function that may be used as templates for the next phase of computational study of cAMP and PKA in the heart, and outline open challenges which are important to consider in future models.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 03 Feb 2021; 154:32-40
McCabe KJ, Rangamani P
J Mol Cell Cardiol: 03 Feb 2021; 154:32-40 | PMID: 33548239
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Abstract

Spatial N-glycomics of the human aortic valve in development and pediatric endstage congenital aortic valve stenosis.

Angel PM, Drake RR, Park Y, Clift CL, ... Bichell DP, Su YR
Congenital aortic valve stenosis (AS) progresses as an obstructive narrowing of the aortic orifice due to deregulated extracellular matrix (ECM) production by aortic valve (AV) leaflets and leads to heart failure with no effective therapies. Changes in glycoprotein and proteoglycan distribution are a hallmark of AS, yet valvular carbohydrate content remains virtually uncharacterized at the molecular level. While almost all glycoproteins clinically linked to stenotic valvular modeling contain multiple sites for N-glycosylation, there are very few reports aimed at understanding how N-glycosylation contributes to the valve structure in disease. Here, we tested for spatial localization of N-glycan structures within pediatric congenital aortic valve stenosis. The study was done on valvular tissues 0-17 years of age with de-identified clinical data reporting pre-operative valve function spanning normal development, aortic valve insufficiency (AVI), and pediatric endstage AS. High mass accuracy imaging mass spectrometry (IMS) was used to localize N-glycan profiles in the AV structure. RNA-Seq was used to identify regulation of N-glycan related enzymes. The N-glycome was found to be spatially localized in the normal aortic valve, aligning with fibrosa, spongiosa or ventricularis. In AVI diagnosed tissue, N-glycans localized to hypertrophic commissures with increases in pauci-mannose structures. In all valve types, sialic acid (N-acetylneuraminic acid) N-glycans were the most abundant N-glycan group. Three sialylated N-glycans showed common elevation in AS independent of age. On-tissue chemical methods optimized for valvular tissue determined that aortic valve tissue sialylation shows both α2,6 and α2,3 linkages. Specialized enzymatic strategies demonstrated that core fucosylation is the primary fucose configuration and localizes to the normal fibrosa with disparate patterning in AS. This study identifies that the human aortic valve structure is spatially defined by N-glycomic signaling and may generate new research directions for the treatment of human aortic valve disease.

Copyright © 2021 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 28 Jan 2021; 154:6-20
Angel PM, Drake RR, Park Y, Clift CL, ... Bichell DP, Su YR
J Mol Cell Cardiol: 28 Jan 2021; 154:6-20 | PMID: 33516683
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This program is still in alpha version.