Journal: J Mol Cell Cardiol

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Abstract

Validation of quantitative measure of repolarization reserve as a novel marker of drug induced proarrhythmia.

Gaur N, Ortega F, Verkerk AO, Mengarelli I, ... Coronel R, Vigmond EJ

Repolarization reserve, the robustness of a cell to repolarize even when one of the repolarization mechanisms is failing, has been described qualitatively in terms of ionic currents, but has not been quantified by a generic metric that is applicable to drug screening. Prolonged repolarization leading to repolarization failure is highly arrhythmogenic. It may lead to ventricular tachycardia caused by triggered activity from early afterdepolarizations (EADs), or it may promote the occurrence of unidirectional conduction block and reentry. Both types of arrhythmia may deteriorate into ventricular fibrillation (VF) and death. We define the Repolarization Reserve Current (RRC) as the minimum constant current necessary to prevent normal repolarization of a cell. After developing and testing RRC for nine computational ionic models of various species, we applied it experimentally to atrial and ventricular human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM), and isolated guinea-pig ventricular cardiomyocytes. In simulations, repolarization was all-or-none with a precise, model-dependent critical RRC, resulting in a discrete shift in the Action Potential Duration (APD) - RRC relation, in the occurrence of EADs and repolarization failure. These data were faithfully reproduced in cellular experiments. RRC allows simple, fast, unambiguous quantification of the arrhythmogenic propensity in cardiac cells of various origins and species without the need of prior knowledge of underlying currents and is suitable for high throughput applications, and personalized medicine applications.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Jul 2020; 145:122-132
Gaur N, Ortega F, Verkerk AO, Mengarelli I, ... Coronel R, Vigmond EJ
J Mol Cell Cardiol: 30 Jul 2020; 145:122-132 | PMID: 32325153
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Abstract

POPDC2 a novel susceptibility gene for conduction disorders.

Rinné S, Ortiz-Bonnin B, Stallmeyer B, Kiper AK, ... Brand T, Decher N

Despite recent progress in the understanding of cardiac ion channel function and its role in inherited forms of ventricular arrhythmias, the molecular basis of cardiac conduction disorders often remains unresolved. We aimed to elucidate the genetic background of familial atrioventricular block (AVB) using a whole exome sequencing (WES) approach. In monozygotic twins with a third-degree AVB and in another, unrelated family with first-degree AVB, we identified a heterozygous nonsense mutation in the POPDC2 gene causing a premature stop at position 188 (POPDC2), deleting parts of its cAMP binding-domain. Popeye-domain containing (POPDC) proteins are predominantly expressed in the skeletal muscle and the heart, with particularly high expression of POPDC2 in the sinoatrial node of the mouse. We now show by quantitative PCR experiments that in the human heart the POPDC-modulated two-pore domain potassium (K) channel TREK-1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2 causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2 knock-in mice displayed stress-induced sinus bradycardia and pauses, a phenotype that was previously also reported for POPDC2 and TREK-1 knock-out mice. We propose that the POPDC2 loss-of-function mutation contributes to AVB pathogenesis by an aberrant modulation of TREK-1, highlighting that POPDC2 represents a novel arrhythmia gene for cardiac conduction disorders.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2020; 145:74-83
Rinné S, Ortiz-Bonnin B, Stallmeyer B, Kiper AK, ... Brand T, Decher N
J Mol Cell Cardiol: 30 Jul 2020; 145:74-83 | PMID: 32535041
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Abstract

Metformin protects against ischaemic myocardial injury by alleviating autophagy-ROS-NLRP3-mediated inflammatory response in macrophages.

Fei Q, Ma H, Zou J, Wang W, ... Wang N, Wang K

Myocardial ischaemia is usually accompanied by inflammatory response which plays a critical role in the myocardial healing and scar formation, while persistent inflammatory response contributes greatly to the myocardial remodeling and consequent heart failure. Metformin (Met), a widely used hypoglycemic drug, has increasingly been shown to exert remarkable cardioprotective effect on ischaemic myocardial injury such as acute myocardial infarction (AMI). However, the underlying mechanisms are still far from being fully understood. In this study, a mouse model of AMI was established through ligating the left anterior descending coronary artery (LAD), 100 mg/kg Met was given immediately after operation once daily for 3 days. It was demonstrated that Met effectively improved the cardiac haemodynamics (LVSP, LVEDP, +dp/dt, -dp/dt), diminished the infarct size, alleviated the disarrangement of myocardial cells and reduced the infiltration of inflammatory cells (macrophages, neutrophils and lymphocytes) in the heart of AMI mice. Mechanistically, Met decreased the expression of NLRP3 and enhanced the accumulation of LC3 puncta in F4/80-positive macrophages in the heart of AMI mice. Single cell suspension of cardiac macrophages was prepared from AMI mice and exhibited increased NLRP3 mRNA and protein expression. In contrast, Met decreased the expression of NLRP3 and p62, whereas increased the ratio of LC3II/LC3I. Additionally, both conditioned medium from H9c2 cardiomyocytes exposed to hydrogen peroxide (H9c2-HO-CM) and combination of mtDNA and ATP (mtDNA-ATP) increased the expression of NLRP3 and cleaved caspase-1 (p10) as well as intracellular ROS production in RAW264.7 macrophages, which were abrogated by Met treatment. Strikingly, chloroquine (CQ), 3-methyladenine (3-MA) and knockdown of autophagy-related gene (Atg5) abrogated the inhibitory effects of Met on H9c2-HO-CM and mtDNA-ATP-induced NLRP3 expression, release of IL-1β and IL-18 as well as ROS production in RAW264.7 macrophages. Collectively, these findings suggest that Met protects against ischaemic myocardial injury through alleviating autophagy-ROS-NLRP3 axis-mediated inflammatory response in macrophages.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2020; 145:1-13
Fei Q, Ma H, Zou J, Wang W, ... Wang N, Wang K
J Mol Cell Cardiol: 30 Jul 2020; 145:1-13 | PMID: 32470468
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Abstract

Cyclic AMP represses pathological MEF2 activation by myocyte-specific hypo-phosphorylation of HDAC5.

He T, Huang J, Chen L, Han G, ... Hagenmüller M, Backs J

Class IIa histone deacetylases (HDACs) critically regulate cardiac function through the repression of the activity of myocyte enhancer factor 2 (MEF2)-dependent gene programs. Protein kinase D (PKD) and Ca/Calmodulin-dependent kinase II (CaMKII) activate MEF2 by phosphorylating distinct HDAC isoforms and thereby creating 14-3-3 binding sites for nucleo-cytoplasmic shuttling. Recently, it has been shown that this process is counteracted by cyclic AMP (cAMP)-dependent signaling. Here, we investigated the specific mechanisms of how cAMP-dependent signaling regulates distinct HDAC isoforms and determined their relative contributions to the protection from pathological MEF2 activation. We found that cAMP is sufficient to induce nuclear retention and to blunt phosphorylation of the 14-3-3 binding sites of HDAC5 (Ser259/498) and HDAC9 (Ser218/448) but not HDAC4 (Ser246/467/632). These regulatory events could be observed only in cardiomyocytes and myocyte-like cells but not in non-myocytes, pointing to an indirect myocyte-specific mode of action. Consistent with one previous report, we found that blunted phosphorylation of HDAC5 and HDAC9 was mediated by protein kinase A (PKA)-dependent inhibition of PKD. However, we show by the use of neonatal cardiomyocytes derived from genetic HDAC mouse models that endogenous HDAC5 but not HDAC9 contributes specifically to the repression of endogenous MEF2 activity. HDAC4 contributed significantly to the repression of MEF2 activity but based on the mechanistic findings of this study combined with previous results we attribute this to PKA-dependent proteolysis of HDAC4. Consistently, cAMP-induced repression of agonist-driven cellular hypertrophy was blunted in cardiomyocytes deficient for both HDAC5 and HDAC4. In conclusion, cAMP inhibits MEF2 through both nuclear accumulation of hypo-phosphorylated HDAC5 and through a distinct HDAC4-dependent mechanism.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Jul 2020; 145:88-98
He T, Huang J, Chen L, Han G, ... Hagenmüller M, Backs J
J Mol Cell Cardiol: 30 Jul 2020; 145:88-98 | PMID: 32485181
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Abstract

Identifying temporal molecular signatures underlying cardiovascular diseases: A data science platform.

Chung NC, Choi H, Wang D, Mirza B, ... Wang W, Ping P
Objective
During cardiovascular disease progression, molecular systems of myocardium (e.g., a proteome) undergo diverse and distinct changes. Dynamic, temporally-regulated alterations of individual molecules underlie the collective response of the heart to pathological drivers and the ultimate development of pathogenesis. Advances in high-throughput omics technologies have enabled cost-effective, temporal profiling of targeted systems in animal models of human diseases. However, computational analysis of temporal patterns from omics data remains challenging. In particular, bioinformatic pipelines involving unsupervised statistical approaches to support cardiovascular investigations are lacking, which hinders one\'s ability to extract biomedical insights from these complex datasets.
Approach and results
We developed a non-parametric data analysis platform to resolve computational challenges unique to temporal omics datasets. Our platform consists of three modules. Module I preprocesses the temporal data using either cubic splines or principal component analysis (PCA), and it simultaneously accomplishes the tasks on missing data imputation and denoising. Module II performs an unsupervised classification by K-means or hierarchical clustering. Module III evaluates and identifies biological entities (e.g., molecular events) that exhibit strong associations to specific temporal patterns. The jackstraw method for cluster membership has been applied to estimate p-values and posterior inclusion probabilities (PIPs), both of which guided feature selection. To demonstrate the utility of the analysis platform, we employed a temporal proteomics dataset that captured the proteome-wide dynamics of oxidative stress induced post-translational modifications (O-PTMs) in mouse hearts undergoing isoproterenol (ISO)-induced hypertrophy.
Conclusion
We have created a platform, CV.Signature.TCP, to identify distinct temporal clusters in omics datasets. We presented a cardiovascular use case to demonstrate its utility in unveiling biological insights underlying O-PTM regulations in cardiac remodeling. This platform is implemented in an open source R package (https://github.com/UCLA-BD2K/CV.Signature.TCP).

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

J Mol Cell Cardiol: 30 Jul 2020; 145:54-58
Chung NC, Choi H, Wang D, Mirza B, ... Wang W, Ping P
J Mol Cell Cardiol: 30 Jul 2020; 145:54-58 | PMID: 32504647
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Abstract

Glucose fluctuations promote vascular BK channels dysfunction via PKCα/NF-κB/MuRF1 signaling.

Zhang ZY, Qian LL, Wang N, Miao LF, ... Ling TY, Wang RX

Glucose fluctuations may contribute to large conductance calcium activated potassium (BK) channel dysfunction. However, the underlying mechanisms remain elusive. The aim of this study was to investigate the molecular mechanisms involved in BK channel dysfunction as a result of glucose fluctuations. A rat diabetic model was established through the injection of streptozotocin. Glucose fluctuations in diabetic rats were induced via consumption and starvation. Rat coronary arteries were isolated and coronary vascular tensions were measured after three weeks. Rat coronary artery smooth muscle cells were isolated and whole-cell BK channel currents were recorded using a patch clamp technique. Human coronary artery smooth muscle cells in vitro were used to explore the underlying mechanisms. After incubation with iberiotoxin (IBTX), the Δ tensions (% Max) of rat coronary arteries in the controlled diabetes mellitus (C-DM), the uncontrolled DM (U-DM) and the DM with glucose fluctuation (GF-DM) groups were found to be 84.46 ± 5.75, 61.89 ± 10.20 and 14.77 ± 5.90, respectively (P < .05), while the current densities of the BK channels in the three groups were 43.09 ± 4.35 pA/pF, 34.23 ± 6.07 pA/pF and 17.87 ± 4.33 pA/pF, respectively (P < .05). The Δ tensions (% Max) of rat coronary arteries after applying IBTX in the GF-DM rats injected with 0.9% sodium chloride (NaCl) (GF-DM + NaCl) and the GF-DM rats injected with N-acetyl-L-cysteine (NAC) (GF-DM + NAC) groups were found to be 8.86 ± 1.09 and 48.90 ± 10.85, respectively (P < .05). Excessive oxidative stress and the activation of protein kinase C (PKC) α and nuclear factor (NF)-κB induced by glucose fluctuations promoted the decrease of BK-β1 expression, while the inhibition of reactive oxygen species (ROS), PKCα, NF-κB and muscle ring finger protein 1 (MuRF1) reversed this effect. Glucose fluctuations aggravate BK channel dysfunction via the ROS overproduction and the PKCα/NF-κB/MuRF1 signaling pathway.

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

J Mol Cell Cardiol: 30 Jul 2020; 145:14-24
Zhang ZY, Qian LL, Wang N, Miao LF, ... Ling TY, Wang RX
J Mol Cell Cardiol: 30 Jul 2020; 145:14-24 | PMID: 32511969
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Abstract

Type 2 innate lymphoid cells regulation by regulatory T cells attenuates atherosclerosis.

Gao X, Lin J, Zheng Y, Liu S, ... He S, Li D

Regulatory T cells (Tregs) have been shown to attenuate the development and progression of atherosclerosis; however, the exact mechanism is still unclear. In our study, Tregs were adoptively transferred into ApoE mice, and type 2 innate lymphoid cells (ILC2s) were expanded by the IL-2/Jes6-1 complex or depleted by anti-CD90.2 mAb in ApoERag1 mice to study their effects on atherosclerosis. Then, Tregs were cocultured with ILC2s in vitro to analyze ILC2s number and IL-13 production. In vivo, ApoERag1 mice were treated with activated Tregs with or without anti-CD90.2 mAb to explore whether Tregs reduced atherosclerosis through ILC2s. Finally, neutralizing antibodies and Transwell assay were used to investigate how Tregs regulate ILC2s. Our results show that both Tregs and ILC2s reduce atherosclerosis lesions and macrophage infiltration. Moreover, Tregs effectively expanded the number of ILC2s and increased their production of IL-13 in vivo and in vitro. Furthermore, the reductions in plaque size and macrophage infiltration by Tregs were partly reversed by anti-CD90.2 mAb. Mechanistically, our data reveal that IL-10, TGF-β and cell-cell contacts are required for Tregs-ILC2s regulation. These results show that Tregs may play a partial protective role against atherosclerosis by expanding the number of ILC2s and consequently increasing IL-13 production.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2020; 145:99-111
Gao X, Lin J, Zheng Y, Liu S, ... He S, Li D
J Mol Cell Cardiol: 30 Jul 2020; 145:99-111 | PMID: 32526223
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Abstract

Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics.

Bhagwan JR, Mosqueira D, Chairez-Cantu K, Mannhardt I, ... Smith JGW, Denning C
Background
Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics.
Methods
We directly compared the p.β-MHC-R453C and p.ACTC1-E99K HCM-associated mutations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and their healthy isogenic counterparts, generated using CRISPR/Cas9 genome editing technology. By harnessing several state-of-the-art HCM phenotyping techniques, these mutations were investigated to identify similarities and differences in disease progression and hypertrophic signaling pathways, towards establishing potential targets for pharmacological treatment. CRISPR/Cas9 knock-in of the genetically-encoded calcium indicator R-GECO1.0 to the AAVS1 locus into these disease models resulted in calcium reporter lines.
Results
Confocal line scan analysis identified calcium transient arrhythmias and intracellular calcium overload in both models. The use of optogenetics and 2D/3D contractility assays revealed opposing phenotypes in the two mutations. Gene expression analysis highlighted upregulation of CALM1, CASQ2 and CAMK2D, and downregulation of IRF8 in p.β-MHC-R453C mutants, whereas the opposite changes were detected in p.ACTC1-E99K mutants. Contrasting profiles of nuclear translocation of NFATc1 and MEF2 between the two HCM models suggest differential hypertrophic signaling pathway activation. Calcium transient abnormalities were rescued with combination of dantrolene and ranolazine, whilst mavacamten reduced the hyper-contractile phenotype of p.ACTC1-E99K hiPSC-CMs.
Conclusions
Our data show that hypercontractility and molecular signaling within HCM are not uniform between different gene mutations, suggesting that a \'one-size fits all\' treatment underestimates the complexity of the disease. Understanding where the similarities (arrhythmogenesis, bioenergetics) and differences (contractility, molecular profile) lie will allow development of therapeutics that are directed towards common mechanisms or tailored to each disease variant, hence providing effective patient-specific therapy.

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

J Mol Cell Cardiol: 30 Jul 2020; 145:43-53
Bhagwan JR, Mosqueira D, Chairez-Cantu K, Mannhardt I, ... Smith JGW, Denning C
J Mol Cell Cardiol: 30 Jul 2020; 145:43-53 | PMID: 32531470
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Abstract

Global analysis of histone modifications and long-range chromatin interactions revealed the differential cistrome changes and novel transcriptional players in human dilated cardiomyopathy.

Liu CF, Abnousi A, Bazeley P, Ni Y, ... Hu M, Tang WHW
Background
Acetylation and methylation of histones alter the chromatin structure and accessibility that affect transcriptional regulators binding to enhancers and promoters. The binding of transcriptional regulators enables the interaction between enhancers and promoters, thus affecting gene expression. However, our knowledge of these epigenetic alternations in patients with heart failure remains limited.
Methods and results
From the comprehensive analysis of major histone modifications, 3-dimensional chromatin interactions, and transcriptome in left ventricular (LV) tissues from dilated cardiomyopathy (DCM) patients and non-heart failure (NF) donors, differential active enhancer and promoter regions were identified between NF and DCM. Moreover, the genome-wide average promoter signal is significantly lower in DCM than in NF. Super-enhancer (SE) analysis revealed that fewer SEs were found in DCM LVs than in NF ones, and three unique SE-associated genes between NF and DCM were identified. Moreover, SEs are enriched within the genomic region associated with long-range chromatin interactions. The differential enhancer-promoter interactions were observed in the known heart failure gene loci and are correlated with the gene expression levels. Motif analysis identified known cardiac factors and possible novel players for DCM.
Conclusions
We have established the cistrome of four histone modifications and chromatin interactome for enhancers and promoters in NF and DCM tissues. Differential histone modifications and enhancer-promoter interactions were found in DCM, which were associated with gene expression levels of a subset of disease-associated genes in human heart failure.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2020; 145:30-42
Liu CF, Abnousi A, Bazeley P, Ni Y, ... Hu M, Tang WHW
J Mol Cell Cardiol: 30 Jul 2020; 145:30-42 | PMID: 32533974
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Abstract

Enhancing fatty acid oxidation negatively regulates PPARs signaling in the heart.

Liu Z, Ding J, McMillen TS, Villet O, Tian R, Shao D

High fatty acid oxidation (FAO) is associated with lipotoxicity, but whether it causes lipotoxic cardiomyopathy remains controversial. Molecular mechanisms that may be responsible for FAO-induced lipotoxic cardiomyopathy are also elusive. In this study, increasing FAO by genetic deletion of acetyl-CoA carboxylase 2 (ACC2) did not induce cardiac dysfunction after 16 weeks of high fat diet (HFD) feeding. This suggests that increasing FAO, per se, does not cause metabolic cardiomyopathy in obese mice. We compared transcriptomes of control and ACC2 deficient mouse hearts under chow- or HFD-fed conditions. ACC2 deletion had a significant impact on the global transcriptome including downregulation of the peroxisome proliferator-activated receptors (PPARs) signaling and fatty acid degradation pathways. Increasing fatty acids by HFD feeding normalized expression of fatty acid degradation genes in ACC2 deficient mouse hearts to the same level as the control mice. In contrast, cardiac transcriptome analysis of the lipotoxic mouse model (db/db) showed an upregulation of PPARs signaling and fatty acid degradation pathways. Our results suggest that enhancing FAO by genetic deletion of ACC2 negatively regulates PPARs signaling through depleting endogenous PPAR ligands, which can serve as a negative feedback mechanism to prevent excess activation of PPAR signaling under non-obese condition. In obesity, excessive lipid availability negates the feedback mechanism resulting in over activation of PPAR cascade, thus contributes to the development of cardiac lipotoxicity.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2020; 146:1-11
Liu Z, Ding J, McMillen TS, Villet O, Tian R, Shao D
J Mol Cell Cardiol: 30 Aug 2020; 146:1-11 | PMID: 32592696
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Abstract

Hypoxia-induced downregulation of Sema3a and CXCL12/CXCR4 regulate the formation of the coronary artery stem at the proper site.

Narematsu M, Kamimura T, Yamagishi T, Nakajima Y
Background
During the formation of the coronary artery stem, endothelial strands from the endothelial progenitor pool surrounding the conotruncus penetrate into the aortic wall. Vascular endothelial growth factors (VEGFs) as well as CXCL12/CXCR4 signaling are thought to play a role in the formation of the coronary stem. However, the mechanisms regulating how endothelial strands exclusively invade into the aorta remain unknown.
Methods and results
Immunohistochemistry showed that before the formation of endothelial strands, Sema3a was highly expressed in endothelial progenitors surrounding the great arteries. At the onset of/during invasion of endothelial strands into the aorta, Sema3a was downregulated and CXCR4 was upregulated in the endothelial strands. In situ hybridization showed that Cxcl12 was highly expressed in the aortic wall compared with in the pulmonary artery. Using avian embryonic hearts, we established two types of endothelial penetration assay, in which coronary endothelial strands preferentially invaded into the aorta in culture. Sema3a blocking peptide induced an excess number of endothelial strands penetrating into the pulmonary artery, whereas recombinant Sema3a inhibited the formation of endothelial strands. In cultured coronary endothelial progenitors, recombinant VEGF protein induced CXCR4-positive endothelial strands, which were capable of being attracted by CXCL12-impregnated beads. Monoazo rhodamine detected that hypoxia was predominant in aortic/subaortic region in ovo and hypoxic condition downregulated the expression of Sema3a in culture.
Conclusion
Results suggested that hypoxia in the aortic region downregulates the expression of Sema3a, thereby enhancing VEGF activity to induce the formation of CXCR4-positive endothelial strands, which are subsequently attracted into the Cxcl12-positive aortic wall to connect the aortic lumen.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 06 Aug 2020; 147:62-73
Narematsu M, Kamimura T, Yamagishi T, Nakajima Y
J Mol Cell Cardiol: 06 Aug 2020; 147:62-73 | PMID: 32777295
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Abstract

CYLD exaggerates pressure overload-induced cardiomyopathy via suppressing autolysosome efflux in cardiomyocytes.

Qi L, Zang H, Wu W, Nagarkatti P, ... Wang X, Cui T

Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Jul 2020; 145:59-73
Qi L, Zang H, Wu W, Nagarkatti P, ... Wang X, Cui T
J Mol Cell Cardiol: 30 Jul 2020; 145:59-73 | PMID: 32553594
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Abstract

Exogenous IL-4 shuts off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to induce neutrophil phagocytosis following myocardial infarction.

Daseke MJ, Tenkorang-Impraim MAA, Ma Y, Chalise U, ... DeLeon-Pennell KY, Lindsey ML
Introduction
Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While interleukin (IL)-4 is an in vitro anti-inflammatory stimulus, the MI myocardium does not express a considerable amount of IL-4 but does express IL4 receptors. We hypothesized that continuous exogenous IL-4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype.
Methods
C57BL/6J male mice (3-6 months of age) were subcutaneously infused with either saline (n = 17) or IL-4 (20 ng/g/day; n = 17) beginning 24 h after MI and evaluated at MI day 3.
Results
Macrophages and neutrophils were isolated ex vivo from the infarct region and examined. Exogenous IL-4 decreased pro-inflammatory Ccl3, Il12a, Tnfa, and Tgfb1 in neutrophils and increased anti-inflammatory Arg1 and Ym1 in macrophages (all p < .05). Tissue clearance by IL-4 treated neutrophils was not different, while selective phagocytosis of neutrophils doubled in IL-4 treated macrophages (p < .05). Of 24,339 genes examined by RNA-sequencing, 2042 genes were differentially expressed in macrophages from IL-4 stimulated infarct (all FDR p < .05). Pdgfc gene expression was ranked first, increasing 3-fold in macrophages stimulated with IL-4 (p = 1 × 10). Importantly, changes in macrophage physiology and transcriptome occurred in the absence of global LV effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF-CC protein (10 μg/ml) or PDGF-CC blocking antibody (200 ng/ml) did not change Arg1 or Ym1 expression, indicating the in vivo effect of IL-4 to stimulate macrophage anti-inflammatory gene expression was independent of PDGF-CC.
Conclusions
Our results indicate that exogenous IL-4 promotes inflammation resolution by turning off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to mediate removal of apoptotic neutrophils.

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

J Mol Cell Cardiol: 30 Jul 2020; 145:112-121
Daseke MJ, Tenkorang-Impraim MAA, Ma Y, Chalise U, ... DeLeon-Pennell KY, Lindsey ML
J Mol Cell Cardiol: 30 Jul 2020; 145:112-121 | PMID: 32574573
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Abstract

OxLDL-mediated immunologic memory in endothelial cells.

Sohrabi Y, Lagache SMM, Voges VC, Semo D, ... Waltenberger J, Findeisen HM

Trained innate immunity describes the metabolic reprogramming and long-term proinflammatory activation of innate immune cells in response to different pathogen or damage associated molecular patterns, such as oxidized low-density lipoprotein (oxLDL). Here, we have investigated whether the regulatory networks of trained innate immunity also control endothelial cell activation following oxLDL treatment. Human aortic endothelial cells (HAECs) were primed with oxLDL for 24 h. After a resting time of 4 days, cells were restimulated with the TLR2-agonist PAM3cys4. OxLDL priming induced a proinflammatory memory with increased production of inflammatory cytokines such as IL-6, IL-8 and MCP-1 in response to PAM3cys4 restimulation. This memory formation was dependent on TLR2 activation. Furthermore, oxLDL priming of HAECs caused characteristic metabolic and epigenetic reprogramming, including activation of mTOR-HIF1α-signaling with increases in glucose consumption and lactate production, as well as epigenetic modifications in inflammatory gene promoters. Inhibition of mTOR-HIF1α-signaling or histone methyltransferases blocked the observed phenotype. Furthermore, primed HAECs showed epigenetic activation of ICAM-1 and increased ICAM-1 expression in a HIF1α-dependent manner. Accordingly, live cell imaging revealed increased monocyte adhesion and transmigration following oxLDL priming. In summary, we demonstrate that oxLDL-mediated endothelial cell activation represents an immunologic event, which triggers metabolic and epigenetic reprogramming. Molecular mechanisms regulating trained innate immunity in innate immune cells also regulate this sustained proinflammatory phenotype in HAECs with enhanced atheroprone cell functions. Further research is necessary to elucidate the detailed metabolic regulation and the functional relevance for atherosclerosis formation in vivo.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Aug 2020; 146:121-132
Sohrabi Y, Lagache SMM, Voges VC, Semo D, ... Waltenberger J, Findeisen HM
J Mol Cell Cardiol: 30 Aug 2020; 146:121-132 | PMID: 32726647
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Abstract

In vivo knockdown of SK3 channels using antisense oligonucleotides protects against atrial fibrillation in rats.

Saljic A, Soattin L, Trachsel DS, Boddum K, Jespersen T
Introduction
GapmeRs are oligonucleotides that bind to a specific RNA sequence and thereby affecting posttranscriptional gene regulation. They therefore hold the potential to manipulate targets where current pharmacological modulators are inefficient or exhibit adverse side effects. Here, we show that a treatment with a GapmeR, mediating knockdown of small conductance Ca-activated K channels (SK3), has an in vivo protective effect against atrial fibrillation (AF) in rats.
Material and methods
A unique SK3-GapmeR design was selected after thorough in vitro evaluation. 22 rats were randomly assigned to receive either 50 mg/kg SK3-GapmeR or vehicle subcutaneously once a week for two weeks. Langendorff experiments were performed seven days after the last injection, where action potential duration (APD), effective refractory period (ERP) and AF propensity were investigated. SK3 channel activity was evaluated using the SK channel blocker, ICA (N-(pyridin-2-yl)-4-(pyridine-2-yl)thiazol-2-amine). SK3 protein expression was assessed by Western Blot.
Results
The designed GapmeR effectively down-regulate the SK3 protein expression in the heart (48% downregulation, p = 0.0095) and did indeed protect against AF. Duration of AF episodes elicited by burst pacing in the rats treated with SK3-GapmeR was reduced 78% compared to controls (3.7 s vs. 16.8 s, p = 0.0353). The number of spontaneous AF episodes were decreased by 68% in the SK3-GapmeR group (39 episodes versus 123 in the control group, respectively) and were also significantly shorter in duration (7.2 s versus 29.7 s in the control group, p = 0.0327). Refractoriness was not altered at sinus rhythm, but ERP prolongation following ICA application was blunted in the SK3-GapmeR group.
Conclusion
The selected GapmeR silenced the cardiac SK3 channels, thereby preventing AF in rats. Thus, GapmeR technology can be applied as an experimental tool of downregulation of cardiac proteins and could potentially offer a novel modality for treatment of cardiac diseases.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 05 Aug 2020; 147:18-26
Saljic A, Soattin L, Trachsel DS, Boddum K, Jespersen T
J Mol Cell Cardiol: 05 Aug 2020; 147:18-26 | PMID: 32768409
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Impact:
Abstract

Circulating microRNAs differentiate Kawasaki Disease from infectious febrile illnesses in childhood.

Jone PN, Korst A, Karimpour-Fard A, Thomas T, ... Sucharov CC, Miyamoto SD
Background
Kawasaki Disease (KD) is an acute vasculitis of unknown etiology in children that can lead to coronary artery lesions (CAL) in 25% of untreated patients. There is currently no diagnostic test for KD, and the clinical presentation is often difficult to differentiate from other febrile childhood illnesses. Circulating microRNAs (miRNAs) are small noncoding RNA molecules that control gene expression by inducing transcript degradation or by blocking translation. We hypothesize that the expression of circulating miRNAs will differentiate KD from non-KD febrile illnesses in children.
Methods
Circulating miRNA profiles from 84 KD patients and 29 non-KD febrile controls (7 viral and 22 bacterial infections) were evaluated. 3 ul of serum from each subject was submitted to 3 freeze/heat cycles to ensure miRNA release from microvesicles or interaction with serum proteins. miRNAs were reverse transcribed using a pool of primers specific for each miRNA. Real-time PCR reactions were performed in a 384 well plate containing sequence-specific primers and TaqMan probes in the ABI7900. \'.
Results
KD patients (3.6 ± 2.2 yrs., 58% male) were found to have a unique circulating miRNA profile, including upregulation of miRNA-210-3p, -184, and -19a-3p (p < .0001), compared to non-KD febrile controls (8.5 ± 6.1 yrs., 72% male).
Conclusions
Circulating miRNAs can differentiate KD from infectious febrile childhood diseases, supporting their potential as a diagnostic biomarker for KD.

Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Aug 2020; 146:12-18
Jone PN, Korst A, Karimpour-Fard A, Thomas T, ... Sucharov CC, Miyamoto SD
J Mol Cell Cardiol: 30 Aug 2020; 146:12-18 | PMID: 32634388
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Impact:
Abstract

Misoprostol attenuates neonatal cardiomyocyte proliferation through Bnip3, perinuclear calcium signaling, and inhibition of glycolysis.

Martens MD, Field JT, Seshadri N, Day C, ... Ivanco TL, Gordon JW

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

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

J Mol Cell Cardiol: 30 Aug 2020; 146:19-31
Martens MD, Field JT, Seshadri N, Day C, ... Ivanco TL, Gordon JW
J Mol Cell Cardiol: 30 Aug 2020; 146:19-31 | PMID: 32640283
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Impact:
Abstract

Pleiotropic cardiac functions controlled by ischemia-induced lncRNA H19.

Hobuß L, Foinquinos A, Jung M, Kenneweg F, ... Fiedler J, Thum T

Myocardial ischemia induces a multifaceted remodeling process in the heart. Novel therapeutic entry points to counteract maladaptive signalling include the modulation of non-coding RNA molecules such as long non-coding RNA (lncRNA). We here questioned if the lncRNA candidate H19 exhibits regulatory potential in the setting of myocardial infarction. Initial profiling of H19 expression revealed a dynamic expression profile of H19 with upregulation in the acute phase after murine cardiac ischemia. In vitro, we found that oxygen deficiency leads to H19 upregulation in several cardiac cell types. Repression of endogenous H19 caused multiple phenotypes in cultivated murine cardiomyocytes including enhanced cardiomyocyte apoptosis, at least partly through attenuated vitamin D signalling. Unbiased proteome analysis revealed further involvement of H19 in mRNA splicing and translation as well as inflammatory signalling pathways. To study H19 function more precisely, we investigated the phenotype of systemic H19 loss in a genetic mouse model of H19 deletion (H19 KO). Infarcted heart tissue of H19 KO mice showed a massive increase of pro-inflammatory cytokines after ischemia-reperfusion injury (I/R) without significant effects on scar formation or cardiac function but exaggerated cardiac hypertrophy indicating pathological cardiac remodeling. H19-dependent changes in cardiomyocyte-derived extracellular vesicle release and alterations in NF-κB signalling were evident. Cardiac cell fractionation experiments revealed that enhanced H19 expression in the proliferative phase after MI derived mainly from cardiac fibroblasts. Here further research is needed to elucidate its role in fibroblast activation and function. In conclusion, the lncRNA H19 is dynamically regulated after MI and involved in multiple pathways of different cardiac cell types including cardiomyocyte apoptosis and cardiac inflammation.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2020; 146:43-59
Hobuß L, Foinquinos A, Jung M, Kenneweg F, ... Fiedler J, Thum T
J Mol Cell Cardiol: 30 Aug 2020; 146:43-59 | PMID: 32649928
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Impact:
Abstract

Simultaneous quantitative assessment of two distinct cell lineages with a nuclear-localized dual genetic reporter.

Tang M, Liu K, Jin H, Li Y, ... Zhang Z, Zhou B

Genetic lineage tracing has been widely used for studying in vivo cell fate plasticity during embryogenesis, tissue homeostasis, and disease development. Recent applications with multiple site-specific recombinases have been used in complex and sophisticated genetic fate mapping studies. However, the previous multicolor reporters for dual recombinases had limitations of precise in situ quantification of cell number, which is mainly due to the intermingling of cells in condensed tissues. Here, we generated a dual recombinase-mediated nuclear-localized GFP and tdTomato reporter line, which enables clear, simultaneous quantification of two distinct cell lineages in vivo. Combining this dual genetic reporter with Tbx18-Cre and Cdh5-Dre lines, which genetically trace epicardial and endothelial cells, respectively, we obtained high-resolution images for the anatomic distribution of the descendants of these two distinct cell lineages in the valve mesenchyme during development, remodeling, and maturation stages. This new dual genetic reporter is expected to facilitate fate tracing of two cell lineages and their objective quantification in vivo.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2020; 146:60-68
Tang M, Liu K, Jin H, Li Y, ... Zhang Z, Zhou B
J Mol Cell Cardiol: 30 Aug 2020; 146:60-68 | PMID: 32668281
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Impact:
Abstract

Mcl-1-mediated mitochondrial fission protects against stress but impairs cardiac adaptation to exercise.

Moyzis AG, Lally NS, Liang W, Leon LJ, ... Orogo AM, Gustafsson ÅB

Myeloid cell leukemia-1 (Mcl-1) is a structurally and functionally unique anti-apoptotic Bcl-2 protein. While elevated levels of Mcl-1 contribute to tumor cell survival and drug resistance, loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and heart failure development. Although Mcl-1 is an anti-apoptotic protein, previous studies indicate that its functions extend beyond regulating apoptosis. Mcl-1 is localized to both the mitochondrial outer membrane and matrix. Here, we have identified that Mcl-1 in the outer mitochondrial membrane mediates mitochondrial fission, which is independent of its anti-apoptotic function. We demonstrate that Mcl-1 interacts with Drp1 to promote mitochondrial fission in response to various challenges known to perturb mitochondria morphology. Induction of fission by Mcl-1 reduces nutrient deprivation-induced cell death and the protection is independent of its BH3 domain. Finally, cardiac-specific overexpression of Mcl-1, but not Mcl-1, contributes to a shift in the balance towards fission and leads to reduced exercise capacity, suggesting that a pre-existing fragmented mitochondrial network leads to decreased ability to adapt to an acute increase in workload and energy demand. Overall, these findings highlight the importance of Mcl-1 in maintaining mitochondrial health in cells.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2020; 146:109-120
Moyzis AG, Lally NS, Liang W, Leon LJ, ... Orogo AM, Gustafsson ÅB
J Mol Cell Cardiol: 30 Aug 2020; 146:109-120 | PMID: 32717194
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Impact:
Abstract

Systolic overload-induced pulmonary inflammation, fibrosis, oxidative stress and heart failure progression through interleukin-1β.

Shang L, Yue W, Wang D, Weng X, ... Hou M, Chen Y

Chronic heart failure is associated with increased interleukin-1β (IL-1β), leukocyte infiltration, and fibrosis in the heart and lungs. Here we further studied the role of IL-1β in the transition from left heart failure to pulmonary hypertension and right ventricular hypertrophy in mice with existing left heart failure produced by transverse aortic constriction. We demonstrated that transverse aortic constriction-induced heart failure was associated with increased lung inflammation and cleaved IL-1β, and inhibition of IL-1β signaling using blocking antibodies of clone B122 effectively attenuated further decrease of left ventricular systolic function in mice with existing heart failure. We found that inhibition of IL-1β attenuated lung inflammation, inflammasome activation, fibrosis, oxidative stress, and right ventricular hypertrophy. IL-1β blocking antibodies of clone B122 also significantly attenuated lung T cell activation. Together, these data indicate that IL-1β signaling exerts a causal role for heart failure progression, or the transition from left heart failure to lung remodeling and right heart hypertrophy.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2020; 146:84-94
Shang L, Yue W, Wang D, Weng X, ... Hou M, Chen Y
J Mol Cell Cardiol: 30 Aug 2020; 146:84-94 | PMID: 32712269
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Impact:
Abstract

Acute effects of alcohol on cardiac electrophysiology and arrhythmogenesis: Insights from multiscale in silico analyses.

Sutanto H, Cluitmans MJM, Dobrev D, Volders PGA, Bébarová M, Heijman J

Acute excessive ethyl alcohol (ethanol) consumption alters cardiac electrophysiology and can evoke cardiac arrhythmias, e.g., in \'holiday heart syndrome\'. Ethanol acutely modulates numerous targets in cardiomyocytes, including ion channels, Ca-handling proteins and gap junctions. However, the mechanisms underlying ethanol-induced arrhythmogenesis remain incompletely understood and difficult to study experimentally due to the multiple electrophysiological targets involved and their potential interactions with preexisting electrophysiological or structural substrates. Here, we employed cellular- and tissue-level in-silico analyses to characterize the acute effects of ethanol on cardiac electrophysiology and arrhythmogenesis. Acute electrophysiological effects of ethanol were incorporated into human atrial and ventricular cardiomyocyte computer models: reduced I, I, I, I and I, dual effects on I and I (inhibition at low and augmentation at high concentrations), and increased I and SR Ca leak. Multiscale simulations in the absence or presence of preexistent atrial fibrillation or heart-failure-related remodeling demonstrated that low ethanol concentrations prolonged atrial action-potential duration (APD) without effects on ventricular APD. Conversely, high ethanol concentrations abbreviated atrial APD and prolonged ventricular APD. High ethanol concentrations promoted reentry in tissue simulations, but the extent of reentry promotion was dependent on the presence of altered intercellular coupling, and the degree, type, and pattern of fibrosis. Taken together, these data provide novel mechanistic insight into the potential proarrhythmic interactions between a preexisting substrate and acute changes in cardiac electrophysiology. In particular, acute ethanol exposure has concentration-dependent electrophysiological effects that differ between atria and ventricles, and between healthy and diseased hearts. Low concentrations of ethanol can have anti-fibrillatory effects in atria, whereas high concentrations promote the inducibility and maintenance of reentrant atrial and ventricular arrhythmias, supporting a role for limiting alcohol intake as part of cardiac arrhythmia management.

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

J Mol Cell Cardiol: 30 Aug 2020; 146:69-83
Sutanto H, Cluitmans MJM, Dobrev D, Volders PGA, Bébarová M, Heijman J
J Mol Cell Cardiol: 30 Aug 2020; 146:69-83 | PMID: 32710981
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Impact:
Abstract

Cardiomyocyte cell cycling, maturation, and growth by multinucleation in postnatal swine.

Velayutham N, Alfieri CM, Agnew EJ, Riggs KW, ... Zafar F, Yutzey KE
Background
Rodent cardiomyocytes (CM) undergo mitotic arrest and decline of mononucleated-diploid population post-birth, which are implicated in neonatal loss of heart regenerative potential. However, the dynamics of postnatal CM maturation are largely unknown in swine, despite a similar neonatal cardiac regenerative capacity as rodents. Here, we provide a comprehensive analysis of postnatal cardiac maturation in swine, including CM cell cycling, multinucleation and hypertrophic growth, as well as non-CM cardiac factors such as extracellular matrix (ECM), immune cells, capillaries, and neurons. Our study reveals discordance in postnatal pig heart maturational events compared to rodents.
Methods and results
Left-ventricular myocardium from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed. Mature cardiac sarcomeric characteristics, such as fetal TNNI1 repression and Cx43 co-localization to cell junctions, were not evident until P30 in pigs. In CMs, appreciable binucleation is observed by P7, with extensive multinucleation (4-16 nuclei per CM) beyond P15. Individual CM nuclei remain predominantly diploid at all ages. CM mononucleation at ~50% incidence is observed at P7-P15, and CM mitotic activity is measurable up to 2mo. CM cross-sectional area does not increase until 2mo-6mo in pigs, though longitudinal CM growth proportional to multinucleation occurs after P15. RNAseq analysis of neonatal pig left ventricles showed increased expression of ECM maturation, immune signaling, neuronal remodeling, and reactive oxygen species response genes, highlighting significance of the non-CM milieu in postnatal mammalian heart maturation.
Conclusions
CM maturational events such as decline of mononucleation and cell cycle arrest occur over a 2-month postnatal period in pigs, despite reported loss of heart regenerative potential by P3. Moreover, CMs grow primarily by multinucleation and longitudinal hypertrophy in older pig CMs, distinct from mice and humans. These differences are important to consider for preclinical testing of cardiovascular therapies using swine, and may offer opportunities to study aspects of heart regeneration unavailable in other models.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2020; 146:95-108
Velayutham N, Alfieri CM, Agnew EJ, Riggs KW, ... Zafar F, Yutzey KE
J Mol Cell Cardiol: 30 Aug 2020; 146:95-108 | PMID: 32710980
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Impact:
Abstract

BMP9 attenuates occurrence of venous malformation by maintaining endothelial quiescence and strengthening vessel walls via SMAD1/5/ID1/α-SMA pathway.

Li Y, Shang Q, Li P, Yang Z, ... Fan X, Jia R

Venous malformation (VM) is a type of vascular morphogenic defect in humans with an incidence of 1%. Although gene mutation is considered as the most common cause of VM, the pathogenesis of those without gene mutation remains to be elucidated. Here, we aimed to explore the relation of bone morphogenetic protein 9 (BMP9) and development of VM. At first, we found serum and tissue BMP9 expression in VM patients was significantly lower than that in healthy subjects, detected via enzyme-linked immunosorbent assay. Next, with wound healing assay, transwell assay and tube formation assay, we discovered BMP9 could inhibit migration and enhance tube formation activity of human umbilical vein endothelial cells (HUVECs) via receptor activin receptor-like kinase 1 (ALK1). Besides, BMP9 improved the expression of structural proteins alpha-smooth muscle actin (α-SMA) and Desmin in human umbilical vein smooth muscle cells (HUVSMCs) via activation of the SMAD1/5-ID1 pathway, determined by RNA-based next-generation sequencing, qPCR, immunofluorescence and western blotting. Intriguingly, this effect could be blocked by receptor ALK1 inhibitor, SMAD1/5 inhibitor and siRNAs targeting ID1, verifying the BMP9/ALK1/SMAD1/5/ID1/α-SMA pathway. Meanwhile, knocking out BMP9 in C57BL/6 mice embryo led to α-SMA scarcity in walls of lung and mesenteric vessels, as well as walls of small trachea. BMP9-/- zebrafish also exhibited abnormal vascular maturity, indicating a critical role of BMP9 in vascular maturity and remodeling. Finally, a VM mice model revealed that BMP9 might have therapeutic effect in VM progression. Our study discovered that BMP9 might inhibit the occurrence of VM by strengthening the vessel wall and maintaining endothelium quiescence. These findings provide promising evidences of new therapeutic targets that might be used for the management of VM.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 27 Jul 2020; 147:92-107
Li Y, Shang Q, Li P, Yang Z, ... Fan X, Jia R
J Mol Cell Cardiol: 27 Jul 2020; 147:92-107 | PMID: 32730768
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Impact:
Abstract

Sex differences underlying preexisting cardiovascular disease and cardiovascular injury in COVID-19.

Medzikovic L, Cunningham CM, Li M, Amjedi M, ... Ruffenach G, Eghbali M

The novel 2019 coronavirus disease (COVID-19), resulting from severe acute respiratory syndrome coronarvirus-2 (SARS-CoV-2) infection, typically leads to respiratory failure in severe cases; however, cardiovascular injury is reported to contribute to a substantial proportion of COVID-19 deaths. Preexisting cardiovascular disease (CVD) is among the most common risk factors for hospitalization and death in COVID-19 patients, and the pathogenic mechanisms of COVID-19 disease progression itself may promote the development of cardiovascular injury, increasing risk of in-hospital death. Sex differences in COVID-19 are becoming more apparent as mounting data indicate that males seem to be disproportionately at risk of severe COVID-19 outcome due to preexisting CVD and COVID-19-related cardiovascular injury. In this review, we will provide a basic science perspective on current clinical observations in this rapidly evolving field and discuss the interplay sex differences, preexisting CVD and COVID-19-related cardiac injury.

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

J Mol Cell Cardiol: 21 Aug 2020; 148:25-33
Medzikovic L, Cunningham CM, Li M, Amjedi M, ... Ruffenach G, Eghbali M
J Mol Cell Cardiol: 21 Aug 2020; 148:25-33 | PMID: 32835666
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Impact:
Abstract

Regulatory T cells in ischemic cardiovascular injury and repair.

Zhuang R, Feinberg MW

Ischemic injury triggers a heightened inflammatory response that is essential for tissue repair, but excessive and chronic inflammatory responses contribute to the pathogenesis of ischemic cardiovascular disease. Regulatory T cells (Tregs), a major regulator of self-tolerance and immune suppression, control innate and adaptive immune responses, modulate specific immune cell subsets, prevent excessive inflammation, and participate in tissue repair after ischemia. Herein, we summarize the multiple potential mechanisms by which Tregs exert suppressor functions including modulation of cytokine production, alteration of cell-cell interactions, and disruption of metabolic pathways. Furthermore, we review the role of Tregs implicated in ischemic injury and repair including myocardial, limb, and cerebral ischemia. We conclude with a perspective on the therapeutic opportunities and future challenges of Treg biology in understanding the pathogenesis of ischemic cardiovascular disease states.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 06 Aug 2020; 147:1-11
Zhuang R, Feinberg MW
J Mol Cell Cardiol: 06 Aug 2020; 147:1-11 | PMID: 32777294
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Impact:
Abstract

Apoptosis repressor with caspase recruitment domain promotes cell proliferation and phenotypic modulation through 14-3-3ε/YAP signaling in vascular smooth muscle cells.

Liu M, Yu T, Li M, Fang X, ... Liu G, Wang J
Aims
In response to vascular injury, vascular smooth muscle cells (VSMC) may change from a contractile phenotype to a proliferative phenotype and consequently become conducive to neointima formation. Apoptosis repressor with caspase recruitment domain (ARC) was initially discovered as an endogenous apoptosis inhibitor, but whether ARC plays a role in VSMCs and whether it can participate in the regulation of atherosclerosis are unknown.
Methods and results
Protein and mRNA levels of ARC in tissues and cells were detected by western blot and quantitative real-time PCR. Immunofluorescence staining was used to detect the protein location, and immunohistochemistry was used to detect protein expression in tissues. VSMC proliferation was analysed using Cell Counting Kit-8 (CCK-8) and EdU assays, while migration was assessed by Transwell assay. Mechanistically, the direct binding between two proteins was verified by immunoprecipitation. We found that ARC expression was stimulated in VSMCs during cell proliferation. Our results also showed that ARC promoted cell proliferation and induced phenotypic modulation of VSMCs in vitro and vivo. Mechanistic studies demonstrated that ARC increased the nuclear localization of Yes associated protein (YAP) by binding to 14-3-3ε and that ARC played a role in promoting cell proliferation and phenotypic modulation. Additionally, the transcription factor p53 negatively regulated ARC expression at the transcriptional level during cell proliferation and phenotypic modulation.
Conclusions
Our findings define a novel role for ARC in the phenotypic transition of proliferating VSMCs, which may provide a new strategy for regulating neointimal formation.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 05 Aug 2020; 147:35-48
Liu M, Yu T, Li M, Fang X, ... Liu G, Wang J
J Mol Cell Cardiol: 05 Aug 2020; 147:35-48 | PMID: 32771410
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Impact:
Abstract

Sarcomere integrated biosensor detects myofilament-activating ligands in real time during twitch contractions in live cardiac muscle.

Vetter AD, Martin AA, Thompson BR, Thomas DD, Metzger JM

The sarcomere is the functional unit of cardiac muscle, essential for normal heart function. To date, it has not been possible to study, in real time, thin filament-based activation dynamics in live cardiac muscle. We report here results from a cardiac troponin C (TnC) FRET-based biosensor integrated into the cardiac sarcomere via stoichiometric replacement of endogenous TnC. The TnC biosensor provides, for the first time, evidence of multiple thin filament activating ligands, including troponin I interfacing with TnC and cycling myosin, during a cardiac twitch. Results show that the TnC FRET biosensor transient significantly precedes that of peak twitch force. Using small molecules and genetic modifiers known to alter sarcomere activation, independently of the intracellular Ca transient, the data show that the TnC biosensor detects significant effects of the troponin I switch domain as a sarcomere-activating ligand. Interestingly, the TnC biosensor also detected the effects of load-dependent altered myosin cycling, as shown by a significant delay in TnC biosensor transient inactivation during the isometric twitch. In addition, the TnC biosensor detected the effects of myosin as an activating ligand during the twitch by using a small molecule that directly alters cross-bridge cycling, independently of the intracellular Ca transient. Collectively, these results aid in illuminating the basis of cardiac muscle contractile activation with implications for gene, protein, and small molecule-based strategies designed to target the sarcomere in regulating beat-to-beat heart performance in health and disease.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 10 Aug 2020; 147:49-61
Vetter AD, Martin AA, Thompson BR, Thomas DD, Metzger JM
J Mol Cell Cardiol: 10 Aug 2020; 147:49-61 | PMID: 32791214
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Impact:
Abstract

Reading the heart at single-cell resolution.

Zhou B, Wang L

The burgeoning field of single-cell transcriptomics augments our ability to scrutinize organ systems at unprecedented resolutions. Single-cell RNA sequencing (scRNA-seq) and analytical techniques have shed light on the cellular heterogeneity, developmental trajectories, intercellular communications of the cardiac system, and thus contributed much to the understanding of cardiac development, homeostasis and disorders. Although generalized protocols are well established for scRNA-seq pipelines, customized sample preparation, quality control, and data interpretation are still needed in cardiac research. In this article, we highlight major steps that impact data quality in scRNA-seq experiments, with particular focus on sample and data processing of cardiomyocytes. We also summarize popular applications of scRNA-seq, outlining general tools, caveats and examples in cardiac research.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 28 Aug 2020; 148:34-45
Zhou B, Wang L
J Mol Cell Cardiol: 28 Aug 2020; 148:34-45 | PMID: 32871159
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Impact:
Abstract

Contribution of estrogen to the pregnancy-induced increase in cardiac automaticity.

Long V, Fiset C
Background
The heart rate progressively increases throughout pregnancy, reaching a maximum in the third trimester. This elevated heart rate is also present in pregnant mice and is associated with accelerated automaticity, higher density of the pacemaker current I and changes in Ca homeostasis in sinoatrial node (SAN) cells. Strong evidence has also been provided showing that 17β-estradiol (E) and estrogen receptor α (ERα) regulate heart rate. Accordingly, we sought to determine whether E levels found in late pregnancy cause the increased cardiac automaticity associated with pregnancy.
Methods and results
Voltage- and current-clamp experiments were carried out on SAN cells isolated from female mice lacking estrogen receptor alpha (ERKOα) or beta (ERKOβ) receiving chronic E treatment mimicking late pregnancy concentrations. E treatment significantly increased the action potential rate (284 ± 24 bpm, +E 354 ± 23 bpm, p = 0.040) and the density of I (+52%) in SAN cells from ERKOβ mice. However, I density remains unchanged in SAN cells from E-treated ERKOα mice. Additionally, E also increased I density (+67%) in nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), recapitulating in a human SAN cell model the effect produced in mice. However, the L-type calcium current (I) and Ca transients, examined using N-hiPSC-CM and SAN cells respectively, were not affected by E, indicating that other mechanisms contribute to changes observed in these parameters during pregnancy.
Conclusion
The accelerated SAN automaticity observed in E-treated ERKOβ mice is explained by an increased I density mediated by ERα, demonstrating that E plays a major role in regulating SAN function during pregnancy.

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

J Mol Cell Cardiol: 11 Aug 2020; 147:27-34
Long V, Fiset C
J Mol Cell Cardiol: 11 Aug 2020; 147:27-34 | PMID: 32798536
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Impact:
Abstract

Longitudinal correlation of biomarkers of cardiac injury, inflammation, and coagulation to outcome in hospitalized COVID-19 patients.

Li C, Jiang J, Wang F, Zhou N, ... Ammirati E, Wang DW
Background
Cardiac injury, as measured by troponin elevation, has been reported among hospitalized coronavirus disease 2019 (COVID-19) patients and portends a poor prognosis. However, how the dynamics of troponin elevation interplay with inflammation and coagulation biomarkers over time is unknown. We assessed longitudinal follow-up of cardiac injury, inflammation and coagulation markers in relation to disease severity and outcome.
Methods
We retrospectively assessed 2068 patients with laboratory-confirmed COVID-19 between January 29 and April 1, 2020 at Tongji Hospital in Wuhan, China. We defined cardiac injury as an increase in high sensitivity cardiac troponin-I (hs-cTnI) above the 99th of the upper reference limit. We explored the dynamics of elevation in hs-cTnI and the relationship with inflammation (interleukin [IL]-6, IL-8, IL-10, IL-2 receptor, tumor necrosis factor-α, C-reactive protein) and coagulation (d-dimer, fibrinogen, international normalized ratio) markers in non-critically ill versus critically ill patients longitudinally and further correlated these markers to survivors and non-survivors.
Results
Median age was 63 years (first to third quartile 51-70 years), 51.4% of whom were women. When compared to non-critically ill patients (N = 1592, 77.0%), critically ill (defined as requiring mechanical ventilation, in shock or multiorgan failure) patients (N = 476, 23.0%), had more frequent cardiac injury on admission (30.3% vs. 2.3%, p < 0.001), with increased mortality during hospitalization (38.4% vs. 0%, p < 0.001). Among critically ill patients, non-survivors (N = 183) had a continuous increase in hs-cTnI levels during hospitalization, while survivors (N = 293) showed a decrease in hs-cTnI level between day 4 and 7 after admission. Specifically, cardiac injury is an independent marker of mortality among critically ill patients at admission, day 4-7 and 8-14. Consistent positive correlations between hs-cTnI and interleukin (IL)-6 on admission (r = 0.59), day 4-7 (r = 0.66) and day 8-14 (r = 0.61; all p < 0.001) and d-dimer (at the same timepoints r = 0.54; 0.65; 0.61, all p < 0.001) were observed. A similar behavior was observed between hs-cTnI and most of other biomarkers of inflammation and coagulation.
Conclusions
Cardiac injury commonly occurs in critically ill COVID-19 patients, with increased levels of hs-cTnI beyond day 3 since admission portending a poor prognosis. A consistent positive correlation of hs-cTnI with IL-6 and d-dimer at several timepoints along hospitalization could suggest nonspecific cytokine-mediated cardiotoxicity.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 19 Aug 2020; 147:74-87
Li C, Jiang J, Wang F, Zhou N, ... Ammirati E, Wang DW
J Mol Cell Cardiol: 19 Aug 2020; 147:74-87 | PMID: 32827510
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Impact:
Abstract

Exercise training reveals micro-RNAs associated with improved cardiac function and electrophysiology in rats with heart failure after myocardial infarction.

Stølen TO, Høydal MA, Ahmed MS, Jørgensen K, ... Ellingsen Ø, Wisløff U
Aims
Endurance training improves aerobic fitness and cardiac function in individuals with heart failure. However, the underlying mechanisms are not well characterized. Exercise training could therefore act as a tool to discover novel targets for heart failure treatment. We aimed to associate changes in Ca handling and electrophysiology with micro-RNA (miRNA) profile in exercise trained heart failure rats to establish which miRNAs induce heart failure-like effects in Ca handling and electrophysiology.
Methods and results
Post-myocardial infarction (MI) heart failure was induced in Sprague Dawley rats. Rats with MI were randomized to sedentary control (sed), moderate (mod)- or high-intensity (high) endurance training for 8 weeks. Exercise training improved cardiac function, Ca handling and electrophysiology including reduced susceptibility to arrhythmia in an exercise intensity-dependent manner where high intensity gave a larger effect. Fifty-five miRNAs were significantly regulated (up or down) in MI-sed, of which 18 and 3 were changed towards Sham-sed in MI-high and MI-mod, respectively. Thereafter we experimentally altered expression of these \"exercise-miRNAs\" individually in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) in the same direction as they were changed in MI. Of the \"exercise-miRNAs\", miR-214-3p prolonged AP duration, whereas miR-140 and miR-208a shortened AP duration. miR-497-5p prolonged Ca release whereas miR-214-3p and miR-31a-5p prolonged Ca decay.
Conclusion
Using exercise training as a tool, we discovered that miR-214-3p, miR-497-5p, miR-31a-5p contribute to heart-failure like behaviour in Ca handling and electrophysiology and could be potential treatment targets.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 09 Sep 2020; 148:106-119
Stølen TO, Høydal MA, Ahmed MS, Jørgensen K, ... Ellingsen Ø, Wisløff U
J Mol Cell Cardiol: 09 Sep 2020; 148:106-119 | PMID: 32918915
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Impact:
Abstract

Eicosapentaenoic acid ameliorates pulmonary hypertension via inhibition of tyrosine kinase Fyn.

Kurahara LH, Hiraishi K, Yamamura A, Zhang Y, ... Inoue R, Hirano K

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity. This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs). Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-β2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases\' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH\'s pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH.

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

J Mol Cell Cardiol: 01 Sep 2020; 148:50-62
Kurahara LH, Hiraishi K, Yamamura A, Zhang Y, ... Inoue R, Hirano K
J Mol Cell Cardiol: 01 Sep 2020; 148:50-62 | PMID: 32889002
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Abstract

Phosphoproteomic analysis identifies phospho-Threonine-17 site of phospholamban important in low molecular weight isoform of fibroblast growth factor 2-induced protection against post-ischemic cardiac dysfunction.

Manning JR, Wijeratne AB, Oloizia BB, Zhang Y, Greis KD, Schultz JEJ
Rationale
Among its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK.
Objective
Yet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated.
Methods and results
To identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05).
Conclusion
The findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 24 Aug 2020; 148:1-14
Manning JR, Wijeratne AB, Oloizia BB, Zhang Y, Greis KD, Schultz JEJ
J Mol Cell Cardiol: 24 Aug 2020; 148:1-14 | PMID: 32853649
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Abstract

BRD4 inhibition by JQ1 prevents high-fat diet-induced diabetic cardiomyopathy by activating PINK1/Parkin-mediated mitophagy in vivo.

Mu J, Zhang D, Tian Y, Xie Z, Zou MH

BRD4 is a member of the BET family of epigenetic regulators. Inhibition of BRD4 by the selective bromodomain inhibitor JQ1, alleviates thoracic aortic constriction-induced cardiac hypertrophy and heart failure. However, whether BRD4 inhibition by JQ1 has therapeutic effect on diabetic cardiomyopathy, a major cause of heart failure in patients with Type 2 diabetes, remains unknown. Here, we discover a novel link between BRD4 and PINK1/Parkin-mediated mitophagy during diabetic cardiomyopathy. Upregulation of BRD4 in diabetic mouse hearts inhibits PINK1/Parkin-mediated mitophagy, resulting in accumulation of damaged mitochondria and subsequent impairment of cardiac structure and function. BRD4 inhibition by JQ1 improves mitochondrial function, and repairs the cardiac structure and function of the diabetic heart. These effects depended on rewiring of the BRD4-driven transcription and repression of PINK1. Deletion of Pink1 suppresses mitophagy, exacerbates cardiomyopathy, and abrogates the therapeutic effect of JQ1 on diabetic cardiomyopathy. Our results illustrate a valid therapeutic strategy for treating diabetic cardiomyopathy by inhibition of BRD4.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 14 Sep 2020; 149:1-14
Mu J, Zhang D, Tian Y, Xie Z, Zou MH
J Mol Cell Cardiol: 14 Sep 2020; 149:1-14 | PMID: 32941882
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Impact:
Abstract

Gaining insight into cellular cardiac physiology using single particle tracking.

Li Y, Yi J, Liu W, Liu Y, Liu J

Single particle tracking (SPT) is a robust technique to monitor single-molecule behaviors in living cells directly. By this approach, we can uncover the potential biological significance of particle dynamics by statistically characterizing individual molecular behaviors. SPT provides valuable information at the single-molecule level, that could be obscured by simple averaging that is inherent to conventional ensemble measurements. Here, we give a brief introduction to SPT including the commonly used optical implementations, fluorescence labeling strategies, and data analysis methods. We then focus on how SPT has been harnessed to decipher myocardial function. In this context, SPT has provided novel insight into the lateral diffusion of signal receptors and ion channels, the dynamic organization of cardiac nanodomains, subunit composition and stoichiometry of cardiac ion channels, myosin movement along actin filaments, the kinetic features of transcription factors involved in cardiac remodeling, and the intercellular communication by nanotubes. Finally, we speculate on the prospects and challenges of applying SPT to future questions regarding cellular cardiac physiology using SPT.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 28 Aug 2020; 148:63-77
Li Y, Yi J, Liu W, Liu Y, Liu J
J Mol Cell Cardiol: 28 Aug 2020; 148:63-77 | PMID: 32871158
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Abstract

The CnB1 p.D102A variant is linked to dilated cardiomyopathy via impaired Calcineurin activity.

Jinqiang Zhuang MD, Ruijun Yuan MD, Yizeng MD, Congliang Miao MD, ... Hui Xu MD, Hong J
Background
The role of calcineurin (protein phosphatase 2B (PP2B)) in the pathogenesis of human dilated cardiomyopathy (DCM) has not been fully elucidated. We determined the potential involvement of calcineurin in the pathogenesis of DCM caused by mutations in CnB1, a subunit of calcineurin.
Methods
By whole-exome sequencing, we identified a new CnB1 variant in a Han Chinese proband with cardiomyopathy from a 3-generation family with 2 normal individuals and 3 individuals with familial dilated cardiomyopathy. The potential pathogenic variant was validated by Sanger sequencing. We performed functional and mechanistic experiments in a CnB1-knockin (KI) mouse model and at the cellular level.
Results
We detected a rare heterozygous CnB1 variant (p.D102A) in a proband with dilated cardiomyopathy. This variant was localized to the EF hand 3 region of CnB1, where no variants have been previously reported. KI mice harboring the p.D102A variant exhibited decreased cardiac function and cardiac dilatation. Immunoblotting, RT-PCR and immunofluorescence results showed decreased cardiomyocyte size and heart failure-related protein expression. A calcineurin activity assay demonstrated decreased calcineurin activity in the KI mice, accompanied by the decreased ability of CnB1 to bind CnA.
Conclusions
CnB1 p.D102A is a disease-associated variant that confers susceptibility to cardiac dilatation. This variant is associated with impaired calcineurin activity and a subsequent decrease in the ability of CnB1 to bind CnA.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Aug 2020; 148:15-24
Jinqiang Zhuang MD, Ruijun Yuan MD, Yizeng MD, Congliang Miao MD, ... Hui Xu MD, Hong J
J Mol Cell Cardiol: 30 Aug 2020; 148:15-24 | PMID: 32882262
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Impact:
Abstract

Beclin1 Haploinsufficiency accentuates second-hand smoke exposure -induced myocardial Remodeling and contractile dysfunction through a STING-mediated mechanism.

Liu F, Liu Y, Zhuang Z, Ma J, ... Pei Z, Ren J

Second-hand smoking evokes inflammation and cardiovascular diseases. Recent evidence has revealed a pivotal role for deranged autophagy in smoke exposure-induced cardiac anomalies. This study evaluated the impact of haploinsufficiency of the mTOR-independent autophagy protein Beclin1 on side-stream smoke exposure-induced cardiac anomalies and mechanism(s) involved. Adult WT and Beclin1 haploinsufficiency (Becn) mice were exposed to cigarette smoke for 1 h daily for 90 days. Echocardiographic, cardiomyocyte function, intracellular Ca, autophagy, mitophagy, apoptosis and inflammation were examined. DHE staining was employed to evaluate O level. Our data revealed that Beclin1 deficiency exacerbated smoke exposure-induced myocardial anomalies in geometry, fractional shortening, cardiomyocyte function, intracellular Ca handling, TEM ultrastructure, and inflammation along with pronounced apoptosis and O production. Side-stream smoke provoked excessive autophagy/mitophagy, mtDNA release, and activation of innate immune response signals cyclic GMP-AMP synthase (cGAS) and its effector - stimulator of interferon genes (STING), the effect was abolished or unaffected by Becn haploinsufficiency. STING phosphorylation was overtly promoted by smoke exposure in Becn mice. Smoke exposure also suppressed phosphorylation of mTOR although it facilitated that of ULK1 in both groups. In vitro data revealed that inhibition of cGAS or STING failed to affect smoke extract-induced mitophagy although they abrogated smoke extract-induced cardiomyocyte dysfunction except cGAS inhibition in Becn mice. These data suggest that Beclin1 is integral in the maintenance of cardiac homeostasis under side-stream smoke exposure via a STING-mediated mechanism.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 02 Sep 2020; 148:78-88
Liu F, Liu Y, Zhuang Z, Ma J, ... Pei Z, Ren J
J Mol Cell Cardiol: 02 Sep 2020; 148:78-88 | PMID: 32891637
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Abstract

MicroRNAs targeting the SARS-CoV-2 entry receptor ACE2 in cardiomyocytes.

Lu D, Chatterjee S, Xiao K, Riedel I, ... Bär C, Thum T

The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) as a public health emergency of international concern as more than 15 million cases were reported by 24th July 2020. Angiotensin-converting enzyme 2 (ACE2) is a COVID-19 entry receptor regulating host cell infection. A recent study reported that ACE2 is expressed in cardiomyocytes. In this study, we aimed to explore if there are microRNA (miRNA) molecules which target ACE2 and which may be exploited to regulate the SARS-CoV-2 receptor. Our data reveal that both Ace2 mRNA and Ace2 protein levels are inhibited by miR-200c in rat primary cardiomyocytes and importantly, in human iPSC-derived cardiomyocytes. We report the first miRNA candidate that can target ACE2 in cardiomyocytes and thus may be exploited as a preventive strategy to treat cardiovascular complications of COVID-19.

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

J Mol Cell Cardiol: 02 Sep 2020; 148:46-49
Lu D, Chatterjee S, Xiao K, Riedel I, ... Bär C, Thum T
J Mol Cell Cardiol: 02 Sep 2020; 148:46-49 | PMID: 32891636
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Abstract

Under construction: The dynamic assembly, maintenance, and degradation of the cardiac sarcomere.

Martin TG, Kirk JA

The sarcomere is the basic contractile unit of striated muscle and is a highly ordered protein complex with the actin and myosin filaments at its core. Assembling the sarcomere constituents into this organized structure in development, and with muscle growth as new sarcomeres are built, is a complex process coordinated by numerous factors. Once assembled, the sarcomere requires constant maintenance as its continuous contraction is accompanied by elevated mechanical, thermal, and oxidative stress, which predispose proteins to misfolding and toxic aggregation. To prevent protein misfolding and maintain sarcomere integrity, the sarcomere is monitored by an assortment of protein quality control (PQC) mechanisms. The need for effective PQC is heightened in cardiomyocytes which are terminally differentiated and must survive for many years while preserving optimal mechanical output. To prevent toxic protein aggregation, molecular chaperones stabilize denatured sarcomere proteins and promote their refolding. However, when old and misfolded proteins cannot be salvaged by chaperones, they must be recycled via degradation pathways: the calpain and ubiquitin-proteasome systems, which operate under basal conditions, and the stress-responsive autophagy-lysosome pathway. Mutations to and deficiency of the molecular chaperones and associated factors charged with sarcomere maintenance commonly lead to sarcomere structural disarray and the progression of heart disease, highlighting the necessity of effective sarcomere PQC for maintaining cardiac function. This review focuses on the dynamic regulation of assembly and turnover at the sarcomere with an emphasis on the chaperones involved in these processes and describes the alterations to chaperones - through mutations and deficient expression - implicated in disease progression to heart failure.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 09 Sep 2020; 148:89-102
Martin TG, Kirk JA
J Mol Cell Cardiol: 09 Sep 2020; 148:89-102 | PMID: 32920010
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Abstract

Increased mast cell density is associated with decreased fibrosis in human atrial tissue.

Legere SA, Haidl ID, Castonguay MC, Brunt KR, ... Marshall JS,

Fibrotic remodelling of the atria is poorly understood and can be regulated by myocardial immune cell populations after injury. Mast cells are resident immune sentinel cells present in the heart that respond to tissue damage and have been linked to fibrosis in other settings. The role of cardiac mast cells in fibrotic remodelling in response to human myocardial injury is controversial. In this study, we sought to determine the association between mast cells, atrial fibrosis, and outcomes in a heterogeneous population of cardiac surgical patients, including a substantial proportion of coronary artery bypass grafting patients. Atrial appendage from patients was assessed for collagen and mast cell density by histology and by droplet digital polymerase chain reaction (ddPCR) for mast cell associated transcripts. Clinical variables and outcomes were also followed. Mast cells were detected in human atrial tissue at varying densities. Histological and ddPCR assessment of mast cells in atrial tissue were closely correlated. Patients with high mast cell density had less fibrosis and lower severity of heart failure classification or incidence mortality than patients with low mast cell content. Analysis of a homogeneous population of coronary artery bypass graft patients yielded similar observations. Therefore, evidence from this study suggests that increased atrial mast cell populations are associated with decreased clinical cardiac fibrotic remodelling and improved outcomes, in cardiac surgery patients.

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

J Mol Cell Cardiol: 11 Sep 2020; 149:15-26
Legere SA, Haidl ID, Castonguay MC, Brunt KR, ... Marshall JS,
J Mol Cell Cardiol: 11 Sep 2020; 149:15-26 | PMID: 32931784
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Abstract

Nestin represents a potential marker of pulmonary vascular remodeling in pulmonary arterial hypertension associated with congenital heart disease.

Zhou JJ, Li H, Qian YL, Quan RL, ... Jing XL, He JG
Objective
Reportedly, nestin was re-expressed in proliferative synthetic-type pulmonary artery smooth muscle cells (PASMCs) and obligatory for PASMC proliferation in pulmonary arterial hypertension (PAH). Accordingly, nestin is increased in pulmonary vascular lesions of congenital heart disease (CHD)-associated PAH patients. We tested the hypothesis whether nestin was re-expressed in proliferative synthetic-type PASMCs and associated with pulmonary vascular remodeling in CHD-PAH.
Materials and methods
Nestin expression was tested using lung tissues from CHD-PAH patients and monocrotaline (MCT) plus aortocaval (AV) shunt-induced PAH rats, human PASMCs (HPASMCs), and pulmonary artery endothelial cells (PAECs) and PASMCs from MCT-AV-induced PAH rats. The role and possible mechanism of nestin on HPASMC proliferation, apoptosis, cell cycle and migration were investigated by assays of CCK-8, EdU, TUNEL, flow cytometry, transwell chamber and immunoblotting assays.
Results
Nestin was solely expressed in proliferative synthetic-type PASMCs, but rarely detected in PAECs. Nestin was barely detected in normal pulmonary arterioles and occlusive pulmonary vascular lesions. Its expression was robustly increased in developing pulmonary vasculature, but returned to normal levels at the late stage of pulmonary vascular remodeling in lung tissues from CHD-PAH patients and MCT-AV-induced PAH rats. Besides, nestin peaks were consistent with the histological features in lung tissues of MCT-AV-induced PAH rats. Moreover, nestin overexpression effectively promoted HPASMC phenotypic transformation, proliferation, apoptosis resistance and migration via enhancing Wnt/β-catenin activation.
Conclusions
These data indicated that nestin was re-expressed in proliferative synthetic-type PASMCs and might represent a potential marker of pulmonary vascular remodeling in CHD-PAH.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 17 Sep 2020; 149:41-53
Zhou JJ, Li H, Qian YL, Quan RL, ... Jing XL, He JG
J Mol Cell Cardiol: 17 Sep 2020; 149:41-53 | PMID: 32950539
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Impact:
Abstract

Inhibition of Pyk2 and Src activity improves Cx43 gap junction intercellular communication.

Zheng L, Trease AJ, Katsurada K, Spagnol G, ... Patel KP, Sorgen PL

Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLa cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLa cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 17 Sep 2020; 149:27-40
Zheng L, Trease AJ, Katsurada K, Spagnol G, ... Patel KP, Sorgen PL
J Mol Cell Cardiol: 17 Sep 2020; 149:27-40 | PMID: 32956670
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Impact:
Abstract

Circadian influence on the microbiome improves heart failure outcomes.

Mistry P, Reitz CJ, Khatua TN, Rasouli M, ... Allen-Vercoe E, Martino TA

Myocardial infarction (MI) leading to heart failure (HF) is a major cause of death worldwide. Previous studies revealed that the circadian system markedly impacts cardiac repair post-MI, and that light is an important environmental factor modulating the circadian influence over healing. Recent studies suggest that gut physiology also affects the circadian system, but how it contributes to cardiac repair post-MI and in HF is not well understood. To address this question, we first used a murine coronary artery ligation MI model to reveal that an intact gut microbiome is important for cardiac repair. Specifically, gut microbiome disruption impairs normal inflammatory responses in infarcted myocardium, elevates adverse cardiac gene biomarkers, and leads to worse HF outcomes. Conversely, reconstituting the microbiome post-MI in mice with prior gut microbiome disruption improves healing, consistent with the notion that normal gut physiology contributes to cardiac repair. To investigate a role for the circadian system, we initially utilized circadian mutant Clock mice, revealing that a functional circadian mechanism is necessary for gut microbiome benefits on post-MI cardiac repair and HF. Finally, we demonstrate that circadian-mediated gut responses that benefit cardiac repair can be conferred by time-restricted feeding, as wake time feeding of MI mice improves HF outcomes, but these benefits are not observed in MI mice fed during their sleep time. In summary, gut physiology is important for cardiac repair, and the circadian system influences the beneficial gut responses to improve post-MI and HF outcomes.

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

J Mol Cell Cardiol: 18 Sep 2020; 149:54-72
Mistry P, Reitz CJ, Khatua TN, Rasouli M, ... Allen-Vercoe E, Martino TA
J Mol Cell Cardiol: 18 Sep 2020; 149:54-72 | PMID: 32961201
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Impact:
Abstract

CaMKII exacerbates heart failure progression by activating class I HDACs.

Zhang M, Yang X, Zimmerman RJ, Wang Q, ... Jiang H, Feng N
Background
Persistent cardiac Ca/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling. Class I HDACs, including HDAC1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. While it is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, the role of CaMKII in regulating class I HDACs during heart failure progression is unclear.
Methods and results
CaMKII increases the deacetylase activity of recombinant HDAC1, HDAC2 and HDAC3 via in vitro phosphorylation assays. Phosphorylation sites on HDAC1 and HDAC3 are identified with mass spectrometry. HDAC1 activity is also increased in cardiac-specific CaMKIIδ transgenic mice (CaMKIIδ-tg). Beyond post-translational modifications, CaMKII induces HDAC1 and HDAC3 expression. HDAC1 and HDAC3 expression are significantly increased in CaMKIIδ-tg mice. Inhibition of CaMKII by overexpression of the inhibitory peptide AC3-I in the heart attenuates the upregulation of HDAC1 after myocardial infarction surgery. Importantly, a potent HDAC1 inhibitor Quisinostat improves downregulated autophagy genes and cardiac dysfunction in CaMKIIδ-tg mice. In addition to Quisinostat, selective class I HDACs inhibitors, Apicidin and Entinostat, HDAC3 specific inhibitor RGFP966, as well as HDAC1 and HDAC3 siRNA prevent CaMKII overexpression induced cardiac myocyte hypertrophy.
Conclusion
CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression. Selective class I HDACs inhibition may be a novel therapeutic avenue to alleviate CaMKII hyperactivity induced cardiac dysfunction.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 21 Sep 2020; 149:73-81
Zhang M, Yang X, Zimmerman RJ, Wang Q, ... Jiang H, Feng N
J Mol Cell Cardiol: 21 Sep 2020; 149:73-81 | PMID: 32971072
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Impact:
Abstract

FBXW5 acts as a negative regulator of pathological cardiac hypertrophy by decreasing the TAK1 signaling to pro-hypertrophic members of the MAPK signaling pathway.

Hui X, Hu F, Liu J, Li C, ... Wang F, Li S

Pathological cardiac hypertrophy is a crucial cause of cardiac morbidity and mortality worldwide. However, the molecular mechanisms of this disease remain incompletely understood. As a member of E3 ubiquitin ligases, F-box/WD repeat-containing protein 5 (FBXW5) has been implicated in various pathophysiological processes. However, the role of FBXW5 in pathological cardiac hypertrophy remains largely unknown. In this study, decreased expression of FBXW5 was observed in both neonatal rat cardiomyocytes and mouse hearts with hypertrophic remodeling. Gain- and loss-of-function experiments were performed to study the potential function of FBXW5 in pathological cardiac hypertrophy. The in vitro results showed that FBXW5 had a protective effect against cardiac hypertrophy induced by phenylephrine (PE). FBXW5 knockout mice and mice with AAV9-mediated FBXW5 overexpression were generated. Consistent with the in vitro results, FBXW5 deficiency aggravated cardiac hypertrophy induced by pressure overload. FBXW5 overexpression protected mice from hypertrophic stimuli. Remarkably, FBXW5 ameliorated pathological cardiac hypertrophy by directly interacting with the protein transforming growth factor-beta-activated kinase 1 (TAK1) and blocking the mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, inhibition of TAK1 prevented the effects of FBXW5 on agonist- or pressure overload-induced cardiac hypertrophy. These findings imply that FBXW5 is an essential negative regulator and may be a potential therapeutic target for pathological cardiac hypertrophy.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 20 Sep 2020; epub ahead of print
Hui X, Hu F, Liu J, Li C, ... Wang F, Li S
J Mol Cell Cardiol: 20 Sep 2020; epub ahead of print | PMID: 32971071
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Impact:
Abstract

Glycogen synthase kinase-3β inhibition alleviates activation of the NLRP3 inflammasome in myocardial infarction.

Wang S, Su X, Xu L, Chang C, ... Zhang L, Han S

Inflammasome-promoted sterile inflammation following cardiac damage is critically implicated in heart dysfunction after myocardial infarction (MI). Glycogen synthase kinase-3 (GSK-3β) is a prominent mediator of the inflammatory response, and high GSK-3 activity is associated with various heart diseases. We investigated the regulatory mechanisms of GSK-3β in activation of the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in a rat model with successful induction of MI on days 2-28. An in vitro investigation was performed using newborn rat/human cardiomyocytes and fibroblast cultures under typical inflammasome stimulation and hypoxia treatment. GSK-3β inhibition markedly improved myocardial dysfunction and prevented remodeling, with parallel reduction in the parameters of NLRP3 inflammasome activation after MI. GSK-3β inhibition reduced NLRP3 inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes. GSK-3β\'s interaction with activating signal cointegrator (ASC) as well as GSK-3β inhibition reduced ASC phosphorylation and oligomerization at the tissues and cellular levels. Taken together, these data show that GSK-3β directly mediates NLRP3 inflammasome activation, causing cardiac dysfunction in MI.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 27 Sep 2020; 149:82-94
Wang S, Su X, Xu L, Chang C, ... Zhang L, Han S
J Mol Cell Cardiol: 27 Sep 2020; 149:82-94 | PMID: 32991876
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Impact:
Abstract

Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity.

Lyra-Leite DM, Petersen AP, Ariyasinghe NR, Cho N, McCain ML

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 06 Oct 2020; epub ahead of print
Lyra-Leite DM, Petersen AP, Ariyasinghe NR, Cho N, McCain ML
J Mol Cell Cardiol: 06 Oct 2020; epub ahead of print | PMID: 33038389
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Impact:
Abstract

Single-cell protein expression of hiPSC-derived cardiomyocytes using Single-Cell Westerns.

Jabart E, Molho J, Sin K, Stansfield B, ... Wu JC, Churko JM

The ability to reprogram human somatic cells into human induced pluripotent stem cells (hiPSCs) has enabled researchers to generate cell types in vitro that have the potential to faithfully recapitulate patient-specific disease processes and phenotypes. hiPSC-derived cardiomyocytes (hiPSC-CMs) offer the promise of in vitro patient- and disease-specific models for drug testing and the discovery of novel therapeutic approaches for treating cardiovascular diseases. While methods to differentiate hiPSCs into cardiomyocytes have been demonstrated, the heterogeneity and immaturity of these differentiated populations have restricted their potential in reproducing human disease and the associated target cell phenotypes. These barriers may be overcome through comprehensive single-cell characterization to dissect the rich heterogeneity of hiPSC-CMs and to study the source of varying cell fates. In this study, we optimized and validated a new Single-Cell Western method to assess protein expression in hiPSC-CMs. To better understand distinct subpopulations generated from cardiomyocyte differentiations and to track populations at single-cell resolution over time, we measured and quantified the expression of cardiomyocyte subtype-specific proteins (MLC2V and MLC2A) using Single-Cell Westerns. By understanding their heterogeneity through single-cell protein expression and quantification, we may improve upon current cardiomyocyte differentiation protocols, generate hiPSC-CMs that are more representative of in vivo derived cardiomyocytes for disease modeling, and utilize hiPSC-CMs for regenerative medicine purposes. Single-Cell Westerns provide a robust platform for protein expression analysis at single-cell resolution.

Copyright © 2020 Elsevier Ltd. All rights reserved.

J Mol Cell Cardiol: 30 Sep 2020; 149:115-122
Jabart E, Molho J, Sin K, Stansfield B, ... Wu JC, Churko JM
J Mol Cell Cardiol: 30 Sep 2020; 149:115-122 | PMID: 33010256
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Abstract

Excessive maternal salt intake gives rise to vasopressin-dependent salt sensitivity of blood pressure in male offspring.

Kim YB, Jung WW, Lee SW, Jin X, ... Colwell CS, In Kim Y

Salt sensitivity of blood pressure (SSBP) is a trait carrying strong prognostic implications for various cardiovascular diseases. To test the hypothesis that excessive maternal salt intake causes SSBP in offspring through a mechanism dependent upon arginine-vasopressin (AVP), we performed a series of experiments using offspring of the rat dams salt-loaded during pregnancy and lactation with 1.5% saline drink (\"experimental offspring\") and those with normal perinatal salt exposure (\"control offspring\"). Salt challenge, given at 7-8 weeks of age with either 2% saline drink (3 days) or 8% NaCl-containing chow (4 weeks), had little or no effect on systolic blood pressure (SBP) in female offspring, whereas the salt challenge significantly raised SBP in male offspring, with the magnitude of increase being greater in experimental, than control, rats. Furthermore, the salt challenge not only raised plasma AVP level more and caused greater depressor responses to V1a and V2 AVP receptor antagonists to occur in experimental, than control, males, but it also made GABA excitatory in a significant proportion of magnocellular AVP neurons of experimental males by depolarizing GABA equilibrium potential. The effect of the maternal salt loading on the salt challenge-elicited SBP response in male offspring was precluded by maternal conivaptan treatment (non-selective AVP receptor antagonist) during the salt-loading period, whereas it was mimicked by neonatal AVP treatment. These results suggest that the excessive maternal salt intake brings about SSBP in male offspring, both the programming and the expression of which depend on increased AVP secretion that may partly result from excitatory GABAergic action.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 30 Sep 2020; epub ahead of print
Kim YB, Jung WW, Lee SW, Jin X, ... Colwell CS, In Kim Y
J Mol Cell Cardiol: 30 Sep 2020; epub ahead of print | PMID: 33011158
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Abstract

Autotaxin inhibition reduces cardiac inflammation and mitigates adverse cardiac remodeling after myocardial infarction.

Tripathi H, Al-Darraji A, Abo-Aly M, Peng H, ... Smyth SS, Abdel-Latif A
Objective
Acute myocardial infarction (AMI) initiates pathological inflammation which aggravates tissue damage and causes heart failure. Lysophosphatidic acid (LPA), produced by autotaxin (ATX), promotes inflammation and the development of atherosclerosis. The role of ATX/LPA signaling nexus in cardiac inflammation and resulting adverse cardiac remodeling is poorly understood.
Approach and results
We assessed autotaxin activity and LPA levels in relation to cardiac and systemic inflammation in AMI patients and C57BL/6 (WT) mice. Human and murine peripheral blood and cardiac tissue samples showed elevated levels of ATX activity, LPA, and inflammatory cells following AMI and there was strong correlation between LPA levels and circulating inflammatory cells. In a gain of function model, lipid phosphate phosphatase-3 (LPP3) specific inducible knock out (Mx1-Plpp3) showed higher systemic and cardiac inflammation after AMI compared to littermate controls (Mx1-Plpp3); and a corresponding increase in bone marrow progenitor cell count and proliferation. Moreover, in Mx1- Plpp3 mice, cardiac functional recovery was reduced with corresponding increases in adverse cardiac remodeling and scar size (as assessed by echocardiography and Masson\'s Trichrome staining). To examine the effect of ATX/LPA nexus inhibition, we treated WT mice with the specific pharmacological inhibitor, PF8380, twice a day for 7 days post AMI. Inhibition of the ATX/LPA signaling nexus resulted in significant reduction in post-AMI inflammatory response, leading to favorable cardiac functional recovery, reduced scar size and enhanced angiogenesis.
Conclusion
ATX/LPA signaling nexus plays an important role in modulating inflammation after AMI and targeting this mechanism represents a novel therapeutic target for patients presenting with acute myocardial injury.

Published by Elsevier Ltd.

J Mol Cell Cardiol: 01 Oct 2020; 149:95-114
Tripathi H, Al-Darraji A, Abo-Aly M, Peng H, ... Smyth SS, Abdel-Latif A
J Mol Cell Cardiol: 01 Oct 2020; 149:95-114 | PMID: 33017574
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Abstract

Pharmacological inhibition of the mitochondrial Ca uniporter: Relevance for pathophysiology and human therapy.

Márta K, Hasan P, Rodríguez-Prados M, Paillard M, Hajnóczky G

Mitochondrial Ca uptake has long been considered crucial for meeting the fluctuating energy demands of cells in the heart and other tissues. Increases in mitochondrial matrix [Ca] drive mitochondrial ATP production via stimulation of Ca-sensitive dehydrogenases. Mitochondria-targeted sensors have revealed mitochondrial matrix [Ca] rises that closely follow the cytoplasmic [Ca] signals in many paradigms. Mitochondrial Ca uptake is mediated by the Ca uniporter (mtCU). Pharmacological manipulation of the mtCU is potentially key to understanding its physiological significance, but no specific, cell-permeable inhibitors were identified. In the past decade, as the molecular identity of the mtCU was brought to light, efforts have focused on genetic targeting. However, in the cells/animals that are able to survive impaired mtCU function, robust compensatory changes were found in the mtCU as well as other mechanisms. Thus, the discovery, through chemical library screens on normal and mtCU-deficient cells, of new small-molecule inhibitors with improved cell permeability and specificity might offer a better chance to test the relevance of mitochondrial Ca uptake. Success with the development of small molecule mtCU inhibitors is also expected to have clinical impact, considering the growing evidence for the role of mitochondrial Ca uptake in a variety of diseases, including heart attack, stroke and various neurodegenerative disorders. Here, we review the progress in pharmacological targeting of mtCU and illustrate the challenges in this field using data obtained with MCU-i11, a new small molecule inhibitor.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 05 Oct 2020; epub ahead of print
Márta K, Hasan P, Rodríguez-Prados M, Paillard M, Hajnóczky G
J Mol Cell Cardiol: 05 Oct 2020; epub ahead of print | PMID: 33035551
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Abstract

Decreased inspired oxygen stimulates de novo formation of coronary collaterals in adult heart.

Aghajanian A, Zhang H, Buckley BK, Wittchen ES, Ma WY, Faber JE
Rationale
Collateral vessels lessen myocardial ischemia when acute or chronic coronary obstruction occurs. It has long been assumed that although native (pre-existing) collaterals enlarge in obstructive disease, new collaterals do not form in the adult. However, the latter was recently shown to occur after coronary artery ligation. Understanding the signals that drive this process is challenged by the difficulty in studying collateral vessels directly and the complex milieu of signaling pathways, including cell death, induced by ligation. Herein we show that hypoxemia alone is capable of inducing collateral vessels to form and that the novel gene Rabep2 is required.
Objective
Hypoxia stimulates angiogenesis during embryonic development and in pathological states. We hypothesized that hypoxia also stimulates collateral formation in adult heart by a process that involves RABEP2, a recently identified protein required for formation of collateral vessels during development.
Methods and results
Exposure of mice to reduced FiO induced collateral formation that resulted in smaller infarctions following LAD ligation and that reversed on return to normoxia. Deletion of Rabep2 or knockdown of Vegfa inhibited formation. Hypoxia upregulated Rabep2, Vegfa and Vegfr2 in heart and brain microvascular endothelial cells (HBMVECs). Knockdown of Rabep2 impaired migration of HBMVECs. In contrast to systemic hypoxia, deletion of Rabep2 did not affect collateral formation induced by ischemic injury caused by LAD ligation.
Conclusions
Hypoxia induced formation of coronary collaterals by a process that required VEGFA and RABEP2, proteins also required for collateral formation during development. Knockdown of Rabep2 impaired cell migration, providing one potential mechanism for RABEP2\'s role in collateral formation. This appears specific to hypoxia, since formation after acute ischemic injury was unaffected in Rabep2 mice. These findings provide a novel model for studying coronary collateral formation, and demonstrate that hypoxia alone can induce new collaterals to form in adult heart.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 06 Oct 2020; epub ahead of print
Aghajanian A, Zhang H, Buckley BK, Wittchen ES, Ma WY, Faber JE
J Mol Cell Cardiol: 06 Oct 2020; epub ahead of print | PMID: 33038388
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Abstract

Updated and enhanced pig cardiac transcriptome based on long-read RNA sequencing and proteomics.

Müller T, Boileau E, Talyan S, Kehr D, ... Krijgsveld J, Dieterich C

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.

Copyright © 2019. Published by Elsevier Ltd.

J Mol Cell Cardiol: 09 Oct 2020; epub ahead of print
Müller T, Boileau E, Talyan S, Kehr D, ... Krijgsveld J, Dieterich C
J Mol Cell Cardiol: 09 Oct 2020; epub ahead of print | PMID: 33049256
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Abstract

Targeting the population for gene therapy with MYBPC3.

Carrier L

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited myocardial disease characterized by unexplained left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. Clinical heterogeneity is wide, ranging from asymptomatic individuals to heart failure, arrhythmias and sudden death. HCM is often caused by mutations in genes encoding components of the sarcomere. Among them, MYBPC3, encoding cardiac myosin-myosin binding protein C is the most frequently mutated gene. Three quarter of pathogenic or likely pathogenic mutations in MYBPC3 are truncating and the resulting protein was not detected in HCM myectomy samples. The overall prognosis of the patients is excellent if managed with contemporary therapy, but still remains a significant disease-related health burden, and carriers with double heterozygous, compound heterozygous and homozygous mutations often display a more severe clinical phenotype than single heterozygotes. We propose these individuals as a good target population for MYBPC3 gene therapy.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 09 Oct 2020; epub ahead of print
Carrier L
J Mol Cell Cardiol: 09 Oct 2020; epub ahead of print | PMID: 33049255
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Abstract

The role of the PlGF/Flt-1 signaling pathway in the cardiorenal connection.

Saito Y

Although the concept of the cardiorenal connection is widely accepted, athe underlying molecular mechanism has not been clearly defined. Nevertheless, accumulating evidence indicates that the nervous system and both the humoral and cellular immune systems are all involved. This review article focuses on the roles of the signaling pathway of placental growth factor (PlGF) and its receptor, fms-like tyrosine kinase-1 (Flt-1), in the development of the cardiorenal connection. PlGF, a member of the vascular endothelial cell growth factor family, is a specific ligand for Flt-1 and plays roles in the development of atherosclerosis, wound healing after ischemia injury, and angiogenesis through Flt-1 signaling. Flt-1, a tyrosine-kinase type receptor with a single transmembrane domain, has a soluble isoform (sFlt-1) consisting of only extracellular domains, and is an intrinsic antagonist of PlGF. In renal dysfunction, PlGF is upregulated and sFlt-1 is downregulated by oxidative stress or uremic toxins, leading to activation of the PlGF/Flt-1 signaling pathway, which in turn plays a role in the worsening of atherosclerosis and heart failure, both of which are frequently associated with renal dysfunction. Monocyte chemotactic protein-1 (MCP-1) is involved in the process downstream of the Flt-1 signaling pathway. Plasma levels of sFlt-1 correlate with the severity of renal dysfunction in patients with heart failure or myocardial infarction, and are associated with the incidence of cardiovascular events. This is inconsistent with the concept of relative activation of the PlGF/Flt-1 pathway in renal dysfunction. However, the level of circulating sFlt-1 does not always parallel sFlt-1 synthesis, probably because sFlt-1 is stored on cell surfaces through its heparin-binding domains and its quantity is regulated differently in renal dysfunction. This review summarizes a novel concept wherein noninfectious inflammation via PlGF/Flt-1 signaling is involved in the development of renal dysfunction-related cardiovascular complications.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 08 Oct 2020; epub ahead of print
Saito Y
J Mol Cell Cardiol: 08 Oct 2020; epub ahead of print | PMID: 33045252
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Abstract

Aldo-keto reductase family 1 member B induces aortic valve calcification by activating hippo signaling in valvular interstitial cells.

Gao C, Hu W, Liu F, Zeng Z, ... Liu X, Wang J
Aims
Calcific aortic valve disease (CAVD) is a primary cause of cardiovascular mortality; however, its mechanisms are unknown. Currently, no effective pharmacotherapy is available for CAVD. Aldo-keto reductase family 1 member B (Akr1B1) has been identified as a potential therapeutic target for valve interstitial cell calcification. Herein, we hypothesized that inhibition of Akr1B1 can attenuate aortic valve calcification.
Methods and results
Normal and degenerative tricuspid calcific valves from human samples were analyzed by immunoblotting and immunohistochemistry. The results showed significant upregulation of Akr1B1 in CAVD leaflets. Akr1B1 inhibition attenuated calcification of aortic valve interstitial cells in osteogenic medium. In contrast, overexpression of Akr1B1 aggravated calcification in osteogenic medium. Mechanistically, using RNA sequencing (RNAseq), we revealed that Hippo-YAP signaling functions downstream of Akr1B1. Furthermore, we established that the protein level of the Hippo-YAP signaling effector active-YAP had a positive correlation with Akr1B1. Suppression of YAP reversed Akr1B1 overexpression-induced Runx2 upregulation. Moreover, YAP activated the Runx2 promoter through TEAD1 in a manner mediated by ChIP and luciferase reporter systems. Animal experiments showed that the Akr1B1 inhibitor epalrestat attenuated aortic valve calcification induced by a Western diet in LDLR mice.
Conclusion
This study demonstrates that inhibition of Akr1B1 can attenuate the degree of calcification both in vitro and in vivo. The Akr1B1 inhibitor epalrestat may be a potential treatment option for CAVD.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 08 Oct 2020; epub ahead of print
Gao C, Hu W, Liu F, Zeng Z, ... Liu X, Wang J
J Mol Cell Cardiol: 08 Oct 2020; epub ahead of print | PMID: 33045251
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Abstract

Phosphorylation of Troponin I finely controls the positioning of Troponin for the optimal regulation of cardiac muscle contraction.

Kachooei E, Cordina NM, Potluri PR, Guse JA, McCamey D, Brown LJ

Troponin is the Ca molecular switch that regulates striated muscle contraction. In the heart, troponin Ca sensitivity is also modulated by the PKA-dependent phosphorylation of a unique 31-residue N-terminal extension region of the Troponin I subunit (NH-TnI). However, the detailed mechanism for the propagation of the phosphorylation signal through Tn, which results in the enhancement of the myocardial relaxation rate, is difficult to examine within whole Tn. Several models exist for how phosphorylation modulates the troponin response in cardiac cells but these are mostly built from peptide-NMR studies and molecular dynamics simulations. Here we used a paramagnetic spin labeling approach to position and track the movement of the NH-TnI region within whole Tn. Through paramagnetic relaxation enhancement (PRE)-NMR experiments, we show that the NH-TnI region interacts with a broad surface area on the N-domain of the Troponin C subunit. This region includes the Ca regulatory Site II and the TnI switch-binding site. Phosphorylation of the NH-TnI both weakens and shifts this region to an adjacent site on TnC. Interspin EPR distances between NH-TnI and TnC further reveal a phosphorylation induced re-orientation of the TnC N-domain under saturating Ca conditions. We propose an allosteric model where phosphorylation triggered cooperative changes in both the interaction of the NH-TnI region with TnC, and the re-orientation of the TnC interdomain orientation, together promote the release of the TnI switch-peptide. Enhancement of the myocardial relaxation rate then occurs. Knowledge of this unique role of phosphorylation in whole Tn is important for understanding pathological processes affecting the heart.

Copyright © 2020. Published by Elsevier Ltd.

J Mol Cell Cardiol: 16 Oct 2020; epub ahead of print
Kachooei E, Cordina NM, Potluri PR, Guse JA, McCamey D, Brown LJ
J Mol Cell Cardiol: 16 Oct 2020; epub ahead of print | PMID: 33080242
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This program is still in alpha version.