AIMS:Alternative mRNA splicing is a significant part of transcriptome reprogramming during the pathological manifestation of heart diseases. Earlier studies have identified a muscle-specific isoform of RBFox1 (RNA binding fox-1 homolog 1) to be a key RNA splicing regulator in pressure overload induced heart failure. However, the physiological impact of RBFox1 in myocardial infarction (MI), and the downstream mRNA alternative splicing events during MI induced cardiac remodelling remains unknown. METHODS AND RESULTS:Here we found RBFox1 expression was significantly decreased in Sprague-Dawley rat hearts post MI. Restoring the expression of RBFox1 prevented cardiac remodelling and dysfunction post MI characterized by improved cardiac function, reduced hypertrophy and fibrosis, associated with attenuated induction of cardiac stress marker genes. In cultured cardiomyocytes, expression of RBFox1 was sufficient to prevent hypoxia induced cell death measured by TUNEL staining and cleaved caspase 3, while inactivation of RBFox1 aggravated cardiac cell death. Mechanistically, we identified RBFox1 expression affected a broad spectrum of gene expression in post-MI hearts. In addition, a hypoxia-sensitive alternative splicing variant of Mbnl1 (Muscleblind-like 1) mRNA was identified to be regulated by RBFox1, resulting in the expression of a cell death related Mbnl1 isoform with 12 amino-acid deletion at the C-terminus (Mbnl1-ΔExon7). Strikingly, the selective inhibition of Mbnl1 Exon7 inclusion using anti-sense oligo protected the heart from myocardial infarction induced injury in vivo. CONCLUSION:In summary, we have established a cardio-protective role of RBFox1 in myocardial infarction induced cardiac remodelling and dysfunction. Restoration of RBFox1 expression, and targeted modulation of its downstream alternative splicing target Mbnl1, is a potential therapeutic approach for cardiac dysfunction and remodelling in MI injured heart.

Heart failure is a multifactorial disease, the percentage of patients with heart failure caused by metabolic syndrome is increasing year by year. The effect of gut flora dysbiosis on metabolic syndrome and heart failure has received widespread attention in recent years. Drugs to treat the condition urgently need to be discovered. C20DM, as a precursor compound of ginsenoside, is a small molecule compound obtained by biosynthetic means and is not available in natural products. In this project, we found that C20DM could improve the diversity of gut flora and elevate the expression of intestinal tight junction proteins-Occludin, Claudin, ZO-1, which inhibited the activity of the TLR4-MyD88-NF-kB pathway, and as a result, reduced myocardial inflammation and slowed down heart failure in metabolic syndrome mice. In conclusion, our study suggests that C20DM can treat heart failure by regulating gut flora, and it may be a candidate drug for treating metabolic syndrome-induced heart failure.

Introduction: Alternative mRNA splicing affects a broad spectrum of cardiac genes during pathological manifestation of heart diseases. Earlier studies from our lab have identified a muscle-specific isoform of RBFox1 to be a key RNA splicing regulator in pressure overload induced heart failure through regulating cardiac transcription factor alternative splicing. However, the physiological impact of RBFox1 in myocardial infarction (MI), and the RBFox1 downstream mRNA alternative splicing events during MI induced cardiac remodeling remains unknown. Goals: To investigate the functional impact of RBFox1 in MI induced cardiac remodeling. Method and Results: At both mRNA and protein levels, we found RBFox1 to be significantly decreased in Sprague-Dawley Rat hearts post MI. To investigate the functional impact of RBFox1 in myocardial infarction, we utilized AAV9 to achieve cardiac specific expression of RBFox1 in rats. Interestingly, expression of RBFox1 prevented cardiac dysfunction post MI characterized by improved cardiac function based on echocardiography. Expression of RBFox1 further reduced hypertrophy, cell death and fibrotic remodeling at both molecular and histological levels post MI. In vitro, expression of RBFox1 is sufficient to prevent hypoxia induced cardiac cell death based on TUNEL staining and cleaved caspase 3 level, while inactivation of RBFox1 promoted cardiac cell death. To further understand the molecular mechanism underlying RBFox1 mediated cardiac protection, we performed RNA-seq analysis to determine the transcriptome changes in vivo. RNA-seq showed insulin secretion, cAMP signaling and muscle contraction pathways to be top significantly changed pathways in RBFox1 expressed rat hearts post MI. Lastly, we have identified Mbnl1 mRNA alternative splicing to be regulated by RBFox1 through promoting a 36bp exon exclusion at the C-terminal of Mbnl1 protein, potentially affecting the function of Mbnl1. Conclusion: In summary, we have identified a previously uncharacterized role of RBFox1 in myocardial infarction injury. RBFox1 expression is critical to preserve cardiac function post MI, potentially through regulating downstream targets alternative splicing, including Mbnl1.

Adrenal catecholamines, including norepinephrine and epinephrine play an important role in cardiovascular physiology. Serum catecholamine level is tightly regulated in response to metabolic and cardiac stress. The biosynthesis of catecholamine is driven by a series of enzymatic reaction carried out by the key rate-limiting enzymes, including tyrosine hydroxylase (TH), DOPA decarboxylase (DDC) and phenylethanolamine-N-methyltransferase (PNMT). Although the transcription regulation of these enzymes has been described before, the regulatory mechanism at post-transcription or protein translation level has never been explored. Using a system genetics approach, we explored genes associated with the adrenal gland traits in HMDP at basal and post isoproterenol treatment. We have identified EPRS (Glutamyl-Prolyl-tRNA synthetase) to be significantly associated with adrenal gland mass post ISO treatment. EPRS is a tRNA synthetase responsible for charging tRNA during protein translation, EPRS also regulates mRNA translation as an RNA binding protein. To explore the functional impact of EPRS in adrenal gland physiology, we generated an EPRS adrenal gland specific knockout mouse model (EPRS-aKO). Inactivation of EPRS in adrenal gland led to a decrease of adrenal gland mass associated with reduced systemic catecholamine level. Interestingly, we further observed a moderate yet significantly decreased heart rate and ejection fraction in EPRS-aKO mice. Through both in vivo and in vitro analysis, we confirmed inactivation of EPRS led to a reduced protein, but not mRNA expression for both TH and DDC. In adipose tissue, activation of EPRS through mTOR signaling pathway is critical for its function as RNA binding protein. To further explore the mechanism through EPRS mediated catecholamine synthesis, we treated PC12 cells with rapamycin to inhibit mTOR activity. Treatment with Rapamycin diminished EPRS activity and expression, reduced TH protein expression along with a decrease of catecholamine level. Our results identified a novel regulatory scheme of catecholamine synthesis through mTOR-EPRS axis, adding a novel layer of gene regulation in catecholamine production and providing new mechanistic insights for heart-adrenal cross talk.

HomeCirculationVol. 148, No. 16Regulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1 No AccessResearch ArticleRequest AccessFull TextAboutView Full TextView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toNo AccessResearch ArticleRequest AccessFull TextRegulation of Postnatal Cardiomyocyte Maturation by an RNA Splicing Regulator RBFox1 Jijun Huang, Josh Z. Lee, Christoph D. Rau, Arash Pezhouman, Tomohiro Yokota, Hiromi Miwa, Matthew Feldman, Tsz Kin Kong, Ziyue Yang, Woan Ting Tay, Ivan Pushkarsky, Kyungsoo Kim, Shan S. Parikh, Shreya Udani, Boon Seng Soh, Chen Gao, Linsey Stiles, Orian S. Shirihai, Bjorn C. Knollmann, Reza Ardehali, Dino Di Carlo and Yibin Wang Jijun HuangJijun Huang Correspondence to: Yibin Wang, PhD, Duke-NUS Medical School, 8 College Rd, Level 8, Singapore 169857, Singapore. Email E-mail Address: [email protected] https://orcid.org/0000-0002-5520-193X Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Division of Endocrinology (J.H.), University of California, Los Angeles. , Josh Z. LeeJosh Z. Lee Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. , Christoph D. RauChristoph D. Rau Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Department of Genetics and Computational Medicine, University of North Carolina, Chapel Hill (C.D.R.). , Arash PezhoumanArash Pezhouman https://orcid.org/0000-0001-9106-7136 Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles. Section of Cardiology, Department of Internal Medicine, Baylor College of Medicine, Houston, TX (A.P., R.A.). , Tomohiro YokotaTomohiro Yokota Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles. Department of Medicine, Greater Los Angeles VA Healthcare System, CA (T.Y.). , Hiromi MiwaHiromi Miwa Department of Bioengineering, Samueli School of Engineering (H.M., S.U., D.D.), University of California, Los Angeles. , Matthew FeldmanMatthew Feldman School of Medicine, Meharry Medical College, Nashville, TN (M.F.). , Tsz Kin KongTsz Kin Kong Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. , Ziyue YangZiyue Yang Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX (Z.Y.). , Woan Ting TayWoan Ting Tay https://orcid.org/0000-0002-4845-287X Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.). , Ivan PushkarskyIvan Pushkarsky Forcyte Biotechnologies, Inc, Los Angeles, CA (I.P.). , Kyungsoo KimKyungsoo Kim https://orcid.org/0000-0003-2869-0659 Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.). , Shan S. ParikhShan S. Parikh https://orcid.org/0000-0003-1806-9199 Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.). , Shreya UdaniShreya Udani Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.). , Boon Seng SohBoon Seng Soh https://orcid.org/0000-0001-9134-3081 Institute of Molecular and Cell Biology, The Agency for Science, Technology and Research (A*STAR), Singapore (B.S.S.). , Chen GaoChen Gao Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Department of Pharmacology and System Physiology, University of Cincinnati, OH (C.G.). , Linsey StilesLinsey Stiles Department of Medicine, David Geffen School of Medicine (L.S., O.S.S.), University of California, Los Angeles. , Orian S. ShirihaiOrian S. Shirihai Department of Medicine, David Geffen School of Medicine (L.S., O.S.S.), University of California, Los Angeles. , Bjorn C. KnollmannBjorn C. Knollmann https://orcid.org/0000-0003-4956-9735 Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN (K.K., S.S.P., B.C.K.). , Reza ArdehaliReza Ardehali https://orcid.org/0000-0003-1318-4016 Division of Cardiology, Department of Medicine (A.P., T.Y., R.A.), University of California, Los Angeles. Section of Cardiology, Department of Internal Medicine, Baylor College of Medicine, Houston, TX (A.P., R.A.). , Dino Di CarloDino Di Carlo Department of Bioengineering, Samueli School of Engineering (H.M., S.U., D.D.), University of California, Los Angeles. and Yibin WangYibin Wang https://orcid.org/0000-0003-0852-0767 Cardiovascular Laboratory, Division of Molecular Medicine, Department of Anesthesiology and Perioperative Medicine (J.H., J.Z.L., C.D.R., T.Y., T.K.K., C.G., Y.W.), University of California, Los Angeles. Signature Research Program of Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore (W.T.T., Y.W.). Originally published16 Oct 2023https://doi.org/10.1161/CIRCULATIONAHA.122.061602Circulation. 2023;148:1263–1266Footnotes*J. Huang and J.Z. Lee contributed equally.For Sources of Funding and Disclosures, see page 1266.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Yibin Wang, PhD, Duke-NUS Medical School, 8 College Rd, Level 8, Singapore 169857, Singapore. Email yibinwang@duke-nus.edu.sgREFERENCES1. Karbassi E, Fenix A, Marchiano S, Muraoka N, Nakamura K, Yang X, Murry CE. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.Nat Rev Cardiol. 2020; 17:341–359. doi: 10.1038/s41569-019-0331-xCrossrefMedlineGoogle Scholar2. Guo Y, Pu WT. Cardiomyocyte maturation: new phase in development.Circ Res. 2020; 126:1086–1106. doi: 10.1161/CIRCRESAHA.119.315862LinkGoogle Scholar3. Gao C, Ren S, Lee JH, Qiu J, Chapski DJ, Rau CD, Zhou Y, Abdellatif M, Nakano A, Vondriska TM, et al. RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure.J Clin Invest. 2016; 126:195–206. doi: 10.1172/JCI84015CrossrefMedlineGoogle Scholar4. Wang Y, Yao F, Wang L, Li Z, Ren Z, Li D, Zhang M, Han L, Wang SQ, Zhou B, et al. Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation.Nat Commun. 2020; 11:2585. doi: 10.1038/s41467-020-16204-wCrossrefMedlineGoogle Scholar5. Pushkarsky I. FLECS technology for high-throughput single-cell force biology and screening.Assay Drug Dev Technol. 2018; 16:7–11. doi: 10.1089/adt.2017.825CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetails October 17, 2023Vol 148, Issue 16 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.122.061602PMID: 37844148 Originally publishedOctober 16, 2023 KeywordsRNA, messengersequence analysis, RNAmyocytes, cardiacPDF download Advertisement SubjectsMechanismsMyocardial BiologyMyocardial RegenerationStem Cells

Free fatty acids（FFAs） play important roles in cardiovascular disease.Studies have shown that it is an important way for FAs to exert biological effects through their own receptors besides directly participating biochemical reaction in body.Free fatty acid receptor 2（FFA2） can be activated by short-chain FAs and is involved in inflammatory reactions and lipid accumulation.Since the known pathological changes caused by FFA2 are also implicated in cardiac hypertrophy,we hypothesized that FFA2 might be pathogenic in cardiac hypertrophy.This paper showed that FFA2 expression significantly increased in cardiac hypertrophy in vivo and in vitro.FFA2 agonist 4-CMTB or TUG-1375 promoted the expression of the hypertrophy markers ANF and BNP and increased cell surface area in vitro,which was further strengthened by FFA2 overexpression,suggesting that FFA2 might contribute to cardiomyocyte hypertrophy.Furthermore,4-CMTB treatment or FFA2 overexpression combined with 4-CMTB treatment elevated the phosphorylation and transcriptional activity of GATA4 and STAT3,which were inhibited by an ERK1/2 inhibitor,and GATA4 and STAT3 knockdown inhibited the elevation of hypertrophy biomarkers in cardiomyocytes treated with 4-CMTB.Taken together,these data indicate that FFA2 can enhance cardiomyocyte hypertrophy by activating STAT3 and GATA4 via ERK1/2,providing a potential new target for therapy.

Heart failure with preserved ejection fraction (HFpEF) is an emerging form of heart failure worldwide with no effective therapies in contrast with heart failure with reserved ejection fraction (HFrEF). To simulate multiple risk-factors associated with HFpEF in clinic, we developed a HFpEF mouse model by introducing cardiac hypertrophy with transverse aortic constriction (TAC) in ObOb ( Lep ob/ob ) mice, which has intrinsic systemic metabolic dysfunctions including obesity and insulin resistance. We first validated pathological changes in diastolic but not systolic parameters in the Ob-TAC vs. Ob-sham mice up to 10 weeks post-TAC by echocardiography. To evaluate the global transcriptome change in difference cell types, we conducted single nuclei RNA sequencing (snRNA-seq) from whole hearts of lean mice (c57), ObOb, and Ob-TAC mice (male only). 10x genomic 3’ GEM kit was used to generate the cDNA library and sequencing was done by Novaseq SP platform. A total of 13k nuclei were recovered from QC, nFeature RNA (< 2500) and mitochondrial gene (< 5%) filtering. By UMAP dimension reduction analysis, we annotated major cardiac cell types in the integrated snRNA-seq dataset, including 3 clusters of Cardiomyocytes (CMs). By pathway analysis of the differentially expressed genes in each CM clusters, we found that insulin resistance and glucagon pathway were enriched among the up regulated genes in CMs in HFpEF vs. lean control, while cell migration, signal transduction including insulin substrates were down regulated. Thus, we hypothesized that the altered crosstalk between glucagon and insulin signaling might contribute to the development of HFpEF in this mouse modal. This hypothesis was validated in a proof-of-concept study showing significant improvement of HFpEF features by inhibiting the glucagon receptors post-TAC with injection of a glucagon receptor antagonist.

Background Cardiac hypertrophy and fibrosis are common adaptive responses to injury and stress, eventually leading to heart failure. Hypoxia signaling is important to the (patho)physiological process of cardiac remodeling. However, the role of endothelial PHD2 (prolyl‐4 hydroxylase 2)/hypoxia inducible factor (HIF) signaling in the pathogenesis of cardiac hypertrophy and heart failure remains elusive. Methods and Results Mice with Egln1Tie2Cre (Tie2‐Cre‐mediated deletion of Egln1 [encoding PHD2]) exhibited left ventricular hypertrophy evident by increased thickness of anterior and posterior wall and left ventricular mass, as well as cardiac fibrosis. Tamoxifen‐induced endothelial Egln1 deletion in adult mice also induced left ventricular hypertrophy and fibrosis. Additionally, we observed a marked decrease of PHD2 expression in heart tissues and cardiovascular endothelial cells from patients with cardiomyopathy. Moreover, genetic ablation of Hif2a but not Hif1a in Egln1Tie2Cre mice normalized cardiac size and function. RNA sequencing analysis also demonstrated HIF‐2α as a critical mediator of signaling related to cardiac hypertrophy and fibrosis. Pharmacological inhibition of HIF‐2α attenuated cardiac hypertrophy and fibrosis in Egln1Tie2Cre mice. Conclusions The present study defines for the first time an unexpected role of endothelial PHD2 deficiency in inducing cardiac hypertrophy and fibrosis in an HIF‐2α–dependent manner. PHD2 was markedly decreased in cardiovascular endothelial cells in patients with cardiomyopathy. Thus, targeting PHD2/HIF‐2α signaling may represent a novel therapeutic approach for the treatment of pathological cardiac hypertrophy and failure.

Protein phosphorylation and dephosphorylation is central to signal transduction in nearly every aspect of cellular function, including cardiovascular regulation and diseases. While protein kinases are often regarded as the molecular drivers in cellular signaling with high specificity and tight regulation, dephosphorylation mediated by protein phosphatases is also gaining increasing appreciation as an important part of the signal transduction network essential for the robustness, specificity and homeostasis of cell signaling. Metal dependent protein phosphatases (PPM, also known as protein phosphatases type 2C, PP2C) belong to a highly conserved family of protein phosphatases with unique biochemical and molecular features. Accumulating evidence also indicates important and specific functions of individual PPM isoform in signaling and cellular processes, including proliferation, senescence, apoptosis and metabolism. At the physiological level, abnormal PPM expression and activity have been implicated in major human diseases, including cancer, neurological and cardiovascular disorders. Finally, inhibitors for some of the PPM members have been developed as a potential therapeutic strategy for human diseases. In this review, we will focus on the background information about the biochemical and molecular features of major PPM family members, with emphasis on their demonstrated or potential roles in cardiac pathophysiology. The current challenge and potential directions for future investigations will also be highlighted.

Highly regulated transcriptional control of cardiac gene networks is critical for heart development and function. Rtf1 is a critical regulator of multiple transcription processes including elongation and co-transcriptional histone modification. Rtf1 ablation in zebrafish and mice eliminates the cardiac progenitor population, producing embryos without hearts, and knockdown of Rtf1 in neonatal rat ventricular myocytes decreases the expression of sarcomere component genes, suggesting that Rtf1 is indispensable for both establishing the myocardial lineage and maintaining normal physiological homeostasis in differentiated cardiomyocytes. To explore the function of Rtf1 in the adult heart, we generated an inducible knockout of Rtf1 in the adult myocardium. Loss of Rtf1 function in adult cardiomyocytes results in left ventricular systolic dysfunction and pathological features of heart failure. The Rtf1 deficient hearts exhibit a gene expression profile characteristic of heart failure, and display disrupted myofibrils and cell-cell junctions. Intriguingly, and in line with Rtf1’s established role in promoting histone H2B ubiquitination, diminished H2BK120 ubiquitination was also observed in Rtf1 deficient adult hearts. Collectively, our findings show that Rtf1 and Rtf1 mediated H2BK120 ubiquitination are essential epigenetic regulators of both myocardial lineage determination during development as well as maintaining cellular homeostasis, integrity and function in mature cardiomyocytes.

The chemotherapeutic effect of doxorubicin (Dox) is limited by cumulative dose-dependent cardiotoxicity in cancer survivors. Dexrazoxane (DRZ) is approved to prevent Dox-induced cardiotoxicity. Humanin and its synthetic analog HNG have a cytoprotective effect on the heart. To investigate the cardioprotective efficacy of HNG alone or in combination with DRZ against Dox-induced cardiotoxicity, 80 adult male mice were randomly divided into 8 groups to receive the following treatments via intraperitoneal injection: saline daily, HNG (5 mg/kg) daily, DRZ (60 mg/kg) weekly, Dox (3 mg/kg) weekly, DRZ + HNG, Dox + HNG, Dox + DRZ, and Dox + HNG + DRZ. Echocardiograms were performed before and at 4, 8, and 9.5 wk after the beginning of treatment. All mice were euthanized at 10 wk. In the absence of Dox, HNG, DRZ, or DRZ + HNG had no adverse effect on the heart. Dox treatment caused decreases in ejection fraction and cardiac mass and increases in cardiomyocyte apoptosis and intracardiac fibrosis. HNG or DRZ alone blunted the Dox-induced decrease in left ventricle posterior wall thickness and modestly ameliorated the Dox-induced decrease in ejection fraction. HNG + DRZ significantly ameliorated Dox-induced decreases in ejection function, cardiac fibrosis, and cardiac mass. Using a targeted analysis for the mitochondrial gene array and protein expression in heart tissues, we demonstrated that HNG + DRZ reversed DOX-induced altered transcripts that were biomarkers of cardiac damage and uncoupling protein-2. We conclude that HNG enhances the cardiac protective effect of DRZ against Dox-induced cardiotoxicity. HNG + DRZ protects mitochondria from Dox-induced cardiac damage and blunts the onset of cardiac dysfunction. Thus, HNG may be an adjuvant to DRZ in preventing Dox-induced cardiotoxicity. NEW & NOTEWORTHY Doxorubicin (Dox) is commonly used for treating a wide range of human cancers. However, cumulative dosage-dependent carditoxicity often limits its clinical applications. We demonstrated in this study that treating young adult male mice with synthetic humanin analog enhanced the cardiac protective effect of dexrazoxane against chemotherapeutic agent Dox-induced cardiac dysfunction. Thus, humanin analog can potentially serve as an adjuvant to dexrazoxane in more effectively preventing Dox-induced cardiac dysfunction and cardiomyopathy.

Glucagon is an important hormone for glycemic control with counter-balancing function vs. insulin, and treatment of glucagon receptor antibody is shown to be efficacious for type I and type II diabetes. It was reported recently that systemic treatment of glucagon aggravated myocardial injury and pathological remodeling in heart in a glucagon receptor dependent manner, while cardiomyocyte specific inactivation of glucagon receptor protected heart from ischemic injury and remodeling, implicating the therapeutic potential of targeting glucagon receptor for heart failure. In this report, we investigated the functional outcome of glucagon receptor antagonist treatment in mice following myocardial infarction (MI) and pressure-overload using a humanized monoclonal glucagon receptor antagonist antibody REMD2.59. Mice treated with REMD2.59 after myocardial infarction showed significantly reduced scar sizes, blunted cardiac hypertrophy and fibrotic remodeling, and attenuated contractile dysfunction at four weeks after myocardial infarction comparing to mice treated with vehicle as controls. In contrast, treatment of glucagon aggravated these pathological manifestations triggered by myocardial infarction. Furthermore, treatment with REMD2.59 at the onset of pressure-overload significantly suppressed the development of cardiac hypertrophy and chamber dilation with marked preservation of cardiac systolic and diastolic function. More remarkably, initiation of REMD2.59 treatment two weeks after pressure-overload significantly blunted the progression of cardiac pathology with remarkable recovery of hypertrophic gene induction. These results provide the first in vivo proof-of-concept evidence that glucagon receptor antagonism is a potentially efficacious therapy to block both onset and progression of heart failure.

BackgroundChronic myocardial infarction (MI) triggers pathological remodeling in the heart and cardiac nervous system. Abnormal function of the autonomic nervous system (ANS), including stellate ganglia (SG) and dorsal root ganglia (DRG) contribute to increased sympathoexcitation, cardiac dysfunction and arrythmogenesis. ANS modulation is a therapeutic target for arrhythmia associated with cardiac injury. However, the molecular mechanism involved in the pathological remodeling in ANS following cardiac injury remains to be established.Methods and resultsIn this study, we performed transcriptome analysis by RNA-sequencing in thoracic SG and (T1-T4) DRG obtained from Yorkshire pigs following either acute (3 to 5 hours) or chronic (8 weeks) myocardial infarction. By differential expression and weighted gene co-expression network analysis (WGCNA), we identified significant transcriptome changes and specific gene modules in the ANS tissues in response to myocardial infarction at either acute or chronic phases. Both differential expressed genes and the member genes of the WGCNA gene module associated with post-infarct condition were significantly enriched for inflammatory signaling and apoptotic cell death. Targeted validation analysis supported a significant induction of inflammatory and apoptotic signal in both SG and DRG following myocardial infarction, along with cellular evidence of apoptosis induction based on TUNEL analysis. Importantly, these molecular changes were observed specifically in the thoracic segments but not in their counterparts obtained from lumbar sections.ConclusionMyocardial injury leads to time-dependent global changes in gene expression in the innervating ANS. Induction of inflammatory gene expression and loss of neuron cell viability in SG and DRG are potential novel mechanisms contributing to abnormal ANS function which can promote cardiac arrhythmia and pathological remodeling in myocardium.

RBFox1 is known to be an RNA splicing regulator with enriched expression in cardiac muscle. Loss of RBFox1 expression is a molecular hallmark associated with pathological hypertrophy and heart failure. However, much of our current knowledge about RBFox1 focuses on nuclear RBFox1 with a major impact on global alternative splicing changes in the diseased heart. Yet, RBFox1 gene also generates a cytosolic isoform through alternative splicing (RBFox1c), but the specific function of RBFox1c in heart has not been characterized. RBFox1c expression is significantly repressed in the mouse failing heart and hypertrophic cardiomyocytes. We performed RNA-seq combined with GO and IPA analysis to determine the impact of RBFox1c expression in culture. Among the genes suppressed specifically by RBFox1c but not the nucleus RBFox1 are groups of pro-inflammatory genes. Both Motif enrichment analysis and de novo motif discovery identified significant enrichment of RBFox1 binding motif in the 3’UTRs of the RBFox1c regulated genes. Using CLIP analysis followed by RT-PCR, we observed RBFox1c, but not nuclear RBFox1 specifically interacted with targeted inflammatory gene 3’UTR. In the cardiac specific RBFox1 knockout mice, enhanced cardiac fibrosis was observed following TAC, associated with elevated expression of RBFox1c dependent inflammatory genes. In contrast, cardiac specific expression of RBFox1c significantly reduced cardiac fibrosis and inflammatory gene expression following TAC, associated with improved ejection fraction and reduced hypertrophic marker gene expression. Further, we tested the effect of RBFox1c expression on cardiac fibrosis response using NRVM conditioned media. We showed the conditioned media from the hypertrophic cardiomyocytes potently induce fibroblast proliferation. However, RBFox1c expression can suppress phenylephrine and isoproterenol induced fibroblasts proliferation. RBFox1 regulates cardiac transcriptome reprogramming at two post-transcriptional steps. The RBFox1 nuclei isoform regulates global RNA splicing reprogramming in heart, while the RBFox1c regulates inflammatory gene expression and fibrotic remodeling potentially through potential interaction with their 3’UTR and targeted RNA degradation.

A systems approach deconvolutes genes specific to and enriched in endothelium from whole-organ transcriptome data, with applications to other cell types and tissues.

HomeHypertensionVol. 67, No. 5Positive Role for a Negative Calcineurin Regulator in Cardiac Hypertrophy Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBPositive Role for a Negative Calcineurin Regulator in Cardiac Hypertrophy Chen Gao and Yibin Wang Chen GaoChen Gao From the Departments of Anesthesiology (C.G.) and Medicine and Physiology (C.G., Y.W.), David Geffen School of Medicine, University of California, Los Angeles. Search for more papers by this author and Yibin WangYibin Wang From the Departments of Anesthesiology (C.G.) and Medicine and Physiology (C.G., Y.W.), David Geffen School of Medicine, University of California, Los Angeles. Search for more papers by this author Originally published28 Mar 2016https://doi.org/10.1161/HYPERTENSIONAHA.116.07140Hypertension. 2016;67:841–842Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2016: Previous Version 1 See related article, pp 866–877Calcineurin is protein phosphatase with characteristic calcium- and calmodulin-dependent activation through its regulatory subunits.1 Activated calcineurin dephosphorylates downstream transcription factor nuclear factor of activated T cells (NFAT), which leads to its nuclear translocation and transcriptional activation.1 Calcineurin-NFAT signaling axis is initially discovered as an essential pathway for T-cell activation1 but has now been implicated in a broad range of cellular processes and cell types ranging from fungus to plants. In the heart, calcineurin-mediated signaling is recognized as a common intracellular pathway leading to cardiac hypertrophy and pathological remodeling triggered by a plethora of pathological stressors.2 In this issue, Zhu et al3 add a new piece of evidence to a significant body of literature4 and further demonstrate that genetic or pharmacological attenuation of calcineurin signaling can have a significant ameliorative effect on the pathogenesis of cardiac hypertrophy and dysfunction in response to various stresses.Like many stress-induced signaling pathways, calcineurin pathway is also tightly controlled by a cohort of endogenous negative regulators in cells.5 The prototypic regulator of calcinurin-1 (RCAN1, also known as ADAPT78, CSP1, DSC1, DSCR1, MCIP1, RCN1, and calcipressin1) is transcriptionally controlled by NFAT and serves as a key negative feedback regulator for calcineurin signaling by direct binding and inhibiting its phosphatase activities. In addition to RCAN1, many other negative regulators for calcineurin-NFAT signaling have been identified, including cain/cabin1 (calcineurin inhibitor 1), RCAN2 (also known as CSP2, DSCR1L1, MCIP2, RCN2, ZAKI-4, and ZAKI4), RCAN3 (also known as DSCR1L2, MCIP3, RCN3, and hRCN3), four-and-a-half LIM domain protein 2, CHP1 (calcineurin B homologous protein 1, also known as SLC9A1BP, Sid470p, p22, and p24), and CHP2. Most of them function through direct interaction with calcineurin as a scaffold. However, specific inhibitory function for calcineurin has also been identified for muscle-specific RING (Figure) as an E3 ubiquitin ligase through targeted calcineurin degradation6 and plasma membrane calcium ATPase as membrane calcium pump.7 Different from RCAN1, many of these endogenous inhibitors are not necessarily bona fide negative feedback regulators for calcineurin signaling as they are not directly induced by calcineurin-NFAT–mediated transcription on stimulation, but nevertheless modulate calcineurin signaling under different extracellular stimuli. Indeed, like RCAN1, manipulating many of these endogenous calcineurin inhibitors can have a significant effect on cardiac hypertrophy and pathological remodeling.Download figureDownload PowerPointFigure. Illustration of negative regulators of calcineurin (CaN)-nuclear factor of activated T-cell (NFAT) signaling pathway in cardiac hypertrophy. ? indicates genes with no known evidence for their effect on hypertrophy. AKAP79 indicates A kinase anchor protein 79; Cain, calcineurin inhibitor; CHP1,2, calcineurin B homologous protein 1, 2; FHL2, four and half LIM protein 2; MuRF1, muscle-specific RING finger protein 1; PMCA, plasma membrane Ca2+ ATPase; and RCAN, regulator of calcineurin.In 2007, Pan et al8 identified yet another negative feedback regulator for calcineurin, termed carabin or EPI64C, which fulfills the criteria of both negative inhibitory function to calcineurin and induction by calcineurin-mediated signaling after T-cell receptor induction. In addition to its inhibitory effect on calcineurin activity, carabin/EPI64C is also reported to have additional inhibitory role for Ras-mediated mitogen-activated protein kinase activation through an intrinsic Ras GTPase-activating protein activity.8 In a recent report by Bisserier et al9 using both genetic knockout mouse model and adeno-associate virus-mediated cardiac targeted gene transfer, carabin/EPI64C is shown to be both necessary and sufficient to attenuate pressure-overload–induced cardiac hypertrophy and pathological remodeling, thus adding yet another negative regulator of calcineurin into the player list in the cardiac hypertrophy regulatory network. In this issue, Zhu et al3 further advance this notion that carabin/EPI64C is a critical regulator of cardiac hypertrophy by offering several important new lines of evidence.3 First, these investigators generated cardiac specific knockout and cardiomyocyte-specific transgenic mouse models to demonstrate in vivo that carabin/EPI64C-mediated regulation of cardiac hypertrophy and pathological remodeling is a cardiomyocyte cell-autonomous process. Second, the underlying mechanism seems to involve direct interaction and inhibition of calcineurin signaling rather than Ras-mitogen-activated protein kinase pathway as originally reported. Finally, carabin/EPI64C-mediated hypertrophy regulation is conserved across different species, and its expression exerts cardiac protection against pressure-overload–induced cardiac hypertrophy and dysfunction in both mice and nonhuman primates. These findings further demonstrate the translational potential of carabin/EPI64C as a therapeutic target for pathological hypertrophy in the stressed human heart.As an endogenous feedback regulator for calcineurin, carabin/EPI64C is both a downstream target of calcineurin/NFAT-mediated transcriptional induction and an upstream negative inhibitor for calcineurin signaling.8 Because calcineurin/NFAT-mediated signaling is a common pathway significantly elevated in the diseased heart, we should expect to observe an induced expression of carabin/EPI64C in stressed hypertrophic myocardium as observed for RCNA1. Yet, both reports by Bisserier et al9 and Zhu et al3 showed a significantly reduced expression of carabin/EPI64C in the pathologically stressed heart and the human failing heart. Therefore, the loss of carabin/EPI64C (but not RCAN1)–mediated negative feedback may represent an interesting new mechanism underlying the hyperactivity of calcineurin in cardiac hypertrophy and pathological remodeling. It is not clear why carabin/EPI64C expression is downregulated in the diseased heart and whether restoring its expression in established hypertrophic heart is able to reverse the pathogenic progression. Understanding the uncoupling mechanism between calcineurin and carabin/EPI64C and testing the therapeutic effect of restoring carabin/EPI64C expression in established hypertrophy may uncover a truly translational path to treat cardiac hypertrophy and pathological remodeling. It is important to note that although pharmacological inhibition of calcineurin has proved to be effective in clinic to suppress immune response and is widely used for organ transplant and other immune disorders, they have not been demonstrated efficacious to treat heart failure or hypertrophic cardiomyopathy in humans. Considering the hypertensive effect of cyclosporine (a pharmacological inhibitor of calcineurin) at systemic level,10 cautions must be taken to translate these insights learnt from tissue-specific and precision genetic manipulations to clinical applications. Clearly, there are a lot more sciences still waiting to be done.Sources of FundingThis work was supported, in part, by grants from National Institutes of Health (HL103205, HL098954, HL108186, and HL114437) to Y. Wang.DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Yibin Wang, 650 Charles E. Young Dr, Room BH 569, CHS, Los Angeles, CA 90095. E-mail [email protected]References1. Clipstone NA, Crabtree GR. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation.Nature. 1992; 357:695–697. doi: 10.1038/357695a0.CrossrefMedlineGoogle Scholar2. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy.Cell. 1998; 93:215–228.CrossrefMedlineGoogle Scholar3. Zhu X, Fang J, Gong J, Guo JH, Zhao GN, Ji YX, Liu HY, Wei X, Li H. Cardiac-specific EPI64C blunts pressure overload–induced cardiac hypertrophhy.Hypertension. 2016; 67:866–877. doi: 10.1161/HYPERTENSIONAHA.115.07042.LinkGoogle Scholar4. Fiedler B, Wollert KC. Interference of antihypertrophic molecules and signaling pathways with the Ca2+-calcineurin-NFAT cascade in cardiac myocytes.Cardiovasc Res. 2004; 63:450–457. doi: 10.1016/j.cardiores.2004.04.002.CrossrefMedlineGoogle Scholar5. 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Xiao L, Gu Y, Gao L, Shangguan J, Chen Y, Zhang Y and Li L (2017)(2017) Sanggenon C protects against pressure overload-induced cardiac hypertrophy via the calcineurin/NFAT2 pathway, Molecular Medicine Reports, 10.3892/mmr.2017.7288, 16:4, (5338-5346), Online publication date: 1-Oct-2017. Li N, Zhou H and Tang Q (2018) miR-133: A Suppressor of Cardiac Remodeling?, Frontiers in Pharmacology, 10.3389/fphar.2018.00903, 9 May 2016Vol 67, Issue 5 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/HYPERTENSIONAHA.116.07140PMID: 27021013 Originally publishedMarch 28, 2016 PDF download Advertisement SubjectsHeart FailureHypertrophyRemodeling

Background: The complexity of cardiac transcriptome and proteome is significantly contributed by alternative splicing of mRNA. Alternative splicing is regulated by the cis-regulatory elements located in pre-mRNA together with the trans-activating factors guiding the assembling and function of the spliceosome. In our earlier study, we have observed global changes of alternative splicing events during pressure-overload induced heart failure, and identified RBFox1 as a key regulator for cardiac RNA splicing regulation during postnatal development and pathological remodeling. Both RBFox1 and RBFox2 are highly enriched in cardiomyocytes, and their expression are both significantly repressed in response to pathological stress. Loss-of-function studies for RBFox1 and RBFox2 are achieved using cardiac specific but constitutively active Cre. Therefore, the isoform specific contribution of RBFox1 vs. RBFox2 in maintaining cardiac physiology and homeostasis in adult heart is unknown. Methods and Results: We generated mouse models of cardiac specific and inducible knockout of RBFox1 and RBFox2 individually in adult hearts by breeding the individual floxed alleles with the αMHC-Mer-Cre-Mer mice. At baseline, inactivating RBFox1 in adult heart caused a slight but significant decrease of cardiac function without activating hypertrophy gene expression. However, following myocardial infarction, the RBFox1 deficient hearts showed enhanced global fibrosis in non-infarcted areas comparing to the control animals. In contrast, inactivating RBFox2 in adult mouse heart caused overt heart failure associated with chamber dilation without external stress as early as 2 weeks post tamoxifen administration. Conclusion: We have identified differential impact of RBFox1 and RBFox2 deficiency in adult mouse heart. Our in vivo study illustrate the functional importance of the RBFox family RNA splicing regulators in normal physiology of adult heart, and support the pathogenic contribution of loss of RBFox expression to heart failure. Further analysis focusing on the underlying molecular mechanisms for their differential impact would yield new insights on transcriptome regulation and complexity in cardiac physiology and diseases.