Regulator Of Cardiomyocyte Hypertrophy Research Articles

Circular RNA circZFPM2 regulates cardiomyocyte hypertrophy and survival

Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.

Exosomes derived from cardiac fibroblasts with angiotensin II stimulation provoke hypertrophy and autophagy inhibition in cardiomyocytes

Although accumulating evidence has revealed that autophagy inhibition contributes to the development of pathological cardiac hypertrophy, the mechanisms leading to declined autophagy activity in the hypertrophic heart remain to be elucidated. Exosomes are known to be important mediators of intercellular communication, and the involvement of exosomes in cardiovascular abnormities has attracted increasing attentions. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. Here, we investigated the potential role of CFs-derived exosomes in regulating cardiomyocyte hypertrophy and autophagy. Exosomes from rat CFs treated with angiotensin II (Ang II-CFs-exosomes) were collected and characterized. Our experiments showed that these exosomes could induce hypertrophic responses and impair autophagy activity in primary neonatal rat cardiomyocytes (NRCMs). Ang II-CFs-exosomes blocked the autophagic flux of NRCMs via inhibiting the formation of autolysosomes. Moreover, the pro-hypertrophic effects and autophagy inhibition induced by Ang II-CFs-exosomes was validated in mice receiving injection of the exosomes. These findings highlight a novel role of Ang II-CFs-exosomes in suppressing cardiomyocyte autophagy, which may help to better understand the pathogenesis of cardiac hypertrophy.

Fibin regulates cardiomyocyte hypertrophy and causes protein-aggregate-associated cardiomyopathy in vivo.

Despite the identification of numerous molecular pathways modulating cardiac hypertrophy its pathogenesis is not completely understood. In this study we define an unexpected role for Fibin ("fin bud initiation factor homolog") in cardiomyocyte hypertrophy. Via gene expression profiling in hypertrophic murine hearts after transverse aortic constriction we found a significant induction of Fibin. Moreover, Fibin was upregulated in another mouse model of cardiac hypertrophy (calcineurin-transgenics) as well as in patients with dilated cardiomyopathy. Immunoflourescence microscopy revealed subcellular localization of Fibin at the sarcomeric z-disc. Overexpression of Fibin in neonatal rat ventricular cardiomyocytes revealed a strong anti-hypertrophic effect through inhibiting both, NFAT- and SRF-dependent signalling. In contrast, transgenic mice with cardiac-restricted overexpression of Fibin developed dilated cardiomyopathy, accompanied by induction of hypertrophy-associated genes. Moreover, Fibin overexpression accelerated the progression to heart failure in the presence of prohypertrophic stimuli such as pressure overload and calcineurin overexpression. Histological and ultrastructural analyses surprisingly showed large protein aggregates containing Fibin. On the molecular level, aggregate formation was accompanied by an induction of the unfolded protein response subsequent UPR-mediated apoptosis and autophagy. Taken together, we identified Fibin as a novel potent negative regulator of cardiomyocyte hypertrophy in vitro. Yet, heart-specific Fibin overexpression in vivo causes development of a protein-aggregate-associated cardiomyopathy. Because of close similarities to myofibrillar myopathies, Fibin represents a candidate gene for cardiomyopathy and Fibin transgenic mice may provide additional mechanistic insight into aggregate formation in these diseases.

In this Issue.

HomeCirculation ResearchVol. 132, No. 7In this Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn this Issue Originally published30 Mar 2023https://doi.org/10.1161/RES.0000000000000608Circulation Research. 2023;132:791is related toDiscovery of Transacting Long Noncoding RNAs That Regulate Smooth Muscle Cell PhenotypeDestabilization of Atherosclerotic Plaque by Bilirubin DeficiencyIntegrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte HypertrophyDiscovery of Trans-Acting Long Non-Coding RNAs that Regulate Smooth Muscle Cell Phenotype (p 795)Download figureDownload PowerPointShi et al identify long non-coding RNAs that regulate a phenotype switch in smooth muscle cells.Vascular smooth muscle cells (VSMCs) are a major contributing cell type in the progression of coronary artery disease (CAD) and become pathological by transitioning into a highly proliferative and migratory state. In searching for factors that regulate this transition—which could thus reveal ways to stop it—Shi and colleagues focused on the potential role of long non-coding RNAs (lncRNAs), many of which are regulatory in nature. Human coronary artery SMCs (HCASMCs) were subjected to various CAD stimuli and the expression changes of lncRNAs were analyzed. From the many differentially expressed lncRNAs, the team focused on two—ZIPPOR and TNS1-AS2, which were down- and up-regulated respectively relative to unstimulated cells. Interestingly, functional analysis of these two revealed that, despite being differently affected by CAD stimuli, repression of either led to similar effects in HCASMCs—increased migration and proliferation and increased expression of ZEB2, a master regulator of SMC phenotype transition. Both ZIPPOR and TNS1-AS2 were also found to interact directly with the ZEB2 protein to suppress its expression.While it is not known if these lncRNAs influence CAD progression, their suppression of SMC transition suggests such investigations are warranted.Destabilization of Atherosclerotic Plaque by Bilirubin Deficiency (p 812)Download figureDownload PowerPointLow bilirubin aggravates atherosclerosis in mice, report Chen et al.Bilirubin is a yellowish pigment formed during the breakdown of red blood cells and is ordinarily processed by the liver and excreted. Liver dysfunction can cause a severe build-up of bilirubin resulting in jaundice, but a more modest elevation is associated with lower levels of cardiovascular disease (CVD). Indeed, people with higher levels of the pigment have a lower incidence of CVD, while coronary artery disease patients with especially low levels of bilirubin have less stable plaques and worse outcomes. To investigate how bilirubin might protect against CVD, Chen and colleagues engineered atherosclerosis-prone mice to produce less of the pigment, and compared them with control mice whose bilirubin levels were normal. A lack of bilirubin worsened atherosclerosis in multiple ways. For example, the test animals had increased levels of blood lipids, increased systemic inflammation and oxidative stress, evidence of endothelial dysfunction, and increased plaque instability. While the latter appeared to result from infiltration of inflammatory cells and increased expression of the pro-inflammatory enzyme, MPO, the molecular details behind bilirubin’s other effects are yet to be determined. Nonetheless, the findings lead the way for further research into whether modifying levels of bilirubin might help lower CVD risk.Integrated Proteomics Unveils Regulation of Cardiac Myocyte Hypertrophic Growth by a Nuclear cAMP Nanodomain Under the Control of PDE3A2 (p 828)Download figureDownload PowerPointA PDE3A2-controlled cAMP nanodomain regulates cardiomyocyte hypertrophy, say Subramaniam et al.Within cardiomyocytes, cAMP conveys beta-adrenergic receptor (βAR) signals to boost the strength and frequency of the heart’s contractions. But cAMP has other jobs within cells and its interactions with specific factors at specific subcellular nanodomains are thought to regulate task execution. One such factor is the enzyme PDE, which degrades cAMP. There are many different isoforms of PDE and they are thought to occupy and specify different nanodomains. Subramaniam and colleagues investigated the proteins associating with individual PDE isoforms when cardiomyocytes were stimulated with isoproterenol—a βAR agonist—and in this way discovered that βAR-activated PDE3A2 nanodomains associate with the transcription regulators SMAD4 and HDAC1, are located in the nucleus, and normally repress the expression of genes involved in hypertrophy. Sure enough, inhibition of PDE3 enabled cAMP-induced activation of hypertrophy genes and resulted in cardiomyocyte growth. PDE3 inhibitors are inotropic drugs that provide short term benefits to heart failure patients, but can worsen the condition if used long term. The finding that PDE3 inhibition led to cAMP-induced hypertrophy may thus partly explain the detrimental effects of such drugs on the heart, say the authors. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesDiscovery of Transacting Long Noncoding RNAs That Regulate Smooth Muscle Cell PhenotypeHuitong Shi, et al. Circulation Research. 2023;132:795-811Destabilization of Atherosclerotic Plaque by Bilirubin DeficiencyWeiyu Chen, et al. Circulation Research. 2023;132:812-827Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte HypertrophyGunasekaran Subramaniam, et al. Circulation Research. 2023;132:828-848 March 31, 2023Vol 132, Issue 7 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000608PMID: 36996178 Originally publishedMarch 30, 2023 PDF download Advertisement

Rapid diagnosis of acute myocardial infarction based on reverse transcription-accelerated strand exchange amplification of miR-208a.

Acute myocardial infarction (AMI) is a prevalent cardiovascular disease associated with high morbidity and mortality, posing a significant threat to human health. Therefore, early diagnosis of AMI has become a focal point of research. MiR-208 is specifically expressed in the heart and is involved in the regulation of cardiomyocyte hypertrophy, cardiac fibrosis, and other myocardial gene expressions. It is expected to be applied in the clinical detection of AMI due to its release by damaged myocardial cells within 3 hours of AMI. In this study, we developed a denatured bubble-mediated reverse transcription-accelerated strand exchange amplification (RT-ASEA) method to detect the early biomarker miR-208a of AMI. The novel approach allowed rapid amplification of miR-208a in 15 minutes, with good performance in terms of repeatability (CV < 6%), determination limit (1 × 100 pmol L-1), and linearity (R2 = 0.9690). Based on the analysis of 42 clinical samples, a strong correlation was observed between the Ct value of miR-208a detected by the RT-ASEA method and the cTnI concentration, considered the gold standard for diagnosis of AMI. The research suggested that the RT-ASEA method could be applied to distinguish between AMI and healthy groups. The area under the receiver operating characteristic curve (AUC) was 0.9976, with a sensitivity of 96% and a specificity of 100%. Optimized RT-ASEA is a reliable and efficient method for miRNA detection. Furthermore, this study provides crucial data support for the development of miR-208a as an early biomarker for AMI, which is of great significance in life and health.

Interaction between A-kinase anchoring protein 5 and protein kinase A mediates CaMKII/HDAC signaling to inhibit cardiomyocyte hypertrophy after hypoxic reoxygenation

We reported that A-kinase anchoring protein 5 (AKAP5) played a role in cardiomyocyte apoptosis after hypoxia-reoxygenation (H/R). The role of AKAP5 in cardiomyocyte hypertrophy has not been fully elucidated. Herein we investigated whether AKAP5 regulates cardiomyocyte hypertrophy through calcium/calmodulin-dependent protein kinase II (CaMKII). After H/R, deficiency of AKAP5 in H9C2 cardiomyocytes and neonatal rat cardiac myocytes activated CaMKII and stimulated cardiomyocyte hypertrophy. AKAP5 upregulation limited this. Low expression of AKAP5 increased CaMKII interaction with histone deacetylases 4/5 (HDAC4/5) and increased nuclear export of HDAC4/5. In addition, AKAP5 interactions with protein kinase A (PKA) and phospholamban (PLN) were diminished. Moreover, the phosphorylation of PLN was decreased, and intracellular calcium increased. Interference of this process with St-Ht31 increased CaMKII signaling, decreased PLN phosphorylation and promoted post-H/R cell hypertrophy. And PKA-anchoring deficient AKAP5ΔPKA could not attenuate hypoxia-reoxygenation-induced cardiomyocyte hypertrophy, but AKAP5 could. Altogether, AKAP5 downregulation exacerbated H/R-induced hypertrophy in cardiomyocytes. This was due to, in part, to less in AKAP5-PKA interaction and the accumulation of intracellular Ca2+ with a subsequent increase in CaMKII activity.

Angiotensin II Mediates Cardiomyocyte Hypertrophy in Atrial Cardiomyopathy via Epigenetic Transcriptional Regulation.

Aims European Heart Rhythm Association established an expert consensus to define, characterize, and classify atrial cardiomyopathy into four subgroups based on their histopathological features. The predominant pathological feature of classes I and III is the hypertrophy of atrial cardiomyocytes. Here, we aim to investigate the mechanism of epigenetic transcriptional regulation of cardiomyocyte hypertrophy in atrial cardiomyopathy. Methods and Results Compared with that of sinus rhythm control individuals, the myocardium of patients with atrial fibrillation exhibited increased levels of angiotensin II (AngII), chromatin-bound myocyte enhancer factor 2 (MEF2), acetylated histone H4 (H4ac), and H3K27ac; upregulation of hypertrophy-related genes; and decreased levels of histone deacetylase (HDAC) 4 and HDAC5 bound to the promoters of hypertrophy-related genes. Furthermore, incubation of atrial cardiomyocytes with AngII increased their cross-sectional area and improved the expression of hypertrophy-related genes. AngII also promoted the phosphorylation of HDAC4 and HDAC5 and induced their nuclear export. RNA sequencing analyses revealed that AngII significantly upregulated genes associated with cardiac hypertrophy. Chromatin immunoprecipitation showed that this correlated with increased levels of chromatin-bound MEF2, H4ac, and H3K27ac and decreased HDAC4 and HDAC5 enrichment in the promoters of hypertrophy-related genes. Moreover, these AngII-induced prohypertrophic effects could be partially reverted by treatment with the AngII receptor blocker losartan. Conclusions AngII had a prohypertrophic effect on atrial cardiomyopathy which was epigenetic-dependent. Patients with atrial fibrillation manifest an increased susceptibility to hypertrophy and exhibit epigenetic characteristics that are permissive for the transcription of hypertrophy-related genes. AngII induces histone acetylation via the cytoplasmic-nuclear shuttling of HDACs, which constitutes a novel mechanism of atrial hypertrophy regulation and might provide a promising therapeutic strategy for atrial cardiomyopathy.

DEC1 represses cardiomyocyte hypertrophy by recruiting PRP19 as an E3 ligase to promote ubiquitination-proteasome-mediated degradation of GATA4

Although the pro-hypertrophic role of GATA binding protein 4 (GATA4) during cardiac hypertrophy has been well established, the negative regulatory mechanism to counteract its hyperactivation remains elusive. We hypothesized that the hyperactivation of GATA4 could be a result of loss of interaction between GATA4 with specific suppressors. Using high throughput mass spectrometry technology, we carried out a proteomic screen for endogenous suppressor of GATA4, which disassociated with GATA4 during the hypertrophic response in a cultured cardiac myoblast cell line (H9C2 cells). We identified differentiated embryo chondrocyte 1 (DEC1) negatively regulated the function of GATA4 through physical interaction and negatively regulated cardiac hypertrophy both in vivo and in vitro. Particularly, DEC1 promoted the ubiquitination and proteasome-mediated degradation of GATA4, but did not function as an E3 ligase. Again, using mass spectrometry technology, we systematically identified pre-mRNA processing factor 19 (PRP19) as a newfound E3 ligase, which promoted the K6-linked ubiquitination of GATA4 at its lysine 256. Functional experiments performed in cultured neonatal rat ventricular myocytes and H9C2 cells demonstrated that both DEC1 and PRP19 negatively regulated agonist-induced cardiomyocyte hypertrophic responses. Furthermore, rescue experiments performed in these cells revealed that DEC1 and PRP19 suppressed cardiomyocyte hypertrophy by inhibiting the function of GATA4. Our study thus defined the novel DEC1-PRP19-GATA4 axis to be a previously unknown mechanism in regulating cardiomyocyte hypertrophy. Although GATA4 is indispensable for normal cardiac function, harnessing DEC1- or PRP19-mediated negative regulation to counteract the hyperactivation of GATA4 might serve as a novel therapeutic strategy for pathological cardiac hypertrophy.

G3bp1 – microRNA-1 axis regulates cardiomyocyte hypertrophy

Adaptation of gene expression is one of the most fundamental response of cardiomyocytes to hypertrophic stimuli. G3bp1, an RNA binding protein with site-specific endoribonuclease activity regulates the processing of pre-miR-1 stem-loop, and thus levels of cardiomyocyte -enriched mature miR-1. Here, we examine the role of G3bp1 in regulating gene expression in quiescent cardiomyocytes and those undergoing growth-factor induced hypertrophy. Further, we determine if these changes are facilitated through G3bp1-mediated regulation of miR-1 in these cardiomyocytes. Using isolated cardiomyocytes with knockdown of endogenous G3bp1, we performed high throughput RNA sequencing to determine the change in cardiac transcriptome. Then, using gain and loss of function approach for both, G3bp1 and miR-1, alone or in combination we examine the G3bp1-miR-1 signaling in regulating gene expression and Endothelin (ET-1) -induced cardiomyocyte hypertrophy. We show that knockdown of endogenous G3bp1 results in inhibition of genes involved in calcium handling, cardiac muscle contraction, action potential and sarcomeric structure. In addition, there is inhibition of genes that contribute to hypertrophic and dilated cardiomyopathy development. Conversely, an increase is seen in genes that negatively regulate the Hippo signaling, like Rassf1 and Arrdc3, along with inflammatory genes of TGF-β and TNF pathways. Knockdown of G3bp1 restricts ET-1 induced cardiomyocyte hypertrophy. Interestingly, concurrent silencing of G3bp1 and miR-1 rescues the change in gene expression and inhibition of hypertrophy seen with knockdown of G3bp1 alone. Similarly, expression of exogenous G3bp1 reverses the miR-1 induced inhibition of gene expression. Intriguingly, expression of Gfp tagged G3bp1 results in perinuclear accumulations of G3bp1-Gfp, resembling Stress Granules. Based on our results, we conclude that G3bp1 through its regulation of mature miR-1 levels plays a critical role in regulating the expression of essential cardiac-enriched genes and those involved in development of cardiomyocyte hypertrophy.

The cardiac translational landscape reveals that micropeptides are new players involved in cardiomyocyte hypertrophy

Hypertrophic growth of cardiomyocytes is one of the major compensatory responses in the heart after physiological or pathological stimulation. Protein synthesis enhancement, which is mediated by the translation of messenger RNAs, is one of the main features of cardiomyocyte hypertrophy. Although the transcriptome shift caused by cardiac hypertrophy induced by different stimuli has been extensively investigated, translatome dynamics in this cellular process has been less studied. Here, we generated a nucleotide-resolution translatome as well as transcriptome data from isolated primary cardiomyocytes undergoing hypertrophy. More than 10,000 open reading frames (ORFs) were detected from the deep sequencing of ribosome-protected fragments (Ribo-seq), which orchestrated the shift of the translatome in hypertrophied cardiomyocytes. Our data suggest that rather than increase the translational rate of ribosomes, the increased efficiency of protein synthesis in cardiomyocyte hypertrophy was attributable to an increased quantity of ribosomes. In addition, more than 100 uncharacterized short ORFs (sORFs) were detected in long noncoding RNA genes from Ribo-seq with potential of micropeptide coding. In a random test of 15 candidates, the coding potential of 11 sORFs was experimentally supported. Three micropeptides were identified to regulate cardiomyocyte hypertrophy by modulating the activities of oxidative phosphorylation, the calcium signaling pathway, and the mitogen-activated protein kinase (MAPK) pathway. Our study provides a genome-wide overview of the translational controls behind cardiomyocyte hypertrophy and demonstrates an unrecognized role of micropeptides in cardiomyocyte biology.

PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression.

The present study reveals a link between protein arginine methyltransferase 5 (PRMT5) and Homebox A9 (HoxA9) in the regulation of cardiomyocyte hypertrophy. In cardiomyocyte hypertrophy induced by β-adrenergic receptor agonist isoprenaline (ISO), PRMT5 expression was decreased while HoxA9 was upregulated. Silencing of PRMT5 or inhibition of PRMT5 by its pharmacological inhibitor EPZ augmented the expressions of cardiomyocyte hypertrophic genes brain natriuretic peptide (BNP) and β-Myosin Heavy Chain (β-MHC), whereas overexpression of PRMT5 inhibited ISO-induced cardiomyocyte hypertrophy, suggesting that PRMT5 ameliorates cardiomyocyte hypertrophy. On the contrary, HoxA9 promoted cardiomyocyte hypertrophy, as implied by the gain-of-function and loss-of-function experiments. HoxA9 was involved in the regulation of PRMT5 in cardiomyocyte hypertrophy, since HoxA9 knockdown prevented si-RPMT5-induced cardiomyocyte hypertrophy, and HoxA9 expression impaired the anti-hypertrophic effect of PRMT5. Co-immunoprecipitation experiments revealed that there were physical interactions between PRMT5 and HoxA9. The symmetric dimethylation level of HoxA9 was decreased by ISO or EPZ treatment, suggesting that HoxA9 is methylated by PRMT5. Additionally, PRMT5 repressed the expression of HoxA9. Chromatin immunoprecipitation (ChIP) assay demonstrated that HoxA9 could bind to the promoter of BNP, and that this binding affinity was further enhanced by ISO or EPZ. In conclusion, this study suggests that PRMT5 symmetric dimethylates HoxA9 and represses HoxA9 expression, thus impairing its binding to BNP promoter and ultimately protecting against cardiomyocyte hypertrophy. These findings provide a novel insight of the mechanism underlying the cardiac protective effect of PRMT5, and suggest potential therapeutic strategies of PRMT5 activation or HoxA9 inhibition in treatment of cardiac hypertrophy.

Abstract 15577: Mst 4, a Novel Stripak Associated Kinase is Upregulated in Human Cardiomyopathy and Regulates Cardiomyocyte Hypertrophy, Contractility and Apoptosis

Heart failure is one of the major causes of death worldwide. Its pathophysiology is complex and involves alterations in calcium cycling and hypertrophic signaling, as well as subsequent loss of cardiomyocytes due to apoptosis, cardiac fibrosis and progressive contractile dysfunction. The Striatin-interacting phosphatase and kinase (STRIPAK) complex is a mammalian multiprotein complex that consists of phosphatases, kinases and various adaptor proteins. Yet, the STRIPAK complex has so far not been studied in cardiomyocytes. Now we show in cardiomyocytes that the STRIPAK associated kinase MST4 directly interacts with a recently described novel cardiac STRIPAK protein, Myoscape/STRIP2. Multi tissue immunoblot experiments showed that Mst4 abundance is also highly enriched in heart and skeletal muscle. Immunoblot analyses in human biopsy samples form patients with dilated (DCM n=10) or ischemic cardiomyopathy (ICM n=10) revealed that MST4 is strongly upregulated in DCM (12-fold p&lt;0.001) and ICM (9.6 fold p&lt;0.05) vs. controls. Conversely, upon biomechanical stretch we observed a significant downregulation of MST4 in vitro by 30% (p&lt;0.05) as well as in vivo (-45%, n=6; P&lt;0.001) in a murine model for pressure overload (experimental aortic constriction, (TAC) vs. Sham) under Moreover, we also observed downregulation of MST4 in a porcine myocardial infarction model (-48% vs. porcine controls; n=5 p&lt;0.05). Adenoviral overexpression of MST4 in NRVCM results in significant cellular hypertrophy (485μm2 vs 421μm2; p&lt;0.001) compared to LacZ. Overexpression of MST4 also increases cellular and sarcomeric fractional shortening in adult rat cardiac myocytes vs. LacZ (p&lt;0.05). Finally, MST4 overexpression also protects from cardiac apoptosis as shown by 90% reduced PARP cleavage as well as significantly reduced expression of Caspase 3 and 7 (n=3; p&lt;0.05). Taken together we show that MST 4 is a cardiac enriched Kinase that regulates cardiomyocyte hypertrophy, contractility and apoptosis and may thus be a target for future therapeutic intervention

Novel Therapeutic Effects of Pterosin B on Ang II-Induced Cardiomyocyte Hypertrophy.

Pathological cardiac hypertrophy is characterized by an abnormal increase in cardiac muscle mass in the left ventricle, resulting in cardiac dysfunction. Although various therapeutic approaches are being continuously developed for heart failure, several studies have suggested natural compounds as novel potential strategies. Considering relevant compounds, we investigated a new role for Pterosin B for which the potential life-affecting biological and therapeutic effects on cardiomyocyte hypertrophy are not fully known. Thus, we investigated whether Pterosin B can regulate cardiomyocyte hypertrophy induced by angiotensin II (Ang II) using H9c2 cells. The antihypertrophic effect of Pterosin B was evaluated, and the results showed that it reduced hypertrophy-related gene expression, cell size, and protein synthesis. In addition, upon Ang II stimulation, Pterosin B attenuated the activation and expression of major receptors, Ang II type 1 receptor and a receptor for advanced glycation end products, by inhibiting the phosphorylation of PKC-ERK-NF-κB pathway signaling molecules. In addition, Pterosin B showed the ability to reduce excessive intracellular reactive oxygen species, critical mediators for cardiac hypertrophy upon Ang II exposure, by regulating the expression levels of NAD(P)H oxidase 2/4. Our results demonstrate the protective role of Pterosin B in cardiomyocyte hypertrophy, suggesting it is a potential therapeutic candidate.

Involvement of the mitochondrial nuclease EndoG in the regulation of cell proliferation through the control of reactive oxygen species

The apoptotic nuclease EndoG is involved in mitochondrial DNA replication. Previous results suggested that, in addition to regulate cardiomyocyte hypertrophy, EndoG could be involved in cell proliferation. Here, by using in vivo and cell culture models, we investigated the role of EndoG in cell proliferation. Genetic deletion of Endog both in vivo and in cultured cells or Endog silencing in vitro induced a defect in rodent and human cell proliferation with a tendency of cells to accumulate in the G1 phase of cell cycle and increased reactive oxygen species (ROS) production. The defect in cell proliferation occurred with a decrease in the activity of the AKT/PKB-GSK-3β-Cyclin D axis and was reversed by addition of ROS scavengers. EndoG deficiency did not affect the expression of ROS detoxifying enzymes, nor the expression of the electron transport chain complexes and oxygen consumption rate. Addition of the micropeptide Humanin to EndoG-deficient cells restored AKT phosphorylation and proliferation without lowering ROS levels. Thus, our results show that EndoG is important for cell proliferation through the control of ROS and that Humanin can restore cell division in EndoG-deficient cells and counteracts the effects of ROS on AKT phosphorylation.

Miro1 as a novel regulator of hypertrophy in neonatal rat cardiomyocytes

Cardiac hypertrophy is an adaptive response to manage an excessive cardiac workload and maintain normal cardiac function. However, sustained hypertrophy leads to cardiomyopathy, cardiac failure, and death. Adrenergic receptors play a key role in regulating cardiac function under normal and pathological conditions. Mitochondria are responsible for 90% of ATP production in cardiomyocytes. Mitochondrial function is dynamically regulated by fusion and fission processes. Changes in mitochondrial dynamics and metabolism are central issues in cardiac hypertrophy. Stimulating cardiomyocytes with adrenergic agonists generates hypertrophy and increases mitochondrial fission, which in turn is associated with decreased ATP synthesis. Miro1 is a mitochondrial outer membrane protein involved in mitochondrial dynamics and transport in neurons. The objective of this work was to evaluate whether Miro1 regulates cardiomyocyte hypertrophy through changes in mitochondrial dynamics. In neonatal rat ventricular myocytes, we showed that phenylephrine induced cardiomyocyte hypertrophy and increased Miro1 mRNA and protein levels. Moreover, alpha-adrenergic stimulation provoked a mitochondrial fission pattern in the cardiomyocytes. Miro1 knockdown prevented both the cardiomyocyte hypertrophy and mitochondrial fission pattern. Our results suggest that Miro1 participates in phenylephrine-induced cardiomyocyte hypertrophy through mitochondrial fission.

Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis.

Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics. The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload-induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy. Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.

P4998Novel GATA4 binding proteins, RbAp48/46, regulate cardiomyocyte hypertrophy with depending on the phosphorylate State of GATA4

Abstract Introduction Cardiac hypertrophy is being recognized as a critical event during the development of heart failure. A zinc finger protein GATA4 associates with an intrinsic histone acetyltransferase p300 and regulates myocardial transcriptional activities in response to hypertrophic stimuli. Here, we show that Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48, RbAp46) are novel components of p300/GATA4 complex. Both RbAp48 and 46 form a repressor complex with HDACs and has been implicated in chromatin remodeling and transcriptional repression. However, the precise functional relationships among p300, GATA4, RbAp48, and RbAp46 remain unknown. Hypothesis We assessed the hypothesis that RbAp48/46 form a functional complex with p300/GATA4 and regulated hypertrophic responses in cardiomyocytes. Methods and results IP-WB using nuclear extract from rat heart demonstrated that GATA4 formed a complex with RbAp48, RbAp46, HDAC1, and HDAC2. GST pull down assay using recombinant proteins showed that GATA4 physically interacted with both RbAp48 and RbAp46 but not HDAC1 and HDAC2. Deletion mutant assay revealed that N-terminal domain of GATA4 interacted with RbAp48/46. In HEK293T cell, overexpression of RbAp48/46 recruited HDAC1/2 to GATA4, inhibited p300-induced GATA4 acetylation and suppressed p300/GATA4-dependent ANF and ET-1 promoter activations. Conversely, the knockdown of RbAp48/46 reversed these changes. Although overexpression of HDAC1/2 did not change p300/GATA4-induced these promoter activities, co-expression of HDAC1 or HDAC2 with RbAp48/46 enhanced RbAp48/46-mediated inhibitory actions. In cardiomyocytes, overexpression of RbAp48/46 significantly inhibited phenylephrine (PE)-induced GATA4 acetylation, activation of ANF and ET-1 promoters, and cardiomyocyte hypertrophy. The knockdown of RbAp48/46 reversed these changes. Moreover, the knockdown of HDAC1/2 deteriorated PE-induced hypertrophy-responsive events and did not exhibit RbAp48/46-induced inhibitory actions. Finally, MEK1/ERK-mediated S105 phosphorylation of GATA4 by PE stimulus induced the dissociation of RbAp48/46 with GATA4, the increase of p300-induced GATA4-acetylation, the synergistic activation of ANF and ET-1 promoters with p300/GATA4, and the decrease of RbAp48/46 recruitments onto the GATA element of the ANF promoter. Conversely, PD98059, a MEK1 inhibitor, treatment inhibited GATA4-phosphorylation and these changes. Conclusion In this study, we demonstrate that RbAp48/46 mediate the binding between GATA4 and HDAC1/2 and regulate p300/GATA4 axis. The phosphorylation of S105 GATA4 has a critical role on the dissociation of GATA4/RbAp48/46/HDAC repressor complex, the formation of 300/GATA4 activator complex, and the increase of GATA4 acetylation and hypertrophic responses. These findings suggest that RbAp48/46 may regulate hypertrophic responses involved in modulating the posttranslational modification crosstalk of GATA4. Acknowledgement/Funding This work was supported by JSPS KAKENHI Grant.

KLF15-Wnt–Dependent Cardiac Reprogramming Up-Regulates SHISA3 in the Mammalian Heart

BackgroundThe combination of cardiomyocyte (CM) and vascular cell (VC) fetal reprogramming upon stress culminates in end-stage heart failure (HF) by mechanisms that are not fully understood. Previous studies suggest KLF15 as a key regulator of CM hypertrophy. ObjectivesThis study aimed to characterize the impact of KLF15-dependent cardiac transcriptional networks leading to HF progression, amenable to therapeutic intervention in the adult heart. MethodsTranscriptomic bioinformatics, phenotyping of Klf15 knockout mice, Wnt-signaling-modulated hearts, and pressure overload and myocardial ischemia models were applied. Human KLF15 knockout embryonic stem cells and engineered human myocardium, and human samples were used to validate the relevance of the identified mechanisms. ResultsThe authors identified a sequential, postnatal transcriptional repression mediated by KLF15 of pathways implicated in pathological tissue remodeling, including distinct Wnt-pathways that control CM fetal reprogramming and VC remodeling. The authors further uncovered a vascular program induced by a cellular crosstalk initiated by CM, characterized by a reduction of KLF15 and a concomitant activation of Wnt-dependent transcriptional signaling. Within this program, a so-far uncharacterized cardiac player, SHISA3, primarily expressed in VCs in fetal hearts and pathological remodeling was identified. Importantly, the KLF15 and Wnt codependent SHISA3 regulation was demonstrated to be conserved in mouse and human models. ConclusionsThe authors unraveled a network interplay defined by KLF15–Wnt dynamics controlling CM and VC homeostasis in the postnatal heart and demonstrated its potential as a cardiac-specific therapeutic target in HF. Within this network, they identified SHISA3 as a novel, evolutionarily conserved VC marker involved in pathological remodeling in HF.

Sp1-induced LncRNA CTBP1-AS2 is a novel regulator in cardiomyocyte hypertrophy by interacting with FUS to stabilize TLR4.

Cardiomyocyte hypertrophy is a heart response adapting to increasing cardiac load. Prolonged cardiomyocyte hypertrophy indicates a higher risk of heart failure or even cardiac death. Long noncoding RNAs have been largely reported to modulate human diseases. CTPB1-AS2 is a newly discovered lncRNA reported as an oncogene in papillary thyroid cancer, but its function in cardiomyocyte hypertrophy has never been probed. Toll-like receptor 4 (TLR4) is recognized to play important roles in cardiomyocyte hypertrophy. The present study aimed to investigate the role of CTBP1-AS2 in cardiomyocyte hypertrophy. First, we discovered the low expression of CTBP1-AS2 in normal heart tissues in GETx database. Then, we established cardiomyocyte hypertrophy models on mice and cardiomyocytes through transverse aortic constriction surgery and Ang II treatment. We revealed the up-regulation of CTBP1-AS2 and TLR4 in cardiomyocyte hypertrophy models. Also, we confirmed the positive correlation between CTBP1-AS2 and TLR4 expressions in cardiomyocyte hypertrophy tissues. Loss-of-function assays confirmed that inhibiting CTBP1-AS2 attenuated the Ang II-induced cardiomyocyte hypertrophy. Mechanism research showed that CTBP1-AS2 stabilized TLR4 mRNA by recruiting FUS. Rescue assays certified that CTBP1-AS2 regulated cardiomyocyte hypertrophy through TLR4. Finally, we found Sp1 as an upstream activator for CTBP1-AS2 expression. In conclusion, our study uncovered CTBP1-AS2 as a novel regulator of cardiomyocyte hypertrophy through regulating TLR4, providing a new potential treatment target for cardiomyocyte hypertrophy.

Forkhead box protein O1 (FoxO1) is required for exercise-induced, but not PI3K-induced, physiological cardiac hypertrophy

Background: Physiological cardiac hypertrophy is induced by exercise and is mediated by phosphoinositide 3-kinase (PI3K)/Akt signalling. The transcription factor forkhead box protein O1 (FoxO1) is an Akt substrate that regulates cardiomyocyte hypertrophy in vitro, however its role in physiological hypertrophy is unknown.