Regulation Of Cardiac Hypertrophy Research Articles

FBXW7 promotes pathological cardiac hypertrophy by targeting EZH2-SIX1 signaling

F-box and WD repeat domain–containing 7 (FBXW7) is an E3-ubiquitin ligase, which serves as one of the components of the SKP1, CUL1, and F-box protein type ubiquitin ligase (SCF) complex. Previous studies reveal that FBXW7 participates in cancer, inflammation and Parkinson's disease. FBXW7 also contributes to angiogenesis of endothelial cells. However, the function of FBXW7 in cardiac homeostasis remains to elucidate. Here we identified the critical role of FBXW7 during cardiac hypertrophy in humans and rodents. Quantitative real-time PCR (qRT-PCR) and Western blot revealed that the mRNA and protein levels of FBXW7 were upregulated significantly in hypertrophic hearts in human and mouse as well as Angiotensin II (Ang II)-induced hypertrophic neonatal rat cardiomyocytes (NRCM). Gain-of-function (adenovirus) and loss-of-function (siRNA) experiments provided evidence that FBXW7 promoted Ang II-induced cardiomyocyte hypertrophy as demonstrated by the increase in the size of cardiomyocytes and overexpression of hypertrophic fetal genes myosin heavy chain 7 (Myh7) natriuretic peptide a (Nppa), brain natriuretic peptide (Nppb). Further mechanism study revealed that FBXW7 promoted the expression of sine oculis homeobox homolog 1 (SIX1) in cardiomyocytes, which relied on regulation of the stability of the histone methyltransferase EZH2 (Enhancer of zeste homolog 2). Previous work revealed the pro-hypertrophic role of the EZH2-SIX1 axis in rodents. Indeed, our genetic and pharmacological evidence showed that the EZH2-SIX1 signaling was critically involved in FBXW7 functions in Ang II-induced cardiomyocyte hypertrophy. Therefore, we identified FBWX7 as an important regulator of cardiac hypertrophy via modulating the EZH2-SIX1 axis.

Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

Heart failure remains a major cause of hospitalization and death worldwide. Heart failure can be caused by abnormalities in the epigenome resulting from dysregulation of histone-modifying enzymes. While chromatin enzymes catalyzing lysine acetylation and methylation of histones have been the topic of many investigations, the role of arginine methyltransferases has been overlooked. In an effort to understand regulatory mechanisms implicated in cardiac hypertrophy and heart failure, we assessed the expression of protein arginine methyltransferases (PRMTs) in the left ventricle of failing human hearts and control hearts. Our results show a significant up-regulation of protein arginine methyltransferase 6 (PRMT6) in failing human hearts compared to control hearts, which also occurs in the early phase of cardiac hypertrophy in mouse hearts subjected to pressure overload hypertrophy induced by trans-aortic constriction (TAC), and in neonatal rat ventricular myocytes (NRVM) stimulated with the hypertrophic agonist phenylephrine (PE). These changes are associated with a significant increase in arginine 2 asymmetric methylation of histone H3 (H3R2Me2a) and reduced lysine 4 tri-methylation of H3 (H3K4Me3) observed both in NRVM and in vivo. Importantly, forced expression of PRMT6 in NRVM enhances the expression of the hypertrophic marker, atrial natriuretic peptide (ANP). Conversely, specific silencing of PRMT6 reduces ANP protein expression and cell size, indicating that PRMT6 is critical for the PE-mediated hypertrophic response. Silencing of PRMT6 reduces H3R2Me2a, a mark normally associated with transcriptional repression. Furthermore, evaluation of cardiac contractility and global ion channel activity in live NRVM shows a striking reduction of spontaneous beating rates and prolongation of extra-cellular field potentials in cells expressing low-level PRMT6. Altogether, our results indicate that PRMT6 is a critical regulator of cardiac hypertrophy, implicating H3R2Me2a as an important histone modification. This study identifies PRMT6 as a new epigenetic regulator and suggests a new point of control in chromatin to inhibit pathological cardiac remodeling.

DSCAM-AS1 mediates pro-hypertrophy role of GRK2 in cardiac hypertrophy aggravation via absorbing miR-188-5p.

Sustained cardiac hypertrophy, as previously clarified, serves as a critical initiator of heart failure and therefore is acknowledged as an important factor for heart failure treatment. The broadly demonstrated function and participation of long non-coding RNAs (lncRNAs) in tumors are well accepted. However, the underlying mechanism implicating lncRNAs in cardiac hypertrophy is mostly unexplored and deserves to be specifically studied. The devised work was aimed to disclose the function of lncRNA DS cell adhesion molecule antisense RNA 1 (DSCAM-AS1) in angiotensin II (AngII)-induced cardiac hypertrophy. In this study, we discovered the upregulation of DSCAM-AS1 in cardiomyocytes treated with AngII by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot and qRT-PCR suggested that DSCAM-AS1 silencing attenuated the highly expressed hypertrophic biomarkers including β-myosin heavy chain (β-MHC), brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) at mRNA and protein levels. The expanded cell surface in the presence of AngII treatment area was also shrunk by DSCAM-AS1 silencing. Mechanical analysis manifested that DSCAM-AS1 sponged microRNA-188-5p to boost the pro-hypertrophy gene G protein-coupled receptor kinase 2 (GRK2) expression. Rescue experiments unveiled miR-188-5p and GRK2 managed to reverse the anti-hypertrophy impact of DSCAM-AS1 silencing. In summary, DSCAM-AS1 was identified as a positive modulator in cardiac hypertrophy through miR-188-5p decoying and GRK2 augmentation, giving rise to an enriched theoretical basis for finding a promising target in cardiac hypertrophy regulation.

LncRNA AK045171 protects the heart from cardiac hypertrophy by regulating the SP1/MG53 signalling pathway.

Hearts often undergo abnormal remodelling and hypertrophic growth in response to pathological stress. Long non-coding RNAs (LncRNAs) can change cardiac function and participate in regulation of cardiac hypertrophy. The present study aims to identify the role of AK045171 in cardiac hypertrophy and the underlying mechanism in hypertrophic cascades. Mice with cardiac hypertrophy were established through transverse aortic constriction (TAC). Cardiac hypertrophy in cardiomyocytes was induced by angiotensin II (angII). The expression of AK045171 and its target gene SP1 was examined in cardiomyocytes transfected with miRNA. The AK045171 expression level was downregulated in mice after TAC surgery. Overexpression of AK045171 attenuated cardiac hypertrophy both in vitro and in vivo. The mechanism study indicated that AK045171 binds with SP1, which promotes transcription activation of MEG3. It is suggested that overexpression of AK045171 might have clinical potential to suppress cardiac hypertrophy and heart failure.

Cilostazol alleviate nicotine induced cardiomyocytes hypertrophy through modulation of autophagy by CTSB/ROS/p38MAPK/JNK feedback loop.

Nicotine is proved to be an important factor for cardiac hypertrophy. Autophagy is important cell recycling system involved in the regulation of cardiac hypertrophy. Cilostazol, which is often used in the management of peripheral vascular disease. However, the effects of cilostazol on nicotine induced autophagy and cardiac hypertrophy are unclear. Here, we aim to determine the role and molecular mechanism of cilostazol in alleviating nicotine-induced cardiomyocytes hypertrophy through modulating autophagy and the underlying mechanisms. Our results clarified that nicotine stimulation caused cardiomyocytes hypertrophy and autophagy flux impairment significantly in neonatal rat ventricular myocytes (NRVMs), which were evidenced by augments of LC3-II and p62 levels, and impaired autophagosomes clearance. Interestingly, cathepsin B (CTSB) activity decreased dramatically after stimulation with nicotine in NRVMs, which was crucial for substrate degradation in the late stage of autophagy process, and cilostazol could reverse this effect dramatically. Intracellular ROS levels were increased significantly after nicotine exposure. Meanwhile, p38MAPK and JNK were activated after nicotine treatment. By using ROS scavenger N-acetyl-cysteine (NAC) could reverse the effects of nicotine by down-regulation the phosphorylation of p38MAPK and JNK pathways, and pretreatment of specific inhibitors of p38MAPK and JNK could restore the autophagy impairment and cardiomyocytes hypertrophy induced by nicotine. Moreover, CTSB activity of lysosome regained after the treatment with cilostazol. Cilostazol also inhibited the ROS accumulation and the activation of p38MAPK and JNK, which providing novel connection between lysosome CTSB and ROS/p38MAPK/JNK related oxidative stress pathway. This is the first demonstration that cilostazol could alleviate nicotine induced cardiomyocytes hypertrophy through restoration of autophagy flux by activation of CTSB and inhibiting ROS/p38/JNK pathway, exhibiting a feedback loop on regulation of autophagy and cardiomyocytes hypertrophy.

Long Noncoding RNA Ahit Protects Against Cardiac Hypertrophy Through SUZ12 (Suppressor of Zeste 12 Protein Homolog)-Mediated Downregulation of MEF2A (Myocyte Enhancer Factor 2A).

Long noncoding RNA (lncRNA) can regulate various physiological and pathological processes through multiple molecular mechanisms in cis and in trans. However, the role of lncRNAs in cardiac hypertrophy is yet to be fully elucidated. A mouse lncRNA microarray was used to identify differentially expressed lncRNAs in the mouse hearts following transverse aortic constriction-induced pressure overload comparing to the sham-operated samples. The direct impact of one lncRNA, Ahit, on cardiomyocyte hypertrophy was characterized in neonatal rat cardiomyocytes in response to phenylephrine by targeted knockdown and overexpression. The in vivo function of Ahit was analyzed in mouse hearts by using cardiac-specific adeno-associated virus, serotype 9-short hairpin RNA to knockdown Ahit in combination with transverse aortic constriction. Using catRAPID program, an interaction between Ahit and SUZ12 (suppressor of zeste 12 protein homolog) was predicted and validated by RNA immunoprecipitation and immunoblotting following RNA pull-down. Chromatin immunoprecipitation was performed to determine SUZ12 or H3K27me3 occupancy on the MEF2A (myocyte enhancer factor 2A) promoter. Finally, the expression of human Ahit (leukemia-associated noncoding IGF1R activator RNA 1 [LUNAR1]) in the serum samples from patients of hypertrophic cardiomyopathy was tested by quantitative real-time polymerase chain reaction. A previously unannotated lncRNA, antihypertrophic interrelated transcript (Ahit), was identified to be upregulated in the mouse hearts after transverse aortic constriction. Inhibition of Ahit induced cardiac hypertrophy, both in vitro and in vivo, associated with increased expression of MEF2A, a critical transcriptional factor involved in cardiac hypertrophy. In contrast, overexpression of Ahit significantly attenuated stress-induced cardiac hypertrophy in vitro. Furthermore, Ahit was significantly upregulated in serum samples of patients diagnosed with hypertensive heart disease versus nonhypertrophic hearts (1.46±0.17 fold, P=0.0325). Mechanistically, Ahit directly bound and recruited SUZ12, a core PRC2 (polycomb repressive complex 2) protein, to the promoter of MEF2A, triggering its trimethylation on H3 lysine 27 (H3K27me3) residues and mediating the downregulation of MEF2A, thereby preventing cardiac hypertrophy. Ahit is a lncRNA with a significant role in cardiac hypertrophy regulation through epigenomic modulation. Ahit is a potential therapeutic target of cardiac hypertrophy.

The Changes of Heart miR-1 and miR-133 Expressions following Physiological Hypertrophy Due to Endurance Training.

Objective MicroRNAs (miRNAs) play a key role in the development of the heart. Recent studies have shown that miR- 1 and miR-133 are key regulators of cardiac hypertrophy. Therefore, we aimed to evaluate the effect of an endurance training (ET) program on the expressions of these miRNAs and their transcriptional network. Materials and Methods In this experimental study, cardiac hypertrophy was induced by 14 weeks of ET for 1 hour per day, 6 days per week at 75% VO2 max). The rats (221 ± 23 g) in the experimental (n=7) and control (n=7) groups were anesthetized to evaluate heart morphology changes by echocardiography. Next, we evaluated expressions of miR-1 and miR-133, and heart and neural crest derivatives express 2 (Hand2), Mef2c, histone deacetylase 4 (Hdac4) and serum response factor (Srf) gene expressions by real-time polymerase chain reaction (PCR). Finally, the collected data were evaluated by the independent t test to determine differences between the groups Results The echocardiography result confirmed physiological hypertrophy in the experimental group that underwent ET as shown by the increased left ventricular weight/body surface area (LVW/BSA) (P=0.004), LVW/body weight (BW) (P=0.011), left ventricular diameter end-diastolic (LVDd) (P=0.003), and improvements in heart functional indexes such as fractional shortness (FS) (P=0.036) and stroke volume (SV) (P=0.002). There were significant increases in the expressions of miR-1 (P=0.001) and miR-133 (P=0.004). The expressions of Srf, Hdac4, and Hand2 genes significantly increased (P<0.001) in the experimental group Compared with the control group. The expression of Mef2c did not significantly change. ConclusionThe expressions of miR-1 and miR-133 and their target genes appeared to be involved in physiological hypertrophy induced by ET in these rats.

Progranulin deficiency leads to enhanced age-related cardiac hypertrophy through complement C1q-induced β-catenin activation

AimsAge-related cardiac hypertrophy and subsequent heart failure are predicted to become increasingly serious problems in aging populations. Progranulin (PGRN) deficiency is known to be associated with accelerated aging in the brain. We aimed to evaluate the effects of PGRN deficiency on cardiac aging, including left ventricular hypertrophy. Methods and resultsEchocardiography was performed on wild-type (WT) and PGRN-knockout (KO) mice every 3 months from 3 to 18 months of age. Compared to that of WT mice, PGRN KO mice exhibited age-dependent cardiac hypertrophy and cardiac dysfunction at 18 months. Morphological analyses showed that the heart weight to tibia length ratio and cross-sectional area of cardiomyocytes at 18 months were significantly increased in PGRN KO mice relative to those in WT mice. Furthermore, accumulation of lipofuscin and increases in senescence markers were observed in the hearts of PGRN KO mice, suggesting that PGRN deficiency led to enhanced aging of the heart. Enhanced complement C1q (C1q) and activated β-catenin protein expression levels were also observed in the hearts of aged PGRN KO mice. Treatment of PGRN-deficient cardiomyocytes with C1q caused β-catenin activation and cardiac hypertrophy. Blocking C1q-induced β-catenin activation in PGRN-depleted cardiomyocytes attenuated hypertrophic changes. Finally, we showed that C1 inhibitor treatment reduced cardiac hypertrophy and dysfunction in old KO mice, possibly by reducing β-catenin activation. These results suggest that C1q is a crucial regulator of cardiac hypertrophy induced by PGRN ablation. ConclusionThe present study demonstrates that PGRN deficiency enhances age-related cardiac hypertrophy via C1q-induced β-catenin activation. PGRN is a potential therapeutic target to prevent cardiac hypertrophy and dysfunction.

RETRACTED ARTICLE: Inhibition of miR-296-5p protects the heart from cardiac hypertrophy by targeting CACNG6

Heart often undergoes mal-remodeling and hypertrophic growth in response to pathological stress. MiRNAs can regulate the cardiac function and participate in the regulation of cardiac hypertrophy. The present study aims at identifying the role of miR-296-5p in cardiac hypertrophy and further the underlying mechanism in hypertrophic cascades. Mice with cardiac hypertrophy were established by transverse aortic constriction (TAC). Cardiac hypertrophy in cardiomyocytes was induced by angiotensin II. Expression of miR-296-5p and its target gene CACNG6 was examined in cardiomyocytes transfected by miRNA. The expression of miR-296-5p was upregulated in mice with TAC surgery. The inhibition of miR-296-5p attenuated cardiac hypertrophy both in vitro and in vivo. And dual-luciferase reporter assays showed CACNG6 was the direct target of miR-296-5p, which modulated the expression of calcium signaling. MiR-296-5p was found to aggravate cardiac hypertrophy by targeting CACNG6, which suggests inhibition of miR-296-5p might have clinical potential to suppress cardiac hypertrophy and heart failure.

MiR-625-5p Inhibits Cardiac Hypertrophy Through Targeting STAT3 and CaMKII.

Cardiac hypertrophy is an adaptive cardiac response to heart stress. Sustained cardiac hypertrophy indicates higher risk of heart failure. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been proved to be a key regulator of cardiac hypertrophy, but its mechanism remains largely unknown. Our study proposed to explore the regulatory mechanism of CaMKII in cardiac hypertrophy. We validated that CaMKII was upregulated in cardiac hypertrophy models in vivo and in vitro and that knockdown of CaMKII attenuated Ang II-induced cardiac hypertrophy in vitro. Furthermore, we demonstrated that signal transducer and activator of transcription 3 (STAT3) was highly expressed in cardiac hypertrophy and could stimulate the transactivation of CaMKII. Moreover, we predicted through TargetScan and confirmed that miR-625-5p targeted and inhibited STAT3 so as to reduce the expression of CaMKII. Interestingly, we also found that miR-625-5p directly targeted CaMKII and inhibited its expression. Rescue assays suggested that miR-625-5p attenuated Ang II-induced cardiac hypertrophy through CaMKII/STAT3. Consequently, this study elucidated that miR-625-5p inhibited cardiac hypertrophy through targeting STAT3 and CaMKII, suggesting miR-625-5p as a novel negative regulator of cardiac hypertrophy. Graphical abstract [Figure: see text].

Bioactive peptides attenuate cardiac hypertrophy and fibrosis in spontaneously hypertensive rat hearts

Alcalase potato protein hydrolysate (APPH), a nutraceutical food, might an have important role in anti-obesity activity. Recent studies from our lab indicated that APPH treatment had lipolysis stimulating activity and identified was an efficient anti-obesity diet ingredient. In this study we aim to investigate the beneficial effects of pure peptide amino acid sequences (DIKTNKPVIF (DI) and IF) from APPH supplement in the regulation of cardiac hypertrophy and fibrosis on spontaneously hypertensive rats (SHR). We examined hematoxylin and eosin staining, Masson's trichrome staining, echocardiographic parameters, serum parameters, hypertrophy, inflammation and fibrotic marker expression to demonstrate efficacy of bioactive peptides in a SHR model. There was a significant upregulation between SHR and bioactive peptides treated groups in left heart weight (LHW), LHW/WHW, LHW/Tibia, LVIDd, and LVd mass. In addition, the bioactive peptides repress the protein expression of hypertrophy markers (BNP, MYH7), inflammation (TLR-4, p-NFkB, TNF-α, IL-6), and fibrotic markers (uPA, MMP-2, TIMP1, CTGF). In summary, these results indicate that DI and IF bioactive peptides from APPH attenuate cardiac hypertrophy, inflammation and fibrosis in the SHR model.

MicroRNA-200a-3p Is a Positive Regulator in Cardiac Hypertrophy Through Directly Targeting WDR1 as Well as Modulating PTEN/PI3K/AKT/CREB/WDR1 Signaling.

Cardiac hypertrophy is an adaptive expansion of the myocardium due to the overloaded stress of heart. Recently, emerging studies have drawn a conclusion that microRNAs (miRNAs) are involved in myocardial hypertrophy and even heart failure. To figure out the role of microRNA-200a-3p (miR-200a-3p) in cardiac hypertrophy, the in vitro cardiac hypertrophy model was established in H9c2 cells using angiotensin II (Ang-II) as previously described. First of all, we observed a significant increase of miR-200a-3p expression in Ang-II-induced hypertrophic H9c2 cells. Moreover, inhibition of miR-200a-3p dramatically reversed the Ang-II-upregulated expression of hypertrophic markers (atrial natriuretic peptide, brain natriuretic peptide, and β-MHC) and the expanded cell surface area in H9c2 cells. In addition, our results indicated that miR-200a-3p directly targeted both WDR1 and phosphatase and tensin homolog (PTEN). In this regard, miR-200a-3p further activated PI3K/AKT/CREB pathway so as to intensify its negative regulation on WDR1. At length, WDR1 silence, PTEN inhibitor, and PI3K activator recovered the repressive effect of miR-200a-3p suppression on the development of cardiac hypertrophy. Jointly, our study suggested that miR-200a-3p facilitated cardiac hypertrophy by not only directly targeting WDR1 but also through modulating PTEN/PI3K/AKT/CREB/WDR1 signaling, therefore proving novel downstream molecular pathway of miR-200a-3p in cardiac hypertrophy.

Star-PAP controlled alternative polyadenylation coupled poly(A) tail length regulates protein expression in hypertrophic heart.

Alternative polyadenylation (APA)-mediated 3′-untranslated region (UTR) shortening is known to increase protein expression due to the loss of miRNA regulatory sites. Yet, mRNAs with longer 3′-UTR also show enhanced protein expression. Here, we identify a mechanism by which longer transcripts generated by the distal-most APA site leads to increased protein expression compared to the shorter transcripts and the longer transcripts are positioned to regulate heart failure (HF). A Star-PAP target gene, NQO1 has three poly(A) sites (PA-sites) at the terminal exon on the pre-mRNA. Star-PAP selects the distal-most site that results in the expression of the longest isoform. We show that the NQO1 distal-specific mRNA isoform accounts for the majority of cellular NQO1 protein. Star-PAP control of the distal-specific isoform is stimulated by oxidative stress and the toxin dioxin. The longest NQO1 transcript has increased poly(A) tail (PA-tail) length that accounts for the difference in translation potentials of the three NQO1 isoforms. This mechanism is involved in the regulation of cardiac hypertrophy (CH), an antecedent condition to HF where NQO1 downregulation stems from the loss of the distal-specific transcript. The loss of NQO1 during hypertrophy was rescued by ectopic expression of the distal- but not the proximal- or middle-specific NQO1 mRNA isoforms in the presence of Star-PAP expression, and reverses molecular events of hypertrophy in cardiomyocytes.

MicroRNAs in Cardiac Hypertrophy.

Like other organs, the heart undergoes normal adaptive remodeling, such as cardiac hypertrophy, with age. This remodeling, however, is intensified under stress and pathological conditions. Cardiac remodeling could be beneficial for a short period of time, to maintain a normal cardiac output in times of need; however, chronic cardiac hypertrophy may lead to heart failure and death. MicroRNAs (miRNAs) are known to have a role in the regulation of cardiac hypertrophy. This paper reviews recent advances in the field of miRNAs and cardiac hypertrophy, highlighting the latest findings for targeted genes and involved signaling pathways. By targeting pro-hypertrophic genes and signaling pathways, some of these miRNAs alleviate cardiac hypertrophy, while others enhance it. Therefore, miRNAs represent very promising potential pharmacotherapeutic targets for the management and treatment of cardiac hypertrophy.

Expression and bioinformatics analysis of miRNA in ISO-induced rat cardiac hypertrophy

To investigate the expression changes of miRNAs (miR199a-5P, miR206, miR133a-3P, miR499-5P) in rat model of cardiac hypertrophy induced by isoproterenol (ISO), and to explore the main signal pathways and molecular mechanisms which related to that with the way of bioinformatics. Sixteen SD male rats were randomly divided into two groups: control group and ISO model group. The rats in model group were treated with ISO (1 mg/kg) to induce cardiac hypertrophy, the rats in control group were treated with the same amount of saline, and all were injected subcutaneously at the back. After 10 days of continuous administration, interventricular septal thickness at diastole (IVSd), left ventricular posterior wall thickness at diastole (LVPWd) , left ventricular end-diastolic diameter(LVDd), and systolic function (EF%) were measured by echocardiography. Heart weight (HW) and rat body weight (BW) were weighed, and heart/body weight ratio (HW/BW) was calculated. Myocardial tissues were stained with HE, and myocardial cell surface area was measured by Image J analysis software; RT-qPCR was used to detect the expressions of 4 miRNAs in rat myocardial tissues. Targetscan, miRDB and miRwalk databases were used to predict the possible target genes of four kinds of miRNAs in rats, and FunRich software was used to analyze and predict the signal pathways related to the target genes. Compared with the control group, the IVSd and LVPWd in the model group were thickened, the LV was increased, and the EF% was decreased significantly. The HW and HW/BW were increased. The myocardial cell volume in the model group was increased significantly, the arrangement was disordered, and the cell surface area was increased; the expressions of miR199a-5P and miR206 in the model group were up-regulated by RT-qPCR (P＜0.05); the expressions of miR133a-3P and miR499-5P were down-regulated (P＜0.05). Predicted by bioinformatics application, related signal pathways which target genes of 4 miRNAs maybe involved in cardiac hypertrophy mainly are: VEGF/VEGFR signal pathway, ErbB receptor signal pathway and other signal pathways. ISO-induced cardiac hypertrophy leads to changes in miRNA expression, and bioinformatics predicts related target genes of four miRNAs involved in cardiac hypertrophy and their major signaling pathways. These studies will provide new ideas for the regulation of cardiac hypertrophy and its prevention and treatment measures.

Micro-RNAs Are Related to Epicardial Adipose Tissue in Participants With Atrial Fibrillation: Data From the MiRhythm Study.

Introduction: Epicardial adipose tissue (EAT) has been linked to incidence and recurrence of atrial fibrillation (AF), but the underlying mechanisms that mediate this association remain unclear. Circulating microRNAs (miRNAs) contribute to the regulation of gene expression in cardiovascular diseases, including AF. Thus, we sought to test the hypothesis that circulating miRNAs relate to burden of EAT.Methods: We examined the plasma miRNA profiles of 91 participants from the miRhythm study, an ongoing study examining associations between miRNA and AF. We quantified plasma expression of 86 unique miRNAs commonly expressed in cardiomyocytes using quantitative reverse transcriptase polymerase chain reaction (qPCR). From computed tomography, we used validated methods to quantify the EAT area surrounding the left atrium (LA) and indexed it to body surface area (BSA) to calculate indexed LA EAT (iLAEAT). Participants were divided into tertiles of iLAEAT to identify associations with unique miRNAs. We performed logistic regression analyses adjusting for factors associated with AF to examine relations between iLAEAT and miRNA. We performed further bioinformatics analysis of miRNA predicted target genes to identify potential molecular pathways are regulated by the miRNAs.Results: The mean age of the participants was 59 ± 9, 35% were women, and 97% were Caucasian. Participants in the highest tertile of iLAEAT were more likely to have hypertension, heart failure, and thick posterior walls. In regression analyses, we found that miRNAs 155-5p (p < 0.001) and 302a-3p (p < 0.001) were significantly associated with iLAEAT in patients with AF. The predicted targets of the miRNAs identified were implicated in the regulation of cardiac hypertrophy, adipogenesis, interleukin-8 (IL-8), and nerve growth factor (NGF) signaling.Conclusion: miRNA as well as EAT have previously been linked to AF. Our finding that iLAEAT and miRNAs 155-5p and 302a-3p are associated suggest a possible direct link to between these entities in the development and maintenance of AF. Further research is needed to study causal relationships between these biomarkers.

Abstract 800: HECT Domain Containing-3 (HECTD3) is a Novel Regulator of Cardiac Hypertrophy and Inflammation

Objective: In this study, using a Yeast two-hybrid screen, we identified the HECT domain-containing E3 ubiquitin ligase 3 (HECTD3) as one of the novel cardiac binding partners of SUMO2. Present study thus focusses on deciphering the cardiac function of HECTD3. Methods and Results: HECTD3-SUMO2 interaction was further validated by co-immunoprecipitation analysis. Interesting, adenovirus-mediated overexpression of HECTD3 in neonatal rat ventricular cardiomyocytes (NRVCMs) resulted in downregulation of endogenous as well as overexpressed SUMO2. Proteasome inhibitor MG132 treatment however inhibited HECTD3-mediated SUMO2 downregulation, suggesting SUMO2 as a likely substrate of HECTD3 in ubiquitin-proteasome system dependent protein degradation. To get further insights into cardiac substrates of HECTD3, we performed mass-spectrometry analysis of NRVCM protein lysate. Interestingly, several proteins from interferon signaling, including signal transducer and activator of transcription-1 (STAT1), were significantly downregulated by HECTD3 overexpression. Functional dissection of HECTD3-SUMO2-STAT1 interaction revealed attenuation of cellular hypertrophy and LPS mediated activation of inflammatory cascade in NRVCMs. Importantly, in vitro findings were consistent in vivo , where, AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after transverse-aortic constriction or Angiotensin II treatment. Conclusions: In conclusion, we describe here a novel cardioprotective mechanism involving the ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its sumoylation target STAT1. This “dual pathway” inhibition may be exploited for the prevention and/or therapy of cardiac hypertrophy and heart failure.

Abstract 200: Targeting the Highly Abundant Circular RNA, circHeart, in Cardiomyocytes Attenuates Pressure Overload Induced Hypertrophy

Objectives: Circular RNAs (circRNAs) are single-stranded non-coding RNAs that form a covalent closed looped structure and are generated during gene transcription. In our genome-wide analysis of cardiac circRNA expression in both human and mouse hearts we identified a highly abundant circRNA, named here as circHeart and hypothesize an important role for circHeart in cardiomyocyte physiology and homeostasis. Materials and method: We undertook FISH to visualize the localization of circHeart . Online tools have predicted multiple potential miRNA binding sites on circHeart , including the cardiac-enriched microRNA-133a (miR-133a). We assessed the interactions of circHeart with these miRNAs by luciferase and pull-down assays and performed AAV9-mediated RNAi knockdown and overexpression of circHeart in mice in vivo . In vivo phenotype was tracked by echocardiography, electrocardiogram and histological studies. Transverse aortic constriction (TAC) was also performed. Results: FISH imaging showed circHeart localized largely to the cardiomyocyte cytosol. Luciferase and reciprocal pull-down assays suggest circHeart can interact with miR-133a. MiR-133a is a well-known regulator of cardiac hypertrophy and antagomir-133a treatment has been shown to induce cardiac hypertrophy in vivo . In our studies, while AAV9 RNAi-mediated knockdown of circHeart has no apparent baseline effect in the heart it suppresses pathological cardiac hypertrophy post-TAC. More strikingly, AAV9-mediated overexpression of circHeart in vivo resulted in mouse mortality between 3-4 days post-transduction and surviving mice showed evidence of dilated cardiomyopathy. Known targets of miR-133a were concordantly modulated in the knockdown and overexpression mouse model. Conclusion: CircRNAs have huge potential role as therapeutic targets and/or disease biomarkers and represent a new avenue for cardiovascular research. Taken together, our data are consistent with a mechanistic model of circHeart sequestering miR-133a. Inhibiting circHeart abundance presents a potential target for treating cardiac hypertrophy.

Abstract 655: Protein Arginine Methyltransferase 6 Controls Cardiac Hypertrophy by Differential Arginine and Lysine Methylation of Histone H3

Heart failure remains a common cause of hospitalization and death worldwide. Heart failure can be caused by dysregulation of gene expression following abnormal expression of histone modifying enzymes. While lysine acetylation and methylation of histones have been the topic of many investigations, the role of arginine methylation in H3 has been overlooked. In an effort to understand regulatory mechanisms implicated in cardiac hypertrophy and heart failure, we assessed protein arginine methyl transferase (PRMT) members in the left ventricle of failing human hearts and control hearts. Our results show a specific up-regulation of PRMT6 mRNA in failing human hearts (5.95±2.16 fold in failing versus control, p=0.003) which also occurs in the compensatory phase of cardiac hypertrophy in mouse hearts subjected to pressure overload hypertrophy, and in neonatal rat ventricular myocytes (NRVM) stimulated with the hypertrophic agonist phenylephrine (PE). These changes are associated with a significant increase in arginine 2 asymmetric methylation of H3 (H3R2me2a) and reduced lysine 3 tri-methylation of H3 (H3K4me3) both in NRVM and in vivo . Importantly, forced expression of PRMT6 in NRVM stimulated with PE, enhances the expression of atrial natriuretic peptide (ANP). Conversely, silencing of PRMT6 reduces ANP protein expression and cell size, indicating that PRMT6 is critical for PE-mediated cardiac hypertrophy of NRVM. Also, silencing of PRMT6 reduces H3R2me2a, a mark associated with transcriptional repression. To evaluate the role of PRMT6 on cardiac contractility and global ion channel activity, we assessed contractility and global field potentials in live NRVM expressing normal and low level PRMT6 using the RTCA CardioECR system (ACEA Bioscience Inc.). Strikingly, reduced expression of PRMT6 drastically inhibits the contraction rate of NRVM, which is paralleled by a slight increase in the QT interval. All together, our results indicate that PRMT6 is a critical regulator of cardiac hypertrophy, implicating H3R2me2a as an important histone modification. Future studies investigating the specific gene programs regulated by PRMT6 are on their way. This study may help identify novel points of control to design new drugs for the treatment of heart failure.

Effect of a background Ca2+ entry pathway mediated by TRPC1 on myocardial damage of broilers with induced ascites syndrome

ABSTRACTAscites syndrome (AS) in chickens is associated with profound vascular remodelling and increased pulmonary artery pressure as well as right ventricular hypertrophy. Classical transient receptor potential cation channels (TRPCs) are key regulators of cardiac hypertrophy that act via regulation of calcium influx in mammals. We investigated whether classical transient receptor potential channels in chickens with right ventricular hypertrophy still possess this mechanism for regulating Ca2+ flux. Intravenous injection of cellulose particles was successfully used to induce AS in chickens, and tissues were examined 22 days after treatment. The chickens in the test group showed cardiac hypertrophy with oedema of the cardiac muscle and disruption of myofilaments. The right-to-total ventricle weight ratio (RV/TV), the levels of serum aspartate aminotransferase (AST) and creatine kinase (CK) of the test group were significantly higher than in the control group. Intracellular calcium levels were significantly increased in cardiomyocytes from chickens in the test group. Gene expression of TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in heart tissues from the test group showed no significant differences compared with controls. However, TRPC1 protein levels, as well as mRNA levels, were down-regulated in the heart muscle of AS chickens (P < 0.05). Although we observed an increase in calcium concentration, the expression of TRPC1 decreased in cardiac cells. We hypothesized that an increase in intracellular free calcium concentration could inversely regulate calcium channel expression.RESEARCH HIGHLIGHTSIntracellular Ca2+ levels were increased in the myocardium of AS broilers.Expression of TRPC1, which mediates calcium influx, was decreased in the myocardium of AS broilers.The relationship between intracellular Ca2+ levels and expression of TRPC1 requires further study.