Hypertrophic Agonists Research Articles

LncRNA ZNF593-AS inhibits cardiac hypertrophy and myocardial remodeling by upregulating Mfn2 expression.

lncRNA ZNF593 antisense (ZNF593-AS) transcripts have been implicated in heart failure through the regulation of myocardial contractility. The decreased transcriptional activity of ZNF593-AS has also been detected in cardiac hypertrophy. However, the function of ZNF593-AS in cardiac hypertrophy remains unclear. Herein, we report that the expression of ZNF593-AS reduced in a mouse model of left ventricular hypertrophy and cardiomyocytes in response to treatment with the hypertrophic agonist phenylephrine (PE). In vivo, ZNF593-AS aggravated pressure overload-induced cardiac hypertrophy in knockout mice. By contrast, cardiomyocyte-specific transgenic mice (ZNF593-AS MHC-Tg) exhibited attenuated TAC-induced cardiac hypertrophy. In vitro, vector-based overexpression using murine or human ZNF593-AS alleviated PE-induced myocyte hypertrophy, whereas GapmeR-induced inhibition aggravated hypertrophic phenotypes. By using RNA-seq and gene set enrichment analyses, we identified a link between ZNF593-AS and oxidative phosphorylation and found that mitofusin 2 (Mfn2) is a direct target of ZNF593-AS. ZNF593-AS exerts an antihypertrophic effect by upregulating Mfn2 expression and improving mitochondrial function. Therefore, it represents a promising therapeutic target for combating pathological cardiac remodeling.

Abstract 17387: The Ketone Body Beta-Hydroxybutyrate Attenuates Cardiomyocyte Hypertrophy Independent of BDH1-Mediated Oxidation

Previously, we and others identified evidence for increased myocardial ketone body oxidation in failing mouse and human hearts and demonstrated that increased levels of beta-hydroxybutyrate (ßOHB), the most abundant ketone body, reduced pathologic cardiac remodeling in preclinical models of heart failure. To better understand how ßOHB prevents pathologic cardiac hypertrophy, we sought to determine whether: 1) ßOHB has direct effects on cardiomyocyte (CM) hypertrophy; and 2) if ßOHB oxidation is required for this effect. CMs were isolated from hearts of adult male C57BL/6 cardiac-specific beta-hydroxybutyrate dehydrogenase 1 knockout (csBDH1KO) mice and wildtype (WT) littermate controls. csBDH1KO CMs are unable to oxidize ßOHB given that BDH1 catalyzes the first step of R-ßOHB oxidation. CMs were cultured in media containing 5.5mM glucose ± 3mM R-ßOHB in the presence or absence of the hypertrophic agonist phenylephrine (PE, 100μM). CM width and length were quantified to assess hypertrophic growth. Stimulation with PE increased CM width:length ratio compared to glucose control by 31% and 33% in WT and BDH1KO CMs, respectively. Addition of R-ßOHB in the absence of PE had no effect on CM morphology, whereas R-ßOHB limited PE-induced increases in width:length ratio to 2% and 4% in WT and BDH1KO CMs, respectively (p<0.01 vs PE without R-ßOHB). In addition, PE-induced hypertrophy was prevented in WT CMs treated with S-ßOHB (this stereoisomer cannot be oxidized). To confirm that the cultured CMs utilized ßOHB, WT and BDH1KO CMs were cultured in media containing [U- 13 C] R-ßOHB. Stable isotope tracing showed significant fractional enrichment of citrate by R-ßOHB in WT CMs with limited enrichment in BDH1KO CMs (17.8% vs 4.5%, p=0.02). These findings support a role for ßOHB-mediated attenuation of cardiac hypertrophy through direct action on CMs via a mechanism independent of BDH1-mediated oxidation. Taken together with our previous results, these findings strongly suggest that ketone bodies improve heart failure by providing both oxidation-dependent (fuel source) and oxidation-independent as yet undefined cardioprotective mechanisms.

Abstract P1173: Deciphering CITED4-dependent Signaling In The Heart Through Nascent Proteomics And Secretomics

Objective: Neuregulin-1 (Nrg1) promotes cardiomyocyte hypertrophy, survival, and cell cycle activity through ErbB signaling. It is investigated in clinical trials as a cardioprotective agent; however, its therapeutic use might be limited due to its pro-neoplastic potential for non-cardiomyocytes that makes targeted approaches necessary. High-throughput screening of hypertrophic agonists found that Nrg1 induces CITED4 (C4) expression. C4 is also upregulated in both physiological and pathological cardiac growth and necessary to prevent adverse remodeling in vivo. However, how C4 regulates Nrg1-signaling in the heart is yet unknown. Methods: We combined pulsed-SILAC labeling, click-chemistry and mass spectrometry that allows to capture immediate changes in the proteome and secretome after Nrg1 (12h and 24h) stimulation in siRNA-mediated C4-knockdown (C4KD) and control (ctr) NRVM and complemented this with established cell- and molecular biology techniques to validate and investigate cellular function and molecular signaling. Results: We confirmed dose-dependent C4 mRNA upregulation in response to Nrg1 stimulation in NRVM. Nrg1 downstream signaling through AKT was hindered in C4KD. Computational analysis comparing the nascent proteome after Nrg1 stimulation in C4KD and ctr NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo , we found significant upregulation of TGFb2 in C4KD NRVM. We next confirmed the upregulation of TGFb2 and its downstream effectors in C4KD and ctr, while adenoviral overexpression of C4 led to reduced TGFb2 expression. Additionally, we found TGFb2 secretion increased from C4KD NRVM with Nrg1 stimulation in the nascent secretome. Conditioned media from C4KD NRVM led to an increased pro-fibrotic response in cardiac fibroblasts. In conclusion, nascent proteomics and secretomics help to elucidate C4-dependent Nrg1-signaling, which may be an important contribution toward our goal to identify targetable cardioprotective pathways in the heart.

Abstract P499: Histone H4K20 Trimethylation Is Differentially Regulated In Heart Disease

It has been well established that many cardiac pathologies result from dynamic changes in gene expression and conversely that modulating key epigenetic factors in murine models is capable of preventing or abrogating ischemic injury and pathological remodeling. One epigenetic mechanism is the post-translational modification of histones, which are reversibly methylated on lysine (K) residues and can accept up to three methyl groups (Me1, Me2 and Me3). In the heart, significant changes in global levels of histone H3K4Me3 and H3K9Me3 have been previously reported to be upregulated and downregulated, respectively, during hypertrophy and failure in mice. However, the majority of post-translational modifications on histones have never been examined to quantify global abundance in the heart during disease. In particular, histone H4K20Me3 is important in heterochromatin formation and gene repression in non-cardiac cells but has never been evaluated in the heart. Therefore, we utilized cardiac tissue from three animal models of cardiac stress and employed western blotting and mass spectrometry to quantify the global abundance of total histone H4 and H4K20 methylation. We specifically evaluated tissue from mice subjected to LAD ligation, transverse aortic banding and isoproterenol infusion (via mini-osmotic pump). In addition, we also utilized primary neonatal cardiomyocytes treated with the hypertrophic agonist phenylephrine to quantify H4K20 methylation. Our data show that global levels of histone H4K20Me3 are differentially regulated in some models of cardiac dysfunction, but not all (i.e. isoproterenol infusion). In addition, we measured the abundance of histone methyltransferases and demethylases (via western blotting and qPCR) which are responsible for adding or removing this methyl mark in mouse cardiac tissue, and compared this to published data from human heart failure patients. These analyses allowed us to identify two enzymes, the methyltransferase Smyd5 and demethylase KDM7B, which are also differentially expressed in cardiac tissue during disease. Together these results are the first analysis of histone H4K20 methylation in the heart and suggest a novel role for this methylation site in the pathophysiology of cardiovascular disease.

Microtubules Orchestrate Local Translation to Enable Cardiac Growth

Cardiomyocytes are terminally differentiated cells that respond to stress by regulating their size. During hypertrophy, it is well established that cardiomyocytes activate a hypertrophic transcriptional profile and increase protein translation rates, yet how these changes in transcription and translation are coupled to the sarcomerogenesis remains unclear. Concomitantly, the microtubule network of cardiomyocytes proliferates and is dramatically altered by post-translational modifications and decoration by microtubule-associated proteins, but how the microtubule network may regulate hypertrophy is also unknown. Here, we demonstrate that microtubules are critical for proper localization of the translational machinery (mRNA, ribosomes, and nascent proteins) to the z-disk and intercalated discs of adult rat ventricular myocytes (aRVMs). Microtubule disruption or knockdown of the kinesin-1 motor, but not disruption of the actinomyosin network, causes perinuclear accumulation of the translational machinery in aRVMs. Map4 overexpression or tyrosination of the microtubule network by TTL overexpression or VASH knock down led to similar perinuclear mRNA accumulation. In neonatal rat ventricular myocytes treated with the hypertrophic agonist phenylephrine (PE), mRNA and nascent proteins are redistributed to the periphery during cell growth. Microtubule disruption led to perinuclear accumulation of the translational machinery similar to that observed in aRVMs and prevented the PE-induced hypertrophic growth. Importantly, this lack of hypertrophic growth was not due to a defect in upstream transcriptional or translational regulation as the expression of hypertrophic/fetal gene markers and the enhancement of translation rate was preserved with microtubule disruption, suggesting that localization of the translational machinery is the key factor driving hypertrophic growth. Furthermore, PE-induced hypertrophy in mice is abrograted by colchicine treatment despite augmented expression of hypertrophic/fetal gene markers and increased protein translation rates. Overall, the data demonstrate that microtubule-based trafficking of the translational machinery is critical to couple hypertrophic signaling pathways to productive cardiomyocyte growth.

Deubiquitinase Ubiquitin-Specific Protease 10 Deficiency Regulates Sirt6 signaling and Exacerbates Cardiac Hypertrophy.

BackgroundCardiac hypertrophy (CH) is a physiological response that compensates for blood pressure overload. Under pathological conditions, hypertrophy can progress to heart failure as a consequence of the disorganized growth of cardiomyocytes and cardiac tissue. USP10 (ubiquitin‐specific protease 10) is a member of the ubiquitin‐specific protease family of cysteine proteases, which are involved in viral infection, oxidative stress, lipid drop formation, and heat shock. However, the role of USP10 in CH remains largely unclear. Here, we investigated the roles of USP10 in CH.Methods and ResultsCardiac‐specific USP10 knockout (USP10‐CKO) mice and USP10‐transgenic (USP10‐TG) mice were used to examined the role of USP10 in CH following aortic banding. The specific functions of USP10 were further examined in isolated cardiomyocytes. USP10 expression was increased in murine hypertrophic hearts following aortic banding and in isolated cardiomyocytes in response to hypertrophic agonist. Mice deficient in USP10 in the heart exhibited exaggerated cardiac hypertrophy and fibrosis following pressure overload stress, which resulted in worsening of cardiac contractile function. In contrast, cardiac overexpression of USP10 protected against pressure overload‐induced maladaptive CH. Mechanistically, we demonstrated that USP10 activation and interaction with Sirt6 in response to angiotensin II led to a marked increase in the ubiquitination of Sirt6 and resulted in Akt signaling downregulation and attenuation of cardiomyocyte hypertrophy. Accordingly, inactivation of USP10 reduced Sirt6 abundance and stability and diminished Sirt6‐induced downstream signaling in cardiomyocytes.ConclusionsUSP10 functions as a Sirt6 deubiquitinase that induces cardiac myocyte hypertrophy and triggers maladaptive CH.

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.

Cymbopogon Proximus Essential Oil Protects Rats against Isoproterenol-Induced Cardiac Hypertrophy and Fibrosis.

Cardiac hypertrophy is an independent risk factor of many cardiovascular diseases. Several cardiovascular protective properties of Cymbopogon proximus have been reported. However, no reports investigating the direct effect of C. proximus essential oil on the heart are available. The goal of this study was to explore the cardioprotective effect of C. proximus on cardiac hypertrophy and fibrosis. Male albino rats were administered C. proximus essential oil in the presence or absence of hypertrophic agonist isoproterenol. Cardiac hypertrophy and fibrosis were assessed using real-time polymerase chain reaction (PCR) and histological examination. Pre- treatment of rats with C. proximus decreased the ratio of heart weight to body weight and gene expression of hypertrophy markers atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC), which were induced by isoproterenol. Moreover, C. proximus prevented the increase in gene expression of fibrosis markers procollagen I and procollagen III and alleviated the collagen volume fraction caused by isoproterenol. The pre- treatment with C. proximus essential oil conferred cardio-protection against isoproterenol- induced cardiac hypertrophy and fibrosis.

Transcriptional, Post‐transcriptional, and Post‐translational Regulation of MK5 in Cardiac Cells

BackgroundMAP kinase‐activated protein kinase‐5 (MK5) is a protein serine/threonine kinase identified as a putative substrate of both p38α/β MAPKs and atypical MAPKs ERK3 and ERK4. MK5 mRNA is detected in adult cardiac ventricular fibroblasts and myocytes whereas MK5 immunoreactivity is detected in fibroblasts but not myocytes.Little is known about the molecular mechanisms regulating MK5 in the heart. This study was to investigate the regulation of MK5 activity in cardiac fibroblasts and to determine if pro‐hypertrophic agonists such as angiotensin II (Ang II), endothelin 1 (ET1) and norepinephrine (NE) stimulate the translation of MK5 in cardiac myocytes.MethodsCardiac ventricular fibroblasts and myocytes were isolated from MK5+/+ mice and Sprague‐Dawley rats. MK5 was visualized by immunoblot assay following separation by SDS‐PAGE, 2‐dimensional gel electrophoresis, and Phos‐tag PAGE. mRNA abundance was determined by droplet digital PCR and qPCR. The subcellular localization of ERK3 and MK5 was determined by confocal immunocytofluorescence.ResultsThe copy number of MK5 mRNA was similar in both adult cardiac fibroblasts and myocytes. However, the relative expression of MK5 mRNA splice variants differed between the cell types. In both, MK5.1 was the most abundant variant whereas MK5.2 was the least. MK5.2+MK5.5 represented about 35.9% of total MK5 mRNA in cardiac fibroblasts as compared to 22.9% in myocytes. However, the variant MK5.3 was 0.1% in fibroblasts as compared to 1.4% in myocytes. Considering the variants MK5.4+MK5.5, it was 2.9% in fibroblasts and 5.7% in the myocyte. In cardiac myocytes, inhibition of the proteasome inhibition using MG132 failed to rescue MK5 immunoreactivity. Similarly, incubation with hypertrophic agonists Ang II, ET1, or NE did not result in detection of MK5 immunoreactivity. In serum‐starved cardiac myofibroblasts, MK5 immunoreactivity was primarily localized to the nucleus. Activation of MK5 involves phosphorylation at threonine‐182 (Thr182). Following serum stimulation, phospho‐MK5(Thr182) immunoreactivity was observed in the cytoplasm: phospho‐MK5(Thr182)immunoreactivity appeared to be associated with the cytoskeleton and pseudopodia. No phospho‐MK5(Thr182) immunoreactivity was detected in serum‐stimulated myofibroblasts following incubation with an inhibitor of p38αβMAPK activity, SB203580. In actively dividing fibroblasts, Phos‐tag PAGE in the presence of Mn2+, but not in the presence of EDTA, revealed multiple phospho‐forms of MK5. In addition, the Phos‐tag profile of MK5 immunoreactivity in mouse ventricular fibroblasts differed from that of rat fibroblasts.Conclusions1) The absence of MK5 immunoreactivity in cardiac ventricular myocytes was not due to rapid degradation by the proteasome, indicating differences in post‐transcriptional regulation of MK5 expression involve cell type‐specific translation. 2) In cardiac ventricular fibroblasts, phosphorylation of MK5 at threonine‐182 in response to serum stimulation requires p38a/bactivity. 3) Threonine‐182 is not the only site at which MK5 is phosphorylated in vivo.Support or Funding InformationThis study was supported by a Grant‐In‐Aid from the Heart and Stroke Foundation of Canada.

High content screening identifies licoisoflavone A as a bioactive compound of Tongmaiyangxin Pills to restrain cardiomyocyte hypertrophy via activating Sirt3

Background: Cardiac hypertrophy is a prominent feature of heart remodeling, which may eventually lead to heart failure. Tongmaiyangxin (TMYX) pills are a clinically used botanical drug for treating multiple cardiovascular diseases including chronic heart failure. The aim of the current study was to identify the bioactive compounds in Tongmaiyangxin pills that attenuate cardiomyocytes hypertrophy, and to investigate the underlying mechanism of action. Methods and Results: The anti-hypertrophy effect of TMYX was validated in isoproterenol-induced cardiac hypertrophy model in C57BL/6 mice. After TMYX treatment for 2 weeks, the heart ejection fraction and fractional shortening of the mice model was increased by approximately 20% and 15%, respectively, (p < 0.05). Besides, TMYX dose-dependently reduced the cross section area of cardiomyocytes in the angiotensin-II induced hypertrophy H9c2 model (p < 0.01). Combining high content screening and liquid chromatography mass spectrometry, four compounds with anti-cardiac hypertrophy effects were identified from TMYX, which includes emodin, licoisoflavone A, licoricone and glyasperin A. Licoisoflavone A is one of the compounds with most significant protective effect and we continued to investigate the mechanism. Primary cultures of neonatal rat cardiomyocytes were treated with a hypertrophic agonist phenylephrine (PE) in the presence or absence of licoisoflavone A. After 48 h of treatment, cells were harvested and mitochondrial acetylation was analyzed by western blotting and Image analysis. Interestingly, the results suggested that the anti-hypertrophic effects of licoisoflavone A depend on the activation of the deacetylase Sirt3 (p < 0.01). Finally, we showed that licoisoflavone A-treatment was able to decrease relative ANF and BNP levels in the hypertrophic cardiac cells (p < 0.01), but not in cells co-treated with Sirt3 inhibitors (3-TYP) (p > 0.05). Conclusion: TMYX exerts its anti-hypertrophy effect possibly through upregulating Sirt3 expression. Four compounds were identified from TMYX which may be responsible for the anti-hypertrophy effect. Among these compounds, licoisoflavone A was demonstrated to block the hypertrophic response of cardiomyocytes, which required its positive regulation on the expression of Sirt3. These results suggested that licoisoflavone A is a potential Sirt3 activator with therapeutic effect on cardiac hypertrophy.

Abstract 371: Chromatin Remodeling Mechanisms by Bromodomain PHD Finger Transcription Factor in Cardiac Hypertrophy and Heart Failure

Alteration of chromatin conformation has a profound effect on gene expression, and can lead to heart failure, which remains a major cause of death worldwide. ATP-dependent chromatin remodelers utilize the energy of ATP to remodel chromatin in specific regions of the genome. However, the mechanisms of ATP-dependent chromatin remodeling and how they connect to the genome remain poorly understood. We have discovered that the nucleosome remodeler Bromodomain PHD-finger Transcription Factor (BPTF), the largest subunit of the Nucleosome Remodeling Factor (NURF), plays an important role in cardiac hypertrophy. Our preliminary data show that BPTF is enriched in failing human hearts, a phenomenon similarly observed in mouse hearts subjected to pressure overload hypertrophy (TAC). BPTF is up-regulated in primary neonatal rat ventricular myocytes (NRVM) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) stimulated with the hypertrophic agonist phenylephrine (PE). Strikingly, specific silencing of BPTF in NRVM and hiPSC-CMs, inhibits the expression of the atrial natriuretic peptide (ANP), a marker of cardiac hypertrophy. Chromatin immunoprecipitation performed in NRVM show that BPTF binds to the ANP promoter, but not to GAPDH. Proteomics experiments performed in failing human hearts revealed a multi-protein complex formed between BPTF and novel chromatin regulators. At the level of the nucleosome, BPTF in combination with one of its partners, remodels chromatin by facilitating nucleosomes “sliding” along the DNA. This mechanism is mediated by the binding of BPTF to histone H3 trimethylated at Lysine-4 (H3K4me3), an “active” mark of transcription also up-regulated in the early phase of cardiac hypertrophy in NRVM and in mouse TAC hearts. Our study suggests a novel chromatin signature controlled by BPTF, that dictates specific gene patterns implicated in pathological cardiac hypertrophy. Our study uncovers a new epigenetic mechanism controlling pathological cardiac remodeling and heart failure.

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.

The physiological hypertrophic agonist, norepinephrine, is able to induce PLC‐mediated PI4P hydrolysis in cardiac myocytes via a pool of internal β‐adrenergic receptors

Previously, we have shown that PLC∑‐mediated phosphatidylinositol‐4‐phosphate (PI4P) can be activated by raising cAMP in cardiac myocytes, however, activation by Isoproterenol was unable to induce PI4P hydrolysis. In the accompanying abstract by Wei et al, we have demonstrated that the cell permeant β‐agonist, Dobutamine is able to induce PI4P hydrolysis. Here, we seek to determine if the physiological agonist, norepinephrine is able to induce PI4P hydrolysis, despite being membrane impermeant. In contrast to Iso, norepinephrine induced sustained PI4P hydrolysis that was blocked by the cell permeant β‐adrenergic antagonist, metoprolol (100μM). The cell impermeant βAR antagonist, sotalol (5mM) could not block norepinephrine effects, suggesting that a pool of internal β‐adrenergic receptors is responsible for NE‐mediated PI4P hydrolysis. To directly determine if internal β1‐ARs in NRVMs can be activated by norepinephrine, we monitored translocation of a YFP‐tagged mini Gs protein in β1AR transfected NRVMs2. Norepinephrine induced robust and rapid recruitment of mini Gs to both the plasma membrane, and the perinuclear region corresponding to the Golgi apparatus. Golgi localization could be reversed by Metoprolol but not Sotalol. Isoproterenol, on the other hand, induced a strong recruitment to the plasma membrane but not to the Golgi. NE‐mediated PI4P hydrolysis is also abrogated by an inhibitor of a cation transporter Oct3, Corticosterone (100μM). Oct3 has been recently shown to be required for epinephrine‐mediated β1‐AR stimulation at the Golgi envelope in HELA cells3. Taken together, these data suggest that norepinephrine can activate β‐adrenergic receptors in internal membranes, potentially at the Golgi apparatus, stimulating PI4P hydrolysis. These internal GPCRs may be responsible for cardiac hypertrophy and heart failure and targeting them may allow for the development of selective therapies which can treat heart failure with little to no effect on other cardiac functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Caspase Cleavage of Gelsolin Is an Inductive Cue for Pathologic Cardiac Hypertrophy.

BackgroundCardiac hypertrophy is an adaptive remodeling event that may improve or diminish contractile performance of the heart. Physiologic and pathologic hypertrophy yield distinct outcomes, yet both are dependent on caspase‐directed proteolysis. This suggests that each form of myocardial growth may derive from a specific caspase cleavage event(s). We examined whether caspase 3 cleavage of the actin capping/severing protein gelsolin is essential for the development of pathologic hypertrophy.Methods and ResultsCaspase targeting of gelsolin was established through protein analysis of hypertrophic cardiomyocytes and mass spectrometry mapping of cleavage sites. Pathologic agonists induced late‐stage caspase‐mediated cleavage of gelsolin. The requirement of caspase‐mediated gelsolin cleavage for hypertrophy induction was evaluated in primary cardiomyocytes by cell size analysis, monitoring of prohypertrophy markers, and measurement of hypertrophy‐related transcription activity. The in vivo impact of caspase‐mediated cleavage was investigated by echo‐guided intramyocardial injection of adenoviral‐expressed gelsolin. Expression of the N‐terminal gelsolin caspase cleavage fragment was necessary and sufficient to cause pathologic remodeling in isolated cardiomyocytes and the intact heart, whereas expression of a noncleavable form prevents cardiac remodeling. Alterations in myocardium structure and function were determined by echocardiography and end‐stage cardiomyocyte cell size analysis. Gelsolin secretion was also monitored for its impact on naïve cells using competitive antibody trapping, demonstrating that hypertrophic agonist stimulation of cardiomyocytes leads to gelsolin secretion, which induces hypertrophy in naïve cells.ConclusionsThese results suggest that cell autonomous caspase cleavage of gelsolin is essential for pathologic hypertrophy and that cardiomyocyte secretion of gelsolin may accelerate this negative remodeling response.

Mixed lineage kinase-3 prevents cardiac dysfunction and structural remodeling with pressure overload.

Myocardial hypertrophy is an independent risk factor for heart failure (HF), yet the mechanisms underlying pathological cardiomyocyte growth are incompletely understood. The c-Jun NH2-terminal kinase (JNK) signaling cascade modulates cardiac hypertrophic remodeling, but the upstream factors regulating myocardial JNK activity remain unclear. In this study, we sought to identify JNK-activating molecules as novel regulators of cardiac remodeling in HF. We investigated mixed lineage kinase-3 (MLK3), a master regulator of upstream JNK-activating kinases, whose role in the remodeling process had not previously been studied. We observed increased MLK3 protein expression in myocardium from patients with nonischemic and hypertrophic cardiomyopathy and in hearts of mice subjected to transverse aortic constriction (TAC). Mice with genetic deletion of MLK3 (MLK3-/-) exhibited baseline cardiac hypertrophy with preserved cardiac function. MLK3-/- mice subjected to chronic left ventricular (LV) pressure overload (TAC, 4 wk) developed worsened cardiac dysfunction and increased LV chamber size compared with MLK3+/+ littermates ( n = 8). LV mass, pathological markers of hypertrophy ( Nppa, Nppb), and cardiomyocyte size were elevated in MLK3-/- TAC hearts. Phosphorylation of JNK, but not other MAPK pathways, was selectively impaired in MLK3-/- TAC hearts. In adult rat cardiomyocytes, pharmacological MLK3 kinase inhibition using URMC-099 blocked JNK phosphorylation induced by neurohormonal agents and oxidants. Sustained URMC-099 exposure induced cardiomyocyte hypertrophy. These data demonstrate that MLK3 prevents adverse cardiac remodeling in the setting of pressure overload. Mechanistically, MLK3 activates JNK, which in turn opposes cardiomyocyte hypertrophy. These results support modulation of MLK3 as a potential therapeutic approach in HF. NEW & NOTEWORTHY Here, we identified a role for mixed lineage kinase-3 (MLK3) as a novel antihypertrophic and antiremodeling molecule in response to cardiac pressure overload. MLK3 regulates phosphorylation of the stress-responsive JNK kinase in response to pressure overload and in cultured cardiomyocytes stimulated with hypertrophic agonists and oxidants. This study reveals MLK3-JNK signaling as a novel cardioprotective signaling axis in the setting of pressure overload.

Role of inositol 1,4,5-trisphosphate receptor type 1 in ATP-induced nuclear Ca2+ signal and hypertrophy in atrial myocytes

Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is expressed in atrial muscle, but not in ventricle, and they are abundant in the perinucleus. We investigated the role of IP3R1 in the regulations of local Ca2+ signal and cell size in HL-1 atrial myocytes under stimulation by IP3-generating chemical messenger, ATP. Assessment of nuclear and cytosolic Ca2+ signal using confocal Ca2+ imaging revealed that IP3 generation by ATP (1 mM) induced monophasic nuclear Ca2+ increase, followed by cytosolic Ca2+ oscillation. Genetic knock-down (KD) of IP3R1 eliminated the monophasic nuclear Ca2+ signal and slowed the cytosolic Ca2+ oscillation upon ATP exposure. Prolonged application of ATP as well as other known hypertrophic agonists (endothelin-1 and phenylephrine) increased cell size in wild-type cells, but not in IP3R1 KD cells. Our data indicate that IP3R1 mediates sustained elevation in nuclear Ca2+ level and facilitates cytosolic Ca2+ oscillation upon external ATP increase, and further suggests possible role of nuclear IP3R1 in atrial hypertrophy.

MiR-376b-3p attenuates mitochondrial fission and cardiac hypertrophy by targeting mitochondrial fission factor.

Mitochondrial dysfunction contributes to the pathogenesis of cardiac hypertrophy. The disequilibrium of mitochondrial dynamic, which refers to mitochondrial fusion and fission, leads to mitochondrial morphology alteration and dysfunction. Enhanced understanding of the molecular mechanisms in depth may shed light on the therapy of the disease. In this study, we show that mitochondrial fission factor (MFF) is up-regulated upon hypertrophic agonist noradrenaline (NA) treatment. Knockdown of MFF attenuated NA induced mitochondrial fission and cardiac hypertrophy. Mitochondrial fission factor is a direct target of miR-376b-3p, which attenuated expression enhanced MFF expression through binding to its 3'UTR. Expression of miR-376b-3p weakened the fragmentation of mitochondria as well as decreased hypertrophic response through regulating MFF in NA treated neonatal rat ventricular cells (NRVCs). This study suggested that miR-376b-3p is a novel modulator affecting mitochondrial morphology through targeting MFF.

MiR-485-5p modulates mitochondrial fission through targeting mitochondrial anchored protein ligase in cardiac hypertrophy

The pathogenesis of cardiac hypertrophy is tightly associated with mitochondrial dysfunction. Disequilibrium of mitochondrial dynamic is one of the main drivers in the pathological processes during development of various cardiac diseases. However, the effect of mitochondrial dynamics on cardiac hypertrophy remains largely unclear. MicroRNAs (miRNAs) are small noncoding RNAs that can switch off expression of many genes. Mitochondrial anchored protein ligase (MAPL) is a small ubiquitin-like modifier (SUMO) E3 ligase, which is an important contributor in mitochondrial fission process. In this study, we found that hypertrophic agonist phenylephrine (PE) enhanced the expression of MAPL and promoted mitochondrial fission, while it decreased the expression of mitochondrial fusion protein2 (Mfn2) in hypertrophic cardiomyocytes. Silencing expression of MAPL by siRNA attenuated PE-induced depletion of Mfn2 and increase of mitochondrial fission as well as hypertrophic response in cultured primary cardiomyocytes. MiR-485-5p is screened as a candidate inhibitor of MAPL. Overexpression of miR-485-5p blocked mitochondrial fission and hypertrophy induced by PE through inhibiting MAPL expression and increasing the level of Mfn2 in cultured primary cardiomyocytes. In mice model of cardiac hypertrophy induced by PE, the administration of miR-485-5p agomir significantly decreased the PE induced increase in the expression of MAPL and hypertrophic markers (ANP and β-MHC) along with protection of cardiac structure and function. Together, this study exhibits a novel signaling axis composed of miR-485-5p/MAPL/Mfn2, which regulates mitochondrial machinery and cardiac hypertrophy.

Focal Adhesion Kinase-mediated Phosphorylation of Beclin1 Protein Suppresses Cardiomyocyte Autophagy and Initiates Hypertrophic Growth

Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux.

Abstract 137: Ctcf is Essential for Transcriptome Regulation in the Healthy Heart

Heart failure is a syndrome resulting from complex genetic predisposition and multiple environmental factors. It is well established that global changes in gene expression accompany the transition through hypertrophy and on to overt failure producing deterioration of function at the organ level. Unknown, however, are the global changes in chromatin structure that allows for pathological gene expression. The epigenetic factor CTCF is a ubiquitous protein thought to be involved in maintenance of chromatin microenvironments. We found that hypertrophic stimulation down-regulates CTCF in the heart, and CTCF expression is significantly correlated with heart size in disease across genetic backgrounds. To test this relationship further, we have generated an inducible cardiac-specific CTCF knockout animal. These animals quickly develop severe heart failure characterized by a dilated left ventricle, decreased ejection fraction, and reactivation of the fetal gene program. Our analysis of published CTCF ChIP-seq data performed in mouse heart has also shown CTCF binding peaks flanking the fetal genes. Furthermore, we have found that CTCF abundance at these sites changes after hypertrophic agonist isoproterenol treatment, suggesting that CTCF protects from the development of cardiac disease by regulating gene expression. We also used RNA-seq to compare the transcriptome of adult myocytes isolated from control, CTCF KO mice, and mice that underwent transverse aortic constriction (TAC) to induce heart failure. These analyses showed that CTCF depletion remodels gene expression in a manner that mimics aspects of the pathological transcriptome in heart failure. Our data demonstrate that precise control of CTCF levels is required for normal transcriptome regulation in the adult heart and that alterations in CTCF levels (either via genetic, pharmacologic or surgical techniques) lead to cardiac pathology.