Hypertrophic Response Of Cardiomyocytes Research Articles

Inhibition of N-acetylglucosaminyltransferase V alleviates diabetic cardiomyopathy in mice by attenuating cardiac hypertrophy and fibrosis

BackgroundThe pathogenesis of diabetic cardiomyopathy is closely linked to abnormal glycosylation modifications. N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the production of N-linked -1–6 branching of oligosaccharides, is involved in several pathophysiological mechanisms of many disorders, including cardiac hypertrophy and heart failure. However, the mechanism by which GnT-V regulates cardiac hypertrophy in diabetic cardiomyopathy is currently poorly understood. In this study, we investigated the role of GnT-V on myocardial hypertrophy in diabetic cardiomyopathy and elucidated the underlying mechanisms.Material and methodsStreptozotocin (STZ) was intraperitoneally injected into mice to induce diabetic cardiomyopathy. An adeno-associated virus (AAV) carrying negative control small hairpin RNA (shNC) or GnT-V-specifc small hairpin RNA (shGnT-V) was used to manipulate GnT-V expression. In our study, forty male C57BL/6J mice were randomly divided into four groups (10 mice per group): control mice with AAV-shNC, diabetic cardiomyopathy mice with AAV-shNC, control mice with AAV-shGnT-V, and diabetic cardiomyopathy mice with AAV-shGnT-V. In addition, H9C2 cells and primary neonatal cardiac fibroblasts treated with high glucose were used as a cell model of diabetes. Analysis of cardiac hypertrophy and fibrosis, as well as functional studies, were used to investigate the underlying molecular pathways.ResultsAAV-mediated GnT-V silencing dramatically improved cardiac function and alleviated myocardial hypertrophy and fibrosis in diabetic mice. In vitro experiments demonstrated that GnT-V was elevated in cardiomyocytes and induced cardiomyocyte hypertrophy in response to high glucose stimulation. GnT-V knockdown significantly reduced the expression of the integrinβ1 signaling pathway, as evidenced by decreased downstream ERK1/2 activity, which inhibited cardiomyocyte hypertrophy accompanied by reduced ANP, BNP, and β-MHC expression. Furthermore, knocking down GnT-V expression lowered the TGF-β1-Smads signaling pathway, which reduced the expression of α-SMA, collagen I, and collagen III.ConclusionsOverall, our research indicated that GnT-V may be a useful therapeutic target to treat diabetic cardiomyopathy, primarily in the inhibition of myocardial hypertrophy and fibrosis.

The ion channel Trpc6a regulates the cardiomyocyte regenerative response to mechanical stretch.

Myocardial damage caused, for example, by cardiac ischemia leads to ventricular volume overload resulting in increased stretch of the remaining myocardium. In adult mammals, these changes trigger an adaptive cardiomyocyte hypertrophic response which, if the damage is extensive, will ultimately lead to pathological hypertrophy and heart failure. Conversely, in response to extensive myocardial damage, cardiomyocytes in the adult zebrafish heart and neonatal mice proliferate and completely regenerate the damaged myocardium. We therefore hypothesized that in adult zebrafish, changes in mechanical loading due to myocardial damage may act as a trigger to induce cardiac regeneration. Based on this notion we sought to identify mechanosensors which could be involved in detecting changes in mechanical loading and triggering regeneration. Here we show using a combination of knockout animals, RNAseq and in vitro assays that the mechanosensitive ion channel Trpc6a is required by cardiomyocytes for successful cardiac regeneration in adult zebrafish. Furthermore, using a cyclic cell stretch assay, we have determined that Trpc6a induces the expression of components of the AP1 transcription complex in response to mechanical stretch. Our data highlights how changes in mechanical forces due to myocardial damage can be detected by mechanosensors which in turn can trigger cardiac regeneration.

Abstract 12567: Cardiomyocyte Hypertrophy is Regulated by P300 Binding Protein 1, a Novel Binding Protein

Background: Hypertrophic stimuli such as hemodynamic overload activate the cardiac signaling pathway that terminates in cardiomyocyte nuclei, thus changing the pattern of gene expression associated with cardiomyocyte hypertrophy. This hypertrophy eventually leads to systolic dysfunction and decompensated heart failure. Nuclear acetylation is a critical event in the activation of this hypertrophic gene expression and is mediated in part by intrinsic histone acetyltransferases (HAT) such as coactivator p300. p300-HAT activity is a potential pharmacological target for the prevention of heart failure. However, the regulation mechanism of p300-HAT activity is still unclear. In this study, we identified novel p300 binding partners by proteomic analysis and investigated the role of one of these proteins, p300 binding protein 1 (p300BP1), in the hypertrophic responses of cardiomyocytes. Methods and Results: To identify novel p300 binding proteins, cardiac-specific FLAG-p300 overexpressing mice were generated. Protein fractions from heart tissues of these mice and wild-type mice were purified with anti-FLAG-M2 agarose. Mass spectrometry analysis identified 553 novel p300 binding proteins, including kinases, phosphatases, methyltransferases, and transcriptional regulators. p300BP1 was selected for analysis. Binding was found to occur between baculovirus-expressed the recombinant genes FLAG-p300 full length and HA-p300BP1. The aa762-aa1098 fragment of p300 was required for this binding. In cultured cardiomyocytes, knockdown of p300BP1 prevented phenylephrine-induced increases in cardiomyocyte surface area and hypertrophic gene transcription factors such as ANF and BNP. Conversely, the overexpression of p300BP1 increased the surface area of cardiomyocytes and hypertrophic gene transcriptions. Furthermore, overexpression of p300BP1 significantly increased H3K9 acetylation in cultured cardiomyocytes. A reporter gene assay showed that p300BP1 enhanced p300/GATA4-mediated ANF and BNP promoter activation in HEK293T cells. Conclusions: We have identified multiple novel p300 binding proteins using a proteomics approach. p300BP1 may play an important role in p300/GATA4-dependent hypertrophic responses in cardiomyocytes.

Loss of ADAM15 Exacerbates Transition to Decompensated Myocardial Hypertrophy and Dilation Through Activation of the Calcineurin Pathway.

Myocardial hypertrophy and dilation are key features of cardiomyopathies and involve several cellular and molecular events. ADAMs (a disintegrin and metalloproteinases) are membrane-bound proteinases with diverse functions whose role in heart disease remains underexplored. ADAM15 is expressed in the heart and is downregulated in the failing human heart. We investigated the role ADAM15 in pressure overload cardiomyopathy. We assessed ADAM15 levels in myocardial specimens from patients. Its direct role in pressure overload was investigated by subjecting wildtype and Adam15-deficient mice to transverse aortic constriction (TAC). ADAM15 levels did not change in patients with concentric hypertrophy, but markedly decreased in eccentric hypertrophy and heart failure. Loss of ADAM15 alone did not cause cardiomyopathy in mice (1 year old). After TAC, Adam15-/- mice exhibited worsened eccentric hypertrophy and dilation with greater increase in hypertrophy markers (pJNK, pERK1/2; Nppb, Nppa, Myh7, Acta1) compared with wildtype-TAC. Expression of integrin-α7 (but not integrin β1) increased significantly more in Adam15-/--TAC hearts, while the interaction of these integrins with basement membrane (laminin), decreased consistent with worsened left ventricle dilation. In vitro, ADAM15 knockdown increased cardiomyocyte hypertrophy in response to mechanical stretch. Adam15-/--TAC hearts exhibited increased calcineurin activity and de-phosphorylation of nuclear factor of activated T cells. Calcineurin inhibition (cyclosporin-A) blocked the excess hypertrophy and dilation in Adam15-/--TAC mice. Proteome profiling demonstrated the increased abundance of the key proteins linked to worsened DCM in Adam15-/--TAC. This is the first report demonstrating that ADAM15 can suppress hypertrophy through regulating the integrin-laminin interaction and the calcineurin pathway.

Stem cell-derived extracellular vesicles reduce the expression of molecules involved in cardiac hypertrophy-In a model of human-induced pluripotent stem cell-derived cardiomyocytes.

Cardiac pathological hypertrophy is the major risk factor that usually progresses to heart failure. We hypothesized that extracellular vesicles (EVs), known to act as important mediators in regulating physiological and pathological functions, could have the potential to reduce the cardiac hypertrophy and the ensuing cardiovascular diseases. Herein, the effects of mesenchymal stem cell-derived extracellular vesicles (EV-MSCs) on cardiac hypertrophy were investigated. EVs were isolated from the secretome of human adipose tissue-derived stem cells (EV-ADSCs) or bone marrow-derived stem cells (EV-BMMSCs). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were stimulated with AngII and TGF-β1, in absence or presence of EVs. The results showed that exposure of hiPSC-CMs to AngII and TGF-β1 generated in vitro model of hypertrophic cardiomyocytes characterized by increases in surface area, reactive oxygen species production, protein expression of cardiac-specific biomarkers atrial natriuretic factor, migration inhibitory factor, cTnI, COL1A1, Cx43, α-SMA and signalling molecules SMAD2 and NF-kBp50. The presence of EV-ADSCs or EV-BMMSCs in the hiPSC-CM culture along with hypertrophic stimuli reduced the protein expressions of hypertrophic specific markers (ANF, MIF, cTnI, COL1A1) and the gene expressions of IL-6 molecule involved in inflammatory process associated with cardiac hypertrophy and transcription factors SMAD2, SMAD3, cJUN, cFOS with role in cardiomyocyte hypertrophic response induced by AngII and TGF-β1. The EV-ADSCs were more effective in reducing the protein expressions of hypertrophic and inflammatory markers, while EV-BMMSCs in reducing the gene expressions of transcription factors. Notably, neither EV-ADSCs nor EV-BMMSCs induced significant changes in cardiac biomarkers Cx43, α-SMA and fibronectin. These different effects of stem cell-derived EVs could be attributed to their miRNA content: some miRNAs (miR-126-3p, miR-222-3p, miR-30e-5p, miR-181b-5p, miR-124-3p, miR-155-5p, miR-210-3p hsa-miR-221-3p) were expressed in both types of EVs and others only in EV-ADSCs (miR-181a-5p, miR-185-5p, miR-21-5p) or in EV-BMMSCs (miR-143-3p, miR-146a-5p, miR-93-5p), some of these attenuating the cardiac hypertrophy while others enhance it. In conclusion, in hiPSC-CMs the stem cell-derived EVs through their cargo reduced the expression of hypertrophic specific markers and molecules involved in inflammatory process associated with cardiac hypertrophy. The data suggest the EV potential to act as therapeutic mediators to reduce cardiac hypertrophy and possibly the subsequent cardiovascular events.

GDH promotes isoprenaline-induced cardiac hypertrophy by activating mTOR signaling via elevation of α-ketoglutarate level.

Numerous studies reveal that metabolism dysfunction contributes to the development of pathological cardiac hypertrophy. While the abnormal lipid and glucose utilization in cardiomyocytes responding to hypertrophic stimuli have been extensively studied, the alteration and implication of glutaminolysis are rarely discussed. In the present work, we provide the first evidence that glutamate dehydrogenase (GDH), an enzyme that catalyzes conversion of glutamate into ɑ-ketoglutarate (AKG), participates in isoprenaline (ISO)-induced cardiac hypertrophy through activating mammalian target of rapamycin (mTOR) signaling. The expression and activity of GDH were enhanced in cultured cardiomyocytes and rat hearts following ISO treatment. Overexpression of GDH, but not its enzymatically inactive mutant, provoked cardiac hypertrophy. In contrast, GDH knockdown could relieve ISO-triggered hypertrophic responses. The intracellular AKG level was elevated by ISO or GDH overexpression, which led to increased phosphorylation of mTOR and downstream effector ribosomal protein S6 kinase (S6K). Exogenous supplement of AKG also resulted in mTOR activation and cardiomyocyte hypertrophy. However, incubation with rapamycin, an mTOR inhibitor, attenuated hypertrophic responses in cardiomyocytes. Furthermore, GDH silencing protected rats from ISO-induced cardiac hypertrophy. These findings give a further insight into the role of GDH in cardiac hypertrophy and suggest it as a potential target for hypertrophy-related cardiomyopathy.

Piezo1 is the cardiac mechanosensor that initiates the cardiomyocyte hypertrophic response to pressure overload in adult mice

Pressure overload-induced cardiac hypertrophy is a maladaptive response with poor outcomes and limited treatment options. The transient receptor potential melastatin 4 (TRPM4) ion channel is key to activation of a Ca2+/calmodulin-dependent kinase II (CaMKII)-reliant hypertrophic signaling pathway after pressure overload, but TRPM4 is neither stretch-activated nor Ca2+-permeable. Here we show that Piezo1, which is both stretch-activated and Ca2+-permeable, is the mechanosensor that transduces increased myocardial forces into the chemical signal that initiates hypertrophic signaling via a close physical interaction with TRPM4. Cardiomyocyte-specific deletion of Piezo1 in adult mice prevented activation of CaMKII and inhibited the hypertrophic response: residual hypertrophy was associated with calcineurin activation in the absence of its usual inhibition by activated CaMKII. Piezo1 deletion prevented upregulation of the sodium–calcium exchanger and changes in other Ca2+ handling proteins after pressure overload. These findings establish Piezo1 as the cardiomyocyte mechanosensor that instigates the maladaptive hypertrophic response to pressure overload, and as a potential therapeutic target.

The polyunsaturated fatty acids, EPA and DHA, ameliorate myocardial infarction-induced heart failure by inhibiting p300-HAT activity in rats

While the cardioprotective functions of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and omega-3 unsaturated fatty acids have been previously demonstrated, little is known about their effects on cardiomyocyte hypertrophy. In this study, we compared the effects of EPA and DHA on hypertrophic responses in cardiomyocytes and development of heart failure in rats with myocardial infarction (MI). Both EPA and DHA significantly suppressed phenylephrine- and p300-induced cardiomyocyte hypertrophy, transcription of hypertrophy response genes, and acetylation of histone H3K9 in cardiomyocytes. EPA and DHA directly inhibited p300-histone acetyltransferase activity (IC50: 37.8 and 30.6 μM, respectively). Further, EPA- and DHA-induced allosteric inhibition of histones and competitive inhibition of acetyl-CoA, and significantly prevented p300-induced hypertrophic responses. Rats with moderate MI (left ventricular fractional shortening [FS] <40%) were randomly assigned to three groups, namely, vehicle (saline), EPA (1 g/kg), and DHA (1 g/kg). One week after the operation, rats were orally administrated with test agents for 6 weeks. Echocardiographic analysis demonstrated that both EPA and DHA treatments preserved FS and prevented MI-induced left ventricular remodeling. Furthermore, EPA and DHA significantly suppressed the MI-induced increase in myocardial cell diameter, perivascular fibrosis, mRNA levels of hypertrophic markers, fibrosis, and acetylation of histone H3K9. The effects on hypertrophic responses and the development of heart failure were not different between EPA and DHA groups. Both EPA and DHA suppressed hypertrophic responses and the development of heart failure to the same extent through the inhibition of p300-HAT activity.

Loss of talin in cardiac fibroblasts results in augmented ventricular cardiomyocyte hypertrophy in response to pressure overload.

Pressure overload of the heart is characterized by concentric hypertrophy and interstitial fibrosis. Cardiac fibroblasts (CFs) in the ventricular wall become activated during injury and synthesize and compact the extracellular matrix, which causes interstitial fibrosis and stiffening of the ventricular heart walls. Talin1 (Tln1) and Talin2 (Tln2) are mechanosensitive proteins that participate in focal adhesion transmission of signals from the extracellular environment to the actin cytoskeleton of CFs. The aim of the present study was to determine whether the removal of Tln1 and Tln2 from CFs would reduce interstitial fibrosis and cardiac hypertrophy. Twelve-week-old male and female Tln2-null (Tln2-/-) and Tln2-null, CF-specific Tln1 knockout (Tln2-/-;Tln1CF-/-) mice were given angiotensin-II (ANG II) (1.5 mg/kg/day) or saline through osmotic pumps for 8 wk. Cardiomyocyte area and measures of heart thickness were increased in the male ANG II-infused Tln2-/-;Tln1CF-/- mice, whereas there was no increase in interstitial fibrosis. Systolic blood pressure was increased in the female Tln2-/-;Tln1CF-/- mice after ANG II infusion compared with the Tln2-/- mice. However, there was no increase in cardiac hypertrophy in the Tln2-/-;Tln1CF-/- mice, which was seen in the Tln2-/- mice. Collectively, these data indicate that in male mice, the absence of Tln1 and Tln2 in CFs leads to cardiomyocyte hypertrophy in response to ANG II, whereas it results in a hypertrophy-resistant phenotype in female mice. These findings have important implications for the role of mechanosensitive proteins in CFs and their impact on cardiomyocyte function in the pathogenesis of hypertension and cardiac hypertrophy.NEW & NOTEWORTHY The role of talins has been previously studied in cardiomyocytes; however, these mechanotransductive proteins that are members of the focal adhesion complex have not been examined in cardiac fibroblasts previously. We hypothesized that loss of talins in cardiac fibroblasts would reduce interstitial fibrosis in the heart with a pressure overload model. However, we found that although loss of talins did not alter fibrosis, it did result in cardiomyocyte and ventricular hypertrophy.

Ecklonia stolonifera Okamura Extract Suppresses Myocardial Infarction-Induced Left Ventricular Systolic Dysfunction by Inhibiting p300-HAT Activity.

Ecklonia stolonifera Okamura extract (ESE) has been reported to have various bioactive effects, but its effects on cardiovascular disease have not yet been investigated. First, primary neonatal rat cultured cardiomyocytes were treated with ESE and stimulated with phenylephrine (PE) for 48 h. ESE (1000 µg/mL) significantly suppressed PE-induced cardiomyocyte hypertrophy, hypertrophy-related gene transcription, and the acetylation of histone H3K9. An in vitro p300-HAT assay indicated that ESE directly inhibited p300-HAT activity. Next, one week after myocardial infarction (MI) surgery, rats (left ventricular fractional shortening (LVFS) < 40%) were randomly assigned to three groups: vehicle (saline, n = 9), ESE (0.3 g/kg, n = 10), or ESE (1 g/kg, n = 10). Daily oral administration was carried out for 8 weeks. After treatment, LVFS was significantly higher in the ESE (1 g/kg) group than in the vehicle group. The ESE treatments also significantly suppressed MI-induced increases in myocardial cell diameter, perivascular fibrosis, hypertrophy- and fibrosis-related gene transcription, and the acetylation of histone H3K9. These results suggest that ESE suppressed both hypertrophic responses in cardiomyocytes and the development of heart failure in rats by inhibiting p300-HAT activity. Thus, this dietary extract is a potential novel therapeutic strategy for heart failure in humans.

Multimerization of the GATA4 transcription factor regulates transcriptional activity and cardiomyocyte hypertrophic response.

The activation of the GATA-binding factor 4 (GATA4) transcription factor induces cardiac hypertrophy and heart failure. The multimerization of transcription factors often plays an important role in the regulation of transcriptional activity. Here, we report that the GATA4 transcription factor forms a homomultimer and that residues 308-326 of GATA4 are necessary for its multimerization. The acetylation of GATA4 by the transcriptional co-activator p300 induces the multimerization of GATA4 and activates its DNA binding activity. In addition, we found that the suppression of GATA4 multimerization did not reduce its acetylation, but repressed GATA4/p300-induced gene transcription. Furthermore, the inhibition of GATA4 multimerization suppressed phenylephrine (PE)-induced hypertrophic response in cardiomyocytes. This study demonstrates that the multimerization of GATA4 during the p300-induced acetylation of GATA4 activates the transcription of hypertrophic response genes, which leads to cardiomyocyte hypertrophy. Therefore, the inhibition of GATA4 homomultimerization could serve as a potential therapeutic strategy for the development of novel drugs against heart failure.

Involvement of phospholipase C in the norepinephrine-induced hypertrophic response in Cardiomyocytes

Norepinephrine (NE) is known to mediate cardiomyocyte hypertrophy through the G protein coupled a1 -adrenoceptor (a1 -AR) and the activation of the phosphoinositide-specific phospholipase C (PLC). Since the by-products of PLC activity are important downstream signal transducers for cardiac hypertrophy, the role of and the regulatory mechanisms involved in the activation of PLC isozymes in cardiac hypertrophy are highlighted in this review. The discussion is focused to underscore PLC in different experimental models of cardiac hypertrophy, as well as in isolated adult and neonatal cardiomyocytes treated with NE. Particular emphasis is laid concerning the a1 -AR-PLC-mediated hypertrophic signalling pathway. From the information provided, it is evident that the specific activation of PLC isozymes is a primary signalling event in the a1 -AR mediated response to NE as well as initiation and progression of cardiac hypertrophy. Furthermore, the possibility of PLC involvement in the perpetuation of cardiac hypertrophy is also described. It is suggested that specific PLC isozymes may serve as viable targets for the prevention of cardiac hypertrophy in patient population at-risk for the development of heart failure.

Abstract 10978: Ecklonia Stolonifera Okamura Extract Suppresses Cardiac Hypertrophy and Systolic Dysfunction Through P300-HAT Inhibition

Introduction: Heart failure is the leading cause of death in the world. Cardiomyocyte hypertrophy is observed during the development of heart failure, suggesting that its inhibition is a potential target for the prevention and treatment of heart failure. In this study, we screened a natural compound library using cultured cardiomyocytes and found that Ecklonia stolonifera Okamura extract (ESE) suppressed cardiomyocyte hypertrophy. Hypothesis: ESE, a perennial brown alga, has been reported to have various bioactive effects, such as antioxidant and anti-inflammatory activity, but its effect on heart failure is still unclear. Therefore, we investigated whether ESE has an inhibitory effect on cardiomyocyte hypertrophic response and on the progression of heart failure in post-myocardial infarction (MI) rats. Methods and Results: First, primary cultured cardiomyocytes from neonatal rats were treated with ESE and then stimulated with phenylephrine (PE) for 48 hours. ESE (1000 μg/mL) significantly suppressed PE-induced increases in cardiomyocyte surface area, hypertrophic response gene transcription, and acetylation of histone H3K9. An in vitro p300-HAT assay indicated that ESE directly inhibited p300-HAT activity (IC50: 505 μg/mL). Next, one week after the ligation of the left anterior descending artery, rats with moderate MI (left ventricular fractioning shorting (LVFS) &lt;40%) were randomly assigned to three groups: vehicle (saline) (n=9), ESE (0.3 g/kg) (n=10), or ESE (1 g/kg) (n=10). Daily oral administration was repeated for 8 weeks. After treatment, LVFS was significantly higher in the ESE (1 g/kg) group (23.3 ± 0.7%, p&lt;0.05) than in the vehicle group (16.6 ± 1.3%). ESE treatment significantly suppressed MI-induced increases both in myocardial cell diameter and in the mRNA levels of hypertrophic response genes. ESE also inhibited MI-induced perivascular fibrosis and the acetylation of histone H3K9. Conclusion: These results suggest that ESE suppresses both hypertrophic responses in cardiomyocytes and the development of heart failure by inhibiting p300-HAT activity. Further studies are needed to clarify the effectiveness of ESE for heart failure therapy.

The human cell atlas of the hypertrophic heart reveals impaired inter-cellular cross-talks

Abstract Introduction The pathophysiology of cardiac hypertrophy is multifactorial and is accompanied by the dysregulation of various signaling pathways contributing to cardiac dysfunction and heart failure. While the hypertrophic response of cardiomyocytes (CM) has been extensively studied, the interplay of CMs with the non-parenchymal cells in the heart is less explored. Here, we apply high-resolution transcriptomic analysis on single cell level allowing the identification of cellular responses and communication in the hypertrophic human heart. Results We analyzed single nuclei RNA sequencing data of cardiac tissues from five patients with aortic stenosis and cardiac hypertrophy and 13 matched healthy subjects. Bioinformatic data analysis of 88,536 nuclei followed by clustering led to the identification of specific heterogenic cell type signatures. Analyzing cell type specific gene expression signatures, we found the expected up-regulation of the cardiac stress MYH7 (4.15-fold), CMYA5 (4.89-fold) and XIRP2 (6.13-fold) in cardiomyocytes (CM) (all p&amp;lt;0.0001). Fibroblasts showed increased expression of genes associated with fibrosis and activation markers such as periostin (POSTN; 6.84-fold, p&amp;lt;0.0001). In-silico analysis of intercellular communication pathways revealed a striking downregulation of ligand-receptor interactions between CMs and other cells in hypertrophic compared to healthy controls indicating that CMs are less responsive to signals from fibroblasts and endothelial cells (ECs) in the hypertrophied heart. Particularly, CM showed reduced expression of receptor tyrosine kinases of the Ephrin family and FGF-family members. Specifically, Ephrin-B1 was significantly downregulated in CMs of the hypertrophic hearts (0.01-fold, p&amp;lt;0.0001). The down-regulation of Ephrin-B1 was additionally validated on protein level using histological sections of hypertrophic cardiomyopathy patients (n=6) versus healthy controls (n=5) (0.66-fold, p=0.02). In-vitro studies in neonatal cardiomyocytes further demonstrated that activation of the Ephrin-B1 receptor by the agonist Ephrin-B2 induced cardioprotective effects. Thus, Ephrin-B2 inhibited phenylephrine (PE) induced Nppb expression by 0.775-fold (vs. PE) and hypertrophic growth (0.774-fold reduction of cell size vs. PE). Similar findings were observed in PE-stimulated human cardiac organoids, which showed a 0.58-fold reduction of size in response to Ephrin-B2 treatments compared to PE alone. Conclusion Investigating the cross-talk in cardiac hypertrophy reveals novel disturbed communication signatures, with a striking reduction in the intercellular communication pathways of CMs. Reduced expression of receptors of the Ephrin family, particularly Ephrin-B1, in CM may prevent cardioprotective signaling by the agonist Ephrin-B2, which is highly expressed in ECs, leading to inhibition of cardioprotective cross-talk between ECs and CMs in the hypertrophic heart. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Dr. Rolf M. Schwiete StiftungDie Deutsche ForschungsgemeinschaftGerman Center for Cardiovascular Research

Abstract P381: Ecklonia Stolonifera Okamura Extract Suppresses Hypertrophic Responses In Cardiomyocytes And Development Of Heart Failure By Inhibiting P300-HAT Activity

Introduction: Heart failure is the leading cause of death in the world. Cardiomyocyte hypertrophy is observed during the development of heart failure, suggesting that its inhibition is a potential target for the prevention and treatment of heart failure. In this study, we screened a natural compound library using cultured cardiomyocytes and found that Ecklonia stolonifera Okamura extract (ESE) suppressed cardiomyocyte hypertrophy. ESE, a perennial brown alga, has been reported to have various bioactive effects, such as antioxidant and anti-inflammatory activity, but its effect on heart failure is still unclear. Therefore, we investigated whether ESE has an inhibitory effect on cardiomyocyte hypertrophic response and on the progression of heart failure in post-myocardial infarction (MI) rats. Methods and Results: First, primary cultured cardiomyocytes from neonatal rats were treated with ESE and then stimulated with phenylephrine (PE) for 48 hours. ESE (1000 μg/mL) significantly suppressed PE-induced increases in cardiomyocyte surface area, hypertrophic response gene transcription, and acetylation of histone H3K9. An in vitro p300-HAT assay indicated that ESE directly inhibited p300-HAT activity (IC50: 505 μg/mL). Next, one week after the ligation of the left anterior descending artery, rats with moderate MI (left ventricular fractioning shorting (LVFS) &lt;40%) were randomly assigned to three groups: vehicle (saline) (n=9), ESE (0.3 g/kg) (n=10), or ESE (1 g/kg) (n=10). Daily oral administration was repeated for 8 weeks. After treatment, LVFS was significantly higher in the ESE (1 g/kg) group (23.3 ± 0.7%, p&lt;0.05) than in the vehicle group (16.6 ± 1.3%). Next, the hearts were isolated and histological analysis, evaluation of gene transcription, and measurement of histone H3K9 acetylation. were performed. ESE treatment significantly suppressed MI-induced increases both in myocardial cell diameter and in the mRNA levels of hypertrophic response genes. ESE also inhibited MI-induced perivascular fibrosis and the acetylation of histone H3K9. Conclusion: These results suggest that ESE suppresses both hypertrophic responses in cardiomyocytes and the development of heart failure by inhibiting p300-HAT activity. Further studies are needed to clarify the effectiveness of ESE for heart failure therapy.

Abstract 115: Cell-type-specific Gene Expression And Transcriptional Networks Reveal Adamts2 As A Powerful Regulator Of Cardiac Homeostasis During Heart Failure

Background: Heart failure (HF) is a highly heterogeneous disorder characterized by the interactions of multiple genetic and environmental factors as well as the interaction of different cell types in the heart. Although reductionistic approaches have successfully identified many genes involved in HF, heritability studies suggest that many genes have resisted discovery through these approaches. By utilizing cell-type-specific gene expression paired with transcriptomic data from a large cohort of mice, we sought to identify important drivers of HF using a systems genetics approach. Methods and Results: Mice from 93 unique inbred lines of the Hybrid Mouse Diversity Panel were given 30 ug/g/day of isoproterenol for three weeks via osmotic minipump to induce heart failure. Transcriptomes were generated from these mice and the weighted Maximal Information Component Analysis (wMICA) algorithm was applied to generate transcriptomic gene networks. Cardiomyocytes and Fibroblasts were isolated from both control and isoproterenol-treated adult C57BL/6J hearts using a Langendorff apparatus (n=3 per sex/treatment) and transcriptomes were generated. Significantly differentially expressed genes were identified using DESEQ2 and used to query the wMICA-derived network, identifying the gene Adamts2 as a potential regulator of cardiac hypertrophy. Follow-up in vitro and in vivo work has demonstrated that Adamts2 knockdown significantly blunts the hypertrophic effect of isoproterenol on cardiomyocytes while simultaneously reducing fibroblast proliferation and increasing apoptosis as measured by TUNEL staining. Careful examination of the gene network reveals evidence of paracrine signaling between cardiomyocytes and fibroblasts and suggests a key trans-cell-type role of Adamts2 in the regulation of HF after catecholamine stimulation. Conclusion: Co-expression network algorithms combined with cell-type-specific transcriptomics identified Adamts2 as a driver of HF. Adamts2 plays an important role via paracrine signaling in the proliferative response of fibroblasts and the hypertrophic response of cardiomyocytes to catecholamines. Further mechanistic analysis of Adamts2 will further reveal its role in the progression of heart failure.

Leucine-rich repeat kinase-2 deficiency protected against cardiac remodelling in mice via regulating autophagy formation and degradation

IntroductionLeucine-rich repetitive kinase-2 (LRRK2) is a Parkinson's disease-related gene that also participates in many inflammatory diseases. However, the functional role of LRRK2 in cardiovascular disease is not clear. ObjectiveIn this study, we aimed to elucidate the role of LRRK2 in cardiac remodelling under pressure overload. MethodsAortic banding surgery was performed to induce cardiac remodelling in a LRRK2 knockout mouse model. A cardiomyocyte remodelling model was established by phenylephrine (PE) stimulation in neonatal rat cardiomyocytes. ResultsLRRK2 was upregulated in remodelled mouse hearts and cardiomyocytes. Cardiac hypertrophy, fibrosis and dysfunction were ameliorated in LRRK2 knockout mice. LRRK2 silencing protected against the PE-induced cardiomyocyte hypertrophic response, while LRRK2 over-expression worsened the PE-induced hypertrophic response in cardiomyocytes. Decreased autophagy was observed in remodelled cardiomyocytes, whereas LRRK2 silencing increased autophagy levels and LRRK2 overexpression reduced autophagy levels. The autophagy inhibitors 3-MA, bafilomycin and chloroquine reversed the protective effects of LRRK2 deficiency. The autophagy activator rapamycin reversed the deleterious effects of LRRK2 overexpression. We found that LRRK2 inhibited Bcl-2 phosphorylation, thus decreasing the phosphorylation of Beclin1. The protective effects of LRRK2 knockout were partly counteracted by Beclin1(+/−) in vivo and Beclin1 silencing in vitro. We also observed an interaction between LRRK2 and Rab7, an autolysosome degradation-associated protein, which caused Rab7 downregulation. Rab7 knockdown almost completely reversed LRRK2 silencing-induced protection of cardiomyocytes ConclusionLRRK2 deficiency protected against cardiac remodelling under pressure overload by increasing Bcl-2/Beclin1 and Rab7-regulated autophagy levels in the heart.

JMJD6 inhibits NF-κB Activation by Demethylating R149 of p65 Subunit to Attenuate Isoproterenol-Induced Cardiac Hypertrophy

Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. However, the mechanisms underlying pathological cardiac hypertrophy still remain to be elucidated, especially involving histone arginine demethylation. Here, we investigated the roles of histone arginine demethylase jumonji domain-containing protein 6 (JMJD6) in isoproterenol (ISO)-induced pathological cardiac hypertrophy. JMJD6 level was upregulated in hypertrophic and ejection fraction preserved heart failure (EFpHF) rat cardiac tissues, while it was decreased in ejection fraction reduced heart failure (EFrHF) patients. JMJD6 overexpression attenuated ISO-induced cardiac hypertrophy as reflecting by the reduction of cardiomyocyte surface area and hypertrophic genes expression in cardiomyocytes. Meanwhile, cardiac-specific JMJD6 overexpression protected the hearts against ISO-induced cardiac hypertrophy, fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) promoted ISO-induced hypertrophic responses in cardiomyocytes. We further demonstrated that JMJD6 interacts with nuclear factor kappa B (NF-κB) p65 in cytoplasm and reduces the nucleus level of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 binds to p65 and demethylate p65 at arginine (R) 149 to inhibit the nuclear translocation of p65, and then inactivated NF-κB signaling, thereby protecting against pathological cardiac hypertrophy. We also found that JMJD6 demethylates histone H3R8, which may a new histone substrate of JMJD6. These findings indicate that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

Metformin suppresses phenylephrine-induced hypertrophic responses by inhibiting p300-HAT activity in cardiomyocytes

IntroductionHeart failure is the final pathway for a wide spectrum of myocardial stress, including hypertension and myocardial infarction. However, the potential effects of metformin on cardiac hypertrophy are still unclear. PurposeThe purpose of this study was to investigate whether metformin leads to suppression of hypertrophic responses in cardiomyocytes. Methods and resultsTo investigate whether metformin inhibited p300-histone acetyltransferase (HAT), we performed an in vitro HAT assay. Metformin directly inhibited p300-mediated acetylation of histone-H3K9. To examine the effects of metformin on hypertrophic responses, cardiomyocytes prepared from neonatal rats were treated with metformin and stimulated with saline or phenylephrine (PE), a α1-adrenergic agonist for 48 h. PE stimulus showed an increase in cell size, myofibrillar organization, expression of the endogenous atrial natriuretic factor and brain natriuretic peptide genes, and acetylation of histone-H3K9 compared with saline-treated cells. These PE-induced changes were inhibited by metformin. Next, to examine the effect of metformin on p300-mediated hypertrophy, cardiomyocytes were transfected with expression vector of p300. Metformin significantly suppressed p300-induced hypertrophic responses and acetylation of histone-H3K9. ConclusionsThe study demonstrates that metformin can suppress PE-induced and p300-mediated hypertrophic responses. Metformin may be useful for the treatment of patients with diabetes and heart failure.

Gender-specific characteristics of hypertrophic response in cardiomyocytes derived from human embryonic stem cells.

Introduction: Gender-specific phenotypes of the heart were reported with respect to both physiology and pathology. While most differences were associated with the sex hormones, differential expression of genes received special attention, particularly X-Y chromosomes’ genes.Methods: Here, we compared cardiogenesis by gene expression analysis of lineage specific markers and X-Y chromosomes’ genes, during in vitro differentiation of XY and XX human embryonic stem cells (hESC), in a hormone-free setup. Results: Downregulation of pluripotency marker (NANOG) and upregulation of cardiac mesoderm and progenitor markers (GATA4, TBX5, NKX2.5, ISL1) was remained temporally similar in differentiating XY and XX hESCs. Isoproterenol treatment of XY and XX hESC-derived cardiomyocytes (hESCCM) induced hypertrophy in a sex-specific manner, with female cardiomyocytes showing response at higher isoproterenol concentration and a later time point of differentiation. Interestingly, KDM5C as an X-linked gene, was markedly upregulated in both hypertrophied male and female cardiomyocytes. Conclusion: Collectively, our results indicated a temporally identical cardiogenesis, but more susceptibility of XY hESC-CM to hypertrophic stimulus in a hormone-free condition.