Neonatal Rat Ventricular Cardiomyocytes Research Articles

P1599Sodium channel gene therapy with dual adeno-associated viral vectors

Abstract Background Sodium channel gene therapy carries significant potential for treatment of acquired and inherited arrhythmias. However, delivery is challenging to the length of the transgene that exceeds the packaging capacity of Adeno-Associated Virus (AAV), the most advanced long-term gene therapy vector to date. To overcome this issue, we have developed dual AAV vectors for the delivery of the skeletal muscle sodium channel 1 (SkM1). Purpose To achieve cardiac delivery of SkM1 and other large therapeutic genes using dual AAV vectors. Methods Dual AAV vectors were constructed, containing SkM1 gene fragments that allow reconstruction in the target cells by trans-splicing and recombination. An oversized single AAV vector containing SkM1 served as a control. HEK293T cells and neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with dual AAV vectors or oversized single AAV vector at an MOI of 50,000 per vector. Etoposide and Teniposide were added 2h before transduction to improve the efficiency. SkM1 mRNA and protein were isolated 3 days post transduction and the expression was detected by RT-qPCR and Western blot. Results Robust full-length SkM1 protein expression was detected in both HEK cells and NRVMs transduced by dual AAV vectors while no expression was detected in the control. Transduction with dual AAV vectors also showed significantly higher SkM1 mRNA expression in both cell types (4 to 29-fold) comparing to oversized single AAV vector. Moreover, a relatively high level of SkM1 mRNA expression was achieved in NRVM; 22-fold higher than the native cardiac sodium channel. Conclusion Efficient delivery and expression of SkM1 was successfully achieved in vitro by hybrid dual AAV vectors. This approach supports the application of SkM1 and other sodium channel antiarrhythmic gene therapies. In vivo validation and functional testing are currently in on-going.

1181Role of rho-mdia1 signaling to maintain cardiac function in response to pressure overload in mice

Abstract Background Cardiac hypertrophy is a compensatory response to pressure overload that leads to heart failure. Recent studies have shown that Rho signaling has crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. Rho is rapidly activated in response to pressure overload, but the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain unclear. Objective To identify the essential roles of mDia1 (Rho-effector molecule) in pressure overload-induced ventricular hypertrophy. Methods and results Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10–12 weeks old) were subjected to transverse aortic constriction (TAC) or a sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography and the pressure-volume loop indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Microarray gene expression profiling showed that the induction of immediate early genes due to the TAC operation was significantly lower in mDia1KO mice than WT mice, as was the activation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK). We examined the role of mDia1 in neonatal rat ventricular cardiomyocytes (NRVMs) exposed to mechanical stress. The siRNA-mediated silencing of mDia1 attenuated stretch-induced ERK and FAK phosphorylation, and gene expression of c-fos. Importantly, loss of mDia1 suppressed an increase in the F/G-actin ratio in response to pressure overload in the mice. In addition, increases in nuclear myocardin-related transcription factors (MRTFs) and serum response factor (SRF) were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs. Conclusions Rho-mDia1, through actin dynamics, plays critical roles in pressure overload-induced hypertrophy by regulating ERK and FAK phosphorylation and the transcriptional activity of MRTF-SRF.

P5395Exercise-induced miR-17-3p Prevents Pathological Myocardial Dysfunction

Abstract Background Exercise is beneficial for pathological myocardial dysfunction and heart failure. We previously reported that miR-17-3p contributed to exercise-induced cardiac growth and protected against cardiac ischemia/reperfusion injury. However, if exercise-induced miR-17-3p can prevent pathological myocardial dysfunction is undetermined. Purpose To investigate if exercise-induced miR-17-3p can prevent pathological myocardial dysfunction. Methods The miR-17-3p expression was examined in phenylephrine (PE, 50μmol/L, 48 h) and angiotensin II (Ang II, 1μmol/L, 48 h) treated primary neonatal rat ventricular cardiomyocytes (NRCM), and the myocardium of thoracic aortic constriction (TAC, 4weeks) or angiotensin II (1.3 mg/kg/day, 4weeks) induced cardiac hypertrophy murine model. miR-17-3p transgenic mice were generated and subjected to TAC or Ang II to investigate the effect of miR-17-3p overexpression in attenuating pathological cardiac dysfunction by echocardiography. Cell area and hypertrophic genes were determined by Wheat Germ Agglutinin (WGA) staining and qRT-PCRs. In addition, cell area and hypertrophic genes (ANP, BNP, β-MHC) were determined by immunolabeling and qRT-PCR in NRCM after the transfection of miR-17-3p mimic or inhibitor. Furthermore, functional rescue assays were performed to identify phosphatase and tensin homolog (PTEN) as a target gene of miR-17-3p, and myocyte enhancer factor 2C (MEF2C) as an upstream regulator of miR-17-3p in pathological cardiac hypertrophy. Results The expression of miR-17-3p was significantly decreased in the heart from TAC or Ang II mouse model, and in PE or Ang II-induced cardiomyocyte hypertrophy model. miR-17-3p overexpression mice displayed improved cardiac function and reduced cardiac hypertrophy after TAC or Ang II treatment in vivo. miR-17-3p mimic significantly attenuated PE or Ang II-induced cardiomyocyte hypertrophy in vitro. Based on functional rescue experiments, PTEN was identified as a direct target of miR-17-3p that mediated its protective effects in cardiac hypertrophy. Moreover, MEF2C was identified as a negative upstream regulator of miR-17-3p involved in the control of cardiac hypertrophy. Proposed mechanism of miR-17-3p Conclusion Exercise-induced miR-17-3p can prevent pathological myocardial dysfunction by targeting PTEN. MEF2C was an upstream regulator of miR-17-3p. Targeting MEF2C/miR-17-3p/ PTEN represents a novel therapeutic strategy for pathological myocardial dysfunction. Acknowledgement/Funding The grants from National Natural Science Foundation of China (81722008, 91639101 and 81570362 to JJ Xiao)

MicroRNA-122 promotes cardiomyocyte hypertrophy via targeting FoxO3

Objective—microRNAs (miRNAs) have emerged as novel regulators for cardiac hypertrophy. MiR-122 is well recognized as a promising therapeutic target in liver disease, whereas recently plays important roles in cardiovascular diseases. The current study aimed to explore the effect of miR-122 on the pathogenesis of cardiomyocyte hypertrophy. Methods and results—The cardiomyocytes isolated from the neonatal rat ventricular cardiomyocytes (NRVMs) were collected and performed to Angiotensin II (Ang II) administration. We observed a dramatically increased miR-122 expression in hypertrophic cardiomyocytes. The NRVMs transfected with miR-122 mimic or negative control were utilized for the functional analysis. Overexpression of miR-122 increased the morphology size of cardiomyocytes and promoted the pro-hypertrophic genes expression, whereas downregulated the anti-hypertrophic genes upon Ang II stimulation. The bioinformatics analysis and luciferase reporter assays exhibited that miR-122 directly targeted FoxO3 and attenuated its gene level in hypertrophic cardiomyocytes. Moreover, miR-122 negatively regulated FoxO3 but promoted calcineurin signaling pathway activation. Importantly, FoxO3 overexpression significantly reversed the effect of miR-122 on cardiomyocyte hypertrophy. Conclusion—Collected, our finding demonstrated that miR-122 accelerated the development of cardiomyocytes hypertrophy partially via directly regulation of FoxO3-calcineurin pathway.

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 924: The Role of Protein Kinase C Isoforms in Cardiomyocyte Hypertrophy

Background: Protein kinase C (PKC) has been linked to cardiomyocyte hypertrophy. However, the exact role of different PKC isoforms in mediating the hypertrophic response remains controversial and both classical and novel isoforms have been suggested to play a critical role. The aim of this study was to characterize the role of PKC isoforms in cardiomyocyte hypertrophy using pharmacological tools and high content screening (HCS). Methods: Neonatal rat ventricular cardiomyocytes (NRVCs) isolated from 1-3 days old Wistar rats and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) differentiated from iPS(IMR90)-4 line were exposed to endothelin-1 (ET-1) and PKC modulators for 48 h followed by fixing and immunofluorescence staining for nuclei, α-actinin and F-actin. Imaging and morphological analysis of thousands of cells were carried out using automated HCS platform. Results: NRVCs exposed to ET-1 (100 nM) or PKC agonists (HMI-1b11 at 10 μM or bryostatin-1 at 10 nM) or inhibitor of classical PKC isoforms, Gö6976 (1 μM), showed a hypertrophic phenotype measured by increased area and number of alpha-actinin and F-actin fibers in HCS. In contrast, inhibition of all PKC isoforms with Gö6983 (1 μM) attenuated ET-1 and PKC agonist-induced hypertrophy similarly to mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (10 μM). In turn, these HCS parameters did not show ET-1 or PKC agonist -induced hypertrophy in hiPSC-CMs. However, qRT-PCR analysis of the expression of NPPA and NPPB genes showed hypertrophic responses to ET-1 and PKC agonists also in hiPSC-CMs. Conclusions: These results confirm the central role of PKC in cardiomyocyte hypertrophy and indicate that either classical PKC isoforms have a direct anti-hypertrophic role in cardiomyocytes or, when classical PKCs are inhibited, the balance is moved towards activation of novel isoforms, which mediate the hypertrophic response. Due to the morphological differences between NRVCs and hiPSC-CMs, similar phenotypic analysis readouts cannot be reliably applied to compare hypertrophic responses in these cell types. These findings may help in developing new drugs that target specific PKC isoforms to treat cardiac hypertrophy.

Abstract 147: Screening for Developmental and Injury-Induced Genes That Facilitate Heart Regeneration

The adult mammalian heart has a limited capacity to regenerate upon injury, such as myocardial infarction (MI). MI in adult mouse hearts results in a reduced cardiac function, due to extensive loss of cardiomyocytes and formation of scar tissue by activated fibroblasts. In contrast, the newborn postnatal day 1 (P1) mouse heart is able to regenerate and restore its cardiac function following injury. However, this regenerative ability dramatically declines after P7. The molecular properties and mechanisms that facilitate neonatal heart regeneration remain unknown. To gain further insight into the realm of cardiac regeneration, our lab performed transcriptome analysis by bulk and single cell RNA-seq on P1 and P8 hearts at various time points following MI surgery. We also performed enhancer profiling by H3K27ac ChIP-seq. Coupling of both these approaches has uncovered two different gene sets that that may promote the regenerative ability of neonatal hearts: developmental genes and injury-induced genes. Amongst the neonatal-specific developmental genes, Igf2bp3, a RNA-binding protein, and Hic2, a transcriptional regulator, have been shown to promote neonatal rat ventricular cardiomyocyte (NRVM) proliferation and are currently being characterized for their involvement in neonatal heart regeneration by using gain-of-function and loss-of-function in vivo approaches. Amongst the injury-induced genes, we identified mitogens secreted from macrophages that promote NRVM proliferation. This is in line with the differential immune response elicited by P1 hearts upon MI-injury. We have also shown that an epicardial secreted factor in P1 hearts can enhance human umbilical vein endothelial cell tube formation, indicative of its role in angiogenesis. In summary, by using a multi-layered genomic analysis, we identified various genes that are involved in different cellular responses during neonatal heart regeneration. This comprehensive analysis of the neonatal regenerative response provides insights into the molecular basis of neonatal heart regeneration, and can potentially identify genes that can be modulated to facilitate adult heart regeneration.

Abstract 781: Doxorubicin-Induced Cardiotoxicity and Novel GATA4-Targeted Compounds

Doxorubicin (DOX) is a widely used anticancer drug which, unfortunately, induces dose-related cardiotoxicity. A deep understanding of DOX’s toxicity is still unclear, partly because most in vitro studies have evaluated the effects of short-term high-dose DOX treatments. Thus, a more precise model of DOX cardiotoxicity is needed to more accurately simulate clinical scenario. The first aim of this study was to establish an in vitro model of long-term low-dose administration of DOX utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The second aim was to investigate if novel GATA4-targeted compounds can protect CMs from DOX toxicity. Acute toxicity was studied in both primary neonatal rat ventricular cardiomyocytes (NRVCs) and hiPSC-CMs. The cells were exposed to 0.1-3 μM DOX for 48 h, after which the lactate dehydrogenase and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out. To study long-term toxicity, hiPSC-CMs were exposed to 100 nM DOX for up to 14 days. MTT assay and high-content analysis were carried out after 4, 7 and 14-day exposures. The effect of the test compound was studied by exposing the cells simultaneously to DOX and the compound. A 48-hour exposure to DOX at 1 and 3 μM concentrations induced more than 62% reductions in both NRVC and hiPSC-CM viability, whereas a 14-day exposure to 100 nM DOX induced a 26% reduction in hiPSC-CM viability. A 4-day exposure to 100 nM DOX induced a 3.1-fold increase in the percentage of cells positive for pro-B-type natriuretic peptide (proBNP) compared to control. When the cells were exposed simultaneously to 100 nM DOX and the test compound (10 μM), the percentage of proBNP+ cells increased only 1.4-fold compared to control. However, over 14-day exposure the compound at 10 μM concentration reduced hiPSC-CM viability 50% compared to control. A long-term exposure of hiPSC-CMs can be utilized as an in vitro model to investigate the mechanisms of delayed DOX-induced cardiotoxicity. The GATA4-targeted test compound exhibited cardioprotective potential against subacute DOX toxicity. Over chronic exposure the compound was, however, toxic to CMs, indicating that further structural optimization is required to develop non-toxic derivatives.

Abstract 917: Actively Blocking Nuclear Localization of G Protein-Couple Receptor 5 Blunts Hypertrophy and Heart Failure After TAC

Introduction: The induction of heart failure (HF) after aortic banding (TAC) in mice encompasses aberrations in gene regulation, leading to maladaptive cardiac hypertrophy and contractile dysfunction. Previously, our lab has shown that the nuclear targeting of GRK5, which is required for maladaptive hypertrophy, is controlled by its N-terminal (NT) calmodulin-binding domain. Hypothesis: We hypothesize that expression of a peptide encoding the NT of GRK5 will act as a GRK5-calmodulin inhibitor preventing the nuclear accumulation of GRK5 and inhibit its pathological activities. Methods: Neonatal rat ventricular cardiomyocytes (NRCM) overexpressing GRK5-NT or control virus were subjected to α-adrenergic stress with 50μM phenylephrine (PE) or vehicle for 30 minutes. Subcellular fractionation and western blotting were used to detect nuclear GRK5. Novel cardiac-specific transgenic mice were generated expressing HA-tagged GRK5-NT and these mice along with their control mice were characterized 8 weeks after TAC or sham surgery. Echocardiography, fibrosis, and lung and heart weight to tibia length metrics were used to evaluate hypertrophy and heart failure response post-TAC. Other cohorts were subjected to TAC for 1 week followed by Western blotting to assess acute signaling and nuclear accumulation of GRK5. Results: In vitro, expression of the GRK5-NT peptide reduced PE-induced nuclear accumulation of GRK5. In both NRCM and tissue, the GRK5-NT peptide was found to bind to calmodulin, which disrupts the endogenous calmodulin-GRK5 interaction. GRK5-NT expression abrogates nuclear GRK5 accumulation following hypertrophic stress in vitro and in vivo. Following 8 weeks of TAC, cardiac function, hypertrophy, and pulmonary congestion were significantly attenuated in GRK5-NT mice compared to NLC mice. Conclusion: We show that the GRK5-NT peptide binds to calmodulin and prevents nuclear accumulation of GRK5 following hypertrophic stress. Our in vivo data show that cardiac-restricted expression of GRK5-NT prevents HF by partially restoring cardiac dysfunction, hypertrophy, and pulmonary congestion following 8 weeks of TAC. Further in vitro work will reveal the calmodulin-dependence of GRK5-NT and the peptide’s effects on G-protein signaling.

Abstract 280: The Effect of YAP/TAZ on Glycolysis and Mitochondrial Oxidative Phosphorylation in Cardiomyocytes

The heart requires a high rate of adenosine triphosphate (ATP) production to maintain healthy cardiac function and viability. Anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the main metabolic pathways by which ATP is generated in mammalian cells, including cardiomyocytes. Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), which are major downstream effectors of the Hippo signaling pathway, play an important role in the regulation of heart size and cellular homeostasis of cardiomyocytes. However, interplay between YAP/TAZ and cardiac energy metabolism has been poorly understood. Here we examined the effect of YAP/TAZ on glycolysis and OXPHOS in isolated neonatal rat ventricular cardiomyocytes (NRVMs). Extracellular acidification rate (ECAR; an index of glycolysis) and oxygen consumption rate (OCR; an index of OXPHOS) of NRVMs transduced with adenoviruses harboring YAP, TAZ, or LacZ (control) for 6 days were assessed by Seahorse XFe96 analyzer. Both YAP and TAZ significantly increased not only the ECAR of basal glycolysis (1.5-fold and 1.3-fold, respectively, p<0.05), glycolytic maximal capacity (1.4-fold and 1.3-fold, respectively, p<0.05), and glycolytic reserve capacity (1.3-fold and 1.2-fold, respectively, P<0.05) (n=12-15) but also basal OCR (1.3-fold and 1.3-fold, respectively, p<0.05) (n=6-10) compared to control LacZ. Quantitative PCR analysis showed that mRNA levels of Pfkm, a key glycolytic enzyme, and subunits of protein complexes I though V in the electron transport chain are significantly increased (1.3-1.9-fold, n=5-6, p<0.05) by both YAP and TAZ compared with control. These results suggest that YAP/TAZ increase ATP production by accelerating glycolysis and OXPHOS in cardiomyocytes.

Abstract 270: Loss of S100A1 Protein Negatively Impacts Glucose Metabolism and Energy Homeostasis in Cardiomyocytes

Introduction: As a true metabolic omnivore, the heart is able to utilize different substrates for energy production. In the hypertrophied and early failing state, cardiac muscle leans towards glucose as a major carbon substrate to meet its energetic demand. Given that S100A1 gene therapy reverses the compromised energetic balance of the failing heart, we were interested in determining the specific role of the EF-hand calcium sensor S1001A1 in cardiomyocyte energy metabolism. Methods and results: To study metabolic changes in response to different carbon substrates, we used neonatal rat ventricular cardiomyocytes (NRVMs), which were subjected to electrical stimulation (2Hz) in culture and adenoviral-mediated anti-S100A1 shRNA silencing or scr-shRNA, and either fed with glucose, oleate or lactate only for 24hrs. Of note, lack of S100A1 expression only impaired energy metabolism of cardiomyocytes in response to glucose as reflected by a significantly decreased ATP content and subsequently enhanced AMP/ATP and p-AMPK/AMPK ratios with consecutive elevated p-ACC/ACC and Glut4 protein levels. In contrast, energy metabolism appeared to remain normal in response to oleate in S100A1-deprived cells. Targeted metabolomics yielded a tremendous decrease in glucose-6-phosphate levels as the first key step in glucose metabolism despite enhanced AMPK activation in glucose-fed S100A1-defiecient cardiomyocytes. Conclusion: Our novel in vitro data indicate that S100A1 protein may be crucial for cardiomyocytes to meet their energetic demand when glucose is the major substrate. Since the latter becomes the preferred substrate for functionally compromised heart over fatty acids, lack of S100A1 expression - as a result of fetal gene reprogramming - most likely impairs the ability of the heart to sufficiently utilize glucose to meet its energetic demand. These data shed new mechanistic light on the sustained therapeutic efficacy of S100A1 gene therapy of heart failure where S100A1 may be a key factor enabling the heart to sufficiently use glucose to reverse its compromised energetic state. Further studies answering these pressing questions are underway for an informed design of a best-case patient enrollment scenario for a first-in-men clinical study.

Abstract 432: Rapamycin Can Ameliorate Antiretroviral Drug Mediated Cardiotoxicity

Introduction: In current therapy, due to application of combined antiretroviral drugs (cART), HIV related mortality significantly reduced, and AIDS became a chronic disease. Although, cART treatment increases life expectancy of AIDS patients, growing AIDS population suffer due to several organ diseases and heart failure is one of the leading causes of comorbidity and mortality. Hypothesis: We tested the hypothesis that application of cART induces cardiotoxicity through inhibition of cellular protein quality control (PQC) and dysregulation of mitochondrial function. Method and results: Cellular protein quality control was analyzed using cardiac tissue samples from HIV+ patients treated with cART and healthy donors. Western blot shows that there is upregulation of autophagy marker protein LC3 II in ART treated heart compared to healthy donor. Additionally, we found that there are abnormalities in the mitochondrial oxidative phosphorylation complexes (OXOPHOS) in the ART treated patient’s heart, compared to healthy donor heart. Using neonatal rat ventricular cardiomyocytes, we tested the role of ART in cellular PQC and mitochondrial function. Our data shows that cardiomyocytes treated with the cART (Ritonavir+ Atazanavir+ abacavir+ Lamivudine) have increased expression of autophagy proteins LC3 II and P62 and higher level of pELF2α. These suggest that, the cardiomyocytes treated with ART have dysregulation of autophagy and increased ER stress. Further, we found that cardiomyocytes treated with cART have high level of mitochondrial superoxide and decreased mitochondrial membrane potential. Mitochondrial function detected in whole cells shows that in ART treated cardiomyocytes there is significantly low OCR compared to control cells. Interestingly, application of pharmacological drug rapamycin can improve the cellular PQC and mitochondrial function in ART treated cells. Conclusion: ART treatment induces cardiotoxicity through inhibition of cellular PQC and dysregulation of mitochondrial function and rapamycin might be important therapeutic drug to reduce the ART mediated cardiotoxicity.

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 120: Methamphetamine-induced Cardiomyopathy Associated With Mitochondrial Dysfunction, Cardiac Fibrosis and Hypertrophy

Introduction: Methamphetamine (METH) is one of the most commonly abused illicit drugs in the United States, exerting a range of adverse effects upon multiple organ systems. Cardiovascular complications are among the major causes of death in METH users. METH-induced cardiomyopathy is a poorly characterized disease entity as METH-induced molecular perturbations, and histopathological changes in the heart remain under-explored. Objectives: We studied histopathology in the hearts of human METH users. We also observed the histological alteration and changes in mitochondrial function in mice that received ‘binge’ administration of METH. Methods and Results: We obtained 32 autopsy heart samples from humans with positive toxicology for chronic METH use and performed Sirius Red and Masson’s Trichrome (MT) staining on left ventricular (LV) sections. Notably, chronic METH user hearts showed intense perivascular and interstitial fibrosis in LVs. ‘Binge’ METH administration in mice for 4 weeks showed an increase in heart weight-to-tibia length and increase in myocyte cross-sectional area in WGA stained LVs compared to saline-treated mice. Sirius red and MT staining also showed an increase in perivascular and interstitial fibrosis in METH mice heart. Isolated mitochondria from METH-treated mice heart showed suppressed mitochondrial bioenergetics measured by Seahorse Analyzer. Immunoblotting in heart lysates and mitochondrial fractions showed altered mitochondrial dynamics regulatory proteins expression in METH mice compared to control saline group. METH-treated cultured neonatal rat ventricular cardiomyocytes also showed suppression of mitochondrial respiration and mitochondrial network disorganization indicating a direct effect of METH on cardiomyocytes. Conclusions: We report that maladaptive cardiac fibrotic remodeling is typical in a human and pre-clinical mouse model of METH abuse. ‘Binge’ METH exposure in mice induces cardiac hypertrophy, cardiac fibrosis, and suppression of mitochondrial respiration. Thus, chronic METH use induces maladaptive cardiac remodeling associated with mitochondrial dysfunction.

Abstract 540: Sigma 1 Receptor-dependent Regulation of Mitochondrial Respiration and Function in Cardiomyocytes

Background: Sigma 1 receptor (Sigmar1) is an abundantly expressed molecular chaperone protein in cardiomyocytes. Sigmar1 ligands have been shown to have protective function in animal models of cardiac hypertrophy, heart failure, and many other cardiovascular disorders. Despite extensive studies, the physiological and molecular functions of Sigmar1 in cardiomyocytes remains elusive. Objective: We investigated the molecular function of Sigmar1 in regulating mitochondrial respiration and function in cardiomyocytes. Methods and Results: To define the molecular function of Sigmar1 in regulating mitochondrial respiration and function in cardiomyocytes, we used small interfering RNA-mediated Sigmar1 knockdown and adenovirus-mediated overexpression in cultured neonatal rat ventricular cardiomyocytes. Sigmar1 knockdown in neonatal cardiomyocytes showed significantly decreased mitochondrial respiration measured by Seahorse Analyzer. Mitochondria stained with mitoTracker green showed a significantly increased number of hyperfused mitochondria in Sigmar1 knockdown cardiomyocytes. Similarly, mitochondria isolated from Sigmar1 global knockout mouse hearts also showed decreased mitochondrial respiration and the accumulation of larger size mitochondria measured by flowcytometry. Sigmar1 overexpression in cardiomyocytes significantly increased mitochondrial respiration and also showed increased mitochondrial fragmentation. Conclusions: Our findings suggested that Sigmar1 plays an essential role in maintaining normal mitochondrial morphology, respiration, and function in cardiomyocytes.

Cardiac α-Actin (ACTC1) Gene Mutation Causes Atrial-Septal Defects Associated With Late-Onset Dilated Cardiomyopathy.

Familial atrial septal defect (ASD) has previously been attributed primarily to mutations in cardiac transcription factors. Here, we report a large, multi-generational family (78 members) with ASD combined with a late-onset dilated cardiomyopathy and further characterize the consequences of mutant α-actin. We combined a genome-wide linkage analysis with cell biology, microscopy, and molecular biology tools to characterize a novel ACTC1 (cardiac α-actin) mutation identified in association with ASD and late-onset dilated cardiomyopathy in a large, multi-generational family. Using a genome-wide linkage analysis, the ASD disease locus was mapped to chromosome 15q14 harboring the ACTC1 gene. In 15 affected family members, a heterozygous, nonsynonymous, and fully penetrant mutation (p. Gly247Asp) was identified in exon 5 of ACTC1 that was absent in all healthy family members (n=63). In silico tools predicted deleterious consequences of this variant that was found absent in control databases. Ultrastructural analysis of myocardial tissue of one of the mutation carriers showed sarcomeric disarray, myofibrillar degeneration, and increased apoptosis, while cardiac proteomics revealed a significant increase in extracellular matrix proteins. Consistently, structural defects and increased apoptosis were also observed in neonatal rat ventricular cardiomyocytes overexpressing the mutant, but not native human ACTC1. Molecular dynamics studies and additional mechanistic analyses in cardiomyocytes confirmed actin polymerization/turnover defects, thereby affecting contractility. A combined phenotype of ASD and late-onset heart failure was caused by a heterozygous, nonsynonymous ACTC1 mutation. Mechanistically, we found a shared molecular mechanism of defective actin signaling and polymerization in both cardiac development and contractile function. Detection of ACTC1 mutations in patients with ASD may thus have further clinical implications with regard to monitoring for (late-onset) dilated cardiomyopathy.

HDL protects against myocardial ischemia reperfusion injury via miR-34b and miR-337 expression which requires STAT3.

PurposeHigh density lipoprotein (HDL) protects against myocardial infarction via mechanisms that remain unclear. STAT3 (signal transducer and activator of transcription 3) plays a key role in HDL-induced cardioprotection. In the heart, microRNAs (miRNAs) are involved in ischemia reperfusion injury. We therefore investigated whether the cardioprotective effect of HDL modulates miRNAs as a downstream target of STAT3 activation.MethodsSTAT3 cardiomyocyte deficient mice (STAT3-KO) and wildtype littermates (STAT3-WT) were submitted to left coronary ligature and reperfused (IR) with or without injection of HDL. Infarct size (IS) was determined and cardiac miRNA expression was evaluated after reperfusion in sham, IR and IR+HDL hearts by microarray analysis.In vitro, neonatal rat ventricular cardiomyocytes were submitted to hypoxia with or without HDL incubation. Cell viability and miRNA expression were analysed.ResultsIn vivo, HDL reduced IS from 40.5±4.3% to 24.4±2.1% (p<0.05) in STAT3-WT mice. HDL failed to protect in STAT3-KO mice. In STAT3-WT mice, both miR-34b and miR-337 were increased in IR compared to sham and IR+HDL groups (p<0.05). These miRNAs were not modulated in STAT3-KO mice. In vitro, incubation with HDL improved cell viability against hypoxia (p<0.05). The expression of miR-34b and miR-337 was increased by hypoxia and reduced by HDL treatment (p<0.05). In cardiomyocytes transfected with miRNA mimics, HDL failed to improve cell viability against hypoxia.ConclusionsOur study, performed both in vivo and in vitro, delineates a novel cardioprotective signalling pathway activated by HDL, involving STAT3-mediated decrease of miR-34b and miR-337 expression.

Overexpression of lncRNA Gm2691 attenuates apoptosis and inflammatory response after myocardial infarction through PI3K/Akt signaling pathway.

Acute myocardial infarction is one of the most threatening disease in the world. In previous studies, numerous dysregulated lncRNAs exposed to ischemic reperfusion injury have been identified. In this differential lncRNAs, Gm2691 attracted our attention due to its high fold change. The aim of the study was to investigate the function and mechanism of lncRNA Gm2691 in ischemic reperfusion injury. AnaeroPack anaerobic system treated neonatal rat ventricular cardiomyocytes were used to analyze the function of lncRNA Gm2691 in vitro. Tunel, Caspase3, and inflammation markers were detected to evaluate apoptosis and inflammatory response. Rat acute myocardial infarction was performed to elucidate the function of lncRNA Gm2691 in vivo. The results showed that LncRNA Gm2691 improved the cardiac function and attenuated the inflammatory response in vivo. We also found that lncRNA Gm2691 reduced the apoptosis and improved cell survival rates in anaeroPack anaerobic system treated neonatal rat ventricular cardiomyocytes. Western blot analysis revealed that lncRNA Gm2691 decreased Akt and ERK1/2 activities, suggesting that lncRNA Gm2691 may functioned through Akt signaling pathway. We verified the function and mechanism of lncRNA Gm2691 and provide evidence that lncRNA Gm2691 may play important role in ischemic reperfusion injury, and understanding the precise role of Gm2691 will undoubtedly shed new light on the clinical treatment.

P38\u03b1 MAPK inhibition translates to cell cycle re-entry of neonatal rat ventricular cardiomyocytes and de novo nestin expression in response to thrombin and after apex resection

The present study tested the hypothesis that p38α MAPK inhibition leads to cell cycle re-entry of neonatal ventricular cardiomyocytes (NNVMs) and de novo nestin expression in response to thrombin and after apex resection of the neonatal rat heart. Thrombin (1 U/ml) treatment of 1-day old NNVMs did not induce cell cycle re-entry or nestin expression. Acute exposure of NNVMs to thrombin increased p38α MAPK and HSP27 phosphorylation and p38α/β MAPK inhibitor SB203580 abrogated HSP27 phosphorylation. Thrombin and SB203580 co-treatment of NNVMs led to bromodeoxyuridine incorporation and nestin expression. SB203580 (5 mg/kg) administration immediately after apex resection of 1-day old neonatal rat hearts and continued for two additional days shortened the fibrin clot length sealing the exposed left ventricular chamber. SB203580-treatment increased the density of troponin-T(+)-NNVMs that incorporated bromodeoxyuridine and expressed nuclear phosphohistone-3. Nestin(+)-NNVMs were selectively detected at the border of the fibrin clot and SB203580 potentiated the density that re-entered the cell cycle. These data suggest that the greater density of ventricular cardiomyocytes and nestin(+)-ventricular cardiomyocytes that re-entered the cell cycle after SB203580 treatment of the apex-resected neonatal rat heart during the acute phase of fibrin clot formation may be attributed in part to inhibition of thrombin-mediated p38α MAPK signalling.

Role of Bcl2-associated Athanogene 3 in Turnover of Gap Junction Protein, Connexin 43, in Neonatal Cardiomyocytes

Any pathological stress that impairs expression, turnover and phosphorylation of connexin 43 (Cx43), one of the major proteins of gap junctions, can adversely impact myocardial cell behavior, thus leading to the development of cardiac arrhythmias and heart failure. Our results in primary neonatal rat ventricular cardiomyocytes (NRVCs) show that impairment of the autophagy-lysosome pathway dysregulates degradation of Cx43, either by inhibiting lysosomal activity or suppressing the level of Bcl2-associated athanogene 3 (BAG3), a stress-induced pleiotropic protein that is involved in protein quality control (PQC) via the autophagy pathway. Inhibition of lysosomal activity leads to the accumulation of Cx43 aggregates and suppression of BAG3 significantly diminished turnover of Cx43. In addition, knock-down of BAG3 reduced the levels of Cx43 by dysregulating Cx43 protein stability. Under stress conditions, expression of BAG3 affected the state of Cx43 phosphorylation and its degradation. Furthermore, we found that BAG3 co-localized with the cytoskeleton protein, α-Tubulin, and depolymerization of α-Tubulin led to the intracellular accumulation of Cx43. These observations ascribe a novel function for BAG3 that involves control of Cx43 turnover under normal and stress conditions and potentially for optimizing communication of cardiac muscle cells through gap junctions.