Neonatal Rat Ventricular Cells Research Articles

Abstract EC103: A Novel Nuclear Role For REDD1 In Cardiac Insulin Sensitivity And Resistance

Insulin resistance (IR) is a hallmark of type II diabetes (TIID) and causes cardiac dysfunction independent of hypertension and coronary artery disease, or diabetic cardiomyopathy (DC). Impaired metabolism and energetics are a major cause of DC, however the molecular mechanisms underlying these defects remain ill-defined. Our unbiased RNA sequencing (RNA-Seq) studies identified ‘regulated in development and DNA damage (REDD)1’ as a potential critical regulator of cardiac insulin sensitivity, as a 1.5-fold increase in REDD1 expression was observed in the hearts of mice treated with acute insulin (1 hour (h)) versus vehicle (n=3). REDD1 is an insulin-sensitive, negative regulator of mTORC1 and global REDD1 deletion leads to whole body IR. Interestingly, cardiac REDD1 is also upregulated in several models of IR. Thus, we hypothesized that while REDD1 is critical for cardiac insulin sensitivity, sustained elevated levels contribute to IR via chronic inhibition of mTORC1. Our findings confirm that acute insulin stimulation or chronic high fat diet (HFD) (2 to 16 weeks) induce cardiac REDD1 expression in vivo (1.7-fold±0.1, n=6). In neonatal rat ventricular myocytes (NRVM) or Hap1 cells, insulin (1 h) or palmitate (24 h) also induce REDD1 expression (1.3-fold±0.1, n=3 or 1.6-fold±0.1, n=9, respectively), which indeed inhibits mTORC1. Notably, insulin treatment following HFD or palmitate, does not further enhance REDD1. In addition, subcellular fractionation shows that REDD1 is uniquely detected in nuclear and chromatin fractions and increases with insulin (1.5-fold±0.1, n=6). We confirmed REDD1 nuclear localization via microscopy and chromatin binding via chromatin immunoprecipitation-deep sequencing (ChIP-Seq) (n=5, pooled). In addition, we determined that REDD1 primarily binds the transcription start sites of metabolic genes and increases with insulin, corresponding to reduced gene expression. Overall, our findings suggest that insulin-inducible cardiac REDD1 exists as part of negative feedback loops that prevent the overactivation of mTORC1 and metabolic gene expression, contributing to insulin sensitivity. Further, sustained fat-induced REDD1 expression, may drive IR via chronic inhibition of mTORC1 and metabolic gene expression.

Abstract P1012: Tbx18 Drives Nodal-like Self-assembly Of Pacemaker Myocytes And Non-myocytes

Background: We have demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes (CMs) to induced pacemaker cells. Cell-cell interactions and cell-extracellular matrix (ECM) are important regulators of microtissue organization and assembly. Here, we sought to gain a finer understanding of self-organization and assembly of pacemaker microtissue with myocytes and non-myocytes driven by TBX18. Methods: 2D monolayer of neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno- GFP and Adeno-TBX18 in vitro model. AAV9-TBX18 was delivered via tail vein injection of Hcn4 (+/eGFP) transgenic mice to track the induced pacemaker myocytes. Result: TBX18-NRVMs showed more compact nodal-like aggregates with a higher density of CMs and surrounded by nonmyocytes. The number of alpha smooth muscle actin (αSMA) + myofibroblasts producing specific ECM and facilitating cell motility was increased by 190% in TBX18-NRVMs as well. Systemic delivery of AAV9-TBX18 via tail vein of mice created aggregation of de novo Hcn4+ induced pacemaker myocytes (iPMs) surrounded by αSMA+ myofibroblasts in vivo. Then, we treated pre-plated neonatal rat ventricular cells (NRVCs, including CMs and nonmyocytes) to explore the potential role of cell-cell communication and cell-ECM interaction in the formation of nodal-like aggregates through scRNA-seq. The data indicated that TBX18+ CM (28.6% % of all CMs) and TBX18-negative CMs (71.4% of all CMs) presented highly consistent transcriptomic profiles at day 3. No significant difference in Hcn4 transcript levels and the ratio of iPMs (defined as Hcn4+, Gja1 low , Nkx2.5 low , Tnni3 high , Actn2 high CM) was observed between them, as well as other pacemaker genes, suggesting cross-communication between them. Despite of TBX18+ fibroblasts (FBs) accounting for only 5% of all FBs, a higher proportion of activated myofibroblasts (39.3% vs. 25.4%) and lower proportion of quiescent FBs were observed in TBX18-NRVCs compared to control. Enrichment analysis revealed that TBX18-CMs lost ventricular specific electrical coupling and sarcomere organization (down-regulated Gja1, Cdh2, Irx3 ), but reconstructed hemidesmosome assembly and cell-ECM interaction enriched in sinoatrial node myocytes (Upregulated Lamc1, Itgb1, Plec, Des, Actn1, and Acta2 ) and activated Tgfβ signaling. Furthermore, treatment with gap junction inhibitor palmitoleic acid as well as Tgfβ receptor inhibitor A83-01 significantly suppressed the activation of quiescent fibroblasts to αSMA+ myofibroblasts and the formation of nodal-like aggregation of TBX18-NRVMs. Conclusion: TBX18 drives self-organization and nodal-like assembly of pacemaker microtissue with myocytes and non-myocytes through cell-cell communication of gap junction and Tgfβ signal.

Resveratrol, novel application by preconditioning to attenuate myocardial ischemia/reperfusion injury in mice through regulate AMPK pathway and autophagy level.

Myocardial ischemia/reperfusion injury (MI/RI) is the main cause of deaths in the worldwide, leading to severe cardiac dysfunction. Resveratrol (RSV) is a polyphenol plant‐derived compound. Our study aimed to elucidate the underlying molecular mechanism of preconditioning RSV in protecting against MI/RI. Mice were ligated and re‐perfused by the left anterior descending branch with or without RSV (30 mg/kg·ip) for 7 days. Firstly, we found that RSV pretreatment significantly alleviated myocardial infarct size, improved cardiac function and decreased oxidative stress. Furthermore, RSV activated p‐AMPK and SIRT1, ameliorated inflammation including the level of TNF‐α and IL‐1β, and promoting autophagy level. Moreover, neonatal rat ventricular myocytes (NRVMs) and H9c2 cells with knockdown the expression of AMPK, SIRT1 or FOXO1 were used to uncover the underlying molecular mechanism for the cardio‐protection of RSV. In NRVMs, RSV increased cellular viability, decreased LDH release and reduced oxidative stress. Importantly, Compound C(CpC) and EX527 reversed the effect of RSV against MI/RI in vivo and in vitro and counteracted the autophagy level induced by RSV. Together, our study indicated that RSV could alleviate oxidative stress in cardiomyocytes through activating AMPK/SIRT1‐FOXO1 signallingpathway and enhanced autophagy level, thus presenting high potential protection on MI/RI.

Geniposide suppresses NLRP3 inflammasome-mediated pyroptosis via the AMPK signaling pathway to mitigate myocardial ischemia/reperfusion injury

BackgroundNLRP3 inflammasome activation and pyroptosis play a significant role in myocardial ischemia reperfusion injury (MI/RI). Geniposide was reported to show potential therapeutic use for MI/RI with its anti-inflammatory and anti-oxidative properties. However, research on the specific mechanism of geniposide has not been reported.MethodsThe MIRI model of animal was created in male C57BL/6J mice and the hypoxia reoxygenation (H/R) model was established for the in vitro experiments. Neonatal rat ventricular myocytes (NRVMs) and H9c2 cells with knockdown of TXNIP or NLRP3 were used. Geniposide was administered to mice before vascular ligation. HE staining, 2,3,5-triphenyltetrazolium chloride (TTC) staining, echocardiography, oxidative stress and myocardial enzyme detection were used to evaluate the cardioprotective effect of geniposide. Meanwhile, pharmacological approaches of agonist and inhibitor were used to observe potential pathway for geniposide cardioprotective in vitro and in vivo. Moreover, ELISA kits were adopted to detect the levels of inflammatory factors, such as IL-1β and IL-18. The gene and protein expression of NLRP3 and pyroptosis-related factors in heart tissue were performed by RT-PCR, western blotting and immunofluorescence in vivo and in vitro, respectively.ResultsOur results indicate that geniposide can reduce the area of myocardial infarction, improve heart function, and inhibit the inflammatory response in mice after MI/RI. In addition, RT-PCR and western blotting shown geniposide promoting AMPK phosphorylation to activate myocardium energy metabolism and reducing the levels of genes and proteins expression of NLRP3, ASC, N-GSDMD and cleaved caspase-1, IL-1β, IL-18. Meanwhile, geniposide improved NRVMs energy metabolism, which decreased ROS levels and the protein expression of TXNIP and thus suppressed the expression of NLRP3. AMPK antagonist or agonist and siRNA downregulation of TXNIP or NLRP3 were also verify the effect of geniposide against H/R injury. Further research found that geniposide promoted the translocation of TXNIP and reduce the binding of TXNIP and NLRP3.ConclusionsIn our study, geniposide can significantly inhibit NLRP3 inflammasome activation via the AMPK signaling pathway and inhibit pyroptosis of cardiomyocytes in myocardial tissues.

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

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

ATF4 Protects the Heart From Failure by Antagonizing Oxidative Stress.

Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms. We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes' whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

Discovery of a first-in-class inhibitor of sulfide:quinone oxidoreductase that protects against adverse cardiac remodelling and heart failure.

Hydrogen sulfide (H2S) is a potent signalling molecule that activates diverse cardioprotective pathways by post-translational modification (persulfidation) of cysteine residues in upstream protein targets. Heart failure patients with reduced ejection fraction (HFrEF) exhibit low levels of H2S. Sulfide:quinone oxidoreductase (SQOR) catalyses the first irreversible step in the metabolism of H2S and plays a key role in regulating H2S-mediated signalling. Here, the aim of this study was to discover a first-in-class inhibitor of human SQOR and evaluate its cardioprotective effect in an animal model of HFrEF. We identified a potent inhibitor of human SQOR (STI1, IC50 = 29 nM) by high-throughput screening of a small-molecule library, followed by focused medicinal chemistry optimization and structure-based design. STI1 is a competitive inhibitor that binds with high selectivity to the coenzyme Q-binding pocket in SQOR. STI1 exhibited very low cytotoxicity and attenuated the hypertrophic response of neonatal rat ventricular cardiomyocytes and H9c2 cells induced by neurohormonal stressors. A mouse HFrEF model was produced by transverse aortic constriction (TAC). Treatment of TAC mice with STI1 mitigated the development of cardiomegaly, pulmonary congestion, dilatation of the left ventricle, and cardiac fibrosis and decreased the pressure gradient across the aortic constriction. Moreover, STI1 dramatically improved survival, preserved cardiac function, and prevented the progression to HFrEF by impeding the transition from compensated to decompensated left ventricle hypertrophy. We demonstrate that the coenzyme Q-binding pocket in human SQOR is a druggable target and establish proof of concept for the potential of SQOR inhibitors to provide a novel therapeutic approach for the treatment of HFrEF.

Klotho attenuated Doxorubicin-induced cardiomyopathy by alleviating Dynamin-related protein 1 - mediated mitochondrial dysfunction

Doxorubicin (Dox)-induced cardiotoxicity could lead to dilated cardiomyopathy and heart failure. Our previous study reported the protective effects of Klotho against hyperglycemia-induced cardiomyopathy. We investigated whether Klotho alleviated Dox-induced cardiotoxicity. Neonatal rat ventricular cardiomyocytes and H9c2 cells were incubated with 5 μM Dox for 24 h with or without Klotho (0.1 μg/mL). Dox-induced cardiotoxicity model was approached in C57BL/6 mice. Cardiac function and serum enzyme activity, apoptosis and mitochondrial dysfunction were measured. We found that pretreatment with Klotho significantly reduced Dox-induced apoptosis in cardiomyocytes. In Dox-treated mice, Klotho also suppressed cardiac cell death and improved cardiac function. Moreover, the expression of Dynamin-related protein 1 (Drp1) was increased after Dox-treatment both in vitro and in vivo, which was related to apoptosis in cardiomyocytes. In vitro experiments, Drp1 ser 616 phosphorylation post-Dox stimulation could be significantly attenuated by Klotho or Drp1 specific inhibitor Mdivi-1. Overexpression of Drp1 in cardiomyocytes increased Dox-induced heart injury which could also be attenuated by Klotho. This study demonstrated that Klotho alleviated Dox-induced cardiotoxicity by reducing apoptosis and mitochondrial fission through down-regulating Drp1 expression. Our findings highlighted new targets for the therapy of Dox-induced cardiomyopathy.

The anesthetic bupivacaine induces cardiotoxicity by targeting L-type voltage-dependent calcium channels.

ObjectiveBupivacaine is an amide local anesthetic with possible side effects that include an irregular heart rate. However, the mechanism of bupivacaine-induced cardiotoxicity has not been fully elucidated, thus we aimed to examine this mechanism.MethodsWe performed electrocardiogram recordings to detect action potential waveforms in Sprague Dawley rats after application of bupivacaine, while calcium (Ca2+) currents in neonatal rat ventricular cells were examined by patch clamp recording. Western blot and quantitative real-time polymerase chain reaction assays were used to detect the expression levels of targets of interest.ResultsIn the present study, after application of bupivacaine, abnormal action potential waveforms were detected in Sprague Dawley rats by electrocardiogram recordings, while decreased Ca2+ currents were confirmed in neonatal rat ventricular cells by patch clamp recording. These alterations may be attributed to a deficiency of CaV1.3 (L-type) Ca2+ channels, which may be regulated by the multifunctional protein calreticulin.ConclusionsThe present study identifies a possible role of the calreticulin–CaV1.3 axis in bupivacaine-induced abnormal action potentials and Ca2+ currents, which may lead to a better understanding anesthetic drug-induced cardiotoxicity.

Long non-coding RNA cardiac hypertrophy-associated regulator governs cardiac hypertrophy via regulating miR-20b and the downstream PTEN/AKT pathway.

Pathological cardiac hypertrophy (CH) is a key factor leading to heart failure and ultimately sudden death. Long non‐coding RNAs (lncRNAs) are emerging as a new player in gene regulation relevant to a wide spectrum of human disease including cardiac disorders. Here, we characterize the role of a specific lncRNA named cardiac hypertrophy‐associated regulator (CHAR) in CH and delineate the underlying signalling pathway. CHAR was found markedly down‐regulated in both in vivo mouse model of cardiac hypertrophy induced by pressure overload and in vitro cellular model of cardiomyocyte hypertrophy induced by angiotensin II (AngII) insult. CHAR down‐regulation alone was sufficient to induce hypertrophic phenotypes in healthy mice and neonatal rat ventricular cells (NRVCs). Overexpression of CHAR reduced the hypertrophic responses. CHAR was found to act as a competitive endogenous RNA (ceRNA) to down‐regulate miR‐20b that we established as a pro‐hypertrophic miRNA. We experimentally established phosphatase and tensin homolog (PTEN), an anti‐hypertrophic signalling molecule, as a target gene for miR‐20b. We found that miR‐20b induced CH by directly repressing PTEN expression and indirectly increasing AKT activity. Moreover, CHAR overexpression mitigated the repression of PTEN and activation of AKT by miR‐20b, and as such, it abrogated the deleterious effects of miR‐20b on CH. Collectively, this study characterized a new lncRNA CHAR and unravelled a new pro‐hypertrophic signalling pathway: lncRNA‐CHAR/miR‐20b/PTEN/AKT. The findings therefore should improve our understanding of the cellular functionality and pathophysiological role of lncRNAs in the heart.

Apolipoprotein-J blocks increased cell injury elicited by ox-LDL via inhibiting ROS-CaMKII pathway

BackgroundOxidized low-density lipoprotein (ox-LDL) is crucial in cardiac injury. Apolipoprotein-J (ApoJ) contributes to antiapoptotic effects in the heart. We aimed to evaluate the protective effects of ApoJ against ox-LDL cytotoxicity in Neonatal rat ventricular cells (NRVCs).Methods and resultsNRVCs were damaged by exposure to ox-LDL, as shown by increased caspase-3/7 activity, enhanced caspase-3 expression, and decreased cell viability. ApoJ overexpression, using an adenovirus vector, significantly reduced ox-LDL-induced cell injury. ApoJ also prevented ox-LDL from augmenting reactive oxygen species (ROS) production, as demonstrated by elevated Nox2/gp91phox and P47 expression. Furthermore, ApoJ overexpression reduced CaMKIIδ expression elicited by ox-LDL in cultured NRVCs. Upregulating CaMKIIδ activity, mediated by ox-LDL, was significantly inhibited by ApoJ overexpression. A CaMKIIδ inhibitor, KN93, prevented ApoJ’s protective effect against ox-LDL cytotoxicity. A ROS scavenger, Mn (III)meso-tetrakis (4-benzoic acid) porphyrin (Mn (III)TBAP), also attenuated CaMKIIδ’s increased expression and activity, induced by ox-LDL, and showed similar results to ApoJ by attenuating ox-LDL-induced cell damage, as ApoJ did.ConclusionsApoJ confers cytoprotection to NRVCs against ox-LDL cytotoxicity through the ROS-CaMKII pathways.

Prenatal exposure to testosterone induces cardiac hypertrophy in adult female rats through enhanced Pkcδ expression in cardiac myocytes

High circulating androgen in women with polycystic ovary syndrome (PCOS) may increase the risk of cardiovascular disease in offspring. The aim of the present study is to investigate whether maternal androgen excess in the rat PCOS model would lead to cardiac hypertrophy in offspring. Maternal testosterone propionate (maternal-TP)–treated adult female offspring displayed cardiac hypertrophy associated with local high cardiac dihydrotestosterone (DHT). The molecular markers of cardiac hypertrophy along with androgen receptor (AR) and PKCδ, were increased in the Maternal-TP group. Treatment of primary neonatal rat ventricular cardiomyocytes (NRCMs) and H9c2 cells with DHT significantly increased cell size and upregulated PKCδ expression, which could be attenuated by AR antagonist. Treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly increased cell size and upregulated myh7 level. Rottlerin, that may inhibit PKCδ, significantly reduced the hypertrophic effect of DHT and PMA on NRCMs and H9c2 cells. Chromatin immunoprecipitation revealed that AR could bind to Pkcδ promoter. Our results indicate that prenatal exposure to testosterone may induce cardiac hypertrophy in adult female rats through enhanced Pkcδ expression in cardiac myocytes.

Metformin Protects against H2O2-Induced Cardiomyocyte Injury by Inhibiting the miR-1a-3p/GRP94 Pathway

Ischemia-reperfusion (I/R) injury is a major side effect of the reperfusion treatment of the ischemic heart. Few therapies are available for the effective prevention of this injury caused by the oxidative stress-induced cardiomyocyte apoptosis. Metformin was shown to have a potential cardiac protective effect and ability to reduce cardiac events, but the exact mechanism remains unclear. Here, we aimed to confirm and investigate the mechanisms underlying potential metformin activity against I/R injury in response to oxidative stress. We determined that the expression of miR-1a-3p was significantly increased in neonatal rat ventricular cells (NRVCs), which were exposed to H2O2in vitro and in the hearts of mice that underwent the I/R injury. MiR-1a-3p was shown to target the 3′ UTR of GRP94, which results in the accumulation of un- or misfolded proteins, leading to the endoplasmic reticulum (ER) stress. The obtained results demonstrated that C/EBP β directly induces the upregulation of miR-1a-3p by binding to its promoter. Furthermore, as a direct allosteric AMPK activator, metformin was shown to activate AMPK and significantly reduce C/EBP β and miR-1a-3p levels compared with those in the control group. In conclusion, metformin protects cardiomyocytes against H2O2 damage through the AMPK/C/EBP β/miR-1a-3p/GRP94 pathway, which indicates that metformin may be applied for the treatment of I/R injury.

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.

Phosphodiesterase‐5 inhibitors “Sildenafil and Ordonafil” suppress/reverse L‐NAME induced cardiac hypertrophy in isolated rabbit heart and in phenylephrine induced hypertrophy in isolated neonatal rat myocytes

Phosphodiesterase 5 inhibitors (PDE5Is) have been shown to suppress and reverse myocardial hypertrophy induced by pressure overload. Calcineurin, a protein phosphatase, has been suggested to function as an important hypertrophic factor in cardiac myocytes and its signaling regulates the cardiac hypertrophic response in coordination with mitogen‐activated protein kinases (MAPKs). The present study investigated the effects of two PDE5Is i.e. Sildenafil (SILD) ‘ Viagra’ and Ordonafil (ORD) on N‐nitro‐L‐arginine methyl ester (L‐NAME) induced cardiac hypertrophy and assessed their ability to attenuate the activity of calcineurin, p38 and protein synthesis to exert their effect on hypertrophied cardiac myocytes.MethodsL‐NAME (10 mg/kg/day) was used to induce cardiac hypertrophy in rabbits' hearts and left ventricular thicknesses (LVTs) were measured in L‐NAME and PDE5Is treated hearts. Then, in cultured neonatal rat ventricular myocytes, Western Blot technique was used to assess the ability of these PDE5Is (different doses) to attenuate the activation of calcineurin, p38 and protein synthesis in phenylphrine‐induced ventricular hypertrophy as indicated by the cell surface area of cultured neonatal rat cardiomyocytes.ResultsL‐NAME significantly increased LVT to 6.1± 0.18 mm vs. 4.6±0.13 mm in control rabbit hearts, (p<0.05). SILD and ORD significantly regressed LVT to 4.8 ± 0.2 mm and 5.1 ± 0.1 mm, respectively (p<0.05). Phenylphrine significantly increased neonatal rat ventricular myocyte cell surface area to 131% of control values (p<0.05). This hypertrophic effect of phenylphrine was associated with significant increment in protein synthesis to 143+5% of control values, (p<0.05). Both drugs (in a dose dependent, 0.001–1 mg/L) decreased cell surface area to 95% and 90% of control value respectively, and both decreased protein synthesis to 88% of control values. Phosphatase activity of calcineurin significantly decreased from 1.93±0.09 IU to 1.15±0.05 IU after one hour and to 1.2±0.06 IU after 24 hours incubation with ORD (0.1 mg/l) incubation, (p<0.05). Whereas this activity significantly decreased after 24 hours (1.22±0.04 IU, (p<0.05)), but not after one hour (1.84±0.05 IU) of SILD (0.1 mg/l) incubation. Protein synthesis and p38 activation were significantly attenuated by both drugs (0.1 mg/L).ConclusionsThese results suggest that both SILD and ORD have the ability to reverse L‐NAME induced cardiac hypertrophy in rabbits and suppress phenylphrine induce hypertrophic myocytes. The results indicate these effects are mediated through inhibiting protein synthesis and the attenuation of calcineurin and p38 activity signaling pathways in neonatal rat ventricular myocytes which is reported in this study for the first time.Support or Funding InformationDeanship of Scientific research ‐ Jordan University of Science & TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Engineered cardiac tissue patch maintains structural and electrical properties after epicardial implantation

Functional cardiac tissue engineering holds promise as a candidate therapy for myocardial infarction and heart failure. Generation of “strong-contracting and fast-conducting” cardiac tissue patches capable of electromechanical coupling with host myocardium could allow efficient improvement of heart function without increased arrhythmogenic risks. Towards that goal, we engineered highly functional 1 cm × 1 cm cardiac tissue patches made of neonatal rat ventricular cells which after 2 weeks of culture exhibited force of contraction of 18.0 ± 1.4 mN, conduction velocity (CV) of 32.3 ± 1.8 cm/s, and sustained chronic activation when paced at rates as high as 8.7 ± 0.8 Hz. Patches transduced with genetically-encoded calcium indicator (GCaMP6) were implanted onto adult rat ventricles and after 4–6 weeks assessed for action potential conduction and electrical integration by two-camera optical mapping of GCaMP6-reported Ca2+ transients in the patch and RH237-reported action potentials in the recipient heart. Of the 13 implanted patches, 11 (85%) engrafted, maintained structural integrity, and conducted action potentials with average CVs and Ca2+ transient durations comparable to those before implantation. Despite preserved graft electrical properties, no anterograde or retrograde conduction could be induced between the patch and host cardiomyocytes, indicating lack of electrical integration. Electrical properties of the underlying myocardium were not changed by the engrafted patch. From immunostaining analyses, implanted patches were highly vascularized and expressed abundant electromechanical junctions, but remained separated from the epicardium by a non-myocyte layer. In summary, our studies demonstrate generation of highly functional cardiac tissue patches that can robustly engraft on the epicardial surface, vascularize, and maintain electrical function, but do not couple with host tissue. The lack of graft-host electrical integration is therefore a critical obstacle to development of efficient tissue engineering therapies for heart repair.

MicroRNA-449a Inhibition Protects H9C2 Cells Against Hypoxia/Reoxygenation-Induced Injury by Targeting the Notch-1 Signaling Pathway

The present study aimed to detect the expression of miR-449a and investigate the effect of miR-449a on cell injury in cardiomyocytes subjected to hypoxia/ reoxygenation (H/R) and its underlying mechanisms. The expression of miR-449a was determined using reverse transcription-polymerase chain reaction in both neonatal rat ventricular myocytes and H9C2 cells. For gain-of-function and loss-of-function studies, H9C2 cells were transfected with either miR-449a mimics or miR-449a inhibitor. The target gene of miR-449a was confirmed by a dual-luciferase reporter assay. Apoptosis was analyzed by both flow cytometry using Annexin V and propidium iodide and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). Necrosis was confirmed by the detection of lactate dehydrogenase release. The cell viability was measured using the methylthiotetrazole method. The protein levels of Notch-1, Notch-1 intracellular domain, hairy and enhancer of split-1 (Hes-1), and apoptosis-related genes were measured by Western blot analysis. MiR-449a was significantly upregulated in both neonatal rat ventricular myocytes and H9C2 cells subjected to H/R. However, H/R-induced cell apoptosis and necrosis were markedly reduced by miR-449a inhibition. By targeting Notch-1, miR-449a regulated the Notch-1/ Hes-1 signaling pathway. The blockade of the Notch signaling pathway partly abolished the protective effect of miR-449a suppression against H/R injury, whereas the overexpression of Notch-1 intracellular domain partly reversed the effect of miR-449a overexpression on H/R-induced cell injury. The present study suggested that miR-449a inhibition protected H9C2 cells against H/R-induced cell injury by targeting the Notch-1 signaling pathway, providing a novel insight into the molecular basis of myocardial ischemia-reperfusion injury and a potential therapeutic target.

Virtual cardiac monolayers for electrical wave propagation

The complex structure of cardiac tissue is considered to be one of the main determinants of an arrhythmogenic substrate. This study is aimed at developing the first mathematical model to describe the formation of cardiac tissue, using a joint in silico–in vitro approach. First, we performed experiments under various conditions to carefully characterise the morphology of cardiac tissue in a culture of neonatal rat ventricular cells. We considered two cell types, namely, cardiomyocytes and fibroblasts. Next, we proposed a mathematical model, based on the Glazier-Graner-Hogeweg model, which is widely used in tissue growth studies. The resultant tissue morphology was coupled to the detailed electrophysiological Korhonen-Majumder model for neonatal rat ventricular cardiomyocytes, in order to study wave propagation. The simulated waves had the same anisotropy ratio and wavefront complexity as those in the experiment. Thus, we conclude that our approach allows us to reproduce the morphological and physiological properties of cardiac tissue.

Suppression of microRNA-16 protects against acute myocardial infarction by reversing beta2-adrenergic receptor down-regulation in rats.

microRNA-16 (miR-16) has been shown to be up-regulated in ischemic heart. Beta2-adrenoreceptor (β2-AR) exerts cardioprotective property in ischemic injury. This study aims to determine the effect of miR-16 in cardiac injury in rats and the possible involvement of β2-AR in this process. Acute myocardial infarction (AMI) model in rats was induced by ligation of left coronary artery. Neonatal rat ventricular cells (NRVCs) were cultured in vitro tests. The cardiomyocyte model of oxidative injury was mimicked by hydrogen peroxide. The expression of miR-16 was obviously up-regulated and β2-AR was remarkably down-regulated in both AMI rats and NRVCs under oxidative stress. miR-16 over-expression in NRVCs reduced cell viability and increased apoptosis. Conversely, inhibition of endogenous miR-16 with its specific inhibitor reversed these changes. Over-expression of miR-16 using an miR-16 lentivirus in AMI rats markedly increased cardiac infarct area, lactate dehydrogenase and creatine kinase activity, and exacerbated cardiac dysfunction. Lentivirus-mediated knockdown of miR-16 alleviated acute cardiac injury. Moreover, miR-16 over-expression significantly suppressed β2-AR protein expression in both cultured NRVCs and AMI rats, while inhibition of miR-16 displayed opposite effect on β2-AR protein expression. Luciferase assay confirmed that miR-16 could directly target the 3′untranslated region of β2-AR mRNA. miR-16 is detrimental to the infarct heart and suppression of miR-16 protects rat hearts from ischemic injury via up-regulating of β2-AR by binding to the 3′untranslated region of β2-AR gene. This study indicates that targeting miR-16/β2-AR axis may be a promising strategy for ischemic heart disease.

Let-7a regulates expression of β1-adrenoceptors and forms a negative feedback circuit with the β1-adrenoceptor signaling pathway in chronic ischemic heart failure.

BackgroundThe aim of the present study was to investigate the role of microRNA (miRNA) let-7a in down-regulation of β1-adrenoceptors (β1-AR) and elucidate the underlying mechanism of chronic ischemia heart failure (CIHF) in rats.Methods and ResultsCIHF model was established by occlusion of coronary artery for 4 weeks. β1-AR level was obviously down-regulated and let-7a up-regulated in the failing heart 4 weeks after myocardial infarction. Overexpression of let-7a inhibited β1-AR expression in neonatal rat ventricular cells (NRVCs), which was abolished by anti-let-7a antisense inhibitor. The lentivirus vector containing precursor let-7a (len-pre-let-7a) further down-regulated the reduced β1-AR level by CIHF and the effect was reversed by len-AMO-let-7a. Len-negative control did not produce any significant influence on β1-AR expression. Importantly, there exists a negative feedback loop associated with β1-AR regulation through β1-AR/cAMP/PKA/GATA4/let-7a/β1-AR signaling pathway in CIHF. As demonstrated, GATA4 was activated by β1-AR up-regulation through cAMP-PKA signaling pathway in early phase of ischemia, then GATA4 positively regulated let-7a expression which in turn suppressed β1-AR expression.ConclusionsLet-7a regulates β1-AR expression and forms a negative feedback loop with β1-AR signaling pathway in ischemic heart failure. This study provides a new insight into the differential expression of β1-AR in early and later phase of myocardial ischemia.