Reduced Cardiomyocyte Death Research Articles

Increased PHLPP1 expression through ERK-4E-BP1 signaling axis drives nicotine induced oxidative stress related damage of cardiomyocytes

Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury.

A Biomimetic Nanocarrier Strategy Targets Ferroptosis and Efferocytosis During Myocardial Infarction.

Myocardial infarction (MI) is characterized by irreversible cardiomyocyte death resulting from an inadequate supply of oxygenated blood to the myocardium. Recent studies have indicated that ferroptosis, a form of regulated cell death, exacerbates myocardial injury during MI. Concurrently, the upregulation of CD47 on the surface of damaged myocardium following MI impairs the clearance of dead cells by macrophages, thereby hindering efferocytosis. In this context, simultaneously inhibiting ferroptosis and enhancing efferocytosis may represent a promising strategy to mitigate myocardial damage post-MI. In this study, we engineered platelet membrane-coated hollow mesoporous silicon nanoparticles (HMSN) to serve as a drug delivery system, encapsulating ferroptosis inhibitor, Ferrostatin-1, along with an anti-CD47 antibody. We aimed to assess the potential of these nanoparticles (designated as Fer-aCD47@PHMSN) to specifically target the site of MI and evaluate their efficacy in reducing cardiomyocyte death and inflammation. The platelet membrane coating on the nanoparticles significantly enhanced their ability to successfully target the site of myocardial infarction (MI). Our findings demonstrate that treatment with Fer-aCD47@PHMSN resulted in a 38.5% reduction in cardiomyocyte ferroptosis under hypoxia, indicated by decreased lipid peroxidation and increased in vitro. Additionally, Fer-aCD47@PHMSN improved cardiomyocyte efferocytosis by approximately 15% in vitro. In MI mice treated with Fer-aCD47@PHMSN, we observed a substantial reduction in cardiomyocyte death (nearly 30%), decreased inflammation, and significant improvement in cardiac function. Our results demonstrated that the cooperation between the two agents induced anti-ferroptosis effects and enhanced dead cardiomyocyte clearance by macrophage as well as anti-inflammation effects. Thus, our nanoparticle Fer-aCD47@PHMSN provides a new therapeutic strategy for targeted therapy of MI.

Transcriptome analysis reveals ADAMTS15 is a potential inflammation-related gene in remote ischemic postconditioning.

Remote ischemic postconditioning (RIPostC) induced by brief episodes of the limb ischemia is a potential therapeutic strategy for myocardial ischemia/reperfusion injury, achieved by reducing cardiomyocyte death, inflammation and so on. The actual mechanisms underlying cardioprotection conferred by RIPostC remain unclear. Exploring gene expression profiles in myocardium at transcriptional level is helpful to deepen the understanding on the cardioprotective mechanisms of RIPostC. This study aims to investigate the effect of RIPostC on gene expressions in rat myocardium using transcriptome sequencing. Rat myocardium samples from the RIPostC group, the control group (myocardial ischemia/reperfusion group) and the sham group were performed transcriptome analysis using RNA sequencing. The levels of cardiac IL-1β, IL-6, IL-10 and TNFα were analyzed by Elisa. The expression levels of candidate genes were verified by qRT-PCR technique. Infarct size was measured by Evans blue and TTC staining. Apoptosis was assessed by TUNEL assays and caspase-3 levels were detected using western blotting. RIPostC can markedly decrease infarct size and reduce the levels of cardiac IL-1β, IL-6 and increase the level of cardiac IL-10. This transcriptome analysis showed that 2 genes were up-regulated (Prodh1 and ADAMTS15) and 5 genes (Caspase-6, Claudin-5, Sccpdh, Robo4 and AABR07011951.1) were down-regulated in the RIPostC group. Go annotation analysis showed that Go terms mainly included cellular process, metabolic process, cell part, organelle, catalytic activity and binding. The KEGG annotation analysis of DEGs found only one pathway, amino acid metabolism, was up-regulated. The relative mRNA expression levels of ADAMTS15, Caspase-6, Claudin-5 and Prodh1 were verified by qRT-PCR, which were consistent with the RNA-seq results. In addition, the relative expression of ADAMTS15 was negatively correlated with the level of cardiac IL-1β (r = -0.748, P = 0.005) and positively correlated with the level of cardiac IL-10 (r = 0.698, P = 0.012). A negative correlation statistical trend was found between the relative expression of ADAMTS15 and the level of cardiac IL-6 (r = -0.545, P = 0.067). ADAMTS15 may be a potential inflammation-related gene in regulation of cardioprotection conferred by remote ischemic postconditioning and a possible therapeutic target for myocardial ischemia reperfusion injury in the future.

Microarray Analysis Reveals Changes in tRNA-Derived Small RNAs (tsRNAs) Expression in Mice with Septic Cardiomyopathy.

Background: tRNA-derived small RNAs (tsRNAs) as a novel non-coding RNA have been studied in many cardiovascular diseases, but the relationship between tsRNAs and septic cardiomyopathy has not been investigated. We sought to analyze changes of the expression profile of tsRNAs in septic cardiomyopathy and reveal an important role for tsRNAs. Methods: We constructed a sepsis model by cecal ligation and puncture (CLP) in mice, and microarray analysis was used to find differentially expressed tsRNAs. Quantitative real-time PCR was used to verify the expression of tsRNAs and the interference effect of angiogenin (ANG), a key nuclease producing tsRNAs. Bioinformatics analysis was used to predict target genes and functions. CCK-8 and LDH release assays were used to detect cell viability and cell death. Results: A total of 158 tsRNAs were screened, of which 101 were up-regulated and 57 were down-regulated. A total of 8 tsRNAs were verified by qPCR, which was consistent with microarray results. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses suggest that these tsRNAs may be associated with the Wnt signaling pathway and participate in cellular process. The expression of tsRNAs decreased after the interference of the key nuclease ANG, while CCK-8 suggested a corresponding decrease in cell viability and an increase in the release of LDH (cell death), indicating that tsRNAs can protect cardiomyocytes during the development of septic cardiomyopathy, reduced cardiomyocyte death. Conclusions: A total of 158 tsRNAs changed significantly in septic cardiomyopathy, and these tsRNAs may play a protective role in the development of septic cardiomyopathy.

Abstract 14981: Stk25 Regulates Cell Survival in Both Human and Mouse Models of Myocardial Injury

Introduction: Dysregulation of cardiac kinases occurs in cardiovascular diseases including hypertrophy, remodeling and ischemia injury. Serine/threonine protein kinase 25 (STK25) is a stress response kinase and putative tumor suppressor that regulates cell survival and metabolism through several signaling pathways, including LATS/3HIPPO and mTOR pathways. Hypothesis: STK25 regulates cell survival, metabolism and cardiac function in human cardiomyocytes and animal injury models. Methods: CRISPR-Cas9 was used to generate a homozygous knockout (KO) of STK25 in induced pluripotent stem cell (iPSC) which were then differentiated into cardiomyocytes (iPSC-CMs). STK25 was quantified in human left ventricular tissue from heart failure (HF) patients. Stk25 KO mice were generated by CRISPR/Cas9 mediated genome engineering in C57BL/6J background. Role of STK25 in gene expression, cell proliferation/apoptosis, metabolism and cardiac function were characterized. Results: Gene set enrichment analysis (GSEA) of differential gene expression obtained by RNA-seq was used to identify upregulation of several growth and metabolism pathways in STK25KO iPSC-CMs including oxidative phosphorylation, mTORC1 signaling and myogenesis. In functional assays, STK25 modulated cell cycle progression, proliferation, hypoxia induced apoptosis, and mitochondrial respiration in iPSC-CMs. n a myocardial infarction model of cardiac injury, loss of Stk25 in vivo led to improvements in cardiac function, reduced cardiomyocyte death and diminished fibrosis. STK25 expression is increased in HF patients. Furthermore, loss of function mutations in STK25 were discovered in atrial myxomas, demonstrating STK25 altered regulation in human heart diseases. Conclusions: STK25 regulates cardiomyocyte survival in vitro and in vivo and is aberrantly regulated in both hearth failure and atrial myxomas.

Neurotrophin-3 contributes to benefits of human embryonic stem cell-derived cardiovascular progenitor cells against reperfused myocardial infarction.

Acute myocardial infarction (MI) resulting from coronary ischemia is a major cause of disability and death worldwide. Transplantation of human embryonic stem cell (hESC)‐derived cardiovascular progenitor cells (hCVPCs) promotes the healing of infarcted hearts by secreted factors. However, the hCVPC‐secreted proteins contributing to cardiac repair remain largely unidentified. In this study, we investigated protective effects of neurotrophin (NT)‐3 secreted from hCVPCs in hearts against myocardial ischemia/reperfusion (I/R) injury and explored the underlying mechanisms to determine the potential of using hCVPC products as a new therapeutic strategy. The implantation of hCVPCs into infarcted myocardium at the beginning of reperfusion following 1 hour of ischemia improved cardiac function and scar formation of mouse hearts. These beneficial effects were concomitant with reduced cardiomyocyte death and increased angiogenesis. Moreover, hCVPCs secreted a rich abundance of NT‐3. The cardioreparative effect of hCVPCs in the I/R hearts was mimicked by human recombinant NT‐3 (hNT‐3) but canceled by NT‐3 neutralizing antibody (NT‐3‐Ab). Furthermore, endogenous NT‐3 was detected in mouse adult cardiomyocytes and its level was enhanced in I/R hearts. Adenovirus‐mediated NT‐3 knockdown exacerbated myocardial I/R injury. Mechanistically, hNT‐3 and endogenous NT‐3 inhibited I/R‐induced cardiomyocyte apoptosis through activating the extracellular signal‐regulated kinase (ERK) and reducing the Bim level, resulting in the cardioreparative effects of infarcted hearts together with their effects in the improvement of angiogenesis. These results demonstrate for the first time that NT‐3 is a cardioprotective factor secreted by hCVPCs and exists in adult cardiomyocytes that reduces I/R‐induced cardiomyocyte apoptosis via the ERK‐Bim signaling pathway and promotes angiogenesis. As a cell product, NT‐3 may represent as a noncell approach for the treatment of myocardial I/R injury.

Hydralazine reduces myocardial infarct size.

A new study shows that hydralazine, a drug used for the treatment of resistant hypertension and heart failure, can inhibit mitochondrial fission, thereby reducing cardiomyocyte death and myocardial infarct size after ischaemia–reperfusion injury in mice.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AimsGenetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission.Methods and resultsPre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001).ConclusionWe show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

Melatonin Exerts Cardioprotective Effects by Inhibiting NLRP3 Inflammasome-Induced Pyroptosis in Mice following Myocardial Infarction.

Myocardial infarction- (MI-) induced myocardial damage is mainly attributed to the loss of cardiomyocytes. Pyroptosis is a newly recognized form of programmed cell necrosis that is associated with the progression of MI. Melatonin has been shown to exert cardioprotective effects against cardiac damage in multiple cardiovascular diseases. However, the effect of melatonin on pyroptosis-induced cardiac injury in MI has not been elucidated. Herein, we found that melatonin administration ameliorated cardiac dysfunction and reduced cardiomyocyte death both in mice following coronary artery ligation and in H9C2 cells exposed to hypoxia. The results also showed that pyroptosis was induced both in vivo and in vitro, as evidenced by increased NLRP3, cleaved caspase-1, GSDMD-N, and mature IL-1β and IL-18 levels, and these changes were decreased by melatonin treatment. Furthermore, we observed that TLR4 and NF-κB levels were increased by MI or hypoxia, and these increases were reversed by melatonin. The antipyroptotic action of melatonin was abrogated by treatment with an agonist of the TLR4/NF-κB signaling pathway. Our results indicate that melatonin can exert cardioprotective effects by inhibiting NLRP3 inflammasome-induced pyroptosis through modulation of the TLR4/NF-κB signaling pathway and provide strong evidence for the utility of melatonin in the treatment of MI.

Group 2 innate lymphoid cells contribute to IL-33-mediated alleviation of cardiac fibrosis.

Rationale: The major cause of heart failure is myocardium death consequent to detrimental cardiac remodeling and fibrosis following myocardial infarction. The cardiac protective cytokine interleukin (IL)-33, which signals by ST2 receptor binding, is associated with group 2 innate lymphoid cell (ILC2) activation and regulates tissue homeostasis and repair following tissue injury in various tissues. However, the distribution and role of IL-33-responsive ILC2s in cardiac fibrosis remain unclear. In this study, we elucidated the roles of IL-33-responsive cardiac-resident ILC2s and IL-33-mediated immunomodulatory functions in cardiac fibrosis.Methods: We examined the distribution of cardiac ILC2s by using flow cytometry. The roles of IL-33-mediated ILC2 expansion in cardiac fibrosis was evaluated in the mouse model of catecholamine-induced cardiac fibrosis. ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice were implemented to determine the contribution of endogenous ILC in the progression of cardiac fibrosis. Histopathological assessments, speckle tracking echocardiography, and transcriptome profile analysis were performed to determine the effects of IL-33-mediated cardiac protective functions.Results: We identified the resident cardiac ILC2s, which share similar cell surface marker and transcriptional factor expression characteristics as peripheral blood and lung tissue ILC2s. IL-33 treatment induced ILC2 expansion via ST2. In vivo, ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice developed exacerbated cardiac fibrosis following catecholamine-induced stress cardiac injury. IL-33 treatment expanded cardiac ILC2s and revealed protective effects against cardiac tissue damage with reduced cardiomyocyte death, immune cell infiltration, tissue fibrosis, and improved myocardial function. Transcriptome analysis revealed that IL-33 attenuated extracellular matrix synthesis- and fibroblast activation-associated gene expressions. IL13-knockout or epidermal growth factor receptor (EGFR) inhibition abolished IL-33-mediated cardiac protective function, confirming IL-13 and EGFR signaling as crucial for IL-33-mediated cardioprotective responses. Moreover, ILC2-produced BMP-7 served as a novel anti-fibrotic factor to inhibit TGF-β1-induced cardiac fibroblast activation.Conclusion: Our findings indicate the presence of IL-33-responsive ILC2s in cardiac tissue and that IL-33-mediated ILC2 expansion affords optimal cardioprotective function via ILC2-derived factors. IL-33-mediated immunomodulation is thus a promising strategy to promote tissue repair and alleviate cardiac fibrosis following acute cardiac injury.

Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis

Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell–cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.

Melatonin fine-tunes intracellular calcium signals and eliminates myocardial damage through the IP3R/MCU pathways in cardiorenal syndrome type 3

Cardiorenal syndrome type-3 (CRS-3) is characterized by acute cardiac injury induced by acute kidney injury. Here, we investigated the causes of CRS-3 by analyzing cardiac function after renal ischemia–reperfusion injury (IRI) using echocardiography and evaluation of pro-inflammatory markers, calcium balance, mitochondrial function, and cardiomyocyte death. Our results show that renal IRI reduces cardiac diastolic function associated with cardiomyocyte death and inflammatory responses. Renal IRI also disrupts cardiomyocyte energy metabolism, induces calcium overload, and impairs mitochondrial function, as evidenced by reduced mitochondrial membrane potential and increased mitochondrial fission. Further, renal IRI induces phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and expression of mitochondrial calcium uniporter (MCU), resulting in cytoplasmic calcium overload and mitochondrial calcium accumulation. Pretreatment with melatonin attenuates renal IRI-mediated cardiac damage by maintaining myocardial diastolic function and reducing cardiomyocyte death. Melatonin also inhibits IP3R phosphorylation and MCU expression, thereby alleviating cytoplasmic and mitochondrial calcium overload. Blockade of IP3R has similar cardioprotective effects, whereas MCU activation abrogates the melatonin-mediated cardioprotection. These results show that the negative effects of renal IRI on myocardial viability and cardiac function are caused by induced IP3R phosphorylation, MCU upregulation, and calcium overload. Melatonin protects cardiac function against CRS-3 by suppressing IP3R-MCU signaling.

Mst1 deletion reduces septic cardiomyopathy via activating Parkin‐related mitophagy

Cardiomyocyte function and viability are highly modulated by mammalian Ste20-like kinase 1 (Mst1)-Hippo pathway and mitochondria. Mitophagy, a kind of mitochondrial autophagy, is a protective program to attenuate mitochondrial damage. However, the relationship between Mst1 and mitophagy in septic cardiomyopathy has not been explored. In the present study, Mst1 knockout mice were used in alipopolysaccharide (LPS)-induced septic cardiomyopathy model. Mitophagy activity was measured via immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay. Pathway blocker and small interfering RNA were used to perform the loss-of-function assay. The results demonstrated that Mst1 was rapidly increased in response to LPS stress. Knockout of Mst1 attenuated LPS-mediated inflammation damage, reduced cardiomyocyte death, and improved cardiac function. At the molecular levels, LPS treatment activated mitochondrial damage, such as mitochondrial respiratory dysfunction, mitochondrial potential reduction, mitochondrial ATP depletion, and caspase family activation. Interestingly, in response to mitochondrial damage, Mst1 deletion activated mitophagy which attenuated LPS-mediated mitochondrial damage. However, inhibition of mitophagy via inhibiting parkin mitophagy abolished the protective influences of Mst1 deletion on mitochondrial homeostasis and cardiomyocyte viability. Overall, our results demonstrated that septic cardiomyopathy is linked to Mst1 upregulation which is followed by a drop in the protective mitophagy.

Cardioprotective Effect of the Mitochondrial Unfolded Protein Response During Chronic Pressure Overload

BackgroundThe mitochondrial unfolded protein response (UPRmt) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear. ObjectivesThe purpose of this study was to: 1) identify conditions that activate the UPRmt in the heart; and 2) study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function. MethodsCultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis. ResultsDiverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction (N-terminal pro–B-type natriuretic peptide). ConclusionsThese results identify the induction of the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.

FBXL10 regulates cardiac dysfunction in diabetic cardiomyopathy via the PKC β2 pathway

Diabetic cardiomyopathy (DCM) is a condition associated with significant structural changes including cardiac tissue necrosis, localized fibrosis, and cardiomyocyte hypertrophy. This study sought to assess whether and how FBXL10 can attenuate DCM using a rat streptozotocin (STZ)‐induced DCM model system. In the current study, we found that FBXL10 expression was significantly decreased in diabetic rat hearts. FBXL10 protected cells from high glucose (HG)‐induced inflammation, oxidative stress, and apoptosis in vitro. In addition, FBXL10 significantly activated PKC β2 signaling pathway in H9c2 cells and rat model. The cardiomyocyte‐specific overexpression of FBXL10 at 12 weeks after the initial STZ administration attenuated oxidative stress and inflammation, thereby reducing cardiomyocyte death and preserving cardiac function in these animals. Moreover, FBXL10 protected against DCM via activation of the PKC β2 pathway. In conclusion, FBXL has the therapeutic potential for the treatment of DCM.

Restricting mitochondrial GRK2 post-ischemia confers cardioprotection by reducing myocyte death and maintaining glucose oxidation.

Increased abundance of GRK2 [G protein-coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.

Inhibiting Receptor of Advanced Glycation End Products Attenuates Pressure Overload-Induced Cardiac Dysfunction by Preventing Excessive Autophagy.

The receptor for advanced glycation end products (RAGE) is involved in heart failure (HF) by mediating diverse pathologic processes, including the promotion of inflammation and autophagy. However, the role of RAGE in pressure overload-induced HF is not well understood. We found that stimulation of RAGE triggered the death of neonatal rat ventricular myocytes (NRVMs), while cell death was alleviated by ATG5 knockdown. Using transverse aortic constriction (TAC) in mice as a model of pressure overload-induced HF, we demonstrated that RAGE knockout or RAGE blockade attenuated cardiac hypertrophy and fibrosis as well as cardiac dysfunction at 8 weeks after TAC. Importantly, RAGE knockout reversed upregulation of autophagy related proteins (LC3BII/I and Beclin 1) and reduced cardiomyocyte death, indicating that excessive autophagy after TAC was inhibited. Moreover, RAGE knockout or blockade reduced the upregulation of pp65-NFκB and BNIP3, which mediate autophagy. Taken together, these results suggest that RAGE plays an important role in the progression of HF by regulating autophagy. Therefore, inhibition of the RAGE-autophagy axis could be a promising new strategy for treatment of heart failure.

Sirt3 attenuates post-infarction cardiac injury via inhibiting mitochondrial fission and normalization of AMPK-Drp1 pathways

Mitochondrial damage is involved in the pathogenesis of post-infarction cardiac injury. However, the upstream regulators of mitochondrial damage have not yet been identified. The aim of our study is to explore the role of Sirt3 in post-infarction cardiac injury with a particular focus on mitochondrial fission and AMPK-Drp1 pathways. Our results indicated that Sirt3 was downregulated in the progression of post-infarction cardiac injury. Overexpression of Sirt3 attenuated cardiac fibrosis, sustained myocardial function, inhibited the inflammatory response, and reduced cardiomyocyte death. Functional studies illustrated that chronic post-infarction cardiac injury was characterized by increased mitochondrial fission, which triggered mitochondrial oxidative stress, metabolic disorders, mitochondrial potential reduction and caspase-9 apoptosis in cardiomyocytes. However, Sirt3 overexpression attenuated mitochondrial fission and thus preserved mitochondrial homeostasis and cardiomyocyte viability. Furthermore, our results confirmed that Sirt3 repressed mitochondrial fission via normalizing AMPK-Drp1 pathways. Inhibition of AMPK activity re-activated Drp1 and thus abrogated the inhibitory effect of Sirt3 on mitochondrial fission. Altogether, our results indicate that Sirt3 enhancement could be an effective approach to retard the development of post-infarction cardiac injury via disrupting mitochondrial fission and normalizing the AMPK-Drp1 axis.

Modelling ischemia-reperfusion injury (IRI) in vitro using metabolically matured induced pluripotent stem cell-derived cardiomyocytes.

Coronary intervention following ST-segment elevation myocardial infarction (STEMI) is the treatment of choice for reducing cardiomyocyte death but paradoxically leads to reperfusion injury. Pharmacological post-conditioning is an attractive approach to minimize Ischemia-Reperfusion Injury (IRI), but candidate drugs identified in IRI animal models have performed poorly in human clinical trials, highlighting the need for a human cell-based model of IRI. In this work, we show that when we imposed sequential hypoxia and reoxygenation episodes [mimicking the ischemia (I) and reperfusion (R) events] to immature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), they display significant hypoxia resistance and minimal cell death (∼5%). Metabolic maturation of hPSC-CMs for 8 days substantially increased their sensitivity to changes in oxygen concentration and led to up to ∼30% cell death post-hypoxia and reoxygenation. To mimic the known transient changes in the interstitial tissue microenvironment during an IRI event in vivo, we tested a new in vitro IRI model protocol that required glucose availability and lowering of media pH during the ischemic episode, resulting in a significant increase in cell death in vitro (∼60%). Finally, we confirm that in this new physiologically matched IRI in vitro model, pharmacological post-conditioning reduces reperfusion-induced hPSC-CM cell death by 50%. Our results indicate that in recapitulating key aspects of an in vivo IRI event, our in vitro model can serve as a useful method for the study of IRI and the validation and screening of human specific pharmacological post-conditioning drug candidates.

Abstract 416: NF-kB-Mir-23a-27a: a Critical Modulator in Post-myocardial Infarction Remodeling

Myocardial infarction (MI) is one of the leading causes of death around the world. Myocardial ischemia contributes to the development of MI and initiates cardiomyocyte (CM) apoptosis, myofibroblasts differentiation, fibrosis and left ventricle (LV) remodeling leading to cardiac dysfunction. Previously, we have demonstrated the direct involvement of miRNAs (a new class of post-translational regulator) and NF-κB in LV remodeling. However, NF-κB-mediated miRNA modulation and miRNA/gene networks in post-MI remodeling remain elusive. Furthermore, with an emerging interest to combine cell transplantation with gene therapy, mesenchymal stem cells (MSC) are considered most beneficial of the stem cells in regenerative medicine. We have a paucity of data in genetically modified MSC on their mobilization within infarcted myocardium. We identified a panel of novel dysregulated miRNAs in the wild type (WT) mice subjected to MI using conventional miRNA array. These miRNAs were restored in cardiac-specific IκBα triple-mutant mice (3M, resistant to NF-κB activation) mice indicated NF-κB-dependent regulation. Among them, miR-23a and miR-27a were significantly upregulated in MI. Our study has identified PPARγ-GSK3β-Bcl 2 -axis, a bona-fide target for miR-23a/-27a after unbiased in vitro screening. Our hypothesis is that NF-κB-dependent miR-23a/-27a is a pathogenic niche, exacerbates post-MI remodeling through several target pathways; and inhibition of NF-κB, miR-23a/-27a and transplantation of 3M-MSC mitigates the cardiac damage and abrogates the cardiac damage by restoring PPARγ-GSK3β-Bcl 2 -axis. Inhibition of NF-κB normalized miR-23a/-27a expression and reduced CM death in hypoxia/ reoxygenation (H/R) model. Our data also indicated that miR-23a is a key regulator in simulated H/R-induced injury. Inhibition of miR-23a showed reduction of cell death and improved mitochondrial function in H/R model. Interestingly, 3M-MSC showed significant reduction in apoptosis, inflammation and oxidative stress in H/R-induced CM injury, compared to WT-MSC. Our findings conclude that miR-23a/-27a could be a triggering factor and 3M-MSC provides a better protection in post-MI remodelling indicate new mechanistic information for therapeutic benefit.