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Myocyte-specific overexpressing HDAC4 promotes myocardial ischemia/reperfusion injury.

ABSTRACT: BACKGROUND:Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts. METHODS:In this study, we created myocyte-specific active HDAC4 transgenic mice to examine the functional role of active HDAC4 in mediating myocardial I/R injury. Ventricular function was determined in the isovolumetric heart, and infarct size was determined using tetrazolium chloride staining. RESULTS:Myocyte-specific overexpressing activated HDAC4 in mice promoted myocardial I/R injury, as indicated by the increases in infarct size and reduction of ventricular functional recovery following I/R injury. Notably, active HDAC4 overexpression led to an increase in LC-3 and active caspase 3 and decrease in SOD-1 in myocardium. Delivery of chemical HDAC inhibitor attenuated the detrimental effects of active HDAC4 on I/R injury, revealing the pivotal role of active HDAC4 in response to myocardial I/R injury. CONCLUSIONS:Taken together, these findings are the first to define that activated HDAC4 as a crucial regulator for myocardial ischemia and reperfusion injury.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6050730 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Myocyte-specific overexpressing HDAC4 promotes myocardial ischemia/reperfusion injury.

Zhang Ling L Wang Hao H Zhao Yu Y Wang Jianguo J Dubielecka Patrycja M PM Zhuang Shougang S Qin Gangjian G Chin Y Eugene YE Kao Race L RL Zhao Ting C TC

Molecular medicine (Cambridge, Mass.) 20180717 1

<h4>Background</h4>Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts.<h4>Methods</h4>In this study, we created myocyte-specific ...[more]

PMID: 30134825

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Project description:Introduction:The use of comorbidity models is crucial in cardioprotective drug development. Hypercholesterolemia causes endothelial and myocardial dysfunction, as well as aggravates ischemia/reperfusion (I/R)-induced myocardial injury. Endogenous cardioprotective mechanisms against I/R are impaired in hyperlipidemic and hyperglycemic in vivo animal models. Therefore, our aim was to develop a medium throughput comorbidity cell-based test system of myocardial I/R injury, hypercholesterolemia and hyperglycemia that mimics comorbidity conditions. Methods:Cardiac myocytes isolated from neonatal or adult rat hearts were cultured in control or in three different hypercholesterolemic media with increasing cholesterol content (hiChol) or hiChol + hyperglycemic medium, respectively. Each group was then subjected to simulated ischemia/reperfusion (SI/R) or corresponding normoxic condition, respectively. Cholesterol uptake was tested by Filipin staining in neonatal cardiac myocytes. Cell viability, total cell count and oxidative stress, i.e., total reactive oxygen species (ROS) and superoxide level were measured by fluorescent assays. Results:Neonatal cardiac myocytes took up cholesterol from the different hiChol media at a concentration-dependent manner. In normoxia, viability of hiChol neonatal cardiac myocytes was not significantly changed, however, superoxide levels were increased as compared to vehicle. After SI/R, the viability of hiChol neonatal cardiac myocytes was decreased and total ROS level was increased as compared to vehicle. HiChol combined with hyperglycemia further aggravated cell death and oxidative stress in normoxic as well as in SI/R conditions. Viability of hiChol adult cardiac myocytes was significantly decreased and superoxide level was increased in normoxia and these changes were further aggravated by SI/R. HiChol combined with hyperglycemia further aggravated cell death, however level of oxidative stress increased only in normoxic condition. Conclusion:HiChol rat cardiac myocytes showed reduction of cell viability and increased oxidative stress, which were further aggravated by SI/R and with additional hyperglycemia. This is the first demonstration that the combination of the current hypercholesterolemic medium and SI/R in cardiac myocytes mimics the cardiac pathology of the comorbid heart with I/R and hypercholesterolemia.

Project description:ObjectiveThe incidence of acute myocardial infarction (AMI) is increasing yearly. With the use of thrombolysis or percutaneous coronary intervention (PCI), the mortality rate of acute myocardial infarction has been significantly reduced. However, reperfusion can cause additional myocardial injury. There is still a lack of effective drugs to treat I/R injury, and it is urgent to find new therapeutic drugs.MethodsIn this study, network pharmacology was used to predict potential targets and biological processes involved in Muscone-mediated treatment of acute myocardial infarction. To model ischemia‒reperfusion injury, a hypoxia-reoxygenation model and in vivo ischemia‒reperfusion injury C57BL/6 mice model was constructed. Mice were treated with Muscone i.p. for 4 weeks. We detected the cardiac function on day 28.The expression levels of the apoptotic proteins Caspase-3 and Bax and the anti-apoptotic protein Bcl-2 were detected by immunoblotting after Muscone treatment of AC16 cells and in vivo. Additionally, the gene expression levels of the PUMA and p53 were analyzed by qRT‒PCR. Molecular docking was used to evaluate the binding energy between Muscone and NLRP3-related proteins. Immunoblotting and qRT‒PCR were used to assess the expression levels of NLRP3 signaling pathway-related proteins (NLRP3, ASC, and Caspase-1) and the NLRP3 gene, respectively. Moreover, the extracellular acidification rate of AC16 cells was measured using the Seahorse system to evaluate glycolysis levels after Muscone treatment. The expression of the key glycolytic enzyme PKM2 was analyzed by immunoblotting and qRT‒PCR. Finally, ChIP‒qPCR was performed to determine the levels of histone modifications (H3K4me3, H3K27me3, and H2AK119Ub) in the PKM2 promoter region.ResultsGO functional enrichment analysis revealed that muscone was involved in regulating the biological processes (BP) of AMI, which mainly included negative regulation of the apoptosis signaling pathway, the response to lipopolysaccharide, and blood pressure regulation. The cellular components (CC) involved in muscone-mediated regulation of AMI mainly included lipid rafts, membrane microdomains, and membrane regions. The molecular functions (MF) involved in muscone-mediated regulation of AMI mainly included oxidoreductase activity, nuclear receptor activity, and transcription factor activity. In vitro results indicated that muscone treatment could inhibit the expression levels of Bax and Caspase-3 in AC16 cells after ischemia‒reperfusion while increasing the expression level of the antiapoptotic protein Bcl-2. Muscone significantly suppressed the transcription levels of p53 and PUMA in AC16 cells. Molecular docking suggested that muscone could bind well with the Cryo-EM structure of NEK7(PDB ID:6NPY). Further investigation of inflammatory pathways revealed that muscone could inhibit the expression level of NLRP3 in AC16 cells and reduce the expression levels of Caspase-1 and Caspase recruitment domain. Fluorescent quantitative PCR experiments showed that muscone significantly inhibited the transcription of NLRP3. Moreover, we found that muscone could enhance the glycolytic efficiency of AC16 cells, which may be related to the increased protein expression of PKM2 in AC16 cells. Fluorescent quantitative PCR showed that muscone could increase the transcription level of PKM2. Chromatin immunoprecipitation assays showed that muscone treatment increased the expression level of H3K4me3 in the PKM2 promoter region and inhibited the levels of H3K27me3 and H2AK119Ub in the PKM2 promoter region.ConclusionMuscone promoted myocardial glycolysis and inhibited NLRP3 pathway activation to improve myocardial ischemia‒reperfusion injury.

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Myocyte-specific overexpressing HDAC4 promotes myocardial ischemia/reperfusion injury.

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Myocyte-specific overexpressing HDAC4 promotes myocardial ischemia/reperfusion injury.

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