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Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury.

ABSTRACT: Connexin 43 (Cx43), a product of the GJA1 gene, is a gap junction protein facilitating intercellular communication between cardiomyocytes. Cx43 protects the heart from ischemic injury by mechanisms that are not well understood. GJA1 mRNA can undergo alternative translation, generating smaller isoforms in the heart, with GJA1-20k being the most abundant. Here, we report that ischemic and ischemia/reperfusion (I/R) injuries upregulate endogenous GJA1-20k protein in the heart, which targets to cardiac mitochondria and associates with the outer mitochondrial membrane. Exploring the functional consequence of increased GJA1-20k, we found that AAV9-mediated gene transfer of GJA1-20k in mouse hearts increases mitochondrial biogenesis while reducing mitochondrial membrane potential, respiration, and ROS production. By doing so, GJA1-20k promotes a protective mitochondrial phenotype, as seen with ischemic preconditioning (IPC), which also increases endogenous GJA1-20k in heart lysates and mitochondrial fractions. As a result, AAV9-GJA1-20k pretreatment reduces myocardial infarct size in mouse hearts subjected to in vivo ischemic injury or ex vivo I/R injury, similar to an IPC-induced cardioprotective effect. In conclusion, GJA1-20k is an endogenous stress response protein that induces mitochondrial biogenesis and metabolic hibernation, preconditioning the heart against I/R insults. Introduction of exogenous GJA1-20k is a putative therapeutic strategy for patients undergoing anticipated ischemic injury.

SUBMITTER: Basheer WA

PROVIDER: S-EPMC6237442 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury.

Basheer Wassim A WA Fu Ying Y Shimura Daisuke D Xiao Shaohua S Agvanian Sosse S Hernandez Diana M DM Hitzeman Tara C TC Hong TingTing T Shaw Robin M RM

JCI insight 20181018 20

Connexin 43 (Cx43), a product of the GJA1 gene, is a gap junction protein facilitating intercellular communication between cardiomyocytes. Cx43 protects the heart from ischemic injury by mechanisms that are not well understood. GJA1 mRNA can undergo alternative translation, generating smaller isoforms in the heart, with GJA1-20k being the most abundant. Here, we report that ischemic and ischemia/reperfusion (I/R) injuries upregulate endogenous GJA1-20k protein in the heart, which targets to card ...[more]

PMID: 30333316

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Project description:BackgroundInhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored.MethodsC57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining.ResultsMalonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate.ConclusionsCardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.

Project description:AimsThe FDA-approved histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA, Vorinostat) has been shown to induce cardiomyocyte autophagy and blunt ischemia/reperfusion (I/R) injury when administered at the time of reperfusion. However, the precise mechanisms underlying the cardioprotective activity of SAHA are unknown. Mitochondrial dysfunction and oxidative damage are major contributors to myocardial apoptosis during I/R injury. We hypothesize that SAHA protects the myocardium by maintaining mitochondrial homeostasis and reducing reactive oxygen species (ROS) production during I/R injury.MethodsMouse and cultured cardiomyocytes (neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes) I/R models were used to investigate the effects of SAHA on mitochondria. ATG7 knockout mice, ATG7 knockdown by siRNA and PGC-1α knockdown by adenovirus in cardiomyocytes were used to test the dependency of autophagy and PGC-1α-mediated mitochondrial biogenesis respectively.ResultsIntact and total mitochondrial DNA (mtDNA) content and mitochondrial mass were significantly increased in cardiomyocytes by SAHA pretreatment before simulated I/R. In vivo, I/R induced >50% loss of mtDNA content in the border zones of mouse hearts, but SAHA pretreatment and reperfusion treatment alone reverted mtDNA content and mitochondrial mass to control levels. Moreover, pretreatment of cardiomyocytes with SAHA resulted in a 4-fold decrease in I/R-induced loss of mitochondrial membrane potential and a 25%-40% reduction in cytosolic ROS levels. However, loss-of-function of ATG7 in cardiomyocytes or mouse myocardium abolished the protective effects of SAHA on ROS levels, mitochondrial membrane potential, mtDNA levels, and mitochondrial mass. Lastly, PGC-1α gene expression was induced by SAHA in NRVMs and mouse heart subjected to I/R, and loss of PGC-1α abrogated SAHA's mitochondrial protective effects in cardiomyocytes.ConclusionsSAHA prevents I/R induced-mitochondrial dysfunction and loss, and reduces myocardial ROS production when given before or after the ischemia. The protective effects of SAHA on mitochondria are dependent on autophagy and PGC-1α-mediated mitochondrial biogenesis.

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Project description:Understanding the complex pathogenesis in myocardial ischemia/reperfusion (I/R) injury (IRI) is an urgent problem in clinical trials. Increasing pieces of evidence have suggested that miRNAs are involved in the occurrence and development of heart diseases by regulating mitochondria-related gene expression. Mitochondria have been acknowledged as the key triggers of cardiac I/R injury. However, the potential impact of miR-130a on mitochondria remains unclear in myocardial IRI. Exploring the regulatory mechanism of miR-130a on mitochondria may provide a new target for IRI therapy. In the present study, we found that miR-130a significantly increased in acute myocardial infarction (AMI) patients and myocardial I/R rats. MiR-130a could downregulate the viability of cardiomyocytes and the knockdown of miR-130a could protect the viability of cardiomyocytes under hypoxia-reoxygenation (HR). Over-expression of miR-130a resulted in mitochondrial dysfunction. It was evidenced by decreases in mitochondrial ATP production, mitochondrial membrane potential (MMP), and an increase in reactive oxygen species (ROS) production. However, suppression of miR-130a could protect against mitochondrial damage, show elevation of mitochondrial ATP production rate and MMP, and reduce ROS production. We further explored the effect of miR-130a on the mitochondrial quality control (QMC) system by determining mitochondrial-protein-specific proteases and analyzed mitochondrial morphology by fluorescence imaging and electron microscopy, respectively. It was noted that miR-130a could suppress mitochondrial fusion and FUNDC1-mediated mitophagy to accelerate myocardial IRI. Moreover, we investigated the potential miR-130a targeted mitochondria-related genes to understand the regulatory mechanism of miR-130a in the setting of myocardial IRI. It was revealed that miR-130a targeted GJA1, and GJA1 rescued IRI by enhancing ATP production rate and oxidative phosphorylation, meanwhile protecting cell viability, MMP, and activating mitophagy. In addition, the knockdown of miR-130a significantly activated FUNDC1-mediated mitophagy, while the knockdown of GJA1 reversed the relevant response. Collectively, our findings suggest that miR-130a regulates FUNDC1-mediated mitophagy by targeting GJA1 in myocardial IRI.

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Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury.

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Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury.

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