The Possible Mechanism of Mirnas Regulation Endoplamic Reticular Stress Response Duriin Cardioplegia Induced Cardiomyocytic Hypoxia/Reperfusion Injury

Background : It is becoming apparent that the accumulation of misfolded proteins in the ER can contribute to vascular and cardiac diseases [1]. Soon after stresses occur to cells, the folding or synthesis of proteins are disturbed, which can lead to the accumulation of misfolded proteins and potentiate cell and organ dysfunction. The accumulation of these misfolded proteins attract chaperones that serve a refolding function, as well as an important role in activating 3 ER transmembrane proteins, PERK (protein kinase R [PKR]-like ER kinase), inositolrequiring enzyme (IRE)-1, and activating transcription factor (ATF)6, which serve as the proximal effectors of the endoplasmic reticulum stress response (ERSR). Cardioplegic cardiac arrest (CCA) under cardiopulmonary bypass (CPB) during cardiac surgery represents a state-of-the-art technique for myocardial preservation. However, cardiomyocytic apoptosis does occur during CCA. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, three miRNAs (30c, 96, 3545-3p) that were assumed in regulating these three proteins during ERSR. This study was designed to further examine the relationship between cardiomyocytic apoptosis and miRNAs-modulated ERSR during CCA. Objective : miRNAs (30c, 96, 3545-3p) are putatively involved in three ER proximal effectors proteins. Experiments will be designed to determine: 1. whether miRNAs expression could protect or provoke cardiomyocytic apoptosis during CCA via modulating ERSR, 2.if the miRNA antagomirs injection in the failed heart could modulating ERSR during CCA and if there were any difference from that of the normal hearts, and 3. modulating of the ERSR pathway via antagonization of miRNAs also had a role in cardiomyoctic protection from apoptosis during H/R injury in vitro. Methods : This three-year proposal will take isolated heart apparatus (first year), coronary artery ligation with congestive heart failure rat animals (2nd year) and H9c2 cardiomyocytes (3rd year) as experimental model。Hypoxic ischemia/reperfusion injury of H9c2 cardiomyocytes will be produced in hypoxic chamber, and cold (4°C) crystalloid cardioplegia will infused every 20 minutes. The whole ischemia will last for 16 hours then reperfused the H9c2 cells with normal oxygenated culture medium for another 2 hours. In the first year, we will looking for the putative antagomirs (30c, 96, 3545-3p) and their related proteins to clarify that if miRNAs had a role of the ERSR in the cardiomyocytic apoptotic mechanism under CCA. In the second year, we will focus on the miRNAs in the congestive failed heart (using coronary artery ligation model) during CCA under CPB, especially those ERSR-related proteins. In the third year, we will utilize the siRNAs transfection method to check the possible downstream pathway modulators in vitro. The following experiments will be performed to check the hypoxia/reperfusion injury： A. Flow cytometry analysis for apoptosis， B. miRNAs array C. Detect mRNAs expression pattern (qRT-PCR) D. TUNEL method E. Western blot to check protein expression F. ELISA to check cardiomyocytic damage (cTnI, caspases activities) Hypothesis. We hypothesize that 1. after hypothermic CCA, ERSR will occur in cardiomyocytes, which will exacerbate in the coronary artery ligaiton congestive heart failure animal model; 2. the ERSR-mediated cardiomyocytic apoptosis under CCA could be modulated via miRNAs; 3. the expression of miRNAs can affect the down-stream ERSR protein expression pattern ; 4. modulating the miRNAs expressed during H/R injury in the cell model could change the effects of H/R insults on ERSR in vitro; 5. injection of antagomirs could inhibit of the miRNAs activation which might be achieved in vivo and provide cardiac protection. Significance. The major goal the this proposal is to determine the relationship among miRNAs-mediated ERSR in myocardial injury following cardiomyocytic ischemia an perfusion injury and whether specific antagonists used during cardiac surgery will reduce ERSR, minimize mitochondrial leakage of apoptotic factors, and enhance overall myocardial protection. We anticipate that information gained in these studies will clarify the pathophysiology of ischemia/reperfusion injury and apoptosis and might represent a novel therapeutic option or prevention of coronary endothelial and myocardial apoptotic injury following extracorporeal circulation. We hope that this knowledge will lead to new strategies for better myocardial protection and better patients’ survival in cardiac surgery.

Project ID:PC10108-0911
External Project ID:NSC101-2314-B182A-137-MY3