The Mechanisms of Micro Rnas Modulate Cardiommyocytic Intracellular Signal Transduction during Cardioplegia-Induced Cardiac Arrest under Cardiopulmonary Bypass

Background : Cardioplegic cardiac arrest (CCA) under cardiopulmonary bypass (CPB) during cardiac surgery represents a state-of-the-art technique for myocardial preservation. Although certain intracellular mediators have been shown to play a role in CCA, the exact nature and mediator of the CCA is not known. Diverse forms of injury and stress evoke a hypertrophic growth response in adult cardiac myocytes, which is characterized by an increase in cell size, enhanced protein synthesis, assembly of sarcomeres, and reactivation of fetal genes, often culminating in heart failure and sudden death. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs that were regulated during cardiac hypertrophy and heart failure. miRNAs are novel biomarkers, modulators and therapeutic targets for disease. This study was designed to further examine the relationship between cardiomyocytic apoptosis and miRNAs-modulated cardiac protection during CCA. Objective : Since miRNAs are likely to be involved during cardiac insults, experiments will be designed to determine: 1. whether miRNAs expression could protect or provoke cardiomyocytic apoptosis durring CCA under CPB, 2. if the miRNAs expression pattern in the failed heart during CCA under CPB is different from that of the normal hearts, and 3. modulating of the miRNAs pathway also had a role in cardiac protection during CCA under CPB ex vivo. Methods : This three-year proposal will take isolated heart apparatus (first year), coronary artery ligation with congestive heart failure rat animals (2nd and 3rd years) and H9c2 cardiomyocytes (both 1st and 2nd years) 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 60 minutes then reperfused the H9c2 cells with normal oxygenated culture medium for another 16 hours. In the first year, we will looking for the possible miRNAs and their related proteins to clarify that if there is a role of the miRNAs in the cardiomyocytic apoptotic mechanism during CCA under CPB. 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 miRNAs up- and down-regulated from our first year results. In the third year, we will utilize the information that we gain from two years to reverse or strengthen the pro- and anti-apoptotic effects of miRNAs using LND- antimiRNAs in vivo. The following experiments will be performed to check the ischemia/reperfusion injury： A. Flow cytometry analysis for apoptosis， B. miRNAs array C. MALTI-TOF MS D. Detect mRNAs expression pattern (qRT-PCR) E. TUNEL method F. Western blot to check protein expression G. ELISA to check cardiomyocytic damage (cTnI, MPOs, caspases activities, ) Hypothesis. We hypothesize that 1. after hypothermic CCA, miRNAs will be expressed by cardiomyocytes, which will exacerbate in the coronary artery ligaiton congestive heart failure animal model; 2. the occurrence of cardiomyocytic apoptosis under hypothermic CCA could be modulated via miRNAs expression; 3. the expression of miRNAs can affect the down-stream protein expression pattern ; 4. modulating the miRNAs expressed during I/R injury in the cell model could change the effects of I/R insults in vitro; 5. inhibit of the miRNAs activation may be achieved in vivo and provide cardiac protection. Significance. The major goal the this proposal is to determine the relationship among miRNAs in myocardial injury following cardiomyocytic ischemia an perfusion injury and whether specific antagonists used during cardiac surgery will induce transcriptional factors translocation, 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:PC10001-0218
External Project ID:NSC98-2628-B182-001-MY3