Optimization of Dose frequency and concentration of CIR on promotion of Mouse embryonic stem cells and Fibroblasts into cardiomyocytes in-vitro
Abstract: Cardiovascular disease is the number 1 killer and more deadly than all forms of cancer combined. It causes almost 850,000 deaths in the USA and myocardial infarction is the most prevalent form of Cardiovascular disease. Many myocardial infarcted patients are treated by balloon angioplasty, stents, or bypass surgery to improve heart function; however, many to do not significantly recover because connective scar tissue replaces the heart muscle in the infarcted area. In our laboratory, we discovered a unique ribonucleic acid (RNA) initially in Mexican salamander embryos and then in fetal human heart that can turn non-muscle cells in tissue/organ culture into normal-appearing cardiac muscle. We have termed these RNAs, Cardiac-Inducing RNA or CIR. The CIR has been cloned and sequenced from RNA libraries of the axolotl and human producing a total of 596 clones (200 axolotl and 396 human fetal heart). 3 clones (1 axolotl-derived and 2 human-derived) can promote differentiation of non-muscle cells into cardiac muscle. The goal of our current project is: (1) To determine the dose frequency and response effect of the CIR on converting mouse embryonic stem cells(MESCs) and fibroblasts into cardiac myocytes in-vitro;(2) To analyze, quantify and compare the expression of cardiac-specific genes in relation to CIR dose and transfection frequency. We hypothesize that these non-muscle cells will form into cardiomyocytes in vitro in direct proportion to an optimal quantity and frequency of CIR treatment. To test our hypothesis, we will perform quantitative real time PCR(qRT-PCR) to analyze cardiac-specific related gene expression at specific time-points after treatment and perform confocal immunofluorescent staining to identify contractile proteins and myofibrils in the cultured cardiac cells. For qRT-PCR analysis, the interaction between mRNA cardiac protein expression will be investigated using multiple regression analysis, based on the differences between target and reference genes in treated vs. control samples.
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