The objectives of this proposal are driven by the hypothesis that the regulatory, or phosphorylatable light chain 2 (MLC2) plays a critical role in cardiac function. Furthermore, we hypothesize that there are functional consequences which result from the differential expression patterns of the different MLC2 isoforms; these are reflected in the changing patterns of MLC2 expression that are observed in the ventricles and atria of falling hearts. Our objective is to test these hypotheses directly in the whole animal context using transgenic mice. Using promoters that are able to drive varying levels of transgene expression in the murine cardiac compartment the steady state levels and isoform populations of the MLC2's that are normally present in the murine atrium and ventricle will be modified. There are two major MLC2's that are expressed in the heart, an atrial specific and ventricle specific form. In the first set of experiments, we will test the stoichiometry of the system as well as isoform functionality by overexpressing in both the atria and ventricle, at various levels, the endogenous MLC2 that is normally found in the ventricle. Similar experiments using a construct in the antisense orientation will be performed in order to lower the ratio of MLC2/MLC1, a condition that is observed in idiopathic dilated cardiomyopathy. Finally, experiments will be carried out with a cDNA which encodes an isoform that is normally expressed only in skeletal striated muscle (MLC2f) and not in the heart in order to determine if there are functional correlates to MLC2 isoform diversity. Parallel studies on the MLC isoforms present in human heart tissue derived from patients in early (NYHA class 1 and 2) and lat. (NYHA class 3 and 4) systolic and diastolic heart failure will be carried out using quantitative PCR analyses in order to relate changes in MLC2/MLC1 ratios at the RNA level to altered cardiac function in vivo. A genetic approach using overexpression and ablation studies carried out via transgenesis holds the promise of providing an unambiguous assignation of basic function and the physiologic or pathophysiologic significance of differential MLC2 isoform content. By coupling a molecular genetic approach with the ability to generate stable transgenic lines for analyses of contractile function and performance, a rigorous analysis of both major and minor phenotypic effects resulting from perturbations in MLC content can be made.
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