The cytoskeleton and actin-associated proteins provide for cellular structural integrity and function as a critical link between the extracellular matrix and the contractile apparatus of the myocyte. The focal adhesion is the intersection between the extracellular matrix and the cellular cytoskeleton, and may be a pivotal point for transmission of mechanical signals and organization of the actin based cytoskeleton. Vinuclin is a key focal adhesion protein. While vinculin is found in all cells, a muscle-specific splice variant termed metavinculin is found only in smooth and cardiac muscle. The biological role of metavinculin is poorly understood and its deficiency or mutation has been linked to human dilated cardiomyopathy. Homozygous vinculin knockout mice die by mid-gestation with severe neural and cardiac abnormalities via an unknown mechanism. Heterozygous vinculin knockout mice survive and breed normally but have abnormal cardiac function and die suddenly. It is possible that the cardiac abnormalities are due to direct affects of the vinculin deficiency in cardiac myocytes or related to alterations in non-cardiac cells. We hypothesize that normal vinculin expression in cardiac myocytes is crucial for appropriate cardiogenesis, cardiac myofibrillogenesis and function of the mature heart and that distinct biological functions of metavinculin exist. To test this we will manipulate vinculin and metavinculin in cultured cardiac myocytes and in the murine genome. Molecular, biochemical and immunocytochemical studies of cultured cells will be performed. Analysis of intact developing mice and the adult murine heart will likewise be evaluated by molecular, morphological, biochemical and physiological techniques. We propose the following specific aims: ? 1. Examine the role of vinculin in cardiogenesis and post-natal function of the heart by use of currently existing global vinculin """"""""knockout"""""""" mice. ? 2. Study how deletion of the vinculin gene specifically in cardiac myocytes alters cardiac form and function. ? 3. Evaluate the biological function of the muscle-specific splice-variant metavinculin as distinct from vinculin, in cultured cardiac myocytes and the intact mouse. ? ?
