Functional analysis of point mutants of cardiac actin involved in dilative and hypertrophic cardiomyopathies

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The biochemical properties of wild-type and a number of point mutants of cardiac actin causing dilative and hypertrophic cardiomyopathy will be investigated. The mutated cardiac actins will be expressed by using the bacculovirus/Sf9 insect cell system, purified and their ablity to polymerize and to stimulate the cardiac myosin ATPase will be determined. Vectors will be generated allowing their ectopic expression in neonatal (NRC) and adult cardiomyocytes (ARC). Their incorporation into sarcomeric thin filaments and their effect on the mechanical output of ARCs will be determined. In case of diminishing effects on the mechanical output small compounds will be tested in order to identify reagents with a positive ionotropic effect on the transfected ARCs. In addition, we will analyse the effect of the small actin binding peptide thymosin ß4 on the survival of control and transfected ARCs and their mechanical output before and after transfection. Cryo-electron microscopy will be used to obtain high resolution images of cardiac F-actin decorated with tropomyosin and wild-type or mutants of the cardiac troponin complex in the absence and presence of Ca2+-ions and/or myosin motor domains.