Myocardial contractility is enhanced with development. The proposed studies will test whether increases in cytosolic calcium(Cai) and in the sensitivity of the myofilaments to Ca underlie this enhancement, whether the immature cell depends relatively more on extracellular calcium(Cao), and whether changes in troponin T (TnT) isoform expression with development are correlated with changes in the sensitivity of the myofilaments to Ca. Three preparations obtained from rabbit ventricles at different stages of development will be studied: Calcium-tolerant intact single cells, chemically-skinned single cells, and chemically-skinned multicellular bundles. The membrane-intact isolated cell will be loaded with the fura-2 AM> Sarcomere shortening and changes in the 340/380 fluorescence ration will be measured at various stimulus patterns and perturbations that will allow us to examine the effects of changes in Cai and of different Cao, Nao, and Nao/Cao ratios, with and without the use of a calcium channel blocker. The fluorescence data will be calibrated by measuring the minimum and maximum fluorescence ratios, Rmin and Rmax, of the cell. The effects of age on Cai and extent of it modulation, and their relationship to developmental changes in sarcomere shortening will be examined. Sarcomere shortening waveforms will be recorded from intact cells: the cells will then be chemically skinned and the force-pCa relation determined and compared with that obtained from the multicellular preparation. The effects of development on the relation and on Cai will be studied. The force-pCa relation of chemically-skinned multicellular bundles will be measured and analyzed. Polyacrylamide gel electrophoresis will be used to examine the protein profile of the bundle. The proportion of TnT isoforms expressed in each bundle will be obtained from the gel and will be compared to the force-pCa characteristics of the preparation, pCa50 and the Hill constant. The effect of TnT isoform expression and its changes with development on the force-pCa relation will be analyzed. The proposed studies will provide new data that will allow us to test hypotheses that relate the control of Cai, the Ca sensitivity of the myofilaments, and the troponin T isoforms to the maturational increase in contractility.
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