During development myocardial proteins and their isoforms undergo marked changes in expression. During that process cardiac contractility also changes. The proposed studies will test hypotheses relate to the effects of development on the mechanisms that control cytosolic calcium concentration ([Ca]i) transients and the response of the myofilaments to [Ca]i. Isoform expression of the L-type calcium channel alpha1 subunit and of troponin T, two proteins central to excitation-contraction coupling and the development acquisition of the sarcoplasmic reticulum (SR) and t-tubule system provide the basis for studying cardiac myocytes from newborn, three week, and adult rabbits. Using the whole-cell voltage clamp preparation and fura-2 fluorescence, we will measure [Ca]i, sarcomere dynamics, and calcium current (ICa). We will examine the effects of development on ICa's contributions to [Ca]i directly and through inducing SR calcium release, and we will correlate those to changes in alpha1 subunit isoform expression using Western blots probed with alpha1 subunit isoform antibodies. The contribution of the SR to [Ca]i as a function of age and alpha1 subunit isoform expression will be determine by measuring SR release of calcium in response to activation by comparing the transients obtained with the SR active and when it is functionally blocked and SR calcium content from the caffeine-induced current. We will determine the force-pCa relation of chemically skinned cells in which, unlike membrane-intact muscle, the myofilament ionic milieu can be controlled tightly in order to test whether myofilament sensitivity to calcium and its dependence on sarcomere length are affected by development. Cells in short term culture will be used to dissect the effects of alpha1 subunit isoforms on ICa and the effects of troponin T isoforms on the sensitivity of myofilaments to calcium. ICa- voltage relations and single channel characteristics will be related to the expression of specific alpha1 subunit isoforms obtained by exposing cells in culture to antisense oligodeoxynucleotides complimentary to alpha1 subunit isoform-specific sequences. We will change troponin T isoform expression in cells in culture by exposing them to antisense oligodeoxynucleotides complimentary to cardiac troponin T isoform specific sequences and will study the effects of such changes on the force-pCa relations of the cells. Studying how development changes myocardial function through altered expression of these proteins important in controlling ICa and the regulation of thick and thin filament interaction will prove valuable in understanding the mechanisms that control [Ca]i and myofilament sensitivity to calcium. The results of these studies will be fundamental to developing rationales for the treatment of patients at all ages with heart disease. For the sick newborn and infant, better care should come through establishing age- dependent therapeutic approaches that are based on knowing how [Ca]i and myofilament response to calcium are controlled at different ages by altered expression of isoform-specific sequences are similar in the human and rabbit.
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