My long term goals are to understand the physiological regulation of cardiac muscle contraction, particularly with respect to Ca and how it changes with pathology. We have developed a number of techniques to study in detail the regulation of [Ca] via individuals Ca transport mechanisms in normal cardiac myocytes. It is also increasingly clear that defects in cellular Ca regulation are intimately involved in the process of cardiac hypertrophy and/or the transition of decompensation during chronic pressure overload. In addition, several groups have used cultured neonatal myocytes to study gene regulation in relation to hypertrophy (since expression changes are relatively fast and their environment can be easily controlled). The overall aim of the present proposal is to combine the above approaches to provide a more comprehensive picture of hypertrophic changes in cellular Ca regulation (from the level of gene and protein expression to cellular Ca transport and in vivo hemodynamics). We will study three experimental model offering different advantages and disadvantages: 1) chronic aortic banding in adult rats at different times after banding (e.g. 8 & 16 weeks), 2) a similar experimental model in rabbit and 3) cultured neonatal rat ventricular myocytes. We will measure a) in vivo hemodynamics during chronic aortic banding (e.g. LVEDP, dp/dt, relaxation time constant, T...), b) mRNA message and protein levels for key proteins such as the SR Ca-pump, Na.Ca exchange, Ca channels, MHCa/b and phospholamban and c) Ca transients, ion currents and cell shortening in myocytes isolated from the same hearts using specific protocols to determine the individual abilities of Ca transport by the SR Ca-pump, Na/Ca exchange, Ica, mitochondria and the sarcolemmal Ca-pump in intact cardiac myocytes. The specific questions to be addressed in banded rats (1-3.5), cultured cells (4-6) and banded rabbits (7) are: 1) Does hypertrophy reduce SERCa2 message, protein and SR Ca transport in intact cells? 2) Are there concomitant changes in Na/Ca exchange or other Ca transport system? 3)Do Ca transport changes coincide with specific phases of hypertrophy (e.g. transition to failure)? 4) Do changes in gene expression and Ca transport in cultured myocytes mirror hypertrophy? (e.g. after stimulation with phorbol esters or angiotensin II, vs verapamil-induced arrest) 5) Are changes in Ca transport and hypertrophy prevented by verapamil or ACE inhibitors? 6) Is regulation of the SR Ca-pump & Na/Ca exchange coordinated or can changes be compensatory? (e.g. when SR Ca transport in cultured cells is blocked by thapsigargin do both systems increase?) 7) Do similar changes in Ca regulation occur in rabbit heart? (Since the rat may not be the ideal model for human hypertrophy) Answer to these focused, yet comprehensive experimental questions will contribute to our overall understanding of changes in cellular Ca transport and their molecular basis for the development of compensatory cardiac hypertrophy and the transition to failure.
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