Our long-term objective is to understand at a molecular mechanistic level the contribution of 1) the contractile protein system (myosin) and 2) the excitation contraction coupling system (ECC) in particular the sarcoplasmic reticulum to myocardial performance in normal hypertrophied and failing hearts (animal, human) including how alterations in these systems contribute to the transition from one state to another. Our approach to achieving the long term objective is to make measurements using cell biophysical (myothermal measurements of energetics, simultaneous measurement of mechanics), biochemical (enzyme activity, isoforms) and molecular biological (m-RNA, antibodies) techniques. Studies will be carried out on isolated myocardium from normal, hypertrophied and failing hearts and from normal, compensated hypertrophied, moderate failing, and end-stage failing human hearts. Thermopile heat measurements of tension dependent heat (TDH) provide measures of in vitro function of the contractile system by way of monitoring force, work and energetics of crossbridge cycling. From these measurements we will be able to determine the average crossbridge tension time integral and cycling rate during contraction and relaxation. The ECC is evaluated by measuring tension independent heat output (TIH)(index of beat to beat total calcium cycling) in conjunction with aequorin light signals (index of free calcium levels) throughout the course of a contraction-relaxation cycle. The tension dependent heat measurements in isometric and working contraction provide, for. the first time, an assessment of the contribution of changes in isoenzyme composition or myofibrillar ATPase activity to economy (integral of Tdt/TDH) and efficiency (W/(W + TDH)) at the level of the crossbridge cycle. The tension independent heat measurements in conjunction with aequorin light signals provide a measure of calcium cycling in the force-frequency studies, the length dependent activation studies and the non steady state rate experiments. These provide unique insights into the contribution of calcium cycling to altered mechanics. The analysis of specific mRNAs and the associated proteins of the sarcoplasmic reticular system (SR) provides information regarding the contribution of alterations in the SR system to the observed changes in calcium cycling.
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