Persistent stress in the cardiovascular system (heart and smooth muscle)results in an increase in mass and a restructuring of the subcellular elements.The theme of this proposal is directed at understanding the functional consequences of alterations in 1) the contractile system (myosin) and 2) the excitation contraction system (ECC) in particular the sarcoplasmic reticulum. In Project #1 thermopile heat measurements of tension dependent heat (TDH) provide measures of in vitro function of the contractile system in terms of the crossbridge cycle. 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 unique features in Project #1 involve the precision of the partitioning between tension dependent and tension independent heat and the newly developed tissue preparation methodology for carrying out these measurements on human hearts. 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 W"""""""" hearts. In project #2 the subcellular alterations found in hypertrophied and failing hearts are evaluated in terms of ventricular function and oxygen consumption. A very important feature in this proposal is the methodology developed for separating the energy requirements of the contractile and the ECC systems. Projects #3 and #4 add an entirely new dimension to this proposal. They are concerned with how the regulation and modulation of smooth muscle myosin crossbridge cycling (myosin phosphorylation, caldesmon) and employ the exciting new techniques of using fluorescently labelled actin filaments and following their motion as they are propelled by myosin adhered to a glass coverslip. This technique is also applicable to measuring crossbridge force development. Skinned and whole smooth muscle preparations are also used in assessing the role of caldemson in regulating the crossbridge cycle (Project #4). The combination of isolated filament and skinned fiber techniques provide powerful new tools to increase our understanding c,f the regulation of the myosin crossbridge cycle. Project #5 addresses the fundamental question of genetic regulation of specific protein expression (SR) in developing and stressed hearts. In addition studies are planned in collaboration with Project #1 involving the correlation of specific mRNAs and proteins of the SR with the tension independent heat measurements and mechanics. The entire group of projects is directed toward increasing our knowledge of the mechanisms responsible for reorganizing of the cardiovascular system in response to stress and the contribution that alter-
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