In humans and other mammals, the maximum work capacity of myocardium in health and disease is limited by the isoforms of myosin heavy chains (MHC) expressed in the left ventricle-there is a nearly 10- fold range in actin-activated ATPase activity of MHC isoforms in species from mouse to human. Small animals tend to express predominantly a-MHC, a fast isoform, while large animals and humans express predominantly b-MHC, a slower and more efficient isoform. However, a small amount (<10% of total) of a-MHC is expressed in the adult human ventricle, which decreases to zero in old age or in diseases such as heart failure. Based on these observations and results during the present project period, we hypothesize that low-level expression of a-MHC contributes to a greater rates of rise of pressure in healthy ventricles, while the loss of a MHC leads to depressed kinetics of pressure (force) development. With respect to mechanisms underlying kinetic differences between MHC isoforms, we hypothesize that a flexible loop (residues 204-216) adjacent to the pocket modulates the kinetics of nucleotide turnover, and that the effects of the loop on kinetics are influenced in an isoform-specific manner by the sequence of the loop and by electrostatic interactions of the loop with another region of the MHC (residues 323-351), the so- called interactive micro-domain (IMD). The following experiments will be done to test these hypotheses: (1) the basis for differences in turnover kinetics of a and b-MHC isoforms will be investigated using knock- in technology to generate mice expressing mutant MHC's in which the loop and/or IMD are replaced with analogous sequences from other MHC isoforms, and then assessing the effects of these mutations on the kinetics of contraction, nucleotide turnover, and ADP release; (2) effects on kinetics due to interactions between the loop and IMD will be assessed by reversing electrostatic charges in these two regions of myosin; (3) contractile effects of expression of small amounts of a-MHC in human myocardium will be predicted from a model based on rate constants of cross-bridge cycling for a and b-MHC isoforms; (4) effects on kinetics due to variable expression of a-MHC will be determined from mechanical measurements on skinned myocardium from healthy or diseased human hearts; and (5) the possibility that regional function in the left ventricle varies with MHC content will be investigated by determining the regional distribution of MHC isoforms in the apex and the left ventricular free wall from healthy and diseased hearts. These studies promise' to provide new insights into the mechanisms by which myocardial contraction varies in health and disease, and results should identify potential molecular targets for therapeutic interventions in diseases such as heart failure. ? ? ? ? ? ?
