The purpose of this research proposal is to relate the depression of ventricular function in dilated cardiomyopathy to alterations of the mechanical behavior of the cardiac myofibril. Further, we hope to correlate changes in myofibrillar mechanical behavior to alterations of its low-molecular weight components (troponin subunits, myosin light chains and/or C-protein). We will utilize a small animal model of dilated cardiomyopathy produced by chronic rapid pacing. Contractile properties of failing myocardium will be characterized by measuring force-sarcomere- length and force-sarcomere velocity relations over varying levels of activation in isolated living and chemically permeabilized trabeculae, and in chemically skinned single myocytes. Mechanical measurements will be performed using state-of-the art techniques to measure and servo-control sarcomere length. Content and isoform distribution of the protein constituents of the myofibril will be measured in all preparations in which mechanical measurements are made, using SDS-polyacrylamide gel electrophoresis. Phosphorylation status of these proteins will be determined using high- voltage isoelectric focussing. Changes in content, isoform distribution and phosphorylation status of myofibrillar proteins will be related to alterations in mechanical behavior using regression analysis, and will be further explored by repeating mechanical measurements and biochemical analyses after reversal of heart failure (occurring with cessation of rapid pacing). Finally, the relationship between specific low-molecular weight myofibrillar proteins and mechanical alterations in heart failure will be directly tested by extraction of selected proteins from skinned single cardiomyopathic myocytes. If the protein under study mediate changes in contractile properties, subsequent reconstitution of the protein with its equivalent from non-failing myocardium should restore contractile function to control levels. The applicant has prior experience with mechanical studies involving whole hearts and isolated muscles, including studies involving animal models of dilated cardiomyopathy and hypertrophy. The sponsor has extensive experience and expertise in mechanical and biochemical studies of isolated muscle, and has developed many of the isolated, single myocyte techniques and extraction/reconstitution methods described in the research proposal. This proposal represents a novel extension of these powerful techniques to study of cardiac pathophysiology.
| Wolff, M R; Buck, S H; Stoker, S W et al. (1996) Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation. J Clin Invest 98:167-76 |
| Wolff, M R; Whitesell, L F; Moss, R L (1995) Calcium sensitivity of isometric tension is increased in canine experimental heart failure. Circ Res 76:781-9 |
