Cross-Bridge Kinetics of Cardiac Myofibrils Carrying Myopathy-Causing Mutation

Dumka, Disha., Cross-bridge kinetics of cardiac myofibrils carrying myopathy-causing mutation. Doctor of Philosophy (Biochemistry and Molecular Biology), May 2007, 98 pp., 8 tables, 23 illustrations, and bibliography: 102 titles. Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). We have focused on one particular mutation of RLC-substitution of glutamic acid (E) at position 22 for lysine (K). The E22k mutation is located in the RLC Ca2+ -binding site. Earlier work has demonstrated that phosphorylation and Ca2+ binding are significantly altered by the E22K mutation. Studies with transgenic (Tg) mice have demonstrated that E22K-RLC mutation increases Ca2+ sensitivity of myofibrillar ATPase activity and steady-state force. However, the mechanisms for the E22K-mutated myocardium that could potentially trigger hypertrophy as seen in human patients harboring this mutation remain unclear. In order to better understand the impact of the E22K-RLC mutation on cardiac muscle contraction, we have studied the three primary parameters which best reflect the mechanism of actomyosin cross-bridge cycling during force generation. Tau one and two (t1 and t2) are the mechanical parameters which measure the dissociation and rebinding time of myosin heads from actin, respectively. Tau three (t3) is an enzymatic parameter which measures the dissociation time of ADP from the active site of myosin. For this study single-turnover contraction experiments were performed on Tg (wild-type and E22K) and non-Tg mouse cardiac myofibrils. Tau one (t1) was statistically greater in Tg-m (Tg-E22K) than in controls indicating that the E22K mutation slows down the rate of cross-bridge dissociation. However, the in-vitro binding experiments showed no difference in binding properties of T g-m vs. Tg-wt myosin to fluorescently labeled actin suggesting that this was a function of genetic manipulation rather than an intrinsic change to muscle. The slight increase in t1 was probably cause by myofibrillar disarray. Tau two (t2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt indicating that the decrease in Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the transgenic mice. Tau three (t3) was the same in Tg-m and in controls indicating that the E22K mutation had no effect time of ADP dissociation from the myosin active site. Thus the E22K mutation did not affect the three parameters that were used to study the cross-bridge kinetics of the cardiac muscle from the transgenic mice carrying the E22K-RLC mutation. On extrapolating the results of this study with transgenic mice to humans, it is likely that the change in cross-bridge kinetics is not the primary trigger through which E22K-RLC mutation affects muscle contraction. However one possible limitation of this study is that the Tg-E22K mice did not completely recapitulate the human phenotype of E22K-mutation. Overall, in this study, we successfully followed the mechanical and the enzymatic events in a small population of cross-bridges (~400) in contracting Tg-m cardiac myofibrils. The characterization of motion of a small population of cross-bridges is important because the different steps of cross-bridge cycle do not become obscured and thus it becomes easy to detect any changes in the cross-bridge cycle.