Studies in Molecular Mechanisms of Skeletal Muscle Contraction: Applications to Transgenic Mice with Inherited Cardiomyopathies

Muscular contraction impacts virtually every physiological function in a human body. Nearly 50% of the body weight is contributed by muscle. Skeletal muscle contraction is responsible for performing every day general functions such as moving, grabbing things and lifting weights. Cardiac muscle contraction is responsible for blood circulation in the body and smooth muscle contraction is involved in the contraction of hollow organs such as lungs, stomach and kidneys. It also maintains body temperature. Central to these events is the transformation of energy derived by hydrolysis of ATP to mechanical force. The force generating steps of muscle contraction is thought to result from the interaction of actin and myosin proteins. Although much of the general knowledge of the mechanism of contraction has been known for over 50 years, emerging advanced techniques have identified some of the key intermediate steps and regulating parameters. My doctoral research involves utilizing one such high resolution technique – single molecule fluorescence spectroscopy. I have used it to discern the motion and conformation of myosin cross-bridges in ex-vivo muscle. When studying the dynamic behavior of actin and myosin it is essential to reduce the number of molecules under observation because 1. The local concentration of actin and myosin is dense and the information obtained by averaging trillions of molecules doesn’t reflect the true character of the process. 2. The trajectory of an enzyme catalyzed reaction cannot be followed and the associated kinetics of actomyosin interaction is lost. 3. The situation becomes worse when studying mutations in sarcomeric proteins with low expression. 4. Heterogeneity between neighboring sarcomeres can be reduced. An important goal of muscle research is to measure the rate of the power stroke. Therefore, part of my thesis is focused on characterizing the pre- and post- power stroke states of muscle contraction. While the extent of force generated during muscle contraction is proportional to the extent of Ca2+ released into the muscle, some of the recent studies have shown that the phosphorylation of the regulatory light chain (RLC) of myosin also modulates contraction. Considering the importance of the phosphorylation of RLC, I investigated the distribution of orientation and kinetics of myosin cross-bridges in phosphorylated and de-phosphorylated muscle. Lastly, I have applied our fluorescence polarization technique with single molecule sensitivity to unravel the deranged contractile properties of muscle in people afflicted with Familial Hypertrophic Cardiomyopathy (FHC) disease. Some of the significant conclusions drawn from my project include evidence for the existence of distinct pre- and post- power stroke states of myosin cross-bridges during contraction in Ex Vivo muscle, Regulatory Light Chain phosphorylation disturbs cross-bridge organization and enhances the power stroke state of contraction and FHC induced by mutations in Troponin-T protein impairs myosin cross-bridge interaction with actin and alters cross-bridge kinetics. Clinically, drugs can be developed to modulate power stroke and enhance muscle performance in myopathies. Site targeted small molecules (peptides) can now be screened to correct for hypo-contractile or hyper-contractile properties associated with FHC. Our technique may also serve as a diagnosis tool for early identification of FHC disease. Finally increasing the basal ATPase activity of resting muscle by RLC phosphorylation is of therapeutic importance in treating individuals with Obesity, Type II Diabetes and Metabolic syndrome.