Identifying Mechanisms of Thin Filament Activation in Cardiac Muscle Contraction

Myocardial contractions are generated by the binding of myosin motor proteins to cardiac actin in the thin filament. This process is regulated by the binding of calcium ions to troponin C. When Ca2+ binds to site II of troponin C, the troponin complex undergoes a conformational change which displaces tropomyosin and uncovers the myosin-binding sites on actin. This allows myosin to bind, and in the presence of ATP, allows for cross-bridges to cycle in order to generate force. While calcium binding to troponin is considered to be the key regulating mechanism for striated muscle contraction, myosin cross-bridges are also thought to play a role in cooperatively activating the thin filament. This cross-bridge contribution to thin filament activation appears to be more important in cardiac muscle systems than it is in skeletal muscle. However, many of these experiments examined the effects of cross-bridges under non-physiological conditions, leading to questions about the importance of actively cycling cross-bridges. Furthermore, there are questions as to the contribution of cross-bridge activation under conditions of low cross-bridge number, such as during filament sliding, primarily due to the inability to effectively change the number of cross-bridges that bind within a system in a controlled manner. In order to examine this possibility, I developed a porcine cardiac heavy meromyosin fragment that could be used to effectively vary the amount of myosin cross-bridges present in the in vitro motility assay. Additionally, I utilized this fragment at varying concentrations to determine several key characteristics of the cooperative mechanisms that govern thin filament activation. As a result, it was determined that cardiac myosin is sensitive to temperature when being proteolytically cleaved to form pcHMM, and subsequently, the protocol for making pcHMM requires decreasing the reaction temperature and increasing the protease concentration in order to yield a functional fragment. Secondly, it was determined that cardiac myosin cross-bridges number plays a minimal role in enhancing the activation of the thin filament by Ca2+ during unloaded filament sliding, and that this contribution in cardiac systems will be seen in the presence of a high density of actively cycling motors. Lastly, it is suggested that the enhancement that is seen due to increased numbers of cycling cardiac motors may be due to the regulatory proteins modulating the speed of myosin cycling, which is consistent with previously published literature. These findings help to advance our understanding of the contractile process and increase our understanding of the molecular complexities that govern thin filament activation by Ca2+.