Reduced positive feedback regulation between myosin crossbridge and cardiac troponin C in fast skeletal myofibrils

Several studies have shown that substitution of cardiac troponin C into fast skeletal muscle causes a marked reduction in cooperativity of Ca²⁺-activation of both myofibrillar ATPase and tension development. To clarify the underlying mechanisms, in the present study, Ca²⁺ binding to cardiac troponin C inserted into fast skeletal myofibrils was measured. Two classes of binding sites with different affinities (classes 1 and 2) were clearly identified, which were equivalent stoichiometrically to the two high-affinity sites (sites III and IV) and a single low-affinity site (site II) of troponin C, respectively. Ca²⁺ binding to class-2 sites and Ca²⁺-activation of myofibrillar ATPase occurred in roughly the same Ca²⁺ concentration range, indicating that site II is responsible for Ca²⁺-regulation. Myosin crossbridge interactions with actin, both in the presence and absence of ATP, enhanced the Ca²⁺ binding affinity of only class-2 sites. These effects of myosin crossbridges, however, were much smaller than the effects on the Ca²⁺ binding to the low-affinity sites of fast skeletal troponin C, which are responsible for regulating fast skeletal myofibrillar ATPase. These findings provide strong evidence that the reduction in the cooperative response to Ca²⁺ upon substituting cardiac troponin C into fast skeletal myofibrils is due to a decrease in the positive feedback interaction between myosin crossbridge attachment and Ca²⁺ binding to the regulatory site of troponin C.