Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters...

Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca²⁺ and to eliminate one of the inactive states at low Ca²⁺. The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca²⁺, full activation with Δ14-TnT·A8V-TnC decreased the apparent K_M for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca²⁺ with Δ14-TnT; the maximum shortening speed at high Ca²⁺ increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca²⁺ sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca²⁺ and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca²⁺, elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.