Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems.

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 Ca and to eliminate one of the inactive states at low Ca. The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca, full activation with Δ14-TnT·A8V-TnC decreased the apparent for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca with Δ14-TnT; the maximum shortening speed at high Ca increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca 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 Ca and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca, 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.