Key result
Δ14-TnT and A8V-TnC mutants increase maximum cardiac shortening velocity ~1.8-fold alongside enhanced calcium sensitivity.
The study confirms that troponin can stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding, providing insights into cardiac muscle regulation.
Supports troponin stabilization of active thin filament state in vitro; leaves open applicability to human cardiac disease mechanisms.
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 KM 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.
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Johnson et al. (2020) studied this question. Δ14-TnT and A8V-TnC mutants vs. Wild-type troponin or bare actin was evaluated on Actin-activated ATPase activity, in vitro motility, and force in cardiac muscle preparations. Δ14-TnT and A8V-TnC mutants decreased the apparent KM for actin-activated ATPase activity, increased maximum shortening speed 1.8-fold, and increased Ca2+ sensitivity in cardiac muscle preparations.
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