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

Abstract

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.

Hypertrophic Cardiomyopathy Mutations of Troponin Reveal Details of Striated Muscle Regulation

“…Cardiac muscle fiber preparations containing Δ14-TnT produced the same maximum force as fibers with wild type troponin and had an increased Ca 2+ sensitivity by 0.2 pCa units ( Gafurov et al, 2004b ). Fibers containing both Δ14-TnT and A8V-TnC produced about 18% more force than fibers with wild type troponin ( Johnson et al, 2020 ). Both Δ14-TnT and the combination of Δ14-TnT & A8V-TnC increased the basal activity by about 3-fold and increased the Ca 2+ sensitivity by 0.3–0.34 pCa units.…”

“…The basic C-terminal region of TnT attenuates the stabilization of the active state that would otherwise occur at saturating Ca 2+ . Higher animals having the CB region have gained a dual activation system requiring Ca 2+ and force producing crossbridges ( Johnson et al, 2020 ).…”

“…The previous solution studies raise an interesting question of how muscle contraction would change if there were no B state and if Ca 2+ gave ≥ 3-fold greater activation of ATPase activity than normally observed. This question was examined with the in vitro motility assay and with a cardiac fiber preparation ( Johnson et al, 2020 ).…”

“…Actin filaments containing Δ14-TnT had an enhanced rate of movement at both low and high Ca 2+ levels ( Johnson et al, 2020 ). The maximum speed of movement was about 1.8x faster than wild type at saturating Ca 2+ .…”

C-Terminal Basic Region of Troponin T Alters the Ca2+-Dependent Changes in Troponin I Interactions

“…13 Stepwise truncation of residues from the CB region of TnT led to an in increase in the stabilization of the active state of actin at low Ca 2+ and especially at saturating Ca 2+ . 18 Substitution of Ala for the Lys and Arg residues in the Cterminal region of cardiac 19,20 or skeletal 21 TnT led to increases in ATPase activity and Ca 2+ sensitivity.…”