Abstract 16949: Cardiac Troponin I and Myosin Binding Protein C Phosphorylation Independently Eliminate Length-Dependent Activation in Cardiac Muscle
Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for beta-adrenergic modulation of cardiac contractility. PKA target proteins include the N-terminus of cardiac troponin I (cTnI), cardiac myosin-binding protein C (C-protein) and titin. In this study we hypothesis that both cTnI N-terminal phosphorylation and C-protein phosphorylation independently modulate contractility by reducing the Ca2+-sensitivity of force and the effect of sarcomere length (SL) on cardiac force-[Ca2+] relations, an important component of the Frank-Starling mechanism. To differentate cTnI and C-protein phosphorylation effects on force-[Ca2+] relations in skinned cardiac trabeculae from rats, we replaced endogenous cardiac troponin (Tn) with either recombinant WT Tn or Tn that contained either a non-phosphorylatable mutant cTnI(S23/24A) or a phosphomimetic mutant cTnI(S23/24D); neither mutant is phosphorylated by PKA, leaving C-protein as the major target of PKA. Force-[Ca2+] relations were measured at 2.3 and 2.0 um SL; tension redevelopment kinetics (kTR) was measured at each [Ca2+]. Ca2+-sensitivity of force (pCa50) at each SL was similar for trabeculae with WT and cTnI(S23/24A), with pCa50 decreasing at the short SL. PKA treatment reduced pCa50 most at 2.3 um SL, eliminating the SL-dependence of Ca2+-sensitivity. Compared to WT and cTnI(S23/24A), phosphomimetic cTnI(S23/24D) reduced pCa50 at both SL, again with no SL-dependence; PKA slightly reduced pCa50 at both SL. Maximal force (Fmax) at each SL was the same for WT and trabeculae reconstituted with mutant troponins; Fmax was unaltered by PKA at either SL. kTR was not significantly altered by PKA under any of the conditions studied, but kTR was reduced at all level of force development by reconstitution with troponin containing phosphomimetic cTnI(S23/24D). In summary, phosphorylation of the N-terminus of cTnI and cardiac C-protein both independently suppressed cardiac contractility and the Frank-Starling mechanism. This occurs at physiologically relevant [Ca2+] by reducing the Ca2+-sensitivity of force and length-dependence of force-[Ca2+] relations in response to physiological conditions that evoke a beta-adrenergic response and activation of PKA.
- © 2010 by American Heart Association, Inc.