Both PKA Treatment and Cardiac Troponin-I N-Terminal Phosphorylation Alone Decrease Ca-Sensitivity and Eliminate Length-Dependent Activation in Skinned Cardiac Muscle

Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility. PKA targets the cardiac troponin I (cTnI) N-terminus, cardiac myosin-binding protein C (cMyBP-C) and titin. To isolate cTnI and cMyBP-C /titin phosphorylation effects on force-[Ca2+] relations, endogenous cardiac troponin (Tn) was exchanged in rat, skinned trabeculae with either WT Tn or Tn containing a non-phosphorylatable mutant cTnI(S23/24A) or phosphomimetic cTnI(S23/24D). PKA cannot phosphorylate either cTnI mutant, leaving cMyBP-C and titin as sole PKA targets. Force-[Ca2+] relations and Ca2+-sensitivity (pCa50) were measured at 2.3 and 2.0 µm SL. Decreasing steady SL reduced maximal force (Fmax) and pCa50 similarly with WT Tn and Tn containing cTnI(S23/24A). PKA treatment of native, WT and cTnI(S23/24A) exchanged trabeculae reduced pCa50 at 2.3, but not 2.0 um SL, eliminating SL-dependence of pCa50. Reconstitution with Tn containing cTnI(S23/24D) reduced pCa50 at both SL (compared to WT and cTnI(S23,24A) and eliminated pCa50 SL-dependence; PKA did not significantly alter pCa50 at either SL. At each SL Fmax was similar with WT and mutant troponins, and was unaffected by PKA. Low angle x-ray diffraction experiments were performed to determine whether shifts in pCa50 were associated with changes in myofilament spacing (D1,0) or interaction. D1,0 at 2.3 um SL was similar in native trabeculae, with WT Tn and Tn containing either cTnI(S23,24A) or cTnI(S23,24D); PKA increased D1,0 in all cases. The results suggest that PKA phosphorylation of either cTnI or cMyBP-C /titin reduced the Ca2+-sensitivity of force and length-dependent activation. Supported by NIH HL067071-06.