Cardiac Troponin I Phosphorylation Increases the Rate of Cardiac Muscle Relaxation

Abstract

Abstract Cardiac troponin (Tn) I (CTnI), compared with skeletal TnI, contains extra amino acids (32 to 33) at its amino terminus, including two adjacent serine residues. These two serine residues are believed to be phosphorylated by protein kinase A (PKA) upon stimulation of the heart by β-agonists. In this study, we found that phosphorylation of a cardiac skinned muscle preparation by PKA, mainly at CTnI, results in a decrease in the Ca 2+ sensitivity of muscle contraction. The pCa 50 decreased by ≈0.27±0.06 pCa units upon phosphorylation. To study cardiac muscle relaxation, we used diazo-2, a photolabile Ca 2+ chelator with a low Ca 2+ affinity in its intact form that is converted to a high-affinity form after photolysis. We found that the rate of cardiac muscle relaxation increased from a time of half-relaxation (t 1/2 )=110±10 milliseconds to t 1/2 =70±8 milliseconds after CTnI phosphorylation. This result demonstrates that CTnI phosphorylation can be linked with the increased rate of muscle relaxation in a relatively intact muscle preparation. Since CTnI phosphorylation has been shown previously to affect the Ca 2+ affinity and Ca 2+ off-rate of CTnC in vitro, it is likely that the faster relaxation seen here reflects faster dissociation of Ca 2+ from cardiac TnC (CTnC). Model calculations show that increased dissociation of Ca 2+ from CTnC, coupled with the faster uptake of Ca 2+ by the sarcoplasmic reticulum stimulated by PKA phosphorylation of phospholamban, can account for the faster relaxation seen in the inotropic response of the heart to catecholamines.