Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation

Yuanhua Cheng,Vijay Rao,An-Yue Tu,Steffen Lindert,Dan Wang,Lucas Oxenford,Andrew D Mcculloch,J Andrew Mccammon,Michael Regnier

doi:10.1074/jbc.m115.683045

Abstract

Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnI(R146G) or cTnI(R21C) influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca(2+) binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnI(WT). cTnI(WT), cTnI(R146G), and cTnI(R21C) were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (Tmax) was maintained for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils, and Ca(2+) sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnI(WT)-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI(WT) myofibrils, especially at Ca(2+) levels that the heart operates in vivo. Importantly, this effect was blunted for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnI(R146G) and cTnI(R21C) blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide.

Highlights

R146G and R21C mutations in cardiac TnI are associated with hypertrophic cardiomyopathy
Purified cardiac troponin I (cTnI) Phosphorylation Level from cardiac troponin C (cTnC) Column— Both WT, R146G, and R21C cTnI were phosphorylated by protein kinase A (PKA) using the cTnC affinity column, and the extent of cTnI phosphorylation was determined by computing the percentage of Ser-23/Ser-24 phosphorylated versus the total amount of cTnI, using Western blot analysis (7)
We tested whether hypertrophic cardiomyopathy (HCM)-associated mutations located in either the N terminus or inhibitory peptide of cTnI may disrupt the formation of an interaction between these regions that occurs with Ser-23/Ser-24 phosphorylation by PKA, blunting the regulatory effects on cardiac troponin (cTn) that normally occur during ␤-adrenergic stimulation

Summary

Background

R146G and R21C mutations in cardiac TnI are associated with hypertrophic cardiomyopathy. Our results suggest that cTnIR146G and cTnIR21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide. Our computational modeling results demonstrated that introduction of the S23D/ S24D substitutions (bis-phosphomimic substitutions) on cTnI (cTnIS23D/S24D) led to the formation of an intra-subunit interaction between the N terminus and the inhibitory peptide of cTnI (8) We hypothesized that this interaction may be the structural correlate for shortening the duration and increasing the rate of the early phase of relaxation by destabilizing cTnI switch peptide interaction with NcTnC (8). We hypothesized that introduction of an HCM mutation located in either the N terminus or the inhibitory peptide of cTnI may disrupt the formation of this intra-subunit interaction and blunt the effects of Ser-23/Ser-24 phosphorylation by PKA during ␤-adrenergic stimulation. In addition to being hyper-contractile during systole, hearts with these mutations may have impaired initiation of diastole during ␤-adrenergic stimulation

Experimental Procedures

Results

Discussion