期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 290, 期 46, 页码 27749-27766出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M115.683045
关键词
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资金
- National Institutes of Health [R01 HL-111197, P41 GM103426]
- American Heart Association [15POST25080292, 11POST7400069, 12POST11570005]
- National Science Foundation
- Howard Hughes Medical Institution
- National Science Foundation Supercomputer Centers
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 beta-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 Ca2+ binding affinity to cTn (K-Ca) and KC-I. PKA phosphorylation resulted in a similar reduction of K-Ca 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 (T-max) was maintained for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils, and Ca2+ sensitivity of tension (pCa(50)) was increased. PKA phosphorylation decreased pCa(50) for cTnI(WT)-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI(WT) myofibrils, especially at Ca2+ 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.
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