title: The dilated cardiomyopathy-causing mutation ACTC E361G in cardiac muscle myofibrils specifically abolishes modulation of Ca(2+) regulation by phosphorylation of troponin I.
abstract: Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca(2+) sensitivity and increases the rate of Ca(2+) release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca(2+)-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca(2+) regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca(2+) sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca(2+) sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca(2+) sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca(2+) sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca(2+) sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033.
authors.affiliation: National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: Petr G
authors.initials: PG
authors.lastname: Vikhorev
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
authors.forename: Weihua
authors.initials: W
authors.lastname: Song
authors.affiliation: National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: Ross
authors.initials: R
authors.lastname: Wilkinson
authors.affiliation: National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: O'Neal
authors.initials: O
authors.lastname: Copeland
authors.affiliation: National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Messer
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Ferenczi
authors.affiliation: National Heart and Lung Institute, Imperial College London, London, UK. Electronic address: s.marston@imperial.ac.uk.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Marston
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002311:Cardiomyopathy, Dilated / Q000235:genetics*; D002352:Carrier Proteins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009119:Muscle Contraction / Q000187:drug effects; D009154:Mutation; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011433:Propranolol / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D012518:Sarcomeres / Q000187:drug effects / Q000378:metabolism; D013829:Thiadiazines / Q000494:pharmacology; D019210:Troponin I / Q000378:metabolism*; D000099163:Myosin Binding Protein C
chemical_list: D000199:Actins; D002118:Calcium; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D011433:Propranolol; D011804:Quinolines; D013829:Thiadiazines; D019210:Troponin I; D000099163:Myosin Binding Protein C; C070609:EMD 53998