Published online before print October 22, 2009, doi: 10.1161/CIRCRESAHA.109.205427
Submitted on January 16, 2009
Revised on September 28, 2009
Accepted on October 8, 2009
Left Ventricular and Myocardial Function in Mice Expressing Constitutively Pseudophosphorylated Cardiac Troponin I
Jonathan A. Kirk ;
From the Cardiovascular Systems Laboratory (J.A.K., C.E., S.H.S., S.G.S.), Department of Bioengineering, University of Pittsburgh, Pa; Department of Cardiology (G.A.M.), Freeman Hospital and Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom; Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology (R.M., M.C.), Washington State University, Pullman; Department of Physiology and Biophysics (C.M.W., R.J.S.), University of Illinois at Chicago; and Department of Medicine (A.F.R.S.), University of Ottawa Heart Institute, Canada.
* To whom correspondence should be addressed. E-mail: sshroff{at}pitt.edu.
Rationale: Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction.
Objective: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnIPKC-P) expressing mutant cTnI (S43E, S45E, T144E).
Methods and Results: Two-dimensional gel analysis showed 7.2±0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model–based analysis for integrative interpretation. Compared to wild-type mice, cTnIPKC-P mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, Fmax). Additionally, cTnIPKC-P mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnIPKC-P mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnIPKC-P mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state.
Conclusions: A modest increase in PKC-induced cTnI phosphorylation (
7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).
Key words: transgenic mice • protein kinase C • cardiac troponin I • calcium (cellular) • model-based analysis