|About the Book|
This project was aimed at further elucidating the role of protein kinase C (PKC)-induced phosphorylation of troponin-I (cTnI) in cardiac contraction. We created a new transgenic mouse model (TG-E), expressing a mutant cardiac TnI constitutivelyMoreThis project was aimed at further elucidating the role of protein kinase C (PKC)-induced phosphorylation of troponin-I (cTnI) in cardiac contraction. We created a new transgenic mouse model (TG-E), expressing a mutant cardiac TnI constitutively pseudo-phosphorylated at the three PKC phosphorylation sites (S43, S45, T144 mutated to glutamate). 2D-DIGE (Difference in Gel Electrophoresis) gels indicated 7.2 +/- 0.5% replacement, with no change in baseline level of actual phosphorylation of cTnI or other myofilamental proteins. Experiments were conducted in perfused isolated mouse hearts, isolated papillary muscles, and skinned fiber preparations. The mechanical measurements were complemented by biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, TG-E mice exhibited negative inotropy in in vivo echocardiographic studies (9% decrease in fractional shortening), 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, TG-E mice exhibited slowed relaxation in echocardiographic studies, isolated hearts, and intact papillary muscles. The TG-E mice showed no differences in calcium sensitivity, cooperativity, steady-state force-ATPase relationship, and calcium transient (amplitude and relaxation). The four-state model of cardiac contraction was used for a model-based analysis of the data. The model was verified as a priori globally identifiable using a differential algebraic approach- however, automated optimization procedures proved to be unreliable. Instead we performed in silico experiments, altering sets of parameters in an attempt to recreate our experimental data. The model-based analysis revealed that experimental observations in TG-E mice could be reproduced by two simultaneous perturbations: a decrease in the rate constant of crossbridge formation and an increase in calcium-independent persistence of the myofilament active state. In summary, a modest increase in PKC-induced cTnI phosphorylation can significantly regulate cardiac muscle contraction: (1) negative inotropy via decreased crossbridge formation and (2) negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that the effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 vs. T144).