Abstract
Carlos Hidalgo1, Bryan D Hudson1, Michael Gotthardt 2, Henk L Granzier1. 1Department of Physiology, Molecular Cardiovascular Research Program, University of Arizona, Tucson AZ; 2Neuromuscular and Cardiovascular Cell Biology, Max-Delbruck-Center for Molecular Medicine, Berlin, Germany. Protein Kinase C-alpha (PKCα) was recently reported to increase myocardial stiffness, an effect that was proposed to be due to phosphorylation of two highly conserved sites (S11878 and S12022) within the proline-gluatamic acid-valine-lysine (PEVK) rich spring element of titin. To test this proposal we investigated the effect of PKCα on phosphorylation and passive stiffness in a mouse model lacking the titin exons that contain these two phosphorylation sites, the PEVK knockout (KO). We used skinned, gelsolin-extracted, left ventricular, myocardium from wildtype and PEVK KO mice. Consistent with previous work we found that PKCα increased passive stiffness in the WT myocardium by 27.4±6.2%. Importantly this effect was completely abolished in KO myocardium. In addition, increases in the elastic and viscous moduli (properties important in diastolic filling) following PKCα incubation was also ablated in the KO. Back phosphorylation assays showed that titin phosphorylation was significantly reduced by 36.1±12.3% in skinned PEVK KO myocardial tissues following incubation with PKCα. The remaining phosphorylation in the KO suggests that PKCα sites exist in the titin molecule outside the PEVK region; these sites are not involved in increasing passive stiffness. Our results firmly support that the PEVK region of cardiac titin is phosphorylated by PKCα and that this increases passive tension. Thus, the PEVK spring element is the critical site of PKCα's involvement in myocardial stiffness.
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