Excision of titin's cardiac PEVK spring element abolishes PKCalpha-induced increases in myocardial stiffness.
Protein kinase C-alpha (PKCalpha) 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-glutamic acid-valine-lysine (PEVK) rich spring element of titin. To test this proposal we investigated the effect of PKCalpha 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 PKCalpha increased passive stiffness in the WT myocardium by 27+/-6%. Importantly, this effect was completely abolished in KO myocardium. In addition, increases in the elastic and viscous moduli at a wide range of frequencies (properties important in diastolic filling) following PKCalpha incubation (27+/-3% and 20+/-4%, respectively) were also ablated in the KO. Back phosphorylation assays showed that titin phosphorylation following incubation with PKCalpha was significantly reduced by 36+/-12% in skinned PEVK KO myocardial tissues. The remaining phosphorylation in the KO suggests that PKCalpha 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 PKCalpha and that this increases passive tension. Thus, the PEVK spring element is the critical site of PKCalpha's involvement in passive myocardial stiffness.
Hudson
Bryan D
BD
Department of Physiology, Sarver Molecular Cardiovascular Research Program, University of Arizona, PO Box 245217 Tucson, AZ 85724, USA.
Hidalgo
Carlos G
CG
Gotthardt
Michael
M
Granzier
Henk L
HL
D000818:Animals; D064211:Connectin; D003971:Diastole / Q000502:physiology; D005091:Exons / Q000235:genetics; D006321:Heart / Q000187:drug effects; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000494:pharmacology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D011494:Protein Kinases; D051571:Protein Kinase C-alpha