Partial replacement of cardiac troponin I with a non-phosphorylatable mutant at serines 43/45 attenuates the contractile dysfunction associated with PKCε phosphorylation

We have previously reported a transgenic mouse that over-expresses constitutively active PKCε in the myocardium and exhibits a steady progression to heart failure. Associated with the decline in function was an increased phosphorylation of sarcomeric proteins including cardiac troponin I (cTnI). To determine whether PKCε phosphorylation of cTnI is sufficient to induce cardiac maladaptation, we have generated a double transgenic mouse (DbTG) that expresses constitutively active PKCε and cTnI harboring non-phosphorylatable mutations in the putative PKC phosphorylation sites (S43A, S45A). We compared the hemodynamic and biochemical properties of the hearts from the DbTG mice to the non-transgenic and single transgenic lines at both 3 and 12 months of age. While no significant differences in LV function were noted in 3-month groups, the depression of function in the PKCε mice was attenuated in the double transgenic mice at 12 months. The improvement in cardiac function was correlated with decreased β-myosin heavy chain and ANF mRNA expression in the 12m DbTG mice. The extent of cTnI phosphorylation was determined using a novel one-dimensional, non-equilibrium isoelectric focusing technique. At 3 months the migration of cTnI phospho-species was different in the PKCε mice and to a lesser degree in the DbTG compared to all other groups. At 12 months additional phospho-species were observed in both the PKCε and DbTG samples, along with an overall shift in the distribution of phospho-species in all groups due to age. These results suggest that phosphorylation of cTnI by PKCε is associated with contractile dysfunction and partial replacement of serines 43/45 improves cardiac performance. Therefore, we conclude that phosphorylation of cTnI at Ser 43 and 45 may contribute to the progression of failure.