Small Residue Substitutions at Cardiac Troponin I Ser43/Ser45 Produce Distinct Functional Responses

Abstract

Our current studies examine the modulatory role of the protein kinase C (PKC)-targeted Ser 43/45 cluster in cardiac troponin I (cTnI). Previously, gene transfer and sarcomeric replacement of endogenous cTnI with phospho-mimetic Asp substitutions at Ser43/Ser45 significantly reduced the peak amplitude and transiently decreased the rate of shortening. Myocytes expressing cTnISer43/45Asp developed an accelerated time to 75% re-lengthening and Ca2+ decay rate. In the current study, we tested whether cTnISer43/45 substitution with Ala would prevent the peak amplitude and shortening rate changes observed with cTnISer43/45Asp. Western blot and immunohistochemistry confirmed that cTnISer43/45Ala gene transfer in adult rat myocytes produced a time-dependent replacement of endogenous cTnI and sarcomeric expression of this cTnI substitution. Unexpectedly, the amplitude and rate of peak shortening in myocytes expressing cTnISer43/45Ala fell between values observed in myocytes expressing endogenous cTnI and cTnISer43/45Asp. These results indicate that additional amino acid properties may be functionally important for these cTnI residues. Next, a cTnISer43/45Asn construct was utilized to test whether polar residues such as Asn serve as a functionally conservative substitution for Ser at these positions. The temporal increase in endogenous cTnI replacement with cTnISer43/45Asn was comparable to cTnISer43/45Ala based on Western analysis and immunohistochemical labeling of myocytes. In ongoing studies, peak shortening amplitude and rate are comparable to controls in myocytes expressing cTnISer43/45Asn, unlike cTnISer43/45Ala. At present, these recent results support the idea that properties other than residue size at Ser43/45 may be important for mimicking the basal shortening phenotype.