Chimera analysis of troponin I domains that influence Ca2+-activated myofilament tension in adult cardiac myocytes

The goal of this study was to investigate isoform-specific functional domai ns of the inhibitory troponin subunit, troponin I (TnI). as it functions wi thin the intact myofilaments of adult cardiac myocytes. Adenovirus-mediated gene transfer was used to deliver and express a TnI chimera composed of th e amino terminus of cardiac TnI (cTnI) and the carboxy terminus of slow ske letal TnI (ssTnI) in adult rat cardiac myocytes. The TnI chimera, designate d N-card/slow-C TnI, was expressed and incorporated into myofilaments after gene transfer, without detectable changes in contractile protein stoichiom etry or sarcomere architecture. Interestingly, force at submaximal Ca2+ lev els was markedly elevated in single permeabilized myocytes expressing the N -card/slow-C TnI chimera relative to force generated in adult myocytes expr essing ssTnI or cTnI. Based on these results, a hierarchy of myofilament Ca 2+ sensitivity is emerging by use of TnI chimera analysis. with the order o f sensitivity being N-card/slow-C TnI much greater than ssTnI much greater than cTnI. These results also strongly suggest that independent isoform-spe cific domains in both the amino and carboxy portions of TnI influence myofi lament Ca2+ sensitivity. In additional studies carried out under pathophysi ological ionic conditions (pH 6.2), the dramatic acidosis-induced decrease in myofilament Ca2+ sensitivity observed in myocytes expressing cTnI was bl unted in myocytes expressing N-card/slow-C TnI in a manner similar to that in ssTnI-expressing myocytes. These results demonstrate that there is a pH- sensitive domain residing in the carboxy-terminal portion of TnI, The disse ction of isoform-specific functional domains under physiological and acidic pH conditions demonstrates the utility of TnI chimeras for analysis of TnI function and provides important insights into the overall function of TnI within the intact myofilament of adult cardiac myocytes.