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(Circulation Research. 2000;86:470.)
© 2000 American Heart Association, Inc.

Cellular Biology

Chimera Analysis of Troponin I Domains That Influence Ca²⁺-Activated Myofilament Tension in Adult Cardiac Myocytes

Margaret V. Westfall, Faris P. Albayya, Immanuel I. Turner, Joseph M. Metzger

From the Department of Physiology, School of Medicine, University of Michigan, Ann Arbor.

Correspondence to Margaret V. Westfall, Department of Physiology, University of Michigan, 1301 E. Catherine St, 7730 Medical Sciences II, Ann Arbor, MI 48109-0622. E-mail wfall{at}w.imap.itd.umich.edu

Abstract—The goal of this study was to investigate isoform-specific functional domains of the inhibitory troponin subunit, troponin I (TnI), as it functions within the intact myofilaments of adult cardiac myocytes. Adenovirus-mediated gene transfer was used to deliver and express a TnI chimera composed of the amino terminus of cardiac TnI (cTnI) and the carboxy terminus of slow skeletal TnI (ssTnI) in adult rat cardiac myocytes. The TnI chimera, designated N-card/slow-C TnI, was expressed and incorporated into myofilaments after gene transfer, without detectable changes in contractile protein stoichiometry or sarcomere architecture. Interestingly, force at submaximal Ca²⁺ levels was markedly elevated in single permeabilized myocytes expressing the N-card/slow-C TnI chimera relative to force generated in adult myocytes expressing ssTnI or cTnI. Based on these results, a hierarchy of myofilament Ca²⁺ sensitivity is emerging by use of TnI chimera analysis, with the order of sensitivity being N-card/slow-C TnI>>ssTnI>>cTnI. These results also strongly suggest that independent isoform-specific domains in both the amino and carboxy portions of TnI influence myofilament Ca²⁺ sensitivity. In additional studies carried out under pathophysiological ionic conditions (pH 6.2), the dramatic acidosis-induced decrease in myofilament Ca²⁺ sensitivity observed in myocytes expressing cTnI was blunted 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 dissection 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.

Key Words: troponin I • chimera • myofilaments • heart • gene transfer

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