Functional analysis of troponin I regulatory domains in the intact myofilament of adult single cardiac myocytes

Troponin I is the putative molecular switch for Ca2+-activated contraction within the myofilament of striated muscles. To gain insight into functional troponin I domain(s) in the context of the intact myofilament, adenovirus- mediated gene transfer was used to replace endogenous cardiac troponin I wi thin the myofilaments of adult cardiac myocytes with the slow skeletal isof orm or a chimera of the slow skeletal and cardiac isoforms. Efficient expre ssion and myofilament incorporation were observed in myocytes with each exo genous troponin I protein without detected changes in the stoichiometry of other contractile proteins and/or sarcomere architecture. Contractile funct ion studies in single, permeabilized myocytes expressing exogenous troponin I provided support for the presence of a Ca2+-sensitive regulatory domain in the carboxyl terminus of troponin I and a second, newly defined Ca2+-sen sitive domain residing in the amino terminus of troponin I. Additional expe riments demonstrated that the isoform-specific, acidic pH-induced contracti le dysfunction in myocytes appears to lie in the carboxyl terminus of tropo nin I. Functional results obtained from adult cardiac myocytes expressing t he chimera or isoforms of troponin I now define multiple troponin I regulat ory domains operating in the intact myofilament and provide new insight int o the Ca2+-sensitive properties of troponin I during contraction.