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 within the myofilaments of adult cardiac myocytes with the slow skeletal isoform or a chimera of the slow skeletal and cardiac isoforms. Efficient expression and myofilament incorporation were observed in myocytes with each exogenous troponin I protein without detected changes in the stoichiometry of other contractile proteins and/or sarcomere architecture. Contractile function 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⁺-sensitive domain residing in the amino terminus of troponin I. Additional experiments demonstrated that the isoform-specific, acidic pH- induced contractile dysfunction in myocytes appears to lie in the carboxyl terminus of troponin I. Functional results obtained from adult cardiac myocytes expressing the chimera or isoforms of troponin I now define multiple troponin I regulatory domains operating in the intact myofilament and provide new insight into the Ca²⁺-sensitive properties of troponin I during contraction.