Functional analysis of troponin I regulatory domains in the intact myofilament of adult single cardiac myocytes.
Troponin I is the putative molecular switch for Ca(2+)-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 Ca(2+)-sensitive regulatory domain in the carboxyl terminus of troponin I and a second, newly defined Ca(2+)-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 Ca(2+)-sensitive properties of troponin I during contraction.
Westfall
M V
MV
Department of Physiology, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109-0622, USA. wfall@w.imap.itd.umich.edu
Albayya
F P
FP
Metzger
J M
JM
D008841:Actin Cytoskeleton / Q000502:physiology*; D000494:Allosteric Regulation; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D006321:Heart / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007537:Isometric Contraction / Q000502:physiology; D018657:Muscle Fibers, Slow-Twitch / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology*; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D019210:Troponin I / Q000235:genetics* / Q000502:physiology*
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D019210:Troponin I; D002118:Calcium