Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A.
Viral-mediated gene transfer of troponin I (TnI) isoforms and chimeras into adult rat cardiac myocytes was used to investigate the role TnI domains play in the myofilament tension response to protein kinase A (PKA). In myocytes expressing endogenous cardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein C and decreased the Ca2+ sensitivity of myofilament tension. In marked contrast, PKA did not influence Ca2+-activated tension in myocytes expressing the slow skeletal isoform of TnI or a chimera (N-slow/card-C TnI), which lack the unique phosphorylatable amino terminal extension found in cTnI. PKA-mediated phosphorylation of a second TnI chimera, N-card/slow-C TnI, which has the amino terminal region of cTnI, caused a decrease in the Ca2+ sensitivity of tension comparable in magnitude to control myocytes. Based on these results, we propose the amino terminal region shared by cTnI and N-card/slow-C TnI plays a central role in determining the magnitude of the PKA-mediated shift in myofilament Ca2+ sensitivity, independent of the isoform-specific functional domains previously defined within the carboxyl terminal backbone of TnI. Interestingly, exposure of permeabilized myocytes to acidic pH after PKA-mediated phosphorylation of cTnI resulted in an additive decrease in myofilament Ca2+ sensitivity. The isoform-specific, pH-sensitive region within TnI lies in the carboxyl terminus of TnI, and the additive response provides further evidence for the presence of a separate domain that directly transduces the PKA phosphorylation signal.
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
Turner
I
I
Albayya
F P
FP
Metzger
J M
JM
D008841:Actin Cytoskeleton / Q000187:drug effects* / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D005260:Female; D018014:Gene Transfer Techniques; D006352:Heart Ventricles / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects; D019210:Troponin I / Q000187:drug effects* / Q000502:physiology
D011994:Recombinant Proteins; D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium