Alterations in myofibrillar function and protein profiles after complete global ischemia in rat hearts.
We studied changes in myofibrillar function and protein profiles after complete global ischemia with anoxia in rat hearts. Hearts were exposed to global ischemia and anoxia (CGI) for 30 or 60 minutes at 37 degrees C, and myofibrils were prepared for measurement of Ca(2+)-dependent Mg(2+)-ATPase activity at pH 7.0 and 6.5. Hearts incubated in cold saline (1 +/- 1 degrees C) and nonincubated hearts served as controls. Maximum ATPase activity was unchanged at pH 7.0 and pH 6.5 in myofibrils from hearts treated with 30 or 60 minutes of CGI. At pH 7.0, the Hill coefficient, which is an index of cooperative interactions among thin-filament proteins, was unchanged after 30 minutes of CGI but was significantly increased after 60 minutes of CGI. A similar trend for increased cooperativity was observed when myofibrillar ATPase activity was measured at pH 6.5 in myofibrils from rat hearts made ischemic for 30 or 60 minutes. Both 30 and 60 minutes of CGI resulted in increased pCa50 values (half-maximally activating free [Ca2+]) at pH 7.0 and pH 6.5. Densitometric analysis of myofibrillar proteins separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that troponin I and troponin T were degraded during 60 minutes of CGI. Two new protein bands appearing in ischemia-treated myofibrils were identified as partially degraded troponin I and troponin T with Western blots. The troponin I fragment could be phosphorylated by cAMP-dependent protein kinase. In addition, we observed phosphorylation of a protein band that corresponded to myosin light chain-2 in myofibrils from CGI-treated hearts. These results suggest that degradation of thin-filament proteins may contribute to the changes in cooperativity of Ca2+ regulation of ATPase activity observed in the myofibrils from rat hearts exposed to CGI.
Westfall
M V
MV
Department of Physiology and Biophysics, University of Illinois, Chicago 60680.
Solaro
R J
RJ
D000818:Animals; D001345:Autoradiography; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D003327:Coronary Disease / Q000378:metabolism / Q000503:physiopathology*; D004591:Electrophoresis, Polyacrylamide Gel; D006321:Heart / Q000503:physiopathology*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008297:Male; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains
D009124:Muscle Proteins; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases