Role of troponin I proteolysis in the pathogenesis of stunned myocardium.
Myocardial stunning is characterized by decreased myofilament Ca2+ responsiveness. To investigate the molecular basis of stunned myocardium, we performed PAGE and Western immunoblot analysis of the contractile proteins. Isolated rat hearts were retrogradely perfused at 37 degrees C for either 50 minutes (control group) or for 10 minutes, followed by 20-minute global ischemia and 20-minute reperfusion (stunned group), or for 20-minute ischemia without reflow. Another group consisted of hearts subjected to 20-minute ischemia in which stunning was mitigated by 10-minute reperfusion with low Ca2+/low pH solution. Myocardial tissue samples subjected to PAGE revealed no obvious differences among groups. Western immunoblots for actin, tropomyosin, troponin C, troponin T, myosin light chain-1, and myosin light chain-2 showed highly selective recognition of the appropriate full-length molecular weight bands in all groups. Troponin I (TnI) Western blots revealed an additional band (approximately 26 kD, compared with 32 kD for the full-length protein) in stunned myocardial samples only. In parallel experiments, skinned trabeculae were treated with calpain I for 20 minutes; Western blots showed a TnI degradation pattern similar to that observed in stunned myocardium. Such TnI degradation was prevented by calpastatin, a naturally occurring calpain inhibitor. The results show that (1) TnI is partially and selectively degraded in stunned myocardium; (2) this degradation could be prevented by low Ca2+/low pH reperfusion, which also prevented the contractile dysfunction of stunning; and (3) calpain I could similarly degrade TnI, supporting the idea that Ca(2+)-dependent myofilament proteolysis underlies myocardial stunning.
Gao
W D
WD
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md. 21205, USA.
Atar
D
D
Liu
Y
Y
Perez
N G
NG
Murphy
A M
AM
Marban
E
E
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000494:pharmacology; D005260:Female; D006321:Heart / Q000187:drug effects / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D015425:Myocardial Reperfusion / Q000379:methods; D015428:Myocardial Reperfusion Injury / Q000209:etiology*; D017682:Myocardial Stunning / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019210:Troponin I / Q000502:physiology*; D017725:Ventricular Pressure / Q000502:physiology
D002135:Calcium-Binding Proteins; D019210:Troponin I; C033668:calpastatin; D002154:Calpain; D002118:Calcium