Westfall M V, Solaro R J
Department of Physiology and Biophysics, University of Illinois, Chicago 60680.
Circ Res. 1992 Feb;70(2):302-13. doi: 10.1161/01.res.70.2.302.
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.
我们研究了大鼠心脏在完全性全心缺血伴缺氧后肌原纤维功能和蛋白质谱的变化。将心脏在37℃下暴露于全心缺血和缺氧（CGI）30或60分钟，然后制备肌原纤维以测量pH 7.0和6.5时Ca(2+)依赖性Mg(2+)-ATP酶活性。在冷盐水（1±1℃）中孵育的心脏和未孵育的心脏作为对照。在接受30或60分钟CGI处理的心脏的肌原纤维中，pH 7.0和pH 6.5时的最大ATP酶活性未发生变化。在pH 7.0时，作为细肌丝蛋白之间协同相互作用指标的希尔系数，在CGI处理30分钟后未发生变化，但在CGI处理60分钟后显著增加。当在pH 6.5下测量缺血30或60分钟的大鼠心脏肌原纤维中的肌原纤维ATP酶活性时，观察到了类似的协同性增加趋势。30和60分钟的CGI均导致pH 7.0和pH 6.5时的pCa50值（半最大激活游离[Ca2+]）增加。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离的肌原纤维蛋白的光密度分析表明，在CGI处理60分钟期间肌钙蛋白I和肌钙蛋白T被降解。通过蛋白质免疫印迹法鉴定出在缺血处理的肌原纤维中出现的两条新蛋白带为部分降解的肌钙蛋白I和肌钙蛋白T。肌钙蛋白I片段可被cAMP依赖性蛋白激酶磷酸化。此外，我们观察到在CGI处理的心脏的肌原纤维中，与肌球蛋白轻链-2相对应的一条蛋白带发生了磷酸化。这些结果表明，细肌丝蛋白的降解可能导致在暴露于CGI的大鼠心脏肌原纤维中观察到的ATP酶活性Ca2+调节协同性的变化。
