Stamm C, Friehs I, Cowan D B, Cao-Danh H, Noria S, Munakata M, McGowan F X, del Nido P J
Department of Cardiac Surgery, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA.
Cardiovasc Res. 2001 Jul;51(1):108-21. doi: 10.1016/s0008-6363(01)00249-8.
Protein kinase C (PKC) activation impairs contractility in the normal heart but is protective during myocardial ischemia. We hypothesized that PKC remains activated post-ischemia and modulates myocardial excitation-contraction coupling during early reperfusion.
Langendorff-perfused rabbit hearts where subjected to 25 min unmodified ischemia and 30 min reperfusion. Total PKC activity was measured, and the intracellular translocation pattern of PKC-alpha, -delta, -epsilon, and -eta assessed by immunohistochemistry and fractionated Western immunoblotting. The PKC-inhibitors chelerythrine and GF109203X were added during reperfusion and also given to non-ischemic hearts. Measurements included left ventricular function, intracellular calcium handling measured by Rhod-2 spectrofluorometry, myofibrillar calcium responsiveness in beating and tetanized hearts, and metabolic parameters.
Total PKC activity was increased at end-ischemia and remained elevated after 30 min of reperfusion. The translocation pattern indicated PKC-epsilon as the main active isoform during reperfusion. Post-ischemic PKC inhibition affected mainly diastolic relaxation, with lesser effect on contractility. Both PKC inhibitors increased the Ca(2+) responsiveness of the myofilaments as indicated by a leftward shift of the calcium-to-force relationship and increased maximum calcium activated tetanic pressure. Diastolic Ca(2+) removal was delayed and the post-ischemic Ca(2+) overload further exacerbated. Depressed systolic function was associated with a lower amplitude of Ca(2+) transients.
PKC is activated during ischemia and remains activated during early reperfusion. Inhibition of PKC activity post-ischemia impairs functional recovery, delays diastolic Ca(2+) removal, and increases Ca(2+) sensitivity of the contractile apparatus, resulting in impaired diastolic relaxation. Thus, post-ischemic PKC activity may serve to restore post-ischemic Ca(2+) homeostasis and attenuate contractile protein calcium sensitivity during the period of post-ischemic Ca(2+) overload.
蛋白激酶C（PKC）激活会损害正常心脏的收缩性，但在心肌缺血期间具有保护作用。我们推测PKC在缺血后仍保持激活状态，并在早期再灌注期间调节心肌兴奋-收缩偶联。
采用Langendorff灌注兔心脏，使其经历25分钟未处理的缺血和30分钟再灌注。测量总PKC活性，并通过免疫组织化学和分级Western免疫印迹法评估PKC-α、-δ、-ε和-η的细胞内转位模式。在再灌注期间添加PKC抑制剂白屈菜红碱和GF109203X，并给予非缺血心脏。测量指标包括左心室功能、通过Rhod-2荧光分光光度法测量的细胞内钙处理、跳动和强直收缩心脏中的肌原纤维钙反应性以及代谢参数。
缺血末期总PKC活性增加，再灌注30分钟后仍保持升高。转位模式表明PKC-ε是再灌注期间的主要活性亚型。缺血后PKC抑制主要影响舒张期松弛，对收缩性的影响较小。两种PKC抑制剂均增加了肌丝的Ca(2+)反应性，表现为钙-力关系向左移位以及最大钙激活强直压力增加。舒张期Ca(2+)清除延迟，缺血后Ca(2+)过载进一步加剧。收缩功能降低与Ca(2+)瞬变幅度较低有关。
PKC在缺血期间被激活，并在早期再灌注期间保持激活状态。缺血后抑制PKC活性会损害功能恢复，延迟舒张期Ca(2+)清除，并增加收缩装置的Ca(2+)敏感性，导致舒张期松弛受损。因此，缺血后PKC活性可能有助于恢复缺血后Ca(2+)稳态，并在缺血后Ca(2+)过载期间减弱收缩蛋白对钙的敏感性。
