Yang Feng Hua, Pyle W Glen
Department of Biomedical Sciences, Ontario Veterinary College, Guelph, Ontario, Canada.
Cell Physiol Biochem. 2011;27(3-4):263-72. doi: 10.1159/000327952. Epub 2011 Apr 1.
Heart transplantation is associated with cold, cardioplegic arrest that impairs myocardial function. Protein Kinase C (PKC) suppression of myofilaments may contribute to this dysfunction. CapZ-deficient cardiac myofilaments are unresponsive to PKC. We hypothesized that myofilaments from CapZ-deficient transgenic hearts are resistant to cardioplegic dysfunction and that PKC inhibition improves function.
Heart function was assessed using a Langendorff apparatus. Myofilaments isolated from murine hearts were assessed with an actomyosin MgATPase assay and protein phosphorylation gels. PKC activation was examined by immunoblotting.
Wildtype hearts showed impaired function after cardioplegic arrest. CapZ-deficient transgenic mouse hearts performed significantly better after 1 h cardioplegia than wildtype hearts, but not after 4 h cardioplegic arrest. Wildtype myofilaments had depressed activation at 1 and 4 h cardioplegic arrest, as demonstrated by reduced actomyosin MgATPase activity. CapZ-deficient myofilaments showed no reduced actomyosin MgATPase activity at either time. Troponin I (TnI) phosphorylation increased by approximately 20% at 1 and 4 h in wildtype mice. Myosin binding protein C (MyBP-C),and troponin T (TnT) phosphorylation increased by less than 10% at 1 h, and tended to rise at 4 h. Myofilament protein phosphorylation was largely unchanged in CapZ-deficient hearts at 1 h, but MyBP-C tended to be dephosphorylated at 4 h cardioplegic arrest. Myofilament-associated PKC-α, -βII, -δ, and -e increased at 1 and 4 h cardioplegia in wildtype hearts, whereas only PKC-α increased in transgenic myofilaments at 1 h. PKC inhibition abolished the cardioplegic-dependent changes in actomyosin MgATPase activity and TnI phosphorylation of wildtype myofilaments.
We demonstrate a direct link between PKC activation and myofilament dysfunction associated with cold, cardioplegic arrest. Moreover, we show for the first time a cardioprotective benefit of decreased cardiac CapZ.
心脏移植与低温心脏停搏有关，这会损害心肌功能。蛋白激酶C（PKC）对肌丝的抑制作用可能导致这种功能障碍。缺乏CapZ的心肌肌丝对PKC无反应。我们假设来自缺乏CapZ的转基因心脏的肌丝对心脏停搏功能障碍具有抗性，并且PKC抑制可改善功能。
使用Langendorff装置评估心脏功能。用肌动球蛋白MgATP酶测定法和蛋白质磷酸化凝胶评估从小鼠心脏分离的肌丝。通过免疫印迹检查PKC激活情况。
野生型心脏在心脏停搏后功能受损。缺乏CapZ的转基因小鼠心脏在心脏停搏1小时后的表现明显优于野生型心脏，但在心脏停搏4小时后则不然。如肌动球蛋白MgATP酶活性降低所示，野生型肌丝在心脏停搏1小时和4小时时激活受到抑制。缺乏CapZ的肌丝在这两个时间点均未显示肌动球蛋白MgATP酶活性降低。野生型小鼠在心脏停搏1小时和4小时时肌钙蛋白I（TnI）磷酸化增加约20%。肌球蛋白结合蛋白C（MyBP-C）和肌钙蛋白T（TnT）磷酸化在1小时时增加不到10%，在4小时时趋于升高。缺乏CapZ的心脏在1小时时肌丝蛋白磷酸化基本未变，但在心脏停搏4小时时MyBP-C趋于去磷酸化。野生型心脏在心脏停搏1小时和4小时时与肌丝相关的PKC-α、-βII、-δ和-e增加，而转基因肌丝在1小时时仅PKC-α增加。PKC抑制消除了野生型肌丝中肌动球蛋白MgATP酶活性和TnI磷酸化的心脏停搏依赖性变化。
我们证明了PKC激活与低温心脏停搏相关的肌丝功能障碍之间存在直接联系。此外，我们首次展示了心脏CapZ减少的心脏保护益处。
