NCA增加心肌收缩力的作用研究

1-Nitrosocyclohexyl acetate increases contractile force of cardiac muscle

目的:硝酰基(HNO)作为一氧化氮(NO)的单电子还原产物,对活体心脏发挥正性肌力作用。我们对于这些效果是否由于乙酸1-亚硝基环已酯(NCA,HNO供体)对肌原纤维的直接作用进行了研究。方法:将大鼠右心室的完整的梳状肌连接在张力换能器与刺激电极之间,肌小节长度设定在2.2-2.3μm之间,K-H液表面灌流后(pH=7.4,室温),Fura-2经玻璃微电极负载进行离子透入法检测[Ca2+]i,同时测定心肌收缩张力的变化。稳态条件下对最大钙离子活化张力(Fmax)及达到50%活性需要[Ca2+]i(Ca50)进行测定。Western blotting方法检测原肌球蛋白表达的变化。结果:收缩力在NCA作用下呈剂量依赖性增加(20-100μmol/L)。不同频率作用下(0.5-3.0 Hz,NCA 20μmol/L,Ca2+0.5 mmol/L)收缩力的增加(P<0.01)和[Ca2+]i瞬变(P>0.05)没有受到明显的影响。舒张期作用力及[Ca2+]i不受NCA的影响。与对照组相比,稳态活化过程中NCA(20μmol/L)能增加最大钙离子活力Fmax[(124.0±15.0)mN/mm2 vs(90.0±4.2)mN/mm2,P<0.05],降低Ca50[(0.39±0.01)μmol/L vs(0.57±0.03)μmol/L,P<0.01],但不影响希尔系数(3.94±0.18 vs 4.92±0.84,P>0.05)。同对照组相比,NCA处理去肌膜心肌收缩力明显增加(P<0.05)。非还原条件下Western blotting可见肌原纤维出现交联。巯基还原剂二硫苏糖醇(DTT,5.0 mmol/L)能够阻止并逆转NCA的活动,进一步证实氧化还原反应依赖HNO的效应。结论:NCA提供的HNO是心脏钙离子增敏剂,心肌调节蛋白质巯基翻译后修饰可能是其靶点作用所在。

AIM：To investigate the effect of 1-nitrosocyclohexyl acetate（NCA,a newly-developed nitroxyl donor） on myofibrils of rat heart.METHODS： Trabecular muscles were dissected from the right ventricles of the rat hearts and mounted between a force transducer and a motor arm.The muscles were superfused with K-H solution（pH 7.4） at room temperature.Fura-2 was loaded into the trabecular muscles via electrophoresis.The length of the sarcomere was set to 2.2-2.3 μm.During steady-state activations,the maximal Ca2＋-activated force（Fmax） and Ca2＋ required for 50% activation（Ca50） were measured.The change of tropomyosin was detected by Western blotting.RESULTS： Twitch force increased in a dose-dependent manner in the presence of NCA at the concentrations from 20 μmol/L to 100 μmol/L.The force increased（P〈0.01） and i transient remained unchanged（P〉0.05） with various frequencies（0.5-3.0 Hz）,20 μmol/L of NCA and 0.5 mmol/L of Ca2＋.Both diastolic force and i were not affected by NCA.Compared with the control data,NCA at concentration of 20 μmol/L increased Fmax[（124.0±15.0） mN/mm2 vs（90.0±4.2） mN/mm2,P〈0.05] and decreased Ca50[（0.39±0.01） μmol/L vs（0.57±0.03） μmol/L,P〈0.01] during steady-state activations.The Hill coefficient was not affected（3.94±0.18 vs 4.92±0.84,P〉0.05）.The skinned force increased（P〈0.05）.Under non-reducing condition,a cross-linking band in tropomyosin was observed by Western blotting.The thiol reducing agent dithiothreitol both prevented and reversed NCA action,confirming NCA/nitroxyl redox sensitivity.CONCLUSION： NCA,a donor of pure nitroxyl,represents a new class of agents that are capable of directly sensitizing cardiac myofibrils to Ca2＋.Post-translational thiol modification of key regulatory myofilament proteins（i.e.tropomyosin） may underlie the effects of NCA.