NCA对受磷蛋白敲除小鼠心肌兴奋-收缩偶联的作用

 目的: 硝酰基(HNO)在轻微增加细胞内钙的基础上可以显著增加心肌肌丝对钙离子的反应性。本研究中,我们应用崭新的HNO供体乙酸1-亚硝基环己酯(NCA)来观察HNO对受磷蛋白敲除(PLB-KO)小鼠心室梳状肌的作用。方法: 小鼠右心室的完整梳状肌被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,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。结果: PLB-KO小鼠心室梳状肌比野生型(WT)小鼠具有更高的钙瞬变及收缩力,同时展示负性收缩力-收缩频率相关性(FFR)。NCA(2.5μmol/L)在不同浓度细胞外钙(Ca²⁺]_o)条件下增加PLB-KO及WT小鼠心肌收缩力,但并不影响PLB-KO小鼠的负性FFR。稳态条件下2组小鼠去肌膜梳状肌的收缩力-钙离子相关性无显著性差异,NCA则增加去肌膜梳状肌的钙离子的反应性。结论: NCA提供的HNO通过增加PLB-KO及WT小鼠心肌肌丝对钙离子的反应性而增强心肌收缩力;心肌细胞内钙瞬变的增加伴随收缩力的增强表明HNO可改善钙离子活性,进一步证实HNO作为正性肌力药物的作用效果。

Effect of NCA on excitation-contraction coupling of cardiac muscle from phospholamban knockout mice

AIM: Nitroxyl(HNO) increases myofilament Ca²⁺ responsiveness relative to increases in intracellular Ca²⁺ in cardiac muscle. In this study, we further investigated this effect of HNO on trabecular muscles from phospholamban knockout(PLB-KO) and wide-type(WT) mice using a novel HNO donor, 1-nitrosocyclohexyl acetate(NCA). 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 2.2~2.3μm. During steady-state activations, the maximal Ca²⁺-activated force and Ca²⁺ required for 50% activation were measured. RESULTS: The intracellular Ca²⁺ transients and force of the PLB-KO muscles at baseline were higher than those of the WT muscles and exhibited a negative force-frequency relationship(FFR). NCA(2.5μmol/L) increased systolic force in both PLB-KO group and WT group at any givenCa²⁺]_o. However, there was more dramatic increase in the force development due to moderate increases in the intracellular Ca²⁺ transients in the WT muscles when external Ca²⁺ increased from 1.5 to 4.5 mmol/L under NCA. NCA did not affect the negative FFR in PLB-KO muscle. Steady-state force-Ca²⁺ relations obtained from skinned muscles were not different between the 2 groups, while NCA increased Ca²⁺ responsiveness in skinned muscles from both PLB-KO and WT mice.CONCLUSION: HNO increases force development in both PLB-KO and WT muscles as a result of increases in myofilament Ca²⁺ responsiveness. The increased intracellular Ca²⁺ transients are accompanied by greater force development in WT mice, suggesting that HNO improves Ca²⁺ activation and establishes HNO as a positive inotropic agent with novel mechanisms.