麻醉药的心肌肌丝结合靶点与其抑制心肌收缩力的关系

Anesthetics are known to depress cardiac contraction, but the molecular mechanisms involved are not known. Here we hypothesized that decreasing myofilament Ca2+ responsiveness is central to the depression of contraction. Moreover, we also aimed to uncover the molecular targets of the anesthetics which depress myocardial contraction via directly targeting key myofilament proteins. Trabeculae muscles from the right ventricles of SD rats were dissected out, force and intracellular Ca2+ ([Ca2+]i) were measured. Steady-state activations were obtained in the presence of ryanodine at the stimulation of 10 HZ. After that, the same muscle was chemically skinned and the force were recorded like the steady state. Fresh myofibrillar samples were isolated and treated with AziPm and Azi-iso (the photoactive analogs of propofol and isoflurane), photolyzed under UV light, then the proteins bands were analyzed by mass spectrometry (LC-MS/MS). Cardiomyocytes were also dissected out to test the L-Calcium Channel by using patch clamp. Our study would be the first discover the molecular targets of myocardial force depression by anesthetic agents and the structural understanding of anesthetic-myofilament protein interaction will not only enhance our understanding the molecular mechanism of anesthetic-induced myocardial depression, but also provide the foundation for re-designing new anesthetics without cardiac effects.

麻醉药可降低心肌收缩力，减少心输出量，进而影响血流动力学稳定，但其机制未明。对心肌肌丝收缩的调节是心肌收缩的重要机制之一。申请者预实验发现：在不改变心肌细胞内钙离子浓度的条件下，临床浓度的麻醉药异丙酚及异氟醚却降低其收缩力，提示麻醉药可能通过降低肌丝对钙离子敏感性来发挥其抑制心肌收缩的作用。本课题组拟通过所掌握的实验技术，检测麻醉药对心肌细胞L-钙离子通道的影响、全细胞功能状态下肌条及心肌肌丝对钙离子的敏感性、肌丝横桥Mg2+-ATPase的功能，以全方位验证麻醉药通过影响心肌肌丝钙离子敏感性并通过与肌丝的直接作用降低心肌收缩功能这一观点，继而通过应用光敏化异丙酚和光敏化异氟醚，找出麻醉药在肌丝上具体的作用靶位点。这将是首次明确的找出麻醉药在心肌肌丝上的结合位点，从而为麻醉药对心脏收缩功能影响提供新机制，完善以往研究对麻醉药影响心功能的机制发现，并对新型麻醉药的研发提供一定的理论支持。

常用麻醉药丙泊酚对循环功能的影响可引起术中的各种变化，主要起因于丙泊酚抑制血管和心肌收缩功能。近年来，其抗炎和抗氧化作用也越来越引起关注。研究表明它可以预防缺血再灌注期间（I/R）细胞内钙超载，减少脂质过氧化反应，提高红细胞抗氧化能力和再灌注后心脏机械功能恢复，以此减弱心肌缺血再灌注损伤。本研究通过异丙酚作用于大鼠离体灌流心脏，模拟心脏正常及缺血再灌注状态。从而进一步探讨丙泊酚对心脏肥大细胞活性的影响以及在心肌缺血再灌注损伤中产生保护作用的其他途径。 离体心脏预先应用 compound 48/80，经由异丙酚预处理后，在各时间点，类胰蛋白酶水平均降低;心肌组织中肥大细胞脱颗粒的程度显著减轻。丙泊酚还可减少RBL-2H3细胞中，由compound 48/80 介导的类胰蛋白酶和组胺释放。心脏经历 I/R 后，R5min和 R60min 时类胰蛋白酶，LDH，CK-MB 和 cTnI 的表达均显著增加；心脏组织中细胞因子（TNF-α 和 IL-6）同样显著增加。此外，I/R 诱导的心脏也表现出更严重的组织病理学损伤和面积更大的梗死区域。compound 48/80 可以进一步加重I/R 损伤。所有这些效应都能被异丙酚抑制，丙泊酚同时还可以减轻 compound 48/80 介导的 I/R 损伤。探究丙泊酚通过抑制PKA调节的肌丝蛋白 cMyBP-C发挥心肌抑制作用。以原代心肌细胞为研究对象进行实验验证，急性分离成年大鼠心肌细胞， 加入异丙酚刺激， 利用ELISA、免疫印迹实验，免疫共沉淀实验方法监测PKA活性、磷酸化水平及cM yBP-C蛋白的磷酸化水平。采用超声心动图测定大鼠心功能：以上实验结果证明丙泊酚能够通过抑制cMyBP-C磷酸化抑制心肌。本研究为临床麻醉药对心脏作用的机制研究提供了新思路，对探寻麻醉药的改进与应用具有重要意义。