烧伤早期心肌损伤的新机制：钙蛋白酶破损肌膜与线粒体

Patients suffering from severe burn injury experience depression of cardiac contractility and output during the first 24 h following the injury, which is referred to as the early phase. Cardiac dysfunction is one of the main reasons for the development of multiple organ failure during burn trauma. It has been established that sarcolemmal membrane integrity and mitochondria are severely destroyed, and intracellular Ca2+ overload occurs. However, how overloaded Ca2+ causes these injuries remains still unclear. We and others previously demonstrated that calpain mediates intracellular calcium overload-induced injury in the setting of myocardial ischemia and reperfusion. Our preliminary data suggest that calpain is involved in heart injury caused by burn trauma. In this study, using a model of three-degree over 30% of total body surface area we will determine or answer: (1) the calpain activity, and the effect of calpain inhibitor on heart injury (including heart function, cTnI release and cell death); (2) the effects of MDL 28170, an inhibitor of calpain, on cell integrity/cell skeleton proteins including fodrin, ankyrin-B, and dystrophin, and mitochondria function and apoptosis pathway; (3) the heart function and cell death in mice over-expressing calpastatin, an endogenous inhibitor of calpain; (4) which subtype of calpain (m- or u-) is involved in heart injury by using siRNA technique. Completing this study is helpful to understand the role of calpain in the damage of membrane integrity and mitochondria caused by burn trauma, and provide potential strategy to protect heart in clinical practice.

重度烧伤早期心肌出现的应激性损伤是多脏器功能衰竭等并发症发生发展的关键因素。研究发现，肌膜完整性、线粒体破损是心肌损伤的重要特征，烧伤时心肌出现胞内钙超载，但钙超载对二者损伤的机制并不清楚。我们和他人的工作表明，钙蛋白酶介导缺血再灌注时胞内钙超载所致的心肌损伤，预实验结果提示，钙蛋白酶的激活是严重烧伤时应激性心肌损伤发生的重要环节。申请课题拟探讨（1）烧伤后心肌钙蛋白酶活性、亚型分布的变化，干预钙蛋白酶对心肌损伤程度的影响；（2）干预钙蛋白酶对肌膜完整性/膜骨架分子（血影蛋白、锚蛋白-B和抗肌萎缩蛋白）、线粒体功能及凋亡通路的影响；（3）高表达内源性抑制分子calpastatin后小鼠心肌损伤程度的改变；（4）u-和m-钙蛋白酶亚型的作用。完成此课题有助于认识钙蛋白酶在烧伤后应激性心肌损伤中的作用，对肌膜完整性和线粒体的作用与机制，为烧伤后心肌损伤的防治提供新思路。

大面积重度烧伤引发应激性心肌损伤，是烧伤患者发生多器官功能衰竭乃至死亡的重要原因。因此，阐明应激性心肌损伤的分子机制对烧伤危重患者的救治具有重要的临床意义。钙蛋白酶（calpain）是Ca2+依赖的非溶酶体类蛋白水解酶，本课题探讨了钙蛋白酶在大鼠重度烫伤引起的心肌损伤中的作用与机制。主要实验结果有：.（1）大鼠重度烫伤后血浆乳酸脱氢酶和肌钙蛋白I显著升高，心肌组织凋亡相关分子Caspase-3活性增加，胞浆细胞色素C含量增加，多聚腺苷二磷酸核糖聚合酶-1（DNA修复酶）明显水解，证实重度烧伤引起应激性心肌损伤。.（2）u-钙蛋白酶在胞膜中含量增加，膜骨架分子肌萎缩蛋白（dystrophin）在胞膜中的含量减少，a-胞衬蛋白（a-spectrin）和锚蛋白-B（Ankyrin-B）水解增加。抑制钙蛋白酶活性不仅减弱重度烧伤引起的钙蛋白酶及下游分子的改变，还明显改善细胞膜质量，减少胞内伊文思蓝的蓄积。.（3）重度烫伤还引起钙蛋白酶与线粒体标志分子COX IV共定位，减少胞内ATP水平，减弱线粒体探针JC-1信号。透射电镜可见，烧伤组心肌线粒体数目减少，面积低于0.2 um2百分比减少，高于1.5 um2的线粒体百分比增加，提示烧伤引起线粒体动力学障碍。分子机制研究显示，线粒体融合蛋白-1和-2未发生明显改变，但线粒体OPA-1（Optic atrophy protein 1）的含量显著减少。尤为重要的是，抑制钙蛋白酶活性明显逆转烫伤引起的线粒体改变。.（4）采用siRNA抑制u-钙蛋白酶催化亚基CAPN1的表达后，烫伤血清引发胞衬蛋白的水解明显减少，细胞凋亡下降，细胞活性回升。.（5）静脉给与重组人II型肿瘤坏死因子受体-抗体融合蛋白（rhTNFR:Fc）显著降低烫伤大鼠血浆乳酸脱氢酶和肌钙蛋白I水平，改善心功能并降低钙蛋白酶活性。.（6）重度烫伤增加肺组织湿干比，破坏肺组织形态，抑制钙蛋白酶活性拮抗重度烫伤引起的肺损伤。.以上结果表明，重度烧伤激活钙蛋白酶活性，造成心肌损伤和肺损伤，其机制与破坏细胞膜和线粒体功能有关。rhTNFR:Fc可降低钙蛋白酶活性，拮抗重度烧伤引起的心肌损伤，或为临床保护心肌的有效手段。