晚钠电流通过CaMK-II调节跨壁胞内钙离子分布在心肌缺血再灌注心律失常中的作用及机制研究

Abnormal distribution of intracellular calcium promotes myocardial ischemia/reperfusion arrhythmia (IRA), but lacking researches of its transmural distribution at present. CaMK-II phosphorylates RyR2 and PLN, regulates distribution of cytoplamic and sarcoplasmic reticulum calcium, previous study found that enhanced late sodium current (INaL) promoted CaMK-II phosphorylation and intracellular calcium overload; using cardiac transverse slicing and Optical Mapping techniques (synchronously detect transmural action potential duration and cytoplamic calcium on tissue level) showed that enhanced INaL promoted alternans in right ventricular and septum. We hypothesize that INaL causes abnormal transmural distribution of intracellular calcium by regulating phosphorylation of RyR2 and PLN via CaMK-II and participates in IRA. This project aims to: 1, building a model for synchronously detecting transmural action potential and sarcoplasmic reticulum calcium, observe the effect of INaL on transmural distribution of action potential and intracellular calcium during myocardial ischemia/reperfusion; 2, detecting cytoplamic and sarcoplasmic reticulum calcium concentration and phosphorylation of RyR2 and PLN of ventricular myocardiocytes under INaL and CaMK-II intervention, clarify mechanism of INaL regulation on intracellular calcium distribution during myocardial ischemia/reperfusion. Our study will provide new theoretical and experimental basis for the treatment of IRA.

胞内钙离子分布异常促进心肌缺血再灌注心律失常(IRA)，目前缺乏其跨壁分布的研究。CaMK-II磷酸化RyR2和PLN调节胞浆、肌浆网钙离子分布，前期实验发现增强晚钠电流（INaL）促进CaMK-II磷酸化和胞内钙超载；利用心脏横切片和Optical Mapping技术（组织水平上同步检测跨壁动作电位和胞浆钙离子）发现增强INaL促进右心室和室间隔电交替。我们假设：INaL通过CaMK-II调节RyR2和PLN磷酸化导致胞内钙离子跨壁分布异常，参与IRA的发生。本课题拟：1，构建跨壁动作电位和肌浆网钙离子同步检测模型，观察INaL对心肌缺血再灌注动作电位和胞内钙离子跨壁分布的影响；2，检测INaL和CaMK-II干预时心室肌胞浆、肌浆网钙离子浓度和RyR2、PLN磷酸化水平，明确INaL调节心肌缺血再灌注时胞内钙离子分布的机制。本研究将为IRA的治疗提供新的理论基础和实验依据。

钠葡萄糖共转运蛋白2抑制剂（SGLT2i）为心力衰竭（HF）提供了新的有效治疗方法。我们比较了小鼠在主动脉横向缩窄（TAC）后2周开始给予empaglifloxin（Empa）或对照处理4周小鼠和假手术小鼠的心脏心律失常指标。Empa逆转了TAC引起的超声心动图测量射血分数和缩短分数的减少以及舒张前后壁厚度的增加。仅在1μM异丙肾上腺素激发后，Burst而非S1S1起搏仅在Langendorff灌注的TAC心脏中诱发了室性心动过速。双光学电压和Ca2+瞬态测量表明，常规10Hz刺激下，Empa处理部分逆转了TAC处理相比sham组导致的80%恢复时动作电位持续时间（APD80）、80%恢复时Ca2+瞬态持续时间（CaTD80）、Ca2+达峰时间（TTP100）、Ca2+衰减常数（decay30-90）的增加，以及刺激周期长度缩短时Ca2+交替发生率的增加。随后的异丙肾上腺素激发缩短了假手术组和TAC组的APD80，缩短了所有组的CaTD80和decay30-90，不影响TTP100和Ca2+交替。APD80在所有组中的异质性相似，在TAC心脏中CaTD80的异质性无论有无异丙肾上腺素激发都更高。标测还显示了TAC组室性心动过速期间的折返传导模式。WB实验提示，p-CaMK-II和Nav1.5但不是t-CaMK-II和NCX蛋白表达出现了差异。在ATX-II处理的CHO细胞中，Empa还降低了晚钠电流，而门控特性没有进一步改变。这些发现将Empa诱导的收缩功能恢复与TAC心脏中心室心律失常脆弱性的降低以及细胞内Na+和Ca2+稳态的恢复联系起来。

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