有氧运动通过“脑-心”轴抑制交感神经过度激活改善心肌梗死小鼠心功能

Aerobic Exercise Improves Cardiac Function in Myocardial Infarction Mice by Suppressing Sympathetic Overactivation through“Brain-Heart”Axis

目的:探讨有氧运动改善心肌梗死小鼠心功能的中枢机制。方法:C57BL/6J小鼠随机分为假手术组(Sham)、心肌梗死组(MI)、心肌梗死运动组(ME),每组12只;光遗传激活组(ChR2)、光遗传抑制组(eNpHR3.0),每组6只;心肌梗死对照组(MI+Dio)、心肌梗死+初级运动皮层(primary motor cortex,M1)区神经元消融组(MI+taCasp3),每组6只。采用左冠状动脉前降支结扎术制备心肌梗死模型,术后1周对ME组进行6周有氧运动干预。心脏注射伪狂犬病病毒(pseudorabies virus,PRV)逆行跨多突触标记,以解析中枢与心脏的神经通路。通过光遗传激活或抑制初级运动皮层M1区谷氨酸(Glutamate,Glu)能神经元,以及神经元消融技术沉默初级运动皮层M1区Glu能神经元,探究初级运动皮层M1区Glu能神经元对心脏交感神经活动的影响。采用Masson和苏木精-伊红染色评估心脏结构病理变化,心电图检测心率变异性,心动超声评定心功能,试剂盒检测血清去甲肾上腺素(norepinephrine,NE)含量。利用Western blot和免疫荧光检测心脏的交感神经标记物酪氨酸羟化酶(tyrosine hydroxylase,TH)、神经生长因子(neu‐rotrophic factor,NGF)、生长相关蛋白43(growth-associated protein 43,GAP43)的表达,以及M1区神经元裂解型半胱氨酸蛋白酶-3(c-Caspase3)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin 6,IL-6)蛋白表达。采用尼氏染色和微管关联蛋白2(microtubule associated protein 2,MAP-2)免疫荧光染色评估神经元损伤。利用电镜观察初级运动皮层M1区神经元超微结构。结果:左心室注射PRV 5.5天后在正常小鼠初级运动皮层M1区第五层(L5)观察到大量被标记的神经元。光遗传激活或抑制初级运动皮层M1区Glu能神经元,显著升高或降低心率变异性指标低频和高频功率比值(LF/HF)。与MI+Dio组比较,MI+taCsap3组血清NE含量、LF/HF功率比值以及心肌梗死边缘区TH、GAP43和NGF表达显著降低。与Sham组比较,MI组初级运动皮层M1区神经元MAP-2表达降低,Nissl小体密度降低,线粒体出现肿胀、分裂,嵴溶解等超微结构病理改变,c-Caspase3表达显著增加,TNF-α和IL-6表达增加,而有氧运动干预显著逆转该变化。与Sham组比较,MI组LF/HF功率比值显著升高,心肌NGF和TH分布面积、密度以及心肌胶原纤维显著增加,心功能降低,有氧运动干预显著逆转该变化。结论:由于支配心脏活动的神经元链与大脑初级运动皮层M1区Glu能神经元存在投射,抑制大脑初级运动皮层M1区Glu能神经元活动,显著改善心肌梗死后交感神经过度激活和心脏恶性重构。有氧运动可显著改善心肌梗死后大脑初级运动皮层M1区神经元损伤,抑制心交感神经恶性重构,改善心功能。提示,聚焦大脑初级运动皮层M1区神经调控与心脏保护,可能为未来研究无创治疗或预防心肌缺血损伤提供新的思路。

Objective:To explore the central mechanism of aerobic exercise in improving cardiac function in mice with myocardial infarction(MI).Methods:C57BL/6J mice were randomly divided into Sham group,MI group and MI exercise group,with 12 mice in each group;photogenetic activation group(ChR2),photogenetic inhibition group(eNpHR3.0),with 6 mice in each group;myocardial infarction control group(MI+Dio),myocardial infarction+M1 neuronal ablation group(MI+taCasp3),with 6 mice in each group.The MI model was prepared by ligation of the anterior descending branch in the left coronary artery.In the ME group,6-wk aerobic exercise training was performed at 1-wk after operation.Retrograde transsynaptic labeling was performed by cardiac injection of pseudorabies virus.The effect of M1 Glu neurons on cardiac sympathetic nerve was investigated by photogenetic activation or inhibition of M1 Glu neurons and neuronal ablation technique to silence M1 Glu neurons.The pathological changes of cardiac structure were evaluated by Masson and HE staining.ECG was used to detect heart rate variability.Cardiac function was assessed by echocardiography,and serum NE content was detected by the ELISA kit.The expressions of TH,GAP43 and NGF in cardiac,as well as c-Casps3,TNF-αand IL-6 in the M1 region were detected by Western blotting and immunofluorescence.Nissl staining and MAP-2 immunofluorescence staining was used to assess neuronal damage.The ultrastructure of neurons in M1 region was observed by electron microscope.Results:A large number of labeled neurons were observed in L5 layer of M1 region after 5.5 days of PRV injection in left ventricular.Photogenetic activation or inhibition of M1 Glu neurons significantly increased or decreased the LF/HF power ratio.Compared with MI+Dio group,the serum NE content,LF/HF power ratio,TH,GAP43 and NGF expression in MI+taCsap3 group were significantly decreased.Compared with Sham group,MI group showed decreased MAP-2 expression and Nissl body density,the mitochondrial swelling,division,crista lysis and other ultrastructural pathological were changed,and the c-Caspase3,TNF-αand IL-6 expression were increased.However,aerobic exercise intervention significantly reversed the change.Compared with Sham group,the power ratio of LF/HF in MI group was significantly increased,the distribution area and density of myocardial NGF and TH and myocardial collagen fibers were significantly increased,and the cardiac function was decreased,which was significantly reversed after aerobic exercise intervention.Conclusions:Due to the projection link between the neuronal chain governing cardiac activity and M1 Glu neurons,the inhibitionof M1 Glu neuron activity can improve the overactivation of sympathetic nerve and the malignant remodeling of the heart after myocardial infarction.Aerobic exercise can alleviate the damage of M1 neurons,inhibit the malignant remodeling of cardiac sympathetic nerve and improve cardiac function after myocardial infarction.It is suggested that the research focus on the neuroregulation and cardiac protection in the M1 region of the primary motor cortex of the brain may provide new ideas for non-invasive treatment or prevention of myocardial ischemic injury in the future.