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Title: Sevoflurane preconditioning alleviates myocardial ischemia reperfusion injury through mitochondrial NAD⁺-SIRT3 pathway in rats.
Author: Qin X, Qin Q, Ran K, Yuan G, Chang Y, Wang Y, Xiao Y.
Journal: Zhong Nan Da Xue Xue Bao Yi Xue Ban; 2022 Aug 28; 47(8):1108-1119. PubMed ID: 36097779.
Abstract:
OBJECTIVES: Myocardial ischemia reperfusion injury (IRI) occurs occasionally in the process of ischemic heart disease. Sevoflurane preconditioning has an effect on attenuating IRI. Preserving the structural and functional integrity of mitochondria is the key to reduce myocardial IRI. Silent information regulator 3 (SIRT3), a class of nicotinamide adenine dinucleotide (NAD⁺) dependent deacetylases, is an important signal-regulating molecule in mitochondria. This study aims to explore the role of mitochondrial NAD⁺-SIRT3 pathway in attenuating myocardial IRI in rats by sevoflurane preconditioning. METHODS: A total of 60 male Sprague Dawley (SD) rats were randomly divided into 5 groups (n=12): A sham group (Sham group), an ischemia reperfusion group (IR group), a sevoflurane preconditioning group (Sev group, inhaled 2.5% sevoflurane for 30 min), a sevoflurane preconditioning+SIRT3 inhibitor 3-TYP group (Sev+3-TYP group, inhaled 2.5% sevoflurane for 30 min and received 5 mg/kg 3-TYP), and a 3-TYP group (5 mg/kg 3-TYP). Except for the Sham group, the IR model in the other 4 groups was established by ligating the left anterior descending coronary artery. The size of myocardial infarction was determined by double staining. Serum cardiac troponin I (cTnI) level was measured. The contents of NAD⁺ and ATP, the activities of mitochondrial complexes I, II, and IV, the content of MDA, the activity of SOD, and the changes of mitochondrial permeability were measured. The protein expression levels of SIRT3, SOD2, catalase (CAT), and voltage dependent anion channel 1 (VDAC1) were detected by Western blotting. The ultrastructure of myocardium was observed under transmission electron microscope. MAP and HR were recorded immediately before ischemia (T0), 30 min after ischemia (T1), 30 min after reperfusion (T2), 60 min after reperfusion (T3), and 120 min after reperfusion (T4). RESULTS: After ischemia reperfusion, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased (both P<0.01), and an obvious myocardial injury occurred, including the increase of myocardial infarction size and serum cTnI level (both P<0.01). Correspondingly, the mitochondria also showed obvious damage on energy metabolism, antioxidant function, and structural integrity, which was manifested as: the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level and mitochondrial permeability were increased (all P<0.01). Compared with the IR group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were increased in the Sev group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were decreased in the Sev group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were increased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were decreased in the Sev group (all P<0.01). Compared with the Sev group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased in the Sev+3-TYP group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were increased in the Sev+3-TYP group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were increased in the Sev+3-TYP group (all P<0.01). CONCLUSIONS: Sevoflurane preconditioning attenuates myocardial IRI through activating the mitochondrial NAD⁺-SIRT3 pathway to preserve the mitochondrial function. 目的: 缺血再灌注损伤(ischemia reperfusion injury，IRI)常发生于缺血性心脏疾病。七氟醚预处理有减轻心肌IRI的作用。保存线粒体结构和功能的完整是减轻心肌IRI的关键，线粒体内重要的信号调节分子沉默信息调节因子3(silent information regulator 3，SIRT3)是一类依赖于烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide，NAD⁺)的去乙酰化酶。本研究旨在探讨NAD⁺-SIRT3通路在七氟醚预处理减轻大鼠心肌IRI中的作用。方法: 将60只健康成年雄性SD大鼠随机分为5组(n=12)：假手术组(Sham组)、缺血再灌注组(IR组)、七氟醚预处理组(Sev组，给予2.5%七氟醚吸入30 min)、七氟醚预处理+SIRT3抑制剂3-TYP干预组(Sev+3-TYP组，给予尾静脉注射5 mg/kg 3-TYP和2.5%七氟醚吸入30 min)、3-TYP干预组(3-TYP组，给予尾静脉注射5 mg/kg 3-TYP)。Sham组只开胸不结扎，其他4组采用结扎冠状动脉左前降支的方法建立缺血再灌注模型。采用双重染色法测定心肌梗死面积。测定血清大鼠肌钙蛋白I(cardiac troponin I，cTnI)水平。测定心肌组织中NAD⁺、ATP、MDA含量，线粒体复合体Ⅰ、II、IV及SOD的活性，线粒体通透性的改变。采用蛋白质印迹法检测心肌组织中SIRT3、SOD2、过氧化氢酶(catalase，CAT)和电压依赖性阴离子通道1(voltage dependent anion channel 1，VDAC1)的蛋白质表达水平。在透射电子显微镜下观察心肌超微结构。记录缺血前即刻(T0)、缺血30 min(T1)、再灌注30 min(T2)、再灌注60 min(T3)和再灌注120 min(T4)时的HR和MAP。结果: 缺血再灌注后，心肌组织中NAD⁺含量减少，SIRT3蛋白质表达水平降低(均P<0.01)，同时出现明显的心肌损伤，包括心肌梗死面积增加，血清cTnI水平升高(均P<0.01)。相应地，线粒体也出现明显的能量代谢、抗氧化功能和结构完整性的受损，表现为：线粒体复合体I、II、IV活性，ATP含量，SOD2和CAT蛋白质表达水平下降，而MDA含量、VDAC1蛋白质表达水平和线粒体通透性增加(均P<0.01)。与IR组比较，Sev组心肌组织中NAD⁺含量增加，SIRT3蛋白质表达水平升高(均P<0.01)；心肌梗死面积缩小，血清cTnI水平降低(均P<0.01)；线粒体复合体I、II、IV活性，ATP含量，SOD2和CAT蛋白质表达水平升高，而MDA含量、VDAC1蛋白质表达水平和线粒体通透性降低(均P<0.01)。与Sev组比较，Sev+3-TYP组心肌组织中NAD⁺含量减少，SIRT3蛋白质表达水平降低(均P<0.01)；心肌梗死面积增加，血清cTnI水平升高(均P<0.01)；线粒体复合体I、II、IV活性，ATP含量，SOD2和CAT蛋白质表达水平下降，而MDA含量、VDAC1蛋白质表达水平和线粒体通透性增加(均P<0.01)。结论: 七氟醚预处理通过激活线粒体NAD⁺-SIRT3通路，保存线粒体功能，从而减轻心肌IRI。. OBJECTIVE: Myocardial ischemia reperfusion injury (IRI) occurs occasionally in the process of ischemic heart disease. Sevoflurane preconditioning has an effect on attenuating IRI. Preserving the structural and functional integrity of mitochondria is the key to reduce myocardial IRI. Silent information regulator 3 (SIRT3), a class of nicotinamide adenine dinucleotide (NAD⁺) dependent deacetylases, is an important signal-regulating molecule in mitochondria. This study aims to explore the role of mitochondrial NAD⁺-SIRT3 pathway in attenuating myocardial IRI in rats by sevoflurane preconditioning. METHODS: A total of 60 male Sprague Dawley (SD) rats were randomly divided into 5 groups (n=12): A sham group (Sham group), an ischemia reperfusion group (IR group), a sevoflurane preconditioning group (Sev group, inhaled 2.5% sevoflurane for 30 min), a sevoflurane preconditioning+SIRT3 inhibitor 3-TYP group (Sev+3-TYP group, inhaled 2.5% sevoflurane for 30 min and received 5 mg/kg 3-TYP), and a 3-TYP group (5 mg/kg 3-TYP). Except for the Sham group, the IR model in the other 4 groups was established by ligating the left anterior descending coronary artery. The size of myocardial infarction was determined by double staining. Serum cardiac troponin I (cTnI) level was measured. The contents of NAD⁺ and ATP, the activities of mitochondrial complexes I, II, and IV, the content of MDA, the activity of SOD, and the changes of mitochondrial permeability were measured. The protein expression levels of SIRT3, SOD2, catalase (CAT), and voltage dependent anion channel 1 (VDAC1) were detected by Western blotting. The ultrastructure of myocardium was observed under transmission electron microscope. MAP and HR were recorded immediately before ischemia (T0), 30 min after ischemia (T1), 30 min after reperfusion (T2), 60 min after reperfusion (T3), and 120 min after reperfusion (T4). RESULTS: After ischemia reperfusion, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased (both P<0.01), and an obvious myocardial injury occurred, including the increase of myocardial infarction size and serum cTnI level (both P<0.01). Correspondingly, the mitochondria also showed obvious damage on energy metabolism, antioxidant function, and structural integrity, which was manifested as: the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level and mitochondrial permeability were increased (all P<0.01). Compared with the IR group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were increased in the Sev group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were decreased in the Sev group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were increased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were decreased in the Sev group (all P<0.01). Compared with the Sev group, the content of NAD⁺ in cardiac tissues and the expression level of SIRT3 protein were decreased in the Sev+3-TYP group (both P<0.01); the size of myocardial infarction and the level of serum cTnI were increased in the Sev+3-TYP group (both P<0.01); the activities of mitochondrial complexes I, II, and IV, ATP content, protein expression levels of SOD2 and CAT were decreased, while MDA content, VDAC1 protein expression level, and mitochondrial permeability were increased in the Sev+3-TYP group (all P<0.01). CONCLUSION: Sevoflurane preconditioning attenuates myocardial IRI through activating the mitochondrial NAD⁺-SIRT3 pathway to preserve the mitochondrial function.

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