Sevoflurane preconditioning alleviates myocardial ischemia reperfusion injury through mitochondrial NAD+-SIRT3 pathway in rats

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.

Keywords: inhalation anesthetics; ischemia reperfusion injury; mitochondria; myocardium; nicotinamide adenine dinucleotide; sevoflurane; silent information regulator 3.

图1

实验方案示意图 Figure 1 Schematic diagram of experiment protocols

图2

各组大鼠心肌组织中烟酰胺腺嘌呤二核苷酸含量和沉默信息调节因子3蛋白质表达水平的比较(n=6， $\overline{\mathit{x}}$ ±s) Figure 2 Comparison of nicotinamide adenine dinucleotide (NAD⁺) content and protein expression levels of silent information regulator 3 (SIRT3) in myocardial tissues of rats in each group (n=6, $\overline{\mathit{x}}$ ±s) A: NAD⁺ content; B: Representative electropherogram of Western blotting of SIRT3 protein expression; C: Histogram of SIRT3 relative protein expression. **P<0.01 vs the Sham group; ††P<0.01 vs the IR group;‡‡P<0.01 vs the Sev group.

图3

各组大鼠心肌梗死情况比较 Figure 3 Comparison of myocardial infarction of rats in each group A: Evans blue (EB) and triphenyl tetrazolium chloride (TTC) double staining determined the infarct size. The area that is not blue stained indicates area at risk (AAR), while the white area indicates infarct size (IS). B: Comparison of AAR or IS percentage (n=6, $\overline{x}$ ±s). Percentage of AAR=(AAR/Left ventricle)×100%; Percentage of IS=(IS/AAR)×100%. **P<0.01 vs the Sham group; ††P<0.01 vs the IR group; ‡‡P<0.01 vs the Sev group.

图4

各组大鼠血清肌钙蛋白I水平的比较(n=6， $\overline{\mathit{x}}$ ±s) Figure 4 Comparison of serum cardiac troponin I (cTnI) levels of rats in each group (n=6, $\overline{\mathit{x}}$ ±s) **P<0.01 vs the Sham group; ††P<0.01 vs the IR group; ‡‡P<0.01 vs the Sev group.

图5

电子显微镜下各组大鼠心肌组织的超微结构(×8 000) Figure 5 Ultrastructure of myocardial tissues of rats in each group under the electron microscope (×8 000)

图6

各组大鼠心肌线粒体复合体I(A)、II(B)、IV(C)活性的比较(n=6， $\overline{\mathit{x}}$ ±s) Figure 6 Comparison of activity of mitochondrial complex I (A), II (B), and IV (C) of rats in each group (n=6, $\overline{\mathit{x}}$ ±s) **P<0.01 vs the Sham group; ††P<0.01 vs the IR group; ‡‡P<0.01 vs the Sev group.

图7

各组大鼠心肌组织中ATP含量的比较(n=6， $\overline{\mathit{x}}$ ±s) Figure 7 Comparison of content of ATP in myocardial tissues of rats in each group (n=6, $\overline{\mathit{x}}$ ±s) **P<0.01 vs the Sham group; ††P<0.01 vs the IR group; ‡‡P<0.01 vs the Sev group.

图8

各组大鼠心肌组织中MDA含量，SOD活性及SOD2、过氧化氢酶蛋白质表达水平的比较(n=6， $\overline{\mathit{x}}$ ±s) Figure 8 Comparison of MDA content, SOD activity, and protein expression levels of SOD2 and catalase (CAT) in myocardial tissues of rats in each group (n=6, $\overline{\mathit{x}}$ ±s) A: MDA content; B: SOD activity; C: Representative electropherogram of Western blotting of SOD2 and CAT protein expression; D: Histogram of SOD2 relative protein expression; E: Histogram of CAT relative protein expression. **P<0.01 vs the Sham group; ††P<0.01 vs the IR group; ‡‡P<0.01 vs the Sev group.

图9

各组大鼠心肌线粒体通透性的改变和心肌组织中电压依赖性阴离子通道1蛋白质表达水平(n=6， $\overline{\mathit{x}}$ ±s) Figure 9 Change of mitochondrial permeability and protein expression level of voltage dependent anion channel 1 (VDAC1) in myocardial tissues of rats in each group (n=6, $\overline{\mathit{x}}$ ±s) A: Change of mitochondrial permeability; B: Representative electropherogram of Western blotting of VDAC1 protein expression; C: Histogram of VDAC1 relative protein expression. **P<0.01 vs the Sham group; ††P<0.01 vs the IR group; ‡‡P<0.01 vs the Sev group.