Dexmedetomidine Ameliorates Cardiac Ischemia/Reperfusion Injury by Enhancing Autophagy Through Activation of the AMPK/SIRT3 Pathway

Abstract

Objective: Myocardial ischemia-reperfusion (I/R) injury is a detrimental disease, resulting in high morbidity and mortality globally. In this study, we aimed to investigate the role of Dex in mitigating cardiac I/R injury.

Methods: H9c2 cells were treated with Dex (1 μM) for 24 h followed by oxygen-glucose deprivation/re-oxygenation (OGD/R). ANP and BNP mRNA of H9c2 cells and the LDH release were measured. Apoptosis of H9c2 cells was analyzed by flow cytometry. Mitochondrial membrane potential and superoxide production were detected by JC-1 staining and MitoSOX^TM Red, respectively. Cell aerobic respiration was measured using Seahorse analysis. In vivo, mice were injected with Dex (25 μg/kg, i.p., once daily) for 5 days and then subjected to heart I/R. Heart function was analyzed by echocardiography. CK-MB and LDH were measured by Elisa. Infarct size was measured using TTC-Evans blue staining. Mitochondrial ultrastructure was observed using transmission electron microscopy. DHE staining, SOD activity, the content of MDA, and the content of GSH/GSSG of heart were measured to evaluate the oxidative stress. In addition, inflammatory cytokines were measured in vivo and in vitro. Furthermore, AMPK, SIRT3, and autophagy-related protein expression in the heart were detected by Western blot.

Results: Dex reduced the H9c2 cells injury exposed to OGD/R, accompanied by improved mitochondrial function and membrane potential. In vivo, Dex improved heart function, myocardial injury, and the mitochondria ultrastructure following I/R injury. Meanwhile, Dex inhibited myocardial oxidative stress and inflammation in the myocardial I/R. Furthermore, Compound C (an AMPK inhibitor) could inhibit Dex-induced autophagy in the I/R heart and the 3-MA (an autophagy inhibitor) could partially interfere with the effects of Dex on the protection of I/R heart.

Conclusion: Dex suppressed oxidative stress and inflammation by promoting autophagy through activating the AMPK/SIRT3 pathway, thus protecting the heart against the I/R injury.

Keywords: AMPK/SIRT3; autophagy; cardiac ischemia reperfusion; dexmedetomidine.

Figure 1

Dex alleviated OGD/R-induced apoptosis, membrane potential reduction, mitochondrial superoxide production, and improved aerobic respiration in H9c2 cells. (A). Detection the proliferation of H9c2 cells stimulated by different concentrations of Dex using the CCK-8 assay. (B). ANP and BNP mRNA expression in H9c2 cells. (C). Flow cytometry analysis for H9c2 cell apoptosis after Annexin V/PI staining. (D). Quantitative analysis of the apoptotic H9c2 cell. (E). LDH release in the medium of H9c2 cells. (F). Representative images of H9c2 cells stained with JC-1 for visualization of mitochondrial membrane potential. Aggregates were stained red, and monomers were stained green. 20 × magnification; Scale bar, 50 μm. (G). Analysis of the ratio of red/green fluorescent density relative to the control group. (H–J). Seahorse analysis for the cellular oxygen consumption rate (OCR) of H9c2 cells (H) and qualification of basal respiration (I) and maximum respiration (J). (K and L). Flow cytometry analysis (K) and quantification (L) of H9c2 cells stained with MitoSOX^TM Red for mitochondrial superoxide. Data are presented as the mean ± SD (n = 6) of at least three independent experiments. Statistical comparisons were conducted by one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparisons.

Figure 2

Dexmedetomidine protected mice against cardiac I/R injury and improved heart function following I/R. (A). Schematic flowchart of the application of Dex and the construction of the cardiac I/R model. (B–C). Left ventricular ejection fraction (LVEF%) and left ventricular fractional shortening (LVFS%) at baseline. (D). Representative images of M-mode echocardiography from mice. (E and F). LVEF% (E) and LVFS% (F) of mice. (G and H). Plasma CK-MB (G) and LDH (H) levels in mice. (I). Representative images of Evans-blue perfused and TTC-stained heart sections outlining the area at risk (AAR; sum of white and red areas); healthy viable tissue (blue); and infarcted tissue (pale white). Scale bar, 1 mm. (J and K). Quantification of infarct size relative to AAR (J) and AAR relative to LV (K). (L). Representative micrographs of the ventricular myocardium were observed by transmission electron microscopy. Fragmentation of muscle bands pointed by green arrows and abnormal shape of mitochondria, including swollen and absent cristae density pointed by Orange arrows. 6000 X magnification; Scale bar, 1 μm. (M). ATP content in heart tissues. Data are presented as the mean ± SD (n = 6). Statistical comparisons were conducted by an unpaired two-tailed Student’s t-test or one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparisons.

Figure 3

Dexmedetomidine attenuated oxidative stress and inflammation in the cardiac I/R. (A). Representative DHE staining of heart sections in the border zones. 20 X magnification; Scale bar, 100 μm. (B). MDA levels in heart tissues. (C). SDO activity in heart tissues. (D–F). GSH and GSSG content in heart tissues and the ratio of GSH/GSSG. (G–I). qPCR analysis for the mRNA levels of IL-1β, IL-6, and TNF-α in heart tissues. (J–L). Detection of IL-1β, IL-6, and TNF-α in the medium of H9c2 cells. Data are presented as the mean ± SD (n = 6) of at least three independent experiments. Statistical comparisons were conducted by one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparisons.

Figure 4

Dexmedetomidine enhanced autophagy for I/R heart protection through upregulation of the AMPK/SIRT3 pathway. (A). Representative images of immunoblots of hearts for the proteins LC3, p62, AMPK, SIRT3, PINK1, and Parkin. (B and C). Qualification of the ratio of LC3 II/I and p62 protein levels of hearts relative to the control mice. (D). Qualification of the AMPK, SIRT3, PINK1, and Parkin protein levels of hearts relative to the control mice. (E). The ratio of NAD⁺/NADH in heart tissues. (F–I). Representative images of immunoblots of hearts for the proteins AMPK, SIRT3, LC3, and p62 and qualification for the expression relative to the I/R mice, including the additional Compound C-treated group. CC: Compound C. (J–L). Representative images of immunoblots of hearts for the proteins LC3 and p62 and qualification for the expression relative to the I/R mice, including the additional 3-MA-treated group. (M) Representative images of M-mode echocardiography from mice after indicated treatments post-I/R. (N–O). LVEF% (N) and LVFS% (O) from mice after indicated treatments post-I/R. =Data are presented as the mean ± SD (n = 6) and were performed in at least three independent experiments in vitro. Statistical comparisons were conducted by one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparisons.

Figure 5

The underlying mechanism involved in the protective effects of DEX against cardiac I/R injury. Created with BioRender.com.