Irisin Attenuates Myocardial Ischemia/Reperfusion Injury and Improves Mitochondrial Function Through AMPK Pathway in Diabetic Mice

Abstract

Aims: Several recent reports have shown irisin protects the heart against ischemia/reperfusion injury. However, the effect of irisin on I/R injury in diabetic mice has not been described. The present study was designed to investigate the role of irisin in myocardial ischemia-reperfusion (MI/R) injury in diabetic mice.

Methods: A mouse model of diabetes was established by feeding wild type or gene-manipulated adult male mice with a high-fat diet. All the mice received intraperitoneal injection of irisin or PBS. Thirty minutes after injection, mice were subjected to 30 min of myocardial ischemia followed by 3h (for cell apoptosis and protein determination), 24 h (for infarct size and cardiac function).

Results: Knock-out of gene FNDC5 augmented MI/R injury in diabetic mice, while irisin treatment attenuated MI/R injury, improved cardiac function, cellular ATP biogenetics, mitochondria potential, and impaired mitochondrion-related cell death. More severely impaired AMPK pathway was observed in diabetic FNDC5^-/- mice received MI/R. Knock-out of gene AMPK blocks the beneficial effects of irisin on MI/R injury, cardiac function, cellular ATP biogenetics, mitochondria potential, and mitochondrion-related cell death.

Conclusions: Our present study demonstrated that irisin improves the mitochondria function and attenuates MI/R injury in diabetic mice through AMPK pathway.

Keywords: AMPK; diabetes; irisin; ischemia/reperfusion; mitochondria.

Figure 1

Myocardial ischemia-reperfusion (MI/R) injury is increased in diabetic FNDC5^-/- mice, while irisin treatment attenuates MI/R injury. (A, B): left ventricular ejection fraction (LVEF; A) and left ventricular fraction shortening (LVFS; B) were determined by echocardiograph as indexes for cardiac function at the end of the 24-hour reperfusion. (C) myocardial infarct size was assessed by Evans blue/TTC double staining. All values are presented as mean ± SEM. n = 6–8/group. ^*P < 0.05.

Figure 2

Mitochondrial function was more severely impaired in diabetic FNDC5^-/- mice received MI/R and Irisin reversed the mitochondrial dysfunction. (A) ATP production was measured to reflect the mitochondrial function. (B–D) Enzymatic activities of complexes I/III/V were determined in isolated mitochondria. All values are presented as mean ± SEM. n = 6–8/group. ^*P < 0.05.

Figure 3

Apoptosis was increased in diabetic FNDC5^-/- mice compared with WT diabetic mice under MI/R injury and irisin inhibited mitochondrial apoptosis in cardiomyocyte in diabetic mice. Cardiomyocyte apoptosis was determined by TUNEL assay (A, B) and caspase-3 assay (C). Mitochondrial apoptotic parameters (D–G): (E) Bad, (F) Caspase-9, (G) Bcl-2, and (H) survivin) were determined via western blot analysis. All values are presented as mean ± SEM. n = 6–8/group. ^*P < 0.05.

Figure 4

Irisin increases P-AMPK expression in diabetic mice received MI/R. P-AMPK and AMPK expression (A, B) was determined by Western blot analysis. All values are presented as mean ± SEM. n = 6–8/group. ^*P < 0.05.

Figure 5

The effects of irisin on cardiac function in diabetic AMPK^-/- mice were inhibited. (A, B) left ventricular ejection fraction (LVEF; A) and left ventricular fraction shortening (LVFS; B) were determined by echocardiograph as indexes for cardiac function at the end of the 24-h reperfusion. (C) myocardial infarct size was assessed by Evans blue/TTC double staining. All values are presented as mean ± SEM. n = 6–8/group. ^*P < 0.05.

Figure 6

The effects of irisin on mitochondrial function in diabetic AMPK^-/- mice were inhibited. (A) ATP production was measured to reflect the mitochondrial function. (B–D) Enzymatic activities of complexes I/III/V were determined in isolated mitochondria. All values are presented as mean ± SEM. n = 6–8/group. ^*P < 0.05.

Figure 7

The effects of irisin on mitochondrial apoptosis in diabetic AMPK^-/- mice were inhibited. Cardiomyocyte apoptosis was determined by TUNEL assay (A, B) and caspase-3 assay (C). Mitochondrial apoptotic parameters (D–H): (E) Bad, (G) Caspase-9, (G) Bcl-2 and (H) survivin) were determined via western blot analysis. All values are presented as mean ± SEM. n = 6–8/group. ^*P < 0.05.