Serelaxin Protects H9c2 Cardiac Myoblasts against Hypoxia and Reoxygenation-Induced Damage through Activation of AMP Kinase/Sirtuin1: Further Insight into the Molecular Mechanisms of the Cardioprotection of This Hormone

Abstract

Serelaxin (RLX), namely the human recombinant Relaxin-2 hormone, protects the heart from ischemia/reperfusion (I/R)-induced damage due to its anti-inflammatory, anti-apoptotic and antioxidant properties. RLX acts by binding to its specific RXFP1 receptor whereby it regulates multiple transduction pathways. In this in vitro study, we offer the first evidence for the involvement of the AMP kinase/Sirtuin1 (AMPK/SIRT1) pathway in the protection by RLX against hypoxia/reoxygenation (H/R)-induced damage in H9c2 cells. The treatment of the H/R-exposed cells with RLX (17 nmol L^-1) enhanced SIRT1 expression and activity. The inhibition of SIRT1 signaling with EX527 (10 µmol L^-1) reduced the beneficial effect of the hormone on mitochondrial efficiency and cell apoptosis. Moreover, RLX upregulated the AMPK pathway, as shown by the increase in the expression of phospho-AMPK-activated protein. Finally, AMPK pathway inhibition by Compound C (10 and 20 μmol L^-1) abrogated the increase in SIRT1 expression induced by RLX, thus suggesting the involvement of the AMPK pathway in this effect of RLX. These results strengthen the concept that RLX exerts its cardioprotective effects against H/R-induced injury through multiple pathways which also include AMPK/SIRT1. These new findings support the use of RLX or RLX-derived molecules as a promising therapeutic for those diseases in which I/R and oxidative stress play a pathogenic role.

Keywords: AMPK; H9c2 cells; SIRT1; Serelaxin; apoptosis; hypoxia–reoxygenation; oxidative stress.

Figure 1

RLX counteracts the decrease in SIRT1 expression in H9c2 cells exposed to H/R. Representative images of Western blotting and quantitative analysis showing SIRT1 and β-actin expression in H9c2 cells under normoxia (n = 3) (A,B) and upon H/R challenge (n = 6) (C,D). Significance of differences (B: Mann–Whitney nonparametric Test; D: one-way ANOVA and Tukey multiple comparison test): *** p < 0.001 vs. controls (Ctrl); °°° p < 0.001 vs. controls (Ctrl); ## p < 0.01 vs. H/R.

Figure 2

RLX counteracts the decrease in SIRT1 activity in H9c2 cells exposed to H/R. Representative diagrams showing SIRT1 activity in H9c2 in normoxia (n = 9–10) (A) and upon H/R challenge (n = 10–14) (B). Significance of differences (A: Student’s unpaired t-test; B: Kruskal–Wallis test and Dunn multiple comparison test): *** p < 0.001 vs. controls (ctrl); ## p < 0.01 vs. H/R.

Figure 3

Inhibition of SIRT1 signaling counteracts the beneficial effects of RLX on mitochondrial respiratory chain efficiency in H9c2 cells exposed to H/R. Representative diagrams showing mitochondrial activity, assayed by the MTT assay, of H9c2 cells in normoxia (Ctrl) (n = 6) and under H/R in the absence (n = 6) and in the presence of RLX (n = 6) and upon treatment with the selective SIRT1 inhibitor, EX527 1 and 10 µmol L⁻¹ (n = 6–8). Significance of differences (one-way ANOVA and Tukey multiple comparison test): *** p < 0.001 vs. controls (Ctrl); # p < 0.05 vs. H/R; °°° p < 0.001 vs. controls (Ctrl) and RLX alone; § p < 0.05 vs. H/R+RLX.

Figure 4

Inhibition of SIRT1 signaling counteracts the beneficial effects of RLX on apoptosis of H9c2 cells exposed to H/R. Representative image of Western blotting (A) and quantitative analysis (B) showing the expression of cleaved and full-length caspase 3 and β-actin in H9c2 cells under normoxia (Ctrl, n = 3–4) and upon H/R challenge in the presence or absence of the selective SIRT1 inhibitor EX527 (1 and 10 µmol L⁻¹, n = 6). Significance of differences (one-way ANOVA and Tukey multiple comparison test): ### p < 0.001 vs. H/R; §§ p < 0.01 vs. H/R+RLX.

Figure 5

RLX treatment activates AMPK signaling in H9c2 cells exposed to H/R. Representative images of Western blotting (A) and quantitative analysis (B) showing phospho-AMPK, AMPK and β-actin expression in H9c2 cells upon H/R challenge (n = 6). Significance of differences (one-way ANOVA and Tukey multiple comparison test): *** p < 0.001 vs. controls (Ctrl); ## p < 0.01 vs. H/R.

Figure 6

Inhibition of AMPK signaling counteracts the increase in SIRT1 expression induced by RLX in H9c2 cells exposed to H/R. Representative images of Western blotting (A) and quantitative analysis (B) showing SIRT1 and β-actin expression in H9c2 cells upon H/R challenge in the presence or absence of the selective AMPK inhibitor Compound C (10 and 20 µmol L⁻¹) (n = 5–6). Significance of differences (one-way ANOVA and Tukey multiple comparison test): *** p < 0.001 vs. controls (Ctrl); ### p < 0.001 vs. H/R; °°° p < 0.001 vs. controls (Ctrl); §§§ p < 0.001 vs. H/R+RLX.