Irisin improves ROS‑induced mitohormesis imbalance in H9c2 cells

Abstract

Abnormal mitohormesis is a key pathogenic mechanism that induces a variety of cardiac diseases, including cardiac hypertrophy and heart failure. Irisin as a muscle factor serves a cardioprotective role in response to cellular oxidative stress injury. Rat cardiomyocyte cells (H9c2) were treated with 40 µM exogenous H₂O₂ to establish an oxidative stress model, followed by addition of 75 nM exogenous irisin for experiments to determine mitochondrial membrane potential, reactive oxygen species, and Mitohormesis‑related factors by attrition cytometry. Subsequently, the expression of mitochondrial membrane potential, reactive oxygen species and Mitohormesis‑related factors were continued to be determined by establishing a peroxisome proliferator‑activated receptor γ coactivator‑1 alpha (PGC‑1α) siRNA interference model and continuing the treatment with the addition of 75 nM irisin 12 h before the end of interference. When H9c2 cells underwent oxidative stress, irisin partially improved mitochondrial membrane potential and reactive oxygen species levels and partially restored mitochondrial energy metabolism by upregulating fusion proteins optic atrophy 1 (OPA1) mitochondrial dynamin‑like GTPase and mitofusin 2 and downregulating fission protein dynamin‑related protein 1. Following interference with PGC‑1α, irisin promoted mitochondrial biosynthesis by increasing the mRNA levels of OPA1 and protein levels of cytochrome c oxidase subunit 4. These results suggested that irisin acted partially independently of the PGC‑1α signaling pathway to regulate mitohormesis imbalance due to oxidative stress and maintain energy metabolism by improving mitochondrial structure.

Keywords: H9c2; PGC‑1α; irisin; mitohormesis; reactive oxygen species.

Figure 1.

Irisin improves H₂O₂-induced oxidation modeling. (A) Cardiomyocyte viability was measured using MTT assay. MDA measurement in H9c2 cells treated with (B) H₂O₂ and (C) irisin and 40 µM H₂O₂. *P<0.05, **P<0.01 vs. 0. MDA, malondialdehyde; OD, optical density.

Figure 2.

Detection of MMP and ROS in H9c2 cells by flow cytometry. MMP (A) control, (B) H₂O₂ and (C) H₂O₂/irisin group. (D) MMP depolarization. ROS (E) control, (F) H₂O₂ and (G) H₂O₂/irisin. (H) Median ROS content in H9c2 cardiomyocytes. **P<0.01 vs. control; ^#P<0.05, ^##P<0.01 vs. H₂O₂. MMP, mitochondrial membrane potential; ROS, reactive oxygen species.

Figure 3.

H₂O₂ induced oxidative stress in H9c2 cardiomyocytes, mitohormesis-related factors were expressed at the transcriptional and protein levels after the addition of irisin. The housekeeping gene, β-tubulin, was used to normalize the expression level, and compared to the control or H₂O₂ group. (A) OPA1 mRNA-related expression. (B) DRP1 mRNA-related expression. (C) Western blot analysis. (D) The results of the gray-scale analysis are also shown which are expressed as a percentage (%) of β-tubulin. *P<0.05 vs. control; ^#P<0.05 vs. H₂O₂. (S-)OPA1, (short-form) optic atrophy 1; DRP1, dynamin-related protein 1; MFN2, mitofusin 2.

Figure 4.

Small interfering RNAs interfere with PGC-1α mRNA. *P<0.05 vs. Seq.2 NC, negative control; Seq, sequence. PGC, peroxisome proliferator-activated receptor-γ coactivator.

Figure 5.

Detection of MMP and ROS content in H9c2 cardiomyocytes following interference with PGC-1α. MMP (A) NC, (B) siRNA and (C) siRNA/irisin group. (D) MMP depolarization. ROS (E) NC, (F) siRNA and (G) siRNA/irisin group. (H) Median ROS content in H9c2 cells. *P<0.05, **P<0.01 vs. NC; ^#P<0.05, ^##P<0.01 vs. siRNA. MMP, mitochondrial membrane potential; ROS, reactive oxygen species; si, small interfering; NC, negative control; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1 alpha.

Figure 6.

After interfering with peroxisome proliferator-activated receptor-γ coactivator-1α expression in H9c2 cardiomyocytes, mitohormesis-associated factors were expressed at the transcriptional and protein levels after the addition of irisin. (A) OPA1 and (B) COX4 mRNA levels. (C) Western blot analysis and (D) semi-quantification. *P<0.05, **P<0.01 vs. NC; ^#P<0.05 vs. siRNA. NC, negative control; siRNA, small interfering RNA; OPA1, optic atrophy 1; COX4, cytochrome c oxidase subunit 4; UCP2, uncoupling protein 2.