Irisin attenuates type 1 diabetic cardiomyopathy by anti-ferroptosis via SIRT1-mediated deacetylation of p53

Abstract

Background: Diabetic cardiomyopathy (DCM) is a serious complication in patients with type 1 diabetes mellitus (T1DM), which still lacks adequate therapy. Irisin, a cleavage peptide off fibronectin type III domain-containing 5, has been shown to preserve cardiac function in cardiac ischemia-reperfusion injury. Whether or not irisin plays a cardioprotective role in DCM is not known.

Methods and results: T1DM was induced by multiple low-dose intraperitoneal injections of streptozotocin (STZ). Our current study showed that irisin expression/level was lower in the heart and serum of mice with STZ-induced TIDM. Irisin supplementation by intraperitoneal injection improved the impaired cardiac function in mice with DCM, which was ascribed to the inhibition of ferroptosis, because the increased ferroptosis, associated with increased cardiac malondialdehyde (MDA), decreased reduced glutathione (GSH) and protein expressions of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), was ameliorated by irisin. In the presence of erastin, a ferroptosis inducer, the irisin-mediated protective effects were blocked. Mechanistically, irisin treatment increased Sirtuin 1 (SIRT1) and decreased p53 K382 acetylation, which decreased p53 protein expression by increasing its degradation, consequently upregulated SLC7A11 and GPX4 expressions. Thus, irisin-mediated reduction in p53 decreases ferroptosis and protects cardiomyocytes against injury due to high glucose.

Conclusion: This study demonstrated that irisin could improve cardiac function by suppressing ferroptosis in T1DM via the SIRT1-p53-SLC7A11/GPX4 pathway. Irisin may be a therapeutic approach in the management of T1DM-induced cardiomyopathy.

Keywords: Ferroptosis; Irisin; SIRT1; Type 1 diabetic cardiomyopathy; p53.

Fig. 1

The heart tissue and serum levels of irisin are decreased in type 1 diabetic mice. A A schematic diagram showing the treatments of the mice. B Western blot of serum samples obtained from type 1 diabetic mice and control non-diabetic mice (10 μL serum per lane). Ponceau S staining Albumin served as loading control (n = 8 per group). C Irisin protein expression in the heart was determined by Western blot. GAPDH served as the loading control. Data are expressed as the mean ± SD. Student’s t-test.^*P < 0.05, ^**P < 0.01. STZ streptozotocin

Fig. 2

Irisin improves cardiac function and ameliorates cardiac remodeling in type 1 diabetic mice. The mice were treated with STZ for 5 days to induce diabetic cardiomyopathy; similarly managed mice injected vehicle without STZ served as controls. Then the control and diabetic mice were intraperitoneally injected with irisin (10 μg/kg body weight/day) or vehicle for 4 consecutive weeks. A Representative M-mode echocardiography (A1) from a mouse in each of the four groups of mice. Quantification of LVEF% (A2), LVFS% (A3), LVIDd (A4), and LVIDs (A5) in the indicated groups (n = 8 per group). B The histopathological changes of myocardial tissues using H&E staining from a mouse in each of the four groups of mice (Scale bar = 50 μm; n = 6 per group). C. Representative Masson staining (C1) and quantification (C2) of the fibrotic area from a mouse in each of the four groups of mice (Scale bar = 50 μm; n = 6 per group). The serum levels of inflammation-associated biomarkers, such as TNF-α (D), IL-1β (E), and IL-6 (F) were shown (n = 8 per group). Data are presented as the mean ± SD.One-way ANOVA, and Bonferroni’s post-hoc test. ^*P < 0.05, ^**P < 0.01. STZ streptozotocin, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, LVIDd left ventricular internal diameter at end-diastole, and LVIDs left ventricular internal diameter at end-systolic, H&E hematoxylin and eosin, TNF- α tumor necrosis factor-α, IL-1β interleukin-1β, and IL-6 interleukin-6

Fig. 3

Irisin attenuates ferroptosis in the heart of type 1 diabetic mice. A MDA, B LPO, C GSH, and D GSH/GSSH ratio in cardiac tissue lysates were measured from the four groups of mice (n = 6 per group). E Representative images (E1) and the statistical results (E2) of the iron staining (Scale bars = 20 μm; n = 6 per group). F Representative Western blots (F1) and quantification (F2) showing myocardial protein levels of SLC7A11 and GPX4 in the four groups of mice (n = 6 per group). G Representative TEM images (G1) showing the mitochondria of cardiac tissue from a mouse in each of the four groups of mice (Scale bars = 500 nm) and (G2) The corresponding relative Flameng scores were shown (n = 6 per group). Data are expressed as the mean ± SD.One-way ANOVA, and Bonferroni’s post-hoc test. *P < 0.05, **P < 0.01. STZ streptozotocin, MDA malondialdehyde, LPO lipid peroxides, GSH reduced glutathione, GSSG oxidized glutathione, TEM transmission electron microscopy, SLC7A11 solute carrier family 7 member 11, and GPX4 glutathione peroxidase 4

Fig. 4

Erastin abolishes the protective effect of irisin on diabetic cardiomyopathy. Type 1 diabetic mice were treated with irisin (10 μg/kg body weight/day) in the presence or absence of the ferroptosis inducer erastin (40 mg/kg body weight/day, 3 consecutive days for 4 weeks). A1. Representative echocardiographic images from a mouse in each of the five groups of mice. Quantification of (A2) LVEF%, (A3) LVFS%, (A4) LVIDd, and (A5) LVIDs are shown (n = 6 per group). B Representative Masson staining images of heart tissues from a mouse in each of the five groups of mice: (B1) quantification (B2) of the fibrotic area (Scale bar = 50 μm; n = 6 per group). C Representative TEM images from the heart of a mouse in each of the five groups of mice (C1) and corresponding relative Flameng scores (Bottom) (C2) of the mitochondria in myocardial tissues of each group (Scale bars = 500 nm; n = 6 per group). Data are presented as the mean ± SD. One-way ANOVA, and Bonferroni’s post-hoc test. ^*P < 0.05, ^**P < 0.01. STZ streptozotocin, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, LVIDd left ventricular internal diameter at end-diastole, and LVIDs left ventricular internal diameter at end-systole, TEM transmission electron microscopy

Fig. 5

Irisin inhibits HG-induced cell damage and ferroptosis in H9C2 cells. H9C2 cells were incubated with HG (35 mmol/L) for 24 h in the presence or absence of irisin (10–40 nmol/L) before physiological/biochemical assessment. The cell viability of H9C2 cells was determined by the CCK-8 Kit (A) and LDH release assay (B) (n = 6 per group). MDA (C), GSH (D), and GSH/GSSG ratio (E) in H9C2 cells were determined using the relevant kits (n = 6 per group). F Western blots of the proteins SLC7A11 and GPX4 in H9C2 cells. Representative blots from a mouse in each of the four groups of mice (F1) and quantitative analysis of SLC7A11 and GPX4 (F2) are shown (n = 4 per group). GAPDH served as the loading control. G Representative images of fluorescence probe for ROS from H9C2 cells in each of the four groups of H9C2 cells (G1) with quantification (G2) (Scale bar = 50 μm; n = 6 per group). H1 Representative fluorescent images of FerroOrange staining in H9C2 cells in each of the four groups of H9C2 cells (Scale bar = 50 μm). H2 The quantitative results of FerroOrange staining are shown (n = 6 per group). Data are presented as the mean ± SD.One-way ANOVA, and Bonferroni’s post-hoc test. ^*P < 0.05, ^**P < 0.01. HG high glucose, MDA malondialdehyde, GSH reduced glutathione, GSSG oxidized glutathione, SLC7A11 solute carrier family 7 member 11, and GPX4 glutathione peroxidase 4, and LDH lactate dehydrogenase

Fig. 6

Irisin down-regulates p53 to promote the expression of SLC7A11 and consequently inhibits ferroptosis in H9C2 cells. A The effect of the irisin on the time course of p53 degradation in HG-treated H9C2 cells. To inhibit protein synthesis, the cells were treated with 100 μmol/L CHX for the indicated time (n = 4 per group). B Representative fluorescent images of FerroOrange staining in each of the four groups of H9C2 cells (B1) and the quantification (B2) in H9C2 cells (Scale bar = 50 μm; n = 6). C1 Immunoblots of SLC7A11, GPX4, and GAPDH from each of the five groups of H9C2 cells. C2 Quantification of the SLC7A11 and GPX4 bands. All results were normalized to the expression level of GAPDH (n = 4 per group). D Representative Western blotting images (D1) with quantification (D2) from each of the five groups of H9C2 cells showing p53 K382 acetylation andp53 protein levels (n = 4 per group). Data are expressed as the mean ± SD. One-way ANOVA, and Bonferroni’s post-hoc test. ^*P < 0.05, ^**P < 0.01. HG high glucose, p53 tumor suppressor p53, SLC7A11 solute carrier family 7 member 11, and GPX4 glutathione peroxidase 4, CHX cycloheximide, and Ac acetylation

Fig. 7

Irisin attenuates ferroptosis through the SIRT1/p53 Pathway. A SIRT1 protein expression in the heart was detected by western blotting (A1) and quantitative analysis (A2), All results were normalized to the expression level of GAPDH (n = 4 per group). B Representative Western blots in each of the four groups of H9C2 cells (B1) and quantification (B2) of the protein levels of SIRT1 in H9C2 cells, All results were normalized to the expression level of GAPDH (n = 4 per group). C Western blot images (C1) and quantification (C2) in each of the four groups of p53 K382 acetylation and p53 protein in H9C2 cells (n = 4 per group). D1 Representative fluorescent images of FerroOrange staining in each of the five groups of H9C2 cells (scale bar = 50 μm) and (D2) quantification of the FerroOrange fluorescence intensity (n = 6 per group). E1 Representative images of M-mode echocardiography of a mouse in each of the four groups of mice. Statistical analyses of (E2) LVEF%, (E3) LVFS%, (E4) LVIDd, and (E5) LVIDs in the indicated groups (n = 6 per group). F Representative TEM images of the heart tissue from a mouse in each of the four groups of mice (F1) and corresponding relative Flameng scores (Bottom) (F2) for mitochondria cardiac tissue (Scale bars = 500 nm; n = 5 per group). G Representative images of iron staining of the heart from a mouse in each of the four groups of mice (G1) and quantitative analysis (G2) of iron deposition in the four groups of mice (Scale bars = 20 μm; n = 5 per group). H p53 K382 acetylation levels and p53 protein levels were determined by Western blotting of the heart from a mouse in each of the four groups of mice (n = 4). Data are expressed as the mean ± SD. One-way ANOVA, and Bonferroni’s post-hoc test. ^*P < 0.05, ^**P < 0.01. HG high glucose, SIRT1 Sirtuin 1, STZ streptozotocin, LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, LVIDd left ventricular internal diameter at end-diastole, LVIDs left ventricular internal diameter at end-systole, and TEM transmission electron microscopy

Fig. 8

Schematic diagram summarizing the protective effects of irisin treatment in type 1 diabetic cardiomyopathy through anti-ferroptosis via induction of the SIRT1-p53-SLC7A11/GPX4 pathway. High glucose, a key pathogenic factor of DCM of the type 1 diabetic model, increases intracellular Fe²⁺ and lipid peroxidation, along with GSH depletion and SLC7A11(cystine/glutamate antiporter)/GPX4 inhibition, implying a role for ferroptosis in the pathogenesis of T1DM. Irisin suppresses ferroptosis and alleviates cardiac remodeling and dysfunction via activation of the SIRT1-p53-SLC7A11/GPX4 pathway. DCM, diabetic cardiomyopathy, SLC7A11 solute carrier family 7 member 11, GPX4 glutathione peroxidase 4, Ac acetylation, GSH reduced glutathione, and SIRT1 Sirtuin 1