Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

Abstract

Adiponectin (ApN) is a hormone with potent effects on various tissues. We previously demonstrated its ability to counteract Duchenne muscular dystrophy (DMD), a severe muscle disorder. However, its therapeutic use is limited. AdipoRon, an orally active ApN mimic, offers a promising alternative. While cardiomyopathy is the primary cause of mortality in DMD, the effects of ApN or AdipoRon on dystrophic hearts have not been investigated. Our recent findings demonstrated the significant protective effects of AdipoRon on dystrophic skeletal muscle. In this study, we investigated whether AdipoRon effects could be extended to dystrophic hearts. As cardiomyopathy develops late in mdx mice (DMD mouse model), 14-month-old mdx mice were orally treated for two months with AdipoRon at a dose of 50 mg/kg/day and then compared with untreated mdx and wild-type (WT) controls. Echocardiography revealed cardiac dysfunction and ventricular hypertrophy in mdx mice, which were fully reversed in AdipoRon-treated mice. AdipoRon also reduced markers of cardiac inflammation, oxidative stress, hypertrophy, and fibrosis while enhancing mitochondrial biogenesis via ApN receptor-1 and CAMKK2/AMPK pathways. Remarkably, treated mice also showed improved skeletal muscle strength and endurance. By offering protection to both cardiac and skeletal muscles, AdipoRon holds potential as a comprehensive therapeutic strategy for better managing DMD.

Keywords: AMPK; Duchenne muscular dystrophy; adiponectin; cardiomyopathy; fibrosis; inflammation; mitochondria.

Figure 1

Effects of AdipoRon treatment on cardiac dysfunction in adult mdx mice. Cardiac dimensions and function were analysed in vivo by transthoracic echocardiography in mice from the three groups. (A) End-systolic volume (µL). (B) End-diastolic volume (µL). (C) Fractional shortening (%). (D) Heart rate (bpm). (E) Ejection fraction (%). (F) Body weight (g). (G) Left ventricular mass (mg). (H) Left ventricular mass over body weight (LVmass/BW) (mg/g). Data are means ± SD; n = 7–8 mice per group for all tests. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05 vs. WT mice. # p < 0.05 vs. mdx mice.

Figure 2

Effects of AdipoRon treatment on cardiac muscle inflammation and stress. mRNA levels of (A) TNFα and (B) IL-1β, two major inflammatory genes. (C) mRNA levels of IL-10, an anti-inflammatory gene. (D) mRNA levels of PRDX3, an oxidative stress marker. mRNA levels were normalised to cyclophilin, and the following ratios are presented as relative expressions to WT values. ELISA assays were used to quantify the levels of (E) TNFα and (F) IL-1β, two major inflammatory cytokines, (G) IL-10, a strong anti-inflammatory cytokine, and (H) HNE, a lipid peroxidation product. For ELISAs, absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. *** p < 0.001, **** p < 0.0001 vs. WT mice. ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Figure 3

Effects of AdipoRon treatment on cardiac muscle inflammation and stress. (A) Immunohistochemistry was performed on cardiac muscle sections with specific antibodies directed against two pro-inflammatory cytokines (TNFα and IL-1β) and two oxidative stress markers (PRDX3 and HNE). Scale bar = 50 μm. Quantification of (B) TNFα, (C) IL-1β, (D) PRDX3, and (E) HNE. For each immunolabelling of (A), the percentage of DAB deposit areas was calculated in cardiac muscle sections. The subsequent ratios are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. ## p < 0.001, ### p < 0.001 vs. mdx mice.

Figure 4

Effects of AdipoRon treatment on cardiac muscle fibrosis and hypertrophy. (A) Picro-Sirius Red staining. Scale bar = 200 µm. (B) Quantification of Picro-Sirius Red staining. mRNA levels of (C) αSMA and (D) TGF-β1, two markers of fibrosis. ELISA assays were used to quantify (E) TGF-β and (F) the active phosphorylated form of SMAD2 (P¬SMAD2), a transcription factor mainly involved in TGF-β signalling. mRNA levels of (G) BNP and (H) ANP, two markers of hypertrophy. (I) ELISA assay was also used to quantify the levels of ANP. The percentage of stained areas was calculated in cardiac muscle sections, and the subsequent ratios are presented as relative expressions to WT values. mRNA levels were normalised to cyclophilin, and the subsequent ratios were presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Figure 5

Effects of AdipoRon treatment on ApN receptors and signalling in the dystrophic cardiac muscle. mRNA levels of (A) AdipoR1 and (B) AdipoR2, adiponectin main receptors. ELISA assays were used to quantify (C) AdipoR1 and (D) AdipoR2. (E) The ratio of AdpoR1 over AdipoR2 mRNA levels was calculated within the cardiac muscle. ELISA assays were used to quantify (F) the active phosphorylated form of AMPKα (P-AMPK), (G) calcium/calmodulin-dependent protein kinase 2 (CAMKK2), and (H) peroxisome proliferator-activated receptor alpha (PPARα), ApN/AdipoRon, main signalling pathways in muscle. (I) mRNA levels of PGC-1α. ELISA assays were used to quantify (J) PGC-1α, (K) the active phosphorylated form of the p65 subunit of NF-κB (P-p65), a transcription factor mainly involved in inflammation, and (L) utrophin A (UTRN), a dystrophin analogue. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expression to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Figure 6

Effects of AdipoRon treatment on cardiac muscle oxidative capacity, injury, and overall muscle function. mRNA levels of (A) ERRα and (B) mtTFA, two markers of mitochondrial biogenesis. ELISA assay was used to quantify (C) TOMM20, a marker of mitochondrial content. (D) Wire test where mice hanging time was recorded (s). (E) Fore-limb grip test and (F) fore- and hind-limb grip test, measuring muscle strength expressed in Gram-force relative to body weight (gf/gBW). (G) Treadmill running exercise, where the total distance covered on the third day was measured (m). (H) CK and (I) LDH plasma activities assessing muscle injury and expressed as IU/L. (J) ELISA assay was used to quantify the active phosphorylated form of RIP (P-RIP), an important regulator of cellular stress that triggers a regulated pathway for necrotic cell death called necroptosis. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all ex vivo experiments. Data are means ± SD; n = 7–8 mice per group for all in vivo functional tests. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.