Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/−) mice

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  Figure 1. miR-25 is significantly upregulated in DMD animal models and cardiac function is compromised in aged mdx/utrn (+/−) KO mice (A) Western blots were performed against SERCA2a, p-PLN, PLN, and GAPDH in the GRMD canine DMD model, kindly provided by Dr. Dongsheng Duan from the University of Missouri. (B) Expression levels of miR-25 were assessed using qRT-PCR analysis. Data are presented as mean ± SD (n = 2 for each group). (C) Western blots were conducted against SERCA2a, p-PLN, PLN, and GAPDH in the mdx/utrn haploinsufficient DMD mouse model (n = 3 for each group). (D) In addition, qRT-PCR analysis was used to measure mRNA levels of pre-miR-25, and mature miR-25 (n = 3 for each group). (E) The figure displays WT, mdx/utrn haploinsufficient (+/−), and mdx/utrn double KO (−/−) mouse models, with body weight measured at 2 months of age (F) due to high mortality of dko mice. Cardiac function was assessed in the mdx/utrn (+/−) haploinsufficient model using echocardiography over a 1-year period. Fractional shortening was used to represent cardiac function (G). For (F) and (G), n = 15 for each group. In the western blot analysis, 15 μg of proteins was loaded into each well. Data are…

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  Figure 2. AAV9 miR-25 TuD transfer improved cardiac function in aged mdx/utrn (+/−) mice (A) Cardiomyocytes isolated from WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD-treated mdx/utrn (+/−) mice were assessed for Ca2+ amplitude, Tau, peak shortening, and maximal rate of contraction and relaxation. AAV9 miR-25 TuD treatment demonstrated enhanced cardiomyocyte Ca2+ handling and contractility (n = 30 for each group). (B) Echocardiograms of WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD-treated mdx/utrn (+/−) mice were performed after 8 weeks. Notably, AAV9 miR-25 TuD transfer led to a significant improvement in cardiac fractional shortening (n = 15 for each group). (C) Invasive hemodynamic measurements, including ESPVR, and responses to dobutamine through Pmax, Pdt, and HR were assessed in WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD-treated mdx/utrn (+/−) mice. AAV9 miR-25 TuD transfer resulted in a substantial improvement in chronotropic and inotropic competency (n = 5 for each group). (D) Survival analysis of WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD-treated mdx/utrn (+/−) mice demonstrated a significantly enhanced survival…

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  Figure 3. AAV9 miR-25 TuD restores Ca2+ regulating proteins and reduces cardiac hypertrophic markers (A) The expression levels of SERCA2a, phospholamban phosphorylation, RyR, and NCX were analyzed using western blotting in WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD mice. Compared with WT, SERCA2a, and phospholamban phosphorylation levels were significantly downregulated in the AAV9 control group, while both levels exhibited a significant increase with miR-25 decoy transfer. No statistically significant changes were observed in RyR and NCX expression levels (n = 5 for each group). (B) The expression levels of α-SKA, ANF, BNP, and β-MHC were analyzed using qRT-PCR in WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD mice (n = 5 for each group). In the western blot analysis, 20 μg of proteins was loaded into each well. For (A) and (B), the data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.005.

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  Figure 4. AAV9 miR-25 TuD inhibits cardiac MAPK signaling and pro-apoptotic pathways (A) The expression levels of MAPK proteins were analyzed by western blotting in WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD mice using the indicated antibodies (n = 5 for each group). (B) The expression levels of apoptosis-related proteins were analyzed by western blotting in WT, AAV9 control treated mdx/utrn (+/−), and AAV9 miR-25 TuD mice using the indicated antibodies (n = 5 for each group). In the western blot analysis, 25 μg of proteins was loaded into each well. For (A) and (B), the data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.005.

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  Figure 5. Systemic AAV9 miR-25 TuD transfer decreased cardiac muscle fibrosis in aged mdx/utrn (+/−) mice Histological analysis was conducted on frozen sections of cardiac muscles obtained from WT, AAV9 control, and AAV9 miR-25 TuD-treated mdx/utrn (+/−) mice. (A) Representative images of hematoxylin and eosin (H&E) staining depict cardiac muscles from WT, AAV9 control, or AAV9 miR-25 TuD-treated mdx/utrn (+/−) mice. The scale bar indicates 100 μm. (B) Representative images of picrosirius red staining illustrate cardiac muscles from WT, AAV9 control, or AAV9 miR-25 TuD-treated mdx/utrn (+/−) mice. The scale bar indicates 100 μm. (C) Quantification of cardiomyocyte size was performed based on H&E staining. (D) Cardiac fibrosis was quantified using picrosirius red staining. (E) Ultrastructural images were obtained using transmission electron microscopy. In the western blot analysis, 15 μg of proteins was loaded into each well. For (C), data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.005 (n = 3 for each group).

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  Figure 6. Smad7 is identified as an essential target of miR-25 in cardiac fibrosis (A) The alignment results between miR-25 and Smad7 3′-UTR provide insights into the specific binding locations within the genome sequence. (B) Luciferase reporter activity was measured using Smad7 3′UTR in a dose-dependent manner with miR-25 mimic. Additionally, a deletion mutation in the miR-25 binding site of Smad7 3′UTR was applied to validate the luciferase activity. Protein levels of SERCA2a and Smad7 were examined by western blot analysis after AAV9 pre-miR-25 delivery in WT mice (C), or lentiviral shSmad7 vector delivery (D). (E) AAV9 miR-25 TuD was injected into mdx/utrn (+/−) mice, with or without the lentiviral shSmad7 vector, and protein levels of SERCA2a and Smad7 were detected by western blot analysis. (F) Following euthanasia, mouse hearts were cryosectioned and subjected to Masson’s trichrome staining for the evaluation of cardiac fibrosis. In the western blot analysis, 15 μg of proteins was loaded into each well. For (A)–(F), the data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.005 (n = 3 for each group).

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  Figure 7. AAV9 miR-25 TuD reduces fibrosis by inhibiting TGF-β signaling pathways by restoring Smad7 expression Western blotting was performed to evaluate the expression of Smad7 and GAPDH in the canine (GRMD) models (A), with n = 2 for each group. Similarly, in the mdx/utrn (+/−) mouse models, western blot analysis was conducted to assess Smad7 and GAPDH expression (B), with n = 5 for each group. (C) The expression levels of pre- and mature miR-25 were quantified using qRT-PCR in mdx/utrn (+/−) mice treated with AAV9 control or AAV9 miR-25 TuD, with n = 5 for each group. (D) Furthermore, qRT-PCR analysis was employed to analyze the mRNA expression levels of fibrosis markers, such as fibronectin, galectin-3, and TGF-β1, in comparison with the WT group. In the western blot analysis, 20 μg of proteins was loaded into each well. For (A)–(D), the data represent the mean ± SD. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001.

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  Figure 8. Direct intramuscular injection of AAV9 miR-25 TuD improves muscle performance (A) The treadmill exhaustion test involved calculating the average running distance on the treadmill during the exercise program. The bars represent the mean values for each group, with error bars indicating the standard deviation (n = 8 for each group). (B) Representative histological images with H&E and picrosirius red staining depict mononuclear infiltrates and tissue fibrosis. The scale bar indicates 100 μm. (C) Ultrastructural images were obtained using transmission electron microscopy. (D) Representative western blot images evaluating markers such as SERCA2a for cardiac calcium pump, vimentin, fibronectin, and gelsolin for fibrosis, TGF-β1, p-Smad2, total Smad2/3, and Smad7 for TGF-β signaling molecules, and α-SMA for myofibroblast (n = 3 for each group). (E) Pre- and mature forms of miR-25 were measured before and after gene transfer (n = 5 for each group). The mRNA expressions for (F) anti-inflammatory and (G) pro-inflammatory cytokines were measured using the qRT-PCR method in skeletal muscles (n = 5 for each group). In the western blot analysis, 20 μg of proteins was loaded into each well. For…