Pharmacological and physiological activation of AMPK improves the spliceopathy in DM1 mouse muscles

Abstract

Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a triplet repeat expansion in the 3' untranslated region of dystrophia myotonica protein kinase mRNAs. Mutant mRNAs accumulate in the nucleus of affected cells and misregulate RNA-binding proteins, thereby promoting characteristic missplicing events. However, little is known about the signaling pathways that may be affected in DM1. Here, we investigated the status of activated protein kinase (AMPK) signaling in DM1 skeletal muscle and found that the AMPK pathway is markedly repressed in a DM1 mouse model (human skeletal actin-long repeat, HSALR) and patient-derived DM1 myoblasts. Chronic pharmacological activation of AMPK signaling in DM1 mice with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) has multiple beneficial effects on the DM1 phenotype. Indeed, a 6-week AICAR treatment of DM1 mice promoted expression of a slower, more oxidative phenotype, improved muscle histology and corrected several events associated with RNA toxicity. Importantly, AICAR also had a dose-dependent positive effect on the spliceopathy in patient-derived DM1 myoblasts. In separate experiments, we also show that chronic treatment of DM1 mice with resveratrol as well as voluntary wheel running also rescued missplicing events in muscle. Collectively, our findings demonstrate the therapeutic potential of chronic AMPK stimulation both physiologically and pharmacologically for DM1 patients.

Figure 1

AMPK signaling is decreased in DM1 mouse muscles and human myoblasts. (A) Representative western blots of AMPK, phospho-AMPK (Thr172) and PGC-1α in WT (FVB/N) and DM1 (HSA^LR) EDL muscles. (B) Quantifications of expression levels normalized to their respective β-actin controls. Phospho-AMPK was normalized to total AMPK level. N = 4–5 animals per group (9-month-old). (C–D) Representative western blots and quantifications of AMPK and phospho-AMPK (Thr172) over total AMPK in WT and DM1 myoblasts. (N = 2). T-tests were used, and asterisks indicate significance (**P-value ≤ 0.01).

Figure 2

AICAR promotes expression of the slow, oxidative muscle phenotype in DM1 mouse muscle. (A–B) Representative western blots and quantifications of PGC-1α in TA muscles. β-actin was used as a control. N = 3. (C) Representative immunofluorescence of MyHC IIa (red) in EDL muscles. Laminin (green) was used to delimit muscle fibers. Scale bars, 50 μm. (D) Percentage of MyHC I, IIa and IIb positive fibers. N = 6. (E) Representative SDH staining in EDL muscles. Scale bars, 200 μm. (F) Percentage of SDH-positive fibers. N = 6. (G–H) Representative western blots and quantifications of OXPHOS proteins (CI-NDUFB8, CIII-UQCRC2 and CV-ATP5A) in TA muscles. β-actin was used as a control. N = 6. T-tests were used, and asterisks indicate significance (*P-value ≤ 0.05, **P-value ≤ 0.01 and ***P-value ≤ 0.001).

Figure 3

AICAR improves DM1 mouse muscle histology. (A) Representative H&E staining of control, saline- and AICAR-treated DM1 EDL muscles. Inserts show mean CSA and CV. Scale bars, 50 μm. (B) CSA frequency. N = 5. (C) Percentage of central nuclei. N = 5–6. T-tests were used, and asterisks indicate significance (ns P-value> 0.05, *P-value ≤ 0.05, **P-value ≤ 0.01 and ***P-value ≤ 0.001).

Figure 4

AICAR reverses CUG^exp mRNA aggregation, MBNL1 sequestration and reduces CUGBP1, Staufen1 and RBM3 levels. (A) Representative FISH showing nuclear RNA foci in EDL muscles. Scale bars, 10 μm. (B) Percentage of nuclei containing RNA foci. N = 5–6. (C) Representative western blots showing CUGBP1, Staufen1, MBNL1 and RBM3 levels in saline- and AICAR-treated DM1 TA muscles. (D) Quantifications of western blots. N = 6. (E) Representative immunofluorescence showing MBNL1 localization. Scale bars, 10 μm. (F) Percentage of nuclei showing MBNL1 aggregates in EDL muscles. N = 6. T-tests were used, and asterisks indicate significance (*P-value ≤ 0.05, **P-value ≤ 0.01 and ***P-value ≤ 0.001).

Figure 5

AICAR corrects the DM1 spliceopathy and expression of CLCN1. (A) Radioactive RT-PCR analysis showing alternative splicing patterns of SERCA1, RyR1, TNNT3 and CLCN1 in control, saline- and AICAR-treated DM1 EDL muscles. (B) Percentages of SERCA1 E22 exclusion, RyR1 E70 exclusion, TNNT3 F inclusion and CLCN1 Ex7a inclusion, with mean ± SEM. N = 3–5. (C) Representative immunofluorescence showing CLCN1 expression and localization in EDL muscles. Scale bars, 50 μm. T-tests were used, and asterisks indicate significance (ns P-value > 0.05 and ***P-value ≤ 0.001).

Figure 6

AICAR prevents CUG^exp aggregation and shifts alternative splicing towards WT conditions in human DM1 myoblasts. (A) Human DM1 myoblasts were treated with an increasing dose of AICAR (0, 0.5, 1 and 2 mm). Untreated WT myoblasts were used as controls. Representative RT-PCR of SERCA1, ZFAND1, MDM4 and GPCPD1 mRNAs. (B) Quantifications of RT-PCRs. N = 3. (C) Representative FISH showing nuclear RNA foci in DM1 myoblasts. (D) Number of RNA foci per nuclei. N = 4. (E–F) Representative western blots and quantifications of AMPK and phospho-AMPK over total AMPK showing activation of AMPK signaling in human DM1 fibroblasts treated with 1 mm AICAR. (N = 2). T-tests were used, and asterisks indicate significance (ns P-value > 0.05, *P-value ≤ 0.05, **P-value ≤ 0.01 and ***P-value ≤ 0.001).

Figure 7

Chronic RSV administration decreases DM1 spliceopathy. 2-month-old DM1 mice were fed with control diet or chow supplemented with RSV (100 mg/kg per day) for 6 weeks. (A) Radioactive RT-PCR analysis showing alternative splicing patterns of SERCA1, RyR1, TNNT3 and CLCN1 in control, DM1 and RSV-treated DM1 TA muscles. (B) Percentages of SERCA1 E22 exclusion, RyR1 E70 exclusion, TNNT3 F inclusion and CLCN1 Ex7a inclusion, with mean ± SEM. N = 3–5. T-tests were used, and asterisks indicate significance (ns P-value > 0.05, *P-value ≤ 0.05, **P-value ≤ 0.01 and ***P-value ≤ 0.001).

Figure 8

Physical activity has a beneficial impact on alternative splicing in DM1 mouse muscle. 4- to 8-month-old DM1 mice were provided with free access to an exercise wheel for 8 weeks. (A) RT-PCR analysis showing alternative splicing patterns of SERCA1, RyR1, TNNT3 and CLCN1 in ‘sedentary’ controls and DM1, and ‘exercised’-DM1 EDL muscles. (B) Percentages of SERCA1 E22 exclusion, RyR1 E70 exclusion, TNNT3 F inclusion and CLCN1 Ex7a inclusion, with mean ± SEM. N = 10–15. T-tests were used, and asterisks indicate significance (*P-value ≤ 0.05, **P-value ≤ 0.01 and ***P-value ≤ 0.001).

Figure 9

Regulation of AMPK signaling in DM1 muscle. AMPK signaling is repressed in DM1 muscle. Pharmacological (AICAR or RSV) or physiological (exercise) activation of the AMPK pathway has a beneficial impact on DM1. It promotes the slower, more oxidative muscle phenotype, and improves DM1 muscle histology by decreasing muscle fiber hypertrophy and central nucleation. It also triggers a decrease in nuclear CUG^exp RNA aggregation, a rescue in RNA-binding misregulation and a correction of alternative splicing towards wild-type conditions.