Aim: Myotonic dystrophy type 1 (DM1) is the second most common muscular dystrophy after Duchenne and is the most prevalent muscular dystrophy in adults. DM1 patients that participate in aerobic exercise training experience several physiological benefits concomitant with improved muscle mitochondrial function without alterations in typical DM1-specific disease mechanisms, which suggests that correcting organelle health is key to ameliorate the DM1 pathology. However, our understanding of the molecular mechanisms of mitochondrial turnover and dynamics in DM1 skeletal muscle is lacking. Methods: Skeletal muscle tissue was sampled from healthy and DM1 mice under sedentary conditions and at several recovery time points following an exhaustive treadmill run. Results: We demonstrate that DM1 patients exhibit an imbalance in the transcriptional apparatus for mitochondrial turnover and dynamics in skeletal muscle. Additionally, DM1 mice displayed elevated expression of autophagy and mitophagy regulators. A single dose of exercise successfully enhanced canonical exercise molecular pathways and skeletal muscle mitochondrial biogenesis despite failing to alter the cellular pathology in DM1 mice. However, treadmill running stimulated coordinated organelle fusion and fission signaling, as well as improved alternative splicing of Optic atrophy 1. Exercise also evoked autophagy and mitophagy pathways in DM1 skeletal muscle resulting in the normalized expression of autophagy-and lysosome-related machinery responsible for the clearance of dysfunctional organelles. Conclusion: Collectively, our data indicate that mitochondrial dynamics and turnover processes in DM1 skeletal muscle are initiated with a single dose of exercise, which may underlie the adaptive benefits previously documented in DM1 mice and patients.
Duchenne muscular dystrophy (DMD) is a life‐limiting neuromuscular disorder characterized by muscle weakness and wasting. Previous proof‐of‐concept studies demonstrate that the dystrophic phenotype can be mitigated with the pharmacological stimulation of AMP‐activated protein kinase (AMPK). However, first‐generation AMPK activators have failed to translate from bench to bedside due to either their lack of potency or toxic, off‐target effects. The identification of safe and efficacious molecules that stimulate AMPK in dystrophic muscle is of particular importance as it may broaden the therapeutic landscape for DMD patients regardless of their specific dystrophin mutation. Here, we demonstrate that a single dose of the next generation, orally‐bioactive AMPK agonist MK‐8722 (MK) to mdx mice evoked skeletal muscle AMPK and extensive downstream stimulation within 12 h post‐treatment. Specifically, MK elicited a gene expression profile indicative of a more disease‐resistant slow, oxidative phenotype including increased peroxisome proliferator‐activated receptor ɣ coactivator‐1⍺ activity and utrophin levels. In addition, we observed augmented autophagy signaling downstream of AMPK, as well as elevations in critical autophagic genes such as Map1lc3 and Sqstm1 subsequent to the myonuclear accumulation of the master regulator of the autophagy gene program, transcription factor EB. Lastly, we show that pharmacological AMPK stimulation normalizes the expression of myogenic regulatory factors and amends activated muscle stem cell content in mdx muscle. Our results indicate that AMPK activation via MK enhances disease‐mitigating mechanisms in dystrophic muscle and prefaces further investigation on the chronic effects of novel small molecule AMPK agonists.
The neuromuscular junction (NMJ) is the electrochemical signaling apparatus between an ⍺‐motoneuron and the skeletal muscle fibers that it innervates. AMP‐activated protein kinase (AMPK) regulates neuromuscular phenotype. However, the precise role of this kinase at the NMJ has yet to be elucidated. The purpose of the current study was to examine the role of skeletal muscle AMPK on NMJ biology across the lifespan. We utilized 3‐ and 12‐month‐old (mo) skeletal muscle‐specific AMPKβ1β2 knock out (mKO, n = 10) and wild‐type (WT, n = 10) mice, as well as 22 mo WT (n = 3) animals. The extensor digitorum longus (EDL) and soleus (SOL) muscles were examined to represent fast glycolytic and slow oxidative tissues, respectively. Utilizing confocal microscopy, we acquired 20‐25 NMJ images per muscle to conduct 3D NMJ morphology analyses. Contralateral EDL and SOL muscles were also collected for immunofluorescence staining and muscle innervation determination. Quantitative real‐time PCR was employed to examine genes critical for NMJ maintenance and remodeling in tibialis anterior (TA) muscles. Presynaptic axonal blebbing and sprouting was observed more frequently (p < 0.05) in the EDL and SOL muscles of aged WT animals relative to their younger counterparts. Additionally, the proportion of dysmorphic axons was greater (p < 0.05) in mKO animals compared with age‐matched WT mice. As expected, there was a significant main effect of age on the total number of fragmented and ectopic NMJs in both genotypes. mKO animals displayed a greater (p < 0.05) number of dysmorphic NMJs than their age‐matched WT littermates, which suggests that AMPK is required for the development and maintenance of NMJ structure. Indeed, the number of NMJs positive for postsynaptic abnormalities in the EDL muscles of 12 mo mKO animals was significantly greater than 22 mo WT mice. Fiber type grouping was significantly higher in EDL and SOL muscles of 22 mo WT animals relative to their younger counterparts. Statistical trends (p = 0.09) were also observed towards greater fiber type grouping and increased muscle fibers co‐expressing multiple myosin heavy chain isoforms in only the SOL muscles of mKO animals relative to their age‐matched WT controls, which suggests that AMPK elicits muscle‐specific denervation/reinnervation cycling. mRNA expression of the acetylcholine receptor gamma subunit was significantly greater in mKO mice relative to WT animals. Interestingly, additional NMJ transcripts, such as agrin, rapsyn, docking protein 7, and fibroblast growth factor binding protein 1, were significantly lower in mKO animals relative to their WT littermates. Collectively, these data suggest that the absence of skeletal muscle AMPK accelerates age‐associated changes at the NMJ in more glycolytic muscle fibers, potentially due to a greater ability for reinnervation in oxidative muscles. These results reveal a novel role for AMPK in the maintenance and remodeling of the NMJ during aging.
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