Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients\' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.

Lacking PTRF (polymerase I and transcript release factor), an essential caveolae component, causes a secondary deficiency of caveolins resulting in muscular dystrophy. The transcriptome responses of different types of muscle fibers and mononuclear cells in skeletal muscle to muscular dystrophy caused by Ptrf deletion have not been explored. Here, we created muscular dystrophy mice by Ptrf knockout and applied single-nucleus RNA sequencing (snRNA-seq) to unveil the transcriptional changes of the skeletal muscle at single-nucleus resolution. 11 613 muscle nuclei (WT, 5838; Ptrf KO, 5775) were classified into 12 clusters corresponding to 11 nuclear types. Trajectory analysis revealed the potential transition between type IIb_1 and IIb_2 myonuclei upon muscular dystrophy. Functional enrichment analysis indicated that apoptotic signaling and enzyme-linked receptor protein signaling pathway were significantly enriched in type IIb_1 and IIb_2 myonuclei of Ptrf KO, respectively. The muscle structure development and the PI3K-AKT signaling pathway were significantly enriched in type IIa and IIx myonuclei of Ptrf KO. Meanwhile, metabolic pathway analysis showed a decrease in overall metabolic pathway activity of myonuclei subtypes upon muscular dystrophy, with the most decrease in type IIb_1 myonuclei. Gene regulatory network analysis found that the activity of Mef2c, Mef2d, Myf5, and Pax3 regulons was enhanced in type II myonuclei of Ptrf KO, especially in type IIb_2 myonuclei. In addition, we investigated the transcriptome changes in adipocytes and found that muscular dystrophy enhanced the lipid metabolic capacity of adipocytes. Our findings provide a valuable resource for exploring the molecular mechanism of muscular dystrophy due to Ptrf deficiency.

Bethlem myopathy (BM) is a disease that is caused by mutations in the collagen VI genes. It is a mildly progressive disease characterized by proximal muscle weakness and contracture of the fingers, the wrist, the elbow, and the ankle. BM is an autosomal dominant inheritance that is mainly caused by dominant COL6A1, COL6A2, or COL6A3 mutations. However, a few cases of collagen VI mutations with bilateral facial weakness and Beevor\'s sign have also been reported. This study presents a 50-year-old female patient with symptoms of facial weakness beginning in childhood and with the slow progression of the disease with age. At the age of 30 years, the patient presented with asymmetrical proximal muscle weakness, and the neurological examination revealed bilateral facial weakness and a positive Beevor\'s sign. Phosphocreatine kinase was slightly elevated with electromyography showing myopathic changes and magnetic resonance imaging (MRI) of the lower limb muscles showing the muscle MRI associated with collagen VI (COL6)-related myopathy (COL6-RM). The whole-genome sequencing technology identified the heterozygous mutation c.6817-2(IVS27)A>G in the COL6A3 gene, which was in itself a novel mutation. The present study reports yet another case of BM, which is caused by the recessive COL6A3 intron variation, widening the clinical spectrum and genetic heterogeneity of BM.

Dysferlinopathies are autosomal recessive muscular dystrophies resulting from defects in DYSF (MIM: 603009), which is located on chromosome 2p13 and encodes the dysferlin protein.
We performed exome sequencing and subsequent trio-based analysis in a family with dysferlinopathy.
We report a young patient presenting with hyperCKemia and mild muscle weakness of the lower limbs. Exome sequencing of the proband revealed a homozygous frameshift mutation, NM_001130987.2:c.1471dupA(p.M491Nfs*15), in DYSF. The father was heterozygous for the mutation and the mother did not carry the mutation, as determined by genetic analyses, exome sequencing of parental samples, and a trio-based analysis. Further analysis revealed that the DYSF gene was not deleted; instead, the entire chromosome 2 of the proband was inherited from the father. Thus, the child had paternal uniparental isodisomy for chromosome 2 (uniparental disomy [UPD]2 pat).
We report the first case of dysferlinopathy caused by paternal isodisomy for chromosome 2. Furthermore, our findings highlight the importance of exome sequencing of the proband and parents and trio analyses in clinical settings, particularly when Mendelian inheritance cannot be confirmed, to identify the presence of UPD and to rule out large pathogenic deletions.

OBJECTIVE: To summarize the skeletal muscle magnetic resonance imaging (MRI) features of the lower limbs in common subtypes of muscular dystrophy (MD) and the experience in the application of MRI in the diagnosis of MD.
METHODS: A total of 48 children with MD who were diagnosed by genetic testing were enrolled as subjects. The muscle MRI features of the lower limbs were analyzed. Cumulative fatty infiltration score was calculated for each subtype, and the correlation of cumulative fatty infiltration score with clinical indices was analyzed for Duchenne muscular dystrophy (DMD).
RESULTS: DMD was characterized by the involvement of the gluteus maximus and the adductor magnus. Becker muscular dystrophy was characterized by the involvement of the vastus lateralis muscle. Limb-girdle muscular dystrophy was characterized by the involvement of the adductor magnus, the vastus intermedius, the vastus medialis, and the vastus lateralis muscle. For DMD, the cumulative fatty infiltration score of the lower limb muscles was significantly correlated with age, course of the disease, muscle strength, and motor function (P<0.05), while it was not significantly correlated with the serum creatine kinase level (P>0.05).
CONCLUSIONS: Different subtypes of MD have different MRI manifestations, and MRI may help with the diagnosis and assessment of MD.
目的: 总结肌营养不良（muscular dystrophy，MD）常见亚型下肢肌肉骨骼肌磁共振成像（magnetic resonance imaging，MRI）特点，积累应用MRI协助诊断MD的经验。方法: 选取经基因检测确诊的48例MD患儿为研究对象，分析其下肢肌肉MRI特点，统计各亚型肌肉脂肪浸润累计评分，并分析Duchenne型肌营养不良脂肪浸润累计评分与临床指标的相关性。结果: Duchenne型肌营养不良以臀大肌、大收肌受累为著，Becker型肌营养不良以股外侧肌受累为著，肢带型肌营养不良以大收肌、股中间肌、股内侧肌、股外侧肌受累为著。Duchenne型肌营养不良下肢肌肉脂肪浸润累计评分与年龄、病程、肌肉力量及运动功能明显相关（P<0.05），与血清肌酸激酶无相关性（P>0.05）。结论: 不同亚型MD的MRI表现特点不同，MRI有助于MD的诊断与病情评估。.

Impaired biomolecules and cellular organelles are gradually built up during the development and aging of organisms, and this deteriorating process is expedited under stress conditions. As a major lysosome-mediated catabolic process, autophagy has evolved to eradicate these damaged cellular components and recycle nutrients to restore cellular homeostasis and fitness. The autophagic activities are altered under various disease conditions such as ischemia-reperfusion cardiac injury, sarcopenia, and genetic myopathies, which impact multiple cellular processes related to cellular growth and survival in cardiac and skeletal muscles. Thus, autophagy has been the focus for therapeutic development to treat these muscle diseases. To develop the specific and effective interventions targeting autophagy, it is essential to understand the molecular mechanisms by which autophagy is altered in heart and skeletal muscle disorders. Herein, we summarize how autophagy alterations are linked to cardiac and skeletal muscle defects and how these alterations occur. We further discuss potential pharmacological and genetic interventions to regulate autophagy activities and their applications in cardiac and skeletal muscle diseases.

The aging of the immune system, or immunosenescence, was recently verified to have a causal role in driving the aging of solid organs, while the senolytic elimination of senescent immune cells was found to effectively delay systemic aging. Our recent study also showed that immune cells in severely dystrophic muscles develop senescence-like phenotypes, including the increased expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. Here we further investigated whether the specific clearance of senescent immune cells in dystrophic muscle may effectively improve the function of muscle stem cells and the phenotypes of dystrophic muscle. We observed increased percentage of senescent cells in macrophages from mdx/utro(-/-) mice (a murine model for muscular dystrophy disease, dystrophin-/-; utrophin-/-), while the treatment of mdx/utro(-/-) macrophages with senolytic drug fisetin resulted in reduced number of senescent cells. We administrated fisetin to mdx/utro(-/-) mice for 4 weeks, and observed obviously reduced number of senescent immune cells, restored number of muscle cells, and improve muscle phenotypes. In conclusion, our results reveal that senescent immune cells, such as macrophages, are greatly involved in the development of muscle dystrophy by impacting the function of muscle stem cells, and the senolytic ablation of these senescent cells with fisetin can be an effective therapeutic strategy for improving function of muscle stem cells and phenotypes of dystrophic muscles.

Limb girdle muscular dystrophy type R25 (LGMDR25) is a rare genetic disorder due to loss-of-function mutations in BVES, characterized by progressive proximal lower limb weakness and atrioventricular block. Here we report a young Chinese man with LGMDR25 who presented with asymmetrical lower limb weakness, myalgia, palpitations and dyspnea on exertion. Muscle imaging demonstrated fatty infiltration of the long head of biceps femoris, adductor magnus, gastrocnemius and soleus, and myoedema of semitendinosus and quadriceps, sparing rectus femoris. ECG showed only mild sinus tachycardia but pulmonary function test suggested prominent respiratory muscle weakness. Our report expands the phenotypical spectrum and indicates the importance of monitoring respiratory function in LGMDR25 patients.

Skeletal muscle regeneration relies on satellite cells that can proliferate, differentiate, and form new myofibers upon injury. Emerging evidence suggests that misregulation of satellite cell fate and function influences the severity of Duchenne muscular dystrophy (DMD). The transcription factor Pax7 determines the myogenic identity and maintenance of the pool of satellite cells. The circadian clock regulates satellite cell proliferation and self-renewal. Here, we show that the CLOCK-interacting protein Circadian (CIPC) a negative-feedback regulator of the circadian clock, is up-regulated during myoblast differentiation. Specific deletion of Cipc in satellite cells alleviates myopathy, improves muscle function, and reduces fibrosis in mdx mice. Cipc deficiency leads to activation of the ERK1/2 and JNK1/2 signaling pathways, which activates the transcription factor SP1 to trigger the transcription of Pax7 and MyoD. Therefore, CIPC is a negative regulator of satellite cell function, and loss of Cipc in satellite cells promotes muscle regeneration.

BACKGROUND: Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions.
RESULTS: In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation.
CONCLUSIONS: Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.