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Quantitative analysis of myofiber type composition in human and mouse skeletal muscles.

ABSTRACT: Skeletal muscles are composed of different myofiber types characterized by the expression of myosin heavy chain isoforms, which can be affected by physical activity, aging, and pathological conditions. Here, we present a step-by-step high-throughput semi-automated approach for performing myofiber type quantification of entire human or mouse muscle tissue sections, including immunofluorescence staining, image acquisition, processing, and quantification. For complete details on the use and execution of this protocol, please refer to Abbassi-Daloii et al. (2022).¹.

SUBMITTER: Abbassi-Daloii T

PROVIDER: S-EPMC9898062 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Quantitative analysis of myofiber type composition in human and mouse skeletal muscles.

Abbassi-Daloii Tooba T El Abdellaoui Salma S Kan Hermien E HE van den Akker Erik E 't Hoen Peter A C PAC Raz Vered V Voortman Lenard M LM

STAR protocols 20230129 1

Skeletal muscles are composed of different myofiber types characterized by the expression of myosin heavy chain isoforms, which can be affected by physical activity, aging, and pathological conditions. Here, we present a step-by-step high-throughput semi-automated approach for performing myofiber type quantification of entire human or mouse muscle tissue sections, including immunofluorescence staining, image acquisition, processing, and quantification. For complete details on the use and executi ...[more]

PMID: 36853713

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Project description:As a widespread epigenetic RNA modification, N6-methyladenosine (m6A) plays essential regulatory roles in multiple biological processes. However, its function in maintaining and modulating myofiber-type properties remains largely unknown. To investigate the post-transcriptional modification underlying the myofiber type diversity in chicken skeletal muscle, we evaluated the m6A methylation levels of chicken skeletal muscles with different phenotypic traits, and profiled a transcriptome-wide m6A map in the oxidative and glycolytic skeletal muscles by methylated RNA immunoprecipitation sequencing (MeRIP-seq). Our results showed that the levels of m6A methylation in chicken skeletal muscles were closely related to the composition of myofiber types. The m6A methylation level of anterior latissimus dorsi (ALD, typical oxidative skeletal muscle) was the highest among the three muscles and significantly higher than that of the pectoralis major (PM, typical glycolytic skeletal muscle) (P<0.05). We found that about 24.77 % and 33.50 % of genes were modified by m6A methylation in the PM and ALD, respectively, and identified 6,530 and 9,965 m6A peaks, which were mainly located in the coding sequence (CDS) and stop codon. About 3.14 % of m6A modified genes showed significantly differential methylation levels between these two muscles. Intriguingly, the myofiber type-related genes, such as MYOT, TPM3, TPM1, PDK1, MBNL1, and MYH1G, showed differences in m6A methylation and mRNA expression. Further analysis revealed that the m6A methylation was positively correlated with gene expression homeostasis. It is exciting we found that the expression level of ALKBH5 mRNA and protein, was closely related to the composition of myofiber types. ALKBH5 over-expression could regulate the expression levels of genes related to muscle contraction and metabolism, including MYH1E, MYH1G, MYH7B, PDK1, and TPM1, suggesting the effect of ALKBH5 on the formation of myofiber-type properties in chicken skeletal muscle. Our results contribute to a better understanding of epigenetic factors involved in forming chicken myofiber-type properties and provide new targets for further investigation into chicken's growth development and meat quality.

Project description:Parkinson's disease (PD) is a common neurodegenerative disorder impacting cognition, movement, and quality of life in >10 million individuals worldwide. We recently characterized and quantified a skeletal muscle pathology in PD represented by exaggerated type I myofiber grouping presumed to result from denervation-reinnervation processes. Our previous findings indicated that impaired neuromuscular junction integrity may be involved in type I grouping, which is associated with excessive motor unit activation during weight-bearing tasks. In this study, we performed transcriptional profiling to test the hypothesis that type I grouping severity would link to distinct gene expression networks. We generated transcriptome-wide poly(A) RNA-Seq data from skeletal muscle of individuals with PD [n = 12 (9 men, 3 women); 67 ± 2 yr], age- and sex-matched older adults (n = 12; 68 ± 2 yr), and sex-matched young adults (n = 12; 30 ± 1 yr). Differentially expressed genes were evaluated across cohorts. Weighted gene correlation network analysis (WGCNA) was performed to identify gene networks most correlated with indicators of abnormal type I grouping. Among coexpression networks mapping to phenotypes pathologically increased in PD muscle, one network was highly significantly correlated to type I myofiber group size and another to percentage of type I myofibers found in groups. Annotation of coexpressed networks revealed that type I grouping is associated with altered expression of genes involved in neural development, postsynaptic signaling, cell cycle regulation and cell survival, protein and energy metabolism, inflammation/immunity, and posttranscriptional regulation (microRNAs). These transcriptomic findings suggest that skeletal muscle may play an active role in signaling to promote myofiber survival, reinnervation, and remodeling, perhaps to an extreme in PD.NEW & NOTEWORTHY Despite our awareness of the impact of Parkinson's disease (PD) on motor function for over two centuries, limited attention has focused on skeletal muscle. We previously identified type I myofiber grouping, a novel indicator of muscle dysfunction in PD, presumably a result of heightened rates of denervation/reinnervation. Using transcriptional profiling to identify networks associated with this phenotype, we provide insight into potential mechanistic roles of skeletal muscle in signaling to promote its survival in PD.

Dataset Information

Quantitative analysis of myofiber type composition in human and mouse skeletal muscles.

Publications

Quantitative analysis of myofiber type composition in human and mouse skeletal muscles.

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