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

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.

A transcriptome atlas of leg muscles from healthy human volunteers reveals molecular and cellular signatures associated with muscle location.

Skeletal muscles support the stability and mobility of the skeleton but differ in biomechanical properties and physiological functions. The intrinsic factors that regulate muscle-specific characteristics are poorly understood. To study these, we constructed a large atlas of RNA-seq profiles from six leg muscles and two locations from one muscle, using biopsies from 20 healthy young males. We identified differential expression patterns and cellular composition across the seven tissues using three bioinformatics approaches confirmed by large-scale newly developed quantitative immune-histology procedures. With all three procedures, the muscle samples clustered into three groups congruent with their anatomical location. Concomitant with genes marking oxidative metabolism, genes marking fast- or slow-twitch myofibers differed between the three groups. The groups of muscles with higher expression of slow-twitch genes were enriched in endothelial cells and showed higher capillary content. In addition, expression profiles of Homeobox (HOX) transcription factors differed between the three groups and were confirmed by spatial RNA hybridization. We created an open-source graphical interface to explore and visualize the leg muscle atlas (https://tabbassidaloii.shinyapps.io/muscleAtlasShinyApp/). Our study reveals the molecular specialization of human leg muscles, and provides a novel resource to study muscle-specific molecular features, which could be linked with (patho)physiological processes.

Diffusion-tensor magnetic resonance imaging captures increased skeletal muscle fibre diameters in Becker muscular dystrophy.

BACKGROUND: Becker muscular dystrophy (BMD) is an X-linked disorder characterized by slow, progressive muscle damage and muscle weakness. Hallmarks include fibre-size variation and replacement of skeletal muscle with fibrous and adipose tissues, after repeated cycles of regeneration. Muscle histology can detect these features, but the required biopsies are invasive, are difficult to repeat and capture only small muscle volumes. Diffusion-tensor magnetic resonance imaging (DT-MRI) is a potential non-invasive alternative that can calculate muscle fibre diameters when applied with the novel random permeable barrier model (RPBM). In this study, we assessed muscle fibre diameters using DT-MRI in BMD patients and healthy controls and compared these with histology. METHODS: We included 13 BMD patients and 9 age-matched controls, who underwent water-fat MRI and DT-MRI at multiple diffusion times, allowing RPBM parameter estimation in the lower leg muscles. Tibialis anterior muscle biopsies were taken from the contralateral leg in 6 BMD patients who underwent DT-MRI and from an additional 32 BMD patients and 15 healthy controls. Laminin and Sirius-red stainings were performed to evaluate muscle fibre morphology and fibrosis. Twelve ambulant patients from the MRI cohort underwent the North Star ambulatory assessment, and 6-min walk, rise-from-floor and 10-m run/walk functional tests. RESULTS: RPBM fibre diameter was significantly larger in BMD patients (P = 0.015): mean (SD) = 68.0 (25.3) µm versus 59.4 (19.2) µm in controls. Inter-muscle differences were also observed (P ≤ 0.002). Both inter- and intra-individual RPBM fibre diameter variability were similar between groups. Laminin staining agreed with the RPBM, showing larger median fibre diameters in patients than in controls: 72.5 (7.9) versus 63.2 (6.9) µm, P = 0.006. However, despite showing similar inter-individual variation, patients showed more intra-individual fibre diameter variability than controls-mean variance (SD) = 34.2 (7.9) versus 21.4 (6.9) µm, P < 0.001-and larger fibrosis areas: median (interquartile range) = 21.7 (5.6)% versus 14.9 (3.4)%, P < 0.001. Despite good overall agreement of RPBM and laminin fibre diameters, they were not associated in patients who underwent DT-MRI and muscle biopsy, perhaps due to lack of colocalization of DT-MRI with biopsy samples. CONCLUSIONS: DT-MRI RPBM metrics agree with histology and can quantify changes in muscle fibre size that are associated with regeneration without the need for biopsies. They therefore show promise as imaging biomarkers for muscular dystrophies.

The metabolic landscape in chronic rotator cuff tear reveals tissue-region-specific signatures.

BACKGROUND: Degeneration of shoulder muscle tissues often result in tearing, causing pain, disability and loss of independence. Differential muscle involvement patterns have been reported in tears of shoulder muscles, yet the molecules involved in this pathology are poorly understood. The spatial distribution of biomolecules across the affected tissue can be accurately obtained with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The goal of this pilot study was to decipher the metabolic landscape across shoulder muscle tissues and to identify signatures of degenerated muscles in chronic conditions. METHODS: Paired biopsies of two rotator cuff muscles, torn infraspinatus and intact teres minor, together with an intact shoulder muscle, the deltoid, were collected during an open tendon transfer surgery. Five patients, average age 65.2 ± 3.8 years, were selected for spatial metabolic profiling using high-spatial resolution (MALDI-TOF) and high-mass resolution (MALDI-FTICR) MSI in negative or positive ion mode. Metabolic signatures were identified using data-driven analysis. Verifications of spatial localization for selected metabolic signatures were carried out using antibody immunohistology. RESULTS: Data-driven analysis revealed major metabolic differences between intact and degenerated regions across all muscles. The area of degenerated regions, encompassed of fat, inflammation and fibrosis, significantly increased in both rotator cuff muscles, teres minor (27.9%) and infraspinatus (22.8%), compared with the deltoid (8.7%). The intact regions were characterized by 49 features, among which lipids were recognized. Several of the identified lipids were specifically enriched in certain myofiber types. Degenerated regions were specifically marked by the presence of 37 features. Heme was the most abundant metabolite in degenerated regions, whereas Heme oxygenase-1 (HO-1), which catabolizes heme, was found in intact regions. Higher HO-1 levels correlated with lower heme accumulation. CONCLUSIONS: Degenerated regions are distinguished from intact regions by their metabolome profile. A muscle-specific metabolome profile was not identified. The area of tissue degeneration significantly differs between the three examined muscles. Higher HO-1 levels in intact regions concurred with lower heme levels in degenerated regions. Moreover, HO-1 levels discriminated between dysfunctional and functional rotator cuff muscles. Additionally, the enrichment of specific lipids in certain myofiber types suggests that lipid metabolism differs between myofiber types. The signature metabolites can open options to develop personalized treatments for chronic shoulder muscles degeneration.

Alternative Polyadenylation Utilization Results in Ribosome Assembly and mRNA Translation Deficiencies in a Model for Muscle Aging.

Aging-associated muscle wasting is regulated by multiple molecular processes, whereby aberrant mRNA processing regulation induces muscle wasting. The poly(A)-binding protein nuclear 1 (PABPN1) regulates polyadenylation site (PAS) utilization, in the absence of PABPN1 the alternative polyadenylation (APA) is utilized. Reduced PABPN1 levels induce muscle wasting where the expression of cellular processes regulating protein homeostasis, the ubiquitin-proteasome system, and translation, are robustly dysregulated. Translation is affected by mRNA levels, but PABPN1 impact on translation is not fully understood. Here we show that a persistent reduction in PABPN1 levels led to a significant loss of translation efficiency. RNA-sequencing of rRNA-depleted libraries from polysome traces revealed reduced mRNA abundance across ribosomal fractions, as well as reduced levels of small RNAs. We show that the abundance of translated mRNAs in the polysomes correlated with PAS switches at the 3'-UTR. Those mRNAs are enriched in cellular processes that are essential for proper muscle function. This study suggests that the effect of PABPN1 on translation efficiency impacts protein homeostasis in aging-associated muscle atrophy.