Molecular signatures of age-associated chronic degeneration of shoulder muscles

Abstract

Chronic muscle diseases are highly prevalent in the elderly causing severe mobility limitations, pain and frailty. The intrinsic molecular mechanisms are poorly understood due to multifactorial causes, slow progression with age and variations between individuals. Understanding the underlying molecular mechanisms could lead to new treatment options which are currently limited. Shoulder complaints are highly common in the elderly, and therefore, muscles of the shoulder's rotator cuff could be considered as a model for chronic age-associated muscle degeneration. Diseased shoulder muscles were characterized by muscle atrophy and fatty infiltration compared with unaffected shoulder muscles. We confirmed fatty infiltration using histochemical analysis. Additionally, fibrosis and loss of contractile myosin expression were found in diseased muscles. Most cellular features, including proliferation rate, apoptosis and cell senescence, remained unchanged and genome-wide molecular signatures were predominantly similar between diseased and intact muscles. However, we found down-regulation of a small subset of muscle function genes, and up-regulation of extracellular region genes. Myogenesis was defected in muscle cell culture from diseased muscles but was restored by elevating MyoD levels. We suggest that impaired muscle functionality in a specific environment of thickened extra-cellular matrix is crucial for the development of chronic age-associated muscle degeneration.

Keywords: Gerotarget; atrophy; deep RNA-seq; fatty infiltration; muscle satellite cells; shoulder disease.

Figure 1. Clinical description of deltoid and subscapularis muscles

Anatomical landmarks of shoulder muscles in intact and torn conditions. Representative MRA images from the control group (panels A. and D., transversal and coronal view, respectively) and from torn SSc muscle (panels B. and E., transversal and sagittal view, respectively). A schematic illustration of muscle cross-sectional surface area (CSA) is shown in panels C. and F. (transversal and coronal view, respectively). Muscle atrophy is indicated by the dashed line of the SSc. The long head of the biceps tendon is dislocated out of the bicipital groove, medially (black arrow). Subscapularis muscle (SSc), glenoid (Gl), humeral head (HH), infraspinatus (ISp), deltoid (DM), clavicle (cla), coracoid (cor), acromion (Acr), supraspinatus (SSp) and the teres minor (Tmi). Box plot (G) shows CSA of the DM and SSC between control subjects (n = 52) and patients with a diseased SSc (n = 28). Statistical significance between DM and SSc using unpaired t-test: ** p < 0.001.

Figure 2. Histological analyses in deltoid and subscapularis muscles

A. Representative images of deltoid (DM) and subscapularis muscle (SSc) stained with: a. H&E; b. Nile red (in red) for fatty droplets; c. C12-resazurin (in red) for oxidative metabolic activity. Nuclei are counterstained with DAPI in b and c ; d. Immunofluorescence with an antibody mix for MyHC-2b (green),-2a (red),-1 (blue) and laminin (white). For the SSc two conditions are shown: tissue containing myofibers (middle column) and highly fibrotic tissue without myofibers (right column). Images were taken with light microscope (a), fluorescence microscopes (b and d), or with a confocal microscope (c). Scale bars are 200 (a), 100 (b and d) or 50 (c) μm. B. and C. Analyses in paired samples for DM and SSc in two patients. B: Bar charts depict proportion of myofibers expressing MyHC-isotypes and those that were unstained. C: Cumulative distribution plot of the CSA in SSc, DM and vastus lateralis (VL) reference.

Figure 3. Cellular activities in deltoid (DM) and subscapularis (SSc) -derived muscle cell culture (CC)

A. Nile red staining in deltoid (DM) and subscapularis muscle (SSc) cell culture (CC), nuclei are counterstained with DAPI (blue). Bar chart shows the percentage of cells containing fatty droplets. B. Bar chart shows the percentage of proliferating cells. C. Box plot shows the percentage of apoptotic cells. D. Box plot shows cell senescence (normalized to the reference vastus lateralis cell culture). E. Box plot shows the mitochondrial metabolic rate (normalized to the reference vastus lateralis cell culture). F. Representative images of fused cell cultures. Myoblasts are stained with Desmin (red) and fused cells are stained with MyHC (green). Nuclei are counterstained with DAPI (blue). Box plot shows the percentage of fused cells. Averages and standard deviations are from n = 5 (DM-CC) and n = 8 (SSc-CC). Statistical significance between DM-CC and SSc-CC using unpaired t-tests: * p < 0.05, *** p < 0.001.

Figure 4. RNA expression profiles in deltoid and subscapularis muscle-derived cell cultures

A. Volcano plot shows log2 fold change (FC) versus -log10 of the p-value of all genes found in the RNA-seq (pair-wise analyses, n = 7). Red dots indicate significantly dysregulated genes (p < 0.05 FDR). Blue dots indicate dysregulated genes with a nominal p-value < 0.01. Positive or negative FC indicates higher or lower expression in subscapularis (SSc) compared with the deltoid (DM) cell cultures (CC). B. Functional gene ontology (GO) of dysregulated genes (p < 0.01), daughter GO clusters are connected with a line. GO clusters of muscle development are depicted in blue, in red the extracellular region, in green the inflammatory system and in yellow calcium binding. N indicates the number of genes. C. Box plot shows expression levels in SSc-CC or DM-CC for the most significantly dysregulated genes (p < 0.05 FDR). Fold changes between DM and SSc are depicted. Genes of the GO muscle development are marked in blue and those of the extracellular region in red. D. Representative images of fused SSc cell cultures transduced with either mock or MyoD lentivirus (LV). Myoblasts are stained with MyHC (white) and segmented in green. Nuclei are counterstained with DAPI (blue) and segmented in blue. Nuclei within segmented MyHC objects are marked in red. Scale bar is 200 μm. E. Plots show paired analyses of % of fused cells and total fused area between mock and MyoD LV in 4 SSc cell cultures. *: p-value < 0.05, using a paired T-test.