Infiltration of intramuscular adipose tissue impairs skeletal muscle contraction

Abstract

Key points: Muscle infiltration with adipose tissue (IMAT) is common and associated with loss of skeletal muscle strength and physical function across a diverse set of pathologies. Whether the association between IMAT and muscle weakness is causative or simply correlative remains an open question that needs to be addressed to effectively guide muscle strengthening interventions in people with increased IMAT. In the present studies, we demonstrate that IMAT deposition causes decreased muscle strength using mouse models. These findings indicate IMAT is a novel therapeutic target for muscle dysfunction.

Abstract: Intramuscular adipose tissue (IMAT) is associated with deficits in strength and physical function across a wide array of conditions, from injury to ageing to metabolic disease. Due to the diverse aetiologies of the primary disorders involving IMAT and the strength of the associations, it has long been proposed that IMAT directly contributes to this muscle dysfunction. However, infiltration of IMAT and reduced strength could both be driven by muscle disuse, injury and systemic disease, making IMAT simply an 'innocent bystander.' Here, we utilize novel mouse models to evaluate the direct effect of IMAT on muscle contraction. First, we utilize intramuscular glycerol injection in wild-type mice to evaluate IMAT in the absence of systemic disease. In this model we find that, in isolation from the neuromuscular and circulatory systems, there remains a muscle-intrinsic association between increased IMAT volume and decreased contractile tension (r² > 0.5, P < 0.01) that cannot be explained by reduction in contractile material. Second, we utilize a lipodystrophic mouse model which cannot generate adipocytes to 'rescue' the deficits. We demonstrate that without IMAT infiltration, glycerol treatment does not reduce contractile force (P > 0.8). Taken together, this indicates that IMAT is not an inert feature of muscle pathology but rather has a direct impact on muscle contraction. This finding suggests that novel strategies targeting IMAT may improve muscle strength and function in a number of populations.

Keywords: IMAT; fat infiltration; intermuscular adipose; muscle contraction.

Figure 1.. Intramuscular glycerol injection induces IMAT infiltration at 21 days post-treatment

A, Oil Red O staining of decellularized EDL muscle treated with saline (SAL) and glycerol (GLY) depicting increased quantity and dispersion of IMAT adipocytes (red) following glycerol treatment. B–F, quantification of IMAT infiltration metrics by confocal analysis as a function of treatment (S: saline, G: glycerol) and sex. Quantification confirms increased total and relative lipid volumes, total adipocyte count and nearest neighbour index (dispersion) with glycerol treatment in both sexes. N = 6, *P < 0.05, **P < 0.01, ***P < 0.005 by Bonferroni post-test on two-way ANOVA.

Figure 2.. Intramuscular glycerol injection causes reductions in peak contractile tension at 21 days post-treatment

A, EDL physiological cross-sectional area (PCSA), adjusted for the quantity of IMAT, is not different between treatment groups (S: saline, G: glycerol). B–C, peak tension (normalized to adjusted PCSA) is reduced with glycerol treatment in both twitch and tetanic contractions in both sexes. D–E, measures of calcium handling – time to peak tension and half-relaxation time – are not affected by glycerol treatment. F, time to fatigue is not affected by glycerol treatment. N = 6; *P < 0.05, **P < 0.01 by Bonferroni post-test on two-way ANOVA.

Figure 3.. IMAT metrics are correlated with contractile deficits in male and female EDL muscles

A and D, pairwise regression matrices for male and female mice, respectively. The strength of individual correlations is indicated by the circle size and colour in each box with red and blue shades indicating positive and negative correlations, respectively. Internal asterisks denote significant relationships. B–C, linear regressions of peak twitch and tetanic tensions against lipid volume in male mice. Lipid volume was the strongest linear predictor of both tensions by stepwise multilinear regression. E, linear regression of peak tetanic tension against a linear combination of lipid volume and average adipocyte volume in female mice. Lipid volume and average adipocyte volume in linear combination were the strongest predictors of peak tetanic tension by stepwise multilinear regression. F, linear regressions of time to fatigue against lipid volume in female mice. Lipid volume was the strongest predictor of time to fatigue by stepwise multilinear regression. N = 12.

Figure 4.. Intramuscular glycerol injection increases collagen content and decreases fibre area at 21 days post-treatment

A, representative images of saline- (SAL) and glycerol (GLY)-treated EDL sections stained with haematoxylin & eosin (H&E) to visualize tissue morphology, Picrosirius red to visualize collagen (red) and laminin to visualize fibre areas (red-rimmed areas). Note the appearance of intramuscular adipocytes (unstained areas) and centrally placed nuclei (blue in fibre centres) in GLY-treated muscles. B, quantification of Picrosirius red staining indicates increased collagen deposition with glycerol treatment (S: saline, G: glycerol) in both sexes. C, quantification of fibre cross-sectional area (CSA) on laminin-stained sections indicates decreased fibre CSA in glycerol-treated male muscle only. D, representative images of myosin heavy chain (MHC) isoform immunostaining to identify fibre types. E, quantification of MHC isoform distribution in stained sections reveals no significant shifts in fibre type distributions with glycerol treatment. N = 6; *P < 0.05, **P < 0.01, ***P < 0.005 by Bonferroni post-test on two-way ANOVA.

Figure 5.. Intact muscles exhibit a residual contractile tension deficit at day 21 post-treatment while permeabilized fibres do not

A, peak tetanic tension measured in isolated EDL muscles at day 14 (D14) and day 21 (D21) post-treatment (S: saline, G: glycerol) in male and female mice. Glycerol injection reduces peak tetanic tension at both time points in both sexes. B, peak tension measured in permeabilized fibres isolated from muscles in the indicated groups in A. Glycerol injection reduces peak tension only in male mice and only at day 14. Full recovery of tension is seen in both sexes at day 21. N = 8; **P < 0.01, ***P < 0.005, ****P < 0.001 by Sidak post-test on three-way ANOVA.

Figure 6.. Lipodystrophic muscle develops neither IMAT nor a contractile deficit at day 21 post-glycerol treatment

A, representative images of PDGFRα immunostaining identifying fibro/adipogenic progeitors (FAPs). B, quantification of FAPs per unit area indicates an increase with glycerol (G) treatment compared with saline (S) in both wildtype (WT) and lipodystrophic (LD) genotypes. C, Oil Red O staining of decellularized male glycerol-treated EDL depicting absence of IMAT in lipodystrophic (LD) muscle compared with littermate wildtype (WT) controls. D, predicted lipid volume from Oil Red O extraction indicates a significant increase with glycerol treatment (S: saline, G: glycerol) in WT mice only. E, peak tetanic tension is significantly reduced with glycerol treatment in WT mice only. F, representative images of WT and LD glycerol-treated EDL sections stained with haematoxylin & eosin (H&E) to visualize tissue morphology, Picrosirius red to visualize collagen (red) and laminin to visualize fibre areas (red-rimmed areas). Note the appearance of intramuscular adipocytes (unstained areas) in WT only but centrally placed nuclei (blue in fibre centres) in both genotypes. G, quantification of Picrosirius red staining indicates increased collagen deposition with glycerol treatment in both genotypes. H, quantification of fibre cross-sectional area (CSA) on laminin-stained sections indicates decreased fibre CSA with glycerol treatment in both genotypes. N = 6; *P < 0.05, **P < 0.01 by Bonferroni post-test on two-way ANOVA.