Aberrant RhoA activation in macrophages increases senescence-associated secretory phenotypes and ectopic calcification in muscular dystrophic mice

Abstract

Duchenne Muscular Dystrophy (DMD) patients often suffer from both muscle wasting and osteoporosis. Our previous studies have revealed reduced regeneration potential in skeletal muscle and bone, concomitant with ectopic calcification of soft tissues in double knockout (dKO, dystrophin-/-; utrophin-/-) mice, a severe murine model for DMD. We found significant involvement of RhoA/ROCK (Rho-Associated Protein Kinase) signaling in mediating ectopic calcification of muscles in dKO mice. However, the cellular identity of these RhoA+ cells, and the role that RhoA plays in the chronic inflammation-associated pathologies has not been elucidated. Here, we report that CD68+ macrophages are highly prevalent at the sites of ectopic calcification of dKO mice, and that these macrophages highly express RhoA. Macrophages from dKO mice feature a shift towards a more pro-inflammatory M1 polarization and an increased expression of various senescence-associated secretory phenotype (SASP) factors that was reduced with the RhoA/ROCK inhibitor Y-27632. Further, systemic inhibition of RhoA activity in dKO mice led to reduced number of RhoA+/CD68+ cells, as well as a reduction in fibrosis and ectopic calcification. Together, these data revealed that RhoA signaling may be a key regulator of imbalanced mineralization in the dystrophic musculoskeletal system and consequently a therapeutic target for the treatment of DMD or other related muscle dystrophies.

Keywords: cellular senescence; chronic inflammation; heterotopic ossification; muscle dystrophy; muscle stem cell.

Figure 1

Increased macrophage accumulation at sites of ectopic calcification in skeletal muscle of dKO mice. (A). Micro-CT scanning results indicating increased ectopic calcification and osteoporosis in the hindlimb of dKO mice, compared to WT mice. (B) Alizarin Red staining of hindlimb tissue sections validate the presence of ectopic calcification in dKO mice. (C) Percent of mice that exhibit ectopic calcification (EC) in hindlimbs of dKO mice and WT mice. (D) Alizarin Red staining of skeletal muscle (gastrocnemius) and cardiac muscle (septum) showing ectopic calcification (EC). (E) Immunostaining of dKO skeletal muscle and heart sections with CD68 antibody and bright field imaging showing the extensive accumulation of CD68+ macrophages at the sites of ectopic calcification (EC). (F) Immunostaining of dKO hindlimb sections with RhoA and CD31 antibodies and bright field imaging showing the increased accumulation of RhoA+ cells at the sites of ectopic calcification (EC). n=8 for both WT and dKO mice (8-week old).*=p<0.05.

Figure 2

CD68+ cells at the sites of muscle calcification in dKO mice are positive for RhoA expression. (A) Immunostaining of dKO skeletal muscle (gastrocnemius) sections with RhoA and CD68 antibodies indicating that many of the accumulated CD68+ cells at sites of ectopic calcification (EC) are also RhoA+. (B) Immunostaining of dKO skeletal muscle and bone marrow sections with RhoA and CD68 antibodies showing that CD68+ cells distant from ectopic calcification or those found in bone marrow are negative for RhoA expression. (C) Immunostaining of dKO skeletal muscle with RhoA/F4-80 or RhoA/CD163 antibodies indicating RhoA+ cells are usually positive for F4-80, but negative for CD163 (a marker for M2 macrophages). (D) Quantification of RhoA+/CD68+ cells among CD68+ cells at locations of muscle with or without EC formation. * indicates p<0.05.

Figure 3

CD68+ cells at calcification sites in the heart of dKO mice co-express RhoA. (A) Immunostaining of dKO heart sections (cardiac muscle at septum) with RhoA and CD68 antibodies showing that many of the accumulated CD68+ cells at the sites of ectopic calcification are RhoA+ (white arrows); whereas the CD68+ cells away from the calcification sites are RhoA- (dotted circle). (B) Immunostaining of dKO heart sections (interface of right ventricle and septum) with RhoA and CD68 antibodies showing accumulation of RhoA+/CD68+ cells at the endothelial barrier (cardiac endothelium, dotted lines) which are infiltrating through the endothelium from circulation towards the pathological area of the cardiac muscle (sub-image 2); whereas the CD68+ cells in the circulating blood are RhoA- (sub-image 3). N=6 for both WT and dKO mice (8-week old).

Figure 4

Macrophages accumulating in dystrophic muscles have increased expression of senescence markers. (A) SA-β-Gal staining of gastrocnemius muscle sections from 1, 4, and 8 week old dKO mice with quantification. Significant changes in SA-β-Gal staining was observed by 8 weeks. (B) Immunofluorescent staining of gastrocnemius muscle sections from 8-week old dKO mice indicating co-localization of the senescence marker C₁₂FDG and CD68. ~26% of CD68+ cells are C₁₂FDG+. (C) Immunofluorescent staining of muscle sections from 8 week old dKO mice indicating co-localization of the senescence marker p21 and RhoA. ~78% of RhoA+ cells are p21+. n=6 for 1-week and 4-week old mice, and n=8 for 8-week old mice. * indicates p<0.05.

Figure 5

In vitro inhibition of RhoA/ROCK in dKO macrophages reduced expression of SASP factors. (A) Cultured macrophages isolated from WT and dKO mice obtained by FACS with CD68 immunostaining to validate macrophage isolation in vitro. (B) qPCR results indicating dKO macrophages exhibit up-regulated expression of SASP factors that was reduced by treatment with the RhoA/ROCK inhibitor Y-27632 (10 μM, 2 days). * indicates p<0.05 vs. WT, * indicates p<0.05 vs. dKO.

Figure 6

Systemic inhibition of RhoA/ROCK in dKO mice reduced calcification in skeletal muscle by repressing the accumulation of RhoA+/CD68+ cells. (A) Alizarin Red staining indicating reduction in calcification in dystrophic muscles of dKO mice treated with the RhoA/ROCK inhibitor Y-27632 for 3 times a week, from 3 weeks to 8 weeks of age. Immunostaining of skeletal muscle tissues showed that the accumulation of CD68+/RhoA+ cells in dKO muscle was also reduced by RhoA/ROCK inhibition, while the number of CD163+ cells (M2 macrophages) was increased. (B) quantification of the area of ectopic calcification (EC) in skeletal muscle of dKO mice with or without Y-27632 treatment. (C) Quantification of the number of CD68+, RhoA+, and CD68+/RhoA+ cells with and without Y-27632 treatment (number of cells per area of 100 myofibers). (D) qPCR results of mRNA isolated from dystrophic muscles of dKO mice showing that Y-27632 treatment significantly down-regulated the expression of pro-inflammatory/fibrosis genes (TGF-β1, BMP2, TNF-α, and IL-1β), and up-regulated the expression of anti-inflammatory/fibrosis genes (Klotho, Sirt1, and IL-10). n=8 for both dKO mice with or without Y-27632 treatment, * indicates p<0.05.