Myogenesis in dysferlin-deficient myoblasts is inhibited by an intrinsic inflammatory response

Abstract

Limb-girdle muscular dystrophy type 2B results from mutations in dysferlin, a membrane-associated protein involved in cellular membrane repair. Primary myoblast cultures derived from dysferlinopathy patients show reduced myogenic potential, suggesting that dysferlin may regulate myotube fusion and be required for muscle regeneration. These observations contrast with the findings that muscle develops normally in pre-symptomatic dysferlinopathy patients. To better understand the role of dysferlin in myogenesis, we investigated this process in vitro using cells derived from two mouse models of dysferlinopathy: SJL/J and A/J mice. We observed that myotubes derived from dysferlin-deficient muscle were of significantly smaller diameters, contained fewer myonuclei, and displayed reduced myogenic gene expression compared to dysferlin-sufficient cells. Together, these findings suggest that the absence of dysferlin from myoblasts is detrimental to myogenesis. Pro-inflammatory NFκB signaling was upregulated in dysferlin-deficient myotubes; the anti-inflammatory agent celastrol reduced the NFκB activation and improved myogenesis in dysferlin-deficient cultures. The results suggest that decreased myotube fusion in dysferlin deficiency is attributable to intrinsic inflammatory activation and can be improved using anti-inflammatory mediators.

Fig. 1

SJL/J muscle fuse to form myotubes in culture. (A and B) Muscle satellite cells were derived from SJL/J (left) or C57Bl/6J (right) mice, cultured for 7 days in growth media, then 2 days in fusion medium. Cultures were immunostained with anti-desmin (green) and MyoD (red) antibodies (A) or anti-MHC (red) and counterstained with DAPI (blue) (B). C57Bl/6J cells formed large-diameter fibers containing many nuclei, whereas SJL/J cells formed many thin fibers containing a few nuclei each. (C) Cumulative frequency distribution of myotube width plotted against normalized rank in tissue cultures of SJL/J and C57BL/6J muscle cells. Fiber diameters (y-axis) were quantitated in each of up to 26 muscle fibers of muscle cultures grown and differentiated as described above. Graph represents the range of fiber diameters (in microns) from SJL/J (black) and C57Bl/6J (red) cultures. (D) Summary data of (C), showing reduced myofiber width in SJL/J cultures compared to wild-type muscle cultures. (E) Cumulative frequency distribution of myotube nuclei number per fiber plotted against normalized rank in SJL/J and C57Bl/6J cultures. (F) Summary data of (E), showing reduced nuclei per fiber in SJL/J cultures compared to C57Bl/6J. (G) Plot of myotube width against myonuclear number, showing reduced numbers of nuclei per fiber and decreased fiber thickness for SJL/J derived muscle cultures. (H) Fusion index was decreased in SJL/J myotubes compared to C57Bl/6J. n = 202 SJL/J and 226 C57Bl/6J total nuclei counted in three fields; ^*P < 0.05. Scale bar = 20 μm.

Fig. 2

A/J deficient myoblasts do not show proliferation defects. (A) Dysferlin-sufficient (WT) and dysferlin-deficient (A/J) H-2K cells were plated at equal density and immunostained with indicated antibodies. (B) After 3 days in culture, total number of cells was counted. There was no significant difference in proliferation rates between A/J and WT H-2K cells. (C) Cell counts were taken after 5 days of culture in three independent A/J H-2K clones. There were no differences in proliferation. (D) MTT assay showing no differences in proliferation between A/J H-2K and WT cells. Scale bar = 25 μm.

Fig. 3

A/J myotubes form thinner myotubes and show a reduced myofusion index. (A) Immunostaining of A/J myotubes with antibodies for myosin heavy chain (MHC), calreticulin and Hoechst. (B) Quantitation of the fusion index. n = 386 (WT), 448 (A/J) total nuclei were counted containing 186 (WT) and 63 (A/J) myotubes in four non-overlapping fields. P < 0.05. (C–F) Real-time expression of myogenic genes during the time course of differentiation. Cultures from three independent H-2K A/J or WT clones were differentiated for up to 6 days in culture. Myogenic gene expression was measured using qPCR for MyoD (Myod) (C), myogenin (Myog) (D), myosin heavy chain 1 (Myh1) (E) and myosin heavy chain 4 (Myh4) (F). (G) Top: Western blot of MyoD expression between 2 and 5 days in culture (DIC) in WT and A/J H-2K cells. Bottom: Vinculin was used as a protein loading control. Scale bar = 25 μm.

Fig. 4

Celastrol blocks NFκB signaling. (A) Celastrol inhibits p65 activation and increases fiber diameters. Myoblasts were cultured in 6-well dishes. Twenty-four hours later, media was switched to differentiation media containing 200 nM celastrol. After 5 days in culture, myotubes were stained with antibodies to phospho-p65 (green) and anti-MHC (red). Phospho-p65 levels were higher in cultures of A/J cells compared to wild-type cells. Incubation of differentiating myoblasts with celastrol decreased phospho-p65 in the cultures and increased diameters of cells, shown with MHC staining. Inset shows higher magnification of area indicated by arrowhead. Scale bar = 20 and 5 μm in inset. (B) Cells were plated in 6-well dishes and celastrol was added at indicated concentrations after 24 h, the myotubes were harvested after 5 days. Western blot showing that phosphorylated p65 subunit of NFκB is upregulated in A/J cells and decreases with celastrol treatment (top). Total expression of p105, p50, and IKK is not altered with celastrol treatment. (C and D) Fusion index and nuclei per fiber were calculated from six random fields in two independent experiments. Celastrol significantly increased fusion index (C) and nuclei per fiber (D) in A/J cultures. Scale bar = 20 μm. ^**P < 0.05. ^***P < 0.01.