Fig 2: Overexpression of lipin1 attenuated dystrophic histology in gastrocnemius of muscle-specific mdx:lipin1Tg/0 mice. A Schematic strategy for generating mdx:lipin1Tg/0 mice. B Representative immunoblot and C quantitative densitometry analysis of dystrophin and lipin1 protein in the gastrocnemius muscle of B10, mdx, and mdx:lipin1Tg/0 mice. D Representative H&E staining, E quantification analysis of centrally nucleated myofibers, F cross-sectional areas of muscle fibers, G average cross-sectional area, H picrosirius red staining, and I quantification analysis of collagen deposition of the gastrocnemius muscle in B10, mdx, and mdx:lipin1Tg/0 mice (Scale bars = 100um). *p < 0.05; ****p < 0.0001. J NADH staining of the gastrocnemius muscle in B10, mdx, and mdx:lipin1Tg/0 mice (Scale bars = 500um). n = 3–5 mice per group

Fig 3: The effects of lipin1 overexpression on dystrophic muscle histology through IM injection. A Representative immunofluorescence staining of gastrocnemius muscle from mdx mice 1-month intramuscular post-injection of AAV1-lipin1. Lipin1 was detected by anti-HA antibody (red) and laminin by anti-laminin antibody (green). DAPI was used to stain nucleus (blue). Arrows in the right panels indicate centrally nucleated myofibers, which are characteristic of muscle regeneration (scale bar = 100 μm). B The percentage of centrally nucleated myofibers in the lipin1 overexpressed regions compared with the non-overexpressed regions (n = 3 mice/group, **p < 0.01). C Western blot and D quantification analysis of necroptotic markers in mdx mice treated with either saline or AAV-lipin1 (n = 3 mice/group, *p < 0.05; **p < 0.01)

Fig 4: Increasing lipin1 expression levels in dystrophic muscle did not result in obvious side effects. A Oil Red O staining of gastrocnemius of 6-month-old WT, mdx, and mdx:lipin1Tg/0 mice (scale bar = 100μm; n = 3 mice/group). B Western blot analysis of perilipin 2 in gastrocnemius of B10, mdx and mdx:lipin1Tg/0 mice (n = 3 mice/group). Body composition including C body fat, D lean body mass, E body weight, F body fat to body weight ratios, and G lean body mass to body weight ratios in 6–7-month-old of B10, mdx, and mdx:lipin1Tg/0 mice was determined using EchoMRI (n = 12 mice/group). Ns, no significant significance; *p < 0.05; ***p < 0.001; ****p < 0.0001

Fig 5: Restoration of lipin1 in dystrophic muscle slowed down the degeneration-regeneration cycle. A Representative immunoblots and B quantitative analysis of necroptotic (RIPK1, RIPK3, and MLKL), apoptotic (BAX, BAK, BID, and cleaved Cas 3 (C.Cas 3)), and autophagy (p62 and LC3) markers in indicated mice. n = 3 mice/group. *p < 0.05; **p < 0.01; ***p < 0.001. C Immunostaining of pax7 (green) and laminin (red), and D pax7 (green) and myoD (red) in gastrocnemius of B10, mdx and mdx:lipin1Tg/0 mice. E Western blot and F quantification analysis of indicated markers in gastrocnemius of B10, mdx and mdx:lipin1Tg/0 mice (n = 3 mice/group; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)