Fig. 5: In vivo MICAL2 CRISPR/Cas9 system induces skeletal muscle degeneration/regeneration, inflammation and fibrosis.

a Timeline illustrating the experiment setup. At day -2, H11^LSL-Cas9 underwent skeletal muscle damage by CTX injections on the right hind limb (TA, GC and Q). At day 0, CRISPR/Cas9 system activation via AAV-Cre with either Sham sgRNA or Mical2 sgRNA in both hind limbs (TA, GC and Q). At day 6, 10, 16, 21 and 30 treadmill exhaustion tests were performed and at day 10, 21 and 30 mice were sacrificed for molecular and histological analyses. b Heat map representing qRT-PCR Z-score of Mical2 and Cas9 for the system activation, Pax7, Pax3 and Ccl2 for skeletal muscle regeneration and remodeling and CD45, Tnf-α and cleaved Casp3 (Casp3) for inflammation and apoptosis. c IF analysis illustrates at day 10, day 21 and day 30 LAMININ (red) and MICAL2 (green) localization on left TA cross-sections of AAV-Sham H11^Cas9 mice compared to AAV-Mical2 H11^Cas9 mice. Nuclei stained with HOECHST (blue). Scale bars 50 μm. d The effect of MICAL2 CRISPR/Cas9 is shown by H&E staining on left TA cross-sections of AAV-Sham H11^Cas9 mice compared to AAV-Mical2 H11^Cas9 mice, along time points of 10, 21 and 30 days. Scale bars 50 μm. e IF analysis of LAMININ (red) and alpha-SARCOMERIC ACTININ (α-SA; green) in AAV-Mical2 muscles showed actin filament disorganization compared to controls at day 30 from infection. Scale bars 50 μm. N = 6. * = p < 0.05 by two-tailed t test. See also Fig. S6.