CAP2 deficiency delays myofibril actin cytoskeleton differentiation and disturbs skeletal muscle architecture and function

Figures

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  Fig. 1.. Motor function deficits in neonatal CAP2 mutants. (A–C) Graphs showing that eye opening, pinna detachment, and activity were unchanged in CAP2 mutants. (D) Instead, CAP2 mutants showed increased levels of tremulous movements. (E–I) Moreover, they performed weaker in various motor function tests. (J–O) No changes were noted in cliff avoidance, auditory startle, tactile startle, visual placing, grasping reflex, or forelimb placing. For A–O: n = 18 WT and 16 KO. If mean values (MV) were significantly different between groups (P < 0.05), squares indicating MV and error bars indicating SEMs are shown in red.

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  Fig. 2.. Myopathy in CAP2 mutants. (A) Immunoblots demonstrating presence of CAP2 in all skeletal muscles tested (GAS, M. gastrocnemius; MAS, M. masseter). GAPDH was used as a loading control. (B) Micrographs showing skinned hind legs from age-matched WT and KO. (C) HE-stained adult QUAD cross-sections. White arrows indicate internalized nuclei in KO. (D) Phalloidin staining revealed frequent occurrence of ring fibers in KO. (E) NADH-TR staining revealed altered mitochondrial distribution in KO (asterisks). (F) Quantification of histopathological changes in adult skeletal muscles and in P21 QUAD. Muscle fibers (%) with internalized nuclei (IN), altered mitochondrial distribution (AMD), and ring fibers (RF). n.a., not analyzed. For each analysis, at least nine micrographs of 0.6 mm2 from three mice per genotype have been used. (G) Antibody staining against desmin (red) in longitudinal TA sections. Sections were counterstained with phalloidin (green) and Hoechst 33342 (blue). (H–K) EM of adult WT and KO QUAD cross- and longitudinal sections. Asterisk in J indicates a ring fiber. Scale bar (µm): 1.0 (H, I, and K), 2.5 (J), 20 (C and G), 50 (D), 30 (E).

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  Fig. 3.. Myopathy onset coincides with delay in α-actin switch. (A) Immunoblots demonstrating CAP2 expression throughout QUAD development. β-tubulin was used as a loading control. (B) Micrographs showing phalloidin-stained QUAD cross-sections. (C) Immunoblots showing levels of total actin (pan-actin), skeletal muscle (α-SKA), smooth muscle (α-SMA), and cardiac α-actin (α-CAA), β-actin, and γ-actin throughout QUAD development. β-tubulin was used as a loading control. (D) Antibody staining against desmin (green) and α-SMA (red) in longitudinal TA sections from P12 mice. Sections were counterstained with DAPI (blue). White box indicates area in KO TA shown at higher magnification. Scale bars (µm): 20 (B and D).

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  Fig. 4.. CAP2 reexpression rescued ring fiber myopathy in KO. (A) Scheme showing experimental approach for rescue experiments in KO QUAD. (B) Micrographs from P21 KO QUAD cross-sections upon viral transduction of either GFP or GFP-CAP2. Sections were counterstained with phalloidin (red) to screen for ring fibers. White boxes indicate areas shown at higher magnification. Asterisks indicate ring fibers. Scale bar (µm): 100. (C) Graph showing fractions of GFP-negative or GFP-positive muscle fibers displaying ring fibers in P21 KO QUAD upon viral induction of either GFP or GFP-CAP2. GFP: negative, 32.8 ± 1.8%; positive, 38.0 ± 5.5%, P = 0.547. GFP-CAP2: negative, 36.5 ± 2.3%; positive, 0.2 ± 0.1, P < 0.001. n = 6 images from three mice. MV ± SEM are shown in C. ns, not significant, ***P < 0.001.