Ccn6 is expressed in zebrafish skeletal muscle and remains associated with mitochondrial respiratory complexes. (A) Immunostaining and confocal microscopy demonstrating Ccn6 expression in zebrafish skeletal muscle. (i) Ccn6 expression in the fiber lining near the sarcolemma and in patches in inter-myofibril spaces, as shown by arrows; (ii) DAPI stain showing nuclei; (iii) only secondary antibody control. (B) Ccn6 expression in skeletal muscle mitochondria in comparison with whole tissue and cytosol. Vdac1 is used as a reference as a mitochondrial protein. (C) Sephacryl S-300 gel filtration of zebrafish muscle mitochondrial lysate demonstrating the presence of Ccn6 in high molecular weights encompassing those of mitochondrial respiratory complexes. (D) Blue native (BN)-PAGE of muscle mitochondria (i) and immunoblotting separately with antibodies to Ndufb8 of complex I (ii), Uqcrc2 of complex III (iii), Cox4 of complex IV (iv), Atp5a1 of complex V (v), and Ccn6 (vi). Symbols on the blots(ii–v) denote the positions of different complexes corresponding with the Ccn6 bands in panel (vi). In-gel nitro blue tetrazolium chloride (NBT) assay after BN-PAGE of the mitochondria demonstrates the activity of assembled complex I (vii). (E) Immunostaining demonstrating the co-localization of Ccn6 with the mitochondrial respiratory complex. (i) Ccn6 expression; (ii) Cox4, representing mitochondrial respiratory complex IV expression; (iii) merged view showing the co-localization of Ccn6 with Cox4; (iv) magnified view (inset) of the co-localization (arrowheads); and (v) secondary antibody control for Cox4. The presented data (A,E) are representative of three independent experiments. For pooled experiments (B–D), the number of fish used were 3, 25, and 10, respectively.

Ccn6 depletion inhibits mitochondrial respiratory complex assembly and activity in zebrafish muscle. (A,B) Immunoblotting of the total muscle lysate and corresponding band densitometry demonstrating about 60% reduction in Ccn6 expression by ccn6 morpholino, but not control morpholino, as compared to the corresponding uninjected control. β-actin is used as a reference for the estimation of Ccn6 expression. (C–F) Blue native (BN)-PAGE of the muscle mitochondrial lysate followed by (i) immunoblotting separately with antibodies to Ndufb8 (complex I), Uqcrc2 (complex III), Cox4 (complex IV), and Atp5a1 (complex V) and (ii) corresponding band densitometry projected by a distribution plot, demonstrating that ccn6 morpholino, but not non-targeted morpholino, inhibits respiratory complex assembly as compared to no injection. Vdac1 expression from equal amounts of mitochondrial protein is used as a reference for this estimation. (G,H) Spectrophotometric and bar graph representation of the decrease in complex I activity upon Ccn6 depletion by ccn6 morpholino, but not control morpholino. No injection is a reference for this estimation. (I) ATP measurement assay demonstrating the decrease in ATP synthesis upon ccn6 morpholino (MO), but not control MO injection, as compared to the uninjected control. Data are presented as the mean ± SEM of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; NS, not significant (Student’s t-test). For pooled experiments (C–I), the number of fish used were 10, 10, 10, and 5, respectively, in each experimental group.

Ccn6 depletion causes loss of muscle mitochondrial abundance and alterations in muscle organization. (A) Immunofluorescence of the muscle sections of ccn6 morpholino-injected and uninjected zebrafish using anti-Vdac1 antibody with Alexa Fluor 546 rabbit secondary antibody, demonstrating loss of Vdac1 (mitochondrial structural protein) expression upon Ccn6 depletion: representation of three different experimental sets. Arrow marks denote the presence of mitochondria along the muscle fiber edges near the sarcolemma and in patches in the inter-myofibril spaces. (B) Immunoblot of the muscle lysate and corresponding densitometry showing a reduction in Vdac1 levels in ccn6 morpholino, but not control morpholino-injected fish, as compared to the uninjected control. β-actin is used as a reference protein for this estimation. Data are presented from two independent experiments; tissue was pooled from 10 fish in each experiment. (C) Histology (hematoxylin and eosin staining) of the muscle sections from ccn6 morpholino/control morpholino-injected and uninjected zebrafish demonstrating an increase in the interstitial gaps and presence of inflammatory infiltrates (arrow marks) in ccn6 morpholino-injected muscle: representation of five different experimental sets.

Depletion of Ccn6 expression in zebrafish skeletal muscle inhibits locomotion in response to stimulus. (A,B) Ccn6 depletion in skeletal muscle by ccn6 morpholino restricts the distance covered by zebrafish in response to stimulus as compared to the corresponding controls (control morpholino-injected and uninjected) at 48 and 72 h post-injection (n = 3). (C,D) Significantly less number of turns by ccn6 morpholino-injected fish as compared to the corresponding controls at 48 and 72 h post-injection (n = 3). Data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; NS, not significant (Student’s t test). (E) Representation of the experimental setup.