Zebrafish and cellular models of SELENON-Related Myopathy exhibit novel embryonic and metabolic phenotypes

Abstract

SELENON-Related Myopathy (SELENON-RM) is a rare congenital myopathy caused by mutations of the SELENON gene characterized by axial muscle weakness and progressive respiratory insufficiency. Muscle histopathology commonly includes multiminicores or a dystrophic pattern but is often non-specific. The SELENON gene encodes selenoprotein N (SelN), a selenocysteine-containing redox enzyme located in the endo/sarcoplasmic reticulum membrane where it colocalizes with mitochondria-associated membranes. However, the molecular mechanism(s) by which SelN deficiency causes SELENON-RM are undetermined. A hurdle is the lack of cellular and animal models that show assayable phenotypes. Here we report deep-phenotyping of SelN-deficient zebrafish and muscle cells. SelN-deficient zebrafish exhibit changes in embryonic muscle function and swimming activity in larvae. Analysis of single cell RNAseq data in a zebrafish embryo-atlas revealed coexpression between selenon and genes involved in glutathione redox pathway. SelN-deficient zebrafish and mouse myoblasts exhibit changes in glutathione and redox homeostasis, suggesting a direct relationship with SelN function. We report changes in metabolic function abnormalities in SelN-null myotubes when compared to WT. These results suggest that SelN has functional roles during zebrafish early development and myoblast metabolism.

Keywords: Selenoprotein N; congenital myopathy; multiminicore myopathy; rigid spine muscular dystrophy; zebrafish model.

Figure 1.. Zebrafish selenon mutants show absent or partial expression of Selenoprotein-N.

(A) Sanger sequencing chromatograms show analysis of the selenon gene exon 2 for wild type (WT), selenon^cl502, selenon^cl503, and selenon^cl504 homozygotes using genomic DNA from zebrafish tail clips. (B) Real Time qPCR analyses show selenon transcript levels at 1dpf and 6dpf in zebrafish knock outs (KO’s) and mutants (Mt): selenon^cl502, selenon^cl503, and selenon^cl504with their correspondent WT. (N=5 per group) “*” = p<0.05, “**” = p<0.01, “***” = p<0.001, “****” = p<0.0001. (C) Western Blot analysis shows SelN expression at the predicted size of ~65kDa in positive control (SelN-transfected HEK cells) but not in negative control (WT HEK cells). Protein lysates from 2dpf zebrafish selenon mutants and their corresponding WT controls show SelN expression in all WT fish, no expression in selenon^cl502 and selenon^cl503 , and reduced expression in selenon^cl504 mutant line. (N=30 per group) (D) Ponceau staining in western blot used to demonstrate equal protein loading throughout the blot.

Figure 2.. Selenoprotein-N deficient zebrafish embryos present with impaired spontaneous contractions.

Spontanous tail and trunk contractions of zebrafish embryos were recorded and quantified at 24hpf in SelN homozygous KO selenon^cl502 (N=45 and 29), selenon^cl503 (N=43,48), and homozygous mutant (Mt) selenon^cl504 (N=131,116) and their correspodent WT controls. (A) Mean duration of spontaneous contractions, percent time of contraction activity, and total number of contractions are reported in each line. “*” = p<0.05, “**” = p<0.01, “***” = p<0.001, “****” = p<0.0001. (B) WT, heterozygous and homozygous embryos were observed for hatching activity and recorded up until 80hpf (N = 41–100 WT, 92–134 heterozygous, and 56–93 homozygous mutant embryos per line).

Figure 3.. Selenon-deficient zebrafish larvae show decreased activity.

(A) 6dpf WT (blue) and homozygous selenon-KO selenon^cl502/cl502(red) zebrafish larvae were tested for swim activity using an activity monitor and a 90-minute protocol that included vibration (dotted lines) and cycles of alternating light (non-shaded areas) and dark (shaded areas) periods. (B) Quantification of activity assays of WT and homozygous selenon-mutant lines shows decreased total distance swum in SelN-deficient zebrafish larvae when compared to their correspondending WT controls (N=24). “*” = p<0.05, “***” = p<0.001.

Figure 4.. Analysis of embryonic single cell RNAseq zebrafish atlas reveals correlation between selenon expression and glutathione redox genes in tailbud presomitic mesoderm (PSM).

(A) Tree-cell cluster diagram from Wagner et al. 2017, shows selenon expression (green) throughout early embryogenesis. Tailbud PSM cell clusters show incremental expression of selenon from 6 to 24hpf (red square). (B) Pathway analysis of genes coexpressed with selenon identified in “oxidoreductase activity” gene ontology reveal overexpression of glutathione redox genes as well as apelin adipocyte signaling pathway. (C) Glutathione redox pathway diagram shows the four highly correlated genes with selenon (purple): gpx7, gpx8, gpx4a, and gpx19.

Figure 5.. Glutathione homeostasis is altered in cell an zebrafish models of SELENON-Related Myopathy.

(A) Selenon Knock Down (KD) cell lines show decreased gluathione ratio (GSH/GSSG) when compared to WT control. (B) GSSG, (C) total GSH, and (D) GSH/GSSG ratio were measured in 6dpf selenon^cl502 (N=9 and 7), selenon^cl503 (N=14), and selenon^cl504 (N=13 and 14) zebrafish lines. Results show altered glutathione homeostasis. “*” = p<0.05, “**” = p<0.01, “***” = p<0.001.

Figure 6.. Selenon knock down cells present increased ROS levels when compared to control.

(A) Flow cytometry assay shows fluorescently labeled ROS levels in selenon knock down (KD1 and KD8) and control cells. (B) Quantification of fluorescence reveals significant increase of ROS levels in selenon KD cell lines when compared to control. (C) Immunoblotting of protein carbonylation and (D) nytrosylation demonstrate increased levels in selenon KD when compared to control cells.

Figure 7.. Selenoprotein-N null myoblasts show impairment in metabolism after differentiation at high cell seeding confluency.

(A) Immunoblot shows absence of ~65kDa SelN in three selenon-null myoblast lines: C2C12 Exon 3, C2C12 Exon 5, and mouse quadriceps primary cells (Quad PC). α-Tubulin was used as a loading control and is present at ~52kDa in all lines. (B) Results from seahorse cell respirometer show changes in Oxygen Consumption Rate (OCR) and Extra Cellular Acidification Rate (ECAR) parameters at different cell densities of selenon-null myoblast lines. (C) Quantification of C2C12 selenon-null exon 5 line’s basal levels of OCR and ECAR show differences in metabolism when compared to wild type at 20,000 cell density (N=10). (D) Quantification of basal OCR and ECAR in two C2C12 and one mouse quadricep primary selenon-null cell lines shows impaired metabolism in KO myoblasts when compared to WT (N=30). (E) Quantification of basal OCR in three 1dpf selenon-mutant zebrafish embryos show no differences in metabolism in mutants when compared to WT (N=48). “**” = p<0.01, “***” = p<0.001, “****” = p<0.0001.