Zebrafish and cellular models of SELENON-Congenital myopathy exhibit novel embryonic and metabolic phenotypes

Abstract

Background: SELENON-Congenital Myopathy (SELENON-CM) is a rare congenital myopathy caused by mutations of the SELENON gene characterized by axial muscle weakness and progressive respiratory insufficiency. Muscle histopathology may be non-specific, but commonly includes multiminicores or a dystrophic pattern. 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 cause SELENON-CM remain poorly understood. A hurdle is the lack of cellular and animal models that show easily assayable phenotypes.

Methods: Using CRISPR-Cas9 we generated three zebrafish models of SELENON-CM, which were then studied by spontaneous coiling, hatching, and activity assays. We also performed selenon coexpression analysis using a single cell RNAseq zebrafish embryo-atlas. SelN-deficient myoblasts were generated and assayed for glutathione, reactive oxygen species, carbonylation, and nytrosylation levels. Finally, we tested Selenon-deficient myoblasts' metabolism using a Seahorse cell respirometer.

Results: 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 of selenon and genes involved in the glutathione redox pathway. SelN-deficient zebrafish and mouse myoblasts exhibit altered glutathione and redox homeostasis, as well as abnormal patterns of energy metabolism, suggesting roles for SelN in these functions.

Conclusions: These data demonstrate a role for SelN in zebrafish early development and myoblast metabolism and provide a basis for cellular and animal model assays for SELENON-CM.

Keywords: Congenital myopathy; Multiminicore myopathy; Rigid spine muscular dystrophy; Selenoprotein N; Zebrafish model.

Fig. 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 1 dpf and 6 dpf 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 ~ 65 kDa in positive control (SelN-transfected HEK cells) but not in negative control (WT HEK cells). Protein lysates from 2 dpf 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

Fig. 2

Selenoprotein-N deficient zebrafish embryos present with impaired spontaneous contractions. Spontanous tail and trunk contractions of zebrafish embryos were recorded and quantified at 24 hpf 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 80 hpf (N = 41–100 WT, 92–134 heterozygous, and 56–93 homozygous mutant embryos per line)

Fig. 3

Selenon-deficient zebrafish larvae show decreased activity. (A) 6 dpf 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

Fig. 4

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

Fig. 5

Selenon knock down cells present increased ROS levels when compared to control. (A) Flow cytometry assay of CM-H₂DCFDA stained cells to measure ROS levels in selenon knock down (KD1 and KD8) and WT control cells. (B) Quantification of fluorescence from (A) 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

Fig. 6

Selenoprotein-N null myoblasts show impairment in metabolism after differentiation at high cell seeding confluency. (A) Immunoblot shows absence of ~ 65 kDa 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 ~ 52 kDa 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 plating densities of selenon-null C2C12 Exon 5 cells. (C) Quantification of OCR and ECAR in increasing concentrations of C2C12 selenon-null exon 5 myoblasts show significant differences when compared to wild type only at 20,000 cell density (N = 10). (D) Quantification of basal OCR and ECAR in C2C12 selenon-null exon 3 and one mouse quadricep primary selenon-null cell lines shows impaired metabolism in KO myoblasts when compared to WT at 20,000 cell density (N = 30). (E) Quantification of basal OCR in three 1 dpf 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