Fig 1.
Scores for muscle fiber size variation, anguloid and angular atrophy, fiber splitting, fiber type grouping and centrally displaced myonuclei in gluteal or semimembranosus muscle of 8 horses with myotonic dystrophy (eMD) and 9 controls.
Scoring system was 0 = not present, 1 = mild alterations were present in<20% of 10x field, 2 = moderate alterations present in 21–50% of 10X field and 3 = severe alterations present in > 50% of 10X fields. P values are shown based on statistical comparisons performed with a Mann Whitney test.
Table 1.
Signalments and phenotype of the horses which presented with myotonia. AP= Appaloosa, QH = Quarter Horse.
Fig 2.
Muscle histopathology in frozen sections of eMD horses.
(A) Normal HE stain of the gluteal muscle of a 4-year-old control horse (C2). (B) Marked fiber size variation, anguloid atrophy, fiber splitting (arrow) internalized myonuclei and increased endomysial connective tissue in semimembranosus muscle of 2-month-old eMD1 (HE 20X). (C) Large myofibers and anguloid and angular (arrow) atrophied fibers, fiber splitting and internalized nuclei in gluteal muscle of 3-year-old eMD3 (HE 20X). (D) Marked variation in fiber sizes, anguloid and angular atrophy (arrow) and internalized nuclei in 3-month-old eMD2 (HE 40X) (E) Areas of marked fiber size variation with a focal area of larger fibers juxtaposed with atrophied fibers in gluteal muscle of 3-month-old eMD2 (HE 20X). (F) Normal appearing triceps muscle in eMD2 (HE 10X) (G) Internalized myonuclei and sarcoplasmic masses (arrow) in 3-year-old eMD5 (modified Gomori Trichrome 40X). (H) Normal gluteal muscle of 3-year-old control horse C2 (modified Gomori Trichrome 40X).
Fig 3.
Oxidative fiber typing. (A) Mosaic distribution of oxidative fibers in gluteal muscle from 5-year-old control horse C1(10X).
(B) Oxidative fiber type grouping in the semimembranosus muscle of 3-year-old eMD3 (NADH-TR 10X) (C) Grouped oxidative fibers in gluteal muscle of 3-month-old eMD 2 (NADH-TR 4X). (bar = 20 μm).
Fig 4.
Immunofluorescent fiber typing.
(A) The mosaic distribution of type 1 (blue) type 2A (green) and type 2X (brown) fibers in a control horse C1. Note that type 1 fibers are smaller relative to type 2 fibers. (B) Grouping of type 1 fibers (blue) in the semimembranosus muscle of eMD2. (C) Grouping of type 1 fibers in semimembranosus muscle of eMD1. Note that type 1 fibers are larger relative to type 2 fibers. (D) Staining for developmental myosin (MYH3) in gluteal muscle of control horse C3 with no MYH3 fibers identified. (E) Presence of developmental myosin in semimembranosus muscle fibers of eMD2. F. Serial section of eMD2 showing that developmental fibers in E stain correspond to type 1 fibers in the type 1, 2A, 2X fiber typing.
Fig 5.
Transcriptomic differential gene expression in eMD horses.
Volcano plot depicting the probability of observing the estimated change in gene expression (-log10 scale) on the Y axis and the degree of fold change differences (log2 scale) on the X axis. Selected genes are labelled.
Table 2.
Top upregulated (>3.4 Log2 Fold change) and down regulated (< 3.5 Log2 Fold change) genes in the transcriptomic analysis.
Fig 6.
(A) GO enrichment bubble plots showing the GO terms for the top 25 upregulated DEG with the lowest adjusted P values on the y axis and gene ratios on the X axis. (B) GO enrichment bubble plots showing the GO terms for the top 25 down regulated DEG genes.
Table 3.
Gene names, the total number of differentially expressed proteins (DEP) with increased or decreased expression in their respective functional categories, DEP with >0.7 Log2 fold change (FC), and P values determined by a Benjamini-Hochberg analysis.
Functional categories with DEP that did not meet the 0.5 Lg 2FC threshold included purine nucleotides (N = 2 DEP), antioxidants (N = 4), smooth muscle (N = 4), red blood cells (N = 3), lipids (N = 3), and miscellaneous (N = 11) for a total of 166 DEP out of 913 total detected proteins.
Fig 7.
Volcano Plot for proteomic analysis and correlation to transcription.
(A) Volcano plot depicting estimated P-values for differential protein expression versus log2 fold change. Significant DEP are shown in red. (B) Significant weak correlation between differentially expressed proteins (DEP, log2fold change) and expression of the encoding genes. Select proteins are labelled.
Fig 8.
mRNA sequences in the 3’UTR of DMPK.
(A) VCF file of mRNA sequences in the 3’ UTR of the DMPK gene in 5 eMD and 5 control horses. The yellow highlighted region represents the untranslated region for exon 1 and did not show evidence of expanded trinucleotide repeats that cause human DM1. (B) Alignment of the 3’ untranslated region (UTR) region of DMPK (EquCab 3.0) for 2 eMD and 2 control horses (RNA sequencing depth > 10 reads), compared to the same region in the human reference genome (GRch38.p14) and humans with myotonic dystrophy 1 (DM1). The highlighted region denotes some reads that had a six base pair insertion found in eMD5 and control 5.
Table 4.
The CLCN1 and SCN4A variants identified in the transcriptomic data listing the chromosomal location, the reference or wild type variant, the number of horses with the variant in control and equine myotonic dystrophy (eMD) horses and the predicted consequence of the variant.
Fig 9.
Expression of exon 7 in CLCN1.
(A) mRNA sequences in the region of CLCN1 exon 7 in 5 eMD and 5 control horses showing no clear evidence of retention of exon 7 exclusively in eMD versus control horses as seen in human DM1. (B) A sashimi plot depicting spicing analysis of exon 7. The read density is expressed as a horizontal histogram and splice junction reads are shown as arcs connecting exons with the thickness representing read counts.
Fig 10.
PCR amplification of CLCN1 and evaluation of alternative splicing.
(A) Agarose gel of PCR products from amplification of CLCN1 exons 5 through 10. WT = control horses, A1-A5 represents individual eMD horses, WT1 through 5 represent controls. (B) The frequency of the CLCN1 isoforms containing variations of exons 5 through 10 in eMD and control horses. Values are expressed as the mean percentage of the total sequenced transcripts.
Table 5.
Breed, age and sex of equine myotonic dystrophy (eMD) and control horses as well as the samples used for histology, fiber type composition, presence of embryonic myosin, transcriptomic and proteomic analyses.
Fig 11.
(A) Diffuse waxing and waning myotonic potentials that never ceased found throughout out all muscles except the semimembranosus muscle in Horse 2. (B) Myotonic discharges in Horse 3.
Fig 12.
Hindquarter muscle development in eMD Horses.
(A) Horse 1 with pronounced muscle development and focal muscle contractures in the semimembranosus muscle at 2 months of age. (B) Horse 2 with prominent development of the middle gluteal and semitendinosus muscles at 3 months of age. (C) Horse 3 with large semitendinosus and semimembranosus muscles at 3 years of age. (D) Horse 4 with contractures in the middle gluteal muscles at 2 years of age. (E) Horse 5 with progressive hypotrophy of the superficial gluteal and biceps femoris muscles at 2 years of age. (F) Horse 6 with progressive atrophy of the middle gluteal muscle and hypertrophy of the superficial gluteal muscle.
Table 6.
PCR primers used to amplify exon 5 - 10 of CLCN1 and exons21 −23 of ATP2A1.