An autosomal recessive variant in PYGM causes myophosphorylase deficiency in Red Angus composite cattle.

BACKGROUND: Between 2020 and 2022, eight calves in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) displayed exercise intolerance during forced activity. In some cases, the calves collapsed and did not recover. Available sire pedigrees contained a paternal ancestor within 2-4 generations in all affected calves. Pedigrees of the calves' dams were unavailable, however, the cows were ranch-raised and retained from prior breeding seasons, where bulls used for breeding occasionally had a common ancestor. Therefore, it was hypothesized that a de novo autosomal recessive variant was causative of exercise intolerance in these calves. RESULTS: A genome-wide association analysis utilizing SNP data from 6 affected calves and 715 herd mates, followed by whole-genome sequencing of 2 affected calves led to the identification of a variant in the gene PYGM (BTA29:g.42989581G > A). The variant, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon (p.Arg650*). The protein product of PYGM, myophosphorylase, breaks down glycogen in skeletal muscle. Glycogen concentrations were fluorometrically assayed as glucose residues demonstrating significantly elevated glycogen concentrations in affected calves compared to cattle carrying the variant and to wild-type controls. The absence of the PYGM protein product in skeletal muscle was confirmed by immunohistochemistry and label-free quantitative proteomics analysis; muscle degeneration was confirmed in biopsy and necropsy samples. Elevated skeletal muscle glycogen persisted after harvest, resulting in a high pH and dark-cutting beef, which is negatively perceived by consumers and results in an economic loss to the industry. Carriers of the variant did not exhibit differences in meat quality or any measures of animal well-being. CONCLUSIONS: Myophosphorylase deficiency poses welfare concerns for affected animals and negatively impacts the final product. The association of the recessive genotype with dark-cutting beef further demonstrates the importance of genetics to not only animal health but to the quality of their product. Although cattle heterozygous for the variant may not immediately affect the beef industry, identifying carriers will enable selection and breeding strategies to prevent the production of affected calves.

Pseudohyperkalemia in horses with rhabdomyolysis reported by an enzymatic chemistry analyzer.

OBJECTIVE: To investigate pseudohyperkalemia occurring in horses experiencing rhabdomyolysis when serum chemistry profiles are run on an VetScan VS2 analyzer (Abaxis). ANIMALS: 18 horses with rhabdomyolysis (creatine kinase [CK] > 1,000 U/L). METHODS: In 3 horses with serum CK activities > 5,800 U/L and persistent serum potassium concentrations of > 8.5 mmol/L (VetScan VS2), potassium concentrations were reevaluated with either i-STAT Alinity Base Station (Abbott), Catalyst (Idexx), or Cobas c501 (Roche) ion-specific analyzers. Paired serum samples from 15 additional horses (median serum CK activity, 7,601 U/L; range, 1,134 to 192,447 U/L) were analyzed on both VetScan VS2 and Cobas c501 machines. Serum potassium concentrations were compared between the VetScan VS2 and ion-specific analyzers by Bland-Altman and Wilcoxon ranked tests and correlated to log10 CK activity via Pearson correlation. RESULTS: Serum potassium concentrations were significantly higher on the VetScan VS2 (6.7 ± 1.6 mmol/L) versus the ion-specific analyzers (4.0 ± 1.1 mmol/L; P < .0001), with high bias shown in Bland-Altman analysis (43.1 ± 27.9). Potassium concentrations positively correlated with log10 CK activity with the VetScan VS2 (R2 = 0.51; P = .003) but not the Cobas (R2 = 0.09; P = .3) analyzer. CLINICAL RELEVANCE: An alternate analyzer to the VetScan VS2 should be used to evaluate serum potassium concentrations in horses with rhabdomyolysis because the VetScan VS2 methodology uses lactate dehydrogenase, which increases in serum with rhabdomyolysis and falsely elevates potassium concentrations.

Novel Expression of GLUT3, GLUT6 and GLUT10 in Equine Gluteal Muscle Following Glycogen-Depleting Exercise: Impact of Dietary Starch and Fat.

Horses have a slow rate of muscle glycogen repletion relative to other species for unknown reasons. Our aim was to determine the expression of glucose transporters (GLUT) and genes impacting GLUT4 expression and translocation in the gluteal muscle. Five fit Thoroughbred horses performed glycogen-depleting exercises on high-starch (HS, 2869 g starch/day) and low-starch, high-fat diets (LS-HF, 358 g starch/d) with gluteal muscle biopsies obtained before and after depletion and during repletion. Muscle glycogen declined by ≈30% on both diets with little increase during repletion on LS-HF. Transcriptomic analysis identified differential expression (DE) of only 2/12 genes impacting GLUT4 translocation (two subunits of AMP protein kinase) and only at depletion on LS-HF. Only 1/13 genes encoding proteins that promote GLUT4 transcription had increased DE (PPARGC1A at depletion LS-HF). GLUT4 comprised ≈30% of total GLUT mRNA expression at rest. Remarkably, by 72 h of repletion expression of GLUT3, GLUT6 and GLUT10 increased to ≈25% of total GLUT mRNA. Expression of GLUT6 and GLUT10 lagged from 24 h of repletion on HS to 72 h on LS-HF. Lacking an increase in GLUT4 gene expression in response to glycogen-depleting exercise, equine muscle increases GLUT3, GLUT6 and GLUT10 expression potentially to enhance glucose transport, resembling responses observed in resistance trained GLUT4-null mice.

Cerebellar axonopathy in Shivers horses identified by spatial transcriptomic and proteomic analyses.

BACKGROUND: Shivers in horses is characterized by abnormal hindlimb movement when walking backward and is proposed to be caused by a Purkinje cell (PC) axonopathy based on histopathology. OBJECTIVES: Define region-specific differences in gene expression within the lateral cerebellar hemisphere and compare cerebellar protein expression between Shivers horses and controls. ANIMALS: Case-control study of 5 Shivers and 4 control geldings ≥16.2 hands in height. METHODS: Using spatial transcriptomics, gene expression was compared between Shivers and control horses in PC soma and lateral cerebellar hemisphere white matter, consisting primarily of axons. Tandem-mass-tag (TMT-11) proteomic analysis was performed on lateral cerebellar hemisphere homogenates. RESULTS: Differences in gene expression between Shivers and control horses were evident in principal component analysis of axon-containing white matter but not PC soma. In white matter, there were 455/1846 differentially expressed genes (DEG; 350 ↓DEG, 105 ↑DEG) between Shivers and controls, with significant gene set enrichment of the Toll-Like Receptor 4 (TLR4) cascade, highlighting neuroinflammation. There were 50/936 differentially expressed proteins (DEP). The 27 ↓DEP highlighted loss of axonal proteins including intermediate filaments (5), myelin (3), cytoskeleton (2), neurite outgrowth (2), and Na/K ATPase (1). The 23 ↑DEP were involved in the extracellular matrix (7), cytoskeleton (7), redox balance (2), neurite outgrowth (1), signal transduction (1), and others. CONCLUSION AND CLINICAL IMPORTANCE: Our findings support axonal degeneration as a characteristic feature of Shivers. Combined with histopathology, these findings are consistent with the known distinctive response of PC to injury where axonal changes occur without a substantial impact on PC soma.

Impact of Coenzyme Q10 Supplementation on Skeletal Muscle Respiration, Antioxidants, and the Muscle Proteome in Thoroughbred Horses.

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transfer system and a potent antioxidant. The impact of CoQ10 supplementation on mitochondrial capacities and the muscle proteome is largely unknown. This study determined the effect of CoQ10 supplementation on muscle CoQ10 concentrations, antioxidant balance, the proteome, and mitochondrial respiratory capacities. In a randomized cross-over design, six Thoroughbred horses received 1600 mg/d CoQ10 or no supplement (control) for 30-d periods separated by a 60-d washout. Muscle samples were taken at the end of each period. Muscle CoQ10 and glutathione (GSH) concentrations were determined using mass spectrometry, antioxidant activities by fluorometry, mitochondrial enzyme activities and oxidative stress by colorimetry, and mitochondrial respiratory capacities by high-resolution respirometry. Data were analyzed using mixed linear models with period, supplementation, and period × supplementation as fixed effects and horse as a repeated effect. Proteomics was performed by tandem mass tag 11-plex analysis and permutation testing with FDR < 0.05. Concentrations of muscle CoQ10 (p = 0.07), GSH (p = 0.75), and malondialdehyde (p = 0.47), as well as activities of superoxide dismutase (p = 0.16) and catalase (p = 0.66), did not differ, whereas glutathione peroxidase activity (p = 0.003) was lower when horses received CoQ10 compared to no supplement. Intrinsic (relative to citrate synthase activity) electron transfer capacity with complex II (ECII) was greater, and the contribution of complex I to maximal electron transfer capacity (FCRPCI and FCRPCIG) was lower when horses received CoQ10 with no impact of CoQ10 on mitochondrial volume density. Decreased expression of subunits in complexes I, III, and IV, as well as tricarboxylic acid cycle (TCA) enzymes, was noted in proteomics when horses received CoQ10. We conclude that with CoQ10 supplementation, decreased expression of TCA cycle enzymes that produce NADH and complex I subunits, which utilize NADH together with enhanced electron transfer capacity via complex II, supports an enhanced reliance on substrates supplying complex II during mitochondrial respiration.

Absence of myofibrillar myopathy in Quarter Horses with a histopathological diagnosis of type 2 polysaccharide storage myopathy and lack of association with commercial genetic tests.

BACKGROUND: Genetic tests for variants in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323 or P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) genes are offered commercially to diagnose myofibrillar myopathy (MFM) and type 2 polysaccharide storage myopathy (PSSM2) in Quarter Horses (QH). OBJECTIVES: To determine if PSSM2-QH has histopathological features of MFM. To compare genotype and allele frequencies of variants P2, P3, P4 between control-QH and PSSM2-QH diagnosed by histopathology. STUDY DESIGN: Retrospective cross-sectional. METHODS: The study includes a total of 229 healthy control-QH, 163 PSSM2-QH GYS1 mutation negative. Desmin stains of gluteal/semimembranosus muscle were evaluated. Purported disease alleles P2, P3a, P3b, P4 were genotyped by pyrosequencing. Genotype, allele frequency and total number of variant alleles or loci were compared between phenotypes using additive/genotypic and dominant models and quantitative effects evaluated by multivariable logistic regression. RESULTS: Histopathological features of MFM were absent in all QH. A P variant allele at any locus was not associated (P > .05) with a histopathological diagnosis of PSSM2 and one or more P variants were common in control-QH (57%) and PSSM2-QH (61%). Allele frequencies (control/PSSM2) were: 0.24/0.21 (P2), 0.07/0.12 (P3a), 0.07/0.11 (P3b) and 0.06/0.08 (P4). P3a and P3b loci were not independent (r2  = 0.894); and not associated with PSSM2 histopathology comparing the haplotype of both P3a and P3b variants to other haplotypes. A receiver operator curve did not accurately predict the PSSM2 phenotype (AUC = 0.67, 95% CI 0.62-0.72), and there was no difference in the total number of variant loci or total variant allele count between control-QH and PSSM2-QH. MAIN LIMITATIONS: P3a and P3b were not in complete linkage disequilibrium. CONCLUSIONS: The P2, P3 and P4 variants in genes associated with human MFM were not associated with PSSM2 in 392 QH. Their use would improperly diagnose PSSM2/MFM in 57% of healthy QH and fail to diagnose PSSM2 in 40% of QH with histopathological evidence of PSSM2.

CONTEXTO: Testes genéticos para detecção das mutações MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323 ou P3b; rs1142918816) e MYOZ3 (P4; rs1142544043) são oferecidos comercialmente para diagnosticar miopatia miofibrilar (MMF) e miopatia por acúmulo de polissacarídeo tipo 2 (PSSM2) em cavalos Quarto de Milha (QM). HIPÓTESES/OBJETIVOS: Determinar se PSSM2-QM tem características similares à MMF. Comparar o genótipo e a frequência dos alelos variantes P2, P3, e P4 entre cavalos QM controle, e PSSM2-QM diagnosticados por histologia. MÉTODOS: 229 cavalos QH saudáveis como controle, e 163 PSSM2-QM positivos na histologia e negativos para a mutação GYS1. METODOLOGIA: Amostras dos músculos glúteo/semimembranoso foram avaliadas após coloração com desmina. Os pretensos genes alelos P2, P3a, P3b e P4 foram genotipados por pirosequenciamento. Genótipo, frequência alélica, e número total de variância alélica ou loci foram comparados entre os fenótipos usado aditivo/genotípico e modelos dominantes e efeitos quantitativos através de regressão logística multivariável. RESULTADOS: Características histopatológicas de MMF não foram encontradas em nenhum QM. Uma variante alélica P em qualquer uma dos loci não foi associada (P > .05) com o diagnóstico histopatológicos de PSSM2 e uma ou mais variante P foram comuns em QM controles (57%) e PSSM2-QM (61%). Frequência alélica (controle/PSSM2) foram: 0.24/0.21 (P2), 0.07/0.12 (P3a), 0.07/0.11 (P3b), e 0.06/0.08 (P4). P3a e P3b loci não foram independentes (r2  = 0.894); e não foram associados com achados histopatológicos de PSSM quando comparando o haplótipo de ambas as variantes P3a e P3b com os outros haplótipos. A curva característica de operação do receptor não previu acuradamente o fenótipo PSSM2 (AUC = 0.67, 95% IC 0.62-0.72), e não houve diferença no número dotal de variantes no loci ou na contagem de variantes alélicas total entre QM controles e PSSM2-QM. PRINCIPAIS LIMITAÇÕES: P3a e P3b não estavam em desequilíbrio de ligação. CONCLUSÕES: As variantes P2, P3 e P4 em genes associados com MMF em humanos não foram associadas com PSSM em 392 QM. O seu uso diagnosticaria impropriamente PSSM2 e MMF em 57% dos cavalos saudáveis utilizados como controle e não diagnosticaria PSSM2 em 40% dos QM com evidência histológica de PSSM2.

Type 2 polysaccharide storage myopathy in Quarter Horses is a novel glycogen storage disease causing exertional rhabdomyolysis.

BACKGROUND: Both type 1 (PSSM1) and type 2 polysaccharide storage myopathy (PSSM2) are characterised by aggregates of abnormal polysaccharide in skeletal muscle. Whereas the genetic basis for PSSM1 is known (R309H GYS1), the cause of PSSM2 in Quarter Horses (PSSM2-QH) is unknown and glycogen concentrations not defined. OBJECTIVES: To characterise the histopathological and biochemical features of PSSM2-QH and determine if an associated monogenic variant exists in genes known to cause glycogenosis. STUDY DESIGN: Retrospective case control. METHODS: Sixty-four PSSM2-QH, 30 PSSM1-QH and 185 control-QH were identified from a biopsy repository and clinical data, histopathology scores (0-3), glycogen concentrations and selected glycolytic enzyme activities compared. Coding sequences of 12 genes associated with muscle glycogenoses were identified from whole genome sequences and compared between seven PSSM2-QH and five control-QH. RESULTS: Exertional rhabdomyolysis in PSSM2-QH occurred predominantly in barrel racing and working cow/roping performance types and improved with regular exercise and a low starch/fat-supplemented diet. Histopathological scores, including the amount of amylase-resistant polysaccharide (PSSM2-QH 1.4 ± 0.6, PSSM1-QH 2.1 ± 0.3, control-QH 0 ± 0, p < 0.001), and glycogen concentrations (PSSM2-QH 129 ± 62, PSSM1-QH 175 ± 9, control-QH 80 ± 27 mmol/kg, p < 0.0001) were intermediate in PSSM2-QH with significant differences among groups. In PSSM2-QH, abnormal polysaccharide had a less filamentous ultrastructure than PSSM1-QH and phosphorylase and phosphofructokinase activities were normal. Seventeen of 30 PSSM2-QH with available pedigrees descended from one of three stallions within four generations. Of the 29 predicted high or moderate impact genetic variants identified in candidate genes, none were present in only PSSM2-QH and absent in control-QH. MAIN LIMITATIONS: Analyses of PSSM2-QH and PSSM1-QH were performed on shipped samples, controls on frozen samples. CONCLUSIONS: PSSM2-QH is a novel glycogen storage disorder that is not the result of a mutation in genes currently known to cause muscle glycogenoses in other species.

CONTEXTO: Ambos os tipos 1 e 2 de miopatia por acúmulo de polissacarídeo (PSSM) são caracterizados por agregados de polissacarídeos anormais no músculo esquelético. Enquanto a base genética do PSSM 1 é conhecida (R309H GYS1), a causa do PSSM2 em cavalos Quarto de Milha (PSSM2-QH) é desconhecida, e a concentração de glicogênio não é definida. OBJETIVOS: Identificar as características histopatológicas e bioquímicas do PSSM-QH e determinar se há uma variante monogênica em genes conhecidos por causar glicogenose. DELINEAMENTO DO ESTUDO: Caso controlado retrospectivo. METODOLOGIA: 64 PSSM2-QH, 30 PSSM1-QH e 185 QH controles foram identificados em um arquivo de dados. Informação clínica, achados histológicos (escala 0-3), concentração de glicogênio e atividade enzimática de algumas enzimas glicolíticas foram comparadas. Sequências codificadas de 12 genes associados com glicogenose muscular foram identificados nas sequências genômicas completas, e comparadas entre 7 PSSM2-QH e 5 QH controles. RESULTADOS: Rabdomiólise por exercício em PSSM2-QH ocorreu predominantemente em cavalos de corrida de tambor e cavalos de team roping/trabalho com gado, e melhorou com exercício regular e uma dieta com baixo amido e alta gordura. A escala histopatológica, incluindo a quantidade de polissacarídeos resistentes à amilase (PSSM2-QH 1.4 ± 0.6, PSSM1-QH 2.1 ± 0.3, controle-QH 0 ± 0, P < 0.001), e concentrações de glicogênio (PSSM2-QH 129 ± 62, PSSM1-QH 175 ± 9, controle-QH 80 ± 27 mmol/kg, P < 0.0001) foram intermediárias em PSSM2-QH com diferença significante entre grupos. Em PSSM2-QH, polissacarídeo anormal teve uma ultraestrutura menos filamentosa do que PSSM1-QH e as atividades de fosforilase e fosfofrutoquinase foram normais. Dezessete dos 30 PSSM2-QH com pedigree disponível descendiam de 1 de 3 garanhões dentro de 4 gerações. Das 29 variações genéticas preditas a terem impacto moderado ou alto como genes candidatos, nenhuma estava presente apenas em PSSM2-QH e ausente no grupo controle-QH. PRINCIPAIS LIMITAÇÕES: As análises feitas nas amostras de PSSM2-QH e PSSM1-QH foram realizadas em amostras enviadas por correio, e as amostras dos animais controles eram amostras congeladas. CONCLUSÕES: PSSM2-QH é uma nova doença por acúmulo de glicogênio que não é o resultado de uma mutação nos genes conhecidos por causarem glicogenose muscular em outras espécies.

Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis.

Certain Standardbred racehorses develop recurrent exertional rhabdomyolysis (RER-STD) for unknown reasons. We compared gluteal muscle histopathology and gene/protein expression between Standardbreds with a history of, but not currently experiencing rhabdomyolysis (N = 9), and race-trained controls (N = 7). Eight RER-STD had a few mature fibers with small internalized myonuclei, one out of nine had histologic evidence of regeneration and zero out of nine degeneration. However, RER-STD versus controls had 791/13,531 differentially expressed genes (DEG). The top three gene ontology (GO) enriched pathways for upregulated DEG (N = 433) were inflammation/immune response (62 GO terms), cell proliferation (31 GO terms), and hypoxia/oxidative stress (31 GO terms). Calcium ion regulation (39 GO terms), purine nucleotide metabolism (32 GO terms), and electron transport (29 GO terms) were the top three enriched GO pathways for down-regulated DEG (N = 305). DEG regulated RYR1 and sarcoplasmic reticulum calcium stores. Differentially expressed proteins (DEP ↑N = 50, ↓N = 12) involved the sarcomere (24% of DEP), electron transport (23%), metabolism (20%), inflammation (6%), cell/oxidative stress (7%), and other (17%). DEP included ↑superoxide dismutase, ↑catalase, and DEP/DEG included several cysteine-based antioxidants. In conclusion, gluteal muscle of RER-susceptible Standardbreds is characterized by perturbation of pathways for calcium regulation, cellular/oxidative stress, inflammation, and cellular regeneration weeks after an episode of rhabdomyolysis that could represent therapeutic targets.

Skeletal Muscle Fiber Type Composition and Citrate Synthase Activity in Fit and Unfit Warmbloods and Quarter Horses.

Selective breeding and discipline specific training has led to equine breeds adept at various athletic disciplines. Breed-specific skeletal muscle adaptations have been studied in many breeds but not Warmbloods (WB). We evaluated gluteal muscle contractile muscle fiber types and citrate synthase activity (CS), a marker for mitochondrial volume density, in WB trained for dressage (second level-Grand Prix) contrasted with Quarter Horses (QH). Gluteus medius muscle biopsies from 14 unfit/18 fit dressage-trained WB and 20 unfit/16 fit reining/working cow QH were analyzed fluorometrically and fiber types determined by ATPase activity. Comparisons were made by one-way ANOVA. Unfit and fit WB had significantly higher % type 1 and lower % type 2X fibers than QH. Unfit WB had significantly higher CS than unfit QH but CS did not differ between fit WB and fit QH. CS was only significantly higher in fit versus unfit QH, not fit versus unfit WB. In conclusion, WB gluteal muscle has an inherently high % type 1/low % type 2X fibers and high mitochondrial content whether unfit or trained for dressage, contrasting QH with an inherently low % type 1/high % type 2X and low mitochondrial content, that was enhanced in fit QH. Similar CS activity in fit WB versus QH despite a two-fold difference in % type 2X fibers indicates that mitochondrial volume density cannot accurately be predicted from contractile fiber type composition.

Movement Disorders and Cerebellar Abiotrophy.

Movement disorders are defined as involuntary movements that are not due to a painful stimulus or associated with changes in consciousness or proprioception. Diagnosis involves ruling out any lameness and neurologic disease and characterizing the gait during walking backward and forward and trotting. Shivers causes abnormal hindlimb hypertonicity during walking backward and, when advanced, a few strides walking forward. Stringhalt causes consistent hyperflexion during walking forward and trotting and variable difficulty when walking backward. Classification and potential causes are discussed as well as other enigmatic movement disorders in horses are presented. Cerebellar abiotrophy is reviewed.

Prevalence of clinical signs and factors impacting expression of myosin heavy chain myopathy in Quarter Horse-related breeds with the MYH1E321G mutation.

BACKGROUND: The prevalence of clinical signs and factors triggering muscle atrophy and rhabdomyolysis associated with an MYH1E321G mutation in Quarter Horses and related breeds (QH) remain poorly understood. HYPOTHESIS/OBJECTIVES: Determine the prevalence and potential triggers of atrophy and stiffness in horses homozygous reference (N/N), heterozygous (My/N), and homozygous (My/My) for the MYH1E321G mutation. ANIMALS: Two-hundred seventy-five N/N, 100 My/N, and 10 My/My QH. METHODS: A retrospective case-control study using a closed-ended questionnaire completed by clients of the Veterinary Genetics Laboratory at the University of California, Davis. History of clinical signs, disease, vaccination and performance were analyzed by genotype using contingency testing. RESULTS: Atrophy occurred in proportionately more horses with MYH1E321G (My) than N/N QH and more frequently in My/My than My/N QH (P < .001; My/My 8/10 [80%], My/N 17/100 [17%], N/N 29/275 [11%]). More My/My horses had rapid atrophy (P < .001), with recurrence in 50%. Fewer My/My horses recovered versus My/N QH (P < .001). Stiffness was common across genotypes (P = .100; My/My 4/10 [40%], My/N 18/100 [18%], N/N 48/275 [17%]). Three months before the observed atrophy and stiffness, 47% of MYH1E321G QH were vaccinated or had respiratory or gastrointestinal disease. Horses achieving 100% expected performance did not differ across genotypes (50% My/My, 71% My/N, 55% N/N), but, only 4/10 My/My QH were competing. My/N horses achieved national or world championships or both. CONCLUSION AND CLINICAL IMPORTANCE: Approximately 20% of My/N QH develop rapid atrophy. Atrophy is more common (80%) in homozygous My/My QH and less likely to resolve. Inciting causes such as vaccination and infection are inapparent in over half of cases.

Serum and cerebrospinal fluid phosphorylated neurofilament heavy protein concentrations in equine neurodegenerative diseases.

BACKGROUND: Currently, there is little information regarding the concentrations of phosphorylated neurofilament heavy protein (pNfH) in the serum and cerebrospinal fluid (CSF) of horses with neurodegenerative diseases. Specifically, pNfH concentrations have not yet been evaluated in horses with equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM). OBJECTIVES: To determine pNfH concentrations using a commercial enzyme-linked immunosorbent assay (ELISA) in serum and CSF from control horses and horses with eNAD/EDM, cervical vertebral compressive myelopathy (CVCM) and Shivers. STUDY DESIGN: Case-control study using biobanked samples from diseased horses and prospective or biobanked samples from control horses. METHODS: The pNfH ELISA was performed on samples from horses diagnosed with eNAD/EDM (n = 64), CVCM (n = 26) and Shivers (n = 9) and 51 neurologically normal control horses. RESULTS: Median and 95% confidence interval (CI) serum pNfH concentrations in control, CVCM, and eNAD/EDM horses were 0.08 ng/mL (0.07-0.15), 0.07 ng/mL (0.07-0.15) and 0.07 ng/mL (0.07-1.13), respectively. Serum pNfH concentrations were below the limit of detection (<0.07 ng/mL) for all Shivers horses. CSF pNfH concentrations in control, CVCM-, eNAD/EDM- and Shivers-affected horses were 1.26 ng/mL (1.06-1.5), 3.07 ng/mL (1.15-29.9), 1.78 ng/mL (1.5-2.28) and 1.39 ng/mL (0.74-3.89), respectively. CSF pNfH concentrations were significantly higher in CVCM (P = .001) and eNAD/EDM (P  = .01) affected horses compared to control horses. Serum pNfH concentrations >1 ng/mL were significantly associated with eNAD/EDM (P = .01) with only 12% sensitivity but 99% specificity. CSF pNfH concentrations >3 ng/mL were significantly associated with CVCM (P = .0002), with 50% sensitivity and 86% specificity. MAIN LIMITATIONS: A limited number of control horses tested were <1 year of age. CONCLUSIONS: Serum pNfH concentrations are specifically increased (>1 ng/mL) in some horses with eNAD/EDM. Increased CSF pNfH concentrations (>3 ng/mL) can be observed with eNAD/EDM or CVCM.

Hand-Held Point-of-Care Ultrasound: A New Tool for Veterinary Student Self-Driven Learning in the Time of COVID-19.

The coronavirus pandemic abruptly halted all in-person clerkships, or clinical rotations, for clinical veterinary students across the United States. Online clerkships in radiology offered the opportunity to expand the student's ability to interpret medical images but did not allow for the development of physical hands-on imaging skills recognized as core competencies in veterinary medicine. The present report highlights the value of providing veterinary students with a smartphone-associated Butterfly iQ point-of-care ultrasound during a 3-week self-driven virtual clerkship. During the virtual rotation, the student was able to develop the skills required to generate sufficient quality images using three horses residing on her property. The affordability, portability, ease of use of the Butterfly iQ and availability of animals made it possible to develop hands-on imaging skills when distance learning was required.

Prevalence of the E321G MYH1 variant in Brazilian Quarter Horses.

BACKGROUND: In the Quarter Horse (QH), myosin heavy chain myopathy (MYHM), which is characterised by nonexertional rhabdomyolysis or immune-mediated myositis (IMM) with acute muscle atrophy, is strongly associated with the missense E321G MYH1 mutation. OBJECTIVES: To document the existence of MYHM in the Brazilian QH population, this study includes a case report of two related QH foals with the E321G MYH1 mutation that had clinical signs of MYHM, with histological confirmation of IMM in one of the foals. This prompted an investigation the aim of which was to determine the allele frequency of the E321G MYH1 variant across QHs using a DNA archive in Brazil. Study design Cross sectional. METHODS: To estimate the allele frequency of the E321G MYH1 variant in Brazilian QHs, 299 DNA samples from QHs used in different disciplines (reining, barrel racing, halter, cutting and racing) were analysed. DNA fragments containing the region with the mutation were amplified by PCR and used for direct genomic sequencing. RESULTS: Of the 299 genotyped QHs, 44 animals (14.7%) were heterozygous (My/N) for the E321G MYH1 variant, and 255 (85.3%) were homozygous for the wild-type allele (N/N), implying an allele frequency of 0.074. Reining horses had a significantly higher prevalence of heterozygosity than horses in other disciplines (P = .008). MAIN LIMITATIONS: The DNA samples were collected from 2010 to 2014. As only registered QHs were evaluated, the results may not reflect the actual incidence in the general population of Brazilian QHs. CONCLUSIONS: The reported cases of MYHM and the high prevalence of the MYH1 mutation found in the assessed Brazilian QH population, particularly in reining QHs, suggests that MYHM should be included in genetic screening. Reasonable control measures are important to prevent an increase in the incidence of MYHM in QHs in Brazil.

Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport.

We have analyzed the enzymatic activity of the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) from the horse gluteal muscle. Horses are bred for peak athletic performance yet exhibit a high incidence of exertional rhabdomyolysis, with elevated levels of cytosolic Ca2+ proposed as a correlative linkage. We recently reported an improved protocol for isolating SR vesicles from horse muscle; these horse SR vesicles contain an abundant level of SERCA and only trace-levels of sarcolipin (SLN), the inhibitory peptide subunit of SERCA in mammalian fast-twitch skeletal muscle. Here, we report that the in vitro Ca2+ transport rate of horse SR vesicles is 2.3 ± 0.7-fold greater than rabbit SR vesicles, which express close to equimolar levels of SERCA and SLN. This suggests that horse myofibers exhibit an enhanced SR Ca2+ transport rate and increased luminal Ca2+ stores in vivo. Using the densitometry of Coomassie-stained SDS-PAGE gels, we determined that horse SR vesicles express an abundant level of the luminal SR Ca2+ storage protein calsequestrin (CASQ), with a CASQ-to-SERCA ratio about double that in rabbit SR vesicles. Thus, we propose that SR Ca2+ cycling in horse myofibers is enhanced by a reduced SLN inhibition of SERCA and by an abundant expression of CASQ. Together, these results suggest that horse muscle contractility and susceptibility to exertional rhabdomyolysis are promoted by enhanced SR Ca2+ uptake and luminal Ca2+ storage.

Myofibre Hyper-Contractility in Horses Expressing the Myosin Heavy Chain Myopathy Mutation, MYH1E321G.

Myosinopathies are defined as a group of muscle disorders characterized by mutations in genes encoding myosin heavy chains. Their exact molecular and cellular mechanisms remain unclear. In the present study, we have focused our attention on a MYH1-related E321G amino acid substitution within the head region of the type IIx skeletal myosin heavy chain, associated with clinical signs of atrophy, inflammation and/or profound rhabdomyolysis, known as equine myosin heavy chain myopathy. We performed Mant-ATP chase experiments together with force measurements on isolated IIx myofibres from control horses (MYH1E321G-/-) and Quarter Horses homozygous (MYH1E321G+/+) or heterozygous (MYH1E321G+/-) for the E321G mutation. The single residue replacement did not affect the relaxed conformations of myosin molecules. Nevertheless, it significantly increased its active behaviour as proven by the higher maximal force production and Ca2+ sensitivity for MYH1E321G+/+ in comparison with MYH1E321G+/- and MYH1E321G-/- horses. Altogether, these findings indicate that, in the presence of the E321G mutation, a molecular and cellular hyper-contractile phenotype occurs which could contribute to the development of the myosin heavy chain myopathy.

The Impact of N-Acetyl Cysteine and Coenzyme Q10 Supplementation on Skeletal Muscle Antioxidants and Proteome in Fit Thoroughbred Horses.

Horses have one of the highest skeletal muscle oxidative capacities amongst mammals, which, combined with a high glycolytic capacity, could perturb redox status during maximal exercise. We determined the effect of 30 d of oral coenzyme Q10 and N-acetyl-cysteine supplementation (NACQ) on muscle glutathione (GSH), cysteine, ROS, and coenzyme Q10 concentrations, and the muscle proteome, in seven maximally exercising Thoroughbred horses using a placebo and randomized cross-over design. Gluteal muscle biopsies were obtained the day before and 1 h after maximal exercise. Concentrations of GSH, cysteine, coenzyme Q10, and ROS were measured, and citrate synthase, glutathione peroxidase, and superoxide dismutase activities analyzed. GSH increased significantly 1 h post-exercise in the NACQ group (p = 0.022), whereas other antioxidant concentrations/activities were unchanged. TMT proteomic analysis revealed 40 differentially expressed proteins with NACQ out of 387 identified, including upregulation of 13 mitochondrial proteins (TCA cycle and NADPH production), 4 Z-disc proteins, and down regulation of 9 glycolytic proteins. NACQ supplementation significantly impacted muscle redox capacity after intense exercise by enhancing muscle glutathione concentrations and increasing expression of proteins involved in the uptake of glutathione into mitochondria and the NAPDH-associated reduction of oxidized glutathione, without any evident detrimental effects on performance.

Integrated proteomic and transcriptomic profiling identifies aberrant gene and protein expression in the sarcomere, mitochondrial complex I, and the extracellular matrix in Warmblood horses with myofibrillar myopathy.

BACKGROUND: Myofibrillar myopathy in humans causes protein aggregation, degeneration, and weakness of skeletal muscle. In horses, myofibrillar myopathy is a late-onset disease of unknown origin characterized by poor performance, atrophy, myofibrillar disarray, and desmin aggregation in skeletal muscle. This study evaluated molecular and ultrastructural signatures of myofibrillar myopathy in Warmblood horses through gluteal muscle tandem-mass-tag quantitative proteomics (5 affected, 4 control), mRNA-sequencing (8 affected, 8 control), amalgamated gene ontology analyses, and immunofluorescent and electron microscopy. RESULTS: We identified 93/1533 proteins and 47/27,690 genes that were significantly differentially expressed. The top significantly differentially expressed protein CSRP3 and three other differentially expressed proteins, including, PDLIM3, SYNPO2, and SYNPOL2, are integrally involved in Z-disc signaling, gene transcription and subsequently sarcomere integrity. Through immunofluorescent staining, both desmin aggregates and CSRP3 were localized to type 2A fibers. The highest differentially expressed gene CHAC1, whose protein product degrades glutathione, is associated with oxidative stress and apoptosis. Amalgamated transcriptomic and proteomic gene ontology analyses identified 3 enriched cellular locations; the sarcomere (Z-disc & I-band), mitochondrial complex I and the extracellular matrix which corresponded to ultrastructural Z-disc disruption and mitochondrial cristae alterations found with electron microscopy. CONCLUSIONS: A combined proteomic and transcriptomic analysis highlighted three enriched cellular locations that correspond with MFM ultrastructural pathology in Warmblood horses. Aberrant Z-disc mechano-signaling, impaired Z-disc stability, decreased mitochondrial complex I expression, and a pro-oxidative cellular environment are hypothesized to contribute to the development of myofibrillar myopathy in Warmblood horses. These molecular signatures may provide further insight into diagnostic biomarkers, treatments, and the underlying pathophysiology of MFM.

Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses.

Recurrent exertional rhabdomyolysis (RER) is a chronic muscle disorder of unknown etiology in racehorses. A potential role of intramuscular calcium (Ca2+) dysregulation in RER has led to the use of dantrolene to prevent episodes of rhabdomyolysis. We examined differentially expressed proteins (DEP) and gene transcripts (DEG) in gluteal muscle of Thoroughbred race-trained mares after exercise among three groups of 5 horses each; 1) horses susceptible to, but not currently experiencing rhabdomyolysis, 2) healthy horses with no history of RER (control), 3) RER-susceptible horses treated with dantrolene pre-exercise (RER-D). Tandem mass tag LC/MS/MS quantitative proteomics and RNA-seq analysis (FDR <0.05) was followed by gene ontology (GO) and semantic similarity of enrichment terms. Of the 375 proteins expressed, 125 were DEP in RER-susceptible versus control, with 52 ↑DEP mainly involving Ca2+ regulation (N = 11) (e.g. RYR1, calmodulin, calsequestrin, calpain), protein degradation (N = 6), antioxidants (N = 4), plasma membranes (N = 3), glyco(geno)lysis (N = 3) and 21 DEP being blood-borne. ↓DEP (N = 73) were largely mitochondrial (N = 45) impacting the electron transport system (28), enzymes (6), heat shock proteins (4), and contractile proteins (12) including Ca2+ binding proteins. There were 812 DEG in RER-susceptible versus control involving the electron transfer system, the mitochondrial transcription/translational response and notably the pro-apoptotic Ca2+-activated mitochondrial membrane transition pore (SLC25A27, BAX, ATP5 subunits). Upregulated mitochondrial DEG frequently had downregulation of their encoded DEP with semantic similarities highlighting signaling mechanisms regulating mitochondrial protein translation. RER-susceptible horses treated with dantrolene, which slows sarcoplasmic reticulum Ca2+ release, showed no DEG compared to control horses. We conclude that RER-susceptibility is associated with alterations in proteins, genes and pathways impacting myoplasmic Ca2+ regulation, the mitochondrion and protein degradation with opposing effects on mitochondrial transcriptional/translational responses and mitochondrial protein content. RER could potentially arise from excessive sarcoplasmic reticulum Ca2+ release and subsequent mitochondrial buffering of excessive myoplasmic Ca2+.

Candidate gene expression and coding sequence variants in Warmblood horses with myofibrillar myopathy.

BACKGROUND: Myofibrillar myopathy (MFM) of unknown aetiology has recently been identified in Warmblood (WB) horses. In humans, 16 genes have been implicated in various MFM-like disorders. OBJECTIVES: To identify variants in 16 MFM candidate genes and compare allele frequencies of all variants between MFM WB and non-MFM WB and coding variants with moderate or severe predicted effects in MFM WB with publicly available data of other breeds. To compare differential gene expression and muscle fibre contractile force between MFM and non-MFM WB. STUDY DESIGN: Case-control. ANIMALS: 8 MFM WB, 8 non-MFM WB, 33 other WB, 32 Thoroughbreds, 80 Quarter Horses and 77 horses of other breeds in public databases. METHODS: Variants were called within transcripts of 16 candidate genes using gluteal muscle mRNA sequences aligned to EquCab3.0 and allele frequencies compared by Fisher's exact test among MFM WB, non-MFM WB and public sequences across breeds. Candidate gene differential expression was determined between MFM and non-MFM WB by fitting a negative binomial generalised log-linear model per gene (false discovery rate <0.05). The maximal isometric force/cross-sectional area generated by isolated membrane-permeabilised muscle fibres was determined. RESULTS: None of the 426 variants identified in 16 candidate genes were associated with MFM including 26 missense variants. Breed-specific differences existed in allele frequencies. Candidate gene differential expression and muscle fibre-specific force did not differ between MFM WB (143.1 ± 34.7 kPa) and non-MFM WB (140.2 ± 43.7 kPa) (P = .8). MAIN LIMITATIONS: RNA-seq-only assays transcripts expressed in skeletal muscle. Other possible candidate genes were not evaluated. CONCLUSIONS: Evidence for association of variants with a disease is essential because coding sequence variants are common in the equine genome. Variants identified in MFM candidate genes, including two coding variants offered as commercial MFM equine genetic tests, did not associate with the WB MFM phenotype.