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.

Stress-deprivation induces an up-regulation of versican and connexin-43 mRNA and protein synthesis and increased ADAMTS-1 production in tendon cells in situ.

Purpose: The proper function of the tenocyte network depends on cell-matrix as well as intercellular communication that is mechanosensitive. Building on the concept that the etiopathogenic stimulus for tendon degeneration is the catabolic response of tendon cells to mechanobiologic under-stimulation, we studied the pericellular matrix rich in versican and its predominant proteolytic enzyme ADAMTS-1, as well as Connexin-43 (Cx43), a major gap junction forming protein in tendons, in stress-deprived rat tail tendon fascicles (RTTfs).Materials and Methods: RTTfs were stress-deprived for up to 7 days under tissue culture conditions. RT-qPCR was used to measure mRNA expression of versican, ADAMTS-1, and Cx43. Protein synthesis was determined using Western blotting and immunohistochemistry.Results: Stress-deprivation (SD) caused a statistically significant up-regulation of versican, ADAMTS-1, and Cx43 mRNA expression that was persistent over the 7-day test period. Western blot analysis and immunohistochemical assessment of protein synthesis revealed a marked increase of the respective proteins with SD. Inhibition of proteolytic enzyme activity with ilomastat prevented the increased versican degradation and Cx43 synthesis in 3 days stress-deprived tendons when compared with non-treated, stress-deprived tendons.Conclusion: In the absence of mechanobiological signaling the immediate pericellular matrix is modulated as tendon cells up-regulate their production of ADAMTS-1, and versican with subsequent proteoglycan degradation potentially leading to cell signaling cues increasing Cx43 gap junctional protein. The results also provide further support for the hypothesis that the cellular changes associated with tendinopathy are a result of decreased mechanobiological signaling and a loss of homeostatic cytoskeletal tension.

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.

Increased susceptibility of skin from HERDA (Hereditary Equine Regional Dermal Asthenia)-affected horses to bacterial collagenase degradation: a potential contributing factor to the clinical signs of HERDA.

BACKGROUND: Hereditary equine regional dermal asthenia (HERDA) is a genetic disorder of collagen resulting in fragile, hyper-extensible skin and ulcerative lesions. The predominance of skin lesions have been shown to occur on the dorsum of HERDA-affected horses. While this has been postulated to be due to increased exposure to sunlight of these areas, the precise pathological mechanism which causes this to occur is unclear. HYPOTHESIS/OBJECTIVES: We hypothesized that an increase in collagenase activity, that has been associated with the exposure of dermal fibroblasts to sunlight, will significantly degrade the material properties of skin from HERDA-affected horses when compared to unaffected controls. ANIMALS: Six unaffected and seven HERDA-affected horses, all euthanized for other reasons. METHODS: Full-thickness skin samples from similar locations on each horse were collected and cut into uniform strips and their material properties (tensile modulus) determined by mechanical testing before (n = 12 samples/horse) or after (n = 12 samples/horse) incubation in bacterial collagenase at 37°C for 6 h. The change in modulus following treatment was then compared between HERDA-affected and unaffected horses using a Student's t-test. RESULTS: The modulus of skin from HERDA-affected horses decreased significantly more than that from unaffected horses following collagenase treatment (54 ± 7% versus 30 ± 16%, P = 0.004). CONCLUSIONS AND CLINICAL IMPORTANCE: The significant decrease in the modulus of skin from HERDA-affected horses following collagenase exposure suggests that their altered collagen microarchitecture is more susceptible to enzymatic degradation and may explain the localization of skin lesions in HERDA-affected horses to those areas of the body most exposed to sunlight. These findings appear to support the previously reported benefits of sunlight restriction in HERDA-affected horses.

Age-related changes in the cellular, mechanical, and contractile properties of rat tail tendons.

Tendon laxity following injury, cyclic creep, or repair has been shown to alter the normal homeostasis of tendon cells, which can lead to degenerative changes in the extracellular matrix. While tendon cells have been shown to have an inherent contractile mechanism that gives them some ability to retighten lax tendons and reestablish a homeostatic cellular environment, the effect of age on this process is unknown. To determine the effect of aging on cell number, cell shape, and tensile modulus on tendons as well as the rate of cell-mediated contraction of lax tendons, tail tendon fascicles from 1-, 3-, and 12-month-old rats were analyzed. Aging results in a decrease (p < 0.001) in cell number per mm(2): 1 m (981 ± 119), 3 m (570 ± 108), and 12 m (453 ± 23), a more flattened (p < 0.001) cell nuclei shape and a higher (p < 0.001) tensile modulus (MPa) of the tendons: 1 m (291 ± 2), 3 m (527 ± 38), and 12 m (640 ± 102). Both the extent and rate of contraction over 7 days decreased with age (p = 0.007). This decrease in contraction rate with age correlates to the observed changes seen in aging tendons [increased modulus (r(2) = 0.95), decreased cell number (r(2) = 0.89)]. The ability of tendons to regain normal tension following injury or exercise-induced laxity is a key factor in the recovery of tendon function. The decreased contraction rate as a function of age observed in the current study may limit the ability of tendon cells to retighten lax tendons in older individuals. This, in turn, may place these structures at further risk for injury or altered function.

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.

Prevalence of the E321G MYH1 variant for immune-mediated myositis and nonexertional rhabdomyolysis in performance subgroups of American Quarter Horses.

BACKGROUND: Immune-mediated myositis (IMM) in American Quarter Horses (QHs) causes acute muscle atrophy and lymphocytic infiltration of myofibers. Recently, an E321G mutation in a highly conserved region of the myosin heavy chain 1 (MYH1) gene was associated with susceptibility to IMM and nonexertional rhabdomyolysis. OBJECTIVES: To estimate prevalence of the E321G MYH1 variant in the QH breed and performance subgroups. ANIMALS: Three-hundred seven elite performance QHs and 146 random registered QH controls. METHODS: Prospective genetic survey. Elite QHs from barrel racing, cutting, halter, racing, reining, Western Pleasure, and working cow disciplines and randomly selected registered QHs were genotyped for the E321G MYH1 variant and allele frequencies were calculated. RESULTS: The E321G MYH1 variant allele frequency was 0.034 ± 0.011 in the general QH population (6.8% of individuals in the breed) and the highest among the reining (0.135 ± 0.040; 24.3% of reiners), working cow (0.085 ± 0.031), and halter (0.080 ± 0.027) performance subgroups. The E321G MYH1 variant was present in cutting (0.044 ± 0.022) and Western Pleasure (0.021 ± 0.015) QHs at lower frequency and was not observed in barrel racing or racing QHs. CONCLUSIONS AND CLINICAL IMPORTANCE: Knowing that reining and working cow QHs have the highest prevalence of the E321G MYH1 variant and that the variant is more prevalent than the alleles for hereditary equine regional dermal asthenia and hyperkalemic periodic paralysis in the general QH population will guide the use of genetic testing for diagnostic and breeding purposes.

Loss of homeostatic tension induces apoptosis in tendon cells: an in vitro study.

Apoptosis (programmed cell death) has been identified as a histopathologic feature of tendinopathy. While the precise mechanism(s) that triggers the apoptotic cascade in tendon cells has not been identified, it has been theorized that loss of cellular homeostatic tension following microscopic damage to individual tendon fibrils could be the stimulus for initiating the pathologic events associated with tendinopathy. To determine if loss of homeostatic tension following stress deprivation could induce apoptosis in tendon cells, rat tail tendons were stress-deprived or cyclically loaded (3% strain at 0.17 Hz) for 24 hours under tissue culture conditions. Caspase-3 (an upstream mediator of apoptosis) mRNA expression was evaluated using quantitative polymerase chain reaction and caspase-3 protein synthesis was identified using immunohistochemistry. Apoptotic cells were identified histologically using an antibody for single-stranded DNA. Stress deprivation for 24 hours resulted in an increase in caspase-3 mRNA expression when compared to fresh controls or cyclically loaded tendons. Stress deprivation also increased the percentage of apoptotic cells (10.59% +/- 2.80) compared to controls (1.87% +/- 1.07) or cyclically loaded tendons (3.73% +/- 0.87). These data suggest loss of homeostatic tension following stress deprivation induces apoptosis in rat tail tendon cells.

Loss of homeostatic strain alters mechanostat "set point" of tendon cells in vitro.

Tendon cells respond to mechanical loads. The character (anabolic or catabolic) and sensitivity of this response is determined by the mechanostat set point of the cell, which is governed by the cytoskeleton and its interaction with the extracellular matrix. To determine if loss of cytoskeletal tension following stress deprivation decreases the mechanoresponsiveness of tendon cells, we cultured rat tail tendons under stress-deprived conditions for 48 hours and then cyclically loaded them for 24 hours at 1%, 3%, or 6% strain at 0.17 Hz. Stress deprivation upregulated MMP-13 mRNA expression and caused progressive loss of cell-matrix contact compared to fresh controls. The application of 1% strain to fresh tendons for 24 hours inhibited MMP-13 mRNA expression compared to stress-deprived tendons over the same period. However, when tendons were stress-deprived for 48 hours and then subjected to the same loading regime, the inhibition of MMP-13 mRNA expression was decreased. In stress-deprived tendons, it was necessary to increase the strain magnitude to 3% to achieve the same level of MMP-13 mRNA inhibition seen in fresh tendons exercised at 1% strain. The data suggest loss of cytoskeletal tension alters the mechanostat set point and decreases the mechanoresponsiveness of tendon cells.

The effect of stress-deprivation and cyclic loading on the TIMP/MMP ratio in tendon cells: an in vitro experimental study.

PURPOSE: To determine the effect of stress deprivation and cyclic loading on TIMP-1/MMP-13 mRNA expression ratio in rat tail tendon (RTT) cells. METHOD: Adult RTTs were stress-deprived for 0, 24, 48, or 72 hours in the presence or absence of a MMP inhibitor (ilomastat), or subjected to 1%, 3%, or 6% strain for 24 h under tissue culture conditions. TIMP-1 and MMP-13 (rat interstitial collagenase) mRNA expression were measured using quantitative PCR and TIMP/MMP ratios were calculated for each group. RESULTS: The ratio of TIMP-1 to MMP-13 in control RTTs was 3.73:1 +/- 0.73. Stress deprivation for 24 h significantly decreased the TIMP-1/MMP-13 ratio (0.25:1 +/- 0.04) and MMP-13 expression continued to increase significantly with time of stress deprivation. Inhibition of MMP-13 mRNA expression with ilomastat in stress-deprived samples did not alter TIMP-1 expression when compared to normal controls. Cyclic loading significantly increased TIMP-1/MMP-13 expression at all strain levels examined. CONCLUSIONS: RTTs normally have a positive TIMP-1/MMP-13 expression ratio. While cyclic loading increased the TIMP-1/MMP-13 ratio, loss of cellular homeostatic tension inversed this ratio through a significant increase in MMP-13 mRNA expression rather than a decrease in TIMP expression. A negative TIMP/MMP ratio has been implicated in the pathogenesis of tendinopathy. Increasing the TIMP/MMP ratios in these patients through exercise may be beneficial in the management of tendinopathy.

An E321G MYH1 mutation is strongly associated with nonexertional rhabdomyolysis in Quarter Horses.

BACKGROUND: An E321G mutation in MYH1 was recently identified in Quarter Horses (QH) with immune-mediated myositis (IMM) defined by a phenotype of gross muscle atrophy and myofiber lymphocytic infiltrates. HYPOTHESIS/OBJECTIVES: We hypothesized that the MYH1 mutation also was associated with a phenotype of nonexertional rhabdomyolysis. The objective of this study was to determine the prevalence of the MYH1 mutation in QH with exertional (ER) and nonexertional (nonER) rhabdomyolysis. ANIMALS: Quarter Horses: 72 healthy controls, 85 ER-no atrophy, 56 ER-atrophy, 167 nonER horses selected regardless of muscle atrophy. METHODS: Clinical and histopathologic information and DNA was obtained from a database for (1) ER > 2 years of age, with or without atrophy and (2) nonER creatine kinase (CK) ≥ 5000 U/L, <5 years of age. Horses were genotyped for E321G MYH1 by pyrosequencing. RESULTS: The MYH1 mutation was present in a similar proportion of ER-no atrophy (1/56; 2%) and in a higher proportion of ER-atrophy (25/85; 29%) versus controls (4/72; 5%). The MYH1 mutation was present in a significantly higher proportion of nonER (113/165; 68%) than controls either in the presence (39/42; 93%) or in absence (72/123; 59%) of gross atrophy. Lymphocytes were present in <18% of muscle samples with the MYH1 mutation. CONCLUSIONS AND CLINICAL IMPORTANCE: Although not associated with ER, the MYH1 mutation is associated with atrophy after ER. The MYH1 mutation is highly associated with nonER regardless of whether muscle atrophy or lymphocytic infiltrates are present. Genetic testing will enhance the ability to diagnose MYH1 myopathies (MYHM) in QH.

A missense mutation in MYH1 is associated with susceptibility to immune-mediated myositis in Quarter Horses.

BACKGROUND: The cause of immune-mediated myositis (IMM), characterized by recurrent, rapid-onset muscle atrophy in Quarter Horses (QH), is unknown. The histopathologic hallmark of IMM is lymphocytic infiltration of myofibers. The purpose of this study was to identify putative functional variants associated with equine IMM. METHODS: A genome-wide association (GWA) study was performed on 36 IMM QHs and 54 breed matched unaffected QHs from the same environment using the Equine SNP50 and SNP70 genotyping arrays. RESULTS: A mixed model analysis identified nine SNPs within a ~ 2.87 Mb region on chr11 that were significantly (Punadjusted < 1.4 × 10- 6) associated with the IMM phenotype. Associated haplotypes within this region encompassed 38 annotated genes, including four myosin genes (MYH1, MYH2, MYH3, and MYH13). Whole genome sequencing of four IMM and four unaffected QHs identified a single segregating nonsynonymous E321G mutation in MYH1 encoding myosin heavy chain 2X. Genotyping of additional 35 IMM and 22 unaffected QHs confirmed an association (P = 2.9 × 10- 5), and the putative mutation was absent in 175 horses from 21 non-QH breeds. Lymphocytic infiltrates occurred in type 2X myofibers and the proportion of 2X fibers was decreased in the presence of inflammation. Protein modeling and contact/stability analysis identified 14 residues affected by the mutation which significantly decreased stability. CONCLUSIONS: We conclude that a mutation in MYH1 is highly associated with susceptibility to the IMM phenotype in QH-related breeds. This is the first report of a mutation in MYH1 and the first link between a skeletal muscle myosin mutation and autoimmune disease.

The location in cartilage of infectious retrovirus in cats infected with feline leukemia virus.

BACKGROUND: Previous studies have suggested that articular cartilage allografts were not likely to transmit infectious retrovirus since viral DNA could not be isolated from chondrocytes of infected individuals. However, the ability of the extracellular matrix of articular cartilage to harbor and transmit a retrovirus has not been examined. We hypothesized that articular cartilage fragments, but not isolated chondrocytes, from cats systemically infected with feline leukemia virus (FeLV) are capable of transmitting infectious retrovirus. METHODS: Fresh cartilage segments and chondrocytes isolated from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cocultured with segments of cartilage, isolated chondrocytes, or fragments of cortical bone from each infected cat. The FeLV p27 antigen was measured in the coculture media by enzyme-linked immunosorbent assay. In addition, FeLV proviral nucleic acids were quantified by real-time quantitative polymerase chain reaction with use of DNA extracted from feline embryonic fibroblast cell cocultures as well as isolated chondrocytes. Immunohistochemistry was used to assess for FeLV p27 antigen in both intact cartilage fragments and isolated chondrocytes. RESULTS: Feline embryonic fibroblast cells cocultured with cartilage fragments from each of the five FeLV-infected cats all demonstrated high levels of proviral DNA, indicating transmission of infective virus. In addition, media from all cocultures of feline embryonic fibroblast cells and chondral fragments became positive for p27 antigen, indicating active viral replication. In contrast, cocultures of feline embryonic fibroblast cells and isolated chondrocytes from all FeLV-infected cats were negative for proviral DNA and p27 antigen. Likewise, no proviral nucleic acids could be detected in isolated chondrocytes from any infected cats. Cocultures of feline embryonic fibroblast cells with cortical bone fragments were positive for proviral DNA and p27 antigen. Immunohistochemical staining of cartilage fragments from FeLV-infected cats demonstrated the presence of p27 antigen throughout the extracellular matrix, but the p27 antigen was not detected in isolated chondrocytes. CONCLUSIONS: Articular cartilage fragments can readily transmit infectious retrovirus, but isolated chondrocytes were likely not the source of the infectious virus because they did not harbor proviral DNA or p27 antigen.

Matrix metalloproteinase inhibitors prevent a decrease in the mechanical properties of stress-deprived tendons: an in vitro experimental study.

BACKGROUND: An increase in matrix metalloproteinases (MMPs) and the resulting degradation of the extracellular matrix have been implicated in the pathogenesis of tendinopathy. Studies have documented the beneficial effects of MMP inhibitors used to treat pathologic conditions in which MMP activity has had a negative effect on connective tissues. HYPOTHESIS: Matrix metalloproteinase inhibitors will prevent the decrease in material properties associated with tendon stress deprivation by inhibiting MMP activity. STUDY DESIGN: Controlled laboratory study. METHODS: Rat tail tendons were subjected to 7 days of in vitro stress deprivation with and without the addition of 1 of 2 broad-spectrum MMP inhibitors (doxycycline and ilomastat). The material properties (ultimate tensile stress, strain, and tensile modulus) of the tendons were compared with each other and with fresh control tendons. In addition, tendons from each group were evaluated for MMP-13 messenger RNA expression, MMP-13 protein synthesis, MMP-13 activity, and pericellular matrix morphology. RESULTS: Both MMP inhibitors resulted in a statistically significant reduction in MMP activity in 7 day stress-deprived tendons when compared with nontreated, stress-deprived tendons. Similarly, tendons treated with either ilomastat or doxycycline had significantly improved material properties. MMP-13 messenger RNA expression and protein synthesis were not significantly affected by either MMP inhibitor. Both MMP inhibitors were able to maintain the integrity of the pericellular matrix when compared with nontreated, stress-deprived tendons. CONCLUSION: Matrix metalloproteinase inhibitors prevented the activation of MMP-13 and significantly inhibited pericellular matrix degeneration and the loss of material properties associated with stress deprivation. CLINICAL RELEVANCE: Matrix metalloproteinase inhibitors may play a supportive role in the treatment of tendinopathy by limiting the MMP-mediated degradation of the extracellular matrix.

Crimp length decreases in lax tendons due to cytoskeletal tension, but is restored with tensional homeostasis.

Collagen crimp morphology is thought to contribute to the material behavior of tendons and may reflect the local mechanobiological environment of tendon cells. Following loss of collagen tension in tendons, tenocytes initiate a contraction response that shortens tendon length which, in turn, may alter crimp patterns. We hypothesized that changes in the crimp pattern of tendons are the result of cell-based contractions which are governed by relative tautness/laxity of the collagen matrix. To determine the relationship between crimp pattern and tensional homeostasis, rat tail tendon fascicles (RTTfs) were either allowed to freely contract or placed in clamps with 10% laxity for 7 days. The freely contracting RTTfs showed a significant decrease in percent crimp length on both day 5 (3.66%) and day 7 (7.70%). This decrease in crimp length significantly correlated with the decrease in freely contracting RTTf length. Clamped RTTfs demonstrated a significant decrease in percent crimp length on day 5 (1.7%), but no significant difference in percent crimp length on day 7 (0.57%). The results demonstrate that the tendon crimp pattern appears to be under cellular control and is a reflection of the local mechanobiological environment of the extracellular matrix. The ability of tenocytes to actively alter the crimp pattern of collagen fibers also suggests that tenocytes can influence the viscoelastic properties of tendon. Understanding the interactions between tenocytes and their extracellular matrix may lead to further insight into the role tendon cells play in maintaining tendon heath and homeostasis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:573-579, 2017.

Isolated fibrillar damage in tendons stimulates local collagenase mRNA expression and protein synthesis.

The etiology of repetitive stress injuries in tendons has not been clearly identified. While minor trauma has been implicated as an inciting factor, the precise magnitude and structural level of tissue injury that initiates this degenerative cascade has not been determined. The purpose of this study was to determine if isolated tendon fibril damage could initiate an upregulation of interstitial collagenase (MMP13) mRNA and protein in tendon cells associated with the injured fibril(s). Rat tail tendon fascicles were subjected to in vitro tensile loading until isolated fibrillar damage was documented. Once fibrillar damage occurred, the tendons were immediately unloaded to 100g and maintained at that displacement for 24h under tissue culture conditions. In addition, non-injured tendon fascicles were maintained under unloaded (stress-deprived) conditions in culture for 24h to act as positive controls. In situ hybridization or immunohistochemistry was then performed to localize collagenase mRNA expression or protein synthesis, respectively. Fibrillar damage occurred at a similar stress (41.13+/-5.94MPa) and strain (13.24+/-1.94%) in the experimental tendons. In situ hybridization and immunohistochemistry demonstrated an upregulation of interstitial collagenase mRNA and protein, respectively, in only those cells associated with the damaged fibril(s). In the control (stress-deprived) specimens, collagenase mRNA expression and protein synthesis were observed throughout the fascicle. The results suggest that isolated fibrillar damage and the resultant upregulation of collagenase mRNA and protein in this damaged area occurs through a mechanobiological understimulation of tendon cells. This collagenase production may weaken the tendon and put more of the extracellular matrix at risk for further damage during subsequent loading.

Hypoxia inhibits primary cilia formation and reduces cell-mediated contraction in stress-deprived rat tail tendon fascicles.

BACKGROUND: Hypoxia, which is associated with chronic tendinopathy, has recently been shown to decrease the mechanosensitivity of some cells. Therefore, the purpose of this study was to determine the effect of hypoxia on the formation of elongated primary cilia (a mechanosensing organelle of tendon cells) in vitro and to determine the effect of hypoxia on cell-mediated contraction of stress-deprived rat tail tendon fascicles (RTTfs). METHODS: Tendon cells isolated from RTTfs were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 hours. The cells were then stained for tubulin and the number of cells with elongated cilia counted. RTTfs from 1-month-old male Sprague-Dawley rats were also cultured under hypoxic and normoxic conditions for three days and tendon length measured daily. RESULTS: A significant (p=0.002) decrease in the percent of elongated cilia was found in cells maintained in hypoxic conditions (54.1%±12.2) when compared in normoxic conditions (71.7%±6.32). RTTfs in hypoxia showed a significant decrease in the amount of contraction compared to RTTfs in normoxia after two (p=0.007) and three (p=0.001) days. CONCLUSION: The decreased incidence of elongated primary cilia in a hypoxic environment, as well as the decreased mechanoresponsiveness of tendon cells under these conditions may relate to the inability of some cases of chronic tendinopathy to respond to strain-based rehabilitation modalities (i.e. eccentric loading).

High magnitude, in vitro, biaxial, cyclic tensile strain induces actin depolymerization in tendon cells.

BACKGROUND: the cytoskeleton is a dynamic arrangement of actin filaments that maintain cell shape and are vital in mediating the mechanobiological response of the cell. METHODS: to determine the cytoskeletal response to varying in vitro, biaxial stretch amplitudes, rat-tail tendon cells were paired into control and cyclically strained groups of 4.75, 9.5, or 12% strain at 1 Hz for 2 hours and the actin cytoskeleton stained. The cells were analyzed for actin staining intensity as a measure of relative depolymerization and for cell shape. Collagenase gene expression was measured in cells undergoing 12% cyclic strain at 1 Hz for 24 hours. RESULTS: there was no significant difference in the degree of actin staining intensity between the control group and cells strained at either 4.75 or 9.5%. However, cells strained at 12% demonstrated a significant decrease in actin staining intensity (depolymerization) compared to control cells, increased collagenase expression by 81%, and a clear shift towards a more rounded cell shape. CONCLUSION: the results of this study demonstrate that the previously reported induction of collagenase activity associated with the application of high magnitude, in vitro, tensile strains may actually be a result of cytoskeletal depolymerization, which causes loss of tensional homeostasis and alteration of cell shape.

Thermal energy enhances cell-mediated contraction of lax rat tail tendon fascicles following exercise.

BACKGROUND: the application of thermal energy (TE) has shown promise in the treatment of tendinopathy. However, the precise mechanism(s) of action of this therapy is unclear. The loss of tendon cell homeostatic tension, due to loading-induced laxity, produces catabolic changes associated with tendinopathy. This catabolic activity can be inhibited through the re-establishment of a normal tensile environment via a cellular contraction mechanism. We hypothesized that application of TE will enhance the contraction rate of lax rat tail tendon fascicles (RTTfs) in an in vitro model. METHODS: following loading, 10 lax RTTfs from each mature rat (n=5) were treated once daily for 7 days with TE by replacing the culture media at 37°C (control) with 42°C media. Using calibrated photographs, RTTf lengths were measured daily. Additional RTTfs were utilized to investigate any changes in material (n=12) and/or histological (n=12) properties with TE. RESULTS: TE significantly increased the contraction rate of RTTfs (p>0.001) without altering the material or histological properties. CONCLUSION: these results demonstrate that TE significantly enhances the contraction rate of previously exercised tendons. The ability to more quickly re-establish a normal mechanobiological environment, thus minimizing any catabolic changes, may explain the beneficial effects reported with applied TE in tendinopathy treatment.