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.

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.

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.

Age-dependent effects of systemic administration of oxytetracycline on the viscoelastic properties of rat tail tendons as a mechanistic basis for pharmacological treatment of flexural limb deformities in foals.

OBJECTIVE: To describe the effect of systemically administered oxytetracycline on the viscoelastic properties of rat tail tendon fascicles (TTfs) to provide a mechanistic rationale for pharmacological treatment of flexural limb deformities in foals. SAMPLE: TTfs from ten 1-month-old and ten 6-month-old male Sprague-Dawley rats. PROCEDURES: 5 rats in each age group were administered oxytetracycline (50 mg/kg, IP, q 24 h) for 4 days. The remaining 5 rats in each age group served as untreated controls. Five days after initiation of oxytetracycline treatment, TTfs were collected and their viscoelastic properties were evaluated via a stress-relaxation protocol. Maximum modulus and equilibrium modulus were compared via a 2-way ANOVA. Collagen fibril size, density, and orientation in TTfs were compared between treated and control rats. RESULTS: Viscoelastic properties were significantly decreased in TTfs from 1-month-old oxytetracycline-treated rats, compared with those in TTfs from 1-month-old control rats. Oxytetracycline had no effect on the viscoelastic properties of TTfs from 6-month-old rats. Collagen fibril size, density, and orientation in TTfs from 1-month-old rats did not differ between oxytetracycline-treated and control rats. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed that systemically administered oxytetracycline decreased the viscoelastic properties of TTfs from 1-month-old rats but not those of TTfs from 6-month-old rats. The decrease in viscoelastic properties associated with oxytetracycline treatment does not appear to be caused by altered collagen fibril diameter or organization. The age-dependent effect of oxytetracycline on the viscoelastic properties of tendons may be related to its effect on the maturation of the extracellular matrix of developing tendons.

Evaluation of the use of an autologous platelet-rich fibrin membrane to enhance tendon healing in dogs.

OBJECTIVE: To examine effects of an autologous platelet-rich fibrin (PRF) membrane for enhancing healing of a defect of the patellar tendon (PT) in dogs. ANIMALS: 8 adult dogs. PROCEDURES: Defects were created in the central third of the PT in both hind limbs of each dog. An autologous PRF membrane was implanted in 1 defect/dog, and the contralateral defect was left empty. Dogs (n = 4/time period) were euthanized at 4 and 8 weeks after surgery, and tendon healing was assessed grossly and histologically via a semiquantitative scoring system. Cross-sectional area of the PTs was also compared. RESULTS: Both treated and control defects were filled with repair tissue by 4 weeks. There was no significant difference in the histologic quality of the repair tissue between control and PRF membrane-treated defects at either time point. At both time points, the cross-sectional area of PRF membrane-treated tendons was significantly greater (at least 2.5-fold as great), compared with that of sham-treated tendons. At 4 weeks, the repair tissue consisted of disorganized proliferative fibrovascular tissue originating predominantly from the fat pad. By 8 weeks, the tissue was less cellular and slightly more organized in both groups. CONCLUSIONS AND CLINICAL RELEVANCE: A PRF membrane did not enhance the rate or quality of tendon healing in PT defects. However, it did increase the amount of repair tissue within and surrounding the defect. These results suggested that a PRF membrane may not be indicated for augmenting the repair of acutely injured tendons that are otherwise healthy.

Growth factor-rich plasma increases tendon cell proliferation and matrix synthesis on a synthetic scaffold: an in vitro study.

Numerous scaffolds have been proposed for use in connective tissue engineering. Although these scaffolds direct cell migration and attachment, many are biologically inert and thus lack the physiological stimulus to attract cells and induce mitogenesis and matrix synthesis. In the current study, a bioactive scaffold was created by combining a synthetic scaffold with growth factor-rich plasma (GFRP), an autologous concentration of growth factors derived from a platelet-rich plasma preparation. In vitro tendon cell proliferation and matrix synthesis on autologous GFRP-enriched scaffolds, autologous serum-enriched scaffolds, and scaffolds alone were compared. The GFRP preparation was found to have a 4.7-fold greater concentration of a sentinel growth factor (transforming growth factor-beta1) compared with serum. When combined with media containing calcium, the GFRP produced a thin fibrin matrix over and within the GFRP-enriched scaffolds. Cell proliferation assays demonstrated that GFRP-enriched scaffolds significantly enhanced cell proliferation over autologous serum and control groups at both 48 and 72 h. Analysis of the scaffolds at 14, 21, and 28 days revealed that GFRP-enriched scaffolds significantly increased the deposition of a collagen-rich extracellular matrix when compared with the other groups. These results indicate that GFRP can be used to enhance in vitro cellular population and matrix deposition of tissue-engineered scaffolds.

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.

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.

In vitro effects of oxytetracycline on matrix metalloproteinase-1 mRNA expression and on collagen gel contraction by cultured myofibroblasts obtained from the accessory ligament of foals.

OBJECTIVE: To determine the effects of oxytetracycline on matrix metalloproteinase-1 (MMP-1) mRNA expression and collagen gel contraction by equine myofibroblasts in an effort to explain the mechanistic basis for the pharmacologic treatment of flexural deformities in foals. SAMPLE POPULATION: Cultured myofibroblasts from the accessory ligament (distal check ligament) of 6 foals. PROCEDURE: Collagen gel scaffolds seeded with equine myofibroblasts were cultured in individual culture dishes containing complete media (Dulbecco's modified Eagle medium with 10% fetal bovine serum) and oxytetracycline (0, 12.5, 25, or 75 microg/mL) for 48 hours. After 24 hours, the gels were released from the bottom of the culture plate and allowed to contract. Photographs were taken at 0, 1, 2, 4, 6, 8, and 24 hours after release to assess the degree of collagen gel contraction. Additional gels were harvested at 2 hours after release for RNA isolation and reverse transcriptase-polymerase chain reaction assessment of the degree of MMP-1 mRNA expression. RESULTS: Oxytetracycline induced a dose-dependent inhibition of collagen gel contraction by equine myofibroblasts. Oxytetracycline also induced a dose-dependent decrease in MMP-1 mRNA expression by equine myofibroblasts. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study indicate that oxytetracycline inhibits tractional structuring of collagen fibrils by equine myofibroblasts through an MMP-1 mediated mechanism. In young foals, oxytetracycline administration may make the developing ligaments and tendons more susceptible to elongation during normal weight-bearing. Inhibition of normal collagen organization may provide the mechanistic explanation for the results seen following the pharmacologic treatment of flexural deformities in foals by oxytetracycline administration.