OBJECTIVE To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions.
SAMPLE Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred.
PROCEDURES Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter. Cells that stably integrated the TAg were selected and expanded to passage 25. For each passage, differentiation and fusion properties of the cells were determined and immunocytochemical analyses were performed to evaluate expression of TAg and other muscle-specific proteins. Optimum conditions that led to cell differentiation into myotubes were also determined.
RESULTS Compared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33°C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37°C or when incubated with doxycycline at 33°C. Expression of TAg was not detected when cells were incubated with doxycycline at 37°C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that production of an immortalized clonal equine skeletal muscle cell line was possible. A clonal equine skeletal muscle cell line will be a valuable in vitro tool for use in equine physiology and disease research.
Objective—To determine the effects of racing and
nontraining on plasma thyroxine (T4), free thyroxine
(fT4), thyroid-stimulating hormone (TSH), and thyroglobulin
autoantibody (TgAA) concentrations in sled
dogs and compare results with reference ranges
established for dogs of other breeds.
Animals—122 sled dogs.
Procedure—Plasma thyroid hormone concentrations
were measured before dogs began and after they finished
or were removed from the Iditarod Trail Sled
Dog Race in Alaska and approximately 3 months after
Results—Concentrations of T4 and fT4 before the race
were less than the reference range for nonsled dogs in
26% and 18% of sled dogs, respectively. Immediately
after racing, 92% of sled dogs had plasma T4 concentrations
less than the reference range. Three months after
the race, 25% of sled dogs had plasma T4 concentrations
less than the reference range. For T4, fT4, TSH, and
TgAA, significant differences were not detected in samples
collected before the race versus 3 months later.
Conclusions and Clinical Relevance—Plasma T4, fT4,
and TSH concentrations decreased in dogs that complete
a long distance sled dog race. Many clinically normal
sled dogs have plasma T4 and fT4 values that are
lower than the reference range for nonsled dogs. We
suggest that the reference ranges for sled dogs are 5.3
to 40.3 nmol/L and 3.0 to 24.0 pmol/L for plasma T4 and
fT4 concentrations, respectively, and 8.0 to 37.0 mU/L
for TSH. (J Am Vet Med Assoc 2004;224:226–231)
Objective—To investigate the influence of simulated contraction of the cricoarytenoideus dorsalis (CAD) muscle on the 3-D motion of the arytenoid cartilage.
Sample Population—5 larynges from equine cadavers.
Procedures—Serial computed tomographic scans of each larynx were conducted at 7 incremental forces simulating contraction of medial, lateral, and combined bellies of the left CAD muscle. Three-dimensional reconstruction of radiopaque markers placed at anatomic landmarks on the left arytenoid and cricoid cartilages enabled quantification of marker displacement according to a Cartesian coordinate system. Rotation (roll, pitch, and yaw) of dorsal and ventral arytenoid planes was calculated relative to a plane formed by the coordinates of 3 markers on the cricoid cartilage by use of Euler angles.
Results—Displacement and rotational data showed that rocking motion occurs throughout arytenoid abduction and most of the rotational component is attributable to pitch; greater pitch was associated with action of the lateral belly. Roll of the ventral arytenoid plane was principally associated with action of the medial belly, which counteracted the tendency of the arytenoid cartilage to rotate medially into the rima glottidis lumen. The distance between markers on the arytenoid cartilage was not constant during contraction because of slight deformation of the corniculate process of the arytenoid cartilage, therefore indicating that the arytenoid cartilage is not a rigid body during abduction.
Conclusions and Clinical Relevance—Arytenoid cartilage abduction was dependent on the rocking motion elicited by the lateral belly of the CAD muscle; therefore, laryngoplasty suture placement should mimic the action of the lateral, rather than the medial, muscle belly. (Am J Vet Res 2010;71:1003–1010)
Objective—To determine whether dietary antioxidants
would attenuate exercise-induced increases in
plasma creatine kinase (CK) activity in sled dogs.
Animals—41 trained adult sled dogs.
Procedure—Dogs, randomly assigned to 2 groups,
received the same base diet throughout the study.
After 8 weeks on that diet, 1 group (21 dogs) received
a daily supplement containing vitamins E (457 U) and
C (706 mg) and β-carotene (5.1 mg), and a control
group (20 dogs) received a supplement containing
minimal amounts of antioxidants. After 3 weeks, both
groups performed identical endurance exercise on
each of 3 days. Blood samples were collected before
and 3 weeks after addition of supplements and after
each day of exercise. Plasma was analyzed for vitamins
E and C, retinol, uric acid, triglyceride, and cholesterol
concentrations, total antioxidant status (TAS),
and CK activity.
Results—Feeding supplements containing antioxidants
caused a significant increase in vitamin E concentration
but did not change retinol or vitamin C concentrations
or TAS. Exercise caused significantly higher
CK activity, but did not cause a significant difference
in CK activity between groups. Exercise was
associated with significantly lower vitamin E, retinol,
and cholesterol concentrations and TAS but significantly
higher vitamin C, triglyceride, and uric acid concentrations
in both groups.
Conclusions and Clinical Relevance—Use of supplements
containing the doses of antioxidants used
here failed to attenuate exercise-induced increases in
CK activity. Muscle damage in sled dogs, as measured
by plasma CK activity, may be caused by a
mechanism other than oxidant stress. (Am J Vet Res
Objective—To evaluate whether biochemical or genetic alterations in AMP-activated protein kinase (AMPK) play a role in the development of polysaccharide storage myopathy (PSSM) in Quarter Horses.
Animals—30 PSSM-affected and 30 unaffected (control) Quarter Horses.
Procedures—By use of an established peptide phosphotransfer assay, basal and maximal AMPK activities were measured in muscle biopsy samples obtained from 6 PSSM-affected and 6 control horses. In 24 PSSM-affected and 24 control horses, microsatellite markers identified from the chromosomal locations of all 7 AMPK subunit genes were genotyped with a fluorescent DNA fragment analyzer. Alleles of 2 of the AMPK γ subunit genes were genotyped via DNA sequencing. Allele frequencies of DNA markers in or near the AMPK subunit genes were measured in isolated genomic DNA.
Results—No differences in basal or maximal muscle AMPK enzyme activities between PSSM-affected and control horses were detected. There were also no differences in allele frequencies for microsatellite markers near any of the 7 AMPK subunit genes between the 2 groups. Furthermore, previously known and newly identified alleles of 2 equine AMPK γ subunit genes were also not associated with PSSM.
Conclusions and Clinical Relevance—These results have provided no evidence to indicate that AMPK plays a causative role in PSSM in American Quarter Horses.
Procedures—The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin–derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid–Schiff staining.
Results—eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen.
Conclusions and Clinical Relevance—Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.