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Myoplasmic calcium regulation in myotubes from horses with recurrent exertional rhabdomyolysis

Linnea R. LentzDepartment of Clinical and Population Sciences, University of Minnesota, St Paul, MN 55108.
Present address is Medtronic Inc, Fridley, MN 55432.

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Stephanie J. ValbergDepartment of Clinical and Population Sciences, University of Minnesota, St Paul, MN 55108.

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Lee V. HeroldDepartment of Veterinary PathoBiology, University of Minnesota, St Paul, MN 55108.
Present address is 415 Muddy Branch Rd, Gaithersburg, MD 20878.

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Gary W. OnanDepartment of Animal and Food Science, University of Wisconsin, River Falls, WI 54022.

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James R. MickelsonDepartment of Veterinary PathoBiology, University of Minnesota, St Paul, MN 55108.

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Esther M. GallantDepartment of Veterinary PathoBiology, University of Minnesota, St Paul, MN 55108.

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Abstract

Objective—To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER).

Animals—6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses.

Procedures—Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses. Fura-2 fluorescence was used to measure resting myoplasmic calcium concentration as well as caffeine- and 4-chloro-m-cresol (4-CMC)-induced increases in myoplasmic calcium. In addition, intact intercostal muscle fiber bundles were prepared from both types of horses, and their sensitivities to caffeine- and 4-CMC-induced contractures were determined.

Results—Myotubes of RER-affected and control horses had identical resting myoplasmic calcium concentrations. Myotubes from RER-affected horses had significantly higher myoplasmic calcium concentrations than myotubes from control horses following the addition of ≥ 2mM caffeine; however, there was no difference in their response to 4-CMC (≥ 1mM). Caffeine contracture thresholds for RER and control intact muscle cell bundles (2 vs 10mM, respectively) were significantly different, but 4-CMC contracture thresholds of muscle bundles from RER-affected and control horses (500µM) did not differ.

Conclusions and Clinical Relevance—An increase in caffeine sensitivity of muscle cells derived from a family of related RER-affected horses was detected in vitro by use of cell culture with calcium imaging and by use of fiber bundle contractility techniques. An alteration in muscle cell calcium regulation is a primary factor in the cause of this heritable myopathy. (Am J Vet Res 2002;63:1724–1731)

Abstract

Objective—To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER).

Animals—6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses.

Procedures—Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses. Fura-2 fluorescence was used to measure resting myoplasmic calcium concentration as well as caffeine- and 4-chloro-m-cresol (4-CMC)-induced increases in myoplasmic calcium. In addition, intact intercostal muscle fiber bundles were prepared from both types of horses, and their sensitivities to caffeine- and 4-CMC-induced contractures were determined.

Results—Myotubes of RER-affected and control horses had identical resting myoplasmic calcium concentrations. Myotubes from RER-affected horses had significantly higher myoplasmic calcium concentrations than myotubes from control horses following the addition of ≥ 2mM caffeine; however, there was no difference in their response to 4-CMC (≥ 1mM). Caffeine contracture thresholds for RER and control intact muscle cell bundles (2 vs 10mM, respectively) were significantly different, but 4-CMC contracture thresholds of muscle bundles from RER-affected and control horses (500µM) did not differ.

Conclusions and Clinical Relevance—An increase in caffeine sensitivity of muscle cells derived from a family of related RER-affected horses was detected in vitro by use of cell culture with calcium imaging and by use of fiber bundle contractility techniques. An alteration in muscle cell calcium regulation is a primary factor in the cause of this heritable myopathy. (Am J Vet Res 2002;63:1724–1731)