Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

Abstract

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.

Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle

“…Our data are consistent with two other reports describing abnormal titin staining and reduced passive tension in WT muscle after lengthening contractions (31,34), as well as with the fact that titin fragmentation is detected in serum and urine, in dystrophindeficient mice as well as Duchenne muscular dystrophy patients (54)(55)(56). Disruption of the DGC is accompanied by compromised lateral force transmission (31,34). Thus, the excessive reduction in titin staining in muscles of the inducible DG KO mice may have been due to excessive strain on titin during contractions, when such transmission was disrupted.…”

“…The extent of disruption of distinct components of the cytoskeleton/sarcomere may be unequal, as suggested by our finding that the expression of titin was disrupted by lengthening contractions whereas that of desmin, nebulin, and α-actinin was unperturbed. Our data are consistent with two other reports describing abnormal titin staining and reduced passive tension in WT muscle after lengthening contractions (31,34), as well as with the fact that titin fragmentation is detected in serum and urine, in dystrophindeficient mice as well as Duchenne muscular dystrophy patients (54)(55)(56). Disruption of the DGC is accompanied by compromised lateral force transmission (31,34).…”

“…Passive elements of the sarcomeric cytoskeleton, such as titin, are susceptible to damage from lengthening contractions (31,34). Fig.…”

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

“…17 In addition, muscle from mdx mice with a dominant negative TRPC transgene show reduced Ca 2+ entry and less severe dystrophy 18 and muscle from TRPC1 knockout mice show less contraction-induced injury than wild-type muscle. 19 On the other hand, several studies show TRPV2 and TRPV4 contribute to Ca 2+ entry and stretch-induced injury in mdx muscle. 16,20 Evidently, the precise molecular identity of the MS channel responsible for Ca 2+ entry in dystrophic skeletal muscle remains uncertain.…”

“…Despite this conclusion, there is good evidence for a role of TRPC1 in Ca 2+ entry and muscle damage in normal and dystrophic muscle from mdx mice. [17][18][19] These observations for a functional role of TRPC1 in the pathogenesis of dystrophin-deficiency suggest either TRPC1 acts as an independent source of Ca 2+ entry contributing to the dystrophic phenotype or that MS channels represent a novel variant formed by heteromeric subunit assembly TRPV4 subunits. 48,49 Ma et al showed TRPV4 and TRPC1 coassemble to form heteromeric TRPV4-C1 channels.…”