Consequence of parvalbumin deficiency in the mdx mouse: histological, biochemical and mechanical phenotype of a new double mutant.

We tested the hypothesis whether the mild dystrophy in mdx mice could result from the contribution of the cytosolic calcium buffer parvalbumin in maintaining a normal cytosolic [Ca2+]i, in spite of an increased passive Ca2+ influx. By crossing mdx mice with parvalbumin-deficient mice, double mutant mice, lacking both dystrophin and parvalbumin, were obtained. Though resting cytosolic [Ca2+]i and total calcium content were similar to that of mdx muscles, this new animal model presented a slightly more severe phenotype than the mdx mouse. Muscle pseudo-hypertrophy, the density of myotubes and of centronucleated fibres as well as the loss of IIB fibres were all increased in parvalbumin-deficient mdx mice. Many of these deficits were overcome in late adulthood, albeit fibrosis was clearly more pronounced than in mdx muscles. At 90 days, parvalbumin-deficient mdx mice showed higher levels of creatine phosphokinase and lower muscle strength, in vivo, than mdx mice. Isometric tension of isolated muscle was reduced, but the susceptibility to eccentric contraction was not increased. The slight aggravation of muscle dystrophy observed in mdx mice deprived of parvalbumin cannot explain the severity of the affection observed in xmd dogs and Duchenne dystrophy patients where parvalbumin is constitutively not expressed.

Intracellular pH regulation in isolated fast-twitch skeletal muscle from dystrophin-deficient mouse.

In muscles from anaesthetized dystrophin-deficient mdx mice, exercise results in a stronger acidification and a slower intracellular pH recovery compared to control mice. We examined whether this observation could be attributed to defective H+-carriers in dystrophin-lacking muscles. Immunohistochemistry and Western blots revealed no defect in mdx muscles for the presence of the lactate-/H+co-transporter MCT4 and of the Na+/H+ antiporter NHE1, the main H+-carriers active in fast-twitch skeletal muscle after exercise. Functional tests of the H+-transporters, on isolated muscles submitted to identical flow of superfusion, were performed in conditions meant to lower intracellular pH: repetitive electrical stimulation or NH4Cl pre-pulse. These revealed no defect in intracellular pH recovery in mdx muscles. Therefore, we conclude that impaired intracellular pH regulation in anaesthetized mdx mice is not attributable to a reduced presence or activity of H+-extruders. We propose that CO2 washout might be slowed down in vivo in mdx muscles because of the defective vascular response in contracting muscles from these mice.

Involvement of trp-2 protein in store-operated influx of calcium in fibroblasts.

Mammalian homologues of the Drosophila melanogaster transient receptor potential (trp) gene have been proposed to encode store-operated channels. This assertion essentially stays on the fact that expression of different trp proteins produces trans-membrane cation currents. However, the selectivity of the expressed channels and their mode of activation, in particular, their dependence to store depletion appears to be quite variable. In the present work, we adopted an anti-sense strategy to study this question in transfected Chinese hamster ovary cells expressing the rat neurotensin receptor (CHO-NTR cells), a cellular model characterized by its very large store-dependent entry of Ca(2+). We identified different trp transcripts by RT-PCR, the trp-1 and trp-2 transcripts being by far the most abundant. CHO-NTR cells were then transfected with a mouse trp-2 anti-sense construct (CHO-NTR-TRP2AS cells). We showed that in these cells, trp-2 mRNA was suppressed in comparison with cells transfected with a control plasmid. The store-operated entry of Ca(2+) was evaluated after store depletion by an IP(3)-dependent mechanism (neurotensin stimulation) or by direct inhibition of the endoplasmic reticulum Ca(2+)ATPase (thapsigargin stimulation). In both cases, store-dependent entry of Ca(2+) was largely reduced in CHO-NTR-TRP2AS cells in comparison with control cells, suggesting that trp-2 protein might constitute a functional subunit of store-operated channels.

Subunit composition of native myosin isoenzymes of some striated mammalian muscles.

Six "native" isomyosins of characteristic electrophoretic mobility are present in various proportions according to the muscle type (fast, slow or mixed). SM2 and SM1 contain MHC1; IM contains MHC2A; FM1, FM2 and FM3 contain MHC2A and MHC2B. SM2, SM1 and IM contain MLC1s; SM1, IM, FM2 and FM3 contain MLC1f; FM2 and FM1 contain MLC3f. The six isomyosins appear to separate on the base of their myosin light chain content.

Implantation of autologous cells in minced and devitalized rat skeletal muscles.

Triceps surae of 3 week-old female Wistar rats were minced and orthotopically autografted. Thirty days later, the regenerates developed a force of 314 +/- 58 mN during an isometric maximal tetanus and the maximal area of the muscle tissue was 3.2 +/- 0.5 mm2 (n = 9) in a cross-section. At 60 days, their mean maximal isometric force was increased (1159 +/- 367 mN) as also were their maximal muscle cross-section area (6.8 +/- 0.7 mm2;n = 6). If the minces were devitalized by a freeze and thaw procedure, the regenerates neither contracted under electrical stimulation nor contained any muscle fibre. If devitalized minces were seeded with myogenic cells isolated from the contralateral triceps surae, orthotopically autografted and allowed to regenerate for 30 days, the regeneration was weak: 46 +/- 14 mN of force, 1.2 +/- 0.3 mm2 of muscle area in minces devitalized by cold (n = 7) and 23 +/- 8 mN of force, 0.2 +/- 0.1 mm2 of muscle area in minces devitalized by cold supplemented by heat (n = 8). If viable minces were seeded with myogenic cells, there was no improvement in the extent of regeneration: 275 +/- 83 mN of force, 2.0 +/- 0.6 mm2 of muscle area at 30 days (n = 7) and 738 +/- 191 mN of force, 5.6 +/- 0.9 mm2 of muscle area at 60 days (n = 5). Consequently, although it is possible to induce regeneration by grafting myogenic cells into a devitalized mince, this procedure has no effect when applied to a viable mince.

Muscle regeneration induced by cells autografted in adult rats.

Right triceps surae of 3-week-old Wistar rats were minced and devitalized with liquid nitrogen, a treatment which completely inhibits their ability to regenerate when they are orthotopically autografted. In a first series of experiments, cells were isolated from the left triceps surae, mixed with the devitalized right mince and autografted; in a second series, cells were moreover allowed to proliferate in vitro for a few weeks before being grafted. The regenerates were examined 60 days after surgery. In the first series, all the regenerates were contractile and developed a maximal isometric tetanic force of 18 +/- 6 mN (n = 5); they contained 152 +/- 80 muscle fibres located proximally, the number of which decreased along the proximo-distal axis, being 24 +/- 24 in the median part of the regenerate. The muscle fibres appeared histologically normal except for their shortness (less than 10 mm) and narrowness (mean luminal diameter: 30 microns). In the second series, 2 out of 5 regenerates were comparable with those of the first series except that their fibres were shorter; the 3 other regenerates were unexcitable. These experiments demonstrate that cells isolated from an adult striated muscle are able to regenerate striated muscle fibres in an adult animal and that these cells can retain this property if they are grown in culture.

The origin of muscle stem cells in rat triceps surae regenerating after mincing.

Rat triceps surae was minced and orthotopically autografted. Twitch time to peak, maxima tetanic tension, lactate dehydrogenase activity and total creatine concentration were measured in muscles regenerating for 30, 60 and 90 days. If the minces were frozen and thawed before grafting, muscle regeneration was suppressed. If they were further heated before grafting, muscle regeneration was also suppressed. If one half of the mince was either frozen and thawed or frozen, thawed and heated, and then recombined with the remaining half, muscle regeneration was delayed. However, at 90 days, 'intensive properties' (twitch time to peak, maximum tetanic tension, total creatine concentration and lactate dehydrogenase activity) of regenerates obtained from such partially treated minces were similar to those of regenerates obtained from untreated minces although their 'extensive properties' (weight and maximal tetanic force) were approximately halved. The extent of regeneration depends on the mass of untreated mince autografted and thus, presumably, on the number of viable muscle stem cells initially present in the mince.