Jean-Marie Gillis

Expression of full-length utrophin prevents muscular dystrophy in mdx mice

Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fiber leading to the gradual depletion of skeletal muscle. The molecular structure of dystrophin is very similar to that of the related protein utrophin. Utrophin is found in all tissues and is confined to the neuromuscular and myotendinous junctions in mature muscle. Sarcolemmal localization of a truncated utrophin transgene in the dystrophin-deficient mdx mouse significantly improves the dystrophic muscle phenotype. Therefore, upregulation of utrophin by drug therapy is a plausible therapeutic approach in the treatment of DMD. Here we demonstrate that expression of full-length utrophin in mdx mice prevents the development of muscular dystrophy. We assessed muscle morphology, fiber regeneration and mechanical properties (force development and resistance to stretch) of mdx and transgenic mdx skeletal and diaphragm muscle. The utrophin levels required in muscle are significantly less than the normal endogenous utrophin levels seen in lung and kidney, and we provide evidence that the pathology depends on the amount of utrophin expression. These results also have important implications for DMD therapies in which utrophin replacement is achieved by delivery using exogenous vectors.

Parvalbumins and Muscle-relaxation - a Computer-simulation Study

SummaryThe distribution of Ca2+ and Mg2+ among the ‘regulatory’ cation binding sites of troponin (T-sites) and the strong, Ca2+-Mg2+ binding sites of troponin and parvalbumins (P-sites) in the sarcoplasm of a muscle was calculated. At rest, 60% of the T-sites were metal free, while 92% of the P-sites were loaded with Mg2+.In response to a Ca2+ pulse, troponin-calcium (T-Ca) complexes were rapidly formed, while the binding of Ca2+ to P-sites was limited by the slow rate of dissociation of the parvalbumin-magnesium (P-Mg) complexes. Muscle activation was not prevented by a high content of parvalbumins.Parvalbumin and the sarcoplasmic reticulum (SR) pump were complementary relaxing factors that removed Ca2+ from the cytosol and from the T-sites. Parvalbumins dominated the first part of relaxation, while the action of the SR was essential to ensure the return to a very low level of free Ca2+ ion and of T-Ca. After relaxation, a large fraction of the Ca2+ pulse was still bound to parvalbumins and returned slowly to the SR during the recovery.When the SR activity was reduced, the presence of parvalbumins preserved a fast rate of relaxation, at least for a few contractions. This may have a high adaptive value in cold-blooded animals.

Relaxation of Vertebrate Skeletal-muscle - a Synthesis of the Biochemical and Physiological Approaches

II. The a c t i n m y o s i n in terac t ion and its control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 A. The ATPase cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 B. The control of the a c t i n m y o s i n in terac t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 C. Quan t i t a t ive aspects of the re la t ion between ca lc ium and a c t i n m y o s i n in terac t ion . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice.

The fluorescent probe Fura-2/AM was used to determine cytosolic free calcium concentration in soleus muscle and in isolated flexor digitorum brevis fibres. This required a precise calibration; therefore, each calibration parameter was studied in situ. The influence of the dye concentration on calcium measurements was also examined. This precise calibration technique was used to compare absolute free calcium concentration in resting preparations from dystrophic (mdx) and control (C57) mice. We showed that the behavior of the dye was not similar in C57 and in mdx muscles. For this reason, we did not confirm the previous results that cytosolic free calcium concentration is increased in mdx muscles.

Intact satellite cells lead to remarkable protection against Smn gene defect in differentiated skeletal muscle

Deletion of murine Smn exon 7, the most frequent mutation found in spinal muscular atrophy, has been directed to either both satellite cells, the muscle progenitor cells and fused myotubes, or fused myotubes only. When satellite cells were mutated, mutant mice develop severe myopathic process, progressive motor paralysis, and early death at 1 mo of age (severe mutant). Impaired muscle regeneration of severe mutants correlated with defect of myogenic precursor cells both in vitro and in vivo. In contrast, when satellite cells remained intact, mutant mice develop similar myopathic process but exhibit mild phenotype with median survival of 8 mo and motor performance similar to that of controls (mild mutant). High proportion of regenerating myofibers expressing SMN was observed in mild mutants compensating for progressive loss of mature myofibers within the first 6 mo of age. Then, in spite of normal contractile properties of myofibers, mild mutants develop reduction of muscle force and mass. Progressive decline of muscle regeneration process was no more able to counterbalance muscle degeneration leading to dramatic loss of myofibers. These data indicate that intact satellite cells remarkably improve the survival and motor performance of mutant mice suffering from chronic myopathy, and suggest a limited potential of satellite cells to regenerate skeletal muscle.

Long-term study of Ca2+ homeostasis and of survival in collagenase-isolated muscle fibres from normal and mdx mice

Skeletal muscles of the mdx mouse lack dystrophin offering the possibility to study the role of intracellular Ca2+ ions in fibre degeneration. Flexor digitorum brevis muscles of 3‐month‐old mdx and normal mice were dissociated with collagenase; fibres were maintained in culture for 6 days (d0 to d5) and their survival was assessed. Cytosolic [Ca2+], passive Mn2+ influx (indicative of Ca2+ influx) and activity of mechanosensitive/voltage‐independent Ca2+ channels were studied over the same period. Survival of normal fibres declined steadily from d0 to d3, but an acceleration of fibre death occurred in mdx fibres from d1 to d2. This could be greatly reduced but not abolished by lowering external [Ca2+] 10‐fold. In the d0‐d5 period, both mdx and normal fibres showed transient increases of Mn2+ influx and activity of the Ca2+ channels; these peaked at d1 and disappeared by d3–d4. Increases were always significantly larger in mdx fibres. Altogether, over the 6 days, 130 paired measurements of [Ca2+]i and Mn2+ influx were made on 68 fibres from mdx and 62 fibres from normal mice. In 90 % of the fibres, [Ca2+]i remained within the 25–85 nm limits while Mn2+ influx varied more than 10‐fold. The median for Mn2+ influx was 45 % greater in fibres from mdx mice than in fibres from control C57 mice. However, there was no significant difference between [Ca2+]i medians in fibres from normal and mdx mice. Addition of 25–75 nm of a Ca2+ ionophore (4‐bromo‐A23187) to the medium did not affect the level of cytosolic [Ca2+] in both types of fibres, while markedly increasing the rate of Mn2+ influx, as expected. Thus, Ca2+ homeostasis was equally robust in mdx and normal fibres. The remaining 10 % of the fibres showed, at d1, high levels of Mn2+ influx and/or elevated [Ca2+]i above 100 nm. This did not affect survival of normal fibres but was probably responsible of the increased death rate in mdx fibres.

Mini‐dystrophin restores L‐type calcium currents in skeletal muscle of transgenic mdx mice

L‐type calcium currents (iCa) were recorded using the two‐microelectrode voltage‐clamp technique in single short toe muscle fibres of three different mouse strains: (i) C57/SV129 wild‐type mice (wt); (ii) mdx mice (an animal model for Duchenne muscular dystrophy; and (iii) transgenically engineered mini‐dystrophin (MinD)‐expressing mdx mice. The activation and inactivation properties of iCa were examined in 2‐ to 18‐month‐old animals. Ca2+ current densities at 0 mV in mdx fibres increased with age, but were always significantly smaller compared to age‐matched wild‐type fibres. Time‐to‐peak (TTP) of iCa was prolonged in mdx fibres compared to wt fibres. MinD fibres always showed similar TTP and current amplitudes compared to age‐matched wt fibres. In all three genotypes, the voltage‐dependent inactivation and deactivation of iCa were similar. Intracellular resting calcium concentration ([Ca2+]i) and the distribution of dihydropyridine binding sites were also not different in young animals of all three genotypes, whereas iCa was markedly reduced in mdx fibres. We conclude, that dystrophin influences L‐type Ca2+ channels via a direct or indirect linkage which may be disrupted in mdx mice and may be crucial for proper excitation–contraction coupling initiating Ca2+ release from the sarcoplasmic reticulum. This linkage seems to be fully restored in the presence of mini‐dystrophin.

Polymerization of myosin on activation of rat anococcygeus smooth muscle

The in vivo state of assembly ofmyosin in vertebrate smooth muscle is controversial. In vitrostudies on purified smooth muscle myosin show that it ismonomeric (10S) under relaxing conditions and filamentous undercontraction conditions. Electron microscopic and antibodylabelling studies of intact smooth muscles, on the other hand,suggest that myosin is filamentous in the relaxed as well as thecontracting state and that 10S myosin occurs only in traceamounts. However, birefringence, conventional EM and X-raydiffraction evidence suggests that in certain smooth muscles invivo (e.g. rat anococcygeus), while myosin filaments exist in therelaxed state, their number increases on contraction. Here, wehave used low temperature electron microscopic techniques (rapidfreezing followed by freeze-substitution), which preserve labilecomponents in close to their in vivo state, to detect any changein filament number on contraction. The results from ratanococcygeus have been compared with those from guinea pig taeniacoli, in which other techniques have revealed no change infilament number. In the anococcygeus, we find evidence for a 23%increase in filament density in transverse sections ofcontracting muscle compared with relaxed muscle. In the taeniacoli we find no change. These results are in qualitativeagreement with earlier findings. They provide evidence forpolymerization of myosin in contracting rat anococcygeus, andsuggest that this process is subtle and occurs only in somesmooth muscles

Tetanus relaxation of fast skeletal muscles of the mouse made parvalbumin deficient by gene inactivation.

1 The effects of tetanus duration on the relaxation rate of extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles were studied in normal (wild‐type, WT) and parvalbumin‐deficient (PVKO) mice, at 20 °C. 2 In EDL of PVKO, the relaxation rate was low and unaffected by tetanus duration (< 3.2 s). In contrast, the relaxation rate of WT muscles decreased when tetanus duration increased from 0.2 to 3.2 s. In WT muscles, fast relaxation recovered as the rest interval increased. 3 Specific effect of parvalbumin was asserted by calculating the difference in relaxation rate between WT and PVKO muscles. For EDL, the rate constant of relaxation slowing was 1.10 s−1 of tetanization; the rate constant of relaxation recovery was 0.05 s−1 of rest. 4 In FDB, the effects of tetanus duration on WT and PVKO muscles were qualitatively similar to those observed in EDL. 5 Relaxation slowing as tetanus duration increases, reflects the progressive saturation of parvalbumin by Ca2+, while recovery as rest interval increases reflects the return to Ca2+‐free parvalbumin. 6 At all tetanus durations, relaxation rate still remained slightly faster in WT muscles. This suggested that parvalbumin facilitates calcium traffic from myofibrils to the SR. 7 No difference was found between WT and PVKO muscles for: (i) the expression of the fast isoforms of myosin heavy chains, (ii) the force‐velocity relationship and maximal shortening velocity and (iii) the Ca2+‐activated ATPase activity from isolated preparations of the sarcoplasmic reticulum (SR).

In situ measurements of calpain activity in isolated muscle fibres from normal and dystrophin-lacking mdx mice

Calpains are Ca2+‐activated proteases that are thought to be involved in muscle degenerative diseases such as Duchenne muscular dystrophy. Status and activity of calpains in adult muscle fibres are poorly documented. We report here in situ measurements of calpain activity in collagenase‐isolated fibres from C57 mice and form two models of dystrophy: dystrophin‐deficient mdx and calpain‐3 knocked‐out mice. Calpain activity was measured using a permeant, fluorogenic substrate and its Ca2+ dependence was studied. A 30‐fold change of activity was observed between the lowest and the highest steady‐state Ca2+ availability. Fast transient changes of [Ca2+]i induced by electrical stimulation or KCl‐dependent depolarization were ineffective in activating calpain. Slow [Ca2+] transients, as elicited during depletion of Ca2+ stores, Ca2+ store repletion and hypo‐osmotic swelling were able to activate calpain. On return to resting conditions, calpain activity recovered its basal rate within 10 min. In resting intact muscle, μ‐calpain was predominantly in the 80 kDa native form, with a small fraction in the 78 kDa autolysed form. The latter is thought to be responsible for the activity measured in our conditions. Calpain activity in mdx fibres showed an average 1.5‐fold increase compared to activity in C57 fibres. This activity was reduced by a 10‐fold lowering of [Ca2+]o. Calpain‐3‐deficient fibres showed about the same increase, thus calpain‐3 did not contribute to the activity measured here and calpain activation is not specific to dystrophin deficiency. In fibres from transgenic mice over‐expressing calpastatin, a 40–50% reduction of calpain activity was observed, as with synthetic drugs (Z‐Leu‐Leu‐CHO and SNT198438). We provide novel information on the physiological factors that control calpain activity in situ, particularly the effect of intracellular Ca2+ transients that occur in excitation–contraction coupling, Ca2+ store depletion and refilling, and activation of mechanosensitive Ca2+ channels.