Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model. Muscle contraction is activated by Ca 2ϩ release from the sarcoplasmic reticulum in response to plasma membrane depolarization. This process, called excitation-contraction coupling, takes place at the skeletal muscle triad junction, where T-tubules and the sarcoplasmic reticulum terminal cisternae are in close contact (1). Calcium release occurs via the calcium release complex, a macromolecular complex specifically localized in the skeletal muscle triad (2). The main components of this calcium release complex are the ryanodine receptor (RyR) 2 and the dihydropyridine receptor (DHPR) (3), both of which are calcium channels. It is now clear that a number of proteins are associated with these two calcium channels to form the calcium release complex, triadin being part of them. Triadin is an integral membrane protein of the sarcoplasmic reticulum, first identified in rabbit skeletal muscle as a 95-kDa glycoprotein specifically located in the triads (4, 5). Because of its co-localization with RyR in the triads, involvement of triadin in excitation-contraction coupling has been postulated (6, 7). Protein interaction studies have shown that the major molecular partners of triadin are RyR (8 -10), calsequestrin (CSQ), a protein that traps calcium inside the sarcoplasmic reticulum (11-13), and junctin (14). Functional studies have shown that triadin by itself r...

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The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 μM and a 53 ± 8% (mean ± SEM, n = 9, cut fibers) attenuation at 0 mV with 25 μM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 ± 8%) and of the early peak component (46 ± 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (ICa) were left, essentially, unaltered. However, the inactivation of ICa was slowed fourfold, and the conductance was reduced from 200 ± 16 to 143 ± 8 SF−1 (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 ± 10% (n = 3) at 12 μM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30–50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.

The major energy-consuming processes in active skeletal muscle are the release and uptake of calcium ions by the sarcoplasmic reticulum (SR) and the actomyosin (AM) interaction, which results in force development. The chemical energy for these processes is provided by the hydrolysis of adenosine 5fi-triphosphate (ATP). It is well established that marked differences exist between fibre types with regard to the rates of energy expenditure associated with the SR and AM ATPase. The variation in SR ATPase is most likely to be due to differences of density in SR Ca 2+ pumps between different fibre types (Zubrzycka-Gaarn et al. 1982). The difference in the isometric AM ATPase activity between fibre types originates from differences between myosin isoforms (see for a review, Reggiani et al. 2000).Little is known about the fibre type dependence of energy expenditure in human muscle fibres. Improvements in 31 P NMR spectroscopy have made it possible to estimate ATP consumption during twitch stimulation and during maximal voluntary contraction in skeletal muscle in vivo (Boska, 1991; Blei et al. 1993a,b;Turner et al. 1993). However, the large heterogeneity in fibre properties and the variability in fibre type distribution make it very difficult to determine the differences in ATP consumption at the cellular level. Using permeabilized muscle fibres obtained from biopsies from healthy volunteers, Stienen et al. (1996) showed that considerable differences exist in myofibrillar ATPase activity (and isometric tension cost) between fibres of different type. However, so far nothing is known about the partitioning of the SR and AM ATPase activity in human skeletal muscle fibres.The present study aimed to determine the maximum rates of ATP consumption for the SR Ca 2+ pump and for the actomyosin interaction in single human skeletal muscle fibres during isometric contraction and their fibre ATP utilization for calcium uptake and force production in different types of human skeletal muscle fibres 1. The contractile properties and ATPase activity of skinned human skeletal muscle fibres from vastus lateralis were examined. Fibre types were resolved from single fibre segments by SDS-polyacrylamide gel electrophoresis. ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH.2. The partitioning of ATPase activity into (a) calcium-activated activity due to actomyosin (AM) interaction, (b) calcium-activated activity of the sarcoplasmic reticular (SR) calcium pump, and (c) basal (calcium independent) activity was investigated by comparing ATP utilization before and after exposure of the preparations for 30 min to a solution containing 0.5 % Triton X-100, which effectively abolished the SR ATPase activity.3. Partitioning of ATPase activity was also determined by measuring ATP utilization and force at different concentrations of butanedione monoxime (BDM), which inhibits AM interaction.4. The results obtained with Triton X-100 and BDM were similar. At saturating Ca 2+concentrations and 20 C, the AM, SR ...

Abstract-In cardiac muscle Ca 2ϩ -induced Ca 2ϩ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca 2ϩ influx via L-type Ca 2ϩ channels. At present, the mechanisms underlying termination of SR Ca 2ϩ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca 2ϩ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca 2ϩ release, we analyzed Na ϩ -Ca 2ϩ exchange currents reflecting cytosolic Ca 2ϩ signals induced by UV-laser flash-photolysis of caged Ca 2ϩ . Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, ␤-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by Ϸ2-fold. Application of cyclopiazonic acid at moderate concentrations (Ͻ10 mol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca 2ϩ refilling mediated by the SR Ca 2ϩ -pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca 2ϩ sensitivity of CICR appears to be regulated by SR Ca 2ϩ content, possibly resulting from a change in the steady-state Ca 2ϩ sensitivity and in the gating kinetics of the SR Ca 2ϩ release channels (ryanodine receptors Materials and Methods Cell IsolationAll experiments were performed according to the guidelines of the Swiss Animal Protection Law and with the permission of The State Veterinary Office, Bern, Switzerland. Cardiac ventricular myocytes were isolated from adult guinea pigs and mice (Schneider, Rubigen, Switzerland) using established enzymatic procedures. 14 The superfusion solution contained (in mmol/L): 140 NaCl, 5 KCl, 0.5 BaCl 2 , 1.8 CaCl 2 , 1 CsCl, 10 HEPES, 10 glucose, pH 7.4 (NaOH). The pipette filling solution contained (in mmol/L): 120 Cs-aspartate, 20 tetraethylammonium chloride, 2 Na 4 -DM-nitrophen (1-(2-nitro-4,5-dimethoxyphenyl)-1,2-diaminoethane-N,N,NЈ,NЈ-tetraacetic acid, 4Na ϩ ), 2 reduced glutathione, 0.05 Fluo-3, 0.5 CaCl 2 , 5 K 2 -ATP, 20 HEPES, pH 7.2 (CsOH). In experiments with mouse cells, the Na 4 -DM-nitrophen concentration was 4 mmol/L. All experiments were performed at room temperature (20°C to 22°C). Where required, the superfusion solution contained isoproterenol (1 mmol/L; Sigma) or cyclopiazonic acid (1, 5 or 10 mol/L; Sigma). Electrophysiological RecordingsCells were voltage-clamped in the whole-cell configuration of the patch-clamp technique and held at Ϫ80 mV. Flash PhotolysisPairs of UV-light flashes were used to photolyse intracellular DM-nitrophen in an epi-illumination arrangement and were generated with a frequency-tripled neodymium: yttrium-aluminum garnet laser (Nd:YAG laser) (wavelength 355 nm, maximal ...