Alteration of excitation‐contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine

Abstract

1 No clear data is available about functional alterations in the calcium-dependent excitationcontraction (e-c) coupling mechanism of dystrophin-de®cient muscle of mdx mice. 2 By means of the intracellular microelectrode`point' voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle ®bres of dystrophic mdx mouse of two di erent ages: 8 ± 12 weeks, during the active regeneration of hind limb muscles, and 6 ± 8 months, when regeneration is complete. 3 The EDL muscle ®bres of 8 ± 12-week-old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction-and relaxation-related calcium movements) with respect to control mice of 6 ± 8 months. However, at both ages, the EDL muscle ®bres of mdx mice contracted at more negative potentials with respect to age-matched controls and had markedly slower time constants to reach the rheobase. 4 The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e-c coupling, was without e ect on 6 ± 8-month-old wildtype EDL muscle, while it signi®cantly ameliorated the MT of mdx mouse. 5 HPLC determination of taurine content at 6 ± 8 months showed a signi®cant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue di culty in retaining appropriate levels of the amino acid. 6 The data is consistent with a permanent alteration of e-c coupling in mdx EDL muscle ®bres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid.

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“…In the fitting algorithm, each point was weighed by the reciprocal of the variance of that mean V and the best fit estimates of the parameters R and 1/ were made. 9,33 We used this procedure to be able to incorporate all of our determination points and their associated errors into our estimate of R under each condition.…”

“…Fitted rheobase (R) and rate constant (1/ ) parameters Ϯ SE were determined from the variance-covariance matrix in the nonlinear least square fitting algorithm. 9,33 For these fitted parameters, the statistical significance between groups was estimated by using the above tests using these standard errors and a number of degrees of freedom equal to the total number of threshold values determining the curves minus the number of means minus two for the free parameters. 9,33 …”

“…We estimated the uncertainty of any single measurement for a given fiber to be 1 to 2 mV. 9,33 The threshold membrane potential V (mV) for each fiber was averaged at each pulse duration t (ms) and then mean values plotted against duration giving us a strength-duration relationship. A fit estimate of the rheobase voltage (R) and of the rate constant (1/ ) to reach the rheobase was obtained by nonlinear least square algorithm using the following equation:…”

“…31,32 The mechanical threshold (MT) of the fibers was determined in EDL muscle fibers in normal physiological solution using a two microelectrode point voltage clamp method as previously described. 9,33 In brief, the two microelectrodes were inserted within 5 m of each other into the central region of a randomly selected superficial fiber that was continuously viewed using a stereomicroscope (ϫ100 magnification). The holding potential was set at Ϫ90 mV and depolarizing command pulses of variable duration were given at a rate of ϳ0.3 Hz.…”

“…Tetrodotoxin (3 mol/L) was continuously present during recordings to prevent action potential generation. 9,33 As a standard protocol the command-pulse duration was usually set sequentially to each of the following values: 500, 50, 5, 200, 20, 100, and 10 ms. At each duration, the command voltage was increased using an analogue control until contraction was just visible, and then backed down until the contraction just disappeared. A digital sample-and-hold millivoltmeter stored the value of the threshold membrane potential at this point.…”