Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation-contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca 2+ release by measuring Ca 2+ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca 2+ release elicited by voltage pulses was reduced by ∼67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca 2+ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca 2+ may be altered in the mdx mouse.

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle 2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach.The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats.The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs.The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6–8.

Potentiometric dyes are useful tools for studying membrane potential changes from compartments inaccessible to direct electrical recordings. In the past, we have combined electrophysiological and optical techniques to investigate, by using absorbance and fluorescence potentiometric dyes, the electrical properties of the transverse tubular system in amphibian skeletal muscle fibers. In this paper we expand on recent observations using the fluorescent potentiometric indicator di-8-ANEPPS to investigate structural and functional properties of the transverse tubular system in mammalian skeletal muscle fibers. Two-photon laser scanning confocal fluorescence images of live muscle fibers suggest that the distance between consecutive rows of transverse tubules flanking the Z-lines remains relatively constant in muscle fibers stretched to attain sarcomere lengths of up to 3.5 microm. Furthermore, the combined use of two-microelectrode electrophysiological techniques with microscopic fluorescence spectroscopy and imaging allowed us to compare the spectral properties of di-8-ANEPPS fluorescence in fibers at rest, with those of fluorescence transients recorded in stimulated fibers. We found that although the indicator has excitation and emission peaks at 470 and 588 nm, respectively, fluorescence transients display optimal fractional changes (13%/100 mV) when using filters to select excitation wavelengths in the 530-550 nm band and emissions beyond 590 nm. Under these conditions, results from tetanically stimulated fibers and from voltage-clamp experiments suggest strongly that, although the kinetics of di-8-ANEPPS transients in mammalian fibers are very rapid and approximate those of the surface membrane electrical recordings, they arise from the transverse tubular system membranes.

Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC4(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC4(5). The peak ΔF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC4(5) exhibit ΔF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.

Ablation of the immunomodulator osteopontin correlates with reduced fibrosis and improved muscle strength in Duchenne muscular dystrophy models. Here, Capote et al. show that osteopontin ablation skews dystrophic macrophages toward a pro-regenerative phenotype, leading to improved and sustained muscle mass and strength in long-term functional testing.