Attenuated Ca(2+) release in a mouse model of limb girdle muscular dystrophy 2A.

BACKGROUND: Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n',n'-tetraacetic acid (EGTA) to measure Ca(2+) fluxes. Muscles were subjected to both current and voltage clamp conditions. METHODS: Standard and confocal fluorescence microscopy was used to study Ca(2+) release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student's test with significance set at p < 0.05. RESULTS: We found that the peak value of Ca(2+) fluxes elicited by single action potentials was significantly reduced by 15-20 % in C3KO fibers, but the kinetics was unaltered. Ca(2+) release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca(2+) release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca(2+) release in C3KO mice but reduced peak Ca(2+) fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca(2+) release at all triads consistent with a homogenous reduction of functional voltage activated Ca(2+) release sites. CONCLUSIONS: Overall, these results suggest that decreased Ca(2+) release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

Muscleblind-Like 1 and Muscleblind-Like 3 Depletion Synergistically Enhances Myotonia by Altering Clc-1 RNA Translation.

Loss of Muscleblind-like 1 (Mbnl1) is known to alter Clc-1 splicing to result in myotonia. Mbnl1(ΔE3/ΔE3)/Mbnl3(ΔE2) mice, depleted of Mbnl1 and Mbnl3, demonstrate a profound enhancement of myotonia and an increase in the number of muscle fibers with very low Clc-1 currents, where gClmax values approach ~ 1 mS/cm(2), with the absence of a further enhancement in Clc-1 splice errors, alterations in polyA site selection or Clc-1 localization. Significantly, Mbnl1(ΔE3/ΔE3)/Mbnl3(ΔE2) muscles demonstrate an aberrant accumulation of Clc-1 RNA on monosomes and on the first polysomes. Mbnl1 and Mbnl3 bind Clc-1 RNA and both proteins bind Hsp70 and eEF1A, with these associations being reduced in the presence of RNA. Thus binding of Mbnl1 and Mbnl3 to Clc-1 mRNA engaged with ribosomes can facilitate an increase in the local concentration of Hsp70 and eEF1A to assist Clc-1 translation. Dual depletion of Mbnl1 and Mbnl3 therefore initiates both Clc-1 splice errors and translation defects to synergistically enhance myotonia. As the HSA(LR) model for myotonic dystrophy (DM1) shows similar Clc-1 defects, this study demonstrates that both splice errors and translation defects are required for DM1 pathology to manifest. RESEARCH IN CONTEXT: Research in context: Myotonic Dystrophy type 1 (DM1) is a dominant disorder resulting from the expression of expanded CUG repeat RNA, which aberrantly sequesters and inactivates the muscleblind-like (MBNL) family of proteins. In mice, inactivation of Mbnl1 is known to alter Clc-1 splicing to result in myotonia. We demonstrate that concurrent depletion of Mbnl1 and Mbnl3 results in a synergistic enhancement of myotonia, with an increase in muscle fibers showing low chloride currents. The observed synergism results from the aberrant accumulation of Clc-1 mRNA on monosomes and the first polysomes. This translation error reflects the ability of Mbnl1 and Mbnl3 to act as adaptors that recruit Hsp70 and eEF1A to the Clc-1 mRNA engaged with ribosomes, to facilitate translation. Thus our study demonstrates that Clc-1 RNA translation defects work coordinately with Clc-1 splice errors to synergistically enhance myotonia in mice lacking Mbnl1 and Mbnl3.

Inward rectifier potassium currents in mammalian skeletal muscle fibres.

Inward rectifying potassium (Kir) channels play a central role in maintaining the resting membrane potential of skeletal muscle fibres. Nevertheless their role has been poorly studied in mammalian muscles. Immunohistochemical and transgenic expression were used to assess the molecular identity and subcellular localization of Kir channel isoforms. We found that Kir2.1 and Kir2.2 channels were targeted to both the surface and the transverse tubular system membrane (TTS) compartments and that both isoforms can be overexpressed up to 3-fold 2 weeks after transfection. Inward rectifying currents (IKir) had the canonical features of quasi-instantaneous activation, strong inward rectification, depended on the external [K(+)], and could be blocked by Ba(2+) or Rb(+). In addition, IKir records show notable decays during large 100 ms hyperpolarizing pulses. Most of these properties were recapitulated by model simulations of the electrical properties of the muscle fibre as long as Kir channels were assumed to be present in the TTS. The model also simultaneously predicted the characteristics of membrane potential changes of the TTS, as reported optically by a fluorescent potentiometric dye. The activation of IKir by large hyperpolarizations resulted in significant attenuation of the optical signals with respect to the expectation for equal magnitude depolarizations; blocking IKir with Ba(2+) (or Rb(+)) eliminated this attenuation. The experimental data, including the kinetic properties of IKir and TTS voltage records, and the voltage dependence of peak IKir, while measured at widely dissimilar bulk [K(+)] (96 and 24 mm), were closely predicted by assuming Kir permeability (PKir) values of ∼5.5 × 10(-6 ) cm s(-1) and equal distribution of Kir channels at the surface and TTS membranes. The decay of IKir records and the simultaneous increase in TTS voltage changes were mostly explained by K(+) depletion from the TTS lumen. Most importantly, aside from allowing an accurate estimation of most of the properties of IKir in skeletal muscle fibres, the model demonstrates that a substantial proportion of IKir (>70%) arises from the TTS. Overall, our work emphasizes that measured intrinsic properties (inward rectification and external [K] dependence) and localization of Kir channels in the TTS membranes are ideally suited for re-capturing potassium ions from the TTS lumen during, and immediately after, repetitive stimulation under physiological conditions.

Action potential-evoked calcium release is impaired in single skeletal muscle fibers from heart failure patients.

BACKGROUND: Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium (Ca2+) release, mandating investigations in skeletal muscle from HF patients. The purpose of this study was to test the hypothesis that Ca2+ release is significantly impaired in the skeletal muscle of HF patients in whom exercise capacity is severely diminished compared to age-matched healthy volunteers. METHODS AND FINDINGS: Using state-of-the-art electrophysiological and optical techniques in single muscle fibers from biopsies of the locomotive vastus lateralis muscle, we measured the action potential (AP)-evoked Ca2+ release in 4 HF patients and 4 age-matched healthy controls. The mean peak Ca2+ release flux in fibers obtained from HF patients (10±1.2 µM/ms) was markedly (2.6-fold) and significantly (p<0.05) smaller than in fibers from healthy volunteers (28±3.3 µM/ms). This impairment in AP-evoked Ca2+ release was ubiquitous and was not explained by differences in the excitability mechanisms since single APs were indistinguishable between HF patients and healthy volunteers. CONCLUSIONS: These findings prove the feasibility of performing electrophysiological experiments in single fibers from human skeletal muscle, and offer a new approach for investigations of myopathies due to HF and other diseases. Importantly, we have demonstrated that one step in the ECC process, AP-evoked Ca2+ release, is impaired in single muscle fibers in HF patients.

A novel synaptic vesicle fusion path in the rat cerebral cortex: the "saddle" point hypothesis.

We improved freeze-fracture electron microscopy to study synapses in the neuropil of the rat cerebral cortex at ∼2 nm resolution and in three-dimensions. In the pre-synaptic axon, we found that "rods" assembled from short filaments protruding from the vesicle and the plasma membrane connects synaptic vesicles to the membrane of the active zone. We equated these "connector rods" to protein complexes involved in "docking" and "priming" vesicles to the active zone. Depending on their orientation, the "rods" define two synaptic vesicle-fusion paths: When parallel to the plasma membrane, the vesicles hemi-fuse anywhere ("randomly") in the active zone following the conventional path anticipated by the SNARE hypothesis. When perpendicular to the plasma membrane, the vesicles hemi-fuse at the base of sharp crooks, called "indentations," that are spaced 75-85 nm center-to-center, arranged in files and contained within gutters. They result from primary and secondary membrane curvatures that intersect at stationary inflection ("saddle") points. Computer simulations indicate that this novel vesicle-fusion path evokes neurotransmitter concentration domains on the post-synaptic spine that are wider, shallower, and that reach higher average concentrations than the more conventional vesicle fusion path. In the post-synaptic spine, large (∼9× âˆ¼15 nm) rectangular particles at densities of 72±10/ µm2 (170-240/spine) match the envelopes of the homotetrameric GluR2 AMPA-sensitive receptor. While these putative receptors join clusters, called the "post-synaptic domains," the overwhelming majority of the rectangular particles formed bands in the "non-synaptic" plasma membrane of the spine. In conclusion, in the neuropil of the rat cerebral cortex, curvatures of the plasma membrane define a novel vesicle-fusion path that preconditions specific regions of the active zone for neurotransmitter release. We hypothesize that a change in the hybridization of the R-SNARE synaptobrevin from parallel to antiparallel swings the synapse into this novel vesicle-fusion path.

Design and construction of a modular low-cost epifluorescence upright microscope for neuron visualized recording and fluorescence detection.

BACKGROUND: One of the limitations when establishing an electrophysiology setup, particularly in low resource settings, is the high cost of microscopes. The average cost for a microscope equipped with the optics for infrared (IR) contrast or microfluorometry is $40,000. We hypothesized that optical elements and features included in commercial microscopes are not necessary to IR video-visualize neurons or for microfluorometry. NEW METHOD: We present instructions for building a low-cost epifluorescence upright microscope suitable for visualized patch-clamp recording and fluorescence detection using mostly catalog-available parts. RESULTS: This microscope supports applications such as visualized whole-cell recording using IR oblique illumination (IR-OI), or more complex applications such as microfluorometry using a photodiode. In both IR-OI and fluorescence, actual resolution measured with 2-µm latex beads is close to theoretical resolution. The lack of movable parts to switch configurations ensures stability when doing intracellular recording. COMPARISON WITH EXISTING METHODS: The low cost is a significant advantage of this microscope compared to existent custom-built microscopes. The cost of the simplest configuration with IR-OI is ∼$2000, whereas the cost of the configuration with epifluorescence is ∼$5000. Since this design does not use pieces discarded from commercial microscopes, it is completely reproducible. CONCLUSIONS: We suggest that this microscope is a viable alternative for doing in vitro electrophysiology and microfluorometry in low-resource settings. Characteristics such as an open box design, easy assembly, and low-cost make this microscope a useful instrument for science education and teaching for topics such as optics, biology, neuroscience, and for scientific "hands-on" workshops.

Age-dependent chloride channel expression in skeletal muscle fibres of normal and HSA(LR) myotonic mice.

Abstract We combine electrophysiological and optical techniques to investigate the role that the expression of chloride channels (ClC-1) plays on the age-dependent electrical properties of mammalian muscle fibres. To this end, we comparatively evaluate the magnitude and voltage dependence of chloride currents (ICl), as well as the resting resistance, in fibres isolated from control and human skeletal actin (HSA)(LR) mice (a model of myotonic dystrophy) of various ages. In control mice, the maximal peak chloride current ([peak-ICl]max) increases from -583 ± 126 to -956 ± 260 µA cm(-2) (mean ± SD) between 3 and 6 weeks old. Instead, in 3-week-old HSA(LR) mice, ICl are significantly smaller (-153 ± 33 µA cm(-2)) than in control mice, but after a long period of ∼14 weeks they reach statistically comparable values. Thus, the severe ClC-1 channelopathy in young HSA(LR) animals is slowly reversed with aging. Frequency histograms of the maximal chloride conductance (gCl,max) in fibres of young HSA(LR) animals are narrow and centred in low values; alternatively, those from older animals show broad distributions, centred at larger gCl,max values, compatible with mosaic expressions of ClC-1 channels. In fibres of both animal strains, optical data confirm the age-dependent increase in gCl, and additionally suggest that ClC-1 channels are evenly distributed between the sarcolemma and transverse tubular system membranes. Although gCl is significantly depressed in fibres of young HSA(LR) mice, the resting membrane resistance (Rm) at -90 mV is only slightly larger than in control mice due to upregulation of a Rb-sensitive resting conductance (gK,IR). In adult animals, differences in Rm are negligible between fibres of both strains, and the contributions of gCl and gK,IR are less altered in HSA(LR) animals. We surmise that while hyperexcitability in young HSA(LR) mice can be readily explained on the basis of reduced gCl, myotonia in adult HSA(LR) animals may be explained on the basis of a mosaic expression of ClC-1 channels in different fibres and/or on alterations of other conductances.

The delayed rectifier potassium conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers.

A two-microelectrode voltage clamp and optical measurements of membrane potential changes at the transverse tubular system (TTS) were used to characterize delayed rectifier K currents (IK(V)) in murine muscle fibers stained with the potentiometric dye di-8-ANEPPS. In intact fibers, IK(V) displays the canonical hallmarks of K(V) channels: voltage-dependent delayed activation and decay in time. The voltage dependence of the peak conductance (gK(V)) was only accounted for by double Boltzmann fits, suggesting at least two channel contributions to IK(V). Osmotically treated fibers showed significant disconnection of the TTS and displayed smaller IK(V), but with similar voltage dependence and time decays to intact fibers. This suggests that inactivation may be responsible for most of the decay in IK(V) records. A two-channel model that faithfully simulates IK(V) records in osmotically treated fibers comprises a low threshold and steeply voltage-dependent channel (channel A), which contributes ∼31% of gK(V), and a more abundant high threshold channel (channel B), with shallower voltage dependence. Significant expression of the IK(V)1.4 and IK(V)3.4 channels was demonstrated by immunoblotting. Rectangular depolarizing pulses elicited step-like di-8-ANEPPS transients in intact fibers rendered electrically passive. In contrast, activation of IK(V) resulted in time- and voltage-dependent attenuations in optical transients that coincided in time with the peaks of IK(V) records. Normalized peak attenuations showed the same voltage dependence as peak IK(V) plots. A radial cable model including channels A and B and K diffusion in the TTS was used to simulate IK(V) and average TTS voltage changes. Model predictions and experimental data were compared to determine what fraction of gK(V) in the TTS accounted simultaneously for the electrical and optical data. Best predictions suggest that K(V) channels are approximately equally distributed in the sarcolemma and TTS membranes; under these conditions, >70% of IK(V) arises from the TTS.

The Na conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers.

Na (and Li) currents and fluorescence transients were recorded simultaneously under voltage-clamp conditions from mouse flexor digitorum brevis fibers stained with the potentiometric dye di-8-ANEPPS to investigate the distribution of Na channels between the surface and transverse tubular system (TTS) membranes. In fibers rendered electrically passive, voltage pulses resulted in step-like fluorescence changes that were used to calibrate the dye response. The effects of Na channel activation on the TTS voltage were investigated using Li, instead of Na, because di-8-ANEPPS transients show anomalies in the presence of the latter. Na and Li inward currents (I(Na), I(Li); using half of the physiological ion concentration) showed very steep voltage dependences, with no reversal for depolarizations beyond the calculated equilibrium potential, suggesting that most of the current originates from a noncontrolled membrane compartment. Maximum peak I(Li) was ∼ 30% smaller than for I(Na), suggesting a Li-blocking effect. I(Li) activation resulted in the appearance of overshoots in otherwise step-like di-8-ANEPPS transients. Overshoots had comparable durations and voltage dependence as those of I(Li). Simultaneously measured maximal overshoot and peak I(Li) were 54 ± 5% and 773 ± 53 µA/cm(2), respectively. Radial cable model simulations predicted the properties of I(Li) and di-8-ANEPPS transients when TTS access resistances of 10-20 Ω cm(2), and TTS-to-surface Na permeability density ratios in the range of 40:60 to 70:30, were used. Formamide-based osmotic shock resulted in incomplete detubulation. However, results from a subpopulation of treated fibers (low capacitance) provide confirmatory evidence that a significant proportion of I(Li), and the overshoot in the optical signals, arises from the TTS in normal fibers. The quantitative evaluation of the distribution of Na channels between the sarcolemma and the TTS membranes, as provided here, is crucial for the understanding of the radial and longitudinal propagation of the action potential, which ultimately govern the mechanical activation of muscle in normal and diseased conditions.

Functional expression of transgenic 1sDHPR channels in adult mammalian skeletal muscle fibres.

We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca2+ currents (ICa), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB)muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-α1sDHPR-wt in quantities comparable to that of native α1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (α1sDHPR) resulted in ∼50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (gCa) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-α1sDHPR-T935Y exhibited up to 70% isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Qand SRCa2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-α1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of gCa, Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of the mworks as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the α1sDHPR leads to the replacement of native channels interacting with the ryanodine receptor 1 (RyR1), thus demonstrating the feasibility of molecular remodelling of the triads in adult skeletal muscle fibres.

Chloride currents from the transverse tubular system in adult mammalian skeletal muscle fibers.

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (I(Cl)) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward I(Cl), and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and I(Cl) acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be I(Cl) dependent since its magnitude varied in close correlation with the amplitude and time course of I(Cl). While the properties of I(Cl), and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (P(Cl)) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if P(Cl) was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded I(Cl) arises from TTS contributions.

Excitation-contraction coupling alterations in mdx and utrophin/dystrophin double knockout mice: a comparative study.

The double knockout mouse for utrophin and dystrophin (utr(-/-)/mdx) has been proposed to be a better model of Duchenne Muscular Dystrophy (DMD) than the mdx mouse because the former displays more similar muscle pathology to that of the DMD patients. In this paper the properties of action potentials (APs) and Ca(2+) transients elicited by single and repetitive stimulation were studied to understand the excitation-contraction (EC) coupling alterations observed in muscle fibers from mdx and utr(-/-)/mdx mice. Based on the comparison of the AP durations with those of fibers from wild-type (WT) mice, fibers from both mdx and utr(-/-)/mdx mice could be divided in two groups: fibers with WT-like APs (group 1) and fibers with significantly longer APs (group 2). Although the proportion of fibers in group 2 was larger in utr(-/-)/mdx (36%) than in mdx mice (27%), the Ca(2+) release elicited by single stimulation was found to be similarly depressed (32-38%) in utr(-/-)/mdx and mdx fibers compared with WT counterparts regardless of the fiber's group. Stimulation at 100 Hz revealed that, with the exception of those from utr(-/-)/mdx mice, group 1 fibers were able to sustain Ca(2+) release for longer than group 2 fibers, which displayed an abrupt limitation even at the onset of the train. The differences in behavior between fibers in groups 1 and 2 became almost unnoticeable at 50 Hz stimulation. In general, fibers from utr(-/-)/mdx mice seem to display more persistent alterations in the EC coupling than those observed in the mdx model.

Dystrophic skeletal muscle fibers display alterations at the level of calcium microdomains.

The spatiotemporal properties of the Ca(2+)-release process in skeletal muscle fibers from normal and mdx fibers were determined using the confocal-spot detection technique. The Ca(2+) indicator OGB-5N was used to record action potential-evoked fluorescence signals at consecutive locations separated by 200 nm along multiple sarcomeres of FDB fibers loaded with 10- and 30-mM EGTA. Three-dimensional reconstructions of fluorescence transients demonstrated the existence of microdomains of increased fluorescence around the Ca(2+)-release sites in both mouse strains. The Ca(2+) microdomains in mdx fibers were regularly spaced along the fiber axis, displaying a distribution similar to that seen in normal fibers. Nevertheless, both preparations differed in that in 10-mM EGTA Ca(2+) microdomains had smaller amplitudes and were wider in mdx fibers than in controls. In addition, Ca(2+)-dependent fluorescence transients recorded at selected locations within the sarcomere of mdx muscle fibers were not only smaller, but also slower than their counterparts in normal fibers. Notably, differences in the spatial features of the Ca(2+) microdomains recorded in mdx and normal fibers, but not in the amplitude and kinetics of the Ca(2+) transients, were eliminated in 30-mM EGTA. Our results consistently demonstrate that Ca(2+)-release flux calculated from release sites in mdx fibers is uniformly impaired with respect to those normal fibers. The Ca(2+)-release reduction is consistent with that previously measured using global detection techniques.

Somatic Ca2+ transients do not contribute to inspiratory drive in preBötzinger Complex neurons.

PreBötzinger Complex (preBötC) neurons are postulated to underlie respiratory rhythm generation. The inspiratory phase of the respiratory cycle in vitro results from preBötC neurons firing synchronous bursts of action potentials (APs) on top of 10-20 mV, 0.3-0.8 s inspiratory drive potentials. Is the inspiratory drive in individual neurons simply the result of the passive integration of inspiratory-modulated synaptic currents or do active processes modulate these currents? As somatic Ca(2+) is known to increase during inspiration, we hypothesized that it affects inspiratory drive. We combined whole cell recording in an in vitro slice preparation with Ca(2+) microfluorometry to detect single inspiratory neuron somatic Ca(2+) transients with high temporal resolution ( approximately mus). In neurons loaded with either Fluo-4 or Oregon Green BAPTA 5 N, we observed Ca(2+) transients associated with each AP. During inspiration, significant somatic Ca(2+) influx was a direct consequence of activation of voltage-gated Ca(2+) channels by APs. However, when we isolated the inspiratory drive potential in active preBötC neurons (by blocking APs with intracellular QX-314 or by hyperpolarization), we did not detect somatic Ca(2+) transients; yet, the parameters of inspiratory drive were the same with or without APs. We conclude that, in the absence of APs, somatic Ca(2+) transients do not shape the somatic inspiratory drive potential. This suggests that in preBötC neurons, substantial and widespread somatic Ca(2+) influx is a consequence of APs during the inspiratory phase and does not contribute substantively to the inspiratory drive potential. Given evidence that the Ca(2+) buffer BAPTA can significantly reduce inspiratory drive, we hypothesize that dendritic Ca(2+) transients amplify inspiratory-modulated synaptic currents.

Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers.

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 DiBAC(4)(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 DiBAC(4)(5). The peak DeltaF/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 DiBAC(4)(5) exhibit DeltaF/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.

Resolving the fast kinetics of cooperative binding: Ca2+ buffering by calretinin.

Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Némethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast fluorescent Ca2+ indicator following rapid (<50-mus rise time) Ca2+ concentration jumps induced by uncaging Ca2+ from DM-nitrophen. To unravel the kinetics of cooperative binding, we devised several approaches based on known cooperative binding models, resulting in a novel and relatively simple model. This model revealed unexpected and highly specific nonlinear properties of cellular Ca2+ regulation by calretinin. The association rate of Ca2+ with calretinin speeds up as the free Ca2+ concentration increases from cytoplasmic resting conditions ( approximately 100 nM) to approximately 1 muM. As a consequence, the Ca2+ buffering speed of calretinin highly depends on the prevailing Ca2+ concentration prior to a perturbation. In addition to providing a novel mode of action of cellular Ca2+ buffering, our model extends the analysis of cooperativity beyond the static steady-state condition, providing a powerful tool for the investigation of the dynamics and functional significance of cooperative binding in general.

Modulation by caffeine of calcium-release microdomains in frog skeletal muscle fibers.

The effects of caffeine on the process of excitation-contraction coupling in amphibian skeletal muscle fibers were investigated using the confocal spot detection technique. This method permits to carefully discriminate between caffeine effects on the primary sources of Ca2+ release at the Z-lines where the triads are located and secondary actions on other potential Ca Release sources. Our results demonstrate that 0.5 mM caffeine potentiates and prolongs localized action-potential evoked Ca2+ transients recorded at the level of the Z-lines, but that 1mM only prolongs them. The effects at both doses are reversible. At the level of the M-line, localized Ca2+ transients displayed more variability in the presence of 1 mM caffeine than in control conditions. At this dose of caffeine, extra-junctional sources of Ca2+ release also were observed occasionally.

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques.

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system.

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.

Modulation by caffeine of calcium-release microdomains in frog skeletal muscle fibers

The effects of caffeine on the process of excitation-contraction coupling in amphibian skeletal muscle fibers were investigated using the confocal spot detection technique. This method permits to carefully discriminate between caffeine effects on the primary sources of Ca2+ release at the Z-lines where the triads are located and secondary actions on other potential Ca Release sources. Our results demonstrate that 0.5 mM caffeine potentiates and prolongs localized action-potential evoked Ca2+ transients recorded at the level of the Z-lines, but that 1mM only prolongs them. The effects at both doses are reversible. At the level of the M-line, localized Ca2+ transients displayed more variability in the presence of 1 mM caffeine than in control conditions. At this dose of caffeine, extra-junctional sources of Ca2+ release also were observed occasionally.