Quantification of t-tubule area and protein distribution in rat cardiac ventricular myocytes

The transverse (t-) tubules of cardiac ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell. Many of the key proteins involved in excitation–contraction coupling appear to be located predominantly at the t-tubule membrane. Despite their importance, the fraction of cell membrane within the t-tubules remains unclear: measurement of cell capacitance following detubulation suggests 32%, whereas optical measurements suggest up to 65%. We have, therefore, investigated the factors that may account for this discrepancy. Calculation of the combinations of t-tubule radius, length and density that produce t-tubular membrane fractions of 32% or 56% suggest that the true fraction is at the upper end of this range. Assessment of detubulation using confocal and electron microscopy suggests that incomplete detubulation can account for some, but not all of the difference. High cholesterol, and a consequent decrease in specific capacitance, in the t-tubule membrane, may also cause the t-tubule fraction calculated from the loss of capacitance following detubulation to be underestimated. Correcting for both of these factors results in an estimate that is still lower than that obtained from optical measurements suggesting either that optical methods overestimate the fraction of membrane in the t-tubules, or that other, unknown, factors, reduce the apparent fraction obtained by detubulation. A biophysically realistic computer model of a rat ventricular myocyte, incorporating a t-tubule network, is used to assess the effect of the altered estimates of t-tubular membrane fraction on the calculated distribution of ion flux pathways.     

Variance in multiplex suspension array assays: microsphere size variation impact

Background

Skeletal muscle fibres contain transverse tubular (t-tubule) networks that allow electrical signals to rapidly propagate into the fibre. These electrical signals are generated by the transport of ions across the t-tubule membranes and this can result in significant changes in ion concentrations within the t-tubules during muscle excitation. During periods of repeated high-frequency activation of skeletal muscle the t-tubule K⁺ concentration is believed to increase significantly and diffusive K⁺ transport from the t-tubules into the interstitial space provides a mechanism for alleviating muscle membrane depolarization. However, the tortuous nature of the highly branched space-filling t-tubule network impedes the diffusion of material through the network. The effective diffusion coefficient for ions in the t-tubules has been measured to be approximately five times lower than in free solution, which is significantly different from existing theoretical values of the effective diffusion coefficient that range from 2–3 times lower than in free solution. To resolve this discrepancy, in this paper we study the process of diffusion within electron microscope scanned sections of the skeletal muscle t-tubule network using mathematical modelling and computer simulation techniques. Our model includes t-tubule geometry, tautness, hydrodynamic and non-planar network factors.

Results

Using our model we found that the t-tubule network geometry reduced the K⁺ diffusion coefficient to 19–27% of its value in free solution, which is consistent with the experimentally observed value of 21% and is significantly smaller than existing theoretical values that range from 32–50%. We also found that diffusion in the t-tubules is anomalous for skeletal muscle fibres with a diameter of less than approximately 10–20 μm as a result of obstructed diffusion. We also observed that the [K⁺] within the interior of the t-tubule network during high-frequency activation is greater for fibres with a larger diameter. Smaller skeletal muscle fibres are therefore more resistant to membrane depolarization. Because the t-tubule network is anisotropic and inhomogeneous, we also found that the [K⁺] distribution generated within the network was irregular for fibres of small diameter.

Conclusion

Our model explains the measured effective diffusion coefficient for ions in skeletal muscle t-tubules.     

A model of the guinea-pig ventricular cardiac myocyte incorporating a transverse-axial tubular system

A model of the guinea-pig cardiac ventricular myocyte has been developed that includes a representation of the transverse–axial tubular system (TATS), including heterogeneous distribution of ion flux pathways between the surface and tubular membranes. The model reproduces frequency-dependent changes of action potential shape and intracellular ion concentrations and can replicate experimental data showing ion diffusion between the tubular lumen and external solution in guinea-pig myocytes. The model is stable at rest and during activity and returns to rested state after perturbation. Theoretical analysis and model simulations show that, due to tight electrical coupling, tubular and surface membranes behave as a homogeneous whole during voltage and current clamp (maximum difference 0.9 mV at peak tubular I_Na of −38 nA). However, during action potentials, restricted diffusion and ionic currents in TATS cause depletion of tubular Ca²⁺ and accumulation of tubular K⁺ (up to −19.8% and +3.4%, respectively, of bulk extracellular values, at 6 Hz). These changes, in turn, decrease ion fluxes across the TATS membrane and decrease sarcoplasmic reticulum (SR) Ca²⁺ load. Thus, the TATS plays a potentially important role in modulating the function of guinea-pig ventricular myocyte in physiological conditions.     

Numerical analysis of Ca2+ signaling in rat ventricular myocytes with realistic transverse-axial tubular geometry and inhibited sarcoplasmic reticulum

The t-tubules of mammalian ventricular myocytes are invaginations of the cell membrane that occur at each Z-line. These invaginations branch within the cell to form a complex network that allows rapid propagation of the electrical signal, and hence synchronous rise of intracellular calcium (Ca(2+)). To investigate how the t-tubule microanatomy and the distribution of membrane Ca(2+) flux affect cardiac excitation-contraction coupling we developed a 3-D continuum model of Ca(2+) signaling, buffering and diffusion in rat ventricular myocytes. The transverse-axial t-tubule geometry was derived from light microscopy structural data. To solve the nonlinear reaction-diffusion system we extended SMOL software tool (http://mccammon.ucsd.edu/smol/). The analysis suggests that the quantitative understanding of the Ca(2+) signaling requires more accurate knowledge of the t-tubule ultra-structure and Ca(2+) flux distribution along the sarcolemma. The results reveal the important role for mobile and stationary Ca(2+) buffers, including the Ca(2+) indicator dye. In agreement with experiment, in the presence of fluorescence dye and inhibited sarcoplasmic reticulum, the lack of detectible differences in the depolarization-evoked Ca(2+) transients was found when the Ca(2+) flux was heterogeneously distributed along the sarcolemma. In the absence of fluorescence dye, strongly non-uniform Ca(2+) signals are predicted. Even at modest elevation of Ca(2+), reached during Ca(2+) influx, large and steep Ca(2+) gradients are found in the narrow sub-sarcolemmal space. The model predicts that the branched t-tubule structure and changes in the normal Ca(2+) flux density along the cell membrane support initiation and propagation of Ca(2+) waves in rat myocytes.     

Modelling the cardiac transverse-axial tubular system

The transverse-axial tubular system (TATS) of cardiac ventricular myocytes is a complex network of tubules that arises as invaginations of the surface membrane; it appears to form a specialised region of cell membrane that is particularly important for excitation–contraction coupling. However, much remains unknown about the structure and role of the TATS. In this brief review we use experimental data and computer modelling to address the following key questions: (i) What fraction of the cell membrane is within the TATS? (ii) Is the composition of the TATS membrane the same as the surface membrane? (iii) How good is electrical coupling between the surface and TATS membranes? (iv) What fraction of each current is within the TATS? (v) How important is the complex structure of the TATS network? (vi) What is the effect of current inhomogeneity on lumenal ion concentrations? (vii) Does the TATS contribute to the functional changes observed in heart failure? Although there are many areas in which experimental evidence is lacking, computer models provide a method to assess and predict the possible function of the TATS; such models suggest that although the surface and TATS membranes are electrically well coupled, concentration of ion flux pathways within the TATS, coupled to restricted diffusion, may result in the ionic composition in the TATS lumen being different from that in the bulk extracellular space, and varying with activity and in pathological conditions.     

The structure and function of cardiac t-tubules in health and disease

The transverse tubules (t-tubules) are invaginations of the cell membrane rich in several ion channels and other proteins devoted to the critical task of excitation-contraction coupling in cardiac muscle cells (cardiomyocytes). They are thought to promote the synchronous activation of the whole depth of the cell despite the fact that the signal to contract is relayed across the external membrane. However, recent work has shown that t-tubule structure and function are complex and tightly regulated in healthy cardiomyocytes. In this review, we outline the rapidly accumulating knowledge of its novel roles and discuss the emerging evidence of t-tubule dysfunction in cardiac disease, especially heart failure. Controversy surrounds the t-tubules' regulatory elements, and we draw attention to work that is defining these elements from the genetic and the physiological levels. More generally, this field illustrates the challenges in the dissection of the complex relationship between cellular structure and function.     

Effect of cytoskeleton disruptors on L-type Ca channel distribution in rat ventricular myocytes

The cytoskeleton plays an important role in many aspects of cardiac cell function, including protein trafficking. However, the role of the cytoskeleton in determining Ca channel location in cardiac myocytes is unknown. In the present study we therefore investigated the effect of the cytoskeletal disruptors cytochalasin D, latrunculin, nocadazole and colchicine on the distribution of Ca channels in rat ventricular myocytes during culture for up to 96 h. During culture in the absence of these agents, cell edges became rounded, t-tubule density decreased, and the normal transverse distribution of the alpha1 (pore-forming) subunit of the L-type Ca channel became more punctate and peri-nuclear; these changes were associated with loss of synchronous Ca release in response to electrical stimulation. Disruption of tubulin using nocadazole or colchicine or sequestration of monomeric actin by latrunculin had no effect on these changes. In contrast, cytochalasin D inhibited these changes: cell shape, t-tubule density, transverse Ca channel staining and synchronous Ca release were maintained during culture. The protein synthesis inhibitor cycloheximide had similar effects to cytochalasin. These data suggest that cytochalasin stabilizes actin in adult ventricular myocytes in culture, thus stabilizing cell structure and function, and that actin is important in trafficking L-type Ca channels from the peri-nuclear region to the t-tubules, where they are normally located and provide the trigger for Ca release.     

Critical role of cardiac t-tubule system for the maintenance of contractile function revealed by a 3D integrated model of cardiomyocytes

T-tubules in mammalian ventricular myocytes constitute an elaborate system for coupling membrane depolarization with intracellular Ca(2+) signaling to control cardiac contraction. Deletion of t-tubules (detubulation) has been reported in heart diseases, although the complex nature of the cardiac excitation-contraction (E-C) coupling process makes it difficult to experimentally establish causal relationships between detubulation and cardiac dysfunction. Alternatively, numerical simulations incorporating the t-tubule system have been proposed to elucidate its functional role. However, the majority of models treat the subcellular spaces as lumped compartments, and are thus unable to dissect the impact of morphological changes in t-tubules. We developed a 3D finite element model of cardiomyocytes in which subcellular components including t-tubules, myofibrils, sarcoplasmic reticulum, and mitochondria were modeled and realistically arranged. Based on this framework, physiological E-C coupling was simulated by simultaneously solving the reaction-diffusion equation and the mechanical equilibrium for the mathematical models of electrophysiology and contraction distributed among these subcellular components. We then examined the effect of detubulation in this model by comparing with and without the t-tubule system. This model reproduced the Ca(2+) transients and contraction observed in experimental studies, including the response to beta-adrenergic stimulation, and provided detailed information beyond the limits of experimental approaches. In particular, the analysis of sarcomere dynamics revealed that the asynchronous contraction caused by a large detubulated region can lead to impairment of myocyte contractile efficiency. These data clearly demonstrate the importance of the t-tubule system for the maintenance of contractile function.     

Density and sub-cellular distribution of cardiac and neuronal sodium channel isoforms in rat ventricular myocytes

In cardiac ventricular myocytes, Na current is generated mainly by the cardiac NaV1.5 isoform, but the presence of "neuronal" Na channel isoforms in the heart has been demonstrated recently. In this study, we quantified the density and sub-cellular distribution of cardiac and neuronal channel isoforms in rat ventricular myocytes. INa was recorded using the patch clamp technique in control and detubulated myocytes. Detubulation reduced cell capacitance (by approximately 29%) but maximum conductance was not altered (1.94+/-0.15, 14 control vs 1.98+/-0.19 nS/pF, 17 detubulated myocytes). The kinetic properties of INa were similar in both cell types suggesting good voltage control of surface and t-tubule membranes. We calculated Na channel densities assuming the sub-cellular current localization we recently provided (neuronal isoform: approximately 11% of total sarcolemmal current, approximately 3% of cell surface, and approximately 31% of t-tubule current). Single channel conductances were assumed to be 2.2 and 2.5 pS for the cardiac and neuronal isoforms, respectively, after accounting for the use of low Na concentration. We calculated that the density of the cardiac Na channel isoform is relatively constant (in channels/microm2: approximately 11 in total sarcolemma, approximately 13 at the cell surface, approximately 10 at the t-tubules). In contrast, neuronal Na channel isoforms are concentrated at the t-tubules (in channels/microm2: approximately 1 in total sarcolemma, approximately 0.3 at the cell surface, approximately 2.5 at the t-tubules). We conclude that, in contrast to skeletal muscle in which Na channel density is higher at the cell surface than the t-tubules, in ventricular cardiac myocytes the sub-cellular distribution of Na channel density is relatively homogeneous (approximately 13 channels/microm2).     

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick’s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.     

Buffered diffusion around a spherical proton pumping cell: a theoretical analysis

H⁺ ions are a substrate of many active and passive membrane transporters in all cells. Absolute proton fluxes are often quantified using intracellular pH sensitive microelectrodes or pH sensitive dyes. These measurements, however, rely on a priori estimates of the intracellular buffer capacity and on the assumption of diffusive equilibrium inside the cell. Here, assuming local equilibrium of protons with a single mobile buffer, we model the diffusion of H⁺ in the extracellular medium around an H⁺ pumping cell to estimate the expected pH changes as a function of time, distance from the cell, extracellular buffer capacity, and the absolute proton flux across the membrane. In particular, using accurate numerical simulation, we gauge the range of validity of an explicit, analytical solution of the linearized, nonstationary diffusion equation. Our results provide a framework to quantify the absolute membrane proton flux, if spatiotemporal information about the extracellular pH change is available, e.g., using imaging of pH dependent fluorescent dyes.     

Plasma membrane removal in rat skeletal muscle fibers reveals caveolin-3 hot-spots at the necks of transverse tubules

Caveolin-3, the muscle-specific isoform of the caveolae-associated protein caveolin, is often thought to be localized exclusively in the surface membrane in mature fibers and associated with transverse (t)-tubular system only transiently during development. Skeletal muscle fibers present a model where the surface membrane (sarcolemma) can be completely separated from the cell by mechanical dissection. Western blotting of matching portions of individual fibers from adult rat muscle in which the sarcolemma was either removed (skinned segment), or left in place (intact segment), revealed that ≥ 70% of caveolin-3 is actually located deeper in the fiber rather than in the sarcolemma itself. Triton solubility of caveolin-3 was no different between sarcolemmal and t-tubule compartments. Confocal immunofluorescence microscopy showed caveolin-3 present throughout the t-system in adult fibers, with ‘hot-spots’ at the necks of the tubules in the sub-sarcolemmal space. A similar representation was seen for the muscle specific voltage-dependent sodium channel Nav1.4 and it was found that at least some Nav1.4 co-immunoprecipitated with caveolin-3 in skinned muscle fibers. The caveolin-3 hot-spots just inside the opening of t-tubules may form regions that localize ion channels and kinases at the key place needed for efficient electrical transmission into the t-tubules as well as for other signaling processes.     

Mathemathical model of Ca2+ diffusion and buffering in extracellular solution after Ca2+ extrusion from a spherical cell

A mathematical model evaluating the changes in the concentration of free extracellular calcium ([Ca²⁺]) as a function of time and radial distance from a spherical cell following uniform calcium release across its external membrane was developed. The space of extracellular solution was represented as concentric shells of uniform thickness. Two simplified approaches were used, which allowed us to describe calcium diffusion and buffering in each extracellular shell by only a single differential equation. The possibility of simulating the spatio-temporal [Ca²⁺] distribution in the case of uniformly located calcium pumps was demonstrated. Developed for pancreatic acinar cells, our model can be also used for studying ion release from other cells under similar experimental conditions and for reconstruction of ion fluxes in the presence of different fluorescent dyes.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 243–249, November–December, 1996.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Insulin transport within skeletal muscle transverse tubule networks

It has recently been observed in situ in mice that insulin takes approximately 10 min to be transported 20 microm into the t-tubule networks of skeletal muscle fibers. The mechanisms for this slow transport are unknown. It has been suggested that the biochemical composition of the t-tubular space that may include large molecules acting as gels and increased viscosity in the narrow tubules may explain this slow diffusion. In this article, we construct a mathematical model of insulin transport within the t-tubule network to determine potential mechanisms responsible for this slow insulin transport process. Our model includes insulin diffusion, insulin binding to insulin receptors, t-tubule network tortuosity, interstitial fluid viscosity, hydrodynamic wall effects, and insulin receptor internalization and recycling. The model predicted that depending on fiber type there is a 2-15 min delay in the arrival time of insulin between the sarcolemma and inner t-tubules (located 20 microm from the sarcolemma) after insulin injection. This is consistent with the experimental data. Increased viscosity in the narrow t-tubules and large molecules acting as gels are not the primary mechanisms responsible for the slow insulin diffusion. The primary mechanisms responsible for the slow insulin transport are insulin binding to insulin receptors and network tortuosity.     

Active Ion Transport Across Canine Blood Vessel Walls

Experiments giving evidence of active Na and Cl ion fluxes across large canine blood vessel walls (aorta, vena cava) in vitro have been presented. The information has been obtained using ion tracer techniques after Ussing and with diffusion cells of the Hogben type. The available data suggest that the membranes are satisfactorily oxygenated by the bathing solutions saturated with oxygen at atmospheric pressure. Evidence is offered which indicates that active ion transport does occur across the aorta and vena cava in in vitro experiments. Under the conditions of the experiment net Na and Cl flux takes place from intima to adventitia across the aorta, and from adventitia to intima across the vena cava at low measured potential differences. The possible relationships of derangement of active ion transport mechanisms, produced by electric currents and tissue injury potential differences, to intravascular thrombosis are alluded to. It would appear that sodium and chloride fluxes across large blood vessel walls in vitro occur at least in part as the result of metabolic processes and cannot be explained simply on the basis of diffusion across a semipermeable membrane.     

Cell volume regulation in the nervous system

There is good evidence that the three main compartments of the brain, i.e. extracellular space, neurones and glial cells, change their volume during physiological and pathophysiological neuronal activity. However, there is strikingly little knowledge about the mechanisms underlying such alterations in cell volume. For this purpose, a better understanding of the electrophysiological behavior of the neurones and glial cells during volume changes is necessary. Examples are discussed for which changes in cell volume can be derived from the underlying changes in membrane permeabilities. Volume regulatory mechanisms in the brain have not been described under isotonic conditions. However, a rapid volume regulatory decrease is occurring in cultured glial cells during exposure to hypotonic solutions. In contrast, in these cells no volume regulatory increase was found during superfusion with hypertonic media. On the other hand, the entire brain is able to compensate chronic hypertonic perturbations within hours to days. Interestingly, not only ion fluxes induce cellular volume changes but, in turn, water movements can also influence ion fluxes in both neurones and glial cells. With respect to this it should be considered that volume regulatory membrane processes might not exclusively be activated by changes in transmembranal ion gradient, but also by changes of membrane surface shape. Future studies on cellular mechanisms of volume regulation in the brain should imply a combined use of recent techniques such as computerized video-imaging, radiotracer flux measurements and ion-sensitive microelectrodes in defined cell cultures. Optical monitoring and ion-sensitive microelectrodes should enable measurements of volume changes in identified cellular elements of intact nervous structures such as brain slices.     

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

Transverse (t)-tubules are invaginations of the plasma membrane that form a complex network of ducts, 200–400 nm in diameter depending on the animal species, that penetrates deep within the cardiac myocyte, where they facilitate a fast and synchronous contraction across the entire cell volume. There is now a large body of evidence in animal models and humans demonstrating that pathological distortion of the t-tubule structure has a causative role in the loss of myocyte contractility that underpins many forms of heart failure. Investigations into the molecular mechanisms of pathological t-tubule remodelling to date have focused on proteins residing in the intracellular aspect of t-tubule membrane that form linkages between the membrane and myocyte cytoskeleton. In this review, we shed light on the mechanisms of t-tubule remodelling which are not limited to the intracellular side. Our recent data have demonstrated that collagen is an integral part of the t-tubule network and that it increases within the tubules in heart failure, suggesting that a fibrotic mechanism could drive cardiac junctional remodelling. We examine the evidence that the linkages between the extracellular matrix, t-tubule membrane and cellular cytoskeleton should be considered as a whole when investigating the mechanisms of t-tubule pathology in the failing heart.     

Time resolved secretion of chloride from a monolayer of mucin-secreting epithelial cells

Short-circuit current (Isc) measurement is used to quantify transepithelial ion flux. This technique provides a direct measure of net charge transport across a cell monolayer. Isc however, lacks chemical selectivity. Chemically resolved ion fluxes may be much greater than Isc, and differ in different biological processes. This work describes a novel experimental approach and deconvolution method to obtain temporally resolved ion fluxes at epithelial cell monolayers. HT29-Cl.16E cells, a sub clone of the human colonic cancer cell line HT29 was used as a model cell line to validate this approach in the context of epithelial transport studies. This cell line is known to secrete chloride in response to purinergic stimulation. Changes in chloride concentration after stimulation with 1 mM ATP plus 50 nM phorbol-myristate acetate (PMA) are recorded with a chloride ion-selective electrode (ISE) at a short distance (∼50 μm) from the monolayer. The recorded concentrations are transformed to corresponding chloride flux across the monolayer using a deconvolution algorithm for extracellular mass transport based on minimization of the shape error function (Nair and Gratzl in Anal Chem 77:2875–2888, 2005). Simultaneous voltage clamp yields the associated net electrical charge flux (Isc). The dynamics of Cl⁻ flux did correlate with that of the electrical flux, but was found to be greater in amplitude. This suggests that Cl⁻ may not be the only ion secreted. The method of simultaneously assessing ionic and electrical fluxes with a temporal resolution of seconds provides unique information about the dynamics of solute fluxes across the apical membrane. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A mouse model of Huntington’s disease shows altered ultrastructure of transverse tubules in skeletal muscle fibers

Huntington’s disease (HD) is a fatal and progressive condition with severe debilitating motor defects and muscle weakness. Although classically recognized as a neurodegenerative disorder, there is increasing evidence of cell autonomous toxicity in skeletal muscle. We recently demonstrated that skeletal muscle fibers from the R6/2 model mouse of HD have a decrease in specific membrane capacitance, suggesting a loss of transverse tubule (t-tubule) membrane in R6/2 muscle. A previous report also indicated that Cav1.1 current was reduced in R6/2 skeletal muscle, suggesting defects in excitation–contraction (EC) coupling. Thus, we hypothesized that a loss and/or disruption of the skeletal muscle t-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle. We used live-cell imaging with multiphoton confocal microscopy and transmission electron microscopy to assess the t-tubule architecture in late-stage R6/2 muscle and found no significant differences in the t-tubule system density, regularity, or integrity. However, electron microscopy images revealed that the cross-sectional area of t-tubules at the triad were 25% smaller in R6/2 compared with age-matched control skeletal muscle. Computer simulation revealed that the resulting decrease in the R6/2 t-tubule luminal conductance contributed to, but did not fully explain, the reduced R6/2 membrane capacitance. Analyses of bridging integrator-1 (Bin1), which plays a primary role in t-tubule formation, revealed decreased Bin1 protein levels and aberrant splicing of Bin1 mRNA in R6/2 muscle. Additionally, the distance between the t-tubule and sarcoplasmic reticulum was wider in R6/2 compared with control muscle, which was associated with a decrease in junctophilin 1 and 2 mRNA levels. Altogether, these findings can help explain dysregulated EC coupling and motor impairment in Huntington’s disease.