Role of t-tubules in the control of trans-sarcolemmal ion flux and intracellular Ca2+ in a model of the rat cardiac ventricular myocyte

The t-tubules of mammalian ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell, with restricted diffusion to the bulk extracellular space. The trans-sarcolemmal flux of many ions, including Ca(2+), occurs predominantly across the t-tubule membrane and thus into and out of this restricted diffusion space. It seems possible, therefore, that ion concentration changes may occur in the t-tubule lumen, which would alter ion flux across the t-tubule membrane. We have used a computer model of the ventricular myocyte, incorporating a t-tubule compartment and experimentally determined values for diffusion between the t-tubule lumen and bulk extracellular space, and ion fluxes across the t-tubule membrane, to investigate this possibility. The results show that influx and efflux of different ion species across the t-tubule membrane are similar, but not equal. Changes of ion concentration can therefore occur close to the t-tubular membrane, thereby altering trans-sarcolemmal ion flux and thus cell function, although such changes are reduced by diffusion to the bulk extracellular space. Slowing diffusion results in larger changes in luminal ion concentrations. These results provide a deeper understanding of the role of the t-tubules in normal cell function, and are a basis for understanding the changes that occur in heart failure as a result of changes in t-tubule structure and ion fluxes.     

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).     

Uniform Action Potential Repolarization within the Sarcolemma of In Situ Ventricular Cardiomyocytes

Previous studies have speculated, based on indirect evidence, that the action potential at the transverse (t)-tubules is longer than at the surface membrane in mammalian ventricular cardiomyocytes. To date, no technique has enabled recording of electrical activity selectively at the t-tubules to directly examine this hypothesis. We used confocal line-scan imaging in conjunction with the fast response voltage-sensitive dyes ANNINE-6 and ANNINE-6plus to resolve action potential-related changes in fractional dye fluorescence (ΔF/F) at the t-tubule and surface membranes of in situ mouse ventricular cardiomyocytes. Peak ΔF/F during action potential phase 0 depolarization averaged −21% for both dyes. The shape and time course of optical action potentials measured with the water-soluble ANNINE-6plus were indistinguishable from those of action potentials recorded with intracellular microelectrodes in the absence of the dye. In contrast, optical action potentials measured with the water-insoluble ANNINE-6 were significantly prolonged compared to the electrical recordings obtained from dye-free hearts, suggesting electrophysiological effects of ANNINE-6 and/or its solvents. With either dye, the kinetics of action potential-dependent changes in ΔF/F during repolarization were found to be similar at the t-tubular and surface membranes. This study provides what to our knowledge are the first direct measurements of t-tubule electrical activity in ventricular cardiomyocytes, which support the concept that action potential duration is uniform throughout the sarcolemma of individual cells.     

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.     

Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes

Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(pump). Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.     

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.     

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.     

Validation of formamide as a detubulation agent in isolated rat cardiac cells

Kawai M, Hussain M, and Orchard CH. Am J Heart Circ Physiol 277: H603-H609, 1999 developed a technique to detubulate rat ventricular myocytes using formamide and showed that detubulation results in a decrease in cell capacitance, Ca(2+) current density, and Ca(2+) transient amplitude. We have investigated the mechanism of this detubulation and possible direct effects of formamide. Staining ventricular cells with di-8-ANEPPS showed that the t tubule membranes remain inside the cell after detubulation; trapping of FITC-labeled dextran within the t tubules showed that detubulation occurs during formamide washout and that the t tubules appear to reseal within the cell. Detubulation had no effect on the microtubule network but resulted in loss of synchronous Ca(2+) release on electrical stimulation. In contrast, formamide treatment of atrial cells did not significantly change cell capacitance, Ca(2+) current amplitude, action potential configuration, the Ca(2+) transient or the response of the Ca(2+) transient to isoprenaline. We conclude that formamide washout induces detubulation of single rat ventricular myocytes, leaving the t tubules within the cell, but without direct effects on cell proteins that might alter cell function.     

Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes

In some forms of cardiac hypertrophy and failure, the gain of Ca(2+)-induced Ca(2+) release [CICR; i.e., the amount of Ca(2+) released from the sarcoplasmic reticulum normalized to Ca(2+) influx through L-type Ca(2+) channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca(2+) content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the "decreased CICR gain" may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca(2+)/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive β-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.     

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.     

Metabolic stress in isolated mouse ventricular myocytes leads to remodeling of t tubules

Cardiac ventricular myocytes possess an extensive t-tubular system that facilitates the propagation of membrane potential across the cell body. It is well established that ionic currents at the restricted t-tubular space may lead to significant changes in ion concentrations, which, in turn, may affect t-tubular membrane potential. In this study, we used the whole cell patch-clamp technique to study accumulation and depletion of t-tubular potassium by measuring inward rectifier potassium tail currents (I(K1,tail)), and inward rectifier potassium current (I(K1)) "inactivation". At room temperatures and in the absence of Mg(2+) ions in pipette solution, the amplitude of I(K1,tail) measured ~10 min after the establishment of whole cell configuration was reduced by ~18%, but declined nearly twofold in the presence of 1 mM cyanide. At ~35°C I(K1,tail) was essentially preserved in intact cells, but its amplitude declined by ~85% within 5 min of cell dialysis, even in the absence of cyanide. Intracellular Mg(2+) ions played protective role at all temperatures. Decline of I(K1,tail) was accompanied by characteristic changes in its kinetics, as well as by changes in the kinetics of I(K1) inactivation, a marker of depletion of t-tubular K(+). The data point to remodeling of t tubules as the primary reason for the observed effects. Consistent with this, detubulation of myocytes using formamide-induced osmotic stress significantly reduced I(K1,tail), as well as the inactivation of inward I(K1). Overall, the data provide strong evidence that changes in t tubule volume/structure may occur on a short time scale in response to various types of stress.     

Structure-function relationships of Ca spark activity in normal and failing cardiac myocytes as revealed by flash photography

We describe the two-dimensional imaging of excitation-induced Ca gradients in isolated myocytes under physiological conditions, using a novel method of flash photography of fluo-3 fluorescence. This method is useful for showing the spatial distribution and reproducibility of rapidly triggered Ca release events, and their relationship to underlying structures. In normal rat myocytes, Ca sparks were evident 6ms after stimulation emerging from around t-tubules, as judged by co-localization with di-8-ANEPPS staining. Gaps in the spark pattern coincided with gaps in di-8-ANEPPS staining. Vacuolar fluo-3 uptake, previously identified as lysosomal, was prominent in some of the gaps, suggesting possible areas of t-tubule turnover. In normal dog myocytes, the beat-to-beat variance of Ca sparks was very low, t-tubular voids were small, and Ca gradients resolved rapidly. In myocytes from dogs with failure induced by rapid pacing, a reduced Ca transient was observed associated with increased areas that were void of sparks and t-tubules, and a greater beat-to-beat spark variance. These abnormalities resulted in a non-uniform spatial distribution of sparks, leading to Ca gradients across the cell that persisted for longer times after stimulation. Such Ca gradients could cause heterogeneous contraction and contribute to contractile failure.     

Kinetic properties and regulation of biliverdin reductase

Transverse tubules (t-tubules) were prepared from muscle by dissociation of intact triads during centrifugation in ion-free sucrose gradients. They were further purified by the removal of contaminating sarcoplasmic reticulum after loading with calcium phosphate. Purification was accompanied by enrichment in markers specific for t-tubules, e.g., nitrendipine binding sites. According to gel electrophoresis the purified t-tubules contained three major protein bands of 104, 70, and 30 kDa. When solubilized with detergents there was a two- to threefold increase in Mg2+-ATPase activity, and a corresponding increase in the 30-kDa protein band. The 104-kDa protein was shown to be a (Na+ + K+)-ATPase because of its phosphorylation by [gamma-32P]ATP in the presence of sodium ions. The orientation of the t-tubule membrane was predominantly inside-out.     

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.     

Changes in the organization of excitation-contraction coupling structures in failing human heart

Background

The cardiac myocyte t-tubular system ensures rapid, uniform cell activation and several experimental lines of evidence suggest changes in the t-tubular system and associated excitation-contraction coupling proteins may occur in heart failure.

Methods and Results

The organization of t-tubules, L-type calcium channels (DHPRs), ryanodine receptors (RyRs) and contractile machinery were examined in fixed ventricular tissue samples from both normal and failing hearts (idiopathic (non-ischemic) dilated cardiomyopathy) using high resolution fluorescent imaging. Wheat germ agglutinin (WGA), Na-Ca exchanger, DHPR and caveolin-3 labels revealed a shift from a predominantly transverse orientation to oblique and axial directions in failing myocytes. In failure, dilation of peripheral t-tubules occurred and a change in the extent of protein glycosylation was evident. There was no change in the fractional area occupied by myofilaments (labeled with phalloidin) but there was a small reduction in the number of RyR clusters per unit area. The general relationship between DHPRs and RyR was not changed and RyR labeling overlapped with 51±3% of DHPR labeling in normal hearts. In longitudinal (but not transverse) sections there was an ∼30% reduction in the degree of colocalization between DHPRs and RyRs as measured by Pearson''s correlation coefficient in failing hearts.

Conclusions

The results show that extensive remodelling of the t-tubular network and associated excitation-contraction coupling proteins occurs in failing human heart. These changes may contribute to abnormal calcium handling in heart failure. The general organization of the t-system and changes observed in failure samples have subtle differences to some animal models although the general direction of changes are generally similar.     

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.     

Quantitative analysis of Na+-Ca2+ exchanger expression in guinea-pig heart.

In previous studies, regional variations in the expression of the Na+-Ca2+ exchanger (NCX) have been examined qualitatively in human heart using the C2C12 monoclonal antibody [Wang, J., Schwinger, R.H., Frank, K., Muller-Ehmsen, J., Martin-Vasallo, P., Pressley, T.A., Xiang, A., Erdmann, E. & McDonough, A.A. (1996) J. Clin. Invest. 98, 1650-1658]. Although NCX expression was found to be significantly lower in the atria compared to the septum, no significant differences were found between atrial and ventricular tissue. NCX has been located in the general sarcolemma and t-tubules of ventricular muscle and as t-tubules are sparse in atrial tissue compared to ventricular tissue, it is surprising that NCX expression was found to be similar in both atria and ventricles [Wang et al. (1996)]. To reinvestigate this, we have used SDS/PAGE and a quantitative Western blotting technique to determine the pattern of expression of NCX in guinea-pig heart in tissue samples from left atrium, right atrium, septum, left ventricle and right ventricle. NCX protein expression was 17.5 +/- 3.9 pmol.mg-1 of protein in the left atrium and 29.2 +/- 6.1 pmol.mg-1 of protein in the right atrium, which were both significantly lower (P < 0.05) than NCX expression in the septum, left ventricle and right ventricle (64.7 +/- 15.2, 76.8 +/- 19.5 and 69.4 +/- 14.1 pmol.mg-1 of protein, respectively, n = 7). These differences in NCX expression may reflect variations in the cellular location of NCX protein in these regions. To study this, we used confocal immunofluorescence of single isolated myocytes to examine differences in the proportion of fluorescent staining on the general surface membrane compared with the interior of the cell (which presumably reflects a t-tubular location). We found that the general membrane staining was 79.0 +/- 1.2% in cells from the atria which was significantly higher (P < 0. 001) than that seen in cells from the septum, left ventricle and right ventricle, with 48.1 +/- 1.1%, 48.2 +/- 1.8% and 45.6 +/- 1.3%, respectively (n = 20). These results illustrate a similar pattern of NCX expression in guinea-pig and human, with expression in atrial tissue significantly lower than in ventricular tissue. However, the cellular location of NCX differs regionally; in atrial tissue, the majority of the NCX protein is located in the general sarcolemma whereas in ventricular and septal tissue, approximately 50% of NCX protein is located within the cell (presumably at the level of the t-tubules).     

Agonists Bay-K8644 and CGP-28392 open calcium channels reconstituted from skeletal muscle transverse tubules.

The recently described calcium channel agonists Bay-K8644 and CGP-28392 have been used to induce long-term opening of calcium channels from purified rat muscle transverse tubules (t-tubules) incorporated into planar phospholipid bilayers. Agonist-open channels are selective for divalent cations (except Mg++), display voltage-dependent kinetics, and are blocked by the calcium channel antagonist, nitrendipine. The sensitivity to dihydropyridine agonists and antagonists indicate that a pool of t-tubule calcium channels remain functional after membrane fractionation and purification.