Do t-tubules play a role in arrhythmogenesis in cardiac ventricular myocytes?

The transverse (t-) tubules of mammalian ventricular myocytes are invaginations of the surface membrane. The function of many of the key proteins involved in excitation-contraction coupling is located predominantly at the t-tubules, which thus form a Ca(2+)-handling micro-environment that is central to the normal rapid activation and relaxation of the ventricular myocyte. Although cellular arrhythmogenesis shares many ion flux pathways with normal excitation-contraction coupling, the role of the t-tubules in such arrhythmogenesis has not previously been considered. In this brief review we consider how the location and co-location of proteins at the t-tubules may contribute to the generation of arrhythmogenic delayed and early afterdepolarisations, and how the loss of t-tubules that occurs during heart failure may alter the generation of such arrhythmias, as well as contributing to other types of arrhythmia as a result of changes of electrical heterogeneity within the whole heart.

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.

Pharmacological inhibition of the hERG potassium channel is modulated by extracellular but not intracellular acidosis.

INTRODUCTION: Human ether-à-go-go related gene (hERG) is responsible for channels that mediate the rapid delayed rectifier K(+) channel current (I(Kr) ), which participates in repolarization of the ventricles and is a target for some antiarrhythmic drugs. Acidosis occurs in the heart in some pathological situations and can modify the function and responses to drugs of ion channels. The aim of this study was to determine the effects of extracellular and intracellular acidosis on the potency of hERG channel current (I(hERG)) inhibition by the antiarrhythmic agents dofetilide, flecainide, and amiodarone at 37 °C. METHODS AND RESULTS: Whole-cell patch-clamp recordings of I(hERG) were made at 37 °C from hERG-expressing Human Embryonic Kidney (HEK293) cells. Half-maximal inhibitory concentration (IC(50)) values for I(hERG) tail inhibition at -40 mV following depolarizing commands to +20 mV were significantly higher at external pH 6.3 than at pH 7.4 for both flecainide and dofetilide, but not for amiodarone. Lowering pipette pH from 7.2 to 6.3 altered neither I(hERG) kinetics nor the extent of observed I(hERG) blockade by any of these drugs. CONCLUSION: Conditions leading to localized extracellular acidosis may facilitate heterogeneity of action of dofetilide and flecainide, but not amiodarone via modification of hERG channel blockade. Such effects depend on the external pH change rather than intracellular acidification.

Enhanced inhibitory effect of acidosis on hERG potassium channels that incorporate the hERG1b isoform.

Extracellular acidosis occurs in the heart during myocardial ischemia and can lead to dangerous arrhythmias. Potassium channels encoded by hERG (human ether-à-go-go-related gene) mediate the cardiac rapid delayed rectifier K+ current (IKr), and impaired hERG function can exacerbate arrhythmia risk. Nearly all electrophysiological investigations of hERG have centred on the hERG1a isoform, although native IKr channels may be comprised of hERG1a and hERG1b, which has a unique shorter N-terminus. This study has characterised for the first time the effects of extracellular acidosis (an extracellular pH decrease from 7.4 to 6.3) on hERG channels incorporating the hERG1b isoform. Acidosis inhibited hERG1b current amplitude to a significantly greater extent than that of hERG1a, with intermediate effects on coexpressed hERG1a/1b. IhERG tail deactivation was accelerated by acidosis for both isoforms. hERG1a/1b activation was positively voltage-shifted by acidosis, and the fully-activated current-voltage relation was reduced in amplitude and right-shifted (by ∼10 mV). Peak IhERG1a/1b during both ventricular and atrial action potentials was both suppressed and positively voltage-shifted by acidosis. Differential expression of hERG isoforms may contribute to regional differences in IKr in the heart. Therefore inhibitory effects of acidosis on IKr could also differ regionally, depending on the relative expression levels of hERG1a and 1b, thereby increasing dispersion of repolarization and arrhythmia risk.

Spontaneous frequency of rabbit atrioventricular node myocytes depends on SR function.

Spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) appears to play an important role in cardiac sinoatrial node pacemaking. However, comparatively little is known about the role of intracellular Ca(2+) in the atrioventricular node (AVN). Intracellular Ca(2+) was therefore monitored in cells isolated from the rabbit AVN, using fluo-3 in conjunction with confocal microscopy. These cells displayed spontaneous Ca(2+) transients and action potentials. Ca(2+) transients were normally preceded by a small, slow increase (ramp) of intracellular Ca(2+) which was sometimes, but not always, accompanied by Ca(2+) sparks. During the Ca(2+) transient, intracellular [Ca(2+)] increased initially at the cell periphery and propagated inhomogeneously to the cell centre. The rate of spontaneous activity was decreased by ryanodine (1muM) and increased by isoprenaline (500nM); these changes were accompanied by a decrease and increase, respectively, in the slope of the preceding Ca(2+) ramp, with no significant change in Ca(2+) spark characteristics. Rapidly reducing bathing [Na(+)] inhibited spontaneous activity. These findings provide the first information on Ca(2+) handling at the sub-cellular level and link cellular Ca(2+) cycling to the genesis of spontaneous activity in the AVN.

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.

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 approximately 32%, whereas optical measurements suggest up to approximately 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.

Quantification of calcium entry at the T-tubules and surface membrane in rat ventricular myocytes.

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by approximately 60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 micromol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 micromol/L cytosol, respectively). However, more Ca entry occurs per microm(2) of junctional membrane at the cell surface than in the T-tubules (in nM/microm(2): 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions ( approximately 35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.

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.

beta-adrenergic stimulation restores the Ca transient of ventricular myocytes lacking t-tubules.

beta-adrenergic stimulation helps to synchronize Ca release in myocytes from failing hearts. Transverse (t-) tubules, which synchronize Ca release in normal cells and contain many of the elements of the beta-adrenergic pathway, may be depleted in such cells. The objective of the present study was to determine whether beta-adrenergic stimulation could reverse the desynchronization of Ca release observed in detubulated ventricular myocytes. The effect of isoprenaline (0.5 microM) on control and detubulated rat ventricular myocytes was investigated. Ca transients were monitored using whole-cell fluorescence and confocal microscopy, and Ca current recorded using the patch-clamp technique. Immunocytochemistry was used to investigate phospholamban (PLB) phosphorylation. Detubulation reduces and slows the Ca transient; these effects were reversed by isoprenaline. This restoration was associated with partial reversal of the desynchronization of Ca release that occurs in detubulated cells. Sarcoplasmic reticulum Ca load increased by the same amount in normal and detubulated cells, but Ca current increased less in detubulated cells (64%) than in control cells (124%) in response to isoprenaline. The pattern and extent of cAMP-dependent protein kinase and CaMKII-induced phosphorylation of PLB in response to isoprenaline was the same in both cell types. Thus, the beta-adrenergic pathway is functional in the absence of t-tubules; such stimulation appears to increase the speed of propagation of Ca via Ca-induced Ca release between adjacent clusters of ryanodine receptors, which may be relevant in pathological conditions, such as heart failure, in which t-tubules are depleted. The data also suggest that the Ca current responds to local signaling pathways, which are better coupled to the channel in the t-tubules than at the surface membrane, whereas PLB responds to whole-cell signaling.

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.

Two pore residues mediate acidosis-induced enhancement of C-type inactivation of the Kv1.4 K(+) channel.

Acidosis inhibits current through the Kv1.4 K(+) channel, perhaps as a result of enhancement of C-type inactivation. The mechanism of action of acidosis on C-type inactivation has been studied. A mutant Kv1.4 channel that lacks N-type inactivation (fKv1.4 Delta2-146) was expressed in Xenopus oocytes, and currents were recorded using two-microelectrode voltage clamp. Acidosis increased fKv1.4 Delta2-146 C-type inactivation. Replacement of a pore histidine with cysteine (H508C) abolished the increase. Application of positively charged thiol-specific methanethiosulfonate to fKv1.4 Delta2-146 H508C increased C-type inactivation, mimicking the effect of acidosis. Replacement of a pore lysine with cysteine (K532C) abolished the acidosis-induced increase of C-type inactivation. A model of the Kv1.4 pore, based on the crystal structure of KcsA, shows that H508 and K532 lie close together. It is suggested that the acidosis-induced increase of C-type inactivation involves the charge on H508 and K532.

Na+-Ca2+ exchange activity is localized in the T-tubules of rat ventricular myocytes.

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.

Compensatory role of CaMKII on ICa and SR function during acidosis in rat ventricular myocytes.

It has been suggested that the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) increases during acidosis in cardiac muscle. Thus we have investigated the role of CaMKII during acidosis by monitoring intracellular Ca2+ (using fura-2) and ICa (using the perforated patch clamp technique) during acidosis, in the absence and presence of the CaMKII inhibitor KN-93, in rat isolated ventricular myocytes. In the absence of KN-93, acidosis (pH 6.5) increased the amplitude of the fura-2 transient and prolonged its decay, but in the presence of KN-93 acidosis did not alter the amplitude and prolonged the decay more. In the absence of KN-93, acidosis increased the amplitude of the caffeine-induced fura-2 transient but did not alter its amplitude in the presence of KN-93. ICa did not change significantly during acidosis in the absence of KN-93 but decreased during acidosis in the presence of KN-93. These results suggest that activation of CaMKII during acidosis helps to compensate for the direct inhibitory effects of acidosis on sarcoplasmic reticular Ca2+ uptake and ICa.

The effect of acidosis on the ECG of the rat heart.

We have investigated the effect of acidosis on the ECG in isolated rat heart to determine whether acidosis has marked effects on the ECG, and have used pharmacological agents to investigate possible mechanisms whereby acidosis alters the ECG. Acidosis produced a marked decrease in heart rate and an increase in P-R interval with little apparent effect on the duration of the QRS complex. The effects of acidosis did not appear to be due to acidosis-induced changes in transmitter release from severed autonomic nerve terminals within the heart. Experimental Physiology (2001) 86.1, 27-31.

The effect of acidosis on systolic Ca2+ and sarcoplasmic reticulum calcium content in isolated rat ventricular myocytes.

We have investigated the mechanisms responsible for the changes of systolic Ca2+ that occur in voltage-clamped rat ventricular myocytes during acidosis produced by application of the weak acid butyrate (30 mM). Intracellular pH regulation was inhibited with dimethylamiloride (bicarbonate-free solution). The application of butyrate produced an intracellular acidification of 0.33 pH units. This was accompanied by a decrease in systolic Ca2+ to about 50% of control. However, within 2 min, systolic Ca2+ returned to control levels. The decrease in systolic Ca2+ was accompanied by a decrease in the Na+-Ca2+ exchange current observed on repolarisation so that the calculated Ca2+ efflux on Na+-Ca2+ exchange was less than the entry on the L-type Ca2+ current. The magnitude of the Na+-Ca2+ exchange current recovered along with systolic Ca2+ until it equalled the Ca2+ entry on the L-type Ca2+ current. From the measurement of Ca2+ fluxes, it was calculated that, during acidosis, the cell gains 121.6+/-16.2 micromol l(-1) of Ca2+. This is equal to the measured increase of sarcoplasmic reticulum (SR) calcium content obtained by applying caffeine (20 mM) and integrating the resulting Na+-Ca2+ exchange current. We conclude that the recovery of the amplitude of the systolic Ca2+ transient is due to decreased SR calcium release, resulting in reduced Ca2+ efflux from the cell leading to increased SR calcium content.

Effects of the Ca2+ sensitizer EMD 57033 on intracellular Ca2+ in rat ventricular myocytes: relevance to arrhythmogenesis during positive inotropy.

We have investigated the effects of the calcium-sensitizing inotropic agent EMD 57033 on Ca(2+) handling in intact and skinned rat ventricular myocytes. Intracellular Ca(2+) was monitored using fura 2. Myocytes were saponin-skinned, allowing study of sarcoplasmic reticulum (SR) function. In intact myocytes EMD 57033 (1-10 micromol/l) produced a concentration-dependent decrease in the amplitude of the Ca(2+) transient and prolonged its declining phase, but had no effect on the rise time. In skinned myocytes, the amplitude of spontaneous Ca(2+) release from the SR was decreased by EMD 57033 (5 and 10 micromol/l), although this agent had no significant effect on the frequency of spontaneous Ca(2+) release. In the presence of the cross-bridge inhibitor 2,3-butanedione monoxime (5 mmol/l), or in a low bathing Ca(2+) concentration (1 mmol/l), EMD 57033 (10 micromol/l) had smaller effects on both the amplitude and time course of the Ca(2+) transient in intact cells than in the absence of 2,3-butanedione monoxime or in the presence of 2 and 5 mmol/l Ca(2+) respectively. These data suggest that the effects of EMD 57033 on Ca(2+) are due to changes in Ca(2+) binding to troponin C, secondary to cross-bridge formation. Thus, during positive inotropy, EMD 57033 is unlikely to provoke arrhythmias due to effects on SR Ca(2+) handling. In intact cells, its effects on Ca(2+) handling would be expected to protect against arrhythmias.

Inhibition of the K+ channel kv1.4 by acidosis: protonation of an extracellular histidine slows the recovery from N-type inactivation.

1. Acidosis alters the transient outward current, ito, in the heart. We have studied the mechanism underlying the effect of acidosis on one of the K+ channels, Kv1.4 (heterologously expressed in Xenopus laevis oocytes), known to underlie ito. 2. At pH 6.5, wild-type Kv1.4 current was inhibited during repetitive pulsing, in part as a result of a slowing of recovery from N-type inactivation. 3. Acidosis still caused slowing of recovery after deletion of just one (either the first or second) of the N-terminal inactivation ball domains. However, deletion of both the N-terminal inactivation ball domains greatly reduced the inhibition. 4. As well as the N-terminus, other parts of the channel are also required for the effect of acidosis, because, whereas the transfer of the N-terminus of Kv1.4 to Kv1.2 conferred N-type inactivation, it did not confer acidosis sensitivity. 5. Replacement of an extracellular histidine with a glutamine residue (H508Q) abolished the slowing of recovery by acidosis. Reduction of C-type inactivation by raising the bathing K+ concentration or by the mutation K532Y also abolished the slowing. 6. It is concluded that binding of protons to H508 enhances C-type inactivation and this causes a slowing of recovery from N-type inactivation and, thus, an inhibition of current during repetitive pulsing.

Effect of acidosis on transient outward potassium current in isolated rat ventricular myocytes.

The effect of acidosis on the transient outward K(+) current (I(to)) of rat ventricular myocytes has been investigated using the perforated patch-clamp technique. When the holding potential was -80 mV, depolarizing pulses to potentials positive to -20 mV activated I(to) in subepicardial cells but activated little I(to) in subendocardial cells. Exposure to an acid solution (pH 6.5) had no significant effect on I(to) activated from this holding potential in either subepicardial or subendocardial cells. When the holding potential was -40 mV, acidosis significantly increased I(to) at potentials positive to -20 mV in subepicardial cells but had little effect on I(to) in subendocardial cells. The increase in I(to) in subepicardial cells was inhibited by 10 mM 4-aminopyridine. In subepicardial cells, acidosis caused a +8.57-mV shift in the steady-state inactivation curve. It is concluded that in subepicardial rat ventricular myocytes acidosis increases the amplitude of I(to) as a consequence of a depolarizing shift in the voltage dependence of inactivation.

Excitation-contraction coupling in rat ventricular myocytes after formamide-induced detubulation.

Formamide-induced osmotic shock has been used to detubulate isolated adult rat ventricular myocytes (i.e., disrupt the surface membrane-T tubule junction). Cell volume, calculated from cell length and width, rapidly decreased and increased upon application and removal of formamide, respectively. After treatment with formamide, membrane capacitance decreased by 26.4% (from 199.4 +/- 18.7 pF in control cells to 146.7 +/- 6.4 pF in formamide-treated cells; n = 13, P < 0.05). However, the amplitude of the L-type Ca(2+) current (I(Ca)) decreased by a greater extent (from 0.75 +/- 0.14 to 0.18 +/- 0.03 nA; n = 5, P < 0.05) so that the density of I(Ca) decreased by 74.5%. Simultaneous measurements of I(Ca) and Ca(2+) transients (monitored using fura 2) showed that both decreased rapidly upon removal of formamide. However, the Ca(2+) content of the sarcoplasmic reticulum showed little change. Cross-striations, visualized with the fluorescent dye di-8-aminonaphthylethenylpyridinium, were sparse or absent in cells that had been treated with formamide, suggesting that formamide can successfully detubulate cardiac cells and that I(Ca) is concentrated in the T tubules, which therefore play an important role in excitation-contraction coupling.