Inhibition of the Na+-H+ exchanger with cariporide abolishes stretch-induced calcium but not sodium accumulation in mouse ventricular myocytes

We address the question whether activation of the sodium-proton exchanger (NHE) does contribute to the stretch-induced accumulation of intracellular sodium and calcium in mouse ventricular myocytes. NHE-blocker cariporide (10 microM) were applied to the bath for 10 min. Axial stretch was applied for 2 min by increasing the distance between an adherent glass stylus and the patch pipette by 20%. Myocytes (stimulated at 3 Hz) were shock-frozen in diastole and the membrane currents monitored till cryofixation. Controls were treated identically, but not stretched. Total sodium and calcium concentrations ([Na], [Ca]=sum of free and bound Na and Ca) were measured by electron probe microanalysis (EPMA) in peripheral and central cytosol, mitochondria, nucleus and nuclear envelope. Cariporide did not reduce the stretch-activated negative current. The stretch-induced rise in [Na] was not different in the presence and in the absence of cariporide. Cariporide significantly reduced diastolic [Ca] in the cytosol of stretched myocytes. Since cariporide does not prevent the stretch-induced [Na] accumulation, we suggest that not NHE but the stretch-activated streptomycin-sensitive current I(SAC) causes the well documented stretch-induced [Na] accumulation. The discovery that cariporide prevents the stretch-induced rise in cytosolic [Ca] demonstrates an important additional effect of the drug on calcium handling.     

Model of intracellular calcium cycling in ventricular myocytes

We present a mathematical model of calcium cycling that takes into account the spatially localized nature of release events that correspond to experimentally observed calcium sparks. This model naturally incorporates graded release by making the rate at which calcium sparks are recruited proportional to the whole cell L-type calcium current, with the total release of calcium from the sarcoplasmic reticulum (SR) being just the sum of local releases. The dynamics of calcium cycling is studied by pacing the model with a clamped action potential waveform. Experimentally observed calcium alternans are obtained at high pacing rates. The results show that the underlying mechanism for this phenomenon is a steep nonlinear dependence of the calcium released from the SR on the diastolic SR calcium concentration (SR load) and/or the diastolic calcium level in the cytosol, where the dependence on diastolic calcium is due to calcium-induced inactivation of the L-type calcium current. In addition, the results reveal that the calcium dynamics can become chaotic even though the voltage pacing is periodic. We reduce the equations of the model to a two-dimensional discrete map that relates the SR and cytosolic concentrations at one beat and the previous beat. From this map, we obtain a condition for the onset of calcium alternans in terms of the slopes of the release-versus-SR load and release-versus-diastolic-calcium curves. From an analysis of this map, we also obtain an understanding of the origin of chaotic dynamics.     

Aldosterone increases voltage-gated sodium current in ventricular myocytes

The role of aldosterone in the pathogenesis of heart failure (HF) is still poorly understood. Recently, aldosterone has been shown to modulate the function of cardiac Ca(2+) and K(+) channels, thus playing a role in the electrical remodeling process. The goal of this work was to investigate the role of aldosterone on the cardiac Na(+) current (I(Na)). We analyzed the effects of aldosterone on I(Na) in isolated adult mouse ventricular myocytes, using the whole cell patch-clamp technique. After 24 h incubation with 1 microM aldosterone, the I(Na) density was significantly increased (+55%), without alteration of the biophysical properties and the cell membrane capacitance. Aldosterone (10 nM) increased the I(Na) by 23%. In 24-h coincubation experiments, with the use of actinomycin D, cycloheximide, or brefeldin A, the effect of aldosterone on I(Na) was abolished. Spironolactone (mineralocorticoid receptor antagonist, 10 microM) prevented the 1 microM aldosterone-dependent I(Na) increase, whereas RU-38486 (glucocorticoid receptor antagonist, 10 microM) did not. The action potential duration (APD) was longer in aldosterone-treated (APD(90): +53%) than in control myocytes. In addition, the L-type Ca(2+) current was also upregulated (+48%). We performed quantitative RT-PCR measurements and Western blots to quantify the mRNA and protein levels of Na(v)1.5 and Ca(v)1.2 (main channels mediating cardiac I(Na) and I(Ca)), but no significant difference was found. In conclusion, this study shows that aldosterone upregulates the cardiac I(Na) and suggest that this phenomenon may contribute to the HF-induced electrical remodeling process that may be reversed by spironolactone.     

Non-mitochondrial calcium ion regulation in rat ventricular myocytes

Ca2+ exchange has been measured in a suspension of rat ventricular myocytes treated with digitonin or saponin to render the sarcolemma permeable to small molecules and ions. Two fractions of exchange were identified, one that was attributed to the mitochondrial component of the cell and the other to a non-mitochondrial fraction. Mitochondrial Ca2+ uptake was blocked by sodium azide and depended on respiratory substrates whereas non-mitochondrial uptake occurred independently of these molecules but was dependent on ATP and creatine phosphate. Non-mitochondrial Ca2+ uptake could be induced at a Ca2+ concentration below 1 microM and the initial rate increased with concentration up to 100 microM. Uptake could be reversed by sulmazole (a caffeine-like substance) and this reversal in turn inhibited by ryanodine. These properties suggest that the major locus for non-mitochondrial Ca2+ exchange is at the sarcoplasmic reticulum. Ca2+ exchange could be modulated by a number of agents, including carnosine, but was unaffected by others, including Na+, inositol trisphosphate and cyclic AMP. A kinetic model of the data is presented, which incorporates similar data of Ca2+ uptake into the mitochondrial fraction. The rates of Ca2+ exchange measured in these experiments suggest that these two components of the cell can reduce the sarcoplasmic Ca2+ concentration rapidly enough to account for the observed transient nature of the isometric twitch. Furthermore, it is suggested that both non-mitochondrial and mitochondrial fractions of the cell could significantly contribute to tension relaxation in rat cardiac muscle.     

The effects of sodium, hydrogen and magnesium ions on mitochondrial calcium sequestration in adult rat ventricular myocytes

The effect of Na+, H+ and Mg2+ ions on net calcium exchange induced in digitonin-treated myocytes has been investigated. Raising the [Na] from 1.4 to 31.4 mM revealed a sodium-sensitive fraction of net calcium exchange with a K1/2 for Na+ ions of 12 mM, alongside the respiration-dependent accumulation of calcium. An acidosis, but not an alkalosis, was found to depress both of these processes. Mg2+ ions exerted an effect solely on the respiration-dependent calcium sequestration. A simple semi-empirical model based on the experimental data was formulated to assess the effects that altering sarcoplasmic [Na+] and [H+] would have on the calcium-handling properties of cardiac mitochondria. It is concluded that part of the inotropic effects of these ions could be mediated via this organelle.     

Analysis of the T-type calcium channel in embryonic chick ventricular myocytes

Summary T-type calcium channels (I _T channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca²⁺ with Na⁺ (120mm) as the charge carrier.I _T channels were distinguished from L-type calcium channels (I _L) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R+40 mV), and 20 msec atR+60 mV. The frequency histogram of channel open times ₀ was fit by a single-exponential curve while that of closed times _c was biexponeintial. _o was the same atR+40 mV andR+60 mV whereas _c was shortened atR+60 mV. The open-state probability (P _o) increased with depolarization: 0.35 atR+40 mV, 0.8 atR+60 mV and 0.88 atR+80 mV. This increase inP _o at depolarized potentials could be accounted for by the decrease in _c.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Intracellular calcium handling in ventricular myocytes from mdx mice

Duchenne muscular dystrophy (DMD) is a lethal degenerative disease of skeletal muscle, characterized by the absence of the cytoskeletal protein dystrophin. Some DMD patients show a dilated cardiomyopathy leading to heart failure. This study explores the possibility that dystrophin is involved in the regulation of a stretch-activated channel (SAC), which in the absence of dystrophin has increased activity and allows greater Ca(2+) into cardiomyocytes. Because cardiac failure only appears late in the progression of DMD, we examined age-related effects in the mdx mouse, an animal model of DMD. Ca(2+) measurements using a fluorescent Ca(2+)-sensitive dye fluo-4 were performed on single ventricular myocytes from mdx and wild-type mice. Immunoblotting and immunohistochemistry were performed on whole hearts to determine expression levels of key proteins involved in excitation-contraction coupling. Old mdx mice had raised resting intracellular Ca(2+) concentration ([Ca(2+)](i)). Isolated ventricular myocytes from young and old mdx mice displayed abnormal Ca(2+) transients, increased protein expression of the ryanodine receptor, and decreased protein expression of serine-16-phosphorylated phospholamban. Caffeine-induced Ca(2+) transients showed that the Na(+)/Ca(2+) exchanger function was increased in old mdx mice. Two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca(2+)](i) in old mdx mice, suggesting that SACs may be involved in the Ca(2+)-handling abnormalities in these animals. This finding was supported by immunoblotting data, which demonstrated that old mdx mice had increased protein expression of canonical transient receptor potential channel 1, a likely candidate protein for SACs. SACs may play a role in the pathogenesis of the heart failure associated with DMD. Early in the disease process and before the onset of clinical symptoms increased, SAC activity may underlie the abnormal Ca(2+) handling in young mdx mice.     

The effects of exogenous calcium buffers on the systolic calcium transient in rat ventricular myocytes

The aim of this work was to characterize the effects that two commonly used "caged" calcium buffers (NP-EGTA and nitr-5) have on the amplitude and time course of decay of the calcium transient. We made quantitative measurements of both free and total calcium using the measured buffering properties of the cell. Intracellular calcium concentration ([Ca(2+)](i)) was measured with fluo-3 in rat ventricular myocytes. Incorporation of the buffer NP-EGTA decreased both the amplitude and rate of decay of the caffeine response. The slowing could be quantitatively accounted for by the measured increased buffering. These effects were removed by photolysis of NP-EGTA. Similar results were obtained with nitr-5 except that the effects were not completely removed by photolysis. This was shown to be due to the persistence of a component of the increased buffering after photolysis. Both buffers decreased the amplitude of the systolic calcium transient. However, although nitr-5 produced a simple slowing of the decay, NP-EGTA resulted in an initial rapid phase of decay. This rapid phase of decay is attributed to calcium binding to NP-EGTA. This work represents the first quantitative analysis of the effects that extra buffering by a fast and a slow calcium chelator may have on the calcium transient.     

Calcium sparks in mouse ventricular myocytes at physiological temperature

In cardiac muscle, Ca2+ is released from the sarcoplasmic reticulum (SR) in units called Ca2+ sparks. Ca2+ spark characteristics have been studied almost entirely at room temperature. This study compares characteristics of spontaneous sparks detected with fluo 3 in resting mouse ventricular myocytes at 22 and 37 degrees C. The incidence and frequency of Ca2+ sparks decreased dramatically at 37 degrees C compared with 22 degrees C. Also, spark amplitudes and times to peak were significantly reduced at 37 degrees C. In contrast, spatial width and decay times were unchanged. During field stimulation, peak spatially averaged transients were similar at 22 and 37 degrees C, and experiments with fura 2 demonstrated that diastolic and systolic Ca2+ concentrations were unchanged. However, SR Ca2+ content decreased significantly at 37 degrees C. Restoration of SR Ca2+ by superfusion with 5 mM Ca2+ increased spark frequency but did not reverse the effects of temperature on spark parameters. Thus effects of temperature on spark frequency may reflect changes in SR stores, whereas changes in spark amplitude and rise time may reflect known effects of temperature on ryanodine receptor function.     

Presence of a calcium-activated chloride current in mouse ventricular myocytes

The properties of several components of outward K(+) currents, including the pharmacological and kinetics profiles as well as the respective molecular correlates, have been identified in mouse cardiac myocytes. Surprisingly little is known with regard to the Ca(2+)-activated ionic currents. We studied the Ca(2+)-activated transient outward currents in mouse ventricular myocytes. We have identified a 4-aminopyridine (4-AP)- and tetraethyl ammonium-resistant transient outward current that is Ca(2+) dependent. The current is carried by Cl(-) and is critically dependent on Ca(2+) influx via voltage-gated Ca(2+) channels and the sarcoplasmic reticulum Ca(2+) store. The current can be blocked by the anion transport blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Single channel recordings reveal small conductance channels (approximately 1 pS in 140 mM Cl(-)) that can be blocked by anion transport blockers. Ensemble-averaged current faithfully mirrors the transient kinetics observed at the whole level. Niflumic acid (in the presence of 4-AP) leads to prolongation of the early repolarization. Thus this current may contribute to early repolarization of action potentials in mouse ventricular myocytes.     

Computer model of action potential of mouse ventricular myocytes

We have developed a mathematical model of the mouse ventricular myocyte action potential (AP) from voltage-clamp data of the underlying currents and Ca2+ transients. Wherever possible, we used Markov models to represent the molecular structure and function of ion channels. The model includes detailed intracellular Ca2+ dynamics, with simulations of localized events such as sarcoplasmic Ca2+ release into a small intracellular volume bounded by the sarcolemma and sarcoplasmic reticulum. Transporter-mediated Ca2+ fluxes from the bulk cytosol are closely matched to the experimentally reported values and predict stimulation rate-dependent changes in Ca2+ transients. Our model reproduces the properties of cardiac myocytes from two different regions of the heart: the apex and the septum. The septum has a relatively prolonged AP, which reflects a relatively small contribution from the rapid transient outward K+ current in the septum. The attribution of putative molecular bases for several of the component currents enables our mouse model to be used to simulate the behavior of genetically modified transgenic mice.     

Effect of temperature on calcium exchange in digitonin-treated rat ventricular myocytes

The effect of a change in temperature on net mitochondrial Ca2+ exchange has been investigated in a suspension of adult rat ventricular myocytes. Temperature was varied between 42 degrees C and 15 degrees C. Hypothermia reduced the initial rate of respiration-dependent Ca2+ uptake and reduced the Na+-sensitivity of Ca2+ efflux. The net result of these alterations is that at low temperatures, the Ca2+ level at which a steady-state between mitochondria and sarcoplasm is maintained, will be raised.     

Underlying mechanisms of symmetric calcium wave propagation in rat ventricular myocytes

Calcium waves in heart cells are mediated by diffusion-coupled calcium-induced calcium release. The waves propagate in circular fashion. This is counterintuitive in view of the accepted ultrastructure of the cardiac myocyte. The density of calcium release sites in the transverse direction is four times higher than in the longitudinal direction. Simulations with release sites localized along Z-lines and isotropic diffusion yielded highly elliptical, nonphysiological waves. We hypothesized that subcellular organelles counteracted the higher release site density along the Z-lines by acting as transverse diffusion barriers and sites of active calcium uptake. We quantified the reduction of transverse diffusion by microinjecting cells with the nonreactive dye fluorescein. The ratio of the radial diffusion coefficient to the longitudinal coefficient was 0.39. Inhibition of mitochondrial uptake by rotenone accelerated the wave in the transverse direction. Simulations with release sites clustered at the Z-lines and a transverse diffusion coefficient 50% of the longitudinal coefficient generated waves of ellipticity 2/1 (major axis along the Z-line). Introducing additional release sites between the Z-lines at a density 20% of that on the Z-lines produced circular waves. The experiments and simulations support the presence of transverse diffusion barriers, additional uptake sites, and possibly intermediate release sites as well.     

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.     

Effects of nanomolar concentration dihydroouabain on calcium current and intracellular calcium in guinea pig ventricular myocytes

The effects of nanomolar concentration of dihydroouabain (DHO) on L-type calcium current (ICa-L), TTX-sensitive calcium current (ICa(TTX)), and intracellular calcium concentration ([Ca2+]i) were investigated in guinea pig ventricular myocytes. The whole-cell patch-clamp technique was used to record ICa-L and ICa(TTX); [Ca2+]i was detected and recorded with the confocal microscopy. The nanomolar concentration of DHO increased the ICa-L, ICa(TTX), and [Ca2+]i, which could be partially inhibited by nisoldipine or TTX, but still appeared in the absence of extracellular K+ and Na+. These data suggest that DHO could increase [Ca2+]i in non-beating myocytes via stimulating the ICa-L and ICa(TTX), or perhaps triggering directly a release of intracellular calcium.     

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.     

Mechanism of fluoride action on the L-type calcium channel in cardiac ventricular myocytes

The modulatory actions of fluoride on the function of the dihydropyridine-sensitive (L-type) Ca2+ channel were studied in rabbit cardiac myocytes. In cell-attached voltage-clamp experiments, using barium as the charge carrier, fluoride increased the activity of the Ca2+ channel dose-dependently. Low concentrations (<10 mM) of fluoride increased the number of traces with channel activities, and decreased the number of traces without channel activities, resulting in a net increase in the open-channel probability. The effect of 5 mM fluoride on the Ca2+ channel was inhibited by the presence of non-hydrolyzable guanosine diphosphate analog in the cell. On the other hand, high concentrations (>10 mM) of fluoride increased the open-channel duration, resulting in a marked increase in open-channel probability. A pretreatment of myocytes with a phosphatase inhibitor, okadaic acid, virtually abolished the additional effect of fluoride on the open-channel duration or open probability. A concentration of up to 75 mM fluoride had no effect on the Ca2+-channel activity when the myocytes were pretreated with a potent inhibitor of protein kinases, indicating that fluoride increased the Ca2+- channel activity via modulation of the phosphorylation state of the myocyte or the channel protein alone.     

Reverse mode of the sarcoplasmic reticulum calcium pump and load-dependent cytosolic calcium decline in voltage-clamped cardiac ventricular myocytes

We have characterized [Ca](i) decline in voltage-clamped rabbit ventricular myocytes with progressive increases in sarcoplasmic reticulum (SR) calcium load. "Backflux" through the SR calcium pump is a critical feature which allows realistically small values for SR calcium leak fluxes to be used. Total cytosolic calcium was calculated from the latter part of [Ca](i) decline using rate constants for cellular calcium buffers. Intra-SR calcium buffering characteristics were also deduced. We found that the net SR calcium pump flux and rate of [Ca](i) decline decreased as the SR free [Ca] rose, with pump parameters held constant. We have therefore characterized for the first time in intact myocytes both forward and reverse SR calcium pump kinetics as well as intra-SR calcium buffering and SR calcium leak. We conclude that the reverse flux through the SR calcium pump is an important factor in comprehensive understanding of dynamic SR calcium fluxes.     

Tricyclic antidepressant amitriptyline alters sarcoplasmic reticulum calcium handling in ventricular myocytes

Tricyclic antidepressants such as amitriptyline (AMT) have been reported to have adverse side effects on cardiac performance. AMT effects on Ca handling in ventricular myocytes, however, are not well understood. Therefore, we investigated AMT action on sarcoplasmic reticulum (SR) Ca release in ventricular myocytes, ryanodine receptor (RyR) activity, and Ca uptake by SR microsomes. In permeabilized myocytes, AMT transiently increased free luminal Ca concentration ([Ca]) followed by marked depletion. AMT (10 microM) caused a rapid and a transient increase of Ca spark frequency, followed by a significant suppression of spark activity. The latter was associated with a decrease of Ca spark amplitude and SR Ca load to 87 and 60%, respectively. AMT (10 microM) completely abolished propagation of spontaneous Ca waves. Higher concentrations of AMT (0.1-1 mM) evoked SR Ca release reminiscent of the effect of caffeine (20 mM) and caused almost complete depletion of SR Ca content. Studies on single calsequestrin-free RyR channels revealed that AMT increased the mean open time and open probability (Po) in a dose-dependent fashion (dissociation constant = 4.2 microM). High concentrations of AMT (> 25 microM) evoked frequent long openings with Po reaching very high levels (> 0.70). In studies with cardiac SR microsomes, AMT slowed the rate of ATP-dependent Ca uptake. We conclude that AMT affects SR Ca handling in ventricular myocytes by multiple mechanisms, including direct stimulation of RyRs and inhibition of SR Ca uptake. These effects could contribute to AMT cardiotoxicity.