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Am J Physiol Heart Circ Physiol 295: H2008-H2016, 2008. First published September 12, 2008; doi:10.1152/ajpheart.00523.2008
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Tricyclic antidepressant amitriptyline alters sarcoplasmic reticulum calcium handling in ventricular myocytes

Aleksey V. Zima, Jia Qin, Michael Fill, and Lothar A. Blatter

Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois

Submitted 16 May 2008 ; accepted in final form 9 September 2008


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
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 µM) 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 µM) 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 µM). High concentrations of AMT (>25 µM) 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.

heart; excitation-contraction coupling; ryanodine receptor; calcium sparks; calsequestrin; antidepressants

IN CARDIAC MUSCLE, surface membrane depolarization activates L-type Ca channels, generating a small Ca flux that activates type 2 ryanodine receptor (RyR) channels located on the membrane of the sarcoplasmic reticulum (SR) through Ca-induced Ca release. Nearly synchronous activation of multiple RyR channels at discrete sites on the SR generates localized Ca release events called sparks (4). These sparks are thought to be the elementary units of RyR-mediated Ca release in cardiac muscle. Summation of sparks is what ultimately generates global intracellular Ca elevations that drive cardiac contractility. The mechanisms that control RyR-mediated Ca release are still being debated, and one of these mechanisms in question is calsequestrin (CSQ)-dependent luminal Ca regulation.

CSQ is localized inside the lumen of the SR near the RyR channel and is thought to act as a low-affinity, high-capacity intra-SR Ca buffer (3, 8, 21, 28). Additionally, Gyorke et al. (12) have proposed that a Ca-dependent physical interaction between CSQ and the RyR channel is important to the control of the SR Ca release process (12). Mutants of CSQ are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a adrenergically mediated familial arrhythmogenic disorder (15, 24, 33). These tachyarrhythmias are typically triggered by physical exercise or emotional stress and can lead to syncope and sudden cardiac death (16). Thus it is reasonable to predict that pharmacological agents that alter CSQ structure-function may trigger adverse cardiovascular side effects and could represent new tools that can be used in the study of the RyR-mediated SR Ca release process.

Tricyclic antidepressants are known to be cardiotoxic, and overdose of these agents is frequently fatal (18). The adverse side effects of these agents have been attributed to nonspecific actions on various unidentified proteins (1). In the heart, side effects include electrocardiographic (e.g., QRS complex) distortions, ventricular ectopic beats, nodal rhythm, bundle branch block, and ventricular tachycardia (25, 36, 38). Park et al. (22) have showed that these drugs bind to CSQ with dissociation constant (KD) in the micromolar range and reduce its Ca-binding capacity, leading to the proposal that the action of tricyclic antidepressants on CSQ contributes to the undesirable cardiovascular toxic side effects of these agents (22).

Amitriptyline (AMT) is a commonly used tricyclic antidepressant. It is thought to exert its clinical action by inhibiting the reuptake of certain neurotransmitters (e.g., norepinephrine and serotonin). Using confocal imaging and single channel recording, we show that AMT has multiple actions on SR Ca handling. At a low dose (10 µM), AMT promotes Ca sparks and eventually depletes SR Ca load. At higher doses (0.1–1 mM), AMT evokes fast global RyR-mediated SR Ca release events like those evoked by a caffeine application. At the single RyR channel level, AMT robustly activates CSQ-free channels (with 0.1 mM nearly tripling its mean open time) without altering their unitary Ca current. We also show that AMT inhibits SR Ca-ATPase (SERCA) function. Thus the undesirable cardiovascular side effects associated with AMT likely arise from its combined actions on the CSQ, RyR, and SERCA proteins. Part of this work has been published in abstract form (41).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Isolation of ventricular myocytes. Cardiac ventricular myocytes were enzymatically isolated from adult cats and rabbits using methods described previously (27, 31). All procedures were approved by the Loyola University Chicago Institutional Animal Care and Use Committee.

Measurements of Ca sparks and waves in permeabilized cat ventricular myocytes. Spontaneous SR Ca release events were studied in saponin-permeabilized cat ventricular myocytes, as described previously (5, 40). After permeabilization, cells were placed in a solution composed of (in mM): 100 potassium aspartate, 15 KCl, 5 KH2PO4, 5 MgATP, 0.35 EGTA, 0.12 CaCl2, 0.75 MgCl2, 10 phosphocreatine, 10 HEPES, and 0.03 fluo 4 pentapotassium salt, 5 U/ml creatine phosphokinase, and 8% dextran [mol wt 40,000%, and pH 7.2 (KOH)]. Free Ca concentration ([Ca]) of this solution was 150 nM for Ca spark measurements. For Ca wave studies, free [Ca] was adjusted to 250 nM by adding appropriate amounts of CaCl2 (calculated using WinMAXC 2.05; Stanford University). All experiments were performed at room temperature (20–23°C). Changes in intracellular [Ca] ([Ca]i) were measured with a laser scanning confocal microscope (Radiance 2000 MP; Bio-Rad) equipped with a x40 magnification oil-immersion objective lens (numeric aperture = 1.3). The Ca indicator fluo 4 was excited with the 488-nm line of an argon ion laser, and fluorescence was measured at wavelengths >515 nm. Images were acquired in linescan mode (3 ms/line; pixel size 0.12 µm).

Ca sparks were detected and analyzed using SparkMaster at a threshold criteria of 3.8 (23). Analysis included spark frequency (sparks·s–1·100 µm–1), amplitude ({Delta}F/F0), full duration at half-maximal amplitude (ms), and full width at half-maximal amplitude (µm). F0 is the initial fluorescence recorded under steady-state conditions and {Delta}F = F – F0. Ca waves were analyzed in terms of amplitude ({Delta}F/F0), frequency (Hz), and wave propagation velocity (µm/s).

SR Ca load was measured from the peak amplitude of the [Ca]i transient induced by the rapid application of 20 mM caffeine. This concentration of caffeine fully activates RyRs (26) and leads to the synchronized release of the total Ca stored in the SR. We found that caffeine (20 mM) decreased fluo 4 fluorescence by 21% due to chemical quenching of the dye. Therefore, we corrected for fluo 4 quench the relative amounts of SR Ca released by caffeine when those were compared with AMT-induced Ca transients.

Measurements of free luminal [Ca] in permeabilized ventricular myocytes. We used the low-affinity Ca indicator fluo 5N to measure changes in free luminal [Ca] ([Ca]SR). Because [Ca]SR in cat ventricular myocytes could not be successfully measured with low-affinity Ca dyes (because of inefficient dye loading or dye deesterification), we used rabbit ventricular myocytes for [Ca]SR measurements. Isolated rabbit ventricular myocytes were loaded with fluo 5N-AM under conditions that promote dye accumulation in the SR, as previously described (31). Fluo 5N-AM-loaded myocytes were permeabilized with saponin to remove any cytosolic fluo 5N and exposed to the experimental solution used for Ca spark measurements (see above). Fluo 5N was excited at 488 nm, and fluorescence was measured at wavelengths >515 nm. Two-dimensional images were acquired at 5-s intervals. The relative changes in [Ca]SR are presented as normalized fluorescence (F/F0). The fluo 5N signal was corrected for the Ca-insensitive component after complete SR Ca depletion by 20 mM caffeine.

RyR single channel recordings. Heavy SR microsomes were prepared from rat ventricle using a standard cellular subfractionation process (32). Planar lipid bilayers were formed across a 100-µm diameter hole in a 12-µm-thick Teflon partition. This partition separated two 1-ml compartments. One (cis) was virtually grounded and filled with a HEPES-Tris solution (250 mM HEPES, 120 mM Tris, pH 7.4). This chamber was always facing the cytosolic side of the channel. The other (trans) chamber was filled with HEPES-Ca solution [250 mM HEPES, 53 mM Ca(OH)2, pH 7.4] and faced the luminal side of the channel. This high luminal [Ca] assured that no CSQ was associated with the channels tested here (12).

Planar bilayers were formed from a 5:4:1 mixture (50 mg/ml total lipids in decane) of bovine brain phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. Next, 500 mM CsCl and heavy SR microsomes were added to the cis chamber. Once ion channels were incorporated into the bilayer, ion currents >100 pA were observed at 0 mV (mediated by RyRs and by other cationic and anionic channels that permeate Cs or Cl). Subsequently, CsCl was removed from the cis chamber. A mixture of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 1,2-bis(2- amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid (dibromo-BAPTA) was used to buffer free [Ca] in the cis chamber to 1 µM. Free [Ca] was verified with a Ca-selective electrode.

Single RyR channel recordings were sampled at 100 µs/pt and filtered (8-pole Bessel) at 1 kHz. Single channel analysis was done using pCLAMP9 software (Axon Instruments/Molecular Devices, Sunnyvale, CA). Single channel recordings were idealized using the half-amplitude threshold method. Unit current, open times, closed times, and open probability (Po) were determined from idealized traces. No special consideration was taken when infrequent sojourns to a subconductance state (i.e., less than full open conductance) were present at high AMT levels. Exponential maximum likelihood fitting of dwell-time histograms assumed all open and closed time distributions included three components. Only dwell times greater than three times the filter dead time were included in the dwell time analysis.

Measurements of Ca uptake rate by SR microsomes. SR Ca uptake was measured with a spectrophotometer (Shimadzu UV-1650PC) using the Ca-sensitive dye antipyrylazo III (APIII), as described previously (39). SR membrane vesicles isolated from cat ventricle (50 µg/ml) were added to 1 ml phosphate buffer medium containing (in mM): 100 KH2PO4, 3 MgCl2, 2 ATP, 0.05 ruthenium red (RuR), and 0.1 APIII, pH 7.0. Ca uptake was initiated by addition of 20 µM Ca to the medium and measured as changes in absorbance of APIII between 710 and 790 nm. RuR (50 µM) was used to block Ca release from the SR. Parameters of SERCA activity were calculated from the changes of total [Ca] based on the known Ca-buffering capacity of the experimental solution. We found the apparent KD of APIII for Ca was 9.6 µM. The rate of SR Ca uptake was measured as the changes of total [Ca] ({Delta}[Ca]total/s) and plotted against the associated free [Ca]. This relationship was fitted with a Hill equation to determine the maximum velocity (Vmax) and Michaelis-Menten constant (Km).

Drugs. Fluo 4 pentapotassium salt and fluo 5N-AM were purchased from Molecular Probes/Invitrogen (Carlsbad, CA). APIII and N-(2-acetamido)iminodiacetic acid (ADA) were purchased from Sigma. BAPTA, 5,5'-dibromo-BAPTA, Ca(OH)2, CsCl, and HEPES were obtained from Fluka (Milwaukee, WI). CaCl2 standards for calibration were from World Precision Instruments (Sarasota, FL). Phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL) and decane from Aldrich Chemical (Milwaukee, WI). All other drugs and chemicals were either from Fluka or Sigma and were reagent grade.

Statistics. Data are presented as means ± SE of n measurements. Statistical comparisons between groups were performed with the Student's t-test. Differences were considered statistically significant at P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of AMT on free luminal [Ca]. AMT has been reported to alter the function of CSQ by reducing its Ca-binding capacity (22). Thus we examined how AMT affects [Ca]SR by monitoring changes in fluorescence of the low-affinity Ca dye fluo 5N entrapped in the SR of permeabilized ventricular myocytes. As reported previously (31), the fluo 5N signal had a characteristic sarcomeric pattern of staining, which is due to the localization of the dye within the SR. Figure 1A shows two-dimensional (x-y) images of the fluo 5N signal in control conditions as well as 0.5 and 3 min after AMT (0.1 mM) application to saponin-permeabilized ventricular myocytes. AMT application produced an initial brief increase in [Ca]SR followed by marked depletion of remaining [Ca]SR (Fig. 1B). On average, 0.1 mM AMT produced an initial rise of [Ca]SR by 18 ± 6% (n = 5) followed by a sustained depletion of stored Ca to 45 ± 9% of control (n = 5). Intra-SR fluo 5N was not saturated under these conditions because fluorescence could be further increased by SERCA stimulation using the protein kinase A catalytic subunit (5 U/ml) and RyR inhibition with 20 µM RuR (Fig. 1B). To completely deplete [Ca]SR, caffeine (20 mM) was applied after RuR washout. This high concentration of caffeine equally emptied the SR in control conditions and after RuR washout (data not shown).


Figure 1
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  		Fig. 1. Effects of amitriptyline (AMT) on free luminal Ca concentration ([Ca]) in permeabilized ventricular myocytes. A: confocal (x-y) images of fluo 5N fluorescence from a permeabilized ventricular myocyte in controls and 0.5 and 3 min after AMT (0.1 mM) addition, respectively. B: time course of changes in free intrasarcoplasmic reticulum [Ca] ([Ca]SR). Data were normalized to the fluo 5N signal recorded at the beginning of the experiment. Protein kinase A (PKA) catalytic subunit (5 U/ml) and ruthenium red (RuR; 20 µM) were added to the experimental solution to obtain maximal sarcoplasmic reticulum (SR) Ca load. Caffeine (20 mM) was applied to completely deplete SR Ca.

 
The initial brief rise of [Ca]SR after AMT application was most likely mediated by the previously reported effect of AMT to reduce the Ca-binding capacity of CSQ (22). Depletion of [Ca]SR during AMT application could be the result of stimulation of Ca release or inhibition of SERCA Ca uptake, or a combination of both. In the subsequent experiments, we studied in detail AMT effects on SR Ca release, load and uptake, as well as RyR gating.

Effects of AMT on Ca sparks and SR Ca load. The effect of AMT on spontaneous Ca sparks was studied in saponin-permeabilized ventricular myocytes under conditions similar to [Ca]SR measurements. Figure 2A shows representative confocal linescan images of Ca sparks and selected subcellular {Delta}F/F0 plots under control conditions, immediately and 3 min after application of AMT (10 µM) as well as after washout of the drug. Application of AMT transiently increased Ca spark activity. On average (within 0.5 min of 10 µM AMT application), spark frequency increased by 20 ± 7% (n = 10; P < 0.05). After 3 min of AMT application, however, spark frequency significantly decreased (to 47 ± 4% of control; n = 10; P < 0.05; Fig. 2Ba). Effects of AMT were partially reversible after washout. During the later phase of AMT action (measured during 3 min of AMT application), spark amplitude (Fig. 2Bb) and width (Fig. 2Bc) were significantly decreased, but not spark duration (Fig. 2Bd). On average, in the presence of AMT, Ca spark amplitude and width decreased to 87 ± 2 and to 89 ± 3% (n = 10; P < 0.05) of control, respectively. The total number of sparks analyzed were as follows: control: 1077; 0.5 min after AMT application: 304; 3 min after AMT application: 297; and after AMT wash out: 347.


Figure 2
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  		Fig. 2. Effects of AMT on SR Ca release and load studied in permeabilized ventricular myocytes. A: confocal linescan images and {Delta}F/F0 profiles of Ca sparks under control conditions, after addition of AMT (10 µM), and following washout of AMT. Local {Delta}F/F0 profiles obtained by averaging fluo 4 fluorescence from 2-µm-wide regions marked by the black boxes. Ba: average effect of AMT on Ca spark frequency; b, spark amplitude; c, spark width; and d, spark duration. Ca spark properties were averaged over 2 min under control conditions (Ctrl), during 3 min after application of AMT, and over 2 min after AMT washout (W). Results were normalized to the levels recorded under control conditions. C: Ca release induced by application of 20 mM caffeine under control conditions, 3 min after addition of 10 µM AMT, and following washout of AMT. D: average amplitudes of caffeine-induced Ca release (SR Ca load) under control conditions, in the presence of AMT, and after washout of AMT. *P < 0.05 vs. control.

 
We examined if the AMT-induced depression of spark activity was associated with changes in SR Ca load. SR Ca load was measured as [Ca]i transient amplitude during fast application of caffeine (20 mM). After 3 min of AMT application, SR Ca load was partially depleted (Fig. 2C). On average, the amplitude of the caffeine response in the presence of AMT (10 µM) was reduced to 60 ± 5% (n = 5; P < 0.05) of control (Fig. 2D). The effect of AMT on SR Ca content was only partially reversible upon washout of AMT.

AMT effects on SR Ca release and load were dose dependent. Application of 0.1 mM AMT evoked Ca release from a majority of individual release sites along the scanned line, producing global cytosolic Ca transient (Fig. 3A). The amplitude of this AMT-induced Ca transient was 45 ± 9% (n = 6) of the control caffeine-induced Ca transient (Fig. 3A; also see Fig. 3C). After the initial release evoked by AMT, Ca spark activity was completely abolished, and SR Ca load was decreased to 26 ± 3% (n = 6) of control (Fig. 3D). At concentrations of 1 mM and higher, AMT evoked SR Ca release comparable to that induced by caffeine (Fig. 3B). On average, AMT (1 mM) released 82 ± 6% (n = 8) of total SR Ca (Fig. 3, B and C). Consecutive caffeine application induced Ca release with an amplitude of only 13 ± 4% (n = 8) of control (Fig. 3D). Similarly, when cells were exposed to 20 mM caffeine before AMT (1 mM) application, AMT did not evoke any additional Ca release (data not shown). We also found that 0.1 and 1 mM AMT (but not 10 µM) increased basal [Ca] after global release by 7 ± 2% (n = 6) and 15 ± 4% (n = 8), respectively.


Figure 3
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  		Fig. 3. Effects of high doses of AMT on SR Ca release and load. A: linescan images and {Delta}F/F0 profiles of (from left to right): caffeine-induced Ca release under control conditions, Ca release induced by AMT (0.1 mM), caffeine-induced Ca release in the presence of AMT (0.1 mM), and caffeine-induced Ca release after washout of AMT. B: linescan images and {Delta}F/F0 profiles of (from left to right): caffeine-induced Ca release under control conditions, Ca release induced by AMT (1 mM), caffeine-induced Ca release in the presence of AMT (1 mM), and caffeine-induced Ca release after washout of AMT. The profiles were obtained by averaging fluorescence from the entire image. C: average amplitude of AMT-induced SR Ca release. Values were normalized to caffeine-induced Ca transient (SR Ca load) under control conditions. D: average amplitude of caffeine-induced SR Ca release (SR Ca load) in the presence of AMT. *P < 0.05 vs. control. In these experiments, caffeine concentration was 20 mM.

 
Effects of AMT on Ca sparks at increased luminal [Ca] buffer capacity. The transient brief elevation of [Ca]SR following AMT application (see Fig. 1B) was attributed to AMT reducing the Ca-binding capacity of CSQ (22). To determine whether AMT action on SR Ca release was a consequence of reduced intra-SR Ca buffering, the low-affinity Ca buffer ADA was used to elevate SR Ca buffer capacity. After incubation of permeabilized myocytes with ADA (5 mM) for 10 min, ADA was washed out, and Ca sparks were studied in control solution. The slow diffusion of ADA out of the SR allows a time frame of 10–15 min during which Ca sparks can be measured at high intraluminal SR Ca buffer capacity (35, 40). Figure 4 shows typical linescan images and selected subcellular {Delta}F/F0 plots obtained under control conditions, after ADA loading and after subsequent AMT application. After ADA incubation, Ca spark amplitude and duration increased significantly (by 18 ± 4 and by 50 ± 9%, respectively; n = 4; P < 0.05). This change in spark properties was associated with increased SR Ca load by 38 ± 4% (n = 4 cells; P < 0.05). Subsequent application of AMT (10 µM; added 2–3 min after removal of ADA) produced effects similar to those observed in control conditions. As before, AMT transiently increased Ca spark frequency by 49 ± 11% (n = 4; P < 0.05). After 3 min of AMT application, spark frequency was suppressed markedly (to 37 ± 9%; n = 4; P < 0.05; Fig. 4). The latter effect was associated with a decrease in SR Ca load to 40 ± 6% compared with the SR Ca load measured after ADA incubation. Thus, when the SR Ca buffer capacity was increased (after ADA loading) to compensate for AMT effects on CSQ, it did not change the AMT effect on spark activity. It seems likely that AMT effects on SR Ca release and load were not solely due to a consequence of decreased SR Ca buffer capacity.


Figure 4
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  		Fig. 4. Effects of AMT on SR Ca release at increased intraluminal SR Ca buffer capacity. Confocal linescan images and corresponding {Delta}F/F0 profiles of Ca sparks and caffeine-induced Ca releases under control conditions, after incubation with ADA (5 mM) for 10 min, with and without AMT (10 µM) present. Local {Delta}F/F0 profiles obtained by averaging fluo 4 fluorescence from 2-µm-wide regions marked by the black boxes.

 
Effects of AMT on single cardiac RyR channel function. Single cardiac RyR channel activity was measured in planar lipid bilayer studies. Single-channel currents were recorded at 0 mV using Ca as the charge carrier. The charge carrier was provided by the presence of 50 mM luminal Ca. Because this luminal [Ca] promotes CSQ dissociation from the luminal side of the RyR channel (2, 12), the RyR channels tested here were all CSQ free. In the presence of 1 µM cytosolic free Ca, Po was <0.1. Elevating the cytosolic free Ca level to 100 µM increased Po, and subsequent addition of ryanodol (a low-affinity ryanodine derivative) induced intermittent long-lived subconductance states (data not shown) demonstrating that these are indeed RyR channels. When 1 µM cytosolic free Ca was present, addition of 1 µM AMT did not substantially alter channel activity. However, elevating the cytosolic AMT concentration to 5 µM and then 75 µM clearly increased the Po of the channel (Fig. 5A). At high AMT levels, occasional subconductance states were observed. The Po diaries presented in Fig. 5B correspond to the recordings above. These diaries each represent 120 s of recording and illustrate the robust activation of CSQ-free RyR channel by AMT.


Figure 5
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  		Fig. 5. Effects of AMT on single ryanodine receptor (RyR) channel activity. RyR single channel recordings were made with Ca as charge carrier at holding potential of 0 mV. Zero current level marked as dotted line. A: sample single channel recordings in 1, 5, and 75 µM AMT (drug applied to cytosolic chamber). B: open probability (Po) diary plots at the AMT concentrations corresponding to the recordings above each plot. Each point in the Po diaries represents 1 s recording time.

 
The AMT dose vs. Po relationship is presented in Fig. 6A and has a KD of 4.2 µM. The average unitary Ca current carried by the channel was not altered by AMT over the range of AMT tested (Fig. 6A, inset). Unitary current values were computed from idealized records; thus, any subconductance states present are included. Figure 6B illustrates how open event frequency and mean open event duration change with AMT concentration. The elevated Po at 5 µM AMT (Fig. 6A) was due to an increase in both frequency and duration of openings. The high Po at 100 µM was due primarily to an increase in open event duration. Note that the stimulatory action of AMT on single RyR channel function shown here was not due to AMT action on CSQ because no CSQ was associated with these channels.


Figure 6
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  		Fig. 6. Effects of AMT on single RyR channel activity. Summary representing 5–8 different single RyR channels. A: mean single channel Po plotted as a function of AMT concentration. The AMT points were fit with the Hill equation, and this fit indicates an apparent dissociation constant (KD) of 4.2 µM (Hill coefficient = 1.4). Inset: mean unit Ca current plotted as a function of drug concentration. B: mean open event frequency ({blacktriangleup}) and open dwell time ({triangleup}) plotted as a function of AMT concentration.

 
Effects of AMT on Ca waves. Permeabilized cardiomyocytes were placed in an intracellular solution containing 250 nM [Ca], which produced spontaneous Ca release from SR that propagated through the cell as repetitive Ca waves. Figure 7A shows representative line-scan images of Ca waves and selected plots of {Delta}F/F0 changes under control conditions, after addition of AMT (10 µM), and after AMT washout. Addition of AMT resulted in an initial small increase in Ca wave frequency (by 12 ± 5%; n = 5; P < 0.05) as well as wave propagation velocity (by 11 ± 4%; n = 5; P < 0.05). After 0.5 min, AMT caused a significant reduction of wave amplitude that led to wave fragmentation. After 3 min of AMT application, Ca waves ceased completely. The effect of AMT on Ca wave frequency and amplitude was partially reversible after washout.


Figure 7
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  		Fig. 7. Effects of AMT on Ca waves and SR Ca-ATPase (SERCA)-mediated Ca uptake. A: confocal linescan images and {Delta}F/F0 profiles of Ca waves under control conditions, after addition of AMT (10 µM) and following washout of AMT. B: confocal linescan images and {Delta}F/F0 profiles of Ca waves under control conditions, after addition of caffeine (0.1 mM) and following washout of caffeine. Local {Delta}F/F0 profiles obtained by averaging fluo 4 fluorescence from 5-µm-wide regions marked by the black boxes. C: Ca uptake by SR microsomes measured in control conditions (Ctrl) and in the presence of 10 and 100 µM of AMT. Ca uptake was initiated by addition of CaCl2 (20 µM). Effect of different concentrations of AMT on SERCA maximal velocity (Vmax; D) and Michaelis-Menten constant (Km; E). *P < 0.05 vs. control.

 
We compared AMT effects on Ca waves with the action of a low dose of caffeine that does not evoke global SR Ca release but substantially sensitizes RyRs to cytosolic Ca. As shown in Fig. 7B, caffeine (0.1 mM) only transiently increased Ca wave frequency (by 39 ± 9%; n = 4; P < 0.05) and propagation velocity (by 21 ± 8%; n = 4; P < 0.05). After 0.5–1 min of caffeine (0.1 mM) application, Ca wave frequency returned to control levels. These effects were associated with a decrease in Ca wave amplitude (by 23 ± 7%; n = 4; P < 0.05). Immediately after caffeine washout, Ca waves completely disappeared but returned to control activity within next 10–20 s. Lack of similarity between AMT and caffeine effects on Ca waves suggests that ATM effects could not be explained solely based on RyR activation.

Effects of AMT on SERCA-mediated Ca uptake. We tested whether AMT affects SERCA activity. For that purpose, we measured Ca uptake by SR microsomes isolated from cat ventricle. The experiments were carried out in the presence of RuR (50 µM) to completely inhibit RyR-mediated Ca release. SR Ca uptake rate after addition of 20 µM CaCl2 was measured before and after AMT addition. Experimental data (APIII signal decline) were fitted by a single exponential function from which the rate of uptake (expressed as the time constant of APIII signal decline) was derived. Figure 7C illustrates that AMT slowed the Ca uptake rate in a dose-dependent manner. On average, 10, 50, and 100 µM of AMT decreased the time constant of uptake (1/{tau}) by 30 ± 6% (n = 6; P < 0.05), by 54 ± 15% (n = 4; P < 0.05), and by 114 ± 15% (n = 4; P < 0.05), respectively. We also analyzed changes of kinetic parameters of SERCA in the presence of AMT. We found that AMT inhibited SERCA activity by decreasing Vmax in dose-dependent manner (Fig. 7D). At high concentrations AMT also decreased the Ca affinity (Km) of SERCA (Fig. 7E). Therefore, the observed depletion of SR Ca in the presence of AMT was the result of inhibition SR Ca uptake and stimulation of RyR-mediated SR Ca release.


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Tricyclic antidepressants including AMT can have devastating side effects on cardiac performance. Electrocardiogram abnormalities are frequent, and a wide variety of cardiac arrhythmias can occur, the most common being sinus tachycardia and intraventricular conduction delay (QRS prolongation) (36). Previous studies have shown that tricyclic antidepressants modulate sarolemmal ion channels involved in cardiac excitation-contraction coupling (6, 13, 19). The aim of the present study was to investigate effects of AMT on SR Ca handling in ventricular myocytes. The main findings are: 1) AMT transiently increases [Ca]SR presumably due to reduction of CSQ Ca-binding capacity, 2) AMT increases the Po of single RyR channels independently from its action on CSQ, 3) AMT inhibits SERCA-mediated Ca uptake, and 4) combined these actions rapidly stimulate SR Ca release, ultimately leaving the SR Ca depleted.

The effective plasma concentration of tricyclic antidepressants has been measured (14, 37). It seems that micromolar levels of these drugs (the drug plus its metabolites) are required to have their therapeutic action. Here, the different actions of 1–1,000 µM AMT were defined. Significant effects on SR Ca release in cells were observed with concentrations of AMT as low as 10 µM. Clear effects on single RyR channel function were observed at an AMT concentration of 2.5–5 µM. There was no detectable action of AMT on single RyR function or RyR-mediated Ca release in cells when these drugs were applied at therapeutic concentrations (<1 µM). The implication is that alteration of RyR-mediated Ca signaling is unlikely to be involved in the normal therapeutic actions of AMT. However, our results show that AMT activates single RyR channels with an EC50 of 4.2 µM. Thus activation of RyR channels or RyR-mediated Ca release may contribute to the adverse cardiac side effects of this drug during overdose.

During excitation-contraction coupling, global SR Ca release relies on the amount of Ca stored inside the SR. A significant fraction of this Ca is bound to the low-affinity, high-capacity Ca buffer CSQ (3, 21). In addition to its role of Ca buffering, CSQ also modulates RyR function. It has been shown that, together with junctin and triadin, CSQ forms the luminal Ca sensor of the RyR (12). Alteration of CSQ function or expression levels leads to ventricular arrhythmias such as CPVT (15, 24) and instability of Ca regulation (33, 34), suggesting an important role of CSQ in cardiac excitation-contraction coupling. Thus it would be rather straight forward to predict that drugs that interact with CSQ may also alter cardiac excitation-contraction coupling. A previous in vitro study showed that AMT and other tricyclic antidepressants bind to CSQ (with KD in the µM range) and reduce its Ca-binding capacity (22). In agreement with this work, we found that AMT evoked an initial brief increase in free luminal Ca in isolated ventricular myocytes (Fig. 1B). The initial brief increase is likely arising from an AMT-evoked change in CSQ Ca buffering. If this were all AMT did, then [Ca]SR would be expected to return to control levels as the balance between SR Ca release and uptake is reestablished (30). However, AMT significantly depleted [Ca]SR (well below control levels) following the initial brief increase (Fig. 1B). This implies that AMT also simultaneously altered the balance between SR Ca release and uptake.

Measurements of spontaneous SR Ca release events in permeabilized ventricular myocytes revealed that AMT caused a transient increase in Ca spark frequency that was followed by a depletion of SR Ca content and a decrease in spark activity. This effect of AMT is reminiscent of the effect of low doses of caffeine. RyR channel activation by caffeine also results in brief increases in Ca spark frequency followed by a decrease in SR Ca load (17). It has been proposed that stimulation of SR Ca release unloads the SR, and this subsequently results in a decrease in RyR channel activity due to a luminal RyR Ca-dependent inhibitory mechanism (7, 17, 20). This may explain the late (inhibitory) phase of AMT action we observed here. Prolonged application of a low dose of AMT (10 µM) resulted in significantly decreased Ca spark amplitude, spatial width, and frequency. Smaller amplitude and width are consistent with a smaller RyR-mediated SR Ca release flux that would occur with depleted SR Ca content. The decrease in spark frequency is consistent with the existence of the aforementioned RyR Ca-dependent inhibitory mechanism coming into play as SR Ca content is lost. High doses of AMT (0.1–1 mM) evoked a global Ca transient (as do high caffeine doses). For example, 1 mM AMT released almost all Ca stored in the SR. When stored SR Ca was depleted by caffeine, however, AMT did not produce any detectable Ca release, indicating that AMT and caffeine activate release from the same store.

We found that AMT directly stimulates single RyR channels incorporated in lipid bilayers. AMT increased Po and mean open time in a dose-dependent manner. High AMT concentrations induced long-lasting openings and occasionally the appearance of a subconductance state. The CSQ protein is closely associated with the luminal aspect of the RyR channel and is thought to modulate the channel (12). AMT action on single RyR channel function could be due to AMT acting on CSQ that may still be associated with the channel. However, the single RyR channel studies here were done with 50 mM luminal Ca present, and this is known to effectively dissociate CSQ from the RyR channel (12). Thus AMT actions on single RyR function shown here are likely independent of AMT actions on CSQ. This may be the first evidence of the relatively potent action of AMT on the cardiac RyR channel.

How might AMT actions at the molecular level result in the observed cellular effects of the drug? Application of 10 µM AMT initially increased spark frequency, and this is very likely due to an AMT-evoked increase in the frequency of opening and/or mean open time of single RyR channels (Fig. 6B). In addition to the direct effect of AMT on the channel gating, the brief rise of free luminal [Ca] during AMT application (Fig. 1) will further stimulate SR Ca release by a luminal Ca-dependent mechanism (11). The subsequent reduction in spark frequency is likely due to luminal Ca-dependent inhibition of single RyR channel activity as [Ca]SR is depleted (during a sustained AMT application). This is consistent with RyR Po (11) as well as the fraction of releasable Ca being steeply dependent on [Ca]SR (29). Thus we conclude that AMT action on sparks is primarily the result of a direct affect of AMT on single RyR channel activity.

We observed that AMT evoked a brief transient rise in [Ca]SR immediately upon AMT application. This brief rise is attributed to AMT-dependent reduction in CSQ Ca-binding capacity. Previous work has shown that reductions in SR Ca buffer capacity (due to decreased CSQ expression) alter [Ca]SR but not spark frequency (34). When a low-affinity Ca buffer (ADA) was loaded in the SR to compensate for the AMT-dependent reduction in CSQ Ca buffering, it did not reverse (or change) AMT action on spark frequency or SR Ca load. This suggests that AMT-mediated modulation of SR Ca release and load was independent from SR Ca buffering and supports the conclusion that our results are a consequence of a direct AMT stimulation of RyR channels.

Generally, low doses of AMT had actions that were reminiscent of low doses of caffeine. The one exception was the affect of AMT on spontaneous Ca waves. When myocytes were exposed to a higher [Ca]i, Ca waves regularly propagated through the cytosol. Application of AMT completely abolished these Ca waves after a few seconds (Fig. 7A). In contrast, caffeine increased wave frequency, and waves continued for minutes (Fig. 7B). The caffeine-dependent increase in wave frequency is usually attributed to sensitization of RyR function (i.e., lower [Ca]SR threshold for spontaneous Ca release to occur). We found that AMT inhibits SERCA activity in a dose-dependent fashion (Fig. 7C). Therefore, the difference between AMT and caffeine actions may be because AMT (unlike caffeine) also inhibits the SERCA pump.

In conclusion, the action of AMT on intracellular Ca handing in cardiac myocytes likely involves direct effects on CSQ, RyR, and SERCA. The immediate action of AMT to increase spontaneous RyR channel opening is most likely to be arrythmogenic. It has been suggested that augmentation of RyR-mediated Ca leak can lead to proarrhythmogenic delayed or early afterdepolarizations that are common features of the failing heart (for review, see Ref. 9). Furthermore, chronic defects in RyR and CSQ due to genetic mutations can also lead to abnormalities of excitation-contraction coupling and arrhythmias (10, 33). The longer-term action of AMT to promote SR Ca depletion would most likely lead to negative inotropy. The combined AMT action on all three proteins likely explains the general cardiotoxicity of trycyclic antidepressants, like AMT.


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 ABSTRACT
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This work was supported by National Institutes of Health Grants HL-80101 and HL-62231 (to L. A. Blatter) and HL-57832 and AR-54098 (to M. Fill) and by American Heart Association Grant AHA0530309Z (to A. V. Zima).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Blatter, Rush Univ. Medical Center, Dept. of Molecular Biophysics and Physiology, 1750 W. Harrison Ave., Chicago, IL 60612 (e-mail: Lothar_Blatter{at}rush.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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