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Caffeine-induced arrhythmias in murine hearts parallel changes in cellular Ca²⁺ homeostasis

¹Physiological Laboratory, ²Section of Cardiovascular Biology, Department of Biochemistry, and ³Department of Pharmacology, University of Cambridge, United Kingdom

Submitted 12 December 2004 ; accepted in final form 25 May 2005

	ABSTRACT

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Heart failure leading to ventricular arrhythmogenesis is a major cause of clinical mortality and has been associated with a leak of sarcoplasmic reticular Ca²⁺ into the cytosol due to increased open probabilities in cardiac ryanodine receptor Ca²⁺-release channels. Caffeine similarly increases such open probabilities, and so we explored its arrhythmogenic effects on intact murine hearts. A clinically established programmed electrical stimulation protocol adapted for studies of isolated intact mouse hearts demonstrated that caffeine (1 mM) increased the frequency of ventricular tachycardia from 0 to 100% yet left electrogram duration and latency unchanged during programmed electrical stimulation, thereby excluding slowed conduction as a cause of arrhythmogenesis. We then used fluorescence measurements of intracellular Ca²⁺ concentration in isolated mouse ventricular cells to investigate parallel changes in Ca²⁺ homeostasis associated with these arrhythmias. Both caffeine (1 mM) and FK506 (30 µM) reduced electrically evoked cytosolic Ca²⁺ transients yet increased the frequency of spontaneous Ca²⁺-release events. Diltiazem (1 µM) but not nifedipine (1 µM) pretreatment suppressed these increases in frequency. Identical concentrations of diltiazem but not nifedipine correspondingly suppressed the arrhythmogenic effects of caffeine in whole hearts. These findings thus directly implicate spontaneous Ca²⁺ waves in triggered arrhythmogenesis in intact hearts.

mouse; ryanodine receptor; programmed electrical stimulation; ventricular tachycardia; arrhythmia

Caffeine similarly increases open probabilities in RyR2s in isolated myocytes in vitro (35) and induces ventricular arrhythmias in canine and rabbit hearts in vivo (12, 24). The present experiments accordingly sought to correlate the effects of caffeine on propagation of excitation in perfused intact mouse hearts with its effects on Ca²⁺ homeostasis in isolated myocytes. Use of mouse hearts additionally provided baseline data for a model system for which experimentally useful arrhythmogenic genetic variants are becoming increasingly available (1, 4, 6, 8, 17, 25, 28, 36). The proarrhythmic effects of caffeine were confirmed by demonstrating the extent to which a technique of programmed electrical stimulation (PES), adapted from cardiological practice and applied for the first time under these pharmacological conditions to experimental systems, induced ventricular arrhythmogenesis. The findings were compared with results from an adaptation of paced electrogram fractionation analysis (PEFA). This technique has hitherto been employed to detect arrhythmogenic propensities resulting from reentrant electrical activity in clinical proarrhythmic conditions (33, 34) and in murine cardiac models (1).

We demonstrated that caffeine exerted marked arrrhythmogenic effects yet did not influence electrogram duration (EGD). This excluded slowed conduction and reentrant electrical activity as their underlying cause. In contrast, the results closely correlated with observed in vitro effects of caffeine in isolated fluo-3-loaded ventricular myocytes imaged using confocal microscopy. Caffeine then increased the frequency of spontaneous Ca²⁺-release events, the background cytosolic Ca²⁺ concentration ([Ca²⁺]) in high extracellular [Ca²⁺] solution, and the amplitude of Ca²⁺ transients after paced electrical stimulation. These effects in turn were mimicked by the immunosuppressant FK506, which is also thought to increase the open probability of RyR2s (3, 14, 15, 38, 39), specifically by dissociating FKBP12.6 from RyR2 in parallel with the consequences of RyR2 hyperphosphorylation by protein kinase A in heart failure (22).

Finally, as recent mouse studies demonstrated that Ca²⁺-channel blockade suppressed arrhythmias initiated by triggered activity (1), we investigated for and demonstrated parallel and contrasting actions of nifedipine and diltiazem at the cellular level and on the whole mouse heart in the presence of caffeine. This was prompted by a recent report that of the Ca²⁺-channel blockers, diltiazem but not nifedipine inhibits the leak of Ca²⁺ through RyR2s from normal canine SR vesicles after addition of FK506 (39).

	MATERIALS AND METHODS

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Cardiac myocytes and whole hearts were obtained from inbred 129/Sv mice (Harlan; Bicester, UK). The mice were 3–6 mo of age and were kept in an animal house at room temperature, subjected to 12:12-h light-dark cycles, and fed with sterile rodent chow with constant access to water. The whole heart experiments adapted a Langendorff perfusion system for the murine heart. Each mouse was given 100 IU ip heparin to prevent thrombosis and was killed 10 min later by cervical dislocation (Schedule 1 of the UK Animals Scientific Procedures Act, 1986). The heart was then carefully but rapidly excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution that contained (in mM) 119 NaCl, 25 NaHCO₃, 4 KCl, 1.2 KH₂PO₄, 1 MgCl₂, 1.8 CaCl₂, 10 glucose, 2 sodium pyruvate, and pH 7.4 maintained by bubbling with 95% O₂-5% CO₂ (British Oxygen; Manchester, UK). Tissues surrounding the heart were removed leaving a small (3–4 mm) section of aorta, which was cannulated under the buffer surface with a 21-gauge cannula that was prefilled with ice-cold buffer solution to prevent air bubbles from entering the system. The aorta was then sutured to the cannula with fine thread and transferred to the perfusion apparatus to which the cannula was attached, and perfusion commenced in a retrograde manner via the aorta with the bicarbonate-buffered Krebs-Henseleit solution. The buffer first passed through 200- and 5-µm filters (Millipore; Watford, UK) and was warmed and maintained at 37°C via a water jacket and circulator (model C-85A; Techne; Cambridge, UK) before it entered the aorta. The pressure of the buffer entering the aorta shut the aortic valve, and hence the buffer passed into the coronary arteries and ultimately drained via the coronary sinus into the right atrium and then into the stumps of the inferior and superior vena cavae. Perfusion was maintained at a flow rate of 2–2.5 ml/min using a peristaltic pump (model 505S; Watson-Marlow Bredel; Falmouth, Cornwall, UK). After the start of perfusion, healthy, viable hearts suitable for subsequent experimentation regained a healthy pink coloration; with warming, the hearts began spontaneous rhythmic contraction.

For electrophysiological studies, we positioned paired platinum stimulating and recording electrodes of 1-mm interpole spacing on the basal epicardial surfaces of the right and left ventricles, respectively. Hearts were initially paced for 20 min at 10 Hz before the PES was started in an adaptation of corresponding clinical techniques (1, 28, 33, 34). The stimulus amplitude was set at three times the excitation threshold, and the heart was paced using 2-ms square-wave stimuli (Grass S48 stimulator; Grass-Telefactor; Slough, UK). In accordance with the stimulation protocols, we initially applied standard pacing stimuli at frequencies of 8 or 10 Hz for 20 s. After this initial short pacing period, a drive train of eight paced beats (S₁), again at 8 or 10 Hz, was followed by an extrastimulus (S₂) every ninth beat, initially at an S₁-S₂ interval equal to the pacing interval and reduced by 1 ms with each nine-beat cycle until ventricular refractoriness was reached, whereupon the S₂ stimulus elicited no electrogram. Frequencies of 8 and 10 Hz corresponding to physiological whole animal heart rates were used, and protocols using 8 Hz in addition to those using 10 Hz were carried out on each heart. The resulting bipolar electrograms were monitored by the recording electrodes, whose signals were amplified, band-pass filtered (30 Hz to 1 kHz; Gould 2400S; Gould-Nicolet Technologies; Ilford, Essex, UK) and digitized (CED 1401plus; Cambridge Electronic Design; Cambridge, UK).

Analysis of electrogram waveforms was achieved using Spike II software (Cambridge Electronic Design). Specific electrogram features were extracted and analyzed on a personal computer using the clinically accepted technique of PEFA adapted for murine hearts (1, 28, 33, 34). Ventricular conduction curves were constructed from sequences not resulting in arrhythmias; electrogram latencies following the S₂ stimulus of successive peaks and troughs on the electrogram were plotted against the interval between the S₁ and S₂ stimuli (S₁-S₂ interval) at that point. The EGD was taken as the time difference between the arrivals of the first and the last peaks or troughs on the electrogram. The same filters were used in all experiments to obtain a consistent and empirical representation of the EGD.

Single-cell experiments used mouse ventricular myocytes that were isolated using an established enzymatic digestion protocol from the Alliance for Cellular Signaling (32). Mice were killed by cervical dislocation as before. Hearts were rapidly excised and cannulated before being connected to a Langendorff perfusion system and perfused for 4 min at 3 ml/min with buffer that contained (in mM) 113 NaCl, 12 NaHCO₃, 4.7 KCl, 0.6 Na₂HPO₄, 0.032 phenol red, 10 KHCO₃, 10 HEPES, 30 taurine, 0.6 KH₂PO₄, 1.2 MgSO₄·7H₂O, 5.5 glucose, and 10 butanedione monoxime heated to 37°C (pH 7.4). The perfusion buffer was then switched to a digestion buffer that contained the perfusion buffer plus 12.5 µM CaCl₂, trypsin (final concentration, 0.14 mg/ml; Invitrogen; Paisley, Scotland, UK), and liberase blendzyme 1 (a mixture of collagenase and other proteases; final concentration, 0.25 mg/ml; Roche Diagnostics), and this was used to perfuse the heart for 8–10 min at a rate of 3 ml/min.

The heart, which should by now have become swollen, pale, and flaccid, thereby indicating good perfusion, was cut from the cannula below the atria and placed in a dish that contained 2.5 ml of digestion buffer. The tissue was then gently teased into small pieces with fine forceps and gently dissociated. The resulting cell suspension was transferred to a conical tube, and 2.5 ml of myocyte stopping buffer (perfusion buffer with 10% bovine calf serum and 12.5 µM CaCl₂) was added to inactivate the digestion buffer. After additional dissociation using sterile plastic transfer pipettes, the resulting myocytes were allowed to sediment by gravity for 10 min. The supernatant was then transferred to a new conical tube and centrifuged for 1 min at 180 g. The new supernatant was discarded, and the pellets from both the centrifuged sample and the original conical tube were resuspended in a second myocyte stopping buffer (this time containing 5% bovine calf serum) and combined to yield a total volume of 10 ml. CaCl₂ was then cautiously reintroduced to the cell suspension (two additions of 50 µl of 10 mM solution followed by 100 µl of 10 mM solution, 30 µl of 100 mM solution, and 50 µl of 100 mM solution, with 4 min between all additions) to result in a final concentration of 1 mM CaCl₂. During this process, myocytes were examined intermittently under the microscope to confirm a good yield of rod-shaped myocytes (approximately >60%) before the experiments were continued.

Cells were then transferred onto laminin-coated (Sigma-Aldrich; Poole, UK) coverslips and loaded with the acetoxymethyl (AM) ester of fluo-3 (Molecular Probes; Leiden, The Netherlands) by incubation with 5 µM fluo-3 AM in perfusion buffer (1 mM CaCl₂) for 30 min at 25°C in the dark. The coverslips were then washed with perfusion buffer without butanedione monoxime, mounted on a glass coverslip held together with vacuum grease in a 300-µl perfusion chamber, and transferred onto the stage of a Zeiss LSM-510 laser scanning confocal system with a x20 air objective (numerical aperture, 0.5; confocal aperture, 1,000 µm; slice thickness, <42.4 µm) on a Zeiss Axiovert 100M inverted microscope. Fluo-3 fluorescence emission was excited with a 488-nm argon laser and measured at 505- to 550-nm wavelengths. Images were analyzed using an in-house custom-made software program. A series of 500 frames sampled at 98 ms/frame or a series of 250 frames sampled at 98 ms/frame (each series, 128 x 128 pixels/frame) were used to monitor fluorescence changes over time. Fluorescence measurements were made within a region of interest (F) and were normalized to resting fluorescence (F₀) values. To ensure that the effects seen were genuine and not a result of undersampling, all experiments in which myocytes were paced were repeated using the line-scan mode with a sampling rate of 960 µs/line, and these results are also presented. For each of the myocytes studied, F/F₀ values were calculated throughout each time series acquired, and a mean peak F/F₀ was calculated for that series. Where indicated, cells were paced at 0.5 Hz (5 V above excitation threshold of 30–60 V for 2 ms) with two field electrodes. Different solutions were perfused through the perfusion chamber as required. All studies were carried out at room temperature.

All drugs and other chemical agents were purchased from Sigma-Aldrich except where indicated. Diltiazem was initially dissolved in distilled water to make a stock concentration of 1 mM, and nifedipine was dissolved in 96% ethanol to make a similar stock concentration. Nifedipine stock solutions were kept wrapped in foil to prevent light degradation and, with diltiazem stock solutions, were kept refrigerated at 4°C. FK506 (Calbiochem; Merck Biosciences; Nottingham, UK) was prepared in ethanol to make a stock concentration of 30 mM and was stored at –20°C. Caffeine was dissolved directly in buffer solution and kept at room temperature. Final drug concentrations were achieved by dilution with buffer solution where required. Statistical analysis was carried out using a repeated measures one-way ANOVA to compare data (SPSS software). Upon identification of a significant trend, Tukey's honestly significant difference test was used as a post hoc test to compare data. A probability of P < 0.05 was taken as statistically significant. Data are expressed as means ± SE.

	RESULTS

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Caffeine induces ventricular tachycardia in isolated perfused mouse hearts subjected to PES. The first experimental series assessed the arrhythmic effects of 1 mM caffeine on Langendorff-perfused whole murine hearts using PES protocols. The bipolar electrograms recorded from such hearts were then examined by deriving conduction curves using PEFA. Hearts in the absence of caffeine proved consistently resistant to arrhythmias when subject to PES (n = 6), but addition of 1 mM caffeine to the buffer perfusing the heart consistently induced ventricular tachycardia (VT) in all hearts studied (n = 6). Figure 1 shows typical experimental bipolar electrogram traces during PES of extracellular voltage plotted against time from a mouse heart before (Fig. 1A) and after (Fig. 1B) addition of caffeine to the perfusion buffer. The single vertical markers below each trace indicate the timings of the S₁ stimuli, and the double vertical markers indicate the S₂ stimuli. Both of the traces illustrate results at similar stages of the PES protocol. In both traces, five individual S₁ stimulus artifacts are followed by their resulting electrograms. In the lower trace, in the presence of 1 mM caffeine (Fig. 1B), the S₂ stimulus triggered a run of sustained (>30 s) VT. This was not observed in the absence of the drug, when the premature S₂ triggered a single electrogram, after which the heart continued to respond to S₁ stimuli as before (Fig. 1A). The present findings thus demonstrate that caffeine produces an arrhythmic tendency in the isolated perfused mouse heart subject to PES and complements reports of a similar arrhythmic tendency in other species (12, 24).

View larger version (30K): [in this window] [in a new window]   Fig. 1. Caffeine induced ventricular tachycardia (VT) in isolated perfused mouse hearts. Typical experimental bipolar electrogram (BEG; in volts) traces are shown from isolated perfused mouse hearts during programmed electrical stimulation (PES) pacing at 10 Hz as indicated below each trace. A: hearts before caffeine addition. B: hearts after 1 mM caffeine addition. Induction of VT is shown to follow an interposed stimulus (S2; delivered at a 45-ms coupling interval) in the presence of caffeine but not at the same point in the pacing sequence in the absence of the drug. Single vertical markers at the base of each trace indicate the timings of the drive-train (S1) stimuli; double vertical lines indicate the timings of S2 stimuli (in seconds). S1EG and S2EG, S1 and S2 electrograms, respectively.

Caffeine reduces electrically evoked cytosolic Ca²⁺ transients in single mouse myocytes. The arrhythmic events induced by caffeine in Langendorff-perfused whole hearts were then correlated with confocal microscopic observations of changes in cytosolic Ca²⁺ homeostasis. Such experiments were carried out on isolated mouse cardiac ventricular myocytes that were loaded with fluo-3 and stimulated using field electrodes at 0.5 Hz. Figure 2A plots fluorescence signals normalized to their baseline levels between stimuli (F/F₀) against time. The observed peaks in fluorescence reflect cytosolic Ca²⁺ transients that resulted from the SR Ca²⁺ release, which follow normal electrical excitation of the cell and normally lead to excitation-contraction coupling. As reported on earlier occasions, there was some variation in the amplitude of individual peaks in each time series (18, 19). Figure 2B demonstrates no significant reduction in mean peak F/F₀ at 2 min after addition of 0.1 mM caffeine to the perfusate (4.1 ± 0.12 vs. 4.6 ± 0.18 in controls; n = 13 cells). However, addition of 1 mM caffeine significantly reduced mean peak F/F₀ during field stimulation (3.5 ± 0.11 vs. 4.6 ± 0.18 in controls; P < 0.05; n = 13 cells) after 2 min and gave rise to traces showing occasional extrasystolic Ca²⁺ transients (Fig. 2C). Similarly, line-scan experiments confirmed that 0.1 mM caffeine did not affect F/F₀ (4.3 ± 0.10 vs. 4.5 ± 0.12 in controls; n = 6 cells), but that 1.0 mM caffeine significantly reduced mean peak F/F₀ (3.6 ± 0.12 vs. 4.5 ± 0.12 in controls; P < 0.05; n = 6 cells). In contrast, the transients showed statistically indistinguishable half-relaxation times (P > 0.05) before (121 ± 6 ms; Fig. 2A) and after (119.0 ± 5 ms; Fig. 2C) addition of 1 mM caffeine; similarly, there were no significant changes in the corresponding base-to-peak times (17.0 ± 1.0 and 16.4 ± 0.7 ms, respectively; n = 6 cells in each).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Caffeine reduced the amplitude of electrically evoked cytosolic Ca2+ transients. Fluorescence measurements were made within a region of interest (F) and were normalized to resting fluorescence (F0) values. A: traces of normalized fluorescence (F/F0) against time were obtained with a cell in perfusion buffer alone; peak F/F0 values occurred when the cell was stimulated. B: addition of 0.1 mM caffeine to the buffer had no significant effect on F/F0. C: a significant reduction in F/F0 occurred after addition of 1 mM caffeine to the perfusate.

View larger version (107K): [in this window] [in a new window]   Fig. 3. Patterns of Ca2+ wave initiation and propagation. A: phase-contrast image of two closely apposed myocytes. B: successive frames at 0.1-s intervals show patterns of fluo-3 fluorescence through a Ca2+ wave. Frame dimensions, 76.8 x 76.8 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Caffeine increased the frequency of spontaneous Ca2+-release events in single mouse myocytes. A: traces of normalized fluorescence (F/F0) against time are shown for a cell in perfusion buffer alone that contained a high Ca2+ concentration ([Ca2+]). Peaks correspond to spontaneous Ca2+-release events. B: addition of 0.5 mM caffeine to the high [Ca2+] buffer resulted in an increase in the frequency of spontaneous Ca2+-release events. C: increase observed in B was maintained even after 2 min. D: spontaneous Ca2+-release events were abolished by 0.25 mM tetracaine.

Effects of diltiazem and nifedipine pretreatment on caffeine and FK506 induced spontaneous Ca²⁺-release events. Caffeine and FK506 thus both enhance generation of spontaneous Ca²⁺-release events in single ventricular myocytes in parallel with the arrhythmic effect of caffeine in ventricles of whole mouse hearts. Such findings are compatible with a role for such release events in triggering of arrhythmogenesis. This possibility prompted investigations for pharmacological maneuvers that would accomplish parallel "rescues" of the effects of caffeine on both Ca²⁺ homeostasis at the myocyte level and arrhythmogenesis in intact hearts. Initial investigations contrasted the effects of diltiazem and nifedipine on caffeine-induced increases in spontaneous Ca²⁺-release events in myocytes.

The benzothiazepines JTV519 and diltiazem have been reported to either completely or partially block SR leaks of Ca²⁺ through cardiac RyRs in normal canine SR vesicles that resulted from the addition of FK506, probably by stabilizing the RyRs (39). The present experiments revealed that pretreatment with similar concentrations of diltiazem similarly reduced the effects of both caffeine and FK506 on spontaneous Ca²⁺-release events in myocytes. This was not seen at lower diltiazem concentrations (0.1 µM; n = 4). Figure 5A shows typical fluorescence (F/F₀) signals from cells studied in HEPES buffer that contained 5 mM Ca²⁺ and were pretreated with 1 µM diltiazem plotted against time. The peaks demonstrate the passage of Ca²⁺ waves across the myocyte. Inclusion of 1 µM diltiazem did not significantly alter their frequency (11.4 ± 0.71 vs. 12.4 ± 1.0 in high [Ca²⁺] controls; n = 12 cells) but nevertheless blocked the increased frequencies otherwise produced by a subsequent addition of 0.5 mM caffeine (12.2 ± 1.5 vs. 11.8 ± 1.3 in controls with diltiazem pretreatment; n = 6 cells; Fig. 5C). Such diltiazem pretreatment exerted similar effects on FK506-induced increases in spontaneous Ca²⁺-release events. Figure 5B illustrates fluorescence peaks corresponding to Ca²⁺ waves in the presence of 1 µM diltiazem and confirms the absence of changes in their frequency (10.3 ± 0.84 vs. 11.0 ± 0.68 in controls with diltiazem pretreatment; n = 6; Fig. 5D) that normally resulted after a further addition of 30 µM FK506.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Diltiazem pretreatment suppressed caffeine- and FK506-induced increases in spontaneous Ca2+-release events. A and B: traces from different cells show normalized fluorescence (F/F0) against time. Each cell was in high [Ca2+] perfusion buffer and was pretreated with 1 µM diltiazem. C: addition of 0.5 mM caffeine to the cell in A revealed no significant increase in the number of spontaneous Ca2+-release events. D: addition of 30 µM FK506 to the cell in B also showed no significant increase in the number of spontaneous Ca2+-release events.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Nifedipine pretreatment failed to suppresses caffeine- and FK506-induced increases in spontaneous Ca2+-release events. A and B: traces from different cells show normalized fluorescence (F/F0) against time. Each cell was in high [Ca2+] perfusion buffer and was pretreated with 1 µM nifedipine. C: addition of 0.5 mM caffeine to the cell in A caused a significant increase in the number of spontaneous Ca2+-release events. D: addition of 30 µM FK506 to the cell in B also caused a significant increase in the number of spontaneous Ca2+-release events.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Diltiazem pretreatment suppressed caffeine-induced VT as indicated by this series of recordings. A: isolated perfused mouse heart in the absence of pharmacological agents. B: same heart after pretreatment with 1 µM diltiazem. C: subsequent addition of 1 µM diltiazem and 1 mM caffeine to the perfusate. D: same heart after washout of the 1 µM diltiazem. Pretreatment with diltiazem appeared to suppress the caffeine-induced VT (as shown in C) with the return of caffeine-induced arrhythmias seen after diltiazem washout (as shown in D). All traces were taken from the same point in the pacing sequence. Pacing was at 10 Hz.

View larger version (32K): [in this window] [in a new window]   Fig. 8. Nifedipine pretreatment failed to suppresses caffeine-induced VT as shown by this series of recordings. A: isolated perfused mouse heart in the absence of pharmacological agents. B: same heart after pretreatment with 1 µM nifedipine. C: subsequent addition of 1 µM nifedipine and 1 mM caffeine to the perfusate. Pretreatment with nifedipine failed to suppress the induction of caffeine-induced VT shown in C. All traces were taken from the same point in the pacing sequence. Pacing was at 8 Hz.

	DISCUSSION

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We first examined patterns of excitation in whole isolated perfused hearts using PES protocols applied to murine cardiac models on previous occasions (1). This confirmed that caffeine indeed exerts proarrhythmic effects in intact mouse hearts (12). However, results from application of PEFA, hitherto used successfully in clinical studies to predict prospective arrhythmic risk (33, 34), appeared to exclude the reentrant excitation that was suggested on previous occasions as the mechanism for arrhythmogenesis in other murine systems (1, 28). Thus caffeine conserved the alterations in EGD during PES with progressive decreases in interval between pacing (S₁) and extrasystolic (S₂) stimuli, which is in sharp contrast with previous reports from genetically modified (KCNE1–/– and Scn5a+/–) mouse hearts, whose arrhythmic risk clearly correlated with significantly increased EGDs at shortening S₁-S₂ intervals (1, 28). The observed arrhythmogenesis in the latter experimental situations as well as in the clinical studies indicated above (33, 34) have been attributed to slowed conduction leading to reentrant excitation. The present findings are thus more consistent with arrhythmogenesis attributable to triggered activity as has been suggested for caffeine administration on earlier occasions in other experimental systems (12).

These findings in whole hearts prompted studies on Ca²⁺ homeostasis in isolated mouse myocytes. Our experiments first examined cytosolic [Ca²⁺] transients after regular electrical pacing. Caffeine either conserved or reduced these peak fluorescence changes consistent with partial depletion of SR Ca²⁺ stores and in parallel with previous studies that reported reduced SR Ca²⁺ in myocytes from human heart failure (16). These results were then replicated using the immunosuppressant FK506, which is thought specifically to increase open probabilities in RyR2s by dissociating FKBP12.6 from RyR2 (3, 14, 15, 38). This dissociation also occurs in heart failure after RyR2 hyperphosphorylation by protein kinase A. Calmodulin kinase II (CaMKII) similarly phosphorylates RyRs and alters RyR activity; transgenic overexpression of the cytosolic isoform CaMKII-_c increases diastolic Ca²⁺ spark frequency and fractional SR Ca²⁺ release despite reduced SR Ca²⁺ content and diastolic [Ca²⁺] and causes the murine transgenic model to develop heart failure (2, 20, 22, 40). The present experiments used concentrations specifically known both to increase the open probability of RyRs and to induce Ca²⁺ leakage from normal SR vesicles (39).

Second, we examined the effects of caffeine on spontaneous as opposed to stimulated Ca²⁺ release. The myocytes were studied under conditions of elevated extracellular [Ca²⁺] that would give rise to spontaneous periodic peaks resulting from propagating Ca²⁺ waves associated with Ca²⁺ overloading of the SR (7, 27, 35, 37). Such Ca²⁺ waves have been attributed to a summation of a large number of elementary Ca²⁺-release events (sparks) characterized on earlier occasions (5). This Ca²⁺ release has previously been suggested as a potential cause of triggered arrhythmogenic activity through activation of inward currents such as the Na⁺/Ca²⁺ exchange current (23). We found that caffeine increased the frequency of such spontaneous events, which complements previous reports in rat as opposed to mouse myocytes (35). In addition, these findings were reproduced by using FK506 and abolished by using tetracaine, which is known to inhibit Ca²⁺ fluxes through RyRs (10).

The third series of experiments explored for pharmacological maneuvers that would demonstrate parallel rescues of the effects of caffeine on both Ca²⁺ homeostasis at the myocyte level and arrhythmogenesis in intact hearts. They first examined the effects of nifedipine and diltiazem on caffeine-induced increases in spontaneous Ca²⁺-release events in myocytes. The dihydropyridine nifedipine is known to block surface L-type Ca²⁺ channels without affecting Ca²⁺ leaks from SR Ca²⁺ stores (39). It suppresses pacing-induced VT that is initiated by triggered activity attributable to inward L-type Ca²⁺ currents in whole mouse hearts (1). In contrast, the benzothiazepine diltiazem (1 µM) additionally inhibits the Ca²⁺ leak through RyRs in normal canine SR vesicles after addition of FK506, probably by stabilizing RyRs (39). In the present experiments, diltiazem correspondingly abolished the increased frequency of spontaneous Ca²⁺-release events produced by either caffeine or FK506, whereas nifedipine had no effect. In contrast, pretreatment with neither 1 µM nifedipine nor 1 µM diltiazem influenced the frequency of Ca²⁺ waves in myocytes spared caffeine treatment. Furthermore, these agents indeed exerted concordant effects on the whole caffeine-treated mouse heart. Thus diltiazem prevented caffeine-induced VT after PES at concentrations similar to those used in the earlier study by Yano et al. (39). In contrast, caffeine-induced VT after PES persisted in nifedipine-treated hearts. These findings fulfill the prediction that arrhythmias mediated by SR Ca²⁺ release are prevented by agents that alter RyR function such as diltiazem; in contrast, arrhythmias mediated by Ca²⁺ entry through the dihydropyridine receptors would have been blocked by either nifedipine or diltiazem.

Taken together, these results thus demonstrated changes in Ca²⁺ homeostasis at the cellular level that could precisely parallel corresponding observations concerning arrhythmogenesis in intact hearts. They corroborate earlier reports in genetically as opposed to pharmacologically modified murine systems that manipulations of Ca²⁺ homeostasis modify the likelihood of triggered arrhythmias (1). In the present experiments on caffeine-treated mouse hearts, such an effect was most readily attributed to alterations in the release of intracellularly stored Ca²⁺ through RyR2 Ca²⁺-release channels as opposed to extracellular Ca²⁺ entry through surface L-type Ca²⁺ channels.

	GRANTS

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This work was supported by the British Heart Foundation, the Medical Research Council, the Wellcome Trust, the Leverhulme Trust, the Helen Kirkland Fund for Cardiac Research, and the Raymond and Beverly Sackler Medical Research Centre. S. Chawla thanks the Biotechnology and Biological Sciences Research Council for a David Phillips Fellowship.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Balasubramaniam, Physiological Laboratory, Univ. of Cambridge, Downing St., Cambridge, CB2 3EG, U.K. (E-mail: rnb25{at}cam.ac.uk)

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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