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(Circulation Research. 1996;79:94-102.)
© 1996 American Heart Association, Inc.

Articles

Activation of Purinergic Receptors Triggers Oscillatory Contractions in Adult Rat Ventricular Myocytes

Bin-Xian Zhang, Xiuye Ma, Bradley K. McConnell, Derek S. Damron, Meredith Bond

the Department of Molecular Cardiology, Research Institute (B.-X.Z., X.M., B.K.M., M.B.) and the Center for Anesthesiology Research (D.S.D.), Cleveland Clinic Foundation, Cleveland, Ohio and the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine (B.K.M., M.B.), Cleveland, Ohio.

Correspondence to Meredith Bond, PhD, Department of Molecular Cardiology/FF10, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail bondm@cesmtp.ccf.org.

	Abstract

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Extracellular ATP is an important neurotransmitter that modulates cardiac function by activation of purinergic receptors. In this study, the effect of P₂ purinergic receptor activation on contractions and on [Ca²⁺]_i was investigated in adult rat ventricular myocytes. Fura 2 was used to measure [Ca²⁺]_i, and video edge detection was used to measure contraction. Superfusion of 2-methylthio-adenosine-5'-triphosphate (2-M-S-ATP) over quiescent myocytes induced oscillations in contraction and in [Ca²⁺]_i. The frequency of the oscillatory contractions increased with increasing concentrations of 2-M-S-ATP, but the amplitude of contractions varied from cell to cell and was independent of the concentration of 2-M-S-ATP. During electrical stimulation, activation of purinergic receptors in myocytes potentiated the amplitude of contraction and induced arrhythmias. In populations of quiescent myocytes, the plateau phase of the [Ca²⁺]_i signal evoked by 2-M-S-ATP could be shown to represent summed oscillations in [Ca²⁺]_i in individual cells. Pretreatment of quiescent myocytes with thapsigargin or caffeine reduced or abolished the oscillations in contractions and in [Ca²⁺]_i triggered by 2-M-S-ATP, indicating a dependence of the oscillations on uptake and release of Ca²⁺ by the sarcoplasmic reticulum. These data demonstrate the novel phenomenon that activation of purinergic receptors in quiescent myocytes stimulates oscillations in [Ca²⁺]_i and contraction. In electrically stimulated myocytes, activation of purinergic receptors triggers oscillatory contractions and potentiates the amplitude of electrically triggered contractions.

Key Words: purinergic receptor • oscillatory contraction • Ca²⁺ transient

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ATP, which is coreleased with norepinephrine from sympathetic nerve terminals, is now recognized as a physiologically important neurotransmitter.^{1 2 3} ATP has been shown to activate P₂ purinergic receptors on endothelial cells and cardiac myocytes and on sympathetic nerve terminals, resulting in an increase in [Ca²⁺]_i.² The concentration of extracellular ATP is known to vary within the heart under physiological and pathological conditions, eg, exercise,⁴ hypoxia, and ischemia^{5 6} ; thus, activation of P₂ purinergic receptors in the myocardium may be an important factor in the regulation of cardiac contraction.

Stimulation of suspensions of adult ventricular myocytes by ATP evokes transient increases in [Ca²⁺]_i.^{7 8 9} Electrophysiological evidence indicates that the increase in [Ca²⁺]_i, in response to purinergic receptor stimulation of ventricular myocytes by ATP, occurs as a result of influx of Na⁺ and Ca²⁺ via ATP-gated nonselective cation channels.^{10 11} The increases in [Ca²⁺]_i, which occur as a result of ATP activation of purinergic receptors on ventricular myocytes, has also been shown to increase the amplitude of electrically stimulated contractions.¹² In addition to ATP-dependent activation of Ca²⁺ influx across the sarcolemma, a role for SR Ca²⁺ release has also been postulated^{9 13} ; however, the precise role for the SR in ATP-dependent increases in [Ca²⁺]_i remains controversial.¹²

In the ischemic heart, arrhythmias and ventricular fibrillations frequently occur as a result of increases in cellular [Ca²⁺] within the cardiac myocytes in response to factors such as metabolic changes, reduction of the resting membrane potential, and loss of intracellular K⁺.^{14 15} In order to better evaluate the role of ATP-dependent increases in [Ca²⁺]_i in the regulation of cardiac muscle contraction under both physiological and pathological conditions, the functional effects of ATP stimulation of cardiac myocytes, as well as the subcellular pathways activated, require further investigation.^{12 15}

As described in the present study, the effects of activation of purinergic receptors on contraction and on [Ca²⁺]_i in isolated rat ventricular myocytes have been investigated. Our results demonstrate that under quiescent conditions, activation of purinergic receptors in single ventricular myocytes induces oscillatory contractions in which the frequency, but not the amplitude, is regulated by the concentration of extracellular ATP. Our results also indicate that Ca²⁺ release and uptake by the SR is necessary for the observed oscillations in [Ca²⁺]_i and in contraction. During electrical stimulation of cardiac myocytes, purinergic receptor activation both potentiates the amplitude of electrically stimulated contractions and induces arrhythmias. Therefore, our studies support the hypothesis that increases in extracellular ATP and the subsequent ATP activation of purinergic receptors in cardiac myocytes may be important factors contributing to the generation of arrhythmias or ventricular fibrillation.¹⁶

	Materials and Methods

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Preparation of Adult Rat Ventricular Myocytes
The protocol for preparation of adult rat ventricular myocytes is similar to that described previously¹⁷ and is a modification of the method of Altschuld et al.¹⁸ The heart was quickly removed from adult male Sprague-Dawley rats, perfused in a retrograde fashion with oxygenated (95% O₂/5% CO₂) KHB containing (mmol/L) NaCl 118, KCl 4.8, MgCl₂ 1.2, NaHCO₃ 16.7, KH₂PO₄ 1.2, glutamine 0.68, glucose 16.5, pyruvate 7.5, and CaCl₂ 1.0, pH 7.4. After a 5-minute equilibration period, the perfusion buffer was switched to Ca²⁺-free KHB (30 µmol/L EGTA was added instead of 1 mmol/L CaCl₂), and the heart was perfused with this buffer for another 7 minutes. Collagenase (type II, 250 U/mg) was then added to the Ca²⁺-free KHB buffer at a concentration of 170 U/mL with 0.1% BSA. After 20 minutes of digestion, CaCl₂ was readded to the medium at 5-minute intervals to achieve final [Ca²⁺] values of 0.25, 0.5, 1.00, and ultimately 1.25 mmol/L. The ventricles were then cleaned, minced, and further dissociated by shaking in KHB with 250 U/mL collagenase at 37°C under O₂ for 5 to 10 minutes. The resulting digest was filtered, and myocytes were harvested by centrifugation at 500 rpm for 2 minutes in a Dynac II centrifuge. The cells were then washed twice and resuspended in HBS, which contained (mmol/L) NaCl 118, KCl 4.8, MgCl₂ 1.2, KH₂PO₄ 1.2, glutamine 0.68, glucose 11, pyruvate 5.0, and CaCl₂ 1.2, pH 7.4. The viability of a sample of the cells from each preparation was checked under the light microscope. The myocyte preparations used for measurement of intracellular Ca²⁺ in cell suspension contained, on average, 83% rod-shaped cells.

Loading of Myocytes With Fura 2 and Measurement of [Ca²⁺]_i
Rat ventricular myocytes in HBS at 10⁵ cells per milliliter were incubated at room temperature for 20 minutes with 2 µmol/L fura 2-AM with gentle shaking at 5-minute intervals. The myocytes were then washed and resuspended with fresh HBS buffer at 10⁵ cells per milliliter and used immediately for measurement of [Ca²⁺]_i in cell suspension or in single cells. The change in fluorescence in cell suspensions was measured as described previously.¹⁷ Briefly, a custom-built fluorimeter designed for single excitation wavelength measurements (University of Pennsylvania Biomedical Instrumentation Group, Philadelphia) was used. Cell suspensions were stabilized at 37°C under constant stirring. The excitation wavelength was filtered at 340 nm (10-nm half-bandwidth), and the emitted signal was collected through a 510-nm filter (4-nm half-bandwidth). [Ca²⁺]_i values were calculated according to the following equation: [Ca²⁺]_i (nmol/L)=224 (F-F_min)/(F_max-F), where F_min and F_max are minimal and maximal fluorescence (F), respectively, and the K_d of fura 2 for Ca²⁺ is taken as 224 nmol/L.¹⁹ Maximal fluorescence was determined by adding 5 µmol/L digitonin to the cell suspension medium. Once the fluorescent signal stabilized, 5 mmol/L EGTA with 25 mmol/L Tris were added to obtain minimal fluorescence.

The [Ca²⁺]_i measurements in single myocytes were performed on a Photon Technology International Delta Scan RFK6 spectrofluorimeter connected to an Olympus inverted fluorescent microscope equipped with a UV x40 oil-immersion objective, which provided dual-wavelength excitation light. The myocytes were superfused with PB, which contained (mmol/L) NaCl 118, KCl 4.8, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25, Na₂EDTA 0.5, glucose 10, pyruvic acid 3.5, and CaCl₂ 2.2, together with 5 mg/L insulin. The excitation light of 340 or 380 nm was selected by a spinning chopper mirror and directed to the sample by a dichroic mirror. The emitted light of 510 nm was continuously monitored at a time resolution of 20 Hz. The signals obtained at 340 and 380 nm were calculated as the 340/380 ratio in the computer, and the resulting wave patterns were used as an index of the relative change in [Ca²⁺]_i.

Measurement of Cell Shortening
Measurement of myocyte cell shortening was performed as previously described.²⁰ Briefly, myocytes were allowed to attach to the bottom of a perfusion chamber, installed on an Olympus CK 12 inverted microscope, for 3 minutes in PB at 28°C. The myocytes were then superfused with PB or test solutions at the rate of 1.5 mL/min. Single myocytes with one end firmly attached on the chamber and the other end freely contracting were selected for study. Contraction was measured by the apparent changes in cell length, as monitored from the bright-field image of the contracting end of the cell, by an optical edge tracking apparatus (VED 103, Crescent Electronics) with a 17-millisecond (60-Hz) time resolution. Each cell from which contractions were recorded was exposed to only one concentration of 2-M-S-ATP. The Petri dish was then thoroughly washed in distilled H₂O and PB before a new batch of cells was added to the dish. The temperature of the perfusion chamber was stabilized at 28°C by a T Culture Dish System (Bioptechs) during superfusion.

Materials
2-M-S-ATP and Tg were purchased from Research Biochemicals International. Fura 2-AM was obtained from Molecular Probes, and collagenase was obtained from Worthington. Caffeine was purchased from Aldrich. All other chemicals were obtained from Sigma Chemical Co.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
2-M-S-ATP Induces Oscillations in Both [Ca²⁺]_i and Contractions in Quiescent Adult Rat Ventricular Myocytes
Superfusion of quiescent myocytes with control PB (no 2-M-S-ATP added) did not produce oscillations in either contraction or [Ca²⁺]_i. However, inclusion of 2-M-S-ATP in the perfusate induced oscillations in both contraction and [Ca²⁺]_i. Fig 1 demonstrates the oscillations observed in myocyte contraction (panels a and c) and in [Ca²⁺]_i (panels b and d), in response to the superfusion of 5 µmol/L 2-M-S-ATP. The measurements of contraction and changes in the fura 2 fluorescence ratio at 340/380 were obtained in separate experiments. A short latency (20 to 40 seconds) from the start of superfusion of 2-M-S-ATP–containing solution (indicated by the arrows in the figure) to the appearance of the first spike was observed in both contractions and in [Ca²⁺]_i. This latency was independent of ATP concentration and was most likely due to the time required for diffusion of 2-M-S-ATP to the myocytes. The intervals between individual oscillations in [Ca²⁺]_i and contraction, as well as the amplitudes of each of these responses, varied from cell to cell. The overall time course of the individual Ca²⁺ transients and cell shortenings triggered by 2-M-S-ATP was slower than the electrically stimulated contractions recorded from the same cell as illustrated in Fig 1 and in Fig 4, where T_1/2 values are compared. Both the rate of Ca²⁺ increase and the initial rate of shortening as well as the rates of relaxation were slower in response to ATP stimulation compared with electrical stimulation. It is also apparent that in response to electrical stimulation, the initially rapid rate of shortening slows before maximal shortening, most likely as a result of resistance from internal load or from "drag" over the bottom of the Petri dish.

View larger version (15K): [in this window] [in a new window]   Figure 1. Representative traces of oscillations in contraction (a and c) and oscillatory intracellular Ca2+ transients (b and d) in response to perfusion of 5 µmol/L 2-M-S-ATP over single rat ventricular myocytes. Panels c and d show a typical contraction and Ca2+ transient, respectively, on an expanded time scale. Myocytes were superfused with PB solution at 28°C as described in "Materials and Methods." The cells were first stimulated electrically at 0.2 Hz in order to select a single myocyte with one end contracting and the other end firmly attached to the chamber during perfusion. Electrical stimulation was then stopped, and the quiescent myocytes were continuously superfused with PB for at least another 2 minutes. Only myocytes without spontaneous contractions or [Ca2+]i spikes during this period were used. Contractions (a) and Ca2+ transients (b) in response to electrical stimulation (EL) are shown at the beginning of these traces and are expanded in panels c and d, respectively. The arrows indicate the time when superfusion was switched from PB to PB plus 5 µmol/L 2-M-S-ATP.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of Tg on T1/2 of contractions triggered by 2-M-S-ATP. Single ventricular myocytes were superfused with PB plus 0, 0.1, or 0.5 µmol/L Tg, as described in the legend to Fig 3. The values of T1/2 were measured, and the average values are shown in the figure. T1/2 for the electrically stimulated shortenings is 104±9 milliseconds (mean±SE) (n=7), which is significantly less than T1/2 for the 2-M-S-ATP–stimulated cell shortenings (P<.05).

Fig 1b also shows that individual spikes in [Ca²⁺]_i oscillations triggered by 2-M-S-ATP were usually preceded by a slow climb in [Ca²⁺]_i from baseline. However, Fig 1a demonstrates that there was no comparable gradual increase in cell shortening preceding the fast cell shortening transient.

Because myocytes that responded to 2-M-S-ATP stimulation did not generate any oscillations in contraction or [Ca²⁺]_i during superfusion with PB in the absence of 2-M-S-ATP, it can be concluded that the observed oscillations resulted from activation of purinergic receptors upon binding of 2-M-S-ATP. These results provide the first direct evidence that activation of purinergic receptors is sufficient to induce contractions in quiescent ventricular myocytes and also demonstrate that the pattern of the contractions in response to 2-M-S-ATP stimulation is oscillatory.

The Frequency of Oscillations in Myocyte Contraction Increases in a Dose-Dependent Manner With Increasing Concentrations of 2-M-S-ATP
Fig 2 demonstrates the relationship between the frequency of oscillations in myocyte contraction and the concentration of 2-M-S-ATP. The frequency was determined from the number of spikes observed, divided by the period of time of 2-M-S-ATP superfusion, in seconds. Each data point in the figure was calculated by averaging results from three to nine experiments, and the average period of data collection was 6 to 7 minutes. The frequency of the contractile oscillations induced by 2-M-S-ATP was found to increase with increasing concentrations of 2-M-S-ATP superfused over the myocytes (Fig 2), with an estimated EC₅₀ of 0.4±0.02 µmol/L. On the other hand, the amplitude of each cell shortening varied from cell to cell but was independent of the concentration of 2-M-S-ATP superfused.

View larger version (15K): [in this window] [in a new window]   Figure 2. Semilogarithmic plot showing the dependence of frequency of oscillatory contractions (determined over a 6- to 7-minute period) on the concentration of 2-M-S-ATP. Isolated myocytes were superfused with different concentrations of 2-M-S-ATP (0.1, 0.5, 1, 5, 10, and 25 µmol/L, as described in Fig 1), and the frequency of oscillations in a single myocyte was calculated, as stated in "Results." Each data point is presented as mean±SE from three to nine experiments. The EC50 was estimated to be 0.4±0.02 µmol/L 2-M-S-ATP.

Ca²⁺ Release and Uptake in the SR Is Necessary for the Oscillations in Myocyte Contraction Induced by 2-M-S-ATP
In adult rat ventricular myocytes, the main active intracellular Ca²⁺ store has been shown to be the caffeine-releasable SR pool.²¹ Ca²⁺-induced Ca²⁺ release from the SR has also been shown to be the principal mechanism for triggering electrically stimulated contractions in both isolated cardiac myocytes²² and in cardiac muscle preparations.²³ Whether or not the oscillatory contractions, in response to 2-M-S-ATP, are triggered by Ca²⁺-induced Ca²⁺ release, as occurs with electrically stimulated contractions, needs to be determined. Therefore, we studied the involvement of the SR Ca²⁺ store in 2-M-S-ATP–induced contractions by modulating the SR Ca²⁺ transport pathways with Tg, which is known to inhibit Ca²⁺ uptake into the SR,²⁴ and caffeine, which activates Ca²⁺ release from the SR.²⁵

Fig 3 shows the effects of Tg and caffeine on the oscillatory contractions induced by 2-M-S-ATP. Before the addition of 2-M-S-ATP, superfusion of the myocytes with low concentrations of Tg (0.1 or 0.5 µmol/L) for 1 minute significantly reduced the frequency of oscillations in cell shortening (Fig 3, top [traces a, b, and c] and bottom). Superfusion with high concentrations of Tg (8 µmol/L), which depletes the SR Ca²⁺ store (as shown in Fig 7), completely blocked the 2-M-S-ATP–induced oscillatory contractions (Fig 3, top [trace d] and bottom).

View larger version (22K): [in this window] [in a new window]   Figure 3. Effect of prior superfusion with Tg or caffeine (Caf) on the frequency of oscillatory contractions induced by 2-M-S-ATP. Top, After 1 minute of electrical stimulation, the quiescent myocytes were continuously superfused with PB. The solution was then changed to PB plus various concentrations of Tg or 4 mmol/L Caf for 1 minute. The myocytes were then superfused for 6 to 7 minutes with PB containing 5 µmol/L 2-M-S-ATP (indicated by the vertical arrows under traces a through d). In trace e, the first arrow indicates the addition of Caf; the second, addition of 2-M-S-ATP. Bottom, Summary of the effect of Caf and different concentrations of Tg on the frequency of oscillatory contractions. The results are represented as mean±SE of four to nine experiments. In the presence of 8 µmol/L Tg or 4 mmol/L Caf, cell shortening in response to 2-M-S-ATP stimulation was completely inhibited; thus, the frequency is 0.

View larger version (26K): [in this window] [in a new window]   Figure 7. The effect of Tg and caffeine (Caf) pretreatment on the SR Ca2+ store in myocyte suspensions. Top, Ventricular myocytes were fully depolarized with 25 mM KCl. Results are expressed as percent of the maximal change in [Ca2+]i of the control KCl responses (±SE of three experiments). Bottom, The myocyte suspensions were pretreated with Tg or Caf, similar to the procedure described for the experiments shown in Fig 5. EGTA (5 mmol/L) was then added to the medium, followed by rapid application of 2 µmol/L ionomycin (indicated by the arrows under the traces). The results are representative of three experiments.

Exposure of myocytes to 4 mmol/L caffeine also depletes the SR Ca²⁺ store (Fig 7), but by a different mechanism than Tg.²⁵ At the concentration of caffeine used in these studies (4 mmol/L), caffeine-induced oscillatory contractions were consistently observed (Fig 3, top panel [trace e]). In Fig 3, trace e in the top panel and the bottom panel demonstrate that, similar to the effect of 8 µmol/L Tg, superfusion of myocytes with 4 mmol/L caffeine for 1 minute completely inhibited 2-M-S-ATP–dependent oscillatory contractions. These results demonstrate that Ca²⁺ uptake and release by the SR store are necessary for purinergic receptor–activated oscillations in myocyte contraction.

Tg Treatment Prolongs the Relaxation Time Course of Myocyte Shortening Triggered by 2-M-S-ATP
In Fig 4, we compared T_1/2 values of contractions elicited in response to electrical stimulation with T_1/2 values of the spontaneous contractions observed in response to the superfusion of 2-M-S-ATP. The effects of Tg on T_1/2 were also investigated. T_1/2 for electrically stimulated contractions (0.2 Hz) was 104±9 milliseconds (mean±SEM) (n=7). This value did not differ at lower frequencies of electrical stimulation; eg, when cells were stimulated at 0.08 Hz (similar to the maximal frequency of 2-M-S-ATP spontaneous contractions), T_1/2 was 97±3.4 (SEM) (n=5). In contrast, T_1/2 for the 2-M-S-ATP–induced contractions was significantly greater, 177±15 milliseconds (mean±SEM) (n=12) (Fig 4). This suggests that purinergic receptor activation results in a slower rate of Ca²⁺ removal from the cytoplasm than contractions that occur in response to electrical stimulation. In electrically stimulated contractions, Tg has been shown to prolong the duration of the contraction.^{24 26} Brief superfusion of the myocytes with 0.1 or 0.5 µmol/L Tg for 1 minute before 2-M-S-ATP addition increased T_1/2 of 2-M-S-ATP–induced contractions from 177±15 to 233±11 (n=8) (P<.05) and 242±27 (n=3) milliseconds, respectively (Fig 4). These data indicate that the SR Ca²⁺ pump is also one of the major pathways for reduction of [Ca²⁺]_i, and thus relaxation, in contractions stimulated by purinergic receptor activation.

Modification of the SR Ca²⁺ Store by Tg or by Caffeine Inhibits the [Ca²⁺]_i Oscillations Stimulated by 2-M-S-ATP in Quiescent Myocytes
A convenient method to study [Ca²⁺]_i oscillations in cell suspension was recently introduced.²⁷ In that study, it was demonstrated that the plateau phase of the [Ca²⁺]_i signal induced by agonist stimulation of suspensions of pancreatic acinar cells represents the summed response of [Ca²⁺]_i oscillations from individual cells. We have applied a similar approach to investigate, in suspensions of myocytes, the contribution of SR Ca²⁺ release and uptake in the [Ca²⁺]_i oscillations that we observed in single cells (Fig 1b). To confirm that the plateau phase of the [Ca²⁺]_i signal obtained from cell suspensions represents the sum of [Ca²⁺]_i oscillations from individual myocytes, we combined the individual [Ca²⁺]_i oscillation signals recorded from single myocytes and compared them with the [Ca²⁺]_i signals obtained from myocyte suspensions. The [Ca²⁺]_i signal summed by computer from 28 single myocytes is shown in Fig 5a. The resulting integrated signal shows an initial rapid increase in [Ca²⁺]_i, followed by a sustained plateau phase. This is similar to the [Ca²⁺]_i signals elicited by 2-M-S-ATP in myocyte cell suspensions (Fig 5b). Therefore, the [Ca²⁺]_i oscillations that we observed in individual myocytes are also represented by an elevated [Ca²⁺]_i plateau in the fura 2 signal from cell suspensions. From this comparison between single cells and cell populations, we can also conclude that the [Ca²⁺]_i signals previously reported from myocyte suspensions in response to ATP^{7 8 13} represent the average of nonsynchronized oscillations, as opposed to a uniform synchronized increase and decrease in [Ca²⁺]_i in all cells within the population. On the basis of this information, the contribution of SR Ca²⁺ cycling to the [Ca²⁺]_i oscillations observed in single myocytes (Fig 1b) can be examined in myocyte populations.

View larger version (9K): [in this window] [in a new window]   Figure 5. Representative traces of the effects of Tg on 2-M-S-ATP–induced oscillations in [Ca2+]i in single cells (a) and suspensions of myocytes (b through f). The cells were loaded with fura 2-AM as described in "Materials and Methods." The final myocyte concentration used was 1 to 2x104 cells per milliliter. Trace a indicates summed responses of records from 28 cells of Ca2+ transients in response to addition of 5 µmol/L 2-M-S-ATP (at arrow), typified by the Ca2+ transients shown in Fig 1b. Traces b through f indicate Ca2+ transients of a population of fura 2–loaded quiescent myocytes in response to the addition of 25 µmol/L 2-M-S-ATP. Trace b indicates control (dimethyl sulfoxide, solvent for Tg); traces c through f, 1 minute after the addition of 0.5 µmol/L Tg (c), 1 µmol/L Tg (d), 4 µmol/L Tg (e), and 8 µmol/L Tg (f) in dimethyl sulfoxide with constant stirring. The results are representative of three similar experiments. The vertical bars show the changes of the 340/380 fluorescence ratio (a) and [Ca2+]i (b through f).

By studying the effects of Tg and caffeine on the plateau phase of the [Ca²⁺]_i signal stimulated by 2-M-S-ATP in myocyte suspensions, we concluded that Ca²⁺ cycling by the SR Ca²⁺ store was necessary for the [Ca²⁺]_i oscillations observed in single ventricular myocytes in response to purinergic receptor stimulation. Pretreatment of myocyte suspensions with low concentrations of Tg (0.25 to 2 µmol/L) for 1 minute potentiated the amplitude of the initial rapid rise in [Ca²⁺]_i. The maximum potentiation (59±13%, mean±SE, n=3) was observed at 0.5 µmol/L Tg (Fig 5c). This enhancement of the initial Ca²⁺ transient may occur as a result of Tg-dependent inhibition of the SR Ca²⁺ pump, thus slowing Ca²⁺ removal from the cytoplasm during transsarcolemmal Ca²⁺ entry and SR Ca²⁺ release. However, the same treatment also significantly depressed the sustained plateau phase of the Ca²⁺ transient (Fig 5c, 5d, and 5e), presumably reflecting a decreased frequency of individual oscillations in [Ca²⁺]_i. Preincubation of myocyte suspensions with 8 µmol/L Tg for 1 minute abolished the plateau phase (Fig 5f), implying complete inhibition of [Ca²⁺]_i oscillations in individual myocytes. These data therefore indicate that uptake of cytosolic Ca²⁺ by the SR Ca²⁺ pump during 2-M-S-ATP stimulation is necessary for the oscillations in [Ca²⁺]_i to occur.

Fig 6 shows the effects on the [Ca²⁺]_i signals of preincubation of myocyte suspensions with 4 mmol/L caffeine for 1 minute, before the addition of 2-M-S-ATP. Caffeine completely inhibited the elevated plateau phase of the [Ca²⁺]_i signal in response to the addition of 2-M-S-ATP (Fig 6b) compared with the control condition (Fig 6a). These results demonstrate that the [Ca²⁺]_i oscillations in single myocytes (represented as the plateau of the [Ca²⁺]_i signal in cell suspensions) are also dependent on Ca²⁺ release from the SR store during 2-M-S-ATP stimulation.

View larger version (8K): [in this window] [in a new window]   Figure 6. The effect of caffeine pretreatment on oscillations in [Ca2+]i induced by 2-M-S-ATP in myocyte suspensions. Ventricular myocytes were pretreated with 4 mmol/L caffeine by a procedure similar to that described in Fig 5. Addition of 25 µmol/L 2-M-S-ATP is indicated by the arrows. The traces are representative of three experiments. The vertical and horizontal bars show changes in [Ca2+]i and time, respectively.

When the effects of both Tg and caffeine on the 2-M-S-ATP–triggered [Ca²⁺]_i signal in population of myocytes are considered, these results indicate that Ca²⁺ release and uptake by the SR is necessary for the observed oscillations.

Effect of Caffeine and Tg Pretreatment on SR Ca²⁺ Content
Because the effects of Tg and caffeine on SR Ca²⁺ are both dose and time dependent, it was important to document the ability of Tg and of caffeine to deplete SR Ca²⁺ content under the conditions of our experiments. These experiments were performed in suspensions of myocytes. In response to maximum depolarization of the cells by KCl (>20 mmol/L), the resultant increase in [Ca²⁺]_i is known to be a combination of Ca²⁺ entry through voltage-sensitive Ca²⁺ channels and Ca²⁺ release from the SR. Therefore, without interfering with Ca²⁺ transport pathways across the sarcolemma, any reduction in the [Ca²⁺]_i signal in response to KCl should reflect decreased Ca²⁺ release from the SR. Fig 7, top, demonstrates that pretreatment of ventricular myocytes with 4 mmol/L caffeine or 8 µmol/L Tg reduced the KCl-triggered [Ca²⁺]_i signal to 48±4.5% and 55±2.3% (mean±SE) of the control value, respectively, indicating that caffeine and Tg pretreatment decrease Ca²⁺ release from the SR in response to depolarization of the cell by KCl.

The Ca²⁺ content of the SR store after Tg and caffeine pretreatment was also directly measured by the addition of 2 µmol/L ionomycin to the myocyte suspension in the presence of 5 mmol/L EGTA (to prevent Ca²⁺ influx), as described previously.^{17 20} Fig 7, bottom, demonstrates that preincubation of the cells with 1 µmol/L Tg for 1 minute reduced but did not fully deplete SR Ca²⁺ content, as indicated by a smaller increase in fluorescence at 340 nm after the addition of ionomycin (Fig 7, bottom, trace b). However, pretreatment of the cell with 8 µmol/L Tg or 4 mmol/L caffeine completely depleted the SR Ca²⁺ store, as indicated by the absence of an increase in fura 2 fluorescence after the addition of ionomycin (Fig 7, bottom, traces c and d, respectively).

Purinergic Receptor Activation Potentiates Electrically Stimulated Contractions and Induces Spontaneous Contractions
Isolated myocytes were activated by repetitive electrical stimulation in order to mimic the cycles of contraction and relaxation that occur in the intact heart. The effect of 2-M-S-ATP on cell shortening under these conditions was then determined. Two different responses to 2-M-S-ATP stimulation were observed to occur with similar probability. A typical response, observed in approximately half of the experiments, is shown in Fig 8. This figure demonstrates the generation of arrhythmias during 2-M-S-ATP superfusion with little or no potentiation of the electrically stimulated contractions. In other experiments, inclusion of 5 µmol/L 2-M-S-ATP in the perfusate both potentiated the amplitude of electrically stimulated contractions and induced arrhythmias. Four of 10 cells demonstrated arrhythmic contractions during electrical stimulation at 0.2 Hz, whereas 8 of the 10 demonstrated potentiation. Average potentiation was 60±9% (SEM) (n=10) with 5 µmol/L 2-M-S-ATP. Arrhythmic contractions were also observed when the pacing frequency was increased from 0.2 to 0.3 Hz. Because of the increased probability of hypercontraction and Ca²⁺ overload in isolated myocytes at high pacing rates, we did not attempt to extend these measurements to physiological stimulation frequencies.

View larger version (26K): [in this window] [in a new window]   Figure 8. Effect of perfusion of 2-M-S-ATP on the shortening of ventricular myocytes during electrical stimulation. The figure represents a continuous trace showing contractions of an isolated myocyte that is electrically stimulated at 0.2 Hz throughout the experiment; the time during which 5 µmol/L 2-M-S-ATP was included in the perfusate is indicated by the horizontal line under the second half of the trace (lower portion of the figure). The vertical and horizontal bars show the scales of myocyte length and time, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we provide the first direct evidence that the change in [Ca²⁺]_i induced by activation of purinergic receptors in adult rat ventricular myocytes is sufficient to trigger oscillatory contractions under quiescent conditions (Fig 1). The responses shown in Fig 1 are unique, because purinergic receptors are the only known receptors that, upon activation, trigger transient contractions in quiescent myocytes. When single myocytes were continuously stimulated electrically, activation of purinergic receptors was found to both potentiate the amplitude of electrically stimulated contractions and induce arrhythmias (Fig 8). It has been proposed that activation of purinergic receptors in ventricular myocytes may be one of the factors that induces cardiac arrhythmias.¹⁶ Our results with both quiescent myocytes (Fig 1) and electrically stimulated cells (Fig 8) provide experimental support for this hypothesis.

It has been demonstrated that SR Ca²⁺ overload leads to spontaneous contractions and decreases in diastolic myocyte cell length.²⁸ Various treatments, such as an increase in [Ca²⁺]_o, incubation with ouabain,²⁸ or a decrease in extracellular Na⁺,²⁹ induce Ca²⁺ overload in cardiac myocytes. However, there is no evidence that the responses illustrated in Fig 1 are due to Ca²⁺ overload. Specifically, during 2-M-S-ATP stimulation, baseline [Ca²⁺]_i (between contractions) remained stable, without elevation, as during superfusions with control buffer (Fig 1). There was also no change in the diastolic length of myocytes during 2-M-S-ATP stimulation (Figs 1a, 8, and 3, top, trace a). In contrast, Ca²⁺ overload can lead to significant decreases in diastolic cell length.^{28 29}

One unanticipated result of these studies was that only the frequency, and not the amplitude, of the oscillatory contractions triggered in quiescent myocytes was regulated by 2-M-S-ATP concentration (Fig 3). The amplitude of the contractions induced by 2-M-S-ATP was similar to that of the electrically stimulated contraction recorded from the same cell (Fig 1a) and was independent of the concentration of 2-M-S-ATP. The amplitude of contractions triggered both by purinergic receptor activation and by electrical stimulation also varied from cell to cell. This diversity in contractile amplitude may relate to different locations of individual myocytes in the intact heart before dissociation. It has been reported that the duration and amplitude of action potentials differs at different locations in the ventricular wall.^{30 31} This implies that the amplitudes of contractions of individual ventricular myocytes may also differ from each other in vivo according to the location in the heart. Another possible reason for heterogeneity in the amplitude of contraction in our experiments is that we measured the absolute change in myocyte length rather than relative shortening; thus, differences in amplitude may be due, in part, to differences in the resting cell length of the myocytes from which contractions were recorded.

It is important to emphasize that the contractions and changes in [Ca²⁺]_i stimulated by 2-M-S-ATP are distinct from those triggered by electrical stimulation and also differ from spontaneous contractions. The response to 2-M-S-ATP is a series of oscillations in both [Ca²⁺]_i and cell shortening, whereas the response to an electrical stimulus is a single event. No similar phenomena have been observed or reported in response to electrical stimulation or the addition of other agonists. In addition, the initial gradual climb in [Ca²⁺]_i before the Ca²⁺ transient induced by 2-M-S-ATP (Fig 1b) is a novel observation and a unique response to purinergic stimulation. We did not observe a similar gradual increase in [Ca²⁺]_i in response to electrical stimulation (data not shown). Also, when comparing the contractions and intracellular Ca²⁺ transients obtained in response to 2-M-S-ATP stimulation of quiescent myocytes, we did not observe any gradual increase in contraction preceding individual cell shortenings (Figs 1a and 3), suggesting that an ATP-activated contraction occurs only when [Ca²⁺]_i increases to a certain threshold. The observed gradual increase in [Ca²⁺]_i preceding the Ca²⁺ transient (Fig 1b) may reflect a slow accumulation of [Ca²⁺]_i, as a result of either Ca²⁺ entry through the sarcolemma or localized Ca²⁺ release from the SR, in response to purinergic receptor activation.

In the preparations of isolated myocytes used for these studies, >80% of the cells were quiescent and responsive to both electrical and purinergic stimulations, whereas <10% of the myocytes were spontaneously contracting. This latter population of cells did not respond to electrical stimulation and were excluded from the study. Although the data from myocyte suspensions (Figs 5 through 7) presumably include a small contribution from spontaneously beating cells, as well as from quiescent myocytes, the similarity of the summed response to 2-M-S-ATP in single cells (Fig 5a) and in populations (Fig 5b) indicates that the contribution from spontaneously beating cells is not significant.

In response to purinergic receptor stimulation, the rates of increase in Ca²⁺ and the initial rate of shortening as well as their rates of relaxation are significantly slower than electrically stimulated contractions (Fig 1c and 1d), indicating that purinergic receptor stimulation not only depolarizes the sarcolemmal membrane and causes Ca²⁺ influx^{10 11} but may also affect other Ca²⁺ transport pathways and possibly the phosphorylation of myofibrillar proteins. It has been reported that purinergic receptor activation of cardiac myocytes stimulates phospholipase C signaling pathways,³² thus leading to activation of protein kinase C. The activation of protein kinase C by -adrenergic stimulation and by endothelin, which is mediated by the phospholipase C pathway, increases the phosphorylation of several proteins in isolated cardiac myocytes and in intact myocardium.^{20 33} Thus, the slower Ca²⁺ and contraction transients observed in response to 2-M-S-ATP may be related to activation of protein kinase C–dependent pathways.

The sources of Ca²⁺ for the ATP-dependent increases in cytosolic Ca²⁺ have been suggested to be Ca²⁺ influx across the sarcolemma,¹² the result of myocyte acidification,³⁴ or contributions both from SR Ca²⁺ release and Ca²⁺ influx.¹³ In the present study, our results clearly established that SR Ca²⁺ release and uptake are necessary for 2-M-S-ATP–activated oscillations in contractions in ventricular myocytes. Tg has been shown to specifically inhibit Ca²⁺ uptake by internal stores.^{24 35 36 37 38} A rapid Ca²⁺ release has also been observed after the addition of Tg to streptolysin O–permeabilized cardiac myocytes (B.-X. Zhang, unpublished data, 1994). In the present study, application of either Tg or caffeine inhibited 2-M-S-ATP–induced oscillations in contraction in single myocytes (Fig 3) and also inhibited [Ca²⁺]_i oscillations in myocyte suspensions (Figs 5 and 6). When SR Ca²⁺ was depleted either by a high concentration of Tg or by caffeine (Fig 7), the 2-M-S-ATP–induced oscillations in contraction and in [Ca²⁺]_i were abolished (Figs 3, 5, and 6). Thus, we can conclude that Ca²⁺ uptake and release by the SR Ca²⁺ store are necessary for the oscillations in [Ca²⁺]_i and in contraction triggered by 2-M-S-ATP in cardiac myocytes.

The observed increase in T_1/2 of the contractions triggered by purinergic receptor stimulation after incubation with Tg (Fig 4) is similar to the reported effect of Tg on the relaxation rate of electrically stimulated contractions.^{24 26} However, whereas Tg reduces the amplitude of electrically stimulated contractions,³⁵ the effect of preincubation with Tg on the ATP-dependent contractions was a reduction or abolition in frequency, with no evidence of a change in amplitude (Fig 3). Our results suggest that after purinergic receptor stimulation, a contraction occurs only when a threshold concentration of Ca²⁺ is reached in the cytoplasm (Fig 1b). Thus, one possible explanation for the observed reduction in frequency, but not amplitude, of the ATP-triggered oscillatory contractions by submaximal concentrations of Tg may be a decreased rate of SR Ca²⁺ release. Ca²⁺ sparks have recently been shown to be elementary stochastic events representing localized SR Ca²⁺ release. Ca²⁺ sparks have been observed in quiescent³⁹ as well as in stimulated^{40 41} myocytes. A whole-cell Ca²⁺ transient is then explained by recruitment of individual Ca²⁺ sparks.⁴⁰ Thus, the oscillatory contractions that we observed in cells in response to 2-M-S-ATP may result from an increased frequency of Ca²⁺ sparks compared with nonstimulated cells. Although Ca²⁺ spark frequency may vary (eg, in response to membrane depolarization and opening of L-type Ca²⁺ channels), the amplitude and duration of Ca²⁺ sparks in a myocyte do not change significantly.^{40 41} Thus, our observation that the frequency of oscillatory contractions in response to purinergic receptor stimulation decreases in the presence of Tg may be explained by a decreased frequency of the underlying elementary events or Ca²⁺ sparks.

Our observation that only the frequency of the oscillations in contraction in response to purinergic receptor stimulation changes with 2-M-S-ATP concentration (Fig 3) resembles previous observations of inositol tris-phosphate–mediated [Ca²⁺]_i oscillations evoked by agonists in nonexcitable cells.⁴² Previous studies in nonexcitable cells have demonstrated that feedback inhibition of inositol tris-phosphate–mediated Ca²⁺ release by cytosolic Ca²⁺ is the underlying mechanism for agonist-evoked [Ca²⁺]_i oscillations.^{27 43 44 45} Although the cardiac ryanodine receptor may be regulated by endogenous factors,⁴⁶ phosphorylation,⁴⁷ and adaptation in vitro,⁴⁸ there is no evidence that Ca²⁺-induced Ca²⁺ release from cardiac SR is inhibited by high cytosolic Ca²⁺.^{49 50} Therefore, the Ca²⁺ changes that result in the oscillations in contraction in myocytes in response to purinergic receptor activation may occur through a mechanism different from the Ca²⁺ oscillations in nonexcitable cells.

	Selected Abbreviations and Acronyms

 

2-M-S-ATP = 2-methylthio-adenosine-5'-triphosphate HBS = HEPES-buffered saline KHB = Krebs-Henseleit buffer PB = perfusion buffer SR = sarcoplasmic reticulum T1/2 = time to half-relaxation Tg = thapsigargin

	Acknowledgments

 
This study was supported by National Institutes of Health grants R01 HL-41883 (Dr Bond), P50 HL-33713 (Dr Bond), and F32 HL-08726 (Dr Damron), a training grant in cardiovascular research (T32 HL-07653 to Dr Zhang), and hypertension training grant (T32 HL-07714 to Mr McConnell). Dr Bond is an Established Investigator of the American Heart Association. The authors would like to thank LeeAnn Murphy, Michelle Lamorgese, and Dr David Van Wagoner for providing us with some of the ventricular myocytes used in this study. We would also like to thank Drs George Dubyak, David Friel, and Toni Scarpa for their critical review of the manuscript.

Received July 24, 1995; accepted March 27, 1996.

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