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(Circulation Research. 1996;79:147-151.)
© 1996 American Heart Association, Inc.

Articles

Cyclic ADP-Ribose Does Not Regulate Sarcoplasmic Reticulum Ca²⁺ Release in Intact Cardiac Myocytes

Xiaoqing Guo, Michael A. Laflamme, Peter L. Becker

the Department of Physiology, Emory University School of Medicine, Atlanta, Ga.

Correspondence to Dr Peter L. Becker, Department of Physiology, Emory University School of Medicine, 1648 Pierce Dr, Atlanta, GA 30322. E-mail plb@physio.emory.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cyclic ADP-ribose (cADPR), an intracellular second messenger known to mobilize Ca²⁺ in sea urchin eggs, has been implicated in modulating Ca²⁺ release in a variety of mammalian tissues. On the basis of studies of isolated cardiac sarcoplasmic reticulum (SR) vesicles and single SR Ca²⁺ release channels, cADPR has also been proposed to be a modulator of SR Ca²⁺ release in heart. In the present study, we directly examined the ability of cADPR to trigger SR Ca²⁺ release and to modulate Ca²⁺-induced Ca²⁺ release (CICR) in intact rat ventricular myocytes. Voltage-clamped myocytes were dialyzed with up to 100 µmol/L caged cADPR and 0.6 µmol/L calmodulin along with the Ca²⁺-sensitive dye fluo 3. A step increase in the cADPR concentration induced by flash photolysis of caged cADPR neither directly triggered SR Ca²⁺ release nor modulated CICR in intact myocytes. In contrast, under similar conditions, extracellular application of caffeine (1 to 2.5 mmol/L) onto myocytes produced both effects. Under equivalent conditions, flash photolysis of caged cADPR-loaded sea urchin eggs resulted in large Ca²⁺ transients. Further, the sustained presence of high cytosolic concentrations of either cADPR or its antagonist, 8-amino-cADPR, was ineffective in altering normal CICR in myocytes. These findings indicate that cADPR does not regulate SR Ca²⁺ release in intact cardiac myocytes.

Key Words: ventricular myocyte • cyclic ADP-ribose • Ca²⁺-induced Ca²⁺ release

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Modulation of SR Ca²⁺ release is the primary mechanism by which the inotropic state of the heart is regulated. The second messenger cADPR has been shown to trigger Ca²⁺ release from a ryanodine-sensitive Ca²⁺ store in sea urchin eggs,^{1 2} and recent reports have suggested that cADPR may also regulate Ca²⁺ release in a number of vertebrate cells, including brain,³ pituitary,⁴ pancreatic islets,⁵ kidney,⁶ dorsal root ganglion,⁷ sympathetic neuron,⁸ and heart.⁹ In the latter report, Meszaros et al⁹ found that cADPR could mobilize Ca²⁺ from an isolated cardiac SR preparation. cADPR also modulated single RyRC activity, suggesting a possible mechanism for its Ca²⁺-releasing activity. However, others^{10 11} have failed to observe this direct RyRC modulation.

Both cADPR and the enzymes for its synthesis and degradation appear to be ubiquitously distributed in a variety of tissues, including heart,^{12 13 14} supporting the hypothesis that cADPR may be an endogenous regulator of Ca²⁺ signaling in general. Nonetheless, the mechanism by which cADPR might mobilize Ca²⁺ is unclear, even in the better-characterized sea urchin egg system. The identity of cADPR-binding proteins has not been established,¹⁵ and cofactors (putative and identified) are probably necessary for its action.^{16 17} Given the uncertainty of whether conditions for cADPR action are preserved in cell-free systems, we decided to directly examine the ability of cADPR to regulate cardiac SR Ca²⁺ release in a more physiological intact ventricular myocyte preparation. Voltage-clamped myocytes were loaded with caged cADPR¹⁸ and a Ca²⁺-sensitive dye, and the effects of flash photolysis-induced change in cADPR concentration on SR Ca²⁺ release were then studied.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Single ventricular myocytes were enzymatically isolated from 150- to 250-g Sprague-Dawley rats described previously.¹⁹ Briefly, rats were heparinized (2.5 U/g IP) and then anesthetized with sodium pentobarbital (50 mg/kg IP). The heart was rapidly removed and perfused on a Langendorff apparatus with oxygenated nominally Ca²⁺-free Tyrode's solution at a rate of 6 to 8 mL/min for 5 minutes at 37°C. Then the heart was perfused for 5 to 6 minutes with the same solution containing 0.5 mg/mL collagenase D (Boehringer Mannheim) and 0.1 mg/mL pronase E (Sigma Chemical Co). The perfusion was then stopped, and tissues were sliced into small pieces and subjected to gentle agitation in warm 0.2 mmol/L Ca²⁺ Tyrode's solution. Cells were harvested every 5 minutes for a half hour and stored in 0.2 mmol/L Ca²⁺ Tyrode's solution until use. All animal procedures were conducted in accordance with institutional guidelines.

Freshly isolated myocytes were bathed in a solution containing (mmol/L) NaCl 115, CsCl 20, CaCl₂ 1.0, MgCl₂ 1.0, HEPES 10, and glucose 10 (pH 7.4 with NaOH). Myocytes were patch-clamped at room temperature (22°C to 24°C) under the ruptured-patch whole-cell configuration using 2- to 4-M Corning 7052 electrodes filled with an intracellular pipette solution containing (mmol/L) cesium aspartate 120, CsCl 25, HEPES 5, tetraethylammonium chloride 5, NaCl 10, and Mg-ATP 3 (pH 7.2 with CsOH). Inward I_Cas were directly measured under voltage-clamp conditions with an AxoPatch-1D amplifier (Axon Instruments). Fast inward Na⁺ current was eliminated by holding resting membrane potential at -40 mV. Rapid brief pulses of caffeine were gently pressure-ejected onto the surface of a myocyte from a micropipette filled with 1 or 2.5 mmol/L caffeine. Conditioning depolarization pulses to 0 mV (0.5 Hz, 300-millisecond duration) were used to load the SR to a steady state before all tests of Ca²⁺ release.

The high time-resolution microfluorimeter consisted of a Zeiss IM-35 inverted microscope, a quartz objective (x100; numerical aperture, 1.20), a photomultiplier tube, and a three-segment filter wheel that allows sequential excitation at 340, 380, and 470 nm (133 Hz). [Ca²⁺]_i was monitored with 100 µmol/L fluo 3 (when using caged compounds) or fura 2. FI measurements made at 510 to 550 nm, when excited at 470 nm (fluo 3) or at 340 and 380 nm (fura 2), were corrected for background, and autofluorescence was measured before rupturing the patch. A xenon flashlamp (Hi-tech) that produces a burst of light 1 millisecond in duration was merged with the excitation light path of the microfluorimeter with a beam splitter. The flashlamp light was filtered through a UG5 filter. Control experiments have shown that a UV flash alone without caged compounds does not induce any changes in either fluorescence or whole-cell currents.

The egg microinjection procedure was modified from that described by Lee et al.² Dejellied Lytechinus pictus eggs were placed in artificial sea water (mmol/L: NaCl 460, MgCl₂ 27, MgSO₄ 28, CaCl₂ 10, KCl 10, and NaHCO₃ 2.5 [pH 8.0 with NaOH]) at room temperature. Fluo 3 (5 mmol/L) and caged cADPR (500 µmol/L) were dissolved in a microinjection buffer (0.5 mol/L KCl, 50 µmol/L EGTA, and 10 mmol/L MOPS [pH 6.7 with KOH]) and pressure-injected (40 to 60 psi) into eggs. The amount of caged cADPR loading was assessed by estimating fluo 3 loading by the method of Moore et al.²⁰ Briefly, the total cellular fluo 3 FI was recorded and compared with similar measurements derived from bubbles of a Ca²⁺-containing fluo 3 solution of known volume created by injection into hydrated oil. On the basis of these calculations, we estimate the maximum injected volume to have been no more than 3% to 5% of the egg volume; thus, the final egg caged cADPR concentrations were 15 to 25 µmol/L.

Sea urchins (Lytechinus pictus) were obtained from Marinus, Inc. Caged cADPR, nitrophenyl-EGTA, 8-amino-cADPR, fura 2, and fluo 3 were from Molecular Probes. Ryanodine was from Calbiochem-Novabiochem Corp. All other reagents were acquired from Sigma. All values reported in the text are mean±SEM. An unpaired Student's t test was used to assess group differences. A value of P<.01 was accepted as significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We first examined whether a step increase in the cADPR concentration could directly trigger SR Ca²⁺ release. Myocytes were equilibrated with up to 100 µmol/L caged cADPR and 0.6 µmol/L calmodulin.^{16 17} Fig 1A shows recordings from a representative experiment. After a train of conditioning stimuli to ensure SR loading, the membrane potential was held at -40 mV, and a single UV flash was triggered 3 seconds later. No change in the fluo 3 FI was observed. In 13 myocytes, the UV flash-induced FI change was 0.0±0.7%, compared with the 302±9% rise in response to depolarization to 0 mV. Identical results (data not shown) were obtained from myocytes loaded with either 100 µmol/L caged cADPR alone (without calmodulin) or with lower concentrations of caged cADPR (0.1 to 10.0 µmol/L). In contrast, extracellular application of caffeine from a micropipette located near the cell did trigger SR Ca²⁺ release (Fig 1B), as expected by this known modulator of the RyRC.²¹ Furthermore, in myocytes loaded with 1 mmol/L nitrophenyl-EGTA²² and 650 µmol/L Ca²⁺ (free [Ca²⁺], 120 nmol/L), a single UV flash could rapidly elevate [Ca²⁺]_i, and this initial [Ca²⁺]_i rise induced an additional rise due to CICR (Fig 1C). Thus, agents known to trigger SR Ca²⁺ release were effective in our preparation, but flash photolysis of caged cADPR was not.

View larger version (20K): [in this window] [in a new window]   Figure 1. A step increase in the cADPR concentration did not directly induce SR Ca2+ release in cardiac myocytes. A, The [Ca2+]i response, indicated by the fluo 3 FI, in a myocyte loaded with 100 µmol/L caged cADPR to a single UV flash (F) triggered 3 seconds after terminating the conditioning stimuli. The PMT-detected flash artifact was removed for clarity. B, The SR Ca2+ release triggered by local extracellular application of 2.5 mmol/L caffeine. C, The SR Ca2+ release triggered by a step change in the [Ca2+] induced by flash photolysis of caged Ca2+ (nitrophenyl-EGTA). The off-scale transient is the flash artifact.

We next examined whether a step change in the cADPR concentration would alter the sensitivity of the SR Ca²⁺ release process to its physiological trigger, the voltage-dependent I_Ca. In preliminary experiments, flash photolysis of caged cADPR immediately before depolarization to 0 mV did not change the resulting intracellular Ca²⁺ transient amplitude (not shown). However, it is likely that the I_Ca elicited at 0 mV was already more than sufficient to trigger a maximal SR Ca²⁺ release. Therefore, further experiments were designed to ascertain whether photolytically generated cADPR could alter the magnitude of a submaximal SR Ca²⁺ release triggered by a smaller I_Ca.

Submaximal I_Ca levels were elicited by depolarization to less positive test potentials chosen so that the intracellular Ca²⁺ transient amplitude was approximately one third of its amplitude after depolarization to 0 mV. After a train of conditioning pulses to 0 mV, a single UV flash was triggered 100 milliseconds before the first depolarization to this test potential. As illustrated in Fig 2A, photolysis of caged cADPR (right) did not change the amplitude of the subsequent intracellular Ca²⁺ transient relative to that observed in an identical control sequence (left). To quantify changes in the sensitivity of the SR to the trigger I_Ca, we have defined a gain factor as equal to the change in the fluorescence signal divided by the peak I_Ca magnitude (both normalized to their maximum values). As shown in Fig 2C, this gain factor remained unchanged after photolysis of caged cADPR under these conditions (control, 7.1±0.7; flash, 7.3±0.7; P=.49, n=8). In contrast, ejection of 1 mmol/L caffeine near a fura 2-loaded myocyte that was repetitively depolarized to a submaximal test potential increased the intracellular Ca²⁺ transient amplitude, without increasing I_Ca (Fig 2B). Consequently, the gain factor increased 43% (control, 5.8±0.7; flash, 8.0±0.5; P<.01, n=4). Note that the values of the gain factor under control conditions are different for the two treatments because a different Ca²⁺ dye was used. Thus, cADPR did not modulate the sensitivity of the SR Ca²⁺ release process to the trigger I_Ca, whereas caffeine did.

View larger version (27K): [in this window] [in a new window]   Figure 2. A, A step increase in the cADPR concentration did not alter the magnitude of a submaximal SR Ca2+ release triggered by submaximal ICa. Shown are the membrane current (Im), membrane potential (Vm), and fluo 3 FI from a caged cADPR loaded myocyte. On the right is shown the UV flash (F) triggered just before the first depolarization to the test potential following the conditioning stimuli; on the left, the control condition (no flash) is shown. B, Caffeine potentiated CICR in a fura 2-loaded myocyte repetitively depolarized (0.5 Hz) to -25 mV. A 1.0 mmol/L caffeine solution was gently pressure-ejected from a pipette located 150 µm from the cell. C, Percent change in the gain factor (GF) is shown after flash photolysis of caged cADPR (n=8) or exposure to caffeine (n=4). *P<.01 vs control.

To verify that physiological amounts of cADPR were indeed generated inside cells, we examined the ability of our caged cADPR lot and photolysis system to induce Ca²⁺ release in sea urchin eggs taken from the sea urchin Lytechinus pictus. Caged cADPR and fluo 3 were microinjected into eggs. We estimated the final caged cADPR concentration to have been 15 to 25 µmol/L (see "Materials and Methods"). A single UV flash induced a large intracellular Ca²⁺ transient (Fig 3). The average fluo 3 FI increase was 333±11%, and the average time to peak FI was 3.0±0.2 seconds (n=8 eggs). Despite the rapid jump in the cADPR concentration, in all cases the initial [Ca²⁺]_i rose slowly, with a mean delay of 660±100 milliseconds before the onset of the rapid phase. These intracellular Ca²⁺ transients are comparable (although faster in onset and time to peak) to those triggered by direct injection of cADPR into the intact eggs.^{2 23} Thus, in sea urchin eggs loaded with caged cADPR, a single flashlamp burst was capable of producing a robust Ca²⁺ release.

View larger version (9K): [in this window] [in a new window]   Figure 3. Flash photolysis of caged cADPR triggered intracellular Ca2+ transients in sea urchin eggs. Arrow indicates the occurrence of the UV flash (F). The inset shows the rising phase of the intracellular Ca2+ transient at higher time resolution and more clearly shows the delay in the onset of the [Ca2+]i rise. The delay was estimated by extrapolating the rapid phase of the FI rise back to the point at which it intersected the preflash FI.

Consequently, it seems reasonable to conclude that equivalent step changes in the cADPR concentration do not regulate SR Ca²⁺ release in cardiac myocytes. Nevertheless, our results to this point do not necessarily exclude cADPR as a modulator of cardiac SR Ca²⁺ release. Based on whole tissue assays, the endogenous level of cADPR in rat heart has been estimated to be 200 nmol/L,¹² a value that, if all cytosolic, would be approximately threefold to fourfold greater than the ED₅₀ for Ca²⁺ release in intact sea urchin eggs.²³ In light of this, since we were unable to control the basal cADPR concentration, we cannot be certain that the flash-induced cADPR rise produced a physiologically relevant increase in the cADPR signal. Further, notwithstanding the positive response observed in sea urchin eggs, we cannot be certain that the kinetics or magnitude of cADPR transient, shaped by the myocyte's normal metabolic processes, would be appropriate for the cascade of events that might be required for cADPR modulation of Ca²⁺ release in heart. Given these uncertainties, our next approach was to determine if high levels of authentic cADPR or its antagonist, 8-amino-cADPR, would alter CICR when present intracellularly for a sustained period. We reasoned that if cADPR were a modulator of SR Ca²⁺ release in heart, at least one of these treatments should affect normal CICR, regardless of the basal conditions or the kinetics of the transduction pathway.

Myocytes were equilibrated with our standard electrode solution containing fura 2 (control) or with the addition of one of the following: 100 µmol/L cADPR, 10 µmol/L 8-amino-cADPR, or 30 µmol/L ryanodine. The cADPR concentration (>1000 times its ED₅₀ in sea urchin eggs²³ ) was chosen in an attempt to overwhelm the endogenous cADPR hydrolase, although we did not independently confirm this. 8-Amino-cADPR has been shown to be a potent competitive antagonist of cADPR-induced Ca²⁺ release in sea urchin egg extracts²⁴ and in mammalian intestinal smooth muscle cells²⁵ at submicromolar concentrations. A set of intracellular Ca²⁺ transients (as indicated by the fura 2 340/380 FI ratio) resulting from different I_Ca amplitudes were evoked by imposing 300-millisecond depolarization pulses to different potentials over a range of -30 to +10 mV. Fig 4A shows the dependence of the intracellular Ca²⁺ transient amplitude on the I_Ca magnitude from these experiments. The data from each myocyte were normalized to their I_max and [Ca²⁺]_i values. Under control conditions, the [Ca²⁺]_i-I_Ca relation is hyperbolic. With ryanodine, the intracellular Ca²⁺ transient amplitude elicited by I_max decreased 69% (Fig 4B), and the [Ca²⁺]_i-I_Ca relation was linear. These effects of ryanodine are an expected consequence of eliminating the SR contribution to the intracellular Ca²⁺ transient.²⁶ In contrast, the [Ca²⁺]_i-I_Ca relations observed in myocytes loaded with cADPR or 8-amino-cADPR were indistinguishable from control (Fig 4A). Neither cADPR nor 8-amino-cADPR affected the maximum [Ca²⁺]_i change evoked by I_max (Fig 4B), nor was the peak I_Ca observed at 0 mV altered (peak I_Ca was 1.41±0.10, 1.45±0.15, and 1.44±0.16 nA for control, cADPR, and 8-amino-cADPR, respectively; n=6 for each). Thus, intracellular application of supermaximal concentrations of either cADPR or 8-amino-cADPR did not alter the normal CICR, whereas ryanodine did.

View larger version (24K): [in this window] [in a new window]   Figure 4. The effects of cADPR, 8-amino-cADPR, and ryanodine on CICR in cardiac myocytes. A, The normalized [Ca2+]i-ICa relation in fura 2-loaded myocytes under control conditions () or when loaded with either 100 µmol/L cADPR (), 10 µmol/L 8-amino-cADPR (), or 30 µmol/L ryanodine () via the patch electrode. The peak ICa was varied by depolarizing myocytes to different potentials. The change in [Ca2+]i, as indexed by the fura 2 340/380 nm FI ratio, was normalized to the maximum value recording in each myocyte and then plotted vs the similarly normalized magnitude of the peak ICa. The individual measurements in each treatment were grouped over each 10% Imax interval. Error bars are SEM. B, Plot of the maximum intracellular Ca2+ transient amplitude induced by Imax in the presence of these agents. *P<.01 vs all other groups.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this investigation, we found that cADPR had no apparent action on SR Ca²⁺ release in intact ventricular myocytes. Our experiments performed on sea urchin eggs clearly demonstrated that physiological levels of cADPR can be effectively released from caged cADPR photochemically. Thus, the absence of the observable effects on cardiac SR Ca²⁺ release by flash photolysis cannot be attributed to some peculiarity of the caged cADPR lot or the efficiency of our photolysis hardware. It also appears unlikely that the inability of photolytically generated cADPR to modulate SR Ca²⁺ release in heart can be accounted for by postulating that basal cADPR levels were already physiologically saturating (because of either endogenous cADPR¹² or contamination from our lot of caged cADPR), since pharmacological blockage of cADPR by 8-amino-cADPR did not modify CICR. Furthermore, although we do not know the kinetics of the photolytically generated cADPR transient, the fact that CICR was not altered by the sustained presence of cADPR strongly suggests that the issue is moot.

Both Lee et al¹⁶ and Tanaka et al¹⁷ have reported that submicromolar calmodulin is a required cofactor for cADPR Ca²⁺-releasing activity in sea urchin egg extracts. Despite the fact that endogenous levels of calmodulin are in the micromolar range in heart,²⁷ we took the precaution of adding 0.6 µmol/L calmodulin in our patch pipette solution to guard against the unlikely possibility that endogenous calmodulin was depleted by intracellular dialysis.

Meszaros et al⁹ previously reported that submicromolar cADPR enhanced ⁴⁵Ca²⁺ efflux from cardiac, but not skeletal muscle, SR preparations. This action was found to be Ca²⁺ dependent and blocked by ryanodine. cADPR also enhanced ³[H]ryanodine binding to cardiac SR and increased the open probability of single RyRCs being reconstituted in the planar lipid bilayers. These later findings suggested that the mechanism of cADPR's action on SR Ca²⁺ release was by modulation of the RyRC. However, the evidence supporting this direct modulation of the RyRC has been challenged. Under similar conditions, Fruen et al¹⁰ failed to replicate these findings. Sitsapesan and colleagues^{11 28} found that although cADPR could modulate cardiac (and skeletal muscle) RyRC gating in the presence of millimolar lumenal [Ca²⁺], this direct stimulatory effect on RyRC was observed only in the absence of cytosolic Mg-ATP (conditions similar to those used by Meszaros et al⁹ ). Since as little as 100 µmol/L Mg-ATP prevented cADPR activation of the RyRC, these authors proposed that cADPR may act by binding to a nucleotide binding site, but only under conditions of extreme ATP depletion. Thus, the mechanism by which cADPR could stimulate Ca²⁺ release in isolated SR vesicles remains controversial. Given the inability of cADPR to modulate Ca²⁺ release in our intact myocyte preparation, we are unable at this point to reconcile our findings with the SR Ca²⁺-releasing activity reported by Meszaros et al.

In summary, neither a step change in the cADPR concentration nor the sustained presence of cADPR or its antagonist 8-amino-cADPR modulated SR Ca²⁺ release in cardiac myocytes. In all cases, we have contrasted the inability of cADPR to modulate SR Ca²⁺ release in our preparation with evidence that under similar conditions agents known to modulate SR Ca²⁺ release (ie, Ca²⁺, caffeine, and ryanodine) were able to do so in a manner consistent with their known mechanisms of action. Therefore, we believe the most reasonable interpretation of our results is that cADPR does not regulate SR Ca²⁺ release in intact cardiac myocytes.

	Selected Abbreviations and Acronyms

 

cADPR = cyclic ADP-ribose FI = fluorescence intensity ICa = Ca2+ current Imax = maximum ICa RyRC = ryanodine receptor channel SR = sarcoplasmic reticulum

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (AR41552) and a Grant-in-Aid from the American Heart Association (Georgia affiliate) to Dr Becker. We thank Dr Hon Chung Lee, University of Minnesota, for helpful discussions and advice, and H. Bindu Vanapalli for technical assistance.

Received April 30, 1996; accepted May 14, 1996.

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