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CARDIOVASCULAR |
1 Department of Physiology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA
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(Received 14 July 2006;
accepted after revision 29 August 2006;
first published online 31 August 2006)
Corresponding author L. A. Blatter: Department of Physiology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA. Email: lblatte{at}lumc.edu
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Introduction |
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There is also evidence that glycolysis regulates the function of the SR to accumulate and release Ca2+ in a more direct way. The SR Ca2+ release channel, the ryanodine receptor (RyR), is sensitive to intermediates and products of glycolysis. Fructose-1,6-bisphosphate, fructose-6-phosphate and glucose-6-phosphate have been reported to activate the cardiac RyR in planar lipid bilayers and to stimulate ryanodine binding to the cardiac RyR (Kermode et al. 1998a; Kockskämper et al. 2005). On the other hand, products of glycolysis, i.e. pyruvate and L-lactate, cause direct inhibition of RyR activity (Zima et al. 2003; Kockskämper et al. 2005). Therefore, alterations of glycolytic flux which occur, for example, during periods of ischaemia-reperfusion are expected to cause fluctuations of the levels of glycolytic intermediates in close proximity to the SR membrane and, thereby, modulate the activity of the RyR channel and the SR Ca2+ pump. This, in turn, will alter EC coupling via changes in SR Ca2+ load and the amplitude of the [Ca2+]i transient.
The aim of this study was to investigate the mechanisms of regulation of SR Ca2+ release by glycolytic sugar phosphate intermediates in ventricular myocytes. In cardiac myocytes, global [Ca2+]i transients result from the summation of elementary release events, Ca2+ sparks (Cheng et al. 1993). Therefore, to gain a better understanding of the mechanisms of regulation of [Ca2+]i by glycolysis, we investigated spontaneous Ca2+ sparks using laser scanning confocal microscopy in permeabilized myocytes. Membrane permeabilization allows the study of SR function under controlled conditions with regard to cytoplasmic ion concentrations and energy supply. Furthermore, this technique also allows studying directly the effect of membrane-impermeable substances such as glycolytic sugar phosphates on SR Ca2+ release. The results revealed that the effects of the glycolytic sugar phosphates on SR function were dependent on the cellular energy status. With normal cellular energy reserves, accumulation of sugar phosphates stimulated SR Ca2+ release directly by activation of RyR channels. However, under conditions of a low ATP/ADP ratio and absence of CrP, which depresses SR Ca2+ uptake, sugar phosphates could be used by glycolytic enzymes for ATP synthesis and support of SR Ca2+ pump activity to normalize SR Ca2+ load and release.
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Methods |
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The procedure for cell isolation was approved by the Institutional Animal Care and Use Committee of Loyola University Chicago, Stritch School of Medicine. Adult mongrel cats of either sex were anaesthetized with thiopental sodium (30 mg kg–1 I.P.). Following thoracotomy hearts were quickly excised, mounted on a Langendorff apparatus, and retrogradely perfused with collagenase-containing solution at 37°C according to the method previously described (Rubenstein & Lipsius, 1995). Twenty hearts were used to isolate ventricular myocytes and SR vesicles.
The cell membrane was permeabilized by exposure to saponin (0.005% for 30 s; Zima et al. 2003). After permeabilization cells were placed in a solution composed of (mM): potassium aspartate 100; KCl 15; KH2PO4 5; MgATP 5; Na2ADP 0.01; NAD+ 2; EGTA 0.4; CaCl2 0.1; MgCl2 0.75; phosphocreatine (CrP) 10; Hepes 10; fluo-3 pentapotassium salt (Molecular Probes Invitrogen, Carlsbad, CA, USA) 0.04; creatine phosphokinase 5 U ml–1; dextran (MW: 40 000) 8%, and pH 7.2 (KOH). Free [Ca2+] and [Mg2+] of this solution were 100 nM and 1 mM, respectively (calculated using WinMAXC 2.05, Stanford University, CA, USA). All experiments were performed at room temperature (22–25°C). Changes in [Ca2+]i were measured with a laser scanning confocal microscope (Radiance 2000 MP, Bio-Rad, UK) equipped with a x 40 oil-immersion objective lens (N.A. = 1.3). The Ca2+ indicator fluo-3 was excited with the 488 nm line of an argon ion laser and fluorescence was measured at wavelengths > 515 nm. Images were acquired in the linescan mode (3 or 6 ms per scan; pixel size 0.1 µm). Ca2+ sparks were quantified in terms of amplitude (
F/F0), duration (ms), spatial width (µm) and frequency (sparks s–1 (100 µm)–1) using an automated detection algorithm (Cheng et al. 1999; Zima et al. 2004). F0 is the initial fluorescence recorded under steady-state conditions and F = F – F0. Duration and width of sparks were measured at half-maximal amplitude. The algorithm detects sparks as areas of elevated fluorescence (F) intensity relative to the standard deviation (S.D.) of background noise of the fluorescence image. The detection threshold was set to 3.0 ± S.D., which translated to the detection of sparks with an amplitude of F/F0 > 0.3. Since detection relies on a threshold F/F0 value changes in Ca2+ spark amplitude potentially affect calculated Ca2+ spark frequencies, such that, e.g. a general decrease of the Ca2+ spark amplitude (cf. for example Figs 1, 3 and 4) would tend to lead to an apparent decrease and underestimation of frequency because a larger fraction of sparks would fall below detection level. No corrections for such missed events were made in the frequency calculations.
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SR vesicles isolated from cat ventricle were incorporated into planar lipid bilayers and RyR channel activity was measured as previously described (Zima et al. 2003). For recording of Cs+ currents through RyRs, the cis- and trans-chambers contained (mM): CsCH3SO3 400; CaCl2 0.1; Hepes 20; pH 7.3. The cis- and trans-chambers corresponded to the cytosolic and luminal side of the RyR channel, respectively. Free [Ca2+] in the cis-chamber was adjusted to 3 µM by adding an appropriate amount of EGTA. Single channel currents were recorded with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) at a holding potential of –20 mV. Currents were filtered at 1 kHz and sampled at 5 kHz. The mean open probabilities (Po) of the channels were used as an index of activity. Po values were calculated from the 50% threshold analysis using pCLAMP software (Axon Instruments).
Drugs
Glucose-6-phosphate (G6P) mono-potassium salt, fructose-6-phosphate (F6P) di-potassium salt, fructose-1,6-bisphosphate (FBP) di-potassium salt, iodoacetic acid (IAA) mono-sodium salt and NAD+ mono-sodium salt were purchased from Sigma-Aldrich. Addition of glycolytic intermediates at the concentrations used in this study (0.2–3 mM) did not change the free [Ca2+] in the experimental solutions for Ca2+ sparks measurements and RyR channel recordings (verified with a Ca2+-sensitive electrode; Orion Research Incorporated) or the fluorescent properties of fluo-3.
Statistics
Data are presented as mean ± S.E.M. of n measurements. Statistical comparisons between groups were performed with the Student's t test. Differences were considered statistically significant at P < 0.05.
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Results |
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We studied the effect of glycolytic sugar phosphate intermediates on spontaneous Ca2+ sparks in saponin-permeabilized ventricular myocytes at a physiological cytosolic ATP/ADP ratio ([ATP] = 5 mM, [ADP] = 0.01 mM) and CrP content ([CrP] = 10 mM). Under these conditions, Ca2+ sparks were detected with a stable frequency of 3.0 ± 0.5 sparks s–1 (100 µm)–1 (n = 13 cells). Figure 1A shows representative confocal linescan images of Ca2+ sparks and plots of F/F0 from selected subcellular regions under control conditions, 2 and 6 min after addition of fructose-1,6-bisphosphate (FBP) and following washout of FBP. Average data of FBP effects on Ca2+ spark frequency indicate that the sugar phosphate caused a transient increase in Ca2+ spark activity (Fig. 1B). Specifically, within 2 min after addition of FBP (1 mM) Ca2+ spark frequency increased by 40 ± 9% (n = 13; P < 0.05). After 4–6 min the frequency almost returned to the control level (4 ± 20%). Upon washout of FBP, the frequency of Ca2+ sparks decreased to 69 ± 9% of control (n = 13; P < 0.05 versus control). During the initial stimulation of SR Ca2+ release by FBP, the spatio-temporal properties of Ca2+ sparks were not affected by the sugar phosphate. However, during the later phase of FBP action (after > 4 min of FBP application), spark amplitude and width (but not duration) significantly decreased (Fig. 1C and D). In control conditions, Ca2+ spark amplitude, duration and width averaged 0.88 ± 0.04 (F/F0), 48.5 ± 2.7 ms and 1.75 ± 0.04 µm, respectively. After 4 min of FBP (1 mM) exposure, these parameters decreased to 0.64 ± 0.03 (F/F0), 45.0 ± 2.3 ms and 1.51 ± 0.06 µm, respectively (n = 13 cells; number of sparks analysed in control conditions and in the presence of FBP was 798 and 592, respectively).
FBP did not cause any detectable changes in [Ca2+]i when RyR channels were inhibited by ryanodine (10 µM) or tetracaine (1 mM) indicating that the sugar phosphate enhanced specifically RyR-mediated SR Ca2+ release (data not shown).
Ca2+ spark frequency is highly dependent on SR Ca2+ load (Lukyanenko et al. 1996; Satoh et al. 1997). Therefore, we examined changes in SR Ca2+ load during application of FBP. SR Ca2+ load was estimated from the [Ca2+]i transient amplitude during fast application of caffeine (20 mM). While FBP (1 mM) did not cause any changes in SR Ca2+ load during the first minutes, a significant decrease in the amplitude of caffeine-induced [Ca2+]i transient was observed after 6 min of FBP application (Fig. 2A). On average, the amplitude of the caffeine response in the presence of FBP was reduced to 87 ± 2% (n = 6; P < 0.05) of control (Fig. 2B).
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These results suggest that at a physiological cytosolic ATP/ADP ratio and CrP levels the glycolytic sugar phosphates FBP and F6P stimulate SR Ca2+ release through RyR channels, thereby leading to a reduction of SR Ca2+ load.
The effect of glycolytic sugar phosphates on Ca2+ sparks during inhibition of glycolysis
The stimulatory effect of the glycolytic intermediates FBP and F6P on SR Ca2+ release could be mediated indirectly through accumulation of glycolytic products (e.g. ATP) as a result of increased activities of downstream glycolytic enzymes. Therefore, we tested the effects of FBP on Ca2+ sparks in the presence of iodoacetate (IAA). IAA at submillimolar concentrations selectively inhibits glycolysis downstream of FBP production at the stage of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reaction (Carlson & Siger, 1959). Pretreatment of cells with IAA (0.5 mM) for 6 min did not significantly affect Ca2+ spark frequency (3.0 ± 0.5 sparks s–1 (100 µm)–1 in control versus 4.0 ± 0.7 sparks s–1 (100 µm)–1 in the presence of (IAA). Furthermore, inhibition of glycolysis by IAA did not prevent the stimulatory effects of FBP on SR Ca2+ release at the normal ATP/ADP ratio. In the presence of IAA, FBP (1 mM) increased Ca2+ spark frequency by 71 ± 12% (n = 7; P < 0.01). Figure 4A shows confocal images of Ca2+ sparks and selected plots of subcellular changes of F/F0 in the presence of IAA (0.5 mM), 2 and 6 min after the subsequent addition of FBP (1 mM), and following washout of FBP. Similar to control conditions (Fig. 1), the effect of FBP on Ca2+ spark frequency in the presence of IAA reached a maximum within the first minutes, followed by recovery to near-control level (Fig. 4B). After 4 min of exposure to FBP the spark frequency remained 23 ± 13% above the initial control value. This degree of stimulation, however, was not significantly different from the effect of IAA alone. Furthermore, in the presence of IAA (0.5 mM), Ca2+ spark amplitude and width were reduced after 4 min exposure to FBP from F/F0 = 0.80 ± 0.03 to 0.71 ± 0.04 (P < 0.05) and from and 1.66 ± 0.06 to 1.44 ± 0.05 µm (P < 0.05), respectively. Spark duration remained unchanged (40.9 ± 1.5 versus 40.3 ± 1.8 ms, P = N.S.; n = 7 cells; number of sparks analysed in control conditions and in the presence of FBP was 538 and 445, respectively). Similar results were obtained with F6P (data not shown).
Therefore, the stimulatory effect of glycolytic sugar phosphate intermediates on SR Ca2+ release was not dependent on an intact glycolytic pathway, but rather appeared to be the result of a direct interaction with the RyR channel.
Effects of glycolytic sugar phosphates on single RyR channel activity
To test further the hypothesis that stimulation of SR Ca2+ release by sugar phosphates was caused by direct stimulation of the RyR, we studied the effects of sugar phosphates on the activity of isolated RyR channels. Heavy SR microsomes were fused with planar lipid bilayers and single channel currents through the RyR were recorded using Cs+ as the charge carrier. The channels were activated by 3 µM cytosolic [Ca2+] (no ADP, ATP and Mg2+). Figure 5A shows effects of various concentrations of F6P and FBP (added to the cis-chamber) on the activity of RyR channel currents recorded at –20 mV. Both sugar phosphates increased the activity of RyR channels in a concentration-dependent manner, acting on the cytosolic side of the channel. This effect was fully established in less than 1 min, stable throughout the recording (2–6 min) and completely reversible upon washout. Figure 5B summarizes the effects of various concentrations of three sugar phosphates on the open probability (Po) of the RyR. The results indicate that FBP was most effective in stimulating RyR channel activity. Po increased from 0.038 ± 0.016 under control conditions (cis[Ca2+] = 3 µM) to 0.132 ± 0.032 (n = 5) in the presence of 3 mM FBP, i.e. reflecting an activation by 340 ± 49% (n = 5; P < 0.01). The same concentration of F6P increased the activity (Po) of the RyR from 0.024 ± 0.009 to 0.039 ± 0.011 (n = 4) or by 69 ± 11% (n = 4; P < 0.05), whereas glucose-6-phosphate (G6P; 3 mM) had no significant effect on the Po (change from 0.033 ± 0.012 to 0.038 ± 0.016 or by 14 ± 12%; n = 4).
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These results indicate that the glycolytic sugar phosphate intermediates F6P and FBP investigated here increased the activity of RyR channels, but with different degrees of efficiency. Furthermore, this direct stimulatory effect of the sugar phosphates on RyR-mediated SR Ca2+ release is largely independent of the cytosolic ATP level.
Effects of glycolytic sugar phosphates on Ca2+ sparks under low cellular energy reserves
Next, we tested how glycolytic sugar phosphates affect SR Ca2+ release and load under conditions of depleted cytosolic energy reserves. At the early stage of ischaemia, the intracellular concentration of ATP remains relatively constant, whereas CrP concentration falls dramatically and the level of ADP rises (for review see Allen & Orchard, 1987). In the first set of experiments, we studied effect of CrP and ADP on Ca2+ sparks in permeabilized ventricular myocytes separately. Withdrawal of CrP from the experimental solution ([ATP] and [ADP] were kept constant throughout the experiment) decreased Ca2+ spark frequency by 29 ± 5% of control (n = 6; P < 0.05; Fig. 6A and C). Removal of CrP caused a reduction of Ca2+ spark amplitude from F/F0 = 0.76 ± 0.03 to 0.60 ± 0.03 (P < 0.01; n = 6 cells; number of sparks analysed in control conditions and in the absence of CrP was 797 and 396, respectively; Fig. 6D). Increasing the cytosolic [ADP] caused qualitatively similar effects on SR Ca2+ release. Increasing [ADP] from 0.01 to 0.4 mM ([CrP] was kept constant at 10 mM) decreased Ca2+ spark frequency by 42 ± 7% (n = 7; P < 0.01; Fig. 6B and C), concomitant with a decrease in spark amplitude (Fig. 6D). In both cases, the inhibition of Ca2+ sparks required 7–8 min to reach a maximum.
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8 min) decrease of Ca2+ spark frequency to 34 ± 7% of the initial control level (n = 7; P < 0.01). The inhibition of spark activity was accompanied by a decrease of Ca2+ spark amplitude from F/F0 = 0.80 ± 0.04 to 0.67 ± 0.03 (P < 0.01; n = 7 cells; number of sparks analysed in control conditions and in low energy reserve solution was 513 and 211, respectively; Fig. 7C). The subsequent addition of FBP (1 mM) led to a gradual recovery of Ca2+ spark activity. After 8 min of FBP application the spark frequency was restored to 84 ± 7% of the control level (Fig. 7B; filled circles). Furthermore, in the presence of FBP the spark amplitude increased to F/F0 = 0.71 ± 0.03 (n = 7 cells; 291 sparks analysed) which was significantly different (P < 0.05) from the amplitude recorded in low energy reserve solution alone.
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F/F0 = 0.83 ± 0.01 to 0.61 ± 0.01 in low energy reserve solution (n = 4 cells; number of sparks analysed in control conditions and in the presence of the low energy reserve solution was 217 and 93, respectively). Addition of FBP caused a further decrease to F/F0 = 0.51 ± 0.04 (61 sparks analysed). The changes in Ca2+ spark frequency and amplitude observed with depleted cytosolic energy reserves could be the result of a decrease in SR Ca2+ content. Figure 8A shows a representative experiment in which caffeine (20 mM) was applied under control conditions, in low energy reserve solution and at 2 and 8 min after the subsequent addition of FBP (1 mM). Average results from six myocytes are presented in Fig. 8B. Reduction of the ATP/ADP ratio and withdrawal of CrP decreased the amplitude of caffeine-induced [Ca2+]i transient to 62 ± 7% (n = 6; P < 0.01) of control. After the first 2 min, the subsequent exposure of FBP (1 mM) did not affect SR Ca2+ load. After 8 min, however, FBP caused a recovery of SR Ca2+ loading to 88 ± 7% of control (n = 6; P < 0.05 versus low energy reserve solution). Similar to the Ca2+ spark experiments, this effect of FBP on SR Ca2+ load in the presence of the solution with depleted cytosolic energy reserves was sensitive to IAA, i.e. in the presence of IAA FBP failed to rescue SR Ca2+ load (Fig. 8C). SR load under control conditions with and without IAA present was not significantly different.
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Discussion |
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Direct effect of glycolytic sugar phosphates on RyR channel activity
It seems likely that under normal aerobic conditions when cellular energy levels are high, direct stimulation of RyR channels would be the predominant mechanism of modulation of SR Ca2+ release by glycolytic intermediates. We found that at a physiological ATP/ADP ratio and CrP content, two fructose phosphates, FBP and F6P, caused a transient increase in Ca2+ spark frequency, which was accompanied by decreases in amplitude and spatial width of Ca2+ sparks (Figs 1 and 3). Potentially, decreases in amplitude and width can be the result of smaller Ca2+ efflux from the SR due to smaller SR Ca2+ content. Experiments with caffeine confirmed that SR Ca2+ load eventually decreased after FBP application (Fig. 2). Therefore, despite the fact that glycolytic sugar phosphates are substrates for glycolytic ATP production and can potentially improve the efficiency of the SR Ca2+ATPase, at a physiological ATP/ADP ratio SR Ca2+ leak through the RyR channels seems to be more sensitive to sugar phosphates than SR Ca2+ uptake. Experiments with an inhibitor of glycolysis (IAA; Fig. 4) indicated that this stimulatory effect of sugar phosphates on SR Ca2+ release was not dependent on an intact glycolytic pathway (and therefore ATP production), but rather the result of a direct stimulation of RyR channels. This conclusion was drawn from the following observations. First, the transient stimulation of Ca2+ sparks by sugar phosphates is reminiscent of the effect of low doses of the RyR agonist caffeine (Lukyanenko et al. 2001). Sensitization of RyRs with caffeine results in brief increases in Ca2+ spark frequency with consequent decreases in SR Ca2+ content. Presumably, the high Ca2+ leak would gradually deplete SR Ca2+ stores and subsequently reduce RyR activity from the luminal side of the channel by a Ca2+-dependent mechanism (Eisner et al. 1998). Second, single-channel experiments revealed that glycolytic sugar phosphate intermediates stimulated the activity of cardiac RyR channels incorporated into lipid bilayers (Fig. 5). Similar effects of sugar phosphates were previously described for sheep cardiac RyR channels (Kermode et al. 1998a). From all glycolytic intermediates studied, FBP was most effective in stimulating the RyR. We found, for example, that FBP was about five times more potent in stimulating RyR channel activity than F6P (+340% by FBP versus +70% by F6P). The large difference between FBP and F6P effects on RyR channel activity observed in lipid bilayer experiments appears to be at variance with the almost identical effects of these compounds on Ca2+ sparks in permeabilized myocytes. In these experiments, FBP and F6P caused very similar increases in Ca2+ spark frequency of 60 and 40%, respectively. This apparent inconsistency could be explained by the fact that in permeabilized cells the glycolytic enzyme phosphofructokinase can metabolize F6P in the presence of all required cofactors leading to accumulation of FBP and therefore comparable effects of F6P and FBP on Ca2+ sparks.
Studies of isolated RyR channels also revealed that the stimulatory effect of FBP was preserved when the RyR was fully activated by cytosolic ATP. This finding indicates that an interaction of sugar phosphates with the RyR involves sites different from the adenine nucleotide binding site. It is not clear yet, however, whether sugar phosphates interact with other known binding sites or with a novel site on the RyR or a closely associated accessory protein. It has been demonstrated that inorganic phosphate (Pi) binds to a distinct domain on the cardiac RyR channel to produce an increase in Po (Kermode et al. 1998b). Since all glycolytic intermediates studied here contain at least one phosphate group, it seems possible that they can bind to the same site as Pi. Even though the site and molecular mechanism of fructose phosphate action remain to be determined, the present results clearly suggest that the stimulatory effect of glycolytic sugar phosphates on RyR channels per se is independent of the metabolic state of the cell.
Glycolytic sugar phosphates improve SR Ca2+ uptake under conditions of low cytosolic energy reserves
A different situation might arise at low cytosolic ATP/ADP ratio and reduced CrP levels, as may occur during periods of ischaemia/hypoxia. During the first minutes of ischaemia the intracellular [ATP] remains relatively constant, while [ADP] rises and [CrP] falls (Allen & Orchard, 1987). We found that both of these factors affect SR Ca2+ release. At constant [ATP], the increase of [ADP] or the removal of CrP caused a slowly developing decrease of Ca2+ spark frequency, which was accompanied by a significant decrease in spark amplitude (Fig. 6). Similar effects on Ca2+ spark activity by CrP were previously observed in rat cardiomyocytes (Yang & Steele, 2002). It seems likely that the effects of changing ATP/ADP ratio and [CrP] were mediated by the same mechanism. As intracellularly the ATP/ADP ratio is effectively controlled by the CrP level through the creatine kinase reaction, CrP removal enhances local ADP accumulation. The simultaneous increase of [ADP] and removal of CrP, which is observed during ischaemia, caused more significant (and nearly additive) inhibition of Ca2+ sparks compared with the effect of each factor alone (Figs 6 and 7). The slow onset of this inhibition together with the significant depletion of SR Ca2+ load is indicative of a reduction of Ca2+ uptake by the SR Ca2+-ATPase. This observation is consistent with previous work showing that SR Ca2+ release and load are critically dependent on the ratio of [ATP] to [ADP] (Yang & Steele, 2002; Zima et al. 2003). The cytosolic ATP/ADP ratio is known to affect Ca2+-ATPase activity not only through the free energy accumulated in the phosphate bonds of ATP, but also through accumulation of ADP. Elevation of ADP markedly reduces the ability of the SR to store Ca2+ by decreasing the rate of the SR Ca2+ pump (Macdonald & Stephenson, 2001). It seems possible that increases in cytosolic [ADP] could also directly affect SR Ca2+ release. It has been shown that cytosolic ADP can potentiate Ca2+-induced Ca2+ release in rat ventricular trabeculae (Xiang & Kentish, 1995). Single-channel studies revealed that ADP can act as a partial agonist at the adenine nucleotide binding site of the cardiac RyR (Kermode et al. 1998b). However, when the adenine nucleotide binding site of the RyR was fully activated by the more specific agonist ATP (like in our study where we used 5 mM ATP), ADP could decrease RyR activity by antagonizing the effect of ATP (Kermode et al. 1998b). Therefore, the inhibition of Ca2+ spark frequency by high [ADP] (Fig. 7) could be due to a direct inhibition of SR Ca2+ release through the RyR. It has been shown, however, that interventions that inhibit RyR, typically cause only a transient inhibition of SR Ca2+ release (Eisner et al. 1998; Lukyanenko et al. 2001). This can be explained by a concomitant increase in SR Ca2+ content with subsequent Ca2+-dependent activation of RyR from the luminal side of the channel. In contrast to RyR inhibitors, in our study high [ADP] caused a slowly developing, however, non-transient decrease in Ca2+ spark frequency which was accompanied by a reduction in SR Ca2+ load. Thus, the inhibitory effect of high cytosolic [ADP] on Ca2+ sparks could not be explained solely based on the depression of RyR activity. Therefore, the observed decrease in Ca2+ spark frequency and amplitude with low cytosolic energy reserves are likely to be secondary to a decrease in the SR Ca2+-ATPase activity and depletion in SR Ca2+ content.
Under conditions of depleted cytosolic energy reserves glycolytic sugar phosphates caused a completely different type of modulation of SR Ca2+ release than at a normal physiological ATP/ADP ratio and CrP levels. First, we did not observe any stimulation of SR Ca2+ release within the first minutes of FBP application (Fig. 7B, filled symbols). As mentioned before, stimulation of RyR channel activity by sugar phosphates was not mediated by an interaction with the adenine nucleotide binding site of the RyR. Therefore, increased cytosolic [ADP] will not significantly affect the ability of FBP to stimulate the RyR channel. The lack of the initial stimulation of Ca2+ spark frequency by FBP under conditions of low cytosolic energy reserves can be explained by a significant depletion of SR Ca2+ content (Fig. 8) and the inability of sugar phosphates to increase Ca2+ spark frequency via stimulation of the RyR after depletion of SR Ca2+ load. In contrast to control conditions, at a low cytosolic ATP/ADP ratio and low [CrP], FBP caused a slowly developing increase in Ca2+ spark frequency (Fig. 7). We suggest that the slow time course reflects the gradual accumulation of Ca2+ in the SR due to stimulation of the SR Ca2+-ATPase. It is well accepted that increased SR Ca2+ load can potentiate SR Ca2+ release via luminal Ca2+-dependent activation of RyRs (Eisner et al. 1998; Lukyanenko et al. 2001). Indeed, we found that augmentation of Ca2+ spark frequency was associated with higher SR Ca2+ content (Fig. 8). Experiments with IAA indicated that these effects of sugar phosphates on SR Ca2+ release and load were dependent on an intact glycolytic pathway. Therefore, at low cytosolic energy reserves sugar phosphates can be utilized by glycolytic enzymes to increase ATP production. Considering that glycolytic enzymes are associated with the SR membrane (Pierce & Philipson, 1985; Xu & Becker, 1998), stimulation of the glycolytic pathway by providing substrate (sugar phosphates) for ATP synthesis will maintain a high ATP/ADP ratio in close proximity to the SR Ca2+-ATPase. A locally high ATP/ADP ratio will normalize the activity of the SR Ca2+ pump and, thereby, eventually increase SR Ca2+ load and release.
Physiological and pathophysiological significance
In the normally oxygenated heart, glycolysis is partially inhibited at the stage of phosphofructokinase (PFK) by high levels of ATP and citrate (Opie, 1991) and does not work at full capacity. As a result of PFK inhibition, levels of FBP (product of PFK reaction) are relatively low under aerobic conditions. The concentration of FBP in the working heart ranges from 8 to 60 nmol (g wet weight)–1 (Depre et al. 1993). Assuming that intracellular water space amounts to 50% of wet weight, this translates into 16–120 µM FBP. These values are in the lower range of concentrations of FBP that can stimulate the cardiac RyR. Glycolytic enzymes are associated with the SR membrane (Pierce & Philipson, 1985) and, hence, are in close proximity to the RyR. Furthermore, the junctional space between T-tubular membrane and terminal SR represents a microcompartment within the cardiac myocyte characterized by restricted diffusion. Therefore, the local concentration of FBP in the junctional space near the RyR is expected to be much higher than the estimates for whole heart tissue mentioned above. Thus, it is likely that concentrations of FBP (and possibly other glycolytic intermediates) are sufficiently high to regulate RyR channel activity and SR Ca2+ release under physiological conditions.
Depending on factors such as workload, extracellular glucose concentration and insulin, concentrations of FBP can vary by a factor of 7 (Depre et al. 1993). For F6P, these variations can be even higher, i.e. up to a factor of 45 (Depre et al. 1993). Interestingly, oscillations of glycolytic intermediates have been observed even during the cardiac cycle (Wikman-Coffelt et al. 1983). Furthermore, glycolysis is enhanced by ischaemia/hypoxia or adrenergic stimulation (Depre et al. 1998). Mild ischaemia significantly enhances glycolysis by stimulation of PFK activity. Glycolytic ATP production is especially important to maintain intracellular Ca2+ homeostasis during ischaemia (Jeremy et al. 1992). Severe ischaemia, however, is associated with accumulation of intracellular lactate, NADH and protons. All these factors inhibit GAPDH (Opie, 1991) and markedly increase FBP levels (substrate for GAPDH reaction). In our study we found that FBP was more effective in stimulating the RyR than other sugar phosphates. Therefore, we suggest that conditions of severe ischaemia can potentially lead to augmentation of SR Ca2+ leak as a result of stimulation of RyR by glycolytic sugar phosphates. It has been shown that accumulation of sugar phosphates due to inhibition of glycolysis caused intracellular Ca2+ overload (Kusuoka & Marban, 1994). Together with a low rate of ATP production, ischaemia would eventually deplete SR Ca2+ content and depress cardiac performance. Thus, FBP and other glycolytic intermediates are important physiologically relevant regulators of RyR-mediated SR Ca2+ release, and therefore cardiac EC coupling.
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References |
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) and absence of iodoacetate (IAA; •). Effect of FBP on Ca2+ spark amplitude in low cytosolic energy reserve solution in the absence (C) and presence of IAA (D). The amplitude of Ca2+ sparks averaged over 2 min under control conditions, from 7 to 10 min after changing to the low cytosolic energy reserve solution and from 6 to 8 min following subsequent application of FBP. *P < 0.05; **P < 0.01 versus control.