Characteristics of Prolonged Ca2+ Release Events Associated With the Nuclei in Adult Cardiac Myocytes
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《循环研究杂志》
From the School of Biomedical Sciences, University of Leeds, Leeds, UK.
Confocal microscopy was used to study the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Prolonged nuclear Ca2+ release (PNCR) events were identified in both intact and permeabilized rat myocytes. PNCR occurred spontaneously and was restricted to localized regions at the ends of the elongated nuclei. Typically, PNCR took the form of a rapid rise in [Ca2+] followed by a maintained plateau. The mean duration of PNCR (1.78±0.19 seconds) was markedly greater than the half decay time for cytosolic Ca2+ sparks (31.2±0.56 ms) obtained under the same conditions. The PNCR width at half maximum amplitude (5.0±0.2 μm) was also significantly greater than that of cytosolic Ca2+ sparks (2.6±0.05 μm) obtained under the same conditions. Experiments involving the use of syto-11 to accurately locate the nuclei demonstrated that PNCR originates from the nuclear envelope or a closely associated structure. The spatial spread of PNCR was asymmetrical, with greater diffusion of Ca2+ toward the center of the nucleus than the cytosol. Both PNCR and Ca2+ sparks were abolished by interventions that deplete SR Ca2+ stores or inhibit RYR activation. Experiments on intact, electrically stimulated cells revealed that diffusion of Ca2+ from the ends of the nucleus toward the center is a prominent feature of the nucleoplasmic Ca2+ transient. The possibility that recruitment of Ca2+ release sites involved in PNCR might influence the temporal and spatial characteristics of the nucleoplasmic [Ca2+] transient is considered.
Key Words: nucleus ， sarcoplasmic reticulum ， Ca2+ release ， sparks
The [Ca2+] within the nucleus regulates a number of important functions including gene transcription and expression, the cell cycle, and apoptosis.1 The double membrane of the nuclear envelope (NE) is spanned by nuclear pore complexes, which allow the passage of solutes and macromolecules <40 kDa. Therefore, nuclear [Ca2+] is at least partly dictated by passive diffusion: global cytosolic [Ca2+] transients or localized Ca2+ release events are typically followed by nuclear [Ca2+] transients, which occur with a delay reflecting diffusion of Ca2+ across the NE.2 However, there is also evidence that Ca2+ can be actively accumulated into the lumen of the NE and then released via Ca2+ channels (ryanodine or IP3 receptors), resulting in a transient rise in nuclear [Ca2+].3–6
The relative influence of these potential sources of nuclear Ca2+ varies widely among cell types: in myocardial cells, differing patterns of nuclear Ca2+ regulation have been reported in embryonic, neonatal, and adult myocytes. In embryonic chick myocytes, nuclear Ca2+ transients can occur without a preceding rise in cytosolic [Ca2+], suggesting that Ca2+ may be released from the NE.7 Neonatal cardiac myocytes exhibited both nuclear Ca2+ transients and high-frequency Ca2+ sparks in the vicinity of the nucleus.8 This study also demonstrated that adult myocytes placed in tissue culture dedifferentiate and develop a pattern of Ca2+ release more typical of neonatal cells. Hence, high-frequency perinuclear Ca2+ sparks and nuclear Ca2+ transients appear characteristic of immature myocardial cells. In part, this may reflect a greater influence of the IP3 signaling pathway in neonatal or cultured cardiac cells.8 In adult ventricular myocytes, Ca2+ sparks arise predominantly at junctional regions of the SR9 and IP3 receptors are expressed at low levels.10 Furthermore, studies on adult ventricular cells have generally concluded that nucleoplasmic [Ca2+] rises passively because of diffusion from the cytosol, without any contribution from NE derived Ca2+.2,11–13 However, two features of nuclear Ca2+ regulation in adult myocytes have yet to be explained. First, the nuclear Ca2+ transient lasts significantly longer than the cytosolic Ca2+ transient.2,11 Second, the rise in [Ca2+] within the nucleus is biphasic,13 suggesting the possible involvement of a secondary active process.
In the present study, confocal microscopy was used to investigate the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Evidence is provided of long-lasting Ca2+ release events, which occur spontaneously in localized regions at the ends of the elongated nuclei in both permeabilized and intact calls. The properties of these Ca2+ release events and their possible influence on the spatial and temporal properties of the nuclear [Ca2+] transient in intact cells are considered.
Adult Wistar rats (220 to 250 g; University of Leeds, Leeds, UK) were killed in accordance with the UK Home Office Guidance on the Operation of Animals (Scientific Procedures) Act of 1986. Ventricular myocytes were isolated by collagenase digestion as described previously.14
Experiments on Permeabilized Cells
The isolated cells were permeabilized by exposure to saponin (10 μg/mL) in a mock intracellular solution for 6-minute, before centrifugation and resuspension. The intracellular solution contained (mmol/L) 100 KCl, 25 HEPES, 0.36 EGTA, 10 phosphocreatine, 5 ATP, and fluo-3 acid (5 μmol/L); pH 7.0, 20°C to 22°C. The free [Ca2+] and [Mg2+] were adjusted to 200 nmol/L and 1 mmol/L, respectively, by addition of CaCl2 and MgCl2. In most experiments on skinned cells, 5 mmol/L azide was present to inhibit mitochondrial activity. However, azide had no apparent influence on the phenomena reported in the present study. Syto-11 (Molecular Probes), was used as a fluorescent marker of the cell nucleus (for further details see the Expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org).
Experiments on Intact Cells
Intact myocytes were perfused with solutions containing (mmol/L) 113 NaCl, 5.4 KCl, 1 MgCl2, 1.0 CaCl2, 0.37 Na2HPO4, 5.5 glucose, and 5 HEPES; 20°C to 22°C, pH 7.1. In some experiments, 5 mmol/L 2,3-butanedione monoxime (BDM) was added to prevent movement artifacts associated with contraction. This level of BDM markedly inhibits the contractile response with minimal effect on SR Ca2+ release.15 However, the properties of the Ca2+ release events reported in the present study appeared similar in the presence or absence of BDM. Changes in cytosolic [Ca2+] were detected by loading the myocytes with fluo-3AM (5 μmol/L) for 5 minutes at room temperature (20°C to 22°C) as previously described.14
Identification of Ca2+ stored within organelles was achieved by loading the cells with fluo-5N as previously described.16 Briefly, intact cells suspended in a solution containing 10 μmol/L fluo-5N AM (Molecular Probes) for 2 hours at 37°C. A 1-hour period was then allowed for de-esterification before commencing the experiment. In some experiments, the Golgi apparatus was identified by exposure of intact cells to 1 μmol/L BODIPY FL C5-ceramide (Molecular Probes) for 1 minute.
Confocal Microscopy
The experimental chamber was placed on the stage of a Nikon Diaphot Eclipse TE2000 inverted microscope, and the cells were viewed using a 60x water immersion lens (Plan Apo, NA 1.2). A confocal laser-scanning unit (Bio-Rad, Microradiance 2000) was attached to the side port of the microscope. The dyes were excited at 488 nm and emitted fluorescence was measured at >515 nm. Image processing and analysis were done using IDL (Research Systems Inc) and Laserpix (Bio-Rad) and ImageJ (http://rsb.info.nih.gov/ij/) software. Further information regarding the optical properties of the confocal system and image analysis methods are published elsewhere.17
Statistics and Curve Fitting
Results are presented as the mean±SEM. Where appropriate, statistical significance was assessed using a t test. Differences between means were considered significant at P<0.05. Statistics and curve fitting were performed using Origin software (Microcal, UK).
Identification of Prolonged Ca2+ Release Events Associated With Nuclei in Permeabilized Cells
Figure 1 shows sequential x-y images collected at 0.5 Hz from a permeabilized ventricular myocyte. At this low frame rate, cytosolic Ca2+ sparks are captured occasionally and, because of their inherently brief nature, occupy only a single frame. However, frames 5 to 8 show a much more prolonged Ca2+ release event lasting at least 1.5 seconds and with a greater spatial spread than typical Ca2+ sparks. Further examination of the cell using interference microscopy suggested that the prolonged Ca2+ release event occurred close to one end of a nucleus.
Figure 1. Sequential x-y images obtained at 0.5 Hz from a saponin permeabilized ventricular myocyte. Occasional brief cytosolic Ca2+ sparks are apparent (S). Frames 5 to 8 indicate the presence of a single prolonged Ca2+ release event (L) lasting at least 1.5 seconds. Bar=10 μm.
To gain more information regarding the temporal and spatial properties of these prolonged nuclear Ca2+ release (PNCR) events, further experiments were performed in line scan mode. Most ventricular myocytes had two elongated nuclei oriented along the mid axis of the cell. Figure 2A (top) shows a line scan (x-t) image obtained by positioning the scan line longitudinally through both nuclei of a permeabilized rat ventricular myocyte. In this example, a single PNCR was detected from a region of the cell corresponding to one of the nuclei. Much briefer cytosolic Ca2+ sparks are also apparent in the same image. Figure 2A (bottom) shows an x-y image of the same cell after exposure to syto-11, which allows the boundary of the nucleoplasm and the NE to be defined. The nucleus on the right of the image is apparent as the region of highest fluorescence and is in sharp focus. The x-y and x-t images have been aligned to illustrate that the peak of PNCR coincides with the left edge of the nuclear fluorescence, suggesting that the site of origin is the NE or a closely associated structure. Figure 2B shows a surface plot and line profiles derived from the data shown in Figure 2A. The initial rise in [Ca2+] peaked at F/Fo=2.0 and was followed by a plateau, which lasted 1.4 seconds, before decreasing to cytosolic levels. In contrast, cytosolic Ca2+ sparks were much briefer and did not exhibit a plateau.
Figure 2. A, Line scan image of fluo-3 fluorescence obtained with the scan line positioned longitudinally through both nuclei of a permeabilized ventricular myocyte (top) and an x-y image from the same cell following exposure to 250 nmol/L syto-11 (bottom). The top and bottom images have been aligned to show that the intersection of the horizontal scan line (white) and the vertical line passing through the peak of the PNCR (red), coincides with the left edge of the nucleus. B, Surface plot of the line scan image shown in A (top), and selected line profiles obtained from PNCR and cytosolic Ca2+ sparks as indicated (bottom). C, left, Surface plot of PNCR event from another cell showing the asymmetric spread of Ca2+ into cytosolic and nuclear compartments. Normalized data illustrating the signal-averaged width of PNCR events from 12 cells is also shown (right). Vertical lines indicate ±1 SEM. Mean value of x1 (3.3±0.08 μm) was significantly different (P<0.01) from that of x2 (4.2±0.08 μm), confirming that Ca2+ diffused further into the nuclear compartment.
PNCR was typically associated with a limited diffusion of Ca2+, both toward the center of the nucleus and toward the cytosol (eg, Figure 2A). However, Ca2+ diffused further into the nucleoplasm than into the cytosol: Figure 2C (left), shows a surface plot of a PNCR event, which has been oriented to show the spatial spread of Ca2+ into the cytosolic and nuclear compartments. In this example, the asymmetry is particularly apparent, with greater spread into the nucleoplasm. As the plateau declined, the spatial spread also decreased gradually, before Ca2+ release suddenly terminated. Normalized data illustrating the signal-averaged spatial profiles of PNCR events from 12 cells is also shown (right). As suggested in relation to other perinuclear Ca2+ release events, the asymmetrical Ca2+ spread probably reflects differences in the density of Ca2+binding sites between the nuclear and cytosolic compartments.18
On average, PNCR was detected with equal frequency at the left and right edges of the nuclei (n=68 cells), and in-focus events were consistently located at or near the nuclear boundary, as defined by syto-11 fluorescence. However, PNCR was not detected when the scan line was positioned transversely across the midpoint of each nucleus (not shown), indicating that the events are localized to specific regions at the ends of the elongated nuclei. PNCR was not observed following inhibition of RyR activation with high levels of ryanodine (20 μmol/L, not shown). Similarly, PNCR was not detected after depletion of intracellular Ca2+ stores by introduction of 10 mmol/L caffeine (to activate RYR) or 20 μmol/L cyclopiazonic acid (to inhibit SERCA). In contrast to results obtained on some other cell types,6,19 application of IP3 did not induce or modify PNCR.
Further Properties of Long-Lasting Ca2+ Release Events
On average, PNCR was detected at a frequency of 2.5±0.3 (n=33) events per minute, per nucleus. However, in some cells, PNCR occurred at a higher frequency from the same focal point (Figure 3A). Typically, in-focus PNCR events exhibited a rapid rising phase, which peaked at F/Fo=2.0 (see next section for cumulative data). However, in approximately 5% of cells, rapid changes between full and half maximum amplitude were observed (Figure 3B). The longest PNCR event detected lasted 8 seconds, indicating the presence of a substantial Ca2+ pool (Figure 4C). Similar prolonged events were observed in intact cells loaded with fluo-3AM (Figure 3D).
Figure 3. A, Example of repetitive PNCR events originating from the same site in a permeabilized myocyte. B, Example of rapid changes between full and half normal amplitude in a permeabilized myocyte. C, PNCR event lasting 8 seconds. D, PNCR event detected in an intact quiescent myocyte loaded with fluo-3. Bar=10 μm.
Figure 4. Cumulative data showing histograms of amplitude (F/Fo), full width at half maximum amplitude (FWHM), and duration of Ca2+ sparks (A) and prolonged Ca2+ release events (B). Duration of Ca2+ sparks is measured as the half decay time, whereas the duration of PNCR is given as the plateau phase. Note the difference in time scales.
Cumulative Data
Histograms illustrating the temporal and spatial properties of PNCR and cytosolic Ca2+ sparks are given in Figure 4. There was no significant difference between the mean amplitude of Ca2+ sparks (F/Fo=2.0±0.03, n=361) and PNCR events (F/Fo=2.0±0.04, n=68). The mean duration of PNCR was significantly greater (1.78±0.19 seconds, n=68) than the half decay time for Ca2+ sparks (31.2±0.56 ms, n=361; P<0.01). The PNCR half width at the point of maximum amplitude (5.0±0.2 μm, n=68) was also significantly greater than that of typical Ca2+ sparks (2.6±0.05 μm, n=311; P<0.01). Whereas the amplitude distribution of Ca2+ sparks exhibited a typical Gaussian relationship (as in most previous studies), PNCR events appeared more bell shaped, with a higher proportion of low amplitude events being detected. This may reflect the fact that low amplitude events are easier to discriminate from background noise when they last seconds as opposed to milliseconds.
As shown in Figure 2, PNCR typically exhibited a rapid rising phase followed by a slow monotonic decrease, before the final sudden decline in amplitude on termination of Ca2+ release. At the point of release termination, the mean PNCR amplitude was 57.3±2.4% (n=68) of the initial peak. The spatial spread of PNCR also decreased as the amplitude declined (eg, Figure 2C). On average, PNCR width (FWHM) decreased by 20.1±2.5% (n=68) at the point of release termination.
Changes in Nuclear [Ca2+]i in Intact Cells During Electrical Stimulation
Further experiments were performed on intact myocytes to establish whether the inward diffusion of Ca2+ from the ends of the nuclei plays a role in the beat-to-beat regulation of nuclear [Ca2+]. Figure 5A shows an x-t image of an intact ventricular myocyte during an electrically stimulated response. The scan line was positioned longitudinally through one of the nuclei. On stimulation, the rapid rise in [Ca2+] resulted in a horizontal line across cytosolic regions of the line scan image. The [Ca2+] also increased within the nucleus, but with a delay reflecting diffusion of Ca2+ across the NE. As reported previously,2,11 the rise in [Ca2+] within the nucleus was markedly prolonged relative to the cytosolic Ca2+ transient. However, one feature not highlighted in earlier studies is the apparent ‘V’ in the fluorescence signal, joining the ends of the nucleus. This is consistent with diffusion of Ca2+ from the ends of the organelle toward the center. The expanded panel (bottom) shows the same image, with the nucleoplasmic region blanked and the time scale compressed. The arrows indicate regions of high localized Ca2+, which appear to spread outwards into the cytosol before slowly receding.
Figure 5. A, top, Line scan image of an intact ventricular myocyte during electrical stimulation, with the scan line positioned longitudinally through the center of a nucleus. The same image is shown on a different aspect ratio with the nuclear region blanked out (bottom). Arrows indicate apparent outward diffusion of Ca2+ toward the cytosol. B, Line profiles obtained from the cytosol (i), just inside the nucleus (ii), and the center of the nucleus (iii). Line profiles ii and iii were obtained by averaging over 3 pixels, whereas i was averaged over a wider region as indicated. Red arrow indicates the beginning of a secondary rising phase in line profile (ii). C, Cumulative data illustrating the mean time to peak (measured at 90% max, TP90) and the mean half time of the descending phase (TD50). *TP90 and TP50 values within the nucleus that are significantly different from the corresponding cytosolic values (P<0.05). D, Sequential x-y images obtained from another cell at 80 ms intervals after electrical stimulation. Note the bright regions at the ends of the nucleus, which then spread toward the center.
Selected line profiles (Figure 5B) show the change in [Ca2+] within the cytosol (i), just inside the nucleus (ii), and at the center of the nucleus (iii). The rise in [Ca2+] just inside the NE was slightly delayed compared with the cytosolic Ca2+ transient and was typically monotonic. However, the rise in [Ca2+] at the center of the nucleus was markedly slower and the maximum level lower than at the ends of the nucleus. In addition, the line profile from the center of the nucleus exhibited a secondary rise in [Ca2+] (red arrow). Comparison of the line profile and the line scan, suggest that the secondary rise in Ca2+ reflects the influence of Ca2+ diffusing inwards from the ends of the nucleus. Similar results were obtained in 11 other preparations.
Cumulative data illustrating the time courses of the rising and falling phases in regions (i), (ii), and (iii) is given in Figure 5C. The mean propagation rate of Ca2+ from the ends of the nucleus toward the center was 49.8±3.7 μm/sec (n=12). The time to peak (measured at 90% max) of the cytosolic Ca2+ transient (i), and regions (ii) and (iii) of the nucleus was 20.4±1.8 ms, 46.5±5.4 ms, and 157.8±15.1 ms (n=12), respectively. The corresponding descending phases declined with half times of 398.3±49.3 ms, 589.2±71.8, and 598.3±82 ms (n=12).
The role of Ca2+ diffusion from the ends of the nucleus toward the center is further highlighted by sequential x-y images obtained from another cell after electrical stimulation (Figure 5D). The selected frames reveal localized regions of high fluorescence at the ends of the nucleus. These regions broaden, producing a characteristic "dumbbell" appearance, before spreading inwards toward the center of the nucleus. Similar results were obtained in 6 other preparations.
Location of Intracellular Ca2+ Storage Sites in Intact Cells
The distribution of stored intracellular Ca2+ was investigated by exposure of intact cardiac cells to fluo-5N AM, under conditions that favor compartmentalization of dye in the organelles. Figure 6A shows an x-y image of fluo-5N fluorescence centered on one of the two nuclei. As previously reported, a characteristic sarcomeric pattern of staining is present, which corresponds to dye trapped within the lumen of the SR.16 The NE is also visible because of Ca2+ stored within the perinuclear space. However, another consistent feature was the presence of localized areas of high fluorescence at the ends of the nuclei, suggesting regions of high organelle Ca2+.
Figure 6. A Intact cardiac cell after loading with Fluo-5N (top). Fluo-5N–loaded cell before (left) and after (right) exposure to 10 mmol/L caffeine and 20 μmol/L CPA (bottom). B, Intact cell after exposure to BODIPY C5-ceramide. The Golgi apparatus is indicated by the regions of high fluorescence at the ends of the nucleus.
Unlike previous reports in rabbit myocytes,16 electrical stimulation did not result in a decrease in Fluo-5N fluorescence (not shown). This might reflect (1) a higher than expected Ca2+ affinity of the de-esterified dye complexes and/or (2) a higher luminal [Ca2+] within the SR of rat compared with rabbit myocytes. However, a pronounced reduction in both sarcomeric and perinuclear fluorescence did occur after maximal depletion of the SR Ca2+ stores by inhibition of SERCA (with CPA) followed by RYR activation by application of 10 mmol/L caffeine (Figure 6B). This effect was present, but less pronounced with caffeine alone (not shown), which may reflect the that fact that the SR can retain Ca2+ in the presence of caffeine, when SERCA is active.20
The bright region at the ends of the nuclei might reflect Ca2+ stored in the NE or adjoining regions of the SR. However, ultrastructural studies suggest that the Golgi apparatus (GA) is also closely associated with the ends of the nucleus.21 Therefore, in some experiments, the GA was identified using BODIPY FL C5-ceramide. As shown in Figure 6B, the GA was clearly located in distinct regions at both ends of the nucleus. Similar results were obtained in 5 other cells.
Based on the cumulative data (Figure 4), typical spontaneous PNCR events peak at around F/Fo=2, has a half width of 5 μm and lasts for 1.7 seconds. To our knowledge, such prolonged Ca2+ release events have not been reported previously in ventricular myocytes in the absence of pharmacological modification. One important consideration is that PNCR might be an artifact of the experimental conditions or the result of localized damage. However, this seems unlikely because (1) similar events were observed in intact cells loaded with fluo-3 AM (eg, Figure 3D), confirming that PNCR is not a consequence of cell permeabilization, and (2) PNCR was detected in x-y images at low frame rates (eg, Figure 1) and at lower laser intensities (not shown). This suggests that laser damage is unlikely to be the underlying cause of PNCR.
Origin of Spontaneous PNCR Events
Previous work has shown that localized Ca2+ release arising in close proximity to the NE can have a major influence on nucleoplasmic [Ca2+].18 Therefore, the possible involvement of juxtanuclear organelles in PNCR must be considered: the cytosolic region immediately beyond the "poles" of the elongated nuclei contains SR, mitochondria, and the GA.21 The mitochondria are an unlikely source of Ca2+ because spontaneous PNCR was observed in the presence or absence of mitochondrial inhibitors. It is less easy to draw firm conclusions about the involvement of the NE, SR, or GA. Both the SR and the NE membranes of cardiac cells contain SERCA2a and RYR2.7,22 In addition, the lumen of the SR/ER is continuous with the lumen of the NE. Therefore, any factor affecting the Ca2+ content of the SR will affect the NE and vice versa. Recent work has also highlighted the possible role of the GA as a functional Ca2+ store23: the GA has two forms of Ca2+ pump (SERCA and a P-type SPCA) and in some cells types IP3 and ryanodine (RYR2) receptors are also associated with the GA membrane.24,25
In the present study, PNCR was abolished by inhibition of SERCA (with CPA), or drugs, which activate or inhibit RYR function (caffeine or ryanodine). Comparison of the images obtained using fluo-5N loading (Figure 6A) and C5-ceramide (Figure 6B) suggest that the regions of high [Ca2+] at the ends of the nuclei may partly reflect Ca2+ localized in the GA. However, exposure to caffeine and thapsigargin appeared to deplete Ca2+ within the SR, NE, and the regions at the nuclear poles (Figure 6A). Therefore, the shared components of the Ca2+ regulatory systems may preclude pharmacological differentiation between these potential sources of Ca2+. Despite this limitation, several features of PNCR are more consistent with the involvement of the SR or NE than the GA. First, PNCR was abolished by high levels of ryanodine (20 μmol/L), but was not induced or modified by IP3. This suggests that PNCR is likely to involve Ca2+ efflux via RYRs. Second, activation of Ca2+ release from the GA in response to RYR agonists has not been demonstrated in any cell type, suggesting that the NE or adjoining regions of the SR are more probable sources of Ca2+. Whatever its origin, the functional importance of PNCR primarily reflects its location and influence on nuclear Ca2+.
Temporal Properties of Ca2+ Sparks and Prolonged Ca2+ Release Events
A number of mechanisms have been proposed to explain the temporal properties of Ca2+ sparks and long-lasting Ca2+ release events observed after pharmacological modification of RYR function. These include (1) Ca2+-dependent channel inactivation or adaptation,26 (2) partial depletion of luminal Ca2+,27 or (3) stochastic attrition.28 Models involving only one of these potential mechanisms have generally failed to describe accurately the properties of Ca2+ sparks. Moreover, such models are limited by uncertainties over the single RYR Ca2+ flux and the number of channels activated during a Ca2+ spark or a prolonged Ca2+ release event. If sparks reflect the activation of tens or perhaps hundreds of RYRs, simple explanations based on stochastic attrition (simultaneous stochastic closing of RYRs within a cluster) can be excluded. However, recent models incorporating evidence that RYRs can be functionally coupled,29 with other features such as Ca2+ depletion of the SR, have proved more successful.30
It has been shown that pharmacological modification of the RyR can result in long-lasting Ca2+ release events, with similar properties to PNCR., eg, Ca2+ sparks with prolonged "embers" can be induced in cardiac cells by the drugs FK-506 or rapamycin,31 which dissociate FKBP from RYR.32 In this case, it was suggested that Ca2+ release is prolonged because of reduced coupling between RYRs, which impairs the ability of closed channels to facilitate the inactivation of others. However, long-lasting events can also be induced by other substances including ryanoids because of induction of long-lasting subconductance states.33 The fact that modification of RYR gating can produce such long-lasting events confirms that complete depletion of local Ca2+ stores cannot explain the normally brief nature of Ca2+ sparks. However, partial depletion may contribute to spark inactivation by reducing the open probability of the RyR.27
The present study shows that long-lasting Ca2+ release events can occur from localized regions at the ends of the nuclei in the absence of pharmacological agents. The prolonged nature of these events might reflect local structural characteristics, eg, the number of RYRs per cluster, the organization/packing of RYRs, or the degree of coupling conferred by FKBP. Other factors, such as the local phosphorylation levels of phospholamban or RyR may also influence channel inactivation characteristics, thereby prolonging Ca2+ release. Interestingly, "hotspots" of colocalized PKA and calcineurin have been reported to exist at the ends of the nuclei in adult cardiac myocytes (see Figure 7 in Santana et al34).
Recent models suggest that a maintained Ca2+ flux of 10% to 20% of that associated with a Ca2+ spark is sufficient to produce a prolonged Ca2+ release event, similar to that shown in Figure 2. 30 The rapid transitions between full and half maximal amplitude that occur in some PNCR events (Figure 3B) could be explained by a number of possible mechanisms, eg, if PNCR involves the maintained activation of a single RyR, then a 50% decrease in amplitude might reflect the adoption of a subconductance state. However, if many RYRs are active during PNCR then such a decrease in amplitude could be explained if tight coupling between active RyRs enables a simultaneous transition to a subconductance state.33
Physiological Relevance
The spontaneous PNCR events observed in skinned cells (Figure 1) are unlikely to have a role under physiological conditions. This is because the mean duration of PNCR is substantially greater than the cardiac cycle and each rise in [Ca2+] during systole would be expected to disrupt or entrain spontaneous PNCR. However, spontaneous PNCR may play a role in controlling Ca2+ dependent nuclear processes under pathological conditions, when the cell becomes quiescent and Ca2+ regulation is impaired, eg, during ischemia or reperfusion.35
One important consideration is whether the Ca2+ release sites, which underlie spontaneous PNCR in skinned and intact cells, are activated during the electrically stimulated Ca2+ transient. Evidence of Ca2+ diffusion from the ends of the nuclei toward the center is consistent with this possibility (Figure 5A). Furthermore, as with spontaneous PNCR (Figure 2), there is evidence of a prolonged outward diffusion of Ca2+ from a region at the edge of the nucleus toward the cytosol (Figure 5A, bottom). This pattern of Ca2+ release cannot readily be explained by simple inward Ca2+ diffusion from the cytosol to the nucleoplasm and suggests that the Ca2+ release sites, which underlie spontaneous PNCR, are activated during the electrically stimulated Ca2+ transient. PNCR might be triggered via CICR, following each global rise in cytosolic [Ca2+], or by Ca2+ entry across closely associated t-tubule membranes.
Assuming the Ca2+ release sites, which underlie PNCR are recruited during each beat, then the inherently prolonged nature of the underlying Ca2+ release process may contribute to the long-lasting nucleoplasmic Ca2+ transient (eg, Figure 5A). Such a mechanism could be of considerable importance given that nuclear Ca2+ is involved in the regulation gene transcription and expression. Of particular interest to the cardiac field, recent work has shown that changes in nucleoplasmic Ca2+ plays a central role in the development of hypertrophy.1
The present study has identified novel long-lasting Ca2+ release events, which occur spontaneously at the ends of the nuclei in both permeabilized and intact myocytes. In intact electrically stimulated cells, the inward diffusion of Ca2+ from the ends of the nuclei is a prominent feature of the nucleoplasmic Ca2+ transient and this appears to explain the biphasic increase in [Ca2+] at the center of the organelle. The association of PNCR events with the ends of the nuclei suggests that recruitment of the underlying Ca2+ release sites during systole may contribute to the beat-to-beat regulation of nuclear Ca2+.
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Confocal microscopy was used to study the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Prolonged nuclear Ca2+ release (PNCR) events were identified in both intact and permeabilized rat myocytes. PNCR occurred spontaneously and was restricted to localized regions at the ends of the elongated nuclei. Typically, PNCR took the form of a rapid rise in [Ca2+] followed by a maintained plateau. The mean duration of PNCR (1.78±0.19 seconds) was markedly greater than the half decay time for cytosolic Ca2+ sparks (31.2±0.56 ms) obtained under the same conditions. The PNCR width at half maximum amplitude (5.0±0.2 μm) was also significantly greater than that of cytosolic Ca2+ sparks (2.6±0.05 μm) obtained under the same conditions. Experiments involving the use of syto-11 to accurately locate the nuclei demonstrated that PNCR originates from the nuclear envelope or a closely associated structure. The spatial spread of PNCR was asymmetrical, with greater diffusion of Ca2+ toward the center of the nucleus than the cytosol. Both PNCR and Ca2+ sparks were abolished by interventions that deplete SR Ca2+ stores or inhibit RYR activation. Experiments on intact, electrically stimulated cells revealed that diffusion of Ca2+ from the ends of the nucleus toward the center is a prominent feature of the nucleoplasmic Ca2+ transient. The possibility that recruitment of Ca2+ release sites involved in PNCR might influence the temporal and spatial characteristics of the nucleoplasmic [Ca2+] transient is considered.
Key Words: nucleus ， sarcoplasmic reticulum ， Ca2+ release ， sparks
The [Ca2+] within the nucleus regulates a number of important functions including gene transcription and expression, the cell cycle, and apoptosis.1 The double membrane of the nuclear envelope (NE) is spanned by nuclear pore complexes, which allow the passage of solutes and macromolecules <40 kDa. Therefore, nuclear [Ca2+] is at least partly dictated by passive diffusion: global cytosolic [Ca2+] transients or localized Ca2+ release events are typically followed by nuclear [Ca2+] transients, which occur with a delay reflecting diffusion of Ca2+ across the NE.2 However, there is also evidence that Ca2+ can be actively accumulated into the lumen of the NE and then released via Ca2+ channels (ryanodine or IP3 receptors), resulting in a transient rise in nuclear [Ca2+].3–6
The relative influence of these potential sources of nuclear Ca2+ varies widely among cell types: in myocardial cells, differing patterns of nuclear Ca2+ regulation have been reported in embryonic, neonatal, and adult myocytes. In embryonic chick myocytes, nuclear Ca2+ transients can occur without a preceding rise in cytosolic [Ca2+], suggesting that Ca2+ may be released from the NE.7 Neonatal cardiac myocytes exhibited both nuclear Ca2+ transients and high-frequency Ca2+ sparks in the vicinity of the nucleus.8 This study also demonstrated that adult myocytes placed in tissue culture dedifferentiate and develop a pattern of Ca2+ release more typical of neonatal cells. Hence, high-frequency perinuclear Ca2+ sparks and nuclear Ca2+ transients appear characteristic of immature myocardial cells. In part, this may reflect a greater influence of the IP3 signaling pathway in neonatal or cultured cardiac cells.8 In adult ventricular myocytes, Ca2+ sparks arise predominantly at junctional regions of the SR9 and IP3 receptors are expressed at low levels.10 Furthermore, studies on adult ventricular cells have generally concluded that nucleoplasmic [Ca2+] rises passively because of diffusion from the cytosol, without any contribution from NE derived Ca2+.2,11–13 However, two features of nuclear Ca2+ regulation in adult myocytes have yet to be explained. First, the nuclear Ca2+ transient lasts significantly longer than the cytosolic Ca2+ transient.2,11 Second, the rise in [Ca2+] within the nucleus is biphasic,13 suggesting the possible involvement of a secondary active process.
In the present study, confocal microscopy was used to investigate the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Evidence is provided of long-lasting Ca2+ release events, which occur spontaneously in localized regions at the ends of the elongated nuclei in both permeabilized and intact calls. The properties of these Ca2+ release events and their possible influence on the spatial and temporal properties of the nuclear [Ca2+] transient in intact cells are considered.
Adult Wistar rats (220 to 250 g; University of Leeds, Leeds, UK) were killed in accordance with the UK Home Office Guidance on the Operation of Animals (Scientific Procedures) Act of 1986. Ventricular myocytes were isolated by collagenase digestion as described previously.14
Experiments on Permeabilized Cells
The isolated cells were permeabilized by exposure to saponin (10 μg/mL) in a mock intracellular solution for 6-minute, before centrifugation and resuspension. The intracellular solution contained (mmol/L) 100 KCl, 25 HEPES, 0.36 EGTA, 10 phosphocreatine, 5 ATP, and fluo-3 acid (5 μmol/L); pH 7.0, 20°C to 22°C. The free [Ca2+] and [Mg2+] were adjusted to 200 nmol/L and 1 mmol/L, respectively, by addition of CaCl2 and MgCl2. In most experiments on skinned cells, 5 mmol/L azide was present to inhibit mitochondrial activity. However, azide had no apparent influence on the phenomena reported in the present study. Syto-11 (Molecular Probes), was used as a fluorescent marker of the cell nucleus (for further details see the Expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org).
Experiments on Intact Cells
Intact myocytes were perfused with solutions containing (mmol/L) 113 NaCl, 5.4 KCl, 1 MgCl2, 1.0 CaCl2, 0.37 Na2HPO4, 5.5 glucose, and 5 HEPES; 20°C to 22°C, pH 7.1. In some experiments, 5 mmol/L 2,3-butanedione monoxime (BDM) was added to prevent movement artifacts associated with contraction. This level of BDM markedly inhibits the contractile response with minimal effect on SR Ca2+ release.15 However, the properties of the Ca2+ release events reported in the present study appeared similar in the presence or absence of BDM. Changes in cytosolic [Ca2+] were detected by loading the myocytes with fluo-3AM (5 μmol/L) for 5 minutes at room temperature (20°C to 22°C) as previously described.14
Identification of Ca2+ stored within organelles was achieved by loading the cells with fluo-5N as previously described.16 Briefly, intact cells suspended in a solution containing 10 μmol/L fluo-5N AM (Molecular Probes) for 2 hours at 37°C. A 1-hour period was then allowed for de-esterification before commencing the experiment. In some experiments, the Golgi apparatus was identified by exposure of intact cells to 1 μmol/L BODIPY FL C5-ceramide (Molecular Probes) for 1 minute.
Confocal Microscopy
The experimental chamber was placed on the stage of a Nikon Diaphot Eclipse TE2000 inverted microscope, and the cells were viewed using a 60x water immersion lens (Plan Apo, NA 1.2). A confocal laser-scanning unit (Bio-Rad, Microradiance 2000) was attached to the side port of the microscope. The dyes were excited at 488 nm and emitted fluorescence was measured at >515 nm. Image processing and analysis were done using IDL (Research Systems Inc) and Laserpix (Bio-Rad) and ImageJ (http://rsb.info.nih.gov/ij/) software. Further information regarding the optical properties of the confocal system and image analysis methods are published elsewhere.17
Statistics and Curve Fitting
Results are presented as the mean±SEM. Where appropriate, statistical significance was assessed using a t test. Differences between means were considered significant at P<0.05. Statistics and curve fitting were performed using Origin software (Microcal, UK).
Identification of Prolonged Ca2+ Release Events Associated With Nuclei in Permeabilized Cells
Figure 1 shows sequential x-y images collected at 0.5 Hz from a permeabilized ventricular myocyte. At this low frame rate, cytosolic Ca2+ sparks are captured occasionally and, because of their inherently brief nature, occupy only a single frame. However, frames 5 to 8 show a much more prolonged Ca2+ release event lasting at least 1.5 seconds and with a greater spatial spread than typical Ca2+ sparks. Further examination of the cell using interference microscopy suggested that the prolonged Ca2+ release event occurred close to one end of a nucleus.
Figure 1. Sequential x-y images obtained at 0.5 Hz from a saponin permeabilized ventricular myocyte. Occasional brief cytosolic Ca2+ sparks are apparent (S). Frames 5 to 8 indicate the presence of a single prolonged Ca2+ release event (L) lasting at least 1.5 seconds. Bar=10 μm.
To gain more information regarding the temporal and spatial properties of these prolonged nuclear Ca2+ release (PNCR) events, further experiments were performed in line scan mode. Most ventricular myocytes had two elongated nuclei oriented along the mid axis of the cell. Figure 2A (top) shows a line scan (x-t) image obtained by positioning the scan line longitudinally through both nuclei of a permeabilized rat ventricular myocyte. In this example, a single PNCR was detected from a region of the cell corresponding to one of the nuclei. Much briefer cytosolic Ca2+ sparks are also apparent in the same image. Figure 2A (bottom) shows an x-y image of the same cell after exposure to syto-11, which allows the boundary of the nucleoplasm and the NE to be defined. The nucleus on the right of the image is apparent as the region of highest fluorescence and is in sharp focus. The x-y and x-t images have been aligned to illustrate that the peak of PNCR coincides with the left edge of the nuclear fluorescence, suggesting that the site of origin is the NE or a closely associated structure. Figure 2B shows a surface plot and line profiles derived from the data shown in Figure 2A. The initial rise in [Ca2+] peaked at F/Fo=2.0 and was followed by a plateau, which lasted 1.4 seconds, before decreasing to cytosolic levels. In contrast, cytosolic Ca2+ sparks were much briefer and did not exhibit a plateau.
Figure 2. A, Line scan image of fluo-3 fluorescence obtained with the scan line positioned longitudinally through both nuclei of a permeabilized ventricular myocyte (top) and an x-y image from the same cell following exposure to 250 nmol/L syto-11 (bottom). The top and bottom images have been aligned to show that the intersection of the horizontal scan line (white) and the vertical line passing through the peak of the PNCR (red), coincides with the left edge of the nucleus. B, Surface plot of the line scan image shown in A (top), and selected line profiles obtained from PNCR and cytosolic Ca2+ sparks as indicated (bottom). C, left, Surface plot of PNCR event from another cell showing the asymmetric spread of Ca2+ into cytosolic and nuclear compartments. Normalized data illustrating the signal-averaged width of PNCR events from 12 cells is also shown (right). Vertical lines indicate ±1 SEM. Mean value of x1 (3.3±0.08 μm) was significantly different (P<0.01) from that of x2 (4.2±0.08 μm), confirming that Ca2+ diffused further into the nuclear compartment.
PNCR was typically associated with a limited diffusion of Ca2+, both toward the center of the nucleus and toward the cytosol (eg, Figure 2A). However, Ca2+ diffused further into the nucleoplasm than into the cytosol: Figure 2C (left), shows a surface plot of a PNCR event, which has been oriented to show the spatial spread of Ca2+ into the cytosolic and nuclear compartments. In this example, the asymmetry is particularly apparent, with greater spread into the nucleoplasm. As the plateau declined, the spatial spread also decreased gradually, before Ca2+ release suddenly terminated. Normalized data illustrating the signal-averaged spatial profiles of PNCR events from 12 cells is also shown (right). As suggested in relation to other perinuclear Ca2+ release events, the asymmetrical Ca2+ spread probably reflects differences in the density of Ca2+binding sites between the nuclear and cytosolic compartments.18
On average, PNCR was detected with equal frequency at the left and right edges of the nuclei (n=68 cells), and in-focus events were consistently located at or near the nuclear boundary, as defined by syto-11 fluorescence. However, PNCR was not detected when the scan line was positioned transversely across the midpoint of each nucleus (not shown), indicating that the events are localized to specific regions at the ends of the elongated nuclei. PNCR was not observed following inhibition of RyR activation with high levels of ryanodine (20 μmol/L, not shown). Similarly, PNCR was not detected after depletion of intracellular Ca2+ stores by introduction of 10 mmol/L caffeine (to activate RYR) or 20 μmol/L cyclopiazonic acid (to inhibit SERCA). In contrast to results obtained on some other cell types,6,19 application of IP3 did not induce or modify PNCR.
Further Properties of Long-Lasting Ca2+ Release Events
On average, PNCR was detected at a frequency of 2.5±0.3 (n=33) events per minute, per nucleus. However, in some cells, PNCR occurred at a higher frequency from the same focal point (Figure 3A). Typically, in-focus PNCR events exhibited a rapid rising phase, which peaked at F/Fo=2.0 (see next section for cumulative data). However, in approximately 5% of cells, rapid changes between full and half maximum amplitude were observed (Figure 3B). The longest PNCR event detected lasted 8 seconds, indicating the presence of a substantial Ca2+ pool (Figure 4C). Similar prolonged events were observed in intact cells loaded with fluo-3AM (Figure 3D).
Figure 3. A, Example of repetitive PNCR events originating from the same site in a permeabilized myocyte. B, Example of rapid changes between full and half normal amplitude in a permeabilized myocyte. C, PNCR event lasting 8 seconds. D, PNCR event detected in an intact quiescent myocyte loaded with fluo-3. Bar=10 μm.
Figure 4. Cumulative data showing histograms of amplitude (F/Fo), full width at half maximum amplitude (FWHM), and duration of Ca2+ sparks (A) and prolonged Ca2+ release events (B). Duration of Ca2+ sparks is measured as the half decay time, whereas the duration of PNCR is given as the plateau phase. Note the difference in time scales.
Cumulative Data
Histograms illustrating the temporal and spatial properties of PNCR and cytosolic Ca2+ sparks are given in Figure 4. There was no significant difference between the mean amplitude of Ca2+ sparks (F/Fo=2.0±0.03, n=361) and PNCR events (F/Fo=2.0±0.04, n=68). The mean duration of PNCR was significantly greater (1.78±0.19 seconds, n=68) than the half decay time for Ca2+ sparks (31.2±0.56 ms, n=361; P<0.01). The PNCR half width at the point of maximum amplitude (5.0±0.2 μm, n=68) was also significantly greater than that of typical Ca2+ sparks (2.6±0.05 μm, n=311; P<0.01). Whereas the amplitude distribution of Ca2+ sparks exhibited a typical Gaussian relationship (as in most previous studies), PNCR events appeared more bell shaped, with a higher proportion of low amplitude events being detected. This may reflect the fact that low amplitude events are easier to discriminate from background noise when they last seconds as opposed to milliseconds.
As shown in Figure 2, PNCR typically exhibited a rapid rising phase followed by a slow monotonic decrease, before the final sudden decline in amplitude on termination of Ca2+ release. At the point of release termination, the mean PNCR amplitude was 57.3±2.4% (n=68) of the initial peak. The spatial spread of PNCR also decreased as the amplitude declined (eg, Figure 2C). On average, PNCR width (FWHM) decreased by 20.1±2.5% (n=68) at the point of release termination.
Changes in Nuclear [Ca2+]i in Intact Cells During Electrical Stimulation
Further experiments were performed on intact myocytes to establish whether the inward diffusion of Ca2+ from the ends of the nuclei plays a role in the beat-to-beat regulation of nuclear [Ca2+]. Figure 5A shows an x-t image of an intact ventricular myocyte during an electrically stimulated response. The scan line was positioned longitudinally through one of the nuclei. On stimulation, the rapid rise in [Ca2+] resulted in a horizontal line across cytosolic regions of the line scan image. The [Ca2+] also increased within the nucleus, but with a delay reflecting diffusion of Ca2+ across the NE. As reported previously,2,11 the rise in [Ca2+] within the nucleus was markedly prolonged relative to the cytosolic Ca2+ transient. However, one feature not highlighted in earlier studies is the apparent ‘V’ in the fluorescence signal, joining the ends of the nucleus. This is consistent with diffusion of Ca2+ from the ends of the organelle toward the center. The expanded panel (bottom) shows the same image, with the nucleoplasmic region blanked and the time scale compressed. The arrows indicate regions of high localized Ca2+, which appear to spread outwards into the cytosol before slowly receding.
Figure 5. A, top, Line scan image of an intact ventricular myocyte during electrical stimulation, with the scan line positioned longitudinally through the center of a nucleus. The same image is shown on a different aspect ratio with the nuclear region blanked out (bottom). Arrows indicate apparent outward diffusion of Ca2+ toward the cytosol. B, Line profiles obtained from the cytosol (i), just inside the nucleus (ii), and the center of the nucleus (iii). Line profiles ii and iii were obtained by averaging over 3 pixels, whereas i was averaged over a wider region as indicated. Red arrow indicates the beginning of a secondary rising phase in line profile (ii). C, Cumulative data illustrating the mean time to peak (measured at 90% max, TP90) and the mean half time of the descending phase (TD50). *TP90 and TP50 values within the nucleus that are significantly different from the corresponding cytosolic values (P<0.05). D, Sequential x-y images obtained from another cell at 80 ms intervals after electrical stimulation. Note the bright regions at the ends of the nucleus, which then spread toward the center.
Selected line profiles (Figure 5B) show the change in [Ca2+] within the cytosol (i), just inside the nucleus (ii), and at the center of the nucleus (iii). The rise in [Ca2+] just inside the NE was slightly delayed compared with the cytosolic Ca2+ transient and was typically monotonic. However, the rise in [Ca2+] at the center of the nucleus was markedly slower and the maximum level lower than at the ends of the nucleus. In addition, the line profile from the center of the nucleus exhibited a secondary rise in [Ca2+] (red arrow). Comparison of the line profile and the line scan, suggest that the secondary rise in Ca2+ reflects the influence of Ca2+ diffusing inwards from the ends of the nucleus. Similar results were obtained in 11 other preparations.
Cumulative data illustrating the time courses of the rising and falling phases in regions (i), (ii), and (iii) is given in Figure 5C. The mean propagation rate of Ca2+ from the ends of the nucleus toward the center was 49.8±3.7 μm/sec (n=12). The time to peak (measured at 90% max) of the cytosolic Ca2+ transient (i), and regions (ii) and (iii) of the nucleus was 20.4±1.8 ms, 46.5±5.4 ms, and 157.8±15.1 ms (n=12), respectively. The corresponding descending phases declined with half times of 398.3±49.3 ms, 589.2±71.8, and 598.3±82 ms (n=12).
The role of Ca2+ diffusion from the ends of the nucleus toward the center is further highlighted by sequential x-y images obtained from another cell after electrical stimulation (Figure 5D). The selected frames reveal localized regions of high fluorescence at the ends of the nucleus. These regions broaden, producing a characteristic "dumbbell" appearance, before spreading inwards toward the center of the nucleus. Similar results were obtained in 6 other preparations.
Location of Intracellular Ca2+ Storage Sites in Intact Cells
The distribution of stored intracellular Ca2+ was investigated by exposure of intact cardiac cells to fluo-5N AM, under conditions that favor compartmentalization of dye in the organelles. Figure 6A shows an x-y image of fluo-5N fluorescence centered on one of the two nuclei. As previously reported, a characteristic sarcomeric pattern of staining is present, which corresponds to dye trapped within the lumen of the SR.16 The NE is also visible because of Ca2+ stored within the perinuclear space. However, another consistent feature was the presence of localized areas of high fluorescence at the ends of the nuclei, suggesting regions of high organelle Ca2+.
Figure 6. A Intact cardiac cell after loading with Fluo-5N (top). Fluo-5N–loaded cell before (left) and after (right) exposure to 10 mmol/L caffeine and 20 μmol/L CPA (bottom). B, Intact cell after exposure to BODIPY C5-ceramide. The Golgi apparatus is indicated by the regions of high fluorescence at the ends of the nucleus.
Unlike previous reports in rabbit myocytes,16 electrical stimulation did not result in a decrease in Fluo-5N fluorescence (not shown). This might reflect (1) a higher than expected Ca2+ affinity of the de-esterified dye complexes and/or (2) a higher luminal [Ca2+] within the SR of rat compared with rabbit myocytes. However, a pronounced reduction in both sarcomeric and perinuclear fluorescence did occur after maximal depletion of the SR Ca2+ stores by inhibition of SERCA (with CPA) followed by RYR activation by application of 10 mmol/L caffeine (Figure 6B). This effect was present, but less pronounced with caffeine alone (not shown), which may reflect the that fact that the SR can retain Ca2+ in the presence of caffeine, when SERCA is active.20
The bright region at the ends of the nuclei might reflect Ca2+ stored in the NE or adjoining regions of the SR. However, ultrastructural studies suggest that the Golgi apparatus (GA) is also closely associated with the ends of the nucleus.21 Therefore, in some experiments, the GA was identified using BODIPY FL C5-ceramide. As shown in Figure 6B, the GA was clearly located in distinct regions at both ends of the nucleus. Similar results were obtained in 5 other cells.
Based on the cumulative data (Figure 4), typical spontaneous PNCR events peak at around F/Fo=2, has a half width of 5 μm and lasts for 1.7 seconds. To our knowledge, such prolonged Ca2+ release events have not been reported previously in ventricular myocytes in the absence of pharmacological modification. One important consideration is that PNCR might be an artifact of the experimental conditions or the result of localized damage. However, this seems unlikely because (1) similar events were observed in intact cells loaded with fluo-3 AM (eg, Figure 3D), confirming that PNCR is not a consequence of cell permeabilization, and (2) PNCR was detected in x-y images at low frame rates (eg, Figure 1) and at lower laser intensities (not shown). This suggests that laser damage is unlikely to be the underlying cause of PNCR.
Origin of Spontaneous PNCR Events
Previous work has shown that localized Ca2+ release arising in close proximity to the NE can have a major influence on nucleoplasmic [Ca2+].18 Therefore, the possible involvement of juxtanuclear organelles in PNCR must be considered: the cytosolic region immediately beyond the "poles" of the elongated nuclei contains SR, mitochondria, and the GA.21 The mitochondria are an unlikely source of Ca2+ because spontaneous PNCR was observed in the presence or absence of mitochondrial inhibitors. It is less easy to draw firm conclusions about the involvement of the NE, SR, or GA. Both the SR and the NE membranes of cardiac cells contain SERCA2a and RYR2.7,22 In addition, the lumen of the SR/ER is continuous with the lumen of the NE. Therefore, any factor affecting the Ca2+ content of the SR will affect the NE and vice versa. Recent work has also highlighted the possible role of the GA as a functional Ca2+ store23: the GA has two forms of Ca2+ pump (SERCA and a P-type SPCA) and in some cells types IP3 and ryanodine (RYR2) receptors are also associated with the GA membrane.24,25
In the present study, PNCR was abolished by inhibition of SERCA (with CPA), or drugs, which activate or inhibit RYR function (caffeine or ryanodine). Comparison of the images obtained using fluo-5N loading (Figure 6A) and C5-ceramide (Figure 6B) suggest that the regions of high [Ca2+] at the ends of the nuclei may partly reflect Ca2+ localized in the GA. However, exposure to caffeine and thapsigargin appeared to deplete Ca2+ within the SR, NE, and the regions at the nuclear poles (Figure 6A). Therefore, the shared components of the Ca2+ regulatory systems may preclude pharmacological differentiation between these potential sources of Ca2+. Despite this limitation, several features of PNCR are more consistent with the involvement of the SR or NE than the GA. First, PNCR was abolished by high levels of ryanodine (20 μmol/L), but was not induced or modified by IP3. This suggests that PNCR is likely to involve Ca2+ efflux via RYRs. Second, activation of Ca2+ release from the GA in response to RYR agonists has not been demonstrated in any cell type, suggesting that the NE or adjoining regions of the SR are more probable sources of Ca2+. Whatever its origin, the functional importance of PNCR primarily reflects its location and influence on nuclear Ca2+.
Temporal Properties of Ca2+ Sparks and Prolonged Ca2+ Release Events
A number of mechanisms have been proposed to explain the temporal properties of Ca2+ sparks and long-lasting Ca2+ release events observed after pharmacological modification of RYR function. These include (1) Ca2+-dependent channel inactivation or adaptation,26 (2) partial depletion of luminal Ca2+,27 or (3) stochastic attrition.28 Models involving only one of these potential mechanisms have generally failed to describe accurately the properties of Ca2+ sparks. Moreover, such models are limited by uncertainties over the single RYR Ca2+ flux and the number of channels activated during a Ca2+ spark or a prolonged Ca2+ release event. If sparks reflect the activation of tens or perhaps hundreds of RYRs, simple explanations based on stochastic attrition (simultaneous stochastic closing of RYRs within a cluster) can be excluded. However, recent models incorporating evidence that RYRs can be functionally coupled,29 with other features such as Ca2+ depletion of the SR, have proved more successful.30
It has been shown that pharmacological modification of the RyR can result in long-lasting Ca2+ release events, with similar properties to PNCR., eg, Ca2+ sparks with prolonged "embers" can be induced in cardiac cells by the drugs FK-506 or rapamycin,31 which dissociate FKBP from RYR.32 In this case, it was suggested that Ca2+ release is prolonged because of reduced coupling between RYRs, which impairs the ability of closed channels to facilitate the inactivation of others. However, long-lasting events can also be induced by other substances including ryanoids because of induction of long-lasting subconductance states.33 The fact that modification of RYR gating can produce such long-lasting events confirms that complete depletion of local Ca2+ stores cannot explain the normally brief nature of Ca2+ sparks. However, partial depletion may contribute to spark inactivation by reducing the open probability of the RyR.27
The present study shows that long-lasting Ca2+ release events can occur from localized regions at the ends of the nuclei in the absence of pharmacological agents. The prolonged nature of these events might reflect local structural characteristics, eg, the number of RYRs per cluster, the organization/packing of RYRs, or the degree of coupling conferred by FKBP. Other factors, such as the local phosphorylation levels of phospholamban or RyR may also influence channel inactivation characteristics, thereby prolonging Ca2+ release. Interestingly, "hotspots" of colocalized PKA and calcineurin have been reported to exist at the ends of the nuclei in adult cardiac myocytes (see Figure 7 in Santana et al34).
Recent models suggest that a maintained Ca2+ flux of 10% to 20% of that associated with a Ca2+ spark is sufficient to produce a prolonged Ca2+ release event, similar to that shown in Figure 2. 30 The rapid transitions between full and half maximal amplitude that occur in some PNCR events (Figure 3B) could be explained by a number of possible mechanisms, eg, if PNCR involves the maintained activation of a single RyR, then a 50% decrease in amplitude might reflect the adoption of a subconductance state. However, if many RYRs are active during PNCR then such a decrease in amplitude could be explained if tight coupling between active RyRs enables a simultaneous transition to a subconductance state.33
Physiological Relevance
The spontaneous PNCR events observed in skinned cells (Figure 1) are unlikely to have a role under physiological conditions. This is because the mean duration of PNCR is substantially greater than the cardiac cycle and each rise in [Ca2+] during systole would be expected to disrupt or entrain spontaneous PNCR. However, spontaneous PNCR may play a role in controlling Ca2+ dependent nuclear processes under pathological conditions, when the cell becomes quiescent and Ca2+ regulation is impaired, eg, during ischemia or reperfusion.35
One important consideration is whether the Ca2+ release sites, which underlie spontaneous PNCR in skinned and intact cells, are activated during the electrically stimulated Ca2+ transient. Evidence of Ca2+ diffusion from the ends of the nuclei toward the center is consistent with this possibility (Figure 5A). Furthermore, as with spontaneous PNCR (Figure 2), there is evidence of a prolonged outward diffusion of Ca2+ from a region at the edge of the nucleus toward the cytosol (Figure 5A, bottom). This pattern of Ca2+ release cannot readily be explained by simple inward Ca2+ diffusion from the cytosol to the nucleoplasm and suggests that the Ca2+ release sites, which underlie spontaneous PNCR, are activated during the electrically stimulated Ca2+ transient. PNCR might be triggered via CICR, following each global rise in cytosolic [Ca2+], or by Ca2+ entry across closely associated t-tubule membranes.
Assuming the Ca2+ release sites, which underlie PNCR are recruited during each beat, then the inherently prolonged nature of the underlying Ca2+ release process may contribute to the long-lasting nucleoplasmic Ca2+ transient (eg, Figure 5A). Such a mechanism could be of considerable importance given that nuclear Ca2+ is involved in the regulation gene transcription and expression. Of particular interest to the cardiac field, recent work has shown that changes in nucleoplasmic Ca2+ plays a central role in the development of hypertrophy.1
The present study has identified novel long-lasting Ca2+ release events, which occur spontaneously at the ends of the nuclei in both permeabilized and intact myocytes. In intact electrically stimulated cells, the inward diffusion of Ca2+ from the ends of the nuclei is a prominent feature of the nucleoplasmic Ca2+ transient and this appears to explain the biphasic increase in [Ca2+] at the center of the organelle. The association of PNCR events with the ends of the nuclei suggests that recruitment of the underlying Ca2+ release sites during systole may contribute to the beat-to-beat regulation of nuclear Ca2+.
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