1Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia; 2Cardiovascular Sciences, BC Research Institute for Children's and Women's Health, Vancouver, British Columbia, Canada; and 3Laboratorio de Fisiología Celular, Servei de Cardiología, Hospital de Sant Pau, Barcelona, Spain
Submitted 12 April 2004 ; accepted in final form 10 August 2004
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ABSTRACT |
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ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content
However, much less is known about the roles of these Ca2+ transporters during cardiac development. Several studies have indicated that NCX mRNA and protein levels as well as peak NCX current (INCX) were two to six times higher at birth compared with adult values (2, 3, 14, 24). Therefore, it has been suggested that the function of NCX in neonate cardiomyocytes is relatively more important than that in adults. For example, in a few recent studies (3, 6, 60), investigators using field stimulation and analysis of the decay of the Ca2+ transient have shown that NCX plays a relatively more substantial role in extrusion of Ca2+ in intact immature ventricular myocytes. However, it is relatively difficult to address the quantitative impact on the function of a living neonate myocyte. The rate of NCX transport depends on the subsarcolemmal [Na+] and [Ca2+], which are difficult, if not impossible, to directly measure. Furthermore, these values are influenced to a large degree by the spatial architecture of the dyadic junction between the transverse (T) tubules and the junctional SR. Recently, a considerable amount of effort has been focused on the "fuzzy space" in the adult cardiomyocyte. However, with an absence of T tubules in the neonate cardiomyocytes (up to 10 days of age), the nature of the fuzzy space is even more poorly understood in the immature heart.
In this study, using the whole cell perforated patch-clamp technique and [Ca2+]i measurements, we attempted to analyze the relationship between NCX activity and subsarcolemmal [Ca2+] in the immature heart and to determine how it changes during ontogeny.
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METHODS |
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Because the hearts from the different age groups varied considerably in terms of their weight, collagen content, and other characteristics, different protocols had to be developed to isolate Ca2+-tolerant myocytes from the different age groups. These differences included variations in the concentration and volume of collagenase, the concentration and volume of protease, and the perfusion pump speed. The details of these differences are shown in Table 1.
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Whole cell perforated-patch voltage clamp.
Isolated myocytes were allowed to settle on the bottom of a recording chamber mounted on the stage of an inverted microscope (Nikon TE 200) and were superfused with the standard external solution. Ionic currents were measured using the whole cell perforated-patch voltage-clamp technique with an Axopatch 200B patch amplifier (Axon Instruments, Foster City, CA). Data acquisition and voltage protocols were controlled using pCLAMP software (version 8.0; Axon Instruments). Standard internal and external solutions were used to eliminate Na+ and K+ currents. The internal pipette solution contained (in mM) 110 CsCl, 5 MgATP, 1 MgCl2, 20 tetraethylammonium (TEA), 0.025 EGTA, 5 sodium phosphocreatine, and 10 HEPES and was adjusted with CsOH to pH 7.1. The standard external solution contained (in mM) 115 NaCl, 20 CsCl, 1 MgCl2, 1.8 CaCl2, 5 sodium pyruvate, 10 glucose, and 10 HEPES and was adjusted with NaOH to pH 7.4. The perforated-patch configuration was used by including 300400 µg/ml amphotericin B (water solubilized; Sigma) in the patch pipette. Patch pipettes were pulled from thin-walled filamented glass capillaries (ID 1.1 mm, OD 1.65 mm; World Precision Instruments, Sarasota, FL). The pipette resistance was 1.52.5 M. The seal resistance was 2.020.0 G
, and access resistance decreased to <20 M
within 1020 min after seal formation. Cells showing a sudden drop in access resistance were discarded. All measurements were determined at room temperature.
Experimental protocols. After seal formation, the cell was lifted from the bottom of the petri dish and placed in front of a quartz capillary tip (ID 200 µm) of a fast-switching perfusion device (QMM Micromanifold; ALA Scientific Instruments, Westbury, NY). A Marzhäuser MM-33 micromanipulator (Fine Science Tools, North Vancouver, BC, Canada) was used to position the quartz tip of the perfusion system within 100 µm of the cell. The gravity-fed solutions coming into the manifold were controlled by a custom-built rapid switching device that employed multiple Lee solenoids (model AA0501418H; Westbrook, CT), each of which was triggered by TTL pulses programmed within the pCLAMP experimental protocol. This allowed for the rapid application of the 10 mM caffeine that was added to the external bath solution used in the experiments for SR Ca2+ release. In control experiments, either KB-R7943 (1040 µM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 100 µM) was added to the caffeine solution to determine the ionic nature of the caffeine-induced current.
Measurement of [Ca2+]i.
The [Ca2+]i was measured with the fluorescent Ca2+ indicator fluo-3 acetoxymethyl ester (fluo-3 AM; Molecular Probes, Eugene, OR). Cells were incubated with 510 µM fluo-3 AM for 3040 min at room temperature and then washed once using the storage solution. Cells were kept in the storage solution for 1 h before experiments were started. Fluo-3 was excited at 488 nm, and fluorescence emission was measured using a 530-nm band-pass filter. The band-pass width was ±20 nm. We used a Fluorescence System Interface device (Ionoptix, Milton, MA) to record the output from photon-counting photomultiplier tubes. A sampling interval of 10 ms was used to achieve a reasonable signal-to-noise ratio. Fluorescence signals were captured unfiltered but displayed after filtering with a low-pass Butterworth filter with a cutoff frequency of 10 Hz. The [Ca2+]i was calculated from the formula [Ca2+]i = Kd·(F Fmin)/(Fmax F), where Fmin is the background fluorescence determined from a cell-free area and Fmax is the fluorescence acquired after the cell was depolarized to +150 mV for 1020 s to maximize [Ca2+]i and kill the cell at the end of each experiment. Kd is the Ca2+ dissociation constant with fluo-3, and a value of 400 nM was used for all age groups (34, 55). It is well recognized that the concentration of Ca2+ indicator dyes such as fluo-3 can influence the results, particularly if the myocyte is overloaded with the dye and constitutes a significant source of cytosolic Ca2+ buffering. Overloading of the cell by fluo-3 can introduce artifacts in both the kinetics and magnitude of the Ca2+ transient and the subsequent contractile properties. In the present study, care was taken not to overload the cell by observing the Ca2+ transient and contractile kinetics and amplitude in response to both step depolarizations and caffeine application. Cells in which the amplitude of the contraction was too small in response to these conditions were rejected. Typically, under control conditions, myocytes contracted by 10% of their resting length when depolarized to +10 mV and by
25% of their resting length when exposed to caffeine.
Data analysis. Data are presented as means ± SE. Curving fitting was carried out using the Origin 6.0 software (Microcal Software, Northampton, MA). Statistical significance of the results was tested using one-way ANOVA (SPSS 11.0) or Student's t-test for paired or unpaired samples. Post hoc tests were performed using Tukey's multiple comparisons. A P value <0.05 was considered to be significant.
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RESULTS |
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Time course between peaks of INCX and [Ca2+]i.
There was a significant time delay between the peak of INCX and the peak of [Ca2+]i, as shown in Fig. 2, A and B (B shows the same data in A with an expanded time scale), in a 3d myocyte. The solid line represents the inverted INCX, and the shaded line reflects the [Ca2+]i calculated from Ca2+ transient induced by caffeine. In this representative experiment record, the peak of INCX preceded the peak of [Ca2+]i by 140 ms. In all instances, the rise of velocity of Ca2+ extrusion of NCX (VNCX) significantly preceded that of [Ca2+]i. As shown in Fig. 2C, there was a significant decrease in the time delay between the two peaks as a function of age. For example, the mean time delay in the 3d group was 287 ± 64 ms, which was more than fourfold that observed in the 20d group (64 ± 13 ms), and the difference was highly significant (P < 0.0001). This substantially longer time delay observed in the 3d group would also result in a greater underestimation of NCX function when expressed as the slope of VNCX as a function of [Ca2+]i. The time delay between peaks was highly correlated with SR Ca2+ content in younger age groups, especially the 3d group (R2 was 0.87, 0.59, and 0.57 for the 3d, 6d, and 10d groups, respectively; P < 0.01). There was no significant correlation between time delay and SR Ca2+ content in the 20d group (R2 = 0.32). In the SR Ca2+ content range of 2540 amol/pF, the time delay was 183 ± 40 and 70 ± 17 ms for the 3d and 20d groups, respectively, and this difference was highly significant (P = 0.003; n = 20), despite the fact that the SR Ca2+ content was not significantly different (29.4 ± 1.3 and 27.5 ± 0.9 amol/pF in 3d and 20d groups, respectively).
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VNCX in the presence of blockers of SR Ca2+ store function. The driving forces for NCX during the contracture and relaxation periods are likely to be quite different even for a given [Ca2+]i. During the caffeine-induced contracture, one would predict that [Na+]SM might increase as a consequence of Ca2+ extrusion by NCX. The effect of this would be to make the NCX reversal potential more negative and reduce the driving force for normal mode NCX activity. During normal twitches it is unlikely that [Na+]SM accumulates to the same degree, if at all. Thus the slope of VNCX vs. [Ca2+]i measured after the peak of the Ca2+ transient probably underestimates NCX function under physiological conditions. To avoid this, we measured VNCX using the blockers of SR Ca2+ store function (SR blockers) cyclopiazonic acid (CPA; 25 µM), a blocker of SERCA2A, and ryanodine (Ry; 10 µM), which locks the ryanodine receptor (RyR) in a subconducting open state, in the 3d and 20d groups only (n = 7). Figure 4A shows the protocol and representative traces of membrane current and fluo-3 fluorescence of a myocyte from a 20d rabbit. In the presence of SR Ca2+ blockers, the cell was depolarized to various voltages (+30 mV in the representative trace from a 20d myocyte shown in Fig. 4A) for 3s, which was intended to make the distribution of Ca2+ homogeneous between the subsarcolemmal and bulk cytosolic compartments by the end of depolarization. It was assumed, therefore, that at this point [Ca2+]i equaled [Ca2+]SM. Neither caffeine-induced currents nor Ca2+ transients in response to both caffeine applications indicated that the SR Ca2+ store function was successfully blocked by those inhibitors. We assumed, in this case, that normal mode NCX activity (shown as a tail current represented by the shaded trace in Fig. 4A) induced by repolarizing the cell to 80 mV was entirely responsible for the Ca2+ transient decline (solid trace) and cell relaxation. Therefore, the relationship between the tail INCX and [Ca2+]SM (or [Ca2+]i) at each corresponding time (indicated by dotted lines) was investigated. Under these conditions, the relationship of VNCX with [Ca2+]i was also linear. As speculated, the slope of VNCX vs. [Ca2+]i dramatically increased compared with that observed in caffeine-induced contractures. Figure 4B clearly shows the slope of the linear regression determined in the presence of the SR blockers (blocker slope), and it was almost twice as great as the caffeine slope in both age groups (3d and 20d). There were significant differences between the caffeine slope and the blocker slope within each of the two age groups (P < 0.001). In addition, there were significant differences for caffeine slope (P < 0.05) and blocker slope (P < 0.001) between the 3d and 20d groups. The peaks of the [Ca2+]i in caffeine-slope and blocker-slope measurements were 0.82 ± 0.34 and 0.87 ± 0.24 µM for the 3d group and 1.31 ± 0.50 and 0.99 ± 0.30 µM for the 20d group, respectively. Statistically, there were no significant differences in peak [Ca2+]i between caffeine slope and blocker slope within each age group and no significant differences in caffeine slope or blocker slope between the two groups.
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DISCUSSION |
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Further evidence that the caffeine-induced inward current is largely INCX is our observation that the current was extremely sensitive to KB-R7943. We found that the decay of this current was best fit by a double exponential equation. For example, in 3d myocytes under control conditions, the fast and slow time constants of decay () were 198 ± 18 and 1,422 ± 201 ms, respectively (n = 6; data not shown). In the presence of 10 µM KB-R7943 (a dose thought to predominately block reverse mode INCX), the fast and slow
were 405 ± 237 and 2,586 ± 359 ms, respectively, and were both significantly different from those of the control group (P < 0.05). The slowing of the caffeine-induced inward current by KB-R7943 was dose dependent, a dose of 40 µM virtually completely (>90%) blocked this current, and the Ca2+ transient remained elevated for a prolonged time in all age groups (data not shown) under these conditions. Thus these observations support our premise that the caffeine-induced inward current was primarily (>90%) INCX and that this parameter served as a reasonable estimation of SR Ca2+ content in all age groups.
Contrary to the prevailing view in the literature, there was actually a greater amount of Ca2+ stored in the SR, when normalized by cell surface area, in the younger age groups. The SR Ca2+ content expressed in this manner was more than twofold larger in the 3d compared with the 20d group. It has been suggested that the neonatal mammalian ventricular myocytes have a relatively sparse and immature SR compared with that in adults on the basis of ultrastructural (26, 39, 40), biochemical (22, 40), and pharmacological studies (37, 38, 52). Therefore, it has been assumed that the SR in neonatal ventricular myocytes would not be able to store and release comparable amounts of Ca2+ on a beat-to-beat basis compared with the adult. However, the observation that caffeine-induced Ca2+ transients and contractures were robust and spatially homogeneous in neonate hearts was reported recently by others (3, 25, 35). Delbridge et al. (18) reported that in adult rabbit ventricular myocytes, the SR Ca2+ content determined by caffeine-induced INCX integral was 1,208 ± 190 amol; the mean steady-state SR Ca2+ load was calculated to be 87 ± 13 µmol/l nonmitochondrial cytosolic volume. In the 20d group in the present study, the SR Ca2+ content was 30 amol/pF. Assuming a surface-to-volume ratio of 6.4 pF/pl nonmitochondrial cytosolic volume derived from the adult rabbit myocyte (47), the SR Ca2+ content in the 20d group would be 180 µmol/l nonmitochondrial cytosolic volume, which is still twice that in steady state in adult myocytes. Balagure et al. (3) reported that there were no significant differences in SR Ca2+ load among the 1- to 2-day-old, 10- to 12-day-old, and adult rabbit groups when field stimulation was used. The different results could be due to the use of different methods. In our study, we used a whole cell perforated patch-clamp technique and were able to control the membrane potential, important for the study of NCX function. Because there is a higher caffeine-releasable Ca2+ stored in the SR in the neonate compared with the adult, the question must be raised whether this Ca2+ participates in excitation-contraction (E-C) coupling under physiological conditions. Haddock et al. (25) demonstrated that in neonatal rabbit ventricular myocytes, the Ca2+ transient appeared to reflect mainly diffusion from the subsarcolemmal region to the myocyte center and did not seem to involve appreciable SR Ca2+ release. Using blockers of L-type Ca2+ channels and SR function, investigators in other studies (15, 16, 35, 51) have suggested that transsarcolemmal Ca2+ fluxes play a larger role in the beat-to-beat regulation of cardiac contraction relative to the Ca2+ released from SR in the developing mammalian heart. However, our laboratory group (50, 53) previously showed that in the neonate heart there is substantial colocalization of dihydropyridine receptor and RyR, albeit in the periphery of the cell, even in 3d myocytes. Indeed, in our experiments the Ca2+ transient was reduced during twitches elicited by depolarization when SR blockers (CPA and Ry) were applied (data not shown), even in the 3d group. Therefore, we speculate that SR Ca2+ release may be involved in E-C coupling in the neonate heart to a greater degree than previously realized but still less than that in the adult heart. Further experimentation is required to clarify this point.
NCX function.
The linear relationship between VNCX and [Ca2+]i after the peak of [Ca2+]i observed in the present study has been reported in trout atrial myocytes (27) but is not consistent with other reports (5, 6, 11). In those studies, the investigators combined the integrated Ca2+ fluxes, Ca2+ passive buffering capacity, and Ca2+ transient and found a sigmoidal relationship between VNCX and [Ca2+]I, and they reported that VNCX was saturated at 600 nM [Ca2+]i. The maximum value of VNCX (Vmax) was found to be 46 and 27 µmol·l cytosol1·s1 for adult rabbits and rats, respectively. In our study, VNCX did not exhibit saturation even up to values of 13 µM [Ca2+]i, and the peak of VNCX (e.g., 26 amol·s1·pF1 in the 20d group, or 166 µM·l cytosol1·s1) was much greater than the Vmax reported in the previous studies from adults, but it is still much smaller than that reported in trout atrial myocytes (
151 amol·s1·pF1).
Rapid caffeine application indirectly disables the SR Ca2+ pump function by increasing RyR open probability. Therefore, the Ca2+ extrusion in the presence of caffeine is likely mediated by forward mode NCX, mitochondria Ca2+ uniporter, and Ca2+ pump on the sarcolemma. We assumed that NCX was the only contributor to the Ca2+ decline induced by application of caffeine because the slow removal system is thought to play a minor role in the Ca2+ decline compared with that of NCX (7, 8, 10, 33, 43). However, there have been some studies showing that in immature myocytes, the slow removal system may play a relatively more important role (4, 6, 36). In the present study, VNCX was downregulated at least twofold from 3 to 20 days of age. This result is consistent with the two- to fourfold higher NCX density in newborn rabbits than in adult rabbits (1, 2, 14, 24, 31, 45, 57), based on immunoreactivity, mRNA and protein levels, and INCX. Thus, we think that the higher VNCX is due mainly to the higher NCX density in the neonate myocyte. Balaguru et al. (3) reported that the time to 75% of cell relaxation for caffeine application in newborn rabbit myocytes (180 ± 20 ms) was not statistically different from that in adult rabbit myocytes (200 ± 30 ms). This discrepant result is possibly related to the fact that SR Ca2+ content was not significantly different between these age groups in their study. Bassani et al. (6) reported that the contribution of NCX to the relaxation declined from 24% to 5% from birth to adulthood in rat cardiomyocytes.
The slope of the linear regression of VNCX as a function of [Ca2+] determined in the presence of the SR blockers was almost twofold that measured after caffeine application. This result demonstrated that there was an underestimation of the slope determined using application of caffeine, which is likely due to subsarcolemmal Na+ accumulation (19, 23, 42, 58), a consequence of Ca2+ extrusion by NCX during the caffeine-induced contracture. In this contracture period, the relationship between NCX and Ca2+ is complicated by the fact that both Ca2+ and Na+ accumulate in the subsarcolemmal space. However, once the Ca2+ reaches a peak and starts to decline, it appears that VNCX is linear with [Ca2+]SM even with the possibility that Na+ accumulated in this restricted microdomain. In vivo, the increased [Ca2+]i and cell repolarization increase the driving force for forward mode NCX activity, which contributes to cell relaxation, a condition similar to that using the SR function blockers. Therefore, we believe that under physiological conditions, VNCX is linear with Ca2+ and does not exhibit saturating kinetics even up to [Ca2+]SM levels of 13 µM. On the basis of the higher capacity of NCX in forward mode in the neonate heart, we and others (30, 44) have speculated that NCX functions to a greater extent in reverse mode as well and may be an important source of Ca2+ in normal E-C coupling.
Time delay of peaks of INCX and [Ca2+]i. Trafford et al. (56) reported that there was a time hysteresis between the peak of INCX and Ca2+ transient induced by application of caffeine in adult rat ventricular myocytes. The time differential for this hysteresis was 133 ms, much longer than the 64 ms observed in the present study for the 20d group. This discrepancy could be due to species or age differences. In the 3d group there was a 286-ms time delay between the two peaks. Although the present study showed a positive correlation between SR Ca2+ content and the time delay, particularly in the youngest groups, age group time lag differences persisted even with the same SR Ca2+ load. The basis for this difference is not clear and appears to be counterintuitive, given the smaller diffusional distances in the myocytes from younger animals. We hypothesize that this significant discrepancy is likely based on developmental differences in the nature of the microdomain in which RyR and NCX exist. In a study by Haddock et al. (25), investigators using line scanning confocal microscopy found that in an adult rabbit ventricular cell, the Ca2+ transients were uniform across the width of the cell in response to electrical stimulation, whereas in the newborn, the Ca2+ transients were much greater at the periphery than at the cell center. However, in the same experiments, the Ca2+ distribution in response to caffeine appeared to be homogeneous in both age groups. The longer time delay and the higher VNCX resulted in a significantly greater amount (50% of total in the 3d group and 17% of total in the 20d group) of Ca2+ already pumped out at the time of the peak of the Ca2+ transient (P < 0.005) in the 3d group. The Ca2+ left in the cytosol at that point in time was similar between the age groups (data not shown).
[Ca2+]SM. It is very difficult to directly measure [Ca2+]SM or [Ca2+] in other restricted microdomains. In recent years some investigators have tried to predict the [Ca2+]SM based on INCX (56, 58). We used a similar approach and calculated the [Ca2+]SM based on the linear regression of VNCX as a function of [Ca2+]i. As shown by these other groups in the adult myocyte and in our study of neonates, [Ca2+]SM is at least fourfold [Ca2+]i.
Limitations of study. In our present study, we used the same fluo-3 Ca2+ dissociation constant (Kd) of 400 nM for all age groups. Because the Kd is protein dependent, it is not clear whether Kd would be differentially affected during development (9). In addition, we normalized VNCX by cell surface (pF) instead of cell volume (pl). Because there is likely to be a higher surface-to-volume ratio in younger age groups (24, 26), the effect of this assumption would be to underestimate VNCX in the younger age groups.
It should be pointed out that all of the experiments were performed in the presence of Cs+ and at 23°C, which is considerably below the physiological core temperature observed in rabbits (38°C). K+ is commonly replaced by Cs+ in the solutions for experiments of this nature to reduce the contaminating effects of an outward K+ current. Despite the prevalence of this protocol, its potential effect cannot be ignored. Kawai et al. (29), for example, using saponin-skinned adult rat cardiomyocytes, demonstrated that the frequency of spontaneous SR Ca2+ release was significantly reduced when K+ was substituted with Cs+. This observation was not examined in the present study, but if we assume that there is no differential effect of Cs+ as a function of ontogeny, then our conclusions remain unchanged. Although the amplitude of both spontaneous and caffeine-induced SR Ca2+ release was only slightly affected by Cs+, the rate of release was slowed.
Although experiments on myocytes are frequently done at less than physiological temperatures to preserve myocyte function, one cannot ignore the impact of these experimental conditions on the derived results. We have determined that the Q10 of the cloned and expressed mammalian NCX1.1 (including both peak and steady state) is 2.42.6 (21). Use of a value of 2.5 would imply that the absolute value of VNCX peak (in amol·s1·pF1) determined at 23°C would be approximately threefold less than that expected at physiological temperatures, all other things being equal. However, the higher INCX values expected at 37°C might also result in higher [Na+]SM affecting both the driving force and inactivation kinetics, thereby reducing the differences in VNCX between the temperatures. Thus it is difficult to extrapolate. Because there is no evidence that isoforms other than NCX1.1 are expressed during ontogeny, we expect and assume that these are the same for all age groups. We believe, therefore, that although there might be quantitative differences if the experiments were conducted at higher temperatures, the conclusions nevertheless would remain the same.
In conclusion, in this study we found that there was a significantly greater SR Ca2+ content, when normalized per unit cell surface area, in younger age groups and that the SR can be rapidly reloaded with Ca2+. We also found that VNCX is linear with [Ca2+] during relaxation and did not exhibit any indication of saturation up to 13 µM [Ca2+]. VNCX decreased at least twofold in the period from 3 to 20 days of age, and this was likely due to a downregulation of NCX density during this period. Finally, there was a significantly greater time delay between the peak of INCX and the peak of [Ca2+]i in the myocytes from immature hearts, and this may be a reflection of developmental differences in subsarcolemmal microdomains. Although the subcellular localization of NCX relative to RyR has not been studied in detail in the developing heart, some inferences can be made from both the present and previous studies. Our group (49) previously found that the RyR are aligned along the Z lines at the earliest stages postpartum that were examined (3 days old) in a manner not different from that observed in adult myocytes. With respect to NCX, two previous studies (17, 25) demonstrated that in myocytes, before the advent of T tubules, the protein is apparently distributed homogeneously on the sarcolemma. Recently, however, our group (13) demonstrated that NCX is distributed on the cardiomyocyte plasma membrane in an organized pattern before T-tubular development. If there is colocalization of NCX and RyR in the immature cardiomyocytes, then it is likely to occur on the sarcolemma near the Z lines. Although colocalization of these proteins in adult rabbit myocytes is questionable (48), this possibility is supported to some degree by the data in the present study and deserves to be examined further using three-dimensional microscopy.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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