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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby, British Columbia, Canada; 2Cardiovascular Sciences, Child and Family Research Institute, Vancouver, British Columbia, Canada; and 3Laboratorio de Fisiología Celular, Servei de Cardiología, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Submitted 12 September 2007 ; accepted in final form 3 December 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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15% in 3-day-old (3d) myocytes to 90% in 56-day-old (56d) myocytes]. The remaining Ca2+ transient in the presence of Nif in younger age groups was eliminated by the inhibition of Na+/Ca2+ exchanger (NCX) with the subsequent addition of 10 µM KB-R7943 (KB-R). Furthermore, Ca2+ transients were significantly reduced in magnitude after the depletion of SR Ca2+ with caffeine in all age groups, although the effect was significantly greater in the older age groups (from 40% in 3d myocytes up to 70% in 56d myocytes). This SR Ca2+-sensitive Ca2+ transient in the earliest developmental stage was insensitive to Nif but was sensitive to the subsequent addition of KB-R, indicating the presence of NCX-mediated CICR that decreased significantly with age (from 37% in 3d myocytes to 0.5% in 56d myocytes). In contrast, the ICa-mediated CICR increased significantly with age (from 10% in 3d myocytes to 70% in 56d myocytes). The CICR gain as estimated by the integral of the CICR Ca2+ transient divided by the integral of its Ca2+ transient trigger was smaller when mediated by NCX (1.0 for 3d myocytes) than when mediated by ICa (3.0 for 56d myocytes). We conclude that the lower-efficiency NCX-mediated CICR is a predominant mode of CICR in the earliest developmental stages that gradually decreases as the more efficient L-type Ca2+ channel-mediated CICR increases in prominence with ontogeny. excitation-contraction coupling; Na+/Ca2+ exchanger; L-type Ca2+ channels; sarcoplasmic reticulum
70% and 30%, respectively, of total Ca2+ in the steady state (11, 18). However, we have observed in 3-day-old (3d) cardiomyocytes that the magnitude of the Ca2+ transients were reduced by about 40% after the depletion of SR Ca2+ (19). Furthermore, there have been several reports that the neonate ventricular myocytes have a greater amount of Ca2+ stored in the SR than previously realized (19, 34). Recently, it was demonstrated that RyR isolated from neonate hearts have gating properties in planar lipid bilayers similar to that from adults (41). The high expression and activity of Na+-Ca2+ exchanger (NCX) and a possible colocalization of NCX and RyR in the sarcolemma near the Z-lines provide the theoretical possibility of NCX-mediated CICR (6, 19). Given these findings, it is clear that the role of CICR in E-C coupling in the neonatal heart required reexamination. In the present study, using the whole cell-perforated patch-clamp technique and Ca2+ transient measurements in rabbit ventricular myocytes, we demonstrated the presence of reverse-mode NCX-mediated CICR in the early developmental stages. With ontogeny this mechanism of CICR gradually disappeared and the more efficient L-type Ca2+ channel-mediated CICR became dominant.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Whole cell-perforated patch voltage clamp. Whole cell amphotericin-perforated voltage-clamp technique was used as described previously (19–21). The internal pipette solution contained (in mM) 110 CsCl, 5 MgATP, 1 MgCl2, 20 tetraethylammonium, 5 Na2 phosphocreatine, and 10 HEPES, and pH was adjusted to 7.1 with CsOH. The standard external solution contained (in mM) 130 NaCl, 5 CsCl, 1 MgCl2, 2.0 CaCl2, 5 Na-pyruvate, 10 glucose, and 10 HEPES, and pH was adjusted to 7.4 with NaOH.
Measurement of Ca2+ fluorescence.
The cytosolic Ca2+ concentration ([Ca2+]i) was measured with the fluorescent Ca2+ indicator fluo-3 AM as described previously (19–21). F0 was the difference in the background fluorescence determined in the absence and presence of a cell in the area of measurement. 
F was the increment measured from baseline or the background fluorescence in the presence of a cell in the area of measurement.
Experimental protocol. A train of 20 repetitive depolarizations at 0.2 Hz from a holding potential of –80 mV to 10 mV (cells were first depolarized to –40 mV for 50 ms to inactivate Na+ and T-type Ca2+ channels and were then depolarized to 10 mV for 400 mV) was used to achieve a steady-state (data not shown). Then, the calcium transient elicited by the last of the 20 repetitive depolarizations (the steady state) was compared with the calcium transient elicited by a depolarization with a depleted SR Ca2+. The SR Ca2+-depleted state was achieved by an 8-s, 10 mM rapid caffeine application (Caf). Figure 1A shows this experimental protocol schematically. The depolarization immediately after the Caf application and in the continuing (3 s) presence of Caf was considered as depolarization in the SR depleted state. The duration of the Caf application was limited to 11 s to prevent a possible increase in [cAMP]i that could potentially result from longer exposures (54) and confound the results. Two inhibitors, 15 µM nifedipine (Nif), an L-type Ca2+ channel blocker, and 10 µM KB-R7943 (KB-R), a blocker primarily of NCX reverse mode under these conditions and to a lesser degree of L-type Ca2+ channel, were used sequentially throughout the study (2). In total, six distinct solutions were applied, as follows: control solution (Con), Nif, and Nif+KB-R in the steady state, and Caf, Caf+Nif, and Caf+Nif+KB-R in the SR depleted state. Figure 1B shows representative Ca2+ transients for 3d myocytes (left) and 56d myocytes (right) elicited by this protocol in the presence of three pairs of solutions. Figure 1, B1–B3, shows (from top to bottom) the calcium transients elicited by the experimental protocol in control conditions (B1), with Nif (B2), and with Nif+KB-R (B3).
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Data analysis.
Data are presented as means ± SE. Statistical significance of the results was tested using a one-way ANOVA (SPSS 11.0) or Student's t-test for paired or unpaired samples. Post hoc tests were taken with Tukey multiple comparisons. P 
0.05 was taken to be significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ICa-CICR in older age groups is more efficient than NCX-CICR in younger age groups.
Figure 6A shows the superimposition of Ca2+ transients of ICa-CICR vs. NCX-CICR. The upstroke of the Ca2+ transient in 56d myocytes preceded that in 3d myocytes significantly. Time to peak (TTP; indicated by arrows in Fig. 6A) represents the time from the starting point to the peak of the bulk phase Ca2+ transient, which were 150 and 360 ms for 56d and 3d myocytes, respectively, in the representative traces. TTPs for Con, KB-R-sensitive, and Nif-sensitive Ca2+ transients as a function of age are shown in Fig. 6B. TTP significantly decreased with age in Con Ca2+ transients. Differences in TTPs of KB-R-sensitive and Nif-sensitive Ca2+ transients did not reach a level of statistical significance, with the exception of that of Nif-sensitive Ca2+ transients between 3d and 56d myocytes.
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F) divided by its corresponding TTP (s)(50). The efficiency of NCX-CICR decreased with age. Conversely, the efficiency of ICa-CICR increased significantly with age. Within the same group (10d, 20d, and 56d), the efficiency of ICa-CICR was greater than that of NCX-CICR; however, the difference in efficiencies of NCX-CICR vs. ICa-CICR did not reach a level of significance in 3d cells. |
DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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70% and 30% of total Ca2+ at the steady state, respectively, have been reported in adult rabbit ventricular myocytes (11, 18). In the neonatal heart, due to lack of T-tubule in the first 2 wk of life and reportedly sparse SR, it has been assumed that the SR is not able to store and release comparable amounts of Ca2+ on a beat-to-beat basis as that of the adult (27, 37). However, the prevailing view has been challenged by recent findings of robust and spatially homogeneous Caf-induced Ca2+ transients and contractures in neonate hearts (19, 34). Indeed, the amount of Ca2+ stored in the SR was greater in the younger age groups (threefold greater in 3d than in the 56d) when normalized per unit of membrane capacitance (19). The RyR2 receptors appear in well-organized arrays in the cytoplasm of myocytes, even in 3d rabbits, using immunolabeling (10, 46). In addition, the gating properties in planar lipid bilayers (41) and pharmacological properties (52) of RyR2 in neonates are similar to those in adults, and the observations of spontaneous Ca2+ oscillations in early-stage embryonic stem cell-derived cardiomyocytes (55) strongly imply that the RyR is capable of releasing Ca2+ at the earliest developmental stages. In the present study, the contribution of a Caf-sensitive Ca2+ transient either measured by its integral (Fig. 2C) or peak value (supplementary data Fig. 2A; the online version of this article contains supplemental data) was significant as early as 3d (40% of total Ca2+) and amounted to 70% of total Ca2+ in 56d myocytes, indicating that the SR Ca2+ plays an appreciable role in E-C coupling, even at the earliest stage in rabbit cardiomyocytes. Moreover, a combination of 25 µM cyclopiazonic acid and 10 µM ryanodine reduced the total Ca2+ transient by 45% in 3d myocytes (see supplementary data Fig. 1). These findings are apparently contrary to those of Haddock et al. (16), who reported that the Ca2+ transient triggered by field stimulation shows a gradient from the subsarcolemmal region to the cell center in neonatal rabbit ventricular myocytes that was not inhibited by the addition of thapsigargin, a blocker of the SR Ca2+ pump, suggesting that there was no appreciable SR Ca2+ release. In contrast, Seki et al. (48) recently observed a significant reduction in the magnitude of the Ca2+ transient in fetal rat myocytes by thapsigargin in both the subsarcolemma and the cell center. These apparent differences may result from differences in methodology and/or animal species used.
ICa and ICa-CICR play an increasingly important role with development.
Here we report that Nif reduces the Ca2+ transient by more than 90% in 56d myocytes (Fig. 3A) and the transsarcolemmal Ca2+ influx is mainly ICa dependent (ICa was 30%, whereas the ICa-CICR was attributed to 70% of total Ca2+ transient; Fig. 4B), which was also supported by the measurement of the peak of the Ca2+ transient (Fig. 2, B and D). This is consistent with the results reported in the adult rabbit heart by others (11, 18). In contrast, Nif only reduced the Ca2+ transient by 20% in 3d myocytes (Fig. 3), and the contributions of ICa (15%) and ICa-CICR (5%) were much smaller than those in 56d myocytes. Thus, ICa does not play a major role in neonatal rabbit E-C coupling, and this is also consistent with other reports (21, 22, 24, 25, 58). The increasing functional importance of ICa and ICa-CICR with development is commensurate with the increasing density of ICa and the higher degree of colocalization between the L-type Ca2+ channel and RyR found in previous studies (10, 46, 47).
NCX and NCX-CICR play an important role in the early developmental stages.
The role of NCX and NCX-CICR has been extensively investigated in adult ventricular myocytes of several species (7, 29–31, 39, 50, 56, 57). Although NCX-CICR has been observed under certain experimental conditions (39), it is generally agreed that the role of reverse-mode NCX is negligible under physiological condition in most mammalian species (7, 33, 50), with guinea pig ventricular myocytes (29) and possibly some other species as the exception. NCX-CICR in the neonate has not been investigated as much as in the adult cell, presumably due to the general consensus of nonfunctional SR in the neonatal heart (28, 37). In the present study, we found that the percentage of the total Ca2+ transient attributable to NCX and NCX-CICR was 43% and 37%, respectively, in 3d myocytes (Figs. 4 and 5), indicating that NCX and NCX-CICR play a significant role in E-C coupling in the earliest developmental stage. It is also supported by the measurement of the peak value of the Ca2+ transient (supplementary data Fig. 2C). The significant portion of the Ca2+ influx via reverse-mode NCX in the early developmental stages in the present study is consistent with other reports (15, 58) and is in accordance with the higher expression and activity of NCX in the neonatal heart (6, 19, 43). The presence of NCX-CICR in the early developmental stages is likely to relate to the greater NCX expression level, longer action potential, and reduced Ca2+ contribution from ICa and ICa-CICR. Other important factors may be the microdomain geometry where NCX and RyR2 reside as well as the proximity between the two proteins. As expected (data not shown) and as reported in our previous studies, both the cell surface area as determined by the cell membrane capacitance and cell length significantly increased with age (19, 21). Page et al. (40) have found that before the development of a T-tubular system, the surface density of dyads, as well as total dyad areas per unit cell volume and per unit myofibrillar volume, increase progressively during embryonic life until they approach constancy at near adult rabbit values 1 day after birth. Our previous finding that there is fourfold greater time delay between the peak of Caf-induced inward NCX current and Ca2+ transient induced by application of caffeine in the neonate myocyte is suggestive of a structural difference in the subsarcolemmal space of the immature cardiac cells (19). Indeed, we have reported that the sarcolemma and its opposing SR form a narrow sheetlike structure (300 nm in length and 20 nm in depth) in the 3d heart, whereas more tubular structure was observed in 56d heart (100 nm in length and 20 nm in depth) (20). As a result, one may speculate that there will be a greater [Ca2+] in the subsarcolemmal space (19) for a given amount of Ca2+ entering the cell, which will favor an increase in the open probability of RyR2. Furthermore, using a new technique of mathematical cell peeling to double immunolabeled images, our group has revealed a distinct pattern of NCX distribution in 3d myocytes, with an orderly spacing of NCX each 0.7 µm rather than a homogeneous distribution on the sarcolemma as previously reported (9, 15). NCX clusters are predominantly located to the cell periphery at the earliest developmental stage (95% in periphery in 3d) and gradually redistribute toward to the cell interior with age (70% in interior in 56d) along with an alteration of periodicity to 2.0 µm. Similarly, the majority of RyRs are located at the cell periphery and are also spaced at narrow intervals of 0.7 µm in 3d myocytes. The colocalization of the two proteins at the cell periphery was 20-fold greater in 3d myocytes than in 56d myocytes (10). Therefore, it is conceivable that NCX-CICR is more likely to occur at the cell periphery (peripheral CICR) in 3d myocytes than in adult myocytes. Indeed, the equally long TTPs for the [Ca2+] transients produced by NCX and NCX-CICR, indicate that the Ca2+ has to diffuse to the contractile elements from the subsarcolemmal space (Fig. 5), and the [Ca2+] gradient from the periphery to the cell center observed in neonatal myocytes is likely the result of both transmembrane Ca2+ influx and peripheral CICR rather than just transmembrane Ca2+ influx as previously suggested (15, 48). Our results also suggest that the existence of a Ca2+ transient gradient from the cell periphery to the cell center does not exclude the existence of CICR in the neonate. In fact, Ca2+ sparks have been reported to predominate at the subsarcolemmal space and were rarely observed in the center of neonatal myocytes (15), giving further support to the importance of peripheral CICR at early stages.
The efficiency and gain of CICR.
The Ca2+ transient produced by ICa-CICR in the older age groups exhibits a rapid upstroke compared with that produced by NCX-CICR in the younger age groups (TTP 110 ms in 56d vs. 380 ms in 3d) (Fig. 6). As a result, the efficiencies to induce CICR (F/s) were much greater for ICa than for NCX (7.5 for ICa-CICR in 56d vs. 1.0 for NCX-CICR in 3d). The value for adult cells is comparable to that reported by others in adult guinea pig ventricular myocytes (efficiency 1.9 for NCX-CICR and 8.8 for ICa-CICR and a TTP of 120 ms) (50). Moreover, a slow upstroke was unmasked in adult cells after inhibition of ICa or the depletion of SR Ca2+, indicating that ICa-CICR through a mature T-tubular network is required for fast and synchronized Ca2+ release (8, 47). In contrast, the lack of T-tubules in neonatal myocytes and a 100–1000x slower Ca2+ unitary influx rate through NCX (3, 17) (as compared with ICa) results in a lower efficiency of NCX-induced CICR and a slower upstroke of the Ca2+ transient.
Another parameter closely related to the efficiency to induce CICR is the CICR gain. This parameter represents the amplification of CICR and is classically calculated as the SR Ca2+ release divided by the Ca2+ trigger signal producing it. Different studies have reported CICR gain in different species, ranging from 16 at a membrane potential of 0 mV, using peak flux rates in rat ventricle (59), to a gain of 3–8 in rabbit for fractional release measurements with a varied SR Ca2+ load (49). In both studies, the SR Ca2+ release fraction was derived from a mathematical integration of the Ca2+ transient and other Ca2+ flux parameters, such as SR Ca2+ pump uptake, ICa, SR Ca2+ leak, Ca2+ buffering, and sarcolemmal SR Ca2+ pump. Since there is limited knowledge about the developmental change in these parameters, we are unable to infer the SR Ca2+ release from the Ca2+ transients. Therefore, we chose to estimate CICR gain in the present study as the ratio of the cytosolic Ca2+ transient produced by SR Ca2+ release and the Ca2+ transient produced by its trigger, without consideration of potential differential Ca2+ buffering, etc. The CICR gain was also measured by the Ca2+ transient peak value of CICR divided by the peak value of its corresponding trigger, and it showed a similar result (supplementary data Fig. 2E). Consequently, the smaller gain for ICa-CICR in the 56d myocytes in the present study as compared with values by Wier et al. (59) and Shannon et al. (49) is likely due to different means of calculating gain.
Independent of the method used to calculate the CICR gain, the calculation is expected to depend on the influx rate of the Ca2+ trigger, the physical distance of the trigger and the RyR (1, 51), and the nature of the microdomain, as well as the SR Ca2+ content (14, 49). A larger SR Ca2+ load as well as a more restricted microdomain observed in the early developmental stage (19, 20) may be expected to favor a larger NCX-CICR gain in 3d myocytes. However, we observed a smaller NCX-CICR gain in 3d myocytes(1) as compared with ICa-CICR gain in 56d myocytes (3), suggesting that the larger Ca2 influx rate for ICa (3, 17) and colocalization of the trigger and the RyR are more important determinants of CICR gain. Indeed, we observed increased ICa-CICR gain and decreased NCX-CICR gain with age (Fig. 5C), which is in accordance with an increased colocalization of the dihydropyridine receptor and the RyR (46, 47) and a decreased colocalization of the NCX and the RyR (10).
Considerations on the model and possible limitations. In the present study, we used 15 µM Nif and 10 µM KB-R, sequentially, to differentiate between the roles of L-type Ca2+ channels and reverse-mode NCX to elicit CICR. Although these compounds at the concentrations used in the present study may also affect some other ion channels, these effects are not expected to affect the conclusions of the study. Thus, Nif has been reported to affect potassium channels (13, 23), but K+ currents were eliminated by replacement with Cs+ and tetraethyl ammonium, making the presence of K+ currents unlikely. Cs+, on the other hand, has been reported to decrease the frequency of spontaneous Ca2+ release from the SR in skinned adult rat cardiac myocytes (26) and to prolong the rise time and decay of SR Ca2+ release without affecting its amplitude. Although we did not examine the effect of Cs+ on SR Ca2+ release, similar RyR gating properties have been reported in neonates and adults (41), and therefore it was assumed that there are no differential effects of Cs+ as a function of age.
Although the inhibitory effect of KB-R7943 on NCX is well established, this compound also affects other ion channels. In our study, its inhibitory effect on the L-type Ca2+ channel (2, 19) and its dependence on [Na]i (38) could be of some concern. However, in the present study, KB-R is always used subsequent to L-type Ca2+ channel inhibition with Nif, and we have previously shown that 10 µM KB-R acts primarily as an inhibitor of reverse-mode NCX activity under our experimental conditions (2, 19). KB-R has also been reported to inhibit inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum in intact nonexcitable HeLa cells (45). However, it is generally accepted that IP3R does not play a major role in cardiac E-C coupling in single isolated ventricular myocytes postpartum due to its very low concentration and the kinetics of IP3R-mediated SR Ca2+ release (35, 36, 44).
Finally, calcium buffering of the Ca2+ indicator fluo-3 AM may affect the kinetics of the calcium transients, and it has been reported that the intrinsic Ca2+ buffering capacity is two to three times lower in neonatal ventricular myocytes derived from ventricular homogenates and permeabilized ventricular myocytes (4). However, the present study is based on comparisons of pharmacological manipulations within the same cell where the intrinsic Ca2+ buffering capacity or buffering by fluo-3 are likely to remain constant.
Conclusions.
In the present study, we report that SR Ca2+ release is an important component of E-C coupling (40%), even at the earliest neonatal stage, and that it plays an increasingly important role with ontogeny. In the early developmental stage, SR Ca2+ release was predominantly triggered by transsubsarcolemmal Ca2+ influx via reverse-mode NCX producing a slow rise of the calcium transient peaking 380 ms after depolarization. In contrast, predominance of high-gain, high-efficiency ICa-induced CICR at later age stages produced a rapidly rising calcium transient peaking at 110 ms. We conclude that the less efficient peripheral NCX-mediated CICR is the predominant mode in the earliest developmental stages, which gradually disappears as the more efficient L-type Ca2+ channel-induced CICR increases in prominence with development.
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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.
* L. Hove-Madsen and G. F. Tibbits are co-senior authors of this article. 
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P < 0.05). n = 12.
