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Am J Physiol Heart Circ Physiol 282: H2406-H2413, 2002. First published January 24, 2002; doi:10.1152/ajpheart.00320.2001
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Vol. 282, Issue 6, H2406-H2413, June 2002

Contribution of Ca2+ transporters to relaxation in intact ventricular myocytes from developing rats

Rosana A. Bassani and José W. M. Bassani

Centro de Engenharia Biomédica and Departamento de Engenharia Biomédica/ Faculdade de Engenharia Elétrica e de Computação, Universidade Estadual de Campinas, Campinas 13083-971, Brazil


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The relative contributions of Ca2+ transporters to intracellular Ca2+ concentration ([Ca2+]i) decline associated with twitch relaxation were analyzed in intact ventricular myocytes from developing and adult rats. This was accomplished by estimation of individual integrated Ca2+ fluxes with the use of kinetic parameters calculated from [Ca2+]i measurements during twitches and caffeine-evoked contractures, and from myocardial passive Ca2+ buffering data. Our main findings were the following: 1) twitch relaxation and [Ca2+]i decline were significantly slower during the first postnatal week than in adults, 2) inhibition of sarcoplasmic reticulum (SR) Ca2+ accumulation resulted in faster [Ca2+]i decline in young cells than in adult cells, 3) the contributions of the SR Ca2+ uptake and Na+/Ca2+ exchange (NCX) to twitch relaxation increased from ~75 to 92%, and decreased from 24 to 5%, respectively, from birth to adulthood, and 4) Ca2+ transport by the sarcolemmal Ca2+-ATPase was apparently increased in neonates. Our data indicate that despite a marked increase in NCX contribution to cell relaxation in immature rats, the SR Ca2+-ATPase appears to be the predominant transporter responsible for relaxation-associated [Ca2+]i decline from birth to adulthood.

sarcoplasmic reticulum Ca2+-ATPase; Na+-Ca2+ exchange; sarcolemmal Ca2+-ATPase; mitochondrial Ca2+ uniporter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CONSIDERABLE STRUCTURAL and functional changes take place in the mammalian ventricle after birth. The cell proliferation rate drops drastically while cells undergo marked differentiation and growth. In the rat, myocardial protein content is more than duplicated, the relative cell volume occupied by certain organelles [i.e., myofilaments, sarcoplasmic reticulum (SR), and mitochondria] is greatly increased, and the cell surface-to-volume ratio decreases at least two times within the first 2 postnatal weeks (10, 21, 28, 35, 36). Development of the t-tubular system during this period (29) allows action potential conduction along more internal regions of the cell, thus compensating for increase in cell volume and permitting synchronous excitation of the cell as a whole.

In the rat heart, expression of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2; both mRNA and protein levels), as well as Ca2+ uptake rates by SR vesicles, are reported to be low just after birth and to increase during postnatal development (17, 22, 35). In contrast, the Na+/Ca2+ exchanger (NCX) appears to undergo opposite developmental changes. Both mRNA and protein levels were found to be much higher in the neonatal heart than in the adult rat heart, with subsequent downregulation as maturation proceeds (11, 35). Accordingly, the rate of Na+-dependent Ca2+ uptake by cardiac sarcolemmal vesicles from neonatal and young rats is considerably higher than in adults (35). Because SR Ca2+ uptake and Ca2+ extrusion via the NCX are the main pathways for [Ca2+]i decrease during relaxation of the adult mammalian ventricle (3, 6, 27), it would be expected that these changes in SR Ca2+-ATPase (SR-ATPase) and NCX expression might result in major change in the individual contribution of each transporter to relaxation. That is, NCX, which is responsible for <10% of the Ca2+ transport during relaxation of adult rat myocytes (3, 27), would play a more prominent role in relaxation and might even equal or surpass the contribution of the SR Ca2+ uptake.

Although marked ontogenetic changes in the relative roles of SR-ATPase and NCX in twitch relaxation might be expected from the available data on cell protein content and Ca2+ transport in subcellular preparations, it is difficult to predict the impact of these changes in the live cardiac cell. The importance of specific Ca2+ transporters to relaxation-associated Ca2+ decline during postnatal development has been examined in only a few studies (2), and quantitative estimation of the relative contribution on these transporters is still lacking. Moreover, possible ontogenetic changes in transporters other than SR-ATPase and NCX have not been addressed so far.

The purpose of the present study was to investigate the following: 1) whether the relative participation of SR-ATPase and NCX in twitch relaxation of intact, isolated rat ventricular myocytes is actually altered during postnatal development, 2) the extent at which the contribution of each transporter is modified, 3) the time course of these changes, and 4) possible developmental variation in the contribution of the mitochondrial Ca2+ uptake via the Ca2+ uniporter (Mito-U) and sarcolemmal Ca2+-ATPase (SL-ATPase) to twitch relaxation. Our results indicate increased activity of SL-ATPase in preweaning rats (especially neonates), and much higher role of NCX in relaxation during the first postnatal week, compared with adults. However, the SR-ATPase is still the main transporter responsible for twitch relaxation, even in the neonatal rat ventricle.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ventricular myocytes. Ventricular myocytes were isolated from Wistar rats (0-180 days) that had been euthanized by decapitation (until 2 wk of age) or cerebral concussion, by 5-15 min of coronary perfusion with collagenase type I (0.5-0.7 mg/ml; Worthington), followed by mechanical cell dispersion (3). Cells were plated on a collagen-coated coverslip and perfused with modified Tyrode's solution (NT) at 23°C. The experimental protocol was approved by the Ethics Committee for Animal Research of the Universidade Estadual de Campinas. In this study, we considered as neonates and adults aged 0-2 days and 4-6 mo, respectively.

Cell shortening and [Ca2+]i measurements. Cell shortening was measured with a video edge detector (Crescent Instruments; Sandy, UT). For [Ca2+]i measurement, cells were loaded with 5-10 µM indo 1-acetoxymethyl ester (Molecular Probes; Eugene, OR) for 10-15 min at room temperature, followed by washout for 30 min. Indo 1 was excited at 360 nm, and emission was collected at 405 nm (F405) and 485 nm (F485). After background correction, the fluorescence ratio (R = F405/F485) was calculated and converted to [Ca2+] according to (19)
[Ca<SUP>2+</SUP>]<IT>=K</IT><SUB>d</SUB> · &bgr; [(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)]
where Kd is the apparent dissociation constant and Rmin and Rmax are the ratios at minimal and saturating [Ca2+], respectively, which were determined in vivo for each age group, as described by Bassani et al. (3). beta  was determined according to the method of Gomes et al. (18), and the indo 1 Kd was 0.844 µM (4). In cells loaded with carboxyeosin, fluorescence was corrected for changes in indo 1 emission (especially at 485 nm) when necessary.

Experimental protocol. [Ca2+]i transients were obtained during steady-state twitches evoked by electric field stimulation at 0.5 Hz. Contractures were evoked in resting cells by rapid and continuous application of NT containing 10 mM caffeine (Cf-NT), for induction of SR Ca2+ release and inhibition of SR Ca2+ accumulation. Because caffeine-evoked changes in membrane potential (Vm) have been reported to occur sometimes in cardiac myocytes (40), we monitored Vm during caffeine application in a set of myocytes under whole cell current clamp, but did not observe significant changes in Vm. To inhibit both SR Ca2+ accumulation and NCX, caffeine was also applied in 0 Na+-0 Ca2+ solution (Cf-00) (3, 6). Caffeine application was preceded by electric stimulation for 5 min to allow for SR Ca2+ loading.

The declining phase of each transient was fitted by a monoexponential function, for estimation of peak [Ca2+]i and the time constant for [Ca2+]i decline (tau ). From cell shortening measurements, the half-time of mechanical relaxation (t0.5) was determined for each type of contraction.

Estimation of Ca2+ fluxes. Ca2+ fluxes mediated by the SR-ATPase, NCX, and slow systems (i.e., Mito-U and SL-ATPase) were estimated according to Bassani et al. (3). Total [Ca2+] was calculated taking into account passive Ca2+ binding to exogenous (indo 1) and endogenous buffers (10). Indo 1 Kd was assumed as 0.844 µM (4) and intracellular [indo 1] as 50 µM (3) at all ages. Ca2+ fluxes and relative contributions of Ca2+ transporters were also calculated assuming indo 1 Kd as a linear function of myocardial protein content (10), from experimental values of 0.35 µM in a protein-free medium and 0.844 µM in adult ventricular myocytes (4). According to this estimate, indo 1 Kd was assumed as 0.555, 0.70, 0.775, and 0.825 µM at ages 0-2 days, and 1, 2, and 3 wk, respectively, and as 0.844 after 1 mo of age, when myocardial protein content reaches the adult level (10). We also performed calculations varying intracellular [indo 1] between 25 and 50 µM. In both cases (i.e., change in either indo 1 Kd or intracellular concentration), variation in Ca2+ fluxes was <15% (in neonates), and relative contributions of the transporters were little modified. For this reason, we chose to assume age-independent values for these parameters.

Mitochondrial Ca2+ uptake rate was estimated after incubation with a cell-permeant form of 5,6-carboxyeosin diacetate (CE) (Molecular Probes) to inhibit SL-ATPase. Carbonyl-cyanide p-(tri-fluoromethoxy)-phenylhydrazone (FCCP) (3 µM) was used to isolate the mitochondrial component (5-7). All integrated fluxes were expressed as moles per liter of nonmitochondrial cell water.

Solutions. NT was composed of the following (in mM): 140 NaCl, 6 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 5 HEPES. In 0 Na+-0 Ca2+ solution, choline chloride, and EGTA replaced NaCl and CaCl2, respectively, and 1 µM atropine was added. Unless otherwise stated, the chemicals were purchased from Sigma (St. Louis, MO).

Statistical analysis. Data are presented as means ± SE, or accompanied by the respective 95% confidence interval (CI95). One-way analysis of variance, followed by a post hoc Student-Newman-Keuls test, was used for interage comparison. Statistical significance was considered to occur if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time course of mechanical relaxation and [Ca2+]i decline. The time course of [Ca2+]i decline and mechanical relaxation during a twitch were significantly affected by the developmental stage (P < 0.01), as shown in Table 1 and Fig. 1A. Both [Ca2+]i decline and relaxation were slower during the first postnatal week (P < 0.05), attaining the mature time course by the end of the second week. Peak [Ca2+]i values corresponding to the twitch time courses shown in Table 1 were 0.59 ± 0.07, 0.82 ± 0.07, 0.91 ± 0.10, 0.82 ± 0.10, 0.72 ± 0.09, 0.59 ± 0.03, and 0.55 ± 0.07 µM for ages 0-2 days, 1, 2, and 3 wk, and 1, 2, and 4-6 mo, respectively (P < 0.05), and diastolic [Ca2+]i was ~0.2 µM at all ages.

                              
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  		Table 1.   Time constant of [Ca2+]i decline and relaxation half-time in ventricular myocytes of developing and adult rats



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  		Fig. 1.   Relaxation-associated intracellular Ca2+ concentration ([Ca2+]i) decline (normalized to peak [Ca2+]i) during steady-state electric stimulation at 0.5 Hz (A) and caffeine (Cf)-evoked contractures in the presence (B) and absence of extracellular Na+-Ca2+ (C), recorded from isolated ventricular myocytes from 1-day-old, 2-wk-old, and adult rats. In B and C, bold traces represent the monoexponential curve fitting of [Ca2+]i decline.

The time course of [Ca2+]i decline during Cf-NT (when net Ca2+ accumulation by the SR was prevented by a high caffeine concentration) was also affected by age (P < 0.01), but in the opposite direction: [Ca2+]i decline was significantly faster (P < 0.05) until the third week of life than in adults (Table 1 and Fig. 1B). Because cytosolic Ca2+ removal during Cf-NT relies on both NCX and the slow transporters (6, 27), we also investigated the role of the latter in [Ca2+]i decay at a caffeine contracture, during which NCX was inhibited by removal of extracellular Na+ and Ca2+ (Cf-00). In this condition, [Ca2+]i decline is attributable to transport via the Mito-U and SL-ATPase (6). The influence of age on the time course of [Ca2+]i decline during Cf-00 (P < 0.01) was qualitatively similar to that during Cf-NT, but the relative changes in tau  were smaller (Table 1 and Fig. 1C). Ontogenetic changes in the time course of mechanical relaxation during caffeine-evoked contractures roughly reflected those observed for the respective [Ca2+]i transient (Table 1).

Ca2+ fluxes and relative contribution of transporters to twitch relaxation. The relationship between the time derivative of total [Ca2+] and [Ca2+]i values during [Ca2+]i decline allowed the determination of empirical kinetic parameters for SR Ca2+ uptake, NCX, and the combination of slow Ca2+ transporters. With the use of these parameters, relaxation-associated Ca2+ fluxes mediated by each of the transporters during a twitch could be estimated (3). We assumed that individual Ca2+ transporters operate independently of each other and that kinetic parameters of the transporters are similar during twitches and caffeine-evoked contractures. However, it must be acknowledged that during a twitch triggered by an action potential, membrane depolarization might limit, at a certain extent, Ca2+ extrusion via NCX, especially in cells from neonates, in which action potentials are longer than in adult myocytes (23).

Figure 2 shows Ca2+ fluxes, integrated over the [Ca2+]i decline phase of a twitch, mediated by individual transporters in myocytes from adult and 1-wk-old rats. In Fig. 3A, Ca2+ fluxes integrated over 1 s after the intracellular Ca2+ transient peak are indicated for all the studied ages, whereas Fig. 3B shows the relative contribution of each transporter to twitch relaxation during development.


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  		Fig. 2.   Integrated Ca2+ fluxes during twitch relaxation, estimated in isolated myocytes from adult (A) and 1-wk-old (B) rats. Total (bold trace) and individual fluxes, mediated by sarcoplasmic reticulum (SR)- ATPase, Na+/Ca2+ exchange (NCX), and the combination of mitochondrial Ca2+ uniporter and sarcolemmal SL-ATPase (slow) are indicated.



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  		Fig. 3.   A: Ca2+ fluxes via SR-ATPase, NCX, and slow transporters, integrated over 1 s after the peak of a twitch-associated [Ca2+]i transient, estimated in ventricular myocytes from adult and developing rats. B: relative contribution of each of these Ca2+ transporters to [Ca2+]i decline during twitch relaxation. Numbers beneath the bars indicate the age of the animal in weeks. Note that left ordinate axes refer to SR Ca2+ uptake only, whereas the right axes refer to NCX and slow. N and A, neonates and adults, respectively.

The estimated total amount of Ca2+ transported over 1 s after the [Ca2+]i peak (i.e., the sum of the integrated fluxes mediated by individual systems) underwent dramatic increase during the first postnatal weeks (26, 55, and 90 µmol/l at 0-2 days, 1 wk, and 2 wk, respectively, vs. ~80 µmol/l in adults).

NCX-mediated Ca2+ fluxes were more than threefold greater in cells from 1-wk-old rats than in adults (13.1 vs. 3.9 µmol/l), even though the total amount of Ca2+ transported during relaxation in young cells was only ~70% of that in adults, as seen in Fig. 2. Ca2+ flux via NCX showed progressive decrease with maturation over the first month of life. The estimated Ca2+ flux mediated by the SR-ATPase was, at all ages, at least threefold greater than that estimated for NCX. During the first 2 wk of life, the former underwent a steep, almost fourfold increase to the adult level, from 20 to ~75 µmol/l (most of it due to developmental increase in total Ca2+ flux). These changes in both fluxes led to increase in the contribution of the SR Ca2+ uptake to relaxation from ~75% to ~92%, whereas that of NCX decreased from 24% to ~5%, with attainment of adult values at age 1 mo (Fig. 3B).

It should be pointed out that the present experiments were carried out at a temperature lower than the physiological range (23°C). However, we have previously observed that, although attained [Ca2+]i values and [Ca2+]i decline kinetics are temperature dependent, the relative participation of SR Ca2+ uptake, NCX, and slow transporters in relaxation is similar in the range of 25-35°C in mammalian ventricular myocytes (30).

Ca2+ transport by slow systems. The combined participation of the slow systems in relaxation was significantly increased until weaning age, but remained small (<5%) throughout development. During the first month of life, [Ca2+]i decline during Cf-00 was significantly faster than in adults (Table 1). Inhibition of the SL-ATPase by CE (leaving only the Mito-U uninhibited to remove intracellular Ca2+) did not modify significantly diastolic [Ca2+]i or the amplitude of the intracellular Ca2+ transient during Cf-00 in cells from 3-wk-old and adult rats. However, CE increased both values in neonates (P < 0.05, Table 2). Although CE prolonged the declining phase of the [Ca2+]i transient in the 3 groups, it did so in an age-dependent fashion (P < 0.01): CE increased tau  less than two times in adults, whereas the increase was ~2.4-fold at age 3 wk, and as large as ~5.5-fold in neonates (Table 2, Fig. 4). Thus CE abolished the difference in tau  of [Ca2+]i decline between weaning and adult rats, but rendered [Ca2+]i decline in neonates almost twice as slow as in adult cells. CE effects on [Ca2+]i decline of Cf-00 could be mimicked by inhibiting the SL-ATPase with 10 mM extracellular [Ca2+] in the presence of caffeine in Na+-free medium, in cells not loaded with CE (6).

                              
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  		Table 2.   Effects of SL-ATPase inhibition by CE on diastolic [Ca2+]i, Delta [Ca2+]i, and tau  during caffeine application in 0 Na+, 0 Ca2+ solution to ventricular myocytes from developing rats



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  		Fig. 4.   Means ± SE of time constants (tau ) of [Ca2+]i decline during caffeine application in Na+- and Ca2+-free medium in the absence (control) and presence of carbonyl-cyanide p-(tri-fluoromethoxy)-phenylhydrazone (FCCP) and/or after 5,6-carboxyeosin diacetate (CE) loading, in myocytes from neonatal (neo), 3-wk-old, and adult rats. Note the compressed scale on the right ordinate axis (CE + FCCP). * P < 0.05 compared with adults.

When FCCP was present in the perfusate during Cf-00 (to block Mito-U in cells not loaded with CE), a similar increase in tau  was observed in cells from 3-wk-old and adult rats, and [Ca2+]i decline remained faster in the younger cells (adult: from 11.13 ± 2.11 to 24.98 ± 3.13 s, n = 4; 3-wk-old: from 7.48 ± 1.20 to 15.31 ± 3.19 s, n = 6). Unfortunately, this kind of experiment could not be performed in neonatal cells, which were highly intolerant to FCCP. Evoking Cf-00 in the presence of FCCP in CE-loaded cells virtually prevented [Ca2+]i decline (tau  >2.5 min at all ages; Fig. 4), indicating that successful inhibition of all the four systems had been achieved.

Further analysis of the contribution of the individual slow transporters to twitch relaxation revealed that in newborns the SL-ATPase was the more prominent transporter, contributing 3.3% (vs. 0.7% for the Mito-U). At age 3 wk, although total contribution of the slow systems remained ~4%, the participation of SL-ATPase in relaxation decreased to 2.2%, whereas that of Mito-U increased to 1.7%. Finally, in adults, the total contribution of the slow systems to twitch relaxation decreased to 2.6%, mainly due to diminished participation of the SL-ATPase (1.2%), whereas that of the mitochondria was little changed (1.4%).

The rate of Ca2+ uptake by Mito-U was estimated as the FCCP-sensitive component of the flux responsible for [Ca2+]i decline under simultaneous inhibition of net SR Ca2+ uptake, NCX and SL-ATPase (i.e., during Cf-00 after CE loading) (5). Here we obtained comparable values inhibiting SL-ATPase with either 10 mM extracellular [Ca2+] (not shown) or CE (6, 7). The rate of mitochondrial Ca2+ influx estimated in intact myocytes from 3-wk-old rats was not statistically different from that in adults: 2.2 (CI95: 1.9-2.5) and 2.4 (CI95: 2.1-2.7) µM/s, respectively. However, in neonatal cells, estimated mitochondrial Ca2+ influx was much lower, only 0.48 (CI95: 0.32-0.63) µM /s.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we observed in intact, ventricular myocytes isolated from rats during the first weeks after birth increased contribution of NCX accompanied by decreased participation of the SR Ca2+ uptake in relaxation-associated Ca2+ fluxes compared with adults. These changes indicate a functional correlate of the previously reported down- and upregulation of the exchanger and SERCA2 gene expression, respectively, in the rat heart during postnatal development. However, the functional change appears to be not as great as it might be expected from the gene expression data.

Developmental changes in Ca2+ transport by fast systems. Diminished SERCA2 expression has been observed in the rat heart during the perinatal period, associated with reduced Ca2+-ATPase protein level, decreased rate of ATP-dependent Ca2+ uptake and slowed relaxation (14, 17, 24, 35). In the present study, we have also observed prolonged twitch relaxation in neonatal cells, with attainment of the typically adult temporal pattern at age 2 wk, as previously observed in rat ventricular muscle (14). Prolonged twitch relaxation in very young rats could not be ascribed solely to increased myofilament sensitivity to Ca2+ (33) because we found that the concurrent Ca2+ transient was also slowed. Accordingly, estimated Ca2+ flux via SR Ca2+ uptake and its relative participation in relaxation underwent a steep increase during this period reaching adult values at age 2 wk. This suggests that just after birth, NCX, although showing higher activity, was not able to compensate for the reduced SR Ca2+ uptake, resulting in slower overall twitch relaxation. Surprisingly, even in neonatal cells, in which SERCA2 protein level and SR Ca2+ uptake rates (as well as our empirical Vmax for the SR-ATPase in this study) have been reported to reach only ~20-30% of adult values (17), and SERCA2-to-phospholamban ratio is lower than in adults (24), SR-mediated Ca2+ fluxes were more than three times greater than those mediated by NCX, amounting to ~75% of the total relaxation-associated Ca2+ flux (Fig. 3).

This relative contribution is similar to that observed in adult rabbit myocytes, although absolute fluxes are higher in the latter (3, 6). However, in adult rabbit cells, [Ca2+]i decline during a twitch is more than twice as slow as in adult rat myocytes, whereas in rat neonatal cells, [Ca2+]i decline was prolonged by only 40%. Part of this difference might reside in higher systolic [Ca2+]i in immature cells (8, 14, present study), probably due to markedly lower passive Ca2+ buffering (10). Higher [Ca2+]i would result in a greater rate of Ca2+ transport by the SR-ATPase (and also by NCX), which might partially compensate for the apparently lower capacity of the enzyme, thus minimizing the prolongation of [Ca2+]i decline. In addition, cell morphology might affect the contributions of the different Ca2+ transporters to relaxation. For instance, in neonatal ventricular myocytes, a large part of the SR is located in the interior of the cell, and only a small fraction of the SR Ca2+ channels appears to be associated to the sarcolemma (37). During excitation, [Ca2+]i increase is not spatially and temporally homogeneous, as in adult cells: [Ca2+]i peak is higher and faster in the subsarcolemmal than in the center of the cell (20). Thus it is possible that the subsarcolemmal cytosolic Ca2+ pool could be removed by sarcolemmal transporters, whereas that in deeper regions of the cell would be more accessible for SR reuptake. With postnatal maturation, development of the t-tubular system and increase in sarcolemmal-SR association would lead to increased spatial homogeneity of the Ca2+ transient and access of cytosolic Ca2+ to sarcolemmal transporters. This, however, appears to be temporally correlated with increase in SERCA2 expression and decrease in the expression of the exchanger (35), so that the SR-ATPase would remain the greater contributor to relaxation-associated intracellular Ca2+ removal. Nevertheless, it must be acknowledged that these speculations cannot be ascertained with our present approach because our measurements are restricted to global changes in [Ca2+]i, and thus cannot discriminate possible cytosolic compartments, which are likely to be present in developing cells.

It is noteworthy that some authors have not observed diminished SR Ca2+ uptake in neonatal rabbit myocardium (25, 34), despite lower SERCA2 protein content than in adults and lack of negative inotropic response to SR-ATPase inhibition by thapsigargin (12, 15), whereas twitch relaxation has been found either slowed (2) or unchanged (34) compared with adult preparations. In addition, recent reports (2, 12) show that SR Ca2+ pump function seems to be comparable in adult and neonatal intact rabbit ventricular myocytes. These observations indicate that not always can one directly predict changes in the SR-ATPase function in intact cells based on changes in SERCA2 expression.

Ontogenetic changes in the expression and activity of the NCX have also been reported in the rat heart, namely higher mRNA and protein content, and sarcolemmal Na+-dependent Ca2+ uptake rates near birth, which decrease gradually and reach adult levels at age 1 mo (11, 35). In the present study, we observed a similar temporal pattern for ontogenetic changes in the estimated Ca2+ flux carried by the NCX during twitch relaxation, with higher NCX contribution to relaxation of intact isolated myocytes from young rats (Fig. 3). This contribution reached its peak in the first week of life (~25% vs. ~5% in adults), gradually declining with maturation.

Again, it might be interesting to compare myocytes from rats aged 0-7 days with myocytes from adult rabbits. In both, NCX expression and activity are higher than in adult rat myocytes and NCX contributes 25-30% of Ca2+ transport during relaxation (3, 11, 31), the action potential is longer and [Ca2+]i influx is 3-6 times higher than in the adult rat myocyte (3, 23, 36, 39). Because cell Ca2+ content should be constant at steady state, the amount of Ca2+ extruded by NCX should match the influx during excitation. Therefore, from the functional point of view, one would expect that both the adult rabbit and neonatal rat cells might need stronger Ca2+ extrusion mechanisms (such as NCX) than adult rat myocytes, to account for the relatively larger Ca2+ influx. Greater exchanger expression and NCX-mediated ionic current have also been described in the neonatal rabbit ventricle compared with adults (e.g., 1, 11). However, unlike what we observed in rats, NCX has been considered as the main mechanism responsible for relaxation in neonatal rabbit cells (2).

Developmental changes in Ca2+ transport by slow systems. After combined inhibition of SR Ca2+ accumulation and NCX (Cf-00), [Ca2+]i still decays, but at a much lower rate. Ca2+ removal from the myoplasm in this condition is attributable to the Mito-U and SL-ATPase (6). In adult cells, additional inhibition of either SL-ATPase or mitochondrial Ca2+ uptake prolonged [Ca2+]i decrease approximately two times, and combined inhibition of both slow systems virtually abolished [Ca2+]i decline. This result was similar to that observed in adult rabbit cells (6), but different from that reported by Choi and Eisner (13), who found abolition of [Ca2+]i decline in adult rat ventricular myocytes loaded with CE. It is possible that this discrepancy might be partially explained by the use of different signals in [Ca2+]i time course analysis (estimated [Ca2+]i vs. fluorescence ratio), in addition to potential change in indo 1 light emission at high CE loads (see MATERIALS AND METHODS). Thus our results indicate that SL-ATPase and mitochondria contribute similarly for intracellular Ca2+ removal under inhibition of the fast transporters in adult rat myocytes. According to our estimates, the latter mediates a Ca2+ flux of ~2 µM/s at [Ca2+]i ~1 µM.

In myocytes from immature rats, [Ca2+]i decay during Cf-00 was significantly faster than in adults, as also observed in developing rabbit ventricular cells (2). Because enhanced Ca2+ uptake rate by isolated ventricular mitochondria has been described in both species during the first postnatal weeks (9, 38), it would be possible that increased mitochondrial Ca2+ transport via Mito-U might be responsible for the overall acceleration of the slow systems in immature myocytes. However, this possibility was not supported by our experimental data, because inhibition of mitochondrial Ca2+ uptake by FCCP failed to suppress the difference in the time course of [Ca2+]i decline in 3-wk-old and adult cells, and estimated values of FCCP-dependent Ca2+ flux during [Ca2+]i decline were similar in intact cells from 3-wk-old and adult rats. On the other hand, the difference in [Ca2+]i decay between these age groups was abolished by SL-ATPase inhibition with CE. Surprisingly, prolongation of [Ca2+]i decline by CE was much greater in neonatal cells, in which mitochondrial Ca2+ influx was estimated as only ~20% of that at weaning and adult ages. However, it must be kept in mind that in the neonatal ventricle passive Ca2+ buffering is much lower than in adults (8), and this implies lower Ca2+ fluxes at a given [Ca2+]i range. Thus our results indicate two findings. First, the increased Ca2+ uptake rate observed in isolated mitochondria was not reflected by enhanced mitochondrial contribution to cytosolic Ca2+ clearance in intact cells. On the contrary, we estimated this contribution to be smaller in neonates, in which Ca2+ uptake rate by isolated mitochondria was the greatest (9). This might be due to lower relative mitochondrial volume (21, 28) and/or difference in mitochondria localization in immature myocytes (26). Alternatively, Ca2+ uptake by Mito-U in young hearts might be partially suppressed by endogenous factors not present in the isolated organelle. Second, the SL-ATPase appears to be more active in developing than adult cardiac myocytes, especially in the neonatal period. This is, to our knowledge, the first report in literature on the role of this enzyme in Ca2+ homeostasis in developing hearts.

Despite the apparent increase in SL-ATPase activity in the early postnatal period, its contribution to [Ca2+]i decline during twitch relaxation is still too low (<5%) to be considered physiologically significant. However, it is possible that our approach, which considers removal of intracellular Ca2+ only after the peak of the global intracellular Ca2+ transient, might have left unaccounted for Ca2+ extrusion by this enzyme in the early phase of the transient. The high-Ca2+ affinity and the sarcolemmal localization of the ATPase might favor early Ca2+ efflux, whereas [Ca2+]i is still rising. Our present data show significant increase in both diastolic [Ca2+]i and intracellular Ca2+ transient amplitude in response to caffeine (in 0Na+, 0Ca2+ solution) in neonatal myocytes after SL-ATPase inhibition with CE. However, it must be considered that CE (the only currently available SL-ATPase inhibitor amenable for use in intact cells) could also block other enzymes related to Ca2+ transport. Although it does not appear to inhibit NCX, it may block SR-ATPase (16). Marked inhibition of this enzyme would be, however, at odds with increased SR Ca2+ content in CE-loaded neonatal myocytes. Eosin has also been shown to block the Na+-K+ pump (32), although at concentrations ~100-fold higher than those necessary to block the SL-ATPase (16). We cannot rule out the possibility that inhibition of Na+ extrusion by the pump might account for part of CE effects on diastolic [Ca2+]i and SR Ca2+ content in neonatal myocytes. However, it would be expected that such inhibition would lead to [Na+]i loading, which would significantly slow down Ca2+i removal by NCX during Cf-NT in these cells. This would also be expected if CE blocked NCX itself. In neonatal cells, CE does not appear to have caused marked inhibition of either transporter because time constants of [Ca2+]i decline during Cf-NT before (1.36 ± 0.30 s) and after CE loading (1.65 ± 0.28 s, n = 5) were not significantly different. Overall, our results strongly suggest that the SL-ATPase activity might be an important factor in the regulation of diastolic [Ca2+]i and SR Ca2+ content in the neonatal myocardium.

In summary, our results in intact rat ventricular myocytes provide a functional basis to confirm the proposal of reciprocal changes in the participation of SR-ATPase and NCX in twitch relaxation. However, an important observation is that even when NCX contribution reaches its peak (first postnatal week), the major Ca2+ transporter responsible for relaxation appears to be the SR-ATPase.


    ACKNOWLEDGEMENTS

We thank Elizângela S. Oliveira and Gilson B. Maia for excellent technical assistance.


    FOOTNOTES

This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo Grant 95/0355-3.

Portions of this study were presented at the 44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, February 2000.

Address for reprint requests and other correspondence: R. A. Bassani, Centro de Engenharia Biomédica, Universidade Estadual de Campinas, Caixa Postal 6040, 13084-971 Campinas SP, Brazil (E-mail: rosana{at}ceb.unicamp.br).

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.

First published January 24, 2002;10.1152/ajpheart.00320.2001

Received 20 April 2001; accepted in final form 21 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 282(6):H2406-H2413
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