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Rest-dependence of Twitch Amplitude and Sarcoplasmic Reticulum Calcium Content in the Developing Rat Myocardium
J Mol Cell Cardiol 33, 711–722 (2001) doi:10.1006/jmcc.2001.1337, available online at http://www.idealibrary.com on
Rest-dependence of Twitch Amplitude and Sarcoplasmic Reticulum Calcium Content in the Developing Rat Myocardium Sandro A. Ferraz, Jose´ W. M. Bassani and Rosana A. Bassani Centro de Engenharia Biome´dica and Departmento de Engenharia Biome´dica/Faculdade de Engenharia Ele´trica e de Computac¸a˜o, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil (Received 12 September 2000, accepted in revised form 2 January 2001, published electronically 7 February 2001) S. A. F, J. W. M. B R. A. B. Rest-dependence of Twitch Amplitude and Sarcoplasmic Reticulum Calcium Content in the Developing Rat Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 711–722. Post-rest contractile response was studied in isolated ventricular muscle from rats aged 1 to 90 days. Amplitude of rapid cooling contractures (RCC) was taken as an index of the sarcoplasmic reticulum (SR) Ca2+ content. We observed that: (a) developed tension (per cross-section area) increased with age; (b) time to peak twitch force and relaxation half-time decreased from 87±6 to 56±2 ms and from 68±6 to 36±1 ms, respectively, from the neonatal period to adulthood; (c) post-rest twitch potentiation was observed at all ages, with greater relative potentiation in younger preparations, although relative potentiation of [Ca2+]i transient amplitude was similar in young and adult isolated ventricular myocytes; (d) rest did not significantly affect the amplitude of RCC in muscle or caffeine-evoked [Ca2+]i transients in myocytes at any studied age; (e) favoring Ca2+ efflux via Na+–Ca2+ exchange (NCX) during rest reversed twitch potentiation and caused a similar decrease in RCC amplitude (>40%) at all ages; (f) stimulation of Ca2+ influx via NCX during rest increased RCC amplitude (>40%) only in immature preparations. However, when this procedure was repeated after partial SR Ca2+ depletion, increase in RCC amplitude was not significantly age-dependent. We conclude that post-rest twitch potentiation is already present early after birth and does not require rest-dependent changes in SR Ca2+ content at any studied age. Our results suggest that NCX is close to equilibrium during rest in both adult and developing rat myocardium, and does not seem to mediate diastolic net Ca2+ fluxes which may affect the SR Ca2+ content. 2001 Academic Press
K W: Post-rest potentiation; Rapid cooling contractures; Calcium transients; Sarcoplasmic reticulum; Sodium–calcium exchange; Post-natal development.
Introduction Contraction and relaxation in the heart depend on Ca2+ cycling between cytosol and the extracellular medium, as well as intracellular Ca2+ stores. Among the latter, the sarcoplasmic reticulum (SR) is the most prominent. It appears to be the major source
of contraction–activation Ca2+ in the adult mammalian myocardium, and the major Ca2+ sink during relaxation.1–3 The SR Ca2+ content depends on the balance of release and uptake Ca2+ fluxes across the SR membrane, as well as on intra-SR passive Ca2+ buffering, mainly by the high-capacity, low-affinity
Please address all correspondence to: Dr Rosana A. Bassani, Centro de Engenharia Biome´dica, Universidade Estadual de Campinas, Caixa Postal 6040, 13083-970 Campinas, SP, Brazil. Tel: 55 19 3788-9261. Fax: 55 19 3289-3346. E-mail: [email protected] These results were presented in part at the XVI World Congress of the International Society for Heart Research (Rhodes, Greece, May 1998).
0022–2828/01/040711+12 $35.00/0
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protein calsequestrin (for a review, see 1 ). SR Ca2+ release takes place not only during electrical activation of the cell, but also during diastole, although at a very low rate.4 Part of the released Ca2+ is actively taken up back to the SR by the SR Ca2+-ATPase, and the remainder becomes available for extrusion via the sarcolemmal Na+–Ca2+ exchange (NCX). Depending on the activities of both transporters, net Ca2+ loss from the SR may or may not occur during rest. For instance, post-rest SR Ca2+ depletion has been consistently observed in rabbit ventricle,5–8 whereas in rat ventricle, in which NCX activity appears to be relatively lower and SR Ca2+ pump density is higher,3,9,10 either increase or no change in SR Ca2+ content during rest has been reported.6–8,11–13 There are several indications that the SR is still immature in the newborn mammalian heart. Lower expression of SR Ca2+ ATPase, diminished ATPdependent SR Ca2+ uptake and SR ATPase activity, decreased density of SR release channels (estimated by ryanodine binding) and lower negative inotropic effect of SR inhibitors in neonatal than in adult hearts have been described in several species.14–20 On the other hand, NCX expression, Na+-dependent sarcolemmal Ca2+ uptake and transmembrane currents mediated by NCX have been shown to be increased in the neonatal ventricle.18,20,21 These observations have led to the proposal that Ca2+ cycling between the extracellular medium and the cytosol is more prominent in hearts from young than from adult mammals, and that the intracellular Ca2+ cycling increases as the SR develops and the NCX undergoes ontogenetic downregulation. If this is the case, diminished role of the SR in the excitation–contraction coupling and in relaxation would be expected in the neonatal ventricle, in addition to greater impact of NCX-mediated Ca2+ fluxes on cell Ca2+ stores and intracellular [Ca2+] ([Ca2+]i) variations. This would mean that neonatal rat ventricle might behave more like the myocardium of adult rabbit than the adult counterpart of its own species. Thus, one might expect that transmembrane Ca2+ flux carried by NCX would affect SR Ca2+ content more markedly in young than in adult rat ventricle, especially during prolonged rest. In the present report, we studied multicellular ventricular preparations from developing rats, aiming at evaluating the effects of rest and NCX-mediated Ca2+ fluxes on twitch amplitude and SR Ca2+ content. We observed that, in spite of large differences in developed force and relative restpotentiation of twitches, there is a considerable similarity in the qualitative effects of rest on twitch
amplitude, and in the absence of rest-induced change in SR Ca2+ content in rat myocardium during post-natal development. Our results suggest that cardiac NCX appears to be close to thermodynamic equilibrium during rest at all studied ages.
Materials and Methods Preparations Ventricular muscle Developing (from 1 to 22 days old) and adult (3 months old) Wistar rats were sacrificed by stunning/ cervical dislocation, or decapitation (10 days old and younger). Hearts were rapidly cannulated and retrogradely perfused with oxygenated (95% O2-5% CO2) normal Krebs–Henseleit solution (NK) at room temperature to remove residual blood from coronary circulation. A free-running papillary muscle was dissected from the right ventricle and mounted in a Plexiglas chamber especially designed for muscle perfusion. The chordae tendinae was tied with a fine silk thread and attached to an isometric force transducer (mod. F-60, Narco Bio-Systems, Houston, TX, USA). The lower end of the muscle was fixed at the chamber bottom. Platinum electrodes (one in contact with the muscle and other placed parallel to the muscle long axis) delivered bipolar, square, voltage pulses (2 ms duration, 1.2times threshold) for electrical stimulation (mod. SI10, Narco Bio-Systems, Houston TX, USA). Due to the difficulty of isolating viable papillary muscles from 2-week-old and younger rats, at these ages we used a narrow (0.6 mm diameter or less) strip of the right ventricle wall, sufficiently thin to allow adequate oxygenation of the tissue. To check whether these preparations displayed similar contractile behavior, we compared papillary muscles and ventricular strips from 3-week-old rats, at which age viable preparations of both kinds can be obtained. We observed that, although strips developed >30% less active force (normalized to the cross-section area), the percent increase in twitch amplitude after rest was similar to that in papillary muscles (see Table 1). The muscle was perfused with NK and stimulated at 0.5 Hz for a period of 40 min, after which the preparation was stretched to produce an active force of 80% of that at its optimal length. An additional 30 min stabilization period was allowed before the experiment was started. Unless otherwise stated, preparations were maintained at 36.5 °C and stimulated at 0.5 Hz throughout the experient.
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Table 1 Cross-section area (CSA) and active force (normalized to CSA) in ventricular preparations (ventricular wall strip or papillary muscle) of adult (90-day-old) and developing rats during a steady-state twitch at 0.5 Hz (SS-TW), the first twitch evoked after 5 min rest (PR-TW), and a steady-state rapid-cooling contracture (SS-RCC). The number of experiments is indicated in parenthesis. Age (days)
Prep
1–2 3–6 7–14 15–22 15–22 90
Strip Strip Strip Strip Papillary Papillary
CSA (mm2) 0.21±0.02 0.22±0.01 0.26±0.01 0.36±0.06 0.15±0.02 0.23±0.01
(4) (15) (21) (9) (22) (28)
Perfusion flow rate was 25 ml/min, except during solution switch, when it was raised to 40–50 ml/ min. Isolated myocytes Ventricular myocytes were enzymatically dissociated from adult and 1-week-old Wistar rats after coronary perfusion with collagenase (Type 1, Worthington Biochem. Corp., Freehold, NJ, USA), as previously described.2 During the experiments, cells were perfused with HEPES-based solution at 23 °C and field-stimulated through a pair of platinum electrodes. Cells were loaded with 5 indo1 AM (Molecular Probes, Eugene, OR, USA) for 10–15 min at room temperature and washed for 30 min to allow for indo-1 deesterification. Indo-1 was excited at 360 nm. The ratio of emission at 405 and 485 nm was obtained after subtraction of the background fluorescence at each emission wavelength. The fluorescence ratio was converted to [Ca2+]i as described elsewhere,3 using calibration parameters determined in vivo for each cell type and an indo-1 apparent dissociation constant (Kd) of 0.844 .22
Experimental protocols Rapid cooling contractures (RCC) Rapid cooling of heart muscle evokes a contracture which is generally accepted to arise from SR Ca2+ channel opening, plus slowing of cytosolic Ca2+ removal processes.5,6,23 Although inference about the actual amount of Ca2+ stored in the SR is not possible, changes in RCC peak amplitude allow the detection of qualitative changes in SR Ca2+ content brought about by a certain experimental maneuver, such as rest.6 For RCC induction, electrical stimulation was interrupted and the heated perfusion solution was
SS-TW (mgf/mm2)
PR-TW (mgf/mm2)
SS-RCC (mgf/mm2)
17±4 (4) 50±7 (15) 48±6 (21) 98±14 (9) 144±28 (11) 239±36 (15)
38±16 (4) 115±14 (15) 129±16 (21) 143±22 (9) 213±35 (11) 275±39 (15)
198±60 (4) 204±25 (13) 279±30 (15) 513±94 (5) 883±134 (22) 876±103 (28)
quickly switched to cold NK (0 °C) with the aid of a solution selector based on solenoid valves (BioChem Valve Corp., Boonton, NJ, USA). According to our measurements, the temperature of the liquid surrounding the muscle could be decreased from 36.5 °C to 5 °C in approximately 0.4 s.
Post-rest contractile responses After stabilization of twitch amplitude at 0.5 Hz, rest periods ranging from 30 to 600 s were randomly applied, after which a twitch or a RCC was evoked. Before each post-rest RCC (PR-RCC), a steady-state RCC (SS-RCC) was obtained, i.e. evoked 2 s after interruption of the steady-state electrical stimulation, which corresponds to the regular stimulation interval at 0.5 Hz. Under control conditions, the muscle was perfused with NK during rest. To compare the ability of NCX to mediate net transmembrane Ca2+ fluxes during rest (thus changing SR Ca2+ content) in ventricular muscle of developing and adult rats, we manipulated Ca2+ and Na+ transmembrane gradients, in order to favor Ca2+ efflux or influx via the exchanger. This was achieved by Ca2+ removal (0Ca solution) or decrease of [Na+] (Low-Na solution), respectively, in the solution perfusing the muscle during rest only. At the end of the rest period in 0Ca solution, NK was switched on 15 s before the first post-rest twitch was evoked (flow rate 40–50 ml/min). Preliminary experiments showed that this period was sufficient to allow equilibration of extracellular [Ca2+], avoiding underestimation of twitch amplitude due to incomplete restoration of Ca2+ in the extracellular medium. The amplitude of post-rest twitches (PR-TW) and PR-RCC was normalized to that of steady-state twitches (SS-TW) and SS-RCC, respectively. SS-TW amplitude was considered as the average of the last five twitches before stimulation was interrupted.
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[Ca2+]i transients Steady-state electrical stimulation (0.5 Hz) was interrupted for rest periods ranging for 30 s to 5 min, after which stimulation was resumed or 10 m caffeine was applied. The amplitude of the [Ca2+]i transients evoked by caffeine (CfC) was used as an additional index of the SR Ca2+ content.4,7,8,24,25 Post-rest caffeine-induced [Ca2+]i transients (PRCfC) were normalized to the amplitude of steadystate transients (SS-CfC, i.e. caffeine was applied 2 s after interruption of stimulation). In another set of experiments, [Ca2+]i transients associated with SSTW and PR-TW after 3 min rest were obtained in isolated myocytes from 1-week-old and adult rats.
Solutions The composition of NK was (m): 129.4 NaCl, 4.5 KCl, 1.2 KH2PO4, 10.6 NaHCO3, 1.2 MgSO4, 1.5 CaCl2 and 11 glucose, pH 7.4 at 36.5 °C. For RCC induction, pH was adjusted to 7.4 at 0 °C with HCl. In 0Ca solution, equimolar MgSO4 replaced for CaCl2 (contaminating [Ca2+] >10 ). In LowNa solution, NaCl concentration was reduced to 9.4 m (20 m total [Na+]) and osmolality was maintained by sucrose addition. HEPES-based solution had the following composition (m): 140 NaCl, 6 KCl, 1.5 MgCl2, 5 HEPES, 1 CaCl2 and 11 glucose, pH 7.4 at 23 °C. All bicarbonate-based solutions were continuously gassed with 95% O25% CO2. When used, caffeine (Sigma Chem. Co., St. Louis, MO, USA) was added as a solid to the solution. All solutions were made up with deionized water and ACS standard salts.
Statistical analysis Data were compared with one-, two- or three-way analysis of variance, followed by Student– Newman–Keuls test for multiple comparisons. Values are presented as mean±... P values <0.05 were considered as indicative of statistical significance.
Results Developed active force For better characterization of the preparations used in this study, Table 1 shows active force values
(normalized to the muscle cross-section area) measured in ventricular muscle preparations from young and adult rats during: (a) SS-TW at 0.5 Hz; (b) a representative PR-TW (after 5 min rest); and (c) SSRCC. Muscle cross-section area increased with age (P<0.01) in both strips and papillary muscles due to developmental cardiac growth. However, agedependent changes in developed force were still evident after normalization to preparation size. Although analysis of variance indicated that amplitudes of SS-TW, PR-TW and SS-RCC significantly increased (P<0.01) during post-natal life, these changes occurred at determined developmental stages and were not similar for all the three types of contraction. During the first week of life (from 1–2 to 3–6 days of age), the amplitude of SS-TW and PR-TW increased >3-fold (P<0.05 for PR-TW), albeit SS-RCC amplitude increased less than 5%. During the second week (3–6 days v 7–14 days), little change occurred in amplitude of the three types of contraction. In the course of the third post-natal week (7–14 days v 15–22 days), SS-TW and SS-RCC amplitude nearly doubled (P<0.05). Subsequently, from weaning age (>3 weeks) to adulthood, data from papillary muscles indicated a greater increase in SS-TW than PR-TW amplitude (>65 v >30%), whereas RCC amplitude remained virtually the same. Although age-dependent statistical differences in force were not observed in papillary muscles, these changes contributed to reduce average relative twitch potentiation (i.e. increase in PR-TW amplitude, as percent of SS-TW amplitude) after 300 s rest from 48 to 15%. Due to the large ontogenetic variations in developed force, the amplitude of all post-rest contractions (both PR-TW and PR-RCC) will be subsequently referred to as relative values (i.e. percent of the amplitude of the respective contraction measured at steadystate). The time-course of SS-TW was abbreviated during post-natal development. The time to peak force decreased with maturation (P<0.01). It was significantly greater at age 1–2 days than in adults (87±6 v 56±2 ms, P<0.01; n=4 and 8, respectively), but statistical difference was no longer present at the end of the first week (68±5 ms, 58±6 ms and 54±2 ms at ages 3–6 days, 7–14 days and 15–21 days, respectively; n=6, 7 and 7, respectively). The time for 50% relaxation (t1/2) was also significantly reduced (P<0.01) with development, especially in the course of the first postnatal week. Values of t1/2 were 68±6 and 50±9 ms in ventricles from rats at ages 1–2 and 3–6 days, respectively (P<0.05 compared to adult). From the second post-natal week on, t1/2 values were com-
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Figure 1 Post-rest twitch potentiation in ventricular muscle from developing and adult rats. Amplitude of the first post-rest twitch (PR-TW) was normalized to that of the average, preceding steady-state twitch (SS-TW). The dashed line indicates SS-TW amplitude. Data as means±..., obtained in a group of 4–8 preparations at each age.
parable: 39±1 ms, 37±3 ms and 36±1 ms at ages 7–14 days, 15–22 days and adults, respectively. Thus, twitch relaxation in adults was nearly twice as fast as in neonates.
Effects of rest on twitch amplitude It is well known that the adult rat myocardium responds to rest with increase in the amplitude of the first post-rest twitch. In the present study, we observed significant (P<0.01) post-rest potentiation of twitch amplitude not only in adults, but at all studied ages, even neonates, over rest periods ranging from 30 to 600 s. The extent of potentiation (with relation to SS-TW), however, varied with age: it was higher in younger animals and gradually declined with maturation (see Fig. 1). This decline appeared to be mainly due to greater developmental increase in the absolute amplitude of SS-TW than of PR-TW. For instance, SS-TW absolute amplitude had an average increase of >4.8-fold from 1 week of age to adulthood (from 50 to 240 mgf/mm2), whereas a representative PR-TW (after 300 s rest) increased only >2.4-fold (from 115 to 275 mgf/ mm2) in the same period (Table 1). As seen in Figure 1, twitch potentiation had already attained a stable level after 30 s rest at all ages. In some experiments, using rest intervals shorter than 30 s, the time constant for full potentiation was determined according to Bers et al.7 The time constants in adult and developing ventricular muscle were not statistically different, and ranged between 6.5 and 8.5 s, which is close to the value determined in isolated myocytes from adult rats (>8 s).7 This suggests a common mechanism for rest-potentiation at all studied ages.
Figure 2 [Ca2+]i transients obtained in ventricular myocytes isolated from adult and 1-week-old rats, under steady-state electrical stimulation (0.5 Hz) and upon stimulation resumption after 3 min rest.
Rest-induced twitch potentiation was maximal at the end of the first post-natal week (PR-TW >300% of SS-TW v >120% in adults, Fig. 1). To investigate whether this increased potentiation was due to a larger relative increase in peak [Ca2+]i during PR-TW, we performed a set of experiments with isolated ventricular myocytes, in which [Ca2+]i was measured with indo-1 (Fig. 2). Diastolic [Ca2+]i was similar in adult and 1-week-old rat myocytes. The [Ca2+]i transient amplitude during SS-TW was 0.32±0.05 (n=11) and 0.47±0.05 (n=21) in cells from adult and 1-week-old rats, respectively. After 3 min rest, [Ca2+]i transient amplitude was potentiated at both ages (0.42±0.04 and 0.61±0.06 in adult and young, respectively, P<0.05 with relation to SS-TW). At both SS- and PR-TW, [Ca2+]i transient amplitude was greater in young cells (P<0.01). However, the relative [Ca2+]i potentiation was not statistically different (PR-TW was 143±13% and 131±9% of SS-TW in adult and young myocytes, respectively). We have previously shown that stimulation of NCX activity in the direct mode (i.e. Ca2+ extrusion
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Figure 3 (A) Original traces of steady-state (0.5 Hz) and post-rest twitches obtained in ventricular muscle from adult and 7-day-old rats, perfused with normal (NK) and Ca2+-free (0Ca) solution during 5 min rest. (B) Restdependence of twitch amplitude determined in adult rat papillary muscle perfused with normal (NK) or Ca2+-free solution (0Ca) during rest. Data are means±... (n= 8). The dashed line indicates SS-TW amplitude.
from the cell) can convert post-rest potentiation into post-rest decay of twitch amplitude in rat ventricular myocytes.8 Using the same procedure (i.e. omission of extracellular Ca2+ during rest), we were able to change twitch rest-potentiation into rest-decay also in multicellular myocardial preparations of adult rats (Fig. 3). Decrease in the extent of potentiation and even rest-decay of twitches at longer rest intervals were also observed in young myocardium when muscle was perfused with 0Ca solution during rest. This may be seen in Figure 3A, which depicts traces obtained in ventricular muscle from 7-day-old rats, in which twitch potentiation is at its peak.
Effects of rest on RCC amplitude To investigate whether age-dependent changes in PR-TW potentiation involved variations in the SR Ca2+ content, we repeated the rest protocols, but evoking PR-RCC instead of PR-TW. Figure 4A shows RCCs obtained in ventricles from adult and 7-dayold rats after different rest intervals: 2 s (SS-RCC) and 300 s (PR-RCC). The latter was evoked in either
Figure 4 (A) Original traces of RCCs obtained after 2 s and 5 min following interruption of electrical stimulation (SS and NK, respectively). The former was considered as an index of the SR Ca2+ content under steady-state stimulation (SS-RCC, see text). RCCs after 5 min rest were also obtained in preparations from adult and 7-day-old rats perfused with Ca2+-free solution during rest (0Ca). (B) Means±... of RCC amplitude (as percentage of the respective SS-RCC) as a function of rest duration. Muscles were perfused with either normal (NK) or Ca2+free (0Ca) solution during rest.
control condition or after stimulation of Ca2+ efflux by NCX (perfusion with NK or 0Ca solution, respectively, during rest). RCCs with similar features (e.g. phasic behaviour, time-course, and presence of rewarming spike) could be evoked at all ages, even in the newborn myocardium. As pointed out previously, only relative values of PR-RCC amplitude (i.e. as percent of the respective SS-RCC amplitude) shall be taken into consideration here. A striking observation was that control PR-RCC amplitude was similar to that of the respective SSRCC in both adult and developing preparations. It is important to notice that no increase in RCC amplitude was observed in the range of rest periods which consistently produced twitch potentiation, as shown in Figure 4B (note that at some ages, PR-TW amplitude was as large as 300% of that of SS-TW). This indicates that rest-potentiation of twitches occurred in the absence of detectable increase in SR Ca2+ content. Analysis of variance did not reveal statistically significant effects of rest on PR-RCC amplitude under control conditions, although PR-RCC amplitude seemed to decrease
Rest, Na+–Ca2+ Exchange and SR Ca2+ in Developing Heart
Figure 5 (A) [Ca2+]i transients obtained in isolated ventricular myocytes from 1-week-old and adult rats in response to 10 m caffeine application (arrowheads) 2 s (steady-state, SS) or 3 min after interruption of electrical stimulation. (B) Rest-dependence of the amplitude of caffeine-evoked [Ca2+]i transients (CfC) in cells from 1week-old and adult rats. Post-rest (PR-CfC) transient amplitude was normalized to that of the respective SSCfC (n=14 for both ages). The dashed line indicates SSCfC amplitude.
slightly in the younger preparations after very long (600 s) rest periods (see Fig. 4B). Additional experiments were performed with isolated myocytes from 1-week-old and adult rats, in which rest-dependent changes in SR Ca2+ content were investigated by analyzing the amplitude of caffeine-evoked [Ca2+]i transients. Figure 5A shows transients obtained after 2 s (SS-CfC) and 3 min rest (PR-CfC) in cells from adult and young rats. Transients in myocytes from developing rats differed from those in adult cells in both amplitude and time-course, as already reported.26,27 As observed for twitch-associated [Ca2+]i transients, SS-CfC transient amplitude was higher in young (1.317±0.182 , n=14) than in adult myocytes (0.830±0.063 , n=14, P<0.05). However, the amplitude of CfC transients was not significantly affected by rest up to 5 min at either of the studied ages (Fig. 5B). Perfusion with 0Ca solution during rest led to similar rest-dependent decrease in relative PR-RCC amplitude at all ages (Fig. 4B). Again, no significant
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age-related difference was observed. This indicates that, in both adult and developing myocardium, net Ca2+ extrusion by NCX during rest seems to be very low under control conditions and that stimulation of this flux produces rest-dependent SR Ca2+ depletion with comparable time-courses. When 1 m caffeine was added to 0Ca solution (to enhance SR Ca2+ release during rest), a greater decrease in PR-RCC after 10 min rest was observed in preparations from both adult (48±7% v 67±7% of SS-RCC in the absence of caffeine; n=5) and 2–3-day-old rats (49±5% v 61±5% of SS-RCC in the absence of caffeine; n=5). This effect was not significantly different between adults and neonates. In another set of experiments, Ca2+ transport by NCX in the reverse mode (i.e. Ca2+ influx) was stimulated by decrease of extracellular [Na+] from 140 to 20 m during rest. After this treatment, PR-TW could not be consistently obtained, since preparations in the presence of Low-Na solution, especially from adult rats, often developed spontaneous contractile activity, in addition to increased diastolic tension (both indicative of increase in cell Ca2+ load). However, PR-RCCs could be evoked. Surprisingly, enhancement of Ca2+ influx during rest produced different effects on PR-RCC in ventricles from adult and developing rats. In the former, although signs of cellular Ca2+ gain were present in most preparations, PR-RCC amplitude was not increased, but showed rather a small, non-significant decrease (88±6% of SS-RCC). In young myocardium, however, significant, rest-dependent increase in PR-RCC amplitude (P<0.01) was observed with the same treatment, as seen in Figure 6. Increase in RCC amplitude was significant as early as after 60 s exposure to Low-Na solution (139±3, 152±25 and 133±13% of SS-RCC in 1–6, 7–14 and 15–22 day old rats, respectively; n=6). To investigate whether the failure of Low-Na solution at increasing RCC amplitude in adult muscle was related to an already high SR Ca2+ load under steady-state stimulation, we repeated this protocol after partial SR Ca2+ unloading, as follows. After determination of SS-RCC amplitude, the preparation rested for 10 min, under perfusion with 0Ca solution containing 10 m caffeine. This solution was expected to decrease the SR Ca2+ content by enhancing SR Ca2+ leakage and facilitating extrusion of the leaked Ca2+. The muscle was then perfused for 60 s with either NK or Low-Na solution, after which a test RCC was evoked. Similar test RCC amplitudes were observed when muscles from developing (2 weeks old) and adult rats were rested in NK after SR Ca2+ unloading (39±9 and 40±4%
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Figure 7 Post-rest RCC relative amplitude in neonatal and adult myocardium. In the left block (loaded SR), preparations were rested for 3 min in the presence of normal solution (NK) or of a solution containing 20 m Na+ (Low-Na), after interruption of electrical stimulation. In the right block (depleted SR), the experiment was repeated after partial SR Ca2+ depletion, induced by prolonged perfusion with Ca2+-free solution + 10 m caffeine. n=5 for both ages. The asterisk indicates significant difference (P<0.05) between ages. Figure 6 (A) Original traces of SS-RCCs (SS) and postrest RCCs obtained after 5 min rest under perfusion with a solution containing 20 m Na+ (Low-Na) in ventricular preparations from adult and 7-day-old rats. (B) Means±... of RCC amplitude (as a percentage of SSRCC) after 5 min rest with Low-Na solution in adult and developing ventricle. Asterisks indicate significant difference (P<0.05) compared to adults (90 days). Results obtained from 6–10 preparations at each age.
of SS-RCC, respectively, n=4), indicating that a comparable degree of partial SR Ca2+-depletion had been achieved in the two kinds of preparation. When muscles were rested in Low-Na solution, test RCC amplitude was higher then after rest in NK (P<0.01), which was probably due to NCX-mediated SR Ca2+ refilling, from a lower initial Ca2+ load. However, in contrast to what happened before SR unloading, RCC amplitude after rest in Low-Na solution was similar at both age groups: 95±12% and 104±5% of SS-RCC in adult and young, respectively. This indicates that stimulation of Ca2+ influx by Low-Na exposure was equi-effective at increasing RCC amplitude in adult and young ventricle, as long as Low-Na solution was applied after the SR had been partially depleted of Ca2+ (see Fig. 7).
Discussion In this study, we used variation of the stimulatory interval as a tool to investigate the influence of
diastolic transsarcolemmal Ca2+ fluxes on the SR Ca2+ content in ventricular myocardium from developing rats. Our results suggest that, despite the previously reported enhancement in expression and function of rat cardiac Na+–Ca2+ exchanger in the first post-natal weeks, NCX appears to be near equilibrium during electrical rest in both adult and young rat ventricles, not mediating Ca2+ fluxes of magnitude sufficient to affect SR Ca2+ content.
Age-dependent increase in developed force and relaxation rate We observed a general increase in isometric peak force per cross-section area (during both twitches and RCCs) as the animal matures, despite the apparently higher sensitivity of myofilaments to Ca2+ in neonatal rat ventricle.28,29 This has also been observed in other species, for both isometric tension in intact and skinned myocardium,30,31 and unloaded shortening of isolated myocytes.32 Maturation-dependent increase in contraction amplitude might be partially explained by the marked increase in relative myofibrillar volume during post-natal development, especially during the first week of life.33 The subsequent increase in force (from the second to the third week) probably also involves age-dependent changes in cytoarchitecture, which would reduce the cell internal
Rest, Na+–Ca2+ Exchange and SR Ca2+ in Developing Heart
load.32 However, we did not find exactly the same pattern of age-dependent increase in peak force for all the three types of contraction shown in Table 1. Although both SS-TW and PR-TW are evoked by action potentials, the increase in absolute amplitude of the former from birth to adulthood was twice as much as for the latter. The significance of this changes still remains to be determined. Inferences on the SR Ca2+ content during development based on absolute RCC amplitude are precluded by several factors, such as myocyte differentiation (increase in the amount of contractile proteins and changes in cytoarchitecture), changes in myofilament sensitivity to Ca2+ and non-linear relationship between [Ca2+]i and force development. The time-course of SS-TW was markedly abbreviated in the course of the first post-natal week. Contraction rising phase, and especially relaxation, were significantly slower in neonates. After the first week, however, both time to peak tension and relaxation half-time attained adult values. This was probably a result of rapid post-natal maturation of the SR. Shortening of time to peak force might be the result of enhanced efficiency of the Ca2+-induced Ca2+ release mechanism,28 whereas acceleration of relaxation might involve a greater role of SR Ca2+ uptake in relaxation,19,27 since this transporter appears to be responsible for the largest fraction of relaxation-associated Ca2+ fluxes in adult rat ventricle.3 Developmental decrease in myofilament sensitivity to Ca2+28,29 may also contribute to accelerate twitch time-course.
Rest and twitch amplitude Potentiation of PR-TW was observed in rat ventricular myocardium at all studied ages, including neonates. In adult preparations, potentiation was small (but significant), and similar to that observed in rat isolated myocytes paced at 0.5 Hz.8 Greater degree of rest-potentiation may be obtained in the same preparation at higher stimulation rates, due to frequency-dependent decrease in amplitude of SS-TW, but not of PR-TW (R.A. Bassani & J.W.M. Bassani, unpublished observations), according to the well-known negative force–interval relationship in rat heart (e.g. 34). In adult rat ventricle, PR-TW potentiation has been observed in both multicellular preparations and isolated myocytes, with increase in the amplitude of the [Ca2+]i transient which accompanies the first PR-TW (e.g. 8,12,34). However, this potentiation does not appear to require increase in SR Ca2+ content, in sarcolemmal Ca2+ current or
719
in action potential duration to occur.7,8 In other species which present PR-TW decay (e.g. rabbit), prevention of rest-dependent SR Ca2+ loss by NCX inhibition also results in PR-TW potentiation, without apparent changes in SR Ca2+ content.8 Using confocal microscopy, Satoh et al.25 observed that myocytes which show post-rest potentiation (e.g. adult rat, adult rabbit under NCX inhibition) exhibit an increase in the frequency of microscopic SR Ca2+ release events (Ca2+ sparks) after the same rest periods required for twitch potentiation to develop. These observations have led to the proposition that PR-TW potentiation might reflect recovery of some activity-suppressed aspect of excitation–contraction coupling, possibly SR Ca2+ release.7,8,25 Although ventricular muscles from developing and adult rats have consistently exhibited potentiated PR-TW in the present study, relative potentiation was variable, depending on the age. While in adults, an average potentiation of 20% was observed, in younger animals, potentiation could reach as much as 200% (end of the first postnatal week). Considering the apparent increase in Na+–Ca2+ exchanger expression in rat developing myocardium,18,21 one might speculate whether this high degree of potentiation in young myocardium might be caused by net Ca2+ influx during rest (via NCX), which would result in increased Ca2+ accumulation in the SR. This, by its turn, would be expected to result in enhanced SR Ca2+ release during the PR-TW, especially because higher SR Ca2+ load has been shown to increase the fractional SR Ca2+ release during a twitch.24 However, this hypothesis does not seem plausible as we had no indication of rest-induced increase in SR Ca2+ load in either young or adult rats, whichever index of SR Ca2+ content was used (peak isometric tension of RCC in multicellular preparations or peak [Ca2+]i of caffeine-evoked transients recorded in isolated myocytes). Thus, the higher rest-dependent twitch potentiation in the developing ventricle does not seem to be caused by increase in SR Ca2+ content. The mechanisms underlying the higher PR-TW potentiation in young ventricle still remain unclear. Although similar relative rest-potentiation of [Ca2+]i transients have been observed in young and adult myocytes, during both SS-TW and PR-TW, absolute systolic [Ca2+]i values were >50% higher in myocytes from 1-week-old rats than in adults. This is probably due to lower myocardial passive Ca2+ binding capacity at this age (>60% of that in adults35). However, Solaro et al.29 reported that the pCa-tension curve in skinned cardiac myofibrils is not only shifted to the left, but also steeper in neonatal than in adult rats (Hill coefficient of 2.81
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v 1.81 in adults, at pH 7.0). This, associated with the greater absolute increase in [Ca2+]i during PRTW, might result in a much greater degree of myofilament activation in young that in adult myocardium, thus giving rise to a highly-potentiated post-rest contraction.
Rest, NCX and SR Ca2+ content RCC amplitude was used as an index of SR Ca2+ content in ventricular muscle. While inter-age comparisons of RCC amplitude are precluded as pointed out earlier, variations in RCC amplitude in the same preparation can be useful to detect changes in SR Ca2+ load.5,6 In our study, evocation of each PR-RCC was preceded by a SS-RCC (control contracture), to correct for possible time-dependent changes in RCC amplitude. Rest up to 600 s, although producing twitch potentiation, did not affect significantly the SR Ca2+ content at any studied age. Comparable results were obtained in isolated myocytes from adult and 1-week-old rats in which [Ca2+]i transients evoked by caffeine were used to estimate changes in SR Ca2+ load. The absence of rest-dependent alteration in SR Ca2+ agrees with previous observations in isolated adult rat myocytes,7,8 although some authors have observed SR Ca2+ gain after rest.6,11–13,36 This discrepancy might have originated from differences in the experimental conditions (e.g. extracellular [Ca2+], intracellular Na+ content). Rabbit myocytes, which normally display post-rest decrease in SR Ca2+ content (although exhibiting diastolic SR Ca2+ efflux similar to that of rats4), do not show rest-induced changes in SR Ca2+ when NCX is inhibited during rest.8 Thus, it seems plausible to conclude that, differently from rabbit myocardium, minimal Ca2+ extrusion via NCX occurs in rested adult rat ventricle. This is probably because, due to higher intracellular [Na+],36 the reversal potential of the NCX (Erev, i.e. the membrane potential at which the net Ca2+ flux mediated by NCX is null) is very close to the diastolic membrane potential. In addition, rat heart displays less NCX activity, even at systolic [Ca2+]i levels, compared to several other species.3,10,37 Since it has been shown that neonatal rat myocardium exhibits increased NCX expression,18,21 one might expect that it would develop post-rest decay of twitches and RCCs, as observed in rabbit ventricle under control conditions. However, this was not observed in the present study. On the contrary, the amplitudes of RCC developed by muscle strips and [Ca2+]i transients evoked by caffeine in isolated
myocytes were not significantly changed by rest at any developmental stage studied. The absence of major rest-dependent changes in SR Ca2+ load in young hearts might indicate that either: (a) diastolic SR Ca2+ leakage and/or uptake is much slower; (b) NCX is not able to mediate significant net Ca2+ fluxes across the sarcolemma; or (c) that the NCX is close to equilibrium at the diastolic membrane potential. To distinguish from these possibilities, we manipulated transmembrane Na+ and Ca2+ gradients so as to increase the driving force for NCX Ca2+ transport in both directions during rest. To stimulate NCX-mediated Ca2+ efflux, we perfused the muscle during rest with 0Ca solution. Assuming 14 m and 0.1 as [Na+]i36 and [Ca2+]i, respectively, and considering Erev= 3ENa−2ECa (where ENa and ECa are Na+ and Ca2+ equilibrium potentials, see e.g. 38), we estimated, for control conditions (i.e. NK), an Erev of −72 mV, which is not far from the typical diastolic membrane potential in rat cardiac cells (>−75 to −80 mV). This, combined with strong reuptake of the Ca2+ released by the SR,4 would result in relatively small net transmembrane Ca2+ efflux during diastole. However, perfusing the muscle during rest with 0Ca solution (>10 contaminating [Ca2+]) would change Erev to 61 mV, generating a driving force for Ca2+ efflux over ten-fold greater than in control conditions. The present results showed that this procedure was almost as effective at depleting SR Ca2+ in multicellular preparations as in isolated myocytes.8 However, we failed to observe increased depletion in hearts from young animals, as it might have been expected, even when SR Ca2+ loss was enhanced by 1 m caffeine. The similar increase in PR-RCC decay by caffeine in adult and young ventricles suggests absence of major ontogenetic changes in the diastolic rate of SR Ca2+ efflux. Conversely, to stimulate Ca2+ influx via NCX, we lowered extracellular Na+ to 20 m (estimated Erev of >−228 mV, with a driving force of >150 mV favoring Ca2+ influx during rest). Surprisingly, this procedure was effective at increasing RCC amplitude in young, but not in adult ventricle. In the latter, perfusion with Low-Na solution often resulted in increased diastolic force and caused spontaneous contractions, but no significant changes in RCC amplitude were seen. Here, we have to consider two processes operating in series: Ca2+ influx via NCX and uptake of this Ca2+ by the SR. Lower SR Ca2+ uptake in adults does not seem reasonable, since all available evidence points out the opposite (see e.g. 19). Higher NCX-mediated influx in developing muscle might be the cause of this behavior, although this would be at odds with our result of
Rest, Na+–Ca2+ Exchange and SR Ca2+ in Developing Heart
similar SR Ca2+ depletion under perfusion with 0Ca solution. An alternative possibility would be that, under our control experimental conditions (i.e. 0.5 Hz, 1.5 m external [Ca2+] at 36.5 °C), the SR would already have reached its saturating Ca2+ content in adult, but not in the young muscle. To test this possibility, we repeated the exposure to Low-Na solution after partial SR Ca2+ depletion. The SR was partially unloaded by prolonged exposure to 10 m caffeine in nominally Ca2+-free solution. The degree of depletion was similar in both adult and developing muscles (>60% decrease in RCC amplitude). In this condition, perfusion with LowNa solution was not only able to enhance RCC amplitude in adults, but did so at a similar extent as that in young ventricle (>65%). It thus seems plausible to conclude that: (a) the ability of the NCX to mediate diastolic Ca2+ influx does not seem to be greatly affected by age; and (b) SR Ca2+ storage capacity might have attained a “ceiling” under our control experimental conditions in adults only. Thus, Ca2+ entering via NCX during rest would, rather than be accumulated in the SR, enhance diastolic SR Ca2+ release. This would explain the increase in diastolic tension and spontaneous contractions during exposure of adult muscle to Low-Na solution. We do not know why this did not happen in young preparations as well. It is possible that the latter might have a larger SR Ca2+ “reserve”, either because the steady-state Ca2+ content was low or because of higher intra-SR Ca2+ binding capacity (increase in calsequestrin expression has been reported in the neonatal rat heart).14 In addition, lower sensitivity to Ca2+-induced Ca2+ release in the young28 might allow for greater SR Ca2+ accumulation before significant diastolic SR Ca2+ release might occur. It must be stressed that our present results do not argue against increased cardiac NCX function during the first post-natal weeks. They rather suggest that, in both young and adult rat ventricle, NCX appears to be close to thermodynamic equilibrium during diastole (when [Ca2+]i is relatively low), mediating net Ca2+ fluxes which are apparently insufficient to produce major changes in SR Ca2+ content. However, at systolic levels of [Ca2+]i, NCXmediated Ca2+ efflux has been estimated as substantially higher in intact, rat ventricular myocytes from developing rats.27 Faster Ca2+ extrusion via NCX in young myocytes may also be inferred from the faster [Ca2+]i decline of CfC transients (see Fig. 5), even though rest failed to affect the SR Ca2+ content in the same set of cells. Thus, albeit NCX function might be increased in rat myocardium during post-natal development, this transporter
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does not appear to promote, under nearly physiological conditions, diastolic Ca2+ fluxes great enough to affect the SR Ca2+ content, as it does in the rabbit ventricle.
Acknowledgements S.A.F. was the recipient of a post-graduation (Master degree) scholarship from CAPES/Brazil. This work was supported by FAPESP/Brazil (Proc. N. 95/ 0355-3). We are indebted to Dr Donald M. Bers for his valuable discussion and suggestions. We are also grateful to Mr Gilson B. Maia Jr, Ms Karen Silva, Ms Fabiana Ku¨hne, Ms Rubia Francchi and Ms Elisangela Oliveira for technical assistance.
References 1. B DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, The Netherlands: Kluwer Academic Press, 1991. 2. B RA, B JWM, B DM. Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes. J Physiol (London) 1992; 453: 591–608. 3. B JWM, B RA, B DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol (London) 1994; 476: 279–293. 4. B RA, B DM. Rate of diastolic Ca release from the sarcoplasmic reticulum of intact rabbit and rat ventricular myocytes. Biophys J 1995; 68: 2015–2022. 5. B JH. Relationships between the sarcoplasmic reticulum and sarcolemmal calcium transport revealed by rapidly cooling rabbit ventricular muscle. J Gen Physiol 1986; 88: 437–473. 6. B DM. SR Ca loading in cardiac muscle preparations based on rapid cooling contractures. Am J Physiol 1989; 256: C109–C120. 7. B DM, B RA, B JWM, B S, H LV. Paradoxical twitch potentiation after rest: increased fractional release of SR calcium. J Mol Cell Cardiol 1993; 25: 1047–1057. 8. B RA, B DM. Na-Ca exchange is required for rest-decay but not for rest-potentiation of twitches in rabbit and rat ventricular myocytes. J Mol Cell Cardiol 1994; 26: 1335–1347. 9. H-M L, B DM. Sarcoplasmic reticulum Ca uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes. Circ Res 1993; 73: 820–828. 10. S JSK, H SN, M M. Species differences in the activity of the Na+-Ca2+ exchanger in mammalian cardiac myocytes. J Physiol (London) 1995; 488: 623–631. 11. L B, Z K. Net Ca2+ influx and sarcoplasmic reticulum Ca2+ uptake in resting myocytes from the rat heart: comparison with guineapig. J Mol Cell Cardiol 1990; 22: 1221–1229.
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12. B HS, G W-D, MI BR, T K HEDJ. Force-interval relations of twitches and cold contractures in rat cardiac trabeculae: effect of ryanodine. Circ Res 1991; 69: 937–948. 13. M LS, B R, P B, B DM. Effects of left ventricular hypertrophy on force and Ca2+ handling in isolated rat myocardium. Am J Physiol 1998; 274 (Heart Circ Physiol 43): H1361–H1370. 14. L´ AM, L F, L EG, S K. Expression of sarcoplasmic reticulum Ca2+-ATPase and calsequestrin genes in rat heart during ontogenetic development and aging. Circ Res 1991; 69: 1380–1388. 15. W M, B G, G T. Postnatal maturation of excitation-contraction coupling in rat ventricle in relation to the subcellular localization and surface density of 1,4-dihydropyridine and ryanodine receptors. Circ Res 1991; 68: 662–673. 16. A N, T H, S K. Possible action of cyclopiazonic acid on myocardial sarcoplasmic reticulum: inotropic effects on neonatal and adult rat heart. Br J Pharmacol 1993; 108: 571–572. 17. B AMB, B S, M A. Developmental and tissue-specific regulation of rabbit skeletal and cardiac muscle calcium channels involved in excitation-contraction coupling. Circ Res 1994; 75: 503–510. 18. V R, S R, R H, K´ F, O´ ´ I, D H. Reciprocal changes in postnatal expression of the sarcolemmal Na+-Ca2+ exchanger and SERCA2 in rat heart. J Mol Cell Cardiol 1995; 27: 1689–1701. 19. M L. Regulation of intracellular calcium concentration in the developing heart. Cardiovasc Res 1996; 31: E61–E67. 20. H PS, C WA, A M. Na+/Ca2+ exchange current and contractions measured under Cl−-free conditions in developing rabbit hearts. Am J Physiol 1997; 273 (Heart Circ Physiol 42): H837– H846. 21. B SR, Z DB, A M. Steady-state mRNA levels of the sarcolemmal Na+-Ca2+ exchanger peak near birth in developing rabbit and rat hearts. Circ Res 1994; 74: 354–359. 22. B JWM, B RA, B DM. A method for calibration of indo-1 and resting [Ca]i in intact rabbit cardiac myocytes. Biophys J 1995; 68: 1453–1460. 23. K S, S T. Effects of rapid cooling on mechanical and electrical responses in ventricular muscle of guinea-pig. J Physiol (London) 1985; 361: 361–378. 24. B JWM, Y W, B DM. Fractional SR Ca release is regulated by trigger Ca and SR Ca content
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in cardiac myocytes. Am J Physiol 1995; 268 (Cell Physiol 37): C1313–C1319. S H, B LA, B DM. Effects of [Ca2+]i, SR Ca2+ load, and rest on Ca2+ spark frequency in ventricular myocytes. Am J Physiol 1997; 272 (Heart Circ Physiol 41): H657–H668. B RA, B JWM. [Ca]i transients in ventricular myocytes from developing rats. Biophys J 2000; 78: 374A (abstract). B RA, B JWM. Ontogenetic changes in the participations of Ca transporters in relaxation of rat cardiac myocytes. Biophys J 2000; 78: 374A (abstract). F A. Calcium release in skinned cardiac cells: variations with species, tissues, and development. Fed Proc 1982; 41: 2238–2244. S RJ, L JA, K JC, A DG. Effects of acidosis on ventricluar muscle from adult and neonatal rats. Circ Res 1988; 63: 779–787. D P, D J, T M, W R. Post-natal developmental changes in length-tension relationship in cat papillary muscles. J Physiol (London) 1975; 253: 95–102. B V, L´ C. Effects of caffeine and cyclopiazonic acid on contractile proteins of adult and newborn ferret cardiac fibers. J Mol Cell Cardiol 1996; 28: 1287–1295. N R, R MC, A PAW. Developmental changes in the ultrastructure and sarcomere shortening of the isolated rabbit ventricular myocyte. Circ Res 1987; 61: 465–483. O G, A P, L AV. Morphometric study of early postnatal development in the left and right ventricular myocardium of the rat. II. Tissue composition, capillary growth, and sarcoplasmic alterations. Circ Res 1980; 46: 503–512. K-W J, B JR. The influence of the interval between beats on myocardial contractility. Pharmacol Rev 1963; 15: 601–652. B RA, S TS, B DM. Passive Ca2+ binding in ventricular myocardium of neonatal and adult rats. Cell Calcium 1998; 23: 433–442. S MJ, B DM. Rat vs. rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes. Am J Physiol 1989; 256: C813– C822. V R, K C, S W. Sarcolemmal Na+Ca2+ exchange and sarcoplasmic reticulum Ca2+ uptake in several cardiac preparations. Biomed Biochim Acta 1987; 46: S375–S381. R JP, H CC. The stoichiometry of the cardiac sodium-calcium exchange system. J Biol Chem 1984; 259: 7733–7739.
Rest-dependence of Twitch Amplitude and Sarcoplasmic Reticulum Calcium Content in the Developing Rat Myocardium Sandro A. Ferraz, Jose´ W. M. Bassani and Rosana A. Bassani Centro de Engenharia Biome´dica and Departmento de Engenharia Biome´dica/Faculdade de Engenharia Ele´trica e de Computac¸a˜o, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil (Received 12 September 2000, accepted in revised form 2 January 2001, published electronically 7 February 2001) S. A. F, J. W. M. B R. A. B. Rest-dependence of Twitch Amplitude and Sarcoplasmic Reticulum Calcium Content in the Developing Rat Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 711–722. Post-rest contractile response was studied in isolated ventricular muscle from rats aged 1 to 90 days. Amplitude of rapid cooling contractures (RCC) was taken as an index of the sarcoplasmic reticulum (SR) Ca2+ content. We observed that: (a) developed tension (per cross-section area) increased with age; (b) time to peak twitch force and relaxation half-time decreased from 87±6 to 56±2 ms and from 68±6 to 36±1 ms, respectively, from the neonatal period to adulthood; (c) post-rest twitch potentiation was observed at all ages, with greater relative potentiation in younger preparations, although relative potentiation of [Ca2+]i transient amplitude was similar in young and adult isolated ventricular myocytes; (d) rest did not significantly affect the amplitude of RCC in muscle or caffeine-evoked [Ca2+]i transients in myocytes at any studied age; (e) favoring Ca2+ efflux via Na+–Ca2+ exchange (NCX) during rest reversed twitch potentiation and caused a similar decrease in RCC amplitude (>40%) at all ages; (f) stimulation of Ca2+ influx via NCX during rest increased RCC amplitude (>40%) only in immature preparations. However, when this procedure was repeated after partial SR Ca2+ depletion, increase in RCC amplitude was not significantly age-dependent. We conclude that post-rest twitch potentiation is already present early after birth and does not require rest-dependent changes in SR Ca2+ content at any studied age. Our results suggest that NCX is close to equilibrium during rest in both adult and developing rat myocardium, and does not seem to mediate diastolic net Ca2+ fluxes which may affect the SR Ca2+ content. 2001 Academic Press
K W: Post-rest potentiation; Rapid cooling contractures; Calcium transients; Sarcoplasmic reticulum; Sodium–calcium exchange; Post-natal development.
Introduction Contraction and relaxation in the heart depend on Ca2+ cycling between cytosol and the extracellular medium, as well as intracellular Ca2+ stores. Among the latter, the sarcoplasmic reticulum (SR) is the most prominent. It appears to be the major source
of contraction–activation Ca2+ in the adult mammalian myocardium, and the major Ca2+ sink during relaxation.1–3 The SR Ca2+ content depends on the balance of release and uptake Ca2+ fluxes across the SR membrane, as well as on intra-SR passive Ca2+ buffering, mainly by the high-capacity, low-affinity
Please address all correspondence to: Dr Rosana A. Bassani, Centro de Engenharia Biome´dica, Universidade Estadual de Campinas, Caixa Postal 6040, 13083-970 Campinas, SP, Brazil. Tel: 55 19 3788-9261. Fax: 55 19 3289-3346. E-mail: [email protected] These results were presented in part at the XVI World Congress of the International Society for Heart Research (Rhodes, Greece, May 1998).
0022–2828/01/040711+12 $35.00/0
2001 Academic Press
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protein calsequestrin (for a review, see 1 ). SR Ca2+ release takes place not only during electrical activation of the cell, but also during diastole, although at a very low rate.4 Part of the released Ca2+ is actively taken up back to the SR by the SR Ca2+-ATPase, and the remainder becomes available for extrusion via the sarcolemmal Na+–Ca2+ exchange (NCX). Depending on the activities of both transporters, net Ca2+ loss from the SR may or may not occur during rest. For instance, post-rest SR Ca2+ depletion has been consistently observed in rabbit ventricle,5–8 whereas in rat ventricle, in which NCX activity appears to be relatively lower and SR Ca2+ pump density is higher,3,9,10 either increase or no change in SR Ca2+ content during rest has been reported.6–8,11–13 There are several indications that the SR is still immature in the newborn mammalian heart. Lower expression of SR Ca2+ ATPase, diminished ATPdependent SR Ca2+ uptake and SR ATPase activity, decreased density of SR release channels (estimated by ryanodine binding) and lower negative inotropic effect of SR inhibitors in neonatal than in adult hearts have been described in several species.14–20 On the other hand, NCX expression, Na+-dependent sarcolemmal Ca2+ uptake and transmembrane currents mediated by NCX have been shown to be increased in the neonatal ventricle.18,20,21 These observations have led to the proposal that Ca2+ cycling between the extracellular medium and the cytosol is more prominent in hearts from young than from adult mammals, and that the intracellular Ca2+ cycling increases as the SR develops and the NCX undergoes ontogenetic downregulation. If this is the case, diminished role of the SR in the excitation–contraction coupling and in relaxation would be expected in the neonatal ventricle, in addition to greater impact of NCX-mediated Ca2+ fluxes on cell Ca2+ stores and intracellular [Ca2+] ([Ca2+]i) variations. This would mean that neonatal rat ventricle might behave more like the myocardium of adult rabbit than the adult counterpart of its own species. Thus, one might expect that transmembrane Ca2+ flux carried by NCX would affect SR Ca2+ content more markedly in young than in adult rat ventricle, especially during prolonged rest. In the present report, we studied multicellular ventricular preparations from developing rats, aiming at evaluating the effects of rest and NCX-mediated Ca2+ fluxes on twitch amplitude and SR Ca2+ content. We observed that, in spite of large differences in developed force and relative restpotentiation of twitches, there is a considerable similarity in the qualitative effects of rest on twitch
amplitude, and in the absence of rest-induced change in SR Ca2+ content in rat myocardium during post-natal development. Our results suggest that cardiac NCX appears to be close to thermodynamic equilibrium during rest at all studied ages.
Materials and Methods Preparations Ventricular muscle Developing (from 1 to 22 days old) and adult (3 months old) Wistar rats were sacrificed by stunning/ cervical dislocation, or decapitation (10 days old and younger). Hearts were rapidly cannulated and retrogradely perfused with oxygenated (95% O2-5% CO2) normal Krebs–Henseleit solution (NK) at room temperature to remove residual blood from coronary circulation. A free-running papillary muscle was dissected from the right ventricle and mounted in a Plexiglas chamber especially designed for muscle perfusion. The chordae tendinae was tied with a fine silk thread and attached to an isometric force transducer (mod. F-60, Narco Bio-Systems, Houston, TX, USA). The lower end of the muscle was fixed at the chamber bottom. Platinum electrodes (one in contact with the muscle and other placed parallel to the muscle long axis) delivered bipolar, square, voltage pulses (2 ms duration, 1.2times threshold) for electrical stimulation (mod. SI10, Narco Bio-Systems, Houston TX, USA). Due to the difficulty of isolating viable papillary muscles from 2-week-old and younger rats, at these ages we used a narrow (0.6 mm diameter or less) strip of the right ventricle wall, sufficiently thin to allow adequate oxygenation of the tissue. To check whether these preparations displayed similar contractile behavior, we compared papillary muscles and ventricular strips from 3-week-old rats, at which age viable preparations of both kinds can be obtained. We observed that, although strips developed >30% less active force (normalized to the cross-section area), the percent increase in twitch amplitude after rest was similar to that in papillary muscles (see Table 1). The muscle was perfused with NK and stimulated at 0.5 Hz for a period of 40 min, after which the preparation was stretched to produce an active force of 80% of that at its optimal length. An additional 30 min stabilization period was allowed before the experiment was started. Unless otherwise stated, preparations were maintained at 36.5 °C and stimulated at 0.5 Hz throughout the experient.
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Table 1 Cross-section area (CSA) and active force (normalized to CSA) in ventricular preparations (ventricular wall strip or papillary muscle) of adult (90-day-old) and developing rats during a steady-state twitch at 0.5 Hz (SS-TW), the first twitch evoked after 5 min rest (PR-TW), and a steady-state rapid-cooling contracture (SS-RCC). The number of experiments is indicated in parenthesis. Age (days)
Prep
1–2 3–6 7–14 15–22 15–22 90
Strip Strip Strip Strip Papillary Papillary
CSA (mm2) 0.21±0.02 0.22±0.01 0.26±0.01 0.36±0.06 0.15±0.02 0.23±0.01
(4) (15) (21) (9) (22) (28)
Perfusion flow rate was 25 ml/min, except during solution switch, when it was raised to 40–50 ml/ min. Isolated myocytes Ventricular myocytes were enzymatically dissociated from adult and 1-week-old Wistar rats after coronary perfusion with collagenase (Type 1, Worthington Biochem. Corp., Freehold, NJ, USA), as previously described.2 During the experiments, cells were perfused with HEPES-based solution at 23 °C and field-stimulated through a pair of platinum electrodes. Cells were loaded with 5 indo1 AM (Molecular Probes, Eugene, OR, USA) for 10–15 min at room temperature and washed for 30 min to allow for indo-1 deesterification. Indo-1 was excited at 360 nm. The ratio of emission at 405 and 485 nm was obtained after subtraction of the background fluorescence at each emission wavelength. The fluorescence ratio was converted to [Ca2+]i as described elsewhere,3 using calibration parameters determined in vivo for each cell type and an indo-1 apparent dissociation constant (Kd) of 0.844 .22
Experimental protocols Rapid cooling contractures (RCC) Rapid cooling of heart muscle evokes a contracture which is generally accepted to arise from SR Ca2+ channel opening, plus slowing of cytosolic Ca2+ removal processes.5,6,23 Although inference about the actual amount of Ca2+ stored in the SR is not possible, changes in RCC peak amplitude allow the detection of qualitative changes in SR Ca2+ content brought about by a certain experimental maneuver, such as rest.6 For RCC induction, electrical stimulation was interrupted and the heated perfusion solution was
SS-TW (mgf/mm2)
PR-TW (mgf/mm2)
SS-RCC (mgf/mm2)
17±4 (4) 50±7 (15) 48±6 (21) 98±14 (9) 144±28 (11) 239±36 (15)
38±16 (4) 115±14 (15) 129±16 (21) 143±22 (9) 213±35 (11) 275±39 (15)
198±60 (4) 204±25 (13) 279±30 (15) 513±94 (5) 883±134 (22) 876±103 (28)
quickly switched to cold NK (0 °C) with the aid of a solution selector based on solenoid valves (BioChem Valve Corp., Boonton, NJ, USA). According to our measurements, the temperature of the liquid surrounding the muscle could be decreased from 36.5 °C to 5 °C in approximately 0.4 s.
Post-rest contractile responses After stabilization of twitch amplitude at 0.5 Hz, rest periods ranging from 30 to 600 s were randomly applied, after which a twitch or a RCC was evoked. Before each post-rest RCC (PR-RCC), a steady-state RCC (SS-RCC) was obtained, i.e. evoked 2 s after interruption of the steady-state electrical stimulation, which corresponds to the regular stimulation interval at 0.5 Hz. Under control conditions, the muscle was perfused with NK during rest. To compare the ability of NCX to mediate net transmembrane Ca2+ fluxes during rest (thus changing SR Ca2+ content) in ventricular muscle of developing and adult rats, we manipulated Ca2+ and Na+ transmembrane gradients, in order to favor Ca2+ efflux or influx via the exchanger. This was achieved by Ca2+ removal (0Ca solution) or decrease of [Na+] (Low-Na solution), respectively, in the solution perfusing the muscle during rest only. At the end of the rest period in 0Ca solution, NK was switched on 15 s before the first post-rest twitch was evoked (flow rate 40–50 ml/min). Preliminary experiments showed that this period was sufficient to allow equilibration of extracellular [Ca2+], avoiding underestimation of twitch amplitude due to incomplete restoration of Ca2+ in the extracellular medium. The amplitude of post-rest twitches (PR-TW) and PR-RCC was normalized to that of steady-state twitches (SS-TW) and SS-RCC, respectively. SS-TW amplitude was considered as the average of the last five twitches before stimulation was interrupted.
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[Ca2+]i transients Steady-state electrical stimulation (0.5 Hz) was interrupted for rest periods ranging for 30 s to 5 min, after which stimulation was resumed or 10 m caffeine was applied. The amplitude of the [Ca2+]i transients evoked by caffeine (CfC) was used as an additional index of the SR Ca2+ content.4,7,8,24,25 Post-rest caffeine-induced [Ca2+]i transients (PRCfC) were normalized to the amplitude of steadystate transients (SS-CfC, i.e. caffeine was applied 2 s after interruption of stimulation). In another set of experiments, [Ca2+]i transients associated with SSTW and PR-TW after 3 min rest were obtained in isolated myocytes from 1-week-old and adult rats.
Solutions The composition of NK was (m): 129.4 NaCl, 4.5 KCl, 1.2 KH2PO4, 10.6 NaHCO3, 1.2 MgSO4, 1.5 CaCl2 and 11 glucose, pH 7.4 at 36.5 °C. For RCC induction, pH was adjusted to 7.4 at 0 °C with HCl. In 0Ca solution, equimolar MgSO4 replaced for CaCl2 (contaminating [Ca2+] >10 ). In LowNa solution, NaCl concentration was reduced to 9.4 m (20 m total [Na+]) and osmolality was maintained by sucrose addition. HEPES-based solution had the following composition (m): 140 NaCl, 6 KCl, 1.5 MgCl2, 5 HEPES, 1 CaCl2 and 11 glucose, pH 7.4 at 23 °C. All bicarbonate-based solutions were continuously gassed with 95% O25% CO2. When used, caffeine (Sigma Chem. Co., St. Louis, MO, USA) was added as a solid to the solution. All solutions were made up with deionized water and ACS standard salts.
Statistical analysis Data were compared with one-, two- or three-way analysis of variance, followed by Student– Newman–Keuls test for multiple comparisons. Values are presented as mean±... P values <0.05 were considered as indicative of statistical significance.
Results Developed active force For better characterization of the preparations used in this study, Table 1 shows active force values
(normalized to the muscle cross-section area) measured in ventricular muscle preparations from young and adult rats during: (a) SS-TW at 0.5 Hz; (b) a representative PR-TW (after 5 min rest); and (c) SSRCC. Muscle cross-section area increased with age (P<0.01) in both strips and papillary muscles due to developmental cardiac growth. However, agedependent changes in developed force were still evident after normalization to preparation size. Although analysis of variance indicated that amplitudes of SS-TW, PR-TW and SS-RCC significantly increased (P<0.01) during post-natal life, these changes occurred at determined developmental stages and were not similar for all the three types of contraction. During the first week of life (from 1–2 to 3–6 days of age), the amplitude of SS-TW and PR-TW increased >3-fold (P<0.05 for PR-TW), albeit SS-RCC amplitude increased less than 5%. During the second week (3–6 days v 7–14 days), little change occurred in amplitude of the three types of contraction. In the course of the third post-natal week (7–14 days v 15–22 days), SS-TW and SS-RCC amplitude nearly doubled (P<0.05). Subsequently, from weaning age (>3 weeks) to adulthood, data from papillary muscles indicated a greater increase in SS-TW than PR-TW amplitude (>65 v >30%), whereas RCC amplitude remained virtually the same. Although age-dependent statistical differences in force were not observed in papillary muscles, these changes contributed to reduce average relative twitch potentiation (i.e. increase in PR-TW amplitude, as percent of SS-TW amplitude) after 300 s rest from 48 to 15%. Due to the large ontogenetic variations in developed force, the amplitude of all post-rest contractions (both PR-TW and PR-RCC) will be subsequently referred to as relative values (i.e. percent of the amplitude of the respective contraction measured at steadystate). The time-course of SS-TW was abbreviated during post-natal development. The time to peak force decreased with maturation (P<0.01). It was significantly greater at age 1–2 days than in adults (87±6 v 56±2 ms, P<0.01; n=4 and 8, respectively), but statistical difference was no longer present at the end of the first week (68±5 ms, 58±6 ms and 54±2 ms at ages 3–6 days, 7–14 days and 15–21 days, respectively; n=6, 7 and 7, respectively). The time for 50% relaxation (t1/2) was also significantly reduced (P<0.01) with development, especially in the course of the first postnatal week. Values of t1/2 were 68±6 and 50±9 ms in ventricles from rats at ages 1–2 and 3–6 days, respectively (P<0.05 compared to adult). From the second post-natal week on, t1/2 values were com-
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Figure 1 Post-rest twitch potentiation in ventricular muscle from developing and adult rats. Amplitude of the first post-rest twitch (PR-TW) was normalized to that of the average, preceding steady-state twitch (SS-TW). The dashed line indicates SS-TW amplitude. Data as means±..., obtained in a group of 4–8 preparations at each age.
parable: 39±1 ms, 37±3 ms and 36±1 ms at ages 7–14 days, 15–22 days and adults, respectively. Thus, twitch relaxation in adults was nearly twice as fast as in neonates.
Effects of rest on twitch amplitude It is well known that the adult rat myocardium responds to rest with increase in the amplitude of the first post-rest twitch. In the present study, we observed significant (P<0.01) post-rest potentiation of twitch amplitude not only in adults, but at all studied ages, even neonates, over rest periods ranging from 30 to 600 s. The extent of potentiation (with relation to SS-TW), however, varied with age: it was higher in younger animals and gradually declined with maturation (see Fig. 1). This decline appeared to be mainly due to greater developmental increase in the absolute amplitude of SS-TW than of PR-TW. For instance, SS-TW absolute amplitude had an average increase of >4.8-fold from 1 week of age to adulthood (from 50 to 240 mgf/mm2), whereas a representative PR-TW (after 300 s rest) increased only >2.4-fold (from 115 to 275 mgf/ mm2) in the same period (Table 1). As seen in Figure 1, twitch potentiation had already attained a stable level after 30 s rest at all ages. In some experiments, using rest intervals shorter than 30 s, the time constant for full potentiation was determined according to Bers et al.7 The time constants in adult and developing ventricular muscle were not statistically different, and ranged between 6.5 and 8.5 s, which is close to the value determined in isolated myocytes from adult rats (>8 s).7 This suggests a common mechanism for rest-potentiation at all studied ages.
Figure 2 [Ca2+]i transients obtained in ventricular myocytes isolated from adult and 1-week-old rats, under steady-state electrical stimulation (0.5 Hz) and upon stimulation resumption after 3 min rest.
Rest-induced twitch potentiation was maximal at the end of the first post-natal week (PR-TW >300% of SS-TW v >120% in adults, Fig. 1). To investigate whether this increased potentiation was due to a larger relative increase in peak [Ca2+]i during PR-TW, we performed a set of experiments with isolated ventricular myocytes, in which [Ca2+]i was measured with indo-1 (Fig. 2). Diastolic [Ca2+]i was similar in adult and 1-week-old rat myocytes. The [Ca2+]i transient amplitude during SS-TW was 0.32±0.05 (n=11) and 0.47±0.05 (n=21) in cells from adult and 1-week-old rats, respectively. After 3 min rest, [Ca2+]i transient amplitude was potentiated at both ages (0.42±0.04 and 0.61±0.06 in adult and young, respectively, P<0.05 with relation to SS-TW). At both SS- and PR-TW, [Ca2+]i transient amplitude was greater in young cells (P<0.01). However, the relative [Ca2+]i potentiation was not statistically different (PR-TW was 143±13% and 131±9% of SS-TW in adult and young myocytes, respectively). We have previously shown that stimulation of NCX activity in the direct mode (i.e. Ca2+ extrusion
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Figure 3 (A) Original traces of steady-state (0.5 Hz) and post-rest twitches obtained in ventricular muscle from adult and 7-day-old rats, perfused with normal (NK) and Ca2+-free (0Ca) solution during 5 min rest. (B) Restdependence of twitch amplitude determined in adult rat papillary muscle perfused with normal (NK) or Ca2+-free solution (0Ca) during rest. Data are means±... (n= 8). The dashed line indicates SS-TW amplitude.
from the cell) can convert post-rest potentiation into post-rest decay of twitch amplitude in rat ventricular myocytes.8 Using the same procedure (i.e. omission of extracellular Ca2+ during rest), we were able to change twitch rest-potentiation into rest-decay also in multicellular myocardial preparations of adult rats (Fig. 3). Decrease in the extent of potentiation and even rest-decay of twitches at longer rest intervals were also observed in young myocardium when muscle was perfused with 0Ca solution during rest. This may be seen in Figure 3A, which depicts traces obtained in ventricular muscle from 7-day-old rats, in which twitch potentiation is at its peak.
Effects of rest on RCC amplitude To investigate whether age-dependent changes in PR-TW potentiation involved variations in the SR Ca2+ content, we repeated the rest protocols, but evoking PR-RCC instead of PR-TW. Figure 4A shows RCCs obtained in ventricles from adult and 7-dayold rats after different rest intervals: 2 s (SS-RCC) and 300 s (PR-RCC). The latter was evoked in either
Figure 4 (A) Original traces of RCCs obtained after 2 s and 5 min following interruption of electrical stimulation (SS and NK, respectively). The former was considered as an index of the SR Ca2+ content under steady-state stimulation (SS-RCC, see text). RCCs after 5 min rest were also obtained in preparations from adult and 7-day-old rats perfused with Ca2+-free solution during rest (0Ca). (B) Means±... of RCC amplitude (as percentage of the respective SS-RCC) as a function of rest duration. Muscles were perfused with either normal (NK) or Ca2+free (0Ca) solution during rest.
control condition or after stimulation of Ca2+ efflux by NCX (perfusion with NK or 0Ca solution, respectively, during rest). RCCs with similar features (e.g. phasic behaviour, time-course, and presence of rewarming spike) could be evoked at all ages, even in the newborn myocardium. As pointed out previously, only relative values of PR-RCC amplitude (i.e. as percent of the respective SS-RCC amplitude) shall be taken into consideration here. A striking observation was that control PR-RCC amplitude was similar to that of the respective SSRCC in both adult and developing preparations. It is important to notice that no increase in RCC amplitude was observed in the range of rest periods which consistently produced twitch potentiation, as shown in Figure 4B (note that at some ages, PR-TW amplitude was as large as 300% of that of SS-TW). This indicates that rest-potentiation of twitches occurred in the absence of detectable increase in SR Ca2+ content. Analysis of variance did not reveal statistically significant effects of rest on PR-RCC amplitude under control conditions, although PR-RCC amplitude seemed to decrease
Rest, Na+–Ca2+ Exchange and SR Ca2+ in Developing Heart
Figure 5 (A) [Ca2+]i transients obtained in isolated ventricular myocytes from 1-week-old and adult rats in response to 10 m caffeine application (arrowheads) 2 s (steady-state, SS) or 3 min after interruption of electrical stimulation. (B) Rest-dependence of the amplitude of caffeine-evoked [Ca2+]i transients (CfC) in cells from 1week-old and adult rats. Post-rest (PR-CfC) transient amplitude was normalized to that of the respective SSCfC (n=14 for both ages). The dashed line indicates SSCfC amplitude.
slightly in the younger preparations after very long (600 s) rest periods (see Fig. 4B). Additional experiments were performed with isolated myocytes from 1-week-old and adult rats, in which rest-dependent changes in SR Ca2+ content were investigated by analyzing the amplitude of caffeine-evoked [Ca2+]i transients. Figure 5A shows transients obtained after 2 s (SS-CfC) and 3 min rest (PR-CfC) in cells from adult and young rats. Transients in myocytes from developing rats differed from those in adult cells in both amplitude and time-course, as already reported.26,27 As observed for twitch-associated [Ca2+]i transients, SS-CfC transient amplitude was higher in young (1.317±0.182 , n=14) than in adult myocytes (0.830±0.063 , n=14, P<0.05). However, the amplitude of CfC transients was not significantly affected by rest up to 5 min at either of the studied ages (Fig. 5B). Perfusion with 0Ca solution during rest led to similar rest-dependent decrease in relative PR-RCC amplitude at all ages (Fig. 4B). Again, no significant
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age-related difference was observed. This indicates that, in both adult and developing myocardium, net Ca2+ extrusion by NCX during rest seems to be very low under control conditions and that stimulation of this flux produces rest-dependent SR Ca2+ depletion with comparable time-courses. When 1 m caffeine was added to 0Ca solution (to enhance SR Ca2+ release during rest), a greater decrease in PR-RCC after 10 min rest was observed in preparations from both adult (48±7% v 67±7% of SS-RCC in the absence of caffeine; n=5) and 2–3-day-old rats (49±5% v 61±5% of SS-RCC in the absence of caffeine; n=5). This effect was not significantly different between adults and neonates. In another set of experiments, Ca2+ transport by NCX in the reverse mode (i.e. Ca2+ influx) was stimulated by decrease of extracellular [Na+] from 140 to 20 m during rest. After this treatment, PR-TW could not be consistently obtained, since preparations in the presence of Low-Na solution, especially from adult rats, often developed spontaneous contractile activity, in addition to increased diastolic tension (both indicative of increase in cell Ca2+ load). However, PR-RCCs could be evoked. Surprisingly, enhancement of Ca2+ influx during rest produced different effects on PR-RCC in ventricles from adult and developing rats. In the former, although signs of cellular Ca2+ gain were present in most preparations, PR-RCC amplitude was not increased, but showed rather a small, non-significant decrease (88±6% of SS-RCC). In young myocardium, however, significant, rest-dependent increase in PR-RCC amplitude (P<0.01) was observed with the same treatment, as seen in Figure 6. Increase in RCC amplitude was significant as early as after 60 s exposure to Low-Na solution (139±3, 152±25 and 133±13% of SS-RCC in 1–6, 7–14 and 15–22 day old rats, respectively; n=6). To investigate whether the failure of Low-Na solution at increasing RCC amplitude in adult muscle was related to an already high SR Ca2+ load under steady-state stimulation, we repeated this protocol after partial SR Ca2+ unloading, as follows. After determination of SS-RCC amplitude, the preparation rested for 10 min, under perfusion with 0Ca solution containing 10 m caffeine. This solution was expected to decrease the SR Ca2+ content by enhancing SR Ca2+ leakage and facilitating extrusion of the leaked Ca2+. The muscle was then perfused for 60 s with either NK or Low-Na solution, after which a test RCC was evoked. Similar test RCC amplitudes were observed when muscles from developing (2 weeks old) and adult rats were rested in NK after SR Ca2+ unloading (39±9 and 40±4%
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Figure 7 Post-rest RCC relative amplitude in neonatal and adult myocardium. In the left block (loaded SR), preparations were rested for 3 min in the presence of normal solution (NK) or of a solution containing 20 m Na+ (Low-Na), after interruption of electrical stimulation. In the right block (depleted SR), the experiment was repeated after partial SR Ca2+ depletion, induced by prolonged perfusion with Ca2+-free solution + 10 m caffeine. n=5 for both ages. The asterisk indicates significant difference (P<0.05) between ages. Figure 6 (A) Original traces of SS-RCCs (SS) and postrest RCCs obtained after 5 min rest under perfusion with a solution containing 20 m Na+ (Low-Na) in ventricular preparations from adult and 7-day-old rats. (B) Means±... of RCC amplitude (as a percentage of SSRCC) after 5 min rest with Low-Na solution in adult and developing ventricle. Asterisks indicate significant difference (P<0.05) compared to adults (90 days). Results obtained from 6–10 preparations at each age.
of SS-RCC, respectively, n=4), indicating that a comparable degree of partial SR Ca2+-depletion had been achieved in the two kinds of preparation. When muscles were rested in Low-Na solution, test RCC amplitude was higher then after rest in NK (P<0.01), which was probably due to NCX-mediated SR Ca2+ refilling, from a lower initial Ca2+ load. However, in contrast to what happened before SR unloading, RCC amplitude after rest in Low-Na solution was similar at both age groups: 95±12% and 104±5% of SS-RCC in adult and young, respectively. This indicates that stimulation of Ca2+ influx by Low-Na exposure was equi-effective at increasing RCC amplitude in adult and young ventricle, as long as Low-Na solution was applied after the SR had been partially depleted of Ca2+ (see Fig. 7).
Discussion In this study, we used variation of the stimulatory interval as a tool to investigate the influence of
diastolic transsarcolemmal Ca2+ fluxes on the SR Ca2+ content in ventricular myocardium from developing rats. Our results suggest that, despite the previously reported enhancement in expression and function of rat cardiac Na+–Ca2+ exchanger in the first post-natal weeks, NCX appears to be near equilibrium during electrical rest in both adult and young rat ventricles, not mediating Ca2+ fluxes of magnitude sufficient to affect SR Ca2+ content.
Age-dependent increase in developed force and relaxation rate We observed a general increase in isometric peak force per cross-section area (during both twitches and RCCs) as the animal matures, despite the apparently higher sensitivity of myofilaments to Ca2+ in neonatal rat ventricle.28,29 This has also been observed in other species, for both isometric tension in intact and skinned myocardium,30,31 and unloaded shortening of isolated myocytes.32 Maturation-dependent increase in contraction amplitude might be partially explained by the marked increase in relative myofibrillar volume during post-natal development, especially during the first week of life.33 The subsequent increase in force (from the second to the third week) probably also involves age-dependent changes in cytoarchitecture, which would reduce the cell internal
Rest, Na+–Ca2+ Exchange and SR Ca2+ in Developing Heart
load.32 However, we did not find exactly the same pattern of age-dependent increase in peak force for all the three types of contraction shown in Table 1. Although both SS-TW and PR-TW are evoked by action potentials, the increase in absolute amplitude of the former from birth to adulthood was twice as much as for the latter. The significance of this changes still remains to be determined. Inferences on the SR Ca2+ content during development based on absolute RCC amplitude are precluded by several factors, such as myocyte differentiation (increase in the amount of contractile proteins and changes in cytoarchitecture), changes in myofilament sensitivity to Ca2+ and non-linear relationship between [Ca2+]i and force development. The time-course of SS-TW was markedly abbreviated in the course of the first post-natal week. Contraction rising phase, and especially relaxation, were significantly slower in neonates. After the first week, however, both time to peak tension and relaxation half-time attained adult values. This was probably a result of rapid post-natal maturation of the SR. Shortening of time to peak force might be the result of enhanced efficiency of the Ca2+-induced Ca2+ release mechanism,28 whereas acceleration of relaxation might involve a greater role of SR Ca2+ uptake in relaxation,19,27 since this transporter appears to be responsible for the largest fraction of relaxation-associated Ca2+ fluxes in adult rat ventricle.3 Developmental decrease in myofilament sensitivity to Ca2+28,29 may also contribute to accelerate twitch time-course.
Rest and twitch amplitude Potentiation of PR-TW was observed in rat ventricular myocardium at all studied ages, including neonates. In adult preparations, potentiation was small (but significant), and similar to that observed in rat isolated myocytes paced at 0.5 Hz.8 Greater degree of rest-potentiation may be obtained in the same preparation at higher stimulation rates, due to frequency-dependent decrease in amplitude of SS-TW, but not of PR-TW (R.A. Bassani & J.W.M. Bassani, unpublished observations), according to the well-known negative force–interval relationship in rat heart (e.g. 34). In adult rat ventricle, PR-TW potentiation has been observed in both multicellular preparations and isolated myocytes, with increase in the amplitude of the [Ca2+]i transient which accompanies the first PR-TW (e.g. 8,12,34). However, this potentiation does not appear to require increase in SR Ca2+ content, in sarcolemmal Ca2+ current or
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in action potential duration to occur.7,8 In other species which present PR-TW decay (e.g. rabbit), prevention of rest-dependent SR Ca2+ loss by NCX inhibition also results in PR-TW potentiation, without apparent changes in SR Ca2+ content.8 Using confocal microscopy, Satoh et al.25 observed that myocytes which show post-rest potentiation (e.g. adult rat, adult rabbit under NCX inhibition) exhibit an increase in the frequency of microscopic SR Ca2+ release events (Ca2+ sparks) after the same rest periods required for twitch potentiation to develop. These observations have led to the proposition that PR-TW potentiation might reflect recovery of some activity-suppressed aspect of excitation–contraction coupling, possibly SR Ca2+ release.7,8,25 Although ventricular muscles from developing and adult rats have consistently exhibited potentiated PR-TW in the present study, relative potentiation was variable, depending on the age. While in adults, an average potentiation of 20% was observed, in younger animals, potentiation could reach as much as 200% (end of the first postnatal week). Considering the apparent increase in Na+–Ca2+ exchanger expression in rat developing myocardium,18,21 one might speculate whether this high degree of potentiation in young myocardium might be caused by net Ca2+ influx during rest (via NCX), which would result in increased Ca2+ accumulation in the SR. This, by its turn, would be expected to result in enhanced SR Ca2+ release during the PR-TW, especially because higher SR Ca2+ load has been shown to increase the fractional SR Ca2+ release during a twitch.24 However, this hypothesis does not seem plausible as we had no indication of rest-induced increase in SR Ca2+ load in either young or adult rats, whichever index of SR Ca2+ content was used (peak isometric tension of RCC in multicellular preparations or peak [Ca2+]i of caffeine-evoked transients recorded in isolated myocytes). Thus, the higher rest-dependent twitch potentiation in the developing ventricle does not seem to be caused by increase in SR Ca2+ content. The mechanisms underlying the higher PR-TW potentiation in young ventricle still remain unclear. Although similar relative rest-potentiation of [Ca2+]i transients have been observed in young and adult myocytes, during both SS-TW and PR-TW, absolute systolic [Ca2+]i values were >50% higher in myocytes from 1-week-old rats than in adults. This is probably due to lower myocardial passive Ca2+ binding capacity at this age (>60% of that in adults35). However, Solaro et al.29 reported that the pCa-tension curve in skinned cardiac myofibrils is not only shifted to the left, but also steeper in neonatal than in adult rats (Hill coefficient of 2.81
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v 1.81 in adults, at pH 7.0). This, associated with the greater absolute increase in [Ca2+]i during PRTW, might result in a much greater degree of myofilament activation in young that in adult myocardium, thus giving rise to a highly-potentiated post-rest contraction.
Rest, NCX and SR Ca2+ content RCC amplitude was used as an index of SR Ca2+ content in ventricular muscle. While inter-age comparisons of RCC amplitude are precluded as pointed out earlier, variations in RCC amplitude in the same preparation can be useful to detect changes in SR Ca2+ load.5,6 In our study, evocation of each PR-RCC was preceded by a SS-RCC (control contracture), to correct for possible time-dependent changes in RCC amplitude. Rest up to 600 s, although producing twitch potentiation, did not affect significantly the SR Ca2+ content at any studied age. Comparable results were obtained in isolated myocytes from adult and 1-week-old rats in which [Ca2+]i transients evoked by caffeine were used to estimate changes in SR Ca2+ load. The absence of rest-dependent alteration in SR Ca2+ agrees with previous observations in isolated adult rat myocytes,7,8 although some authors have observed SR Ca2+ gain after rest.6,11–13,36 This discrepancy might have originated from differences in the experimental conditions (e.g. extracellular [Ca2+], intracellular Na+ content). Rabbit myocytes, which normally display post-rest decrease in SR Ca2+ content (although exhibiting diastolic SR Ca2+ efflux similar to that of rats4), do not show rest-induced changes in SR Ca2+ when NCX is inhibited during rest.8 Thus, it seems plausible to conclude that, differently from rabbit myocardium, minimal Ca2+ extrusion via NCX occurs in rested adult rat ventricle. This is probably because, due to higher intracellular [Na+],36 the reversal potential of the NCX (Erev, i.e. the membrane potential at which the net Ca2+ flux mediated by NCX is null) is very close to the diastolic membrane potential. In addition, rat heart displays less NCX activity, even at systolic [Ca2+]i levels, compared to several other species.3,10,37 Since it has been shown that neonatal rat myocardium exhibits increased NCX expression,18,21 one might expect that it would develop post-rest decay of twitches and RCCs, as observed in rabbit ventricle under control conditions. However, this was not observed in the present study. On the contrary, the amplitudes of RCC developed by muscle strips and [Ca2+]i transients evoked by caffeine in isolated
myocytes were not significantly changed by rest at any developmental stage studied. The absence of major rest-dependent changes in SR Ca2+ load in young hearts might indicate that either: (a) diastolic SR Ca2+ leakage and/or uptake is much slower; (b) NCX is not able to mediate significant net Ca2+ fluxes across the sarcolemma; or (c) that the NCX is close to equilibrium at the diastolic membrane potential. To distinguish from these possibilities, we manipulated transmembrane Na+ and Ca2+ gradients so as to increase the driving force for NCX Ca2+ transport in both directions during rest. To stimulate NCX-mediated Ca2+ efflux, we perfused the muscle during rest with 0Ca solution. Assuming 14 m and 0.1 as [Na+]i36 and [Ca2+]i, respectively, and considering Erev= 3ENa−2ECa (where ENa and ECa are Na+ and Ca2+ equilibrium potentials, see e.g. 38), we estimated, for control conditions (i.e. NK), an Erev of −72 mV, which is not far from the typical diastolic membrane potential in rat cardiac cells (>−75 to −80 mV). This, combined with strong reuptake of the Ca2+ released by the SR,4 would result in relatively small net transmembrane Ca2+ efflux during diastole. However, perfusing the muscle during rest with 0Ca solution (>10 contaminating [Ca2+]) would change Erev to 61 mV, generating a driving force for Ca2+ efflux over ten-fold greater than in control conditions. The present results showed that this procedure was almost as effective at depleting SR Ca2+ in multicellular preparations as in isolated myocytes.8 However, we failed to observe increased depletion in hearts from young animals, as it might have been expected, even when SR Ca2+ loss was enhanced by 1 m caffeine. The similar increase in PR-RCC decay by caffeine in adult and young ventricles suggests absence of major ontogenetic changes in the diastolic rate of SR Ca2+ efflux. Conversely, to stimulate Ca2+ influx via NCX, we lowered extracellular Na+ to 20 m (estimated Erev of >−228 mV, with a driving force of >150 mV favoring Ca2+ influx during rest). Surprisingly, this procedure was effective at increasing RCC amplitude in young, but not in adult ventricle. In the latter, perfusion with Low-Na solution often resulted in increased diastolic force and caused spontaneous contractions, but no significant changes in RCC amplitude were seen. Here, we have to consider two processes operating in series: Ca2+ influx via NCX and uptake of this Ca2+ by the SR. Lower SR Ca2+ uptake in adults does not seem reasonable, since all available evidence points out the opposite (see e.g. 19). Higher NCX-mediated influx in developing muscle might be the cause of this behavior, although this would be at odds with our result of
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similar SR Ca2+ depletion under perfusion with 0Ca solution. An alternative possibility would be that, under our control experimental conditions (i.e. 0.5 Hz, 1.5 m external [Ca2+] at 36.5 °C), the SR would already have reached its saturating Ca2+ content in adult, but not in the young muscle. To test this possibility, we repeated the exposure to Low-Na solution after partial SR Ca2+ depletion. The SR was partially unloaded by prolonged exposure to 10 m caffeine in nominally Ca2+-free solution. The degree of depletion was similar in both adult and developing muscles (>60% decrease in RCC amplitude). In this condition, perfusion with LowNa solution was not only able to enhance RCC amplitude in adults, but did so at a similar extent as that in young ventricle (>65%). It thus seems plausible to conclude that: (a) the ability of the NCX to mediate diastolic Ca2+ influx does not seem to be greatly affected by age; and (b) SR Ca2+ storage capacity might have attained a “ceiling” under our control experimental conditions in adults only. Thus, Ca2+ entering via NCX during rest would, rather than be accumulated in the SR, enhance diastolic SR Ca2+ release. This would explain the increase in diastolic tension and spontaneous contractions during exposure of adult muscle to Low-Na solution. We do not know why this did not happen in young preparations as well. It is possible that the latter might have a larger SR Ca2+ “reserve”, either because the steady-state Ca2+ content was low or because of higher intra-SR Ca2+ binding capacity (increase in calsequestrin expression has been reported in the neonatal rat heart).14 In addition, lower sensitivity to Ca2+-induced Ca2+ release in the young28 might allow for greater SR Ca2+ accumulation before significant diastolic SR Ca2+ release might occur. It must be stressed that our present results do not argue against increased cardiac NCX function during the first post-natal weeks. They rather suggest that, in both young and adult rat ventricle, NCX appears to be close to thermodynamic equilibrium during diastole (when [Ca2+]i is relatively low), mediating net Ca2+ fluxes which are apparently insufficient to produce major changes in SR Ca2+ content. However, at systolic levels of [Ca2+]i, NCXmediated Ca2+ efflux has been estimated as substantially higher in intact, rat ventricular myocytes from developing rats.27 Faster Ca2+ extrusion via NCX in young myocytes may also be inferred from the faster [Ca2+]i decline of CfC transients (see Fig. 5), even though rest failed to affect the SR Ca2+ content in the same set of cells. Thus, albeit NCX function might be increased in rat myocardium during post-natal development, this transporter
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does not appear to promote, under nearly physiological conditions, diastolic Ca2+ fluxes great enough to affect the SR Ca2+ content, as it does in the rabbit ventricle.
Acknowledgements S.A.F. was the recipient of a post-graduation (Master degree) scholarship from CAPES/Brazil. This work was supported by FAPESP/Brazil (Proc. N. 95/ 0355-3). We are indebted to Dr Donald M. Bers for his valuable discussion and suggestions. We are also grateful to Mr Gilson B. Maia Jr, Ms Karen Silva, Ms Fabiana Ku¨hne, Ms Rubia Francchi and Ms Elisangela Oliveira for technical assistance.
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