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Changes in Calcium Uptake Rate by Rat Cardiac Mitochondria during Postnatal Development
J Mol Cell Cardiol 30, 2013–2023 (1998) Article No. mc980762
Changes in Calcium Uptake Rate by Rat Cardiac Mitochondria during Postnatal Development Rosana A. Bassani, Marcia M. Fagian1, Jose´ W. M. Bassani and Anibal E. Vercesi1 Centro de Engenharia Biome´dica and Departmento de Engenharia Biome´dica, Faculdade de Engenharia Ele´trica e de Computac¸a˜o; 1Departamento de Patologia Clinica, Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil (Received 9 February 1998, accepted in revised form 24 June 1998) R. A. B, M. M. F, J. W. M. B A. E. V. Changes in Calcium Uptake Rate by Rat Cardiac Mitochondria during Postnatal Development. Journal of Molecular and Cellular Cardiology (1998) 30, 2013–2023. Ca2+ uptake, transmembrane electrical potential (Dwm) and oxygen consumption were measured in isolated ventricular mitochondria of rats from 3 days to 5 months of age. Estimated values of ruthenium redsensitive, succinate-supported maximal rate of Ca2+ uptake (Vmax, expressed as nmol Ca2+/min/mg protein) were higher in neonates and gradually fell during postnatal development (from 435±24 at 3–6 days, to 156±10 in adults, P<0.001), whereas K0.5 values (>10 l) were not significantly affected by age. Under similar conditions, mitochondria from adults (5 months old) and neonates (4–6 days old) showed comparable state 4 (succinate and a-ketoglutarate as substrates) and state 3ADP (a-ketoglutarate-supported) respiration rates, as well as Dwm values (>−150 mV). Respiration-indpendent Dwm and Ca2+ uptake, supported by valinomycin-induced K+ efflux were also investigated at these ages. A transient Dwm (>−30 mV) was evoked by valinomycin in both neonatal and adult mitochondria. Respiration-independent Ca2+ uptake was also transient, but its initial rate was significantly higher in neonates than in adults (49.4±10.0 v 28.0±5.7 mmol Ca2+/min/mg protein, P<0.01). These results indicate that Ca2+ uptake capacity of rat cardiac mitochondria is remarkably high just after birth and declines over the first weeks of postnatal life, without change in apparent affinity of the transporter. Increased mitochondrial Ca2+ uptake rate in neonates appears to be related to the uniporter itself, rather than 1998 Academic Press to modification of the driving force of the transport. K W: Mitochondria; Ca2+ uptake; Ca2+ uniporter; Electrical potential; Respiration; Ontogeny; Postnatal development.
Introduction Cardiac mitochondria can accumulate significant amounts of Ca2+ in physiological conditions. It is generally accepted that Ca2+ influx into mitochondria is mediated by a Ca2+ uniporter, driven by the electrochemical gradient of the ion. This gradient is mainly accounted for by the establishment of an electrical potential (Dwm) across the mitochondrial inner membrane (>−150 mV),
due to H+ extrusion from the matrix through the electron transport system or via ATP hydrolysis by the F1-ATP synthase. Ca2+ uptake by the uniporter does not appear to be coupled to transport of other ions or to require direct energy expenditure (for review, see e.g. Crompton 1990; Gunter and Pfeiffer, 1990; Gunter et al., 1994). Ca2+ may be extruded from mitochondria via a Na+-dependent pathway and a Na+-independent mechanism. In cardiac mitochondria, the former predominates, with a
Please address all correspondence to: Dr Rosana A. Bassani, Centro de Engenharia Biomedica, Universidade Estadual de Campinas, Caixa Postal 6040, 13083-970 Campinas, SP, Brazil.
0022–2828/98/102013+11 $30.00/0
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maximal velocity of Ca2+ transport (Vmax) of approximately one tenth of that of the Ca2+ uniporter (Crompton et al., 1976). After electrical activation of cardiac cells, cytosolic Ca2+ concentration ([Ca2+]i) oscillates between 0.1 and 1 l over a few hundred milliseconds. Although the Ca2+ uniporter appears to be kinetically incompatible with the generation of intra-mitochondrial Ca2+ transients on a beat-tobeat basis, steady-state intra-mitochondrial [Ca2+] ([Ca2+]m) would be expected to rise in response to increased time-averaged [Ca2+]i (Crompton, 1990). This might result from the increase in the amplitude and/or frequency of the [Ca2+]i transients, which usually happens, for instance, during adrenergic stimulation of the heart. Indeed, these predictions have been experimentally confirmed in isolated rat ventricular mitochondria (Leisey et al., 1993), myocytes (Griffiths et al., 1997) and whole hearts (Scheur et al., 1996). Increase in [Ca2+]m would ultimately lead to an increase in mitochondrial ATP production, since the activity of several intramitochondrial dehydrogenases (i.e. pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and aketoglutarate dehydrogenase) is stimulated by Ca2+ (McCormack and Denton, 1979; Denton and McCormack, 1990; Moreno-Sa´nchez et al., 1990). It has been proposed that F1-ATP synthase and adenine nucleotide translocase are also targets for Ca2+ stimulatory effects (Harris and Das, 1991; Gunter et al., 1994), and there is evidence that Ca2+ can stimulate directly the mitochondrial phosphorylation system (Mildaziene et al., 1996). Thus, increase in time-averaged [Ca2+]i might act, via Ca2+ uptake by the mitochondrial uniporter, as a signal for enhanced respiration and ATP synthesis, to match elevation of ATP consumption (e.g. by myofilament and sarcoplasmic reticulum Ca2+ATPases) during increased cardiac work. Several changes have been described in cardiac mitochondria during postnatal maturation both in the structural (e.g. increase in the number and volume of mitochondria, see Olivetti et al., 1980) and biochemical aspects (e.g. changes in respiration rate, as well as in protein and lipid composition of the inner membrane, see La Noue et al., 1970; Werner et al., 1982; Andres et al., 1984; Wolf et al., 1991; Scha¨gger et al., 1995; Scho¨nfeld et al., 1996). Increased respiration rate has been reported in cardiac mitochondria from newborn rabbits (Werner et al., 1982; Wolf et al., 1991), paralleled by increase in Ca2+ uptake rate (Wolf et al., 1991). However, in the rat, it was observed that oxygen consumption during the perinatal period is lower than in adults (Scho¨nfeld et al., 1996), whereas
information regarding the rate of Ca2+ uptake in this species is scarce in literature. The aims of the present study were: (a) to compare the ability of isolated cardiac mitochondria of developing and adult rats to take up Ca2+; and (b) to investigate whether possible changes are due to age-dependent alteration of mitochondrial respiration.
Materials and Methods Mitochondria isolation Mitochondria were isolated from hearts of developing (3- to 21-day-old) and adult (5-month-old) Wistar rats by differential centrifugation. Briefly, hearts were dissected and immersed in ice-cold isolation buffer with the following composition: 210 m mannitol, 75 m sucrose, 10 m N-2hydroxyethylpiperazine-N′-2 ethanosulfonic acid (HEPES), 1 m ethylene-glycol-bis-(b-aminoethylether)-N-N-N′-N′-tetraacetic acid (EGTA) and 0.1% bovine serum albumin (BSA), pH 7.2 at 4°C. After atria and major blood vessels were discarded, ventricles were washed in ice-cold medium, finely minced and homogenized (Polytron, 1 second at setting 4; Van Potter, 3 strokes) in the same buffer. The homogenate was centrifuged (4°C) at 800×g for 10 min. The supernatant was retrieved and centrifuged at 7000×g for 10 min. The resulting pellet was then resuspended in the buffer above lacking EGTA and recentrifuged at 7000× g for 10 min. The final pellet was resuspended in the EGTA-free buffer to a protein concentration of 30–50 mg/ml and kept on ice. Protein concentration was determined by the biuret method using BSA as standard.
Respiration-dependent measurements
Respiration rates Maximal state 4 respiration rate was estimated by the rate of oxygen consumption at 25°C using a Clark-type electrode in an incubation medium containing 130 m KCl, 15 m HEPES, 2 l rotenone, 2 m Pi and 10 m succinate or 5 m aketoglutarate (pH 7.2). Respiration was started by mitochondria addition (0.5–1 mg protein/ml). aketoglutarate-supported maximal state 3ADP respiration rate was measured, in the absence and presence of 0.5 m EGTA (free [Ca2+] >3.5–4.5 l
Cardiac Mitochondria Ca2+ Uptake and Development
and <5 n, respectively), after addition of 245 nmol ADP per mg protein. Succinate-dependent uncoupled respiration rate was measured after addition of 0.5 l carbonyl-cyanide p-(tri-fluoromethoxy)-phenylhydrazone (FCCP).
Transmembrane electrical potential (Dwm) Changes in Dwm were monitored at 25°C with ˚ ckerman and Wickstro¨m, 1976) using safranin O (A a spectrophotometer (Hitachi, mod. F-4010). Excitation and emission wavelengths were 495 and 586 nm, respectively. Mitochondria (final concentration 0.25–0.5 mg protein/ml) were added to a cuvette containing control incubation medium with the following composition: 130 m KCl, 15 m HEPES, 0.2 m Pi, and 2 l rotenone (pH 7.2), to which 5 l safranin O were added. Development of Dwm was initiated by addition of 10 m potassium succinate. After stabilization of Dwm, the FCCP-sensitive signal was identified by addition of the uncoupler (1 l). Calibration of safranin O signals was based on K+ equilibration across the mitochondrial membrane after addition of valinomycin (Rottenberg, 1979). Mitochondria were incubated in a K+-free medium containing 250 m sucrose, 10 m tris-(hydroxymethyl) aminomethane (pH 7.2 with HCl), 2 l rotenone, 5 l safranin O and 10 m sodium succinate. After stabilization of succinate-dependent Dwm, valinomycin (0.5 l) was applied, followed by several KCl additions (0.5–2 lmol each). Finally, the generated potential was collapsed with 0.12 m nigericin. The value of K+ equilibrium potential (EK) at each extra-mitochondrial [K+] was calculated according to Nernst’s equation, assuming: (a) constant intramitochondrial [K+] of 120 m; (b) similar K+ activity coefficient in intra- and extramitochondrial compartments; and (c) negligible passive K+ binding to mitochondria. Experimental points obtained in adult and neonatal preparations were satisfactorily fitted by Nernst’s equation (r>0.99, P<0.001).
Ca2+ uptake Succinate-supported Ca2+ uptake rates were measured at 25 and 30°C using the metallochromic Ca2+ indicator arsenazo III. Extramitochondrial [Ca2+] was estimated by arsenazo III differential absorbance at 675 and 685 nm (SLM-Aminco spectrophotometer, mod. DW2000). Mitochondrial suspension aliquots (final concentration 0.5 mg protein/ml) were added to a cuvette containing
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control incubation medium with 40 l arsenazo III (pH 7.2). In some experiments, K+-free medium was used (250 m sucrose, 10 mm Tris-HCl and 2 l rotenone, pH 7.2), to which 1 m Pi and oligomycin (2 lg/ml) were added. Ca2+ uptake was started by addition of 10 m succinate. Preliminary experiments showed that comparable uptake rates were obtained with 5–20 m succinate, in neonatal and adult mitochondria. After the arsenazo III signal attained a stable level, Ca2+ release from the mitochondria was induced by addition of FCCP (1 l). Experiments in which the absorbance signal after FCCP was different from that before succinate addition were discarded. Calibration of the arsenazo III signal was achieved by addition of CaCl2 before mitochondria energization, which not only allowed the increase in extramitochondrial [Ca2+] to the desired level, but also provided internal calibration signals in the range of [Ca2+] attained during uptake. Concentration of contaminating Ca2+ in the incubation media (typically 3.5–5.5 l), as well as in the CaCl2 stock solutions, was measured by atomic absorption spectrometry. Maximal rates of Ca2+ uptake (Vmax) and Ca2+ concentrations at which uptake rate is 50% of Vmax (K0.5) were estimated from the uptake rates (V) at different extramitochondrial Ca2+ concentrations ([Ca2+]) using the second-order Hill equation (Gunter and Pfeiffer, 1990): V=(Vmax · [Ca2+]2) / {(K0.5)2+[Ca2+]2} In all cases, the equation above fitted satisfactorily the experimental points of the V–[Ca2+] relationship (P<0.01). Ruthenium red ability to inhibit Ca2+ uptake was assessed in adult and neonatal mitochondria (0.5 mg/ml) at 25°C in separate experiments (extramitochondrial [Ca2+] of >8–9 l). In this case, ruthenium red (1–500 n) was added 10–20 s after mitochondria energization with 10 m succinate. Absence of significant ruthenium red contamination of glass and plastic material was checked with interpolated control assays (in the absence of ruthenium red). Total Ca2+ uptake (i.e. after stabilization of the indicator signal) was expressed as the percentage of control and fitted as a sigmoid function of ruthenium red molar concentration (r>0.98, P<0.01), for estimation of the ruthenium red concentration necessary to inhibit Ca2+ uptake by 50% (IC50). Triplicate assays were performed at each ruthenium red concentration. Ruthenium red solutions were prepared daily.
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Respiration-independent measurements
SUC
In these experiments, Dwm and Ca uptake supported by valinomycin-induced K+ efflux were assessed in de-energized mitochondria incubated in ˚ ckerman, 1978) at 25°C. Besides K+-free medium (A + rotenone, the K -free incubation medium also contained 1 m Pi, 0.5 l antimycin A and 2 lg/ml oligomycin. For Dwm measurements, 5 l safranin O, 0.1 m EGTA and 1 m MgCl2 were included in the medium. For Ca2+-uptake measurements, the medium contained 40 l arsenazo III. Mitochondria (0.5 mg protein/ml) were incubated in this medium for 5 min, after which respirationindependent Dwm was generated by addition of 30 n valinomycin. Valinomycin-dependent Dwm and Ca2+ uptake were compared to those supported by succinate (in which case antimycin A was omitted) or 0.2 m tetramethylenephenylene diamine (TMPD) plus 0.5 m ascorbate in a similar medium. All chemicals were purchased from Sigma Co. (St Louis, MO, USA). 2+
Statistical analysis Data were compared by one- and two-way analysis of variance, followed by Student–Newmann–Keuls test for multiple comparisons. Values are presented as mean±... or accompanied by the 95% confidence interval. P values Ζ0.05 were considered as indicative of statistical significance.
Results Ca2+ uptake during postnatal development The rate of succinate-supported Ca2+ uptake at 30°C was markedly enhanced in cardiac mitochondria from newborn rats, compared with those from adult animals, as seen in Figure 1, which shows traces recorded from mitochondrial suspensions from neonatal and adult rats containing the same amount of Ca2+ (>17 l) and mitochondrial protein (0.4 mg). Estimation of Ca2+ uptake parameters revealed different effects of age on the maximal uptake rate (Vmax) and the Ca2+ concentration for half-maximal uptake rate (K0.5). The analysis of variance showed that K0.5 was not significantly altered by development (3–6 days, 9.3±0.5 l, n=13; 7–10 days, 7.8±0.4 l, n=3; 14–15 days, 12.3±1.1 l, n=
–3
∆Abs (× 10 )
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2.5 µ MCa
30 Adult 20 Neonate 10 MITO 0
30 s
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Figure 1 Succinate-suppported Ca2+ uptake by cardiac mitochondria from adult and neonatal (3–6-day-old) rats. On the ordinates arsenazo III differential absorption (in arbitrary units). Experiments were carried out at 30°C. After mitochondria addition (MITO), extramitochondrial [Ca2+] was increased in 2.5-l steps. Uptake was started by succinate (SUC) addition and intramitochondrial Ca2+ release was induced by FCCP application. Observe the markedly higher uptake rate in neonatal mitochondria, although the content of mitochondrial protein was the same.
11; 19–21 days, 10.0±1.4 l, n=5; and adult, 9.0±1.0 l, n=19). However, Vmax was markedly influenced by age (P<0.001), attaining its highest values just after birth, then declining gradually during maturation (see Fig. 2). Vmax values estimated at these ages were: 3–6 days, 435±24 nmol Ca2+/min/mg protein; 7–10 days, 365±10 nmol Ca2+/min/mg protein; 14–15 days, 305±25 nmol Ca2+/min/mg protein; 19–21 days, 217±15 nmol Ca2+/min/mg protein; and adult, 156±10 nmol Ca2+/min/mg protein. Interestingly, even after 3 weeks of postnatal life, estimated Vmax values were still significantly higher than in adult rats (P<0.05). In both adult and neonatal (5–7-day-old) mitochondria, Ca2+ uptake was sensitive to ruthenium red in the nanomolar range [see Figs 3(a) and (b)]. Complete uptake inhibition was achieved at 300–500 n ruthenium red. Concentration– inhibition curves obtained in neonatal and adult mitochondria were superimposable [Fig. 3(b)] and estimated ruthenium red IC50 values were not statistically different (neonate, 17 n 95% confidence interval from 12–23 n, DF=4; adult, 21 n, 95% confidence interval from 17–25 n, DF=4). In a few experiments, 0.5 l ruthenium red and 10 m NaCl (to block the Ca2+ uniporter and induce Na+/Ca2+ exchange-mediated Ca2+ efflux, respectively) were added to mitochondrial suspensions after Ca2+ accumulation had reached a
Cardiac Mitochondria Ca2+ Uptake and Development (a)
(b) 500
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Figure 2 Vmax and K0.5 values of succinate-supported Ca2+ uptake by isolated cardiac mitochondria from rats during post-natal development (ages in days are indicated). Values are mean±... ∗ indicates statistical difference with relation to the adult group.
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Figure 3 (a) Succinate-supported Ca2+ uptake by cardiac mitochondria from adult rats in the absence (control) and presence of ruthenium red (RR). (b) Relationship between ruthenium red concentration and inhibition of Ca2+ uptake in adult (Χ) and neonatal (Β) mitochondria. Points are means and bars represent ... values (n=3 for each point).
steady level. This caused a slow release of the accumulated Ca2+, as inferred by rise of the arsenazo III signal. Although interference in signal level after NaCl addition (probably due to Na+ binding to the indicator) precluded estimation of Ca2+ efflux, no gross differences in the rate of change of the arsenazo III signal were observed in adult and neonatal mitochondria (data not shown).
Adult v neonate: respiration-dependent and independent Dwm and Ca2+ uptake In this series of experiments, we compared cardiac mitochondria from adult and neonatal (5–7-dayold) ventricles in assays carried out at 25°C, to slow down the time-course of valinomycin-supported Dwm and Ca2+ uptake.
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R. A. Bassani et al. Table 1 Respiration in cardiac mitochondria from adult and neonatal rats. Oxygen consumption rates (nmol O2/min/mg protein) measured in cardiac mitochondria from adult (5 months old) and neonatal (4–6 days old) rats (25°C, 0.5 mg protein/ml). Data are presented as mean±... (3–4 determination). For further details, see Materials and Methods Substrate State State State State
4a 4a 3ADPa 3ADP+EGTAb
Succinate a-Ketoglutarate a-Ketoglutarate a-Ketoglutarate
Neonate
Adult
60.3±6.9 28.0±0.7 107.2±6.4∗ 77.8±9.8†
51.8±3.4 30.1±2.9 100.8±11.8∗ 60.5±6.2†
a Extramitochondrial [Ca2+]≈3.5–4.5 l; b extramitochondrial [Ca2+]<5 n. ∗ Significantly different (P<0.01) from state 4 rate in respective age group; † significantly different (P<0.01) from state 3ADP rate in the absence of EGTA in respective age group.
Figure 4 Transmembrane electrical potential (Dwm) in neonatal (a) and adult (b) cardiac mitochondria. Mitochondria were incubated in K+-free medium at 25°C. The arrow indicates addition of 10 m succinate (SUC) or 30 n valinomycin (VAL). Note that succinate-dependent Dwm is sustained (reverted by FCCP addition), while respiration-independent Dwm developed after valinomycin attains a lower maximum and then dissipates.
Succinate- and a-ketoglutarate-dependent oxygen consumption rates at resting state were not significantly different in neonatal and adult mitochondria (see Table 1). FCCP addition significantly enhanced succinate-driven respiration in both age groups (P<0.01), but no statistical difference was observed in the uncoupled state respiration rates between ages (112.3±15.4 and 98.0±4.8 nmol O2/min/mg protein in neonates and adults, respectively). ADP addition significantly (P<0.01) enhanced a-ketoglutarate-supported respiration, but
state 3ADP respiration rates were still similar at both ages (Table 1). It is interesting to note that incubation with 0.5 m EGTA caused a significant (P<0.01) decrease in state 3ADP respiration rates, which was not statistically different between ages. Succinate-dependent steady-state Dwm was in the range of −150 to −160 mV in both neonatal and adult cardiac mitochondria (see Fig. 4). Comparable magnitudes of FCCP-sensitive Dwm were also observed after addition of TMPD plus ascorbate in K+-free medium containing rotenone, oligomycin
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Figure 5 Respiration-dependent and -independent Ca2+ uptake in isolated cardiac mitochondria. Preparations were incubated in K+-free medium at 25°C (see Materials and Methods). (a) Ca2+ uptake was started by succinate (SUC) or valinomycin (VAL) addition in adult mitochondria. Notice that, in contrast to succinate-supported Ca2+ accumulation, valinomycin-dependent Ca2+ accumulation is not FCCP-sensitive. (b) Initial phase of respiration-dependent Ca2+ uptake in neonatal (N) and adult (A) mitochondria. (c) Initial phase of respiration-independent Ca2+ uptake in both preparations.
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and antimycin A (data not shown). We have also induced respiration-independent Dwm by valinomycin addition to de-energized mitochondria incubated in K+-free medium containing respiration inhibitors and oligomycin. This Dwm was transient, attaining a much lower amplitude (peak around −30 to −35 mV), which was similar in neonatal and adult mitochondria. The time-course of respirationindependent Dwm was also comparable at the two ages, with time to peak of 10–15 s and half-time of decay of approximately 15 s (see Fig. 4). Although not sustained, the Dwm generated by valinomycin was able to support transiently Ca2+ uptake by cardiac mitochondria. Since the amount of Ca2+ taken up was too small at either very low and high extra-mitochondrial [Ca2+], we have chosen to measure the peak rate of Ca2+ uptake (Vp) within the first 5 s after valinomycin addition at 8–10 l extramitochondrial [Ca2+]. Respirationdependent Vp values were obtained after succinate addition in the same medium (in the absence of antimycin A). Figure 5(a) shows traces from experiments in which valinomycin- and succinatesupported Ca2+ uptake was induced in adult preparations. In contrast with the sustained succinatesupported uptake, respiration-independent Ca2+ uptake was transient (with a time-course similar to the respiration-independent Dwm), attaining a maximal uptake of >3 nmol Ca2+, followed by a slow FCCP-insensitive release of the Ca2+ previously taken up. The same pattern of response was observed in neonatal mitochondria. At both ages, respiration-dependent Vp values were significantly greater (P<0.001) than those obtained after valinomycin addition. However, in either condition, Ca2+ uptake was faster in neonatal mitochondria [Figs 5(b) and 5(c). Both succinateand valinomycin-dependent Vp values were significantly higher (P<0.01) in neonatal (91.9±10.3 and 49.4±10.0 nmol Ca2+/min/mg protein after succinate and valinomycin, respectively, n=5) than in adult mitochondria (48.5±2.9 and 28.0±5.7 nmol Ca2+/min/mg protein after succinate and valinomycin, respectively, n=5). The effects of the experimental protocol (i.e. whether in the presence or absence of respiration) and age were independent, as indicated by the absence of statistically-significant interaction between these factors (P>0.25).
Discussion In this study, we observed that Ca2+ uptake rate was markedly increased in cardiac mitochondria of
neonatal rats and progressively declined as maturation proceeded. Enhancement of Ca2+ uptake by cardiac mitochondria from rabbits up to 14 days old has been reported by Wolf et al. (1991). The present results show that this phenomenon does not seem to be species-dependent and that even in hearts from 3-week-old rats, in which most of cell mechanisms involved in [Ca2+]i regulation have practically reached the adult pattern (Wibo et al., 1991; Vonanen, 1992; Vetter et al., 1997), mitochondrial Ca2+ uptake rate was still significantly higher than in adults. The increase in Ca2+ uptake rate by mitochondria of young rats was paralleled by an increase in the estimated Vmax of the transport, without apparent changes in K0.5. This result suggests that enhanced capacity of transport, rather than increased affinity of the transporter, might be responsible for the agedependent changes in Ca2+ uptake rates. Ruthenium red inhibited Ca2+ uptake in a concentrationdependent fashion. At high concentrations (300–500 n), ruthenium red was able to completely block Ca2+ uptake in neonatal and adult mitochondria, which indicates that uptake was mediated via the Ca2+ uniporter, without significant contribution of other influx mechanisms. The similar potency of ruthenium red at inhibiting Ca2+ uptake at both ages (IC50 ranging from 12–25 n; compare with Ki of >30 n found in liver mitochondria by Reed and Bygrave, 1974), in addition to similar K0.5 values for Ca2+ uptake, suggests absence of marked changes in the uniporter conformation during postnatal development. Increased Vmax with unchanged K0.5 was also reported by Wolf et al. (1991) in neonatal rabbit cardiac mitochondria. Additionally, these authors observed increased phosphorylating respiration rate in these preparations, as previously observed by Werner et al. (1982), who also described augmented resting-state respiration rates in rabbit aged 1 and 10 days. In rat cardiac mitochondria, however, Scho¨enfeld et al. (1996) reported lower oxygen utilization in both phosphorylating and resting states just after birth, with attainment of adult values by the 5th day. At this age, we have also observed state 4 respiration rates similar to those of adult mitochondria, albeit, under the same conditions (i.e. using succinate as substrate in the absence of ADP), Ca2+ uptake rates were higher in the neonate. Comparable rates at both age groups were also observed when a-ketoglutarate was used as substrate, either at state 4 or state 3ADP. Respiration supported by a-ketoglutarate relies on NAD reduction by a-ketoglutarate dehydrogenase, a Ca2+-stimulated enzyme. When state 3ADP
Cardiac Mitochondria Ca2+ Uptake and Development
respiration rates were determined in the presence of 0.5 m EGTA (which was estimated to produce a drop in extramitochondrial [Ca2+] from 3.5–4.5 l to <5 n), a decrease of 30–40% in oxygen utilization rate in both preparations took place. This observation is in agreement with those of McCormack and Denton (1979) and MorenoSa´nchez et al. (1990), which indicate decline in enzyme activity, NAD reduction and state 3ADP respiration after decrease of extramitochondrial Ca2+ availability, and indicates that the enzyme appears to be Ca2+-sensitive also in neonatal mitochondria. In addition to comparable succinate-driven respiration, succinate-supported Dwm was similar at both ages. Although Dwm estimation with safranin O may not be highly accurage (Jung et al., 1988), the values obtained here are in agreement with previous reports (see Gunter and Pfeiffer, 1990) and allowed to discard gross differences in Dwm which might be present at different ages. To investigate whether respiration might in some way be involved in the age-dependent differences in Ca2+ uptake rates, we attempted to induce respiration-independent Ca2+ uptake. The generation of Dwm by valinomycin addition to de-energized mitochondria in K+-free medium was previously shown to support Ca2+ uptake in liver mitochondria ˚ ckerman, 1978; Bragadin et al., 1979; Fiskum (A et al., 1979). Here, we show that respiration-independent Ca2+ uptake can also be induced in cardiac mitochondria. In our conditions, the Dwm developed after valinomycin addition was transient, as expected for transient K+ efflux from mitochondria, which declines as transmembrane [K+] gradient dissipates. Accordingly, valinomycin-dependent Ca2+ uptake was also transient. However, the initial rate of Ca2+ uptake was significantly higher in neonatal mitochondria, despite the similar magnitude and time-course of the Dwm generated with this procedure. This indicates that the driving force for Ca2+ uptake does not appear to be the factor responsible for the differences in Ca2+ transport presently observed during post-natal maturation. One possibility would be that higher net Ca2+ uptake rates measured in young animals might be due to diminished mitochondrial Ca2+ extrusion by mitochondrial Na+/Ca2+ exchange, rather than enhanced Ca2+ influx via the Ca2+ uniporter. However, this seems to be unlikely, since our experiments were conducted in the absence of extramitochondrial Na2+. Moreover, addition of 10 m Na+ to Ca2+-loaded, ruthenium red-exposed mitochondria resulted in apparently similar rates of Ca2+ release in neonatal and adult preparations. Wolf et al. (1991) also failed to observe changes in
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Na+-evoked Ca2+ efflux in cardiac mitochondria free from neonatal rabbits which showed enhanced Ca2+ uptake rates. Although the Na+-independent mechanism mediating mitochondrial Ca2+ efflux has not been investigated in this study, the increase in Ca2+ uptake rates in young animals is too large (up to 2.7-fold) to be accounted for by a mechanism which typically transports Ca2+ at a rate less than 3 orders of magnitude lower than that of the Ca2+ uniporter (Gunter and Pfeiffer, 1990). Thus, a more likely explanation might be the occurrence of agedependent changes in the Ca2+ uniporter capacity to transport Ca2+, perhaps due to modification of lipid composition (Wolf et al., 1991) and/or of density of the uniporter molecules in the inner mitochondrial membrane. During the first weeks after birth, the mammalian heart undergoes dramatic structural development. Left-ventricular wall thickness increases up to fivefold and myocytes undergo marked hypertrophy, with a three- to five-fold increase in sarcoplasmic reticulum and myofibril relative volumes (Olivetti et al., 1980). These changes are accompanied by increase in sarcoplasmic reticulum and myofibrillar. ATPase activity, as well as in muscle contractility (Friedman, 1972; Nakanishi and Jarmakani, 1984; Kaufman et al., 1990). Both growth and enhanced contractile function represent a significant increase in energy demand. However, oxidative phosphorylation does not seem to be completely developed in rat heart at birth and there is evidence that at this age respiration control is exerted mainly (>80%) by the adenine nucleotide translocase and the F1-ATP synthase (Scho¨nfeld et al., 1996). During the first postnatal week, the role of the translocase in the respiration control decreases markedly, while expression and activity of the adenine nucleotide translocase increase with a longer time-course (Scho¨enfeld et al., 1996). Other enzymes also undergo postnatal development in rat heart, such as cytochromes and citric acid cycle enzymes (La Noue et al., 1970; Andres et al., 1984; Scha¨gger et al., 1995). It would be tempting to speculate whether increased mitochondrial Ca2+ uptake in the neonatal period might be important to assure adequate ATP production, via stimulation of Ca2+-sensitive mitochondrial enzymes (i.e. matrix dehydrogenases, F1-ATP synthase, adenine nucleotide translocase) by elevated [Ca2+]m. It seems reasonable to suppose the enhanced mitochondrial Ca2+ uptake in the first days after birth might play a role in metabolic compensation, since cardiac myocytes are rather poor in mitochondria (mitochondrial volume of 20% of cell volume in 1-day-old rat v 30% in adult rat, Olivetti
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et al., 1980). However, from the 10th day on, when relative mitochondrial volume is comparable to that in adults, but Ca2+ uptake rate is still higher, mitochondria might play a greater role in cell Ca2+ homeostasis. It has been estimated that mitochondrial Ca2+ uptake accounts for less than 1–2% of the Ca2+ fluxes during [Ca2+]i decline associated with cell relaxation in adult mammalian cardiac myocytes (Bassani RA et al., 1992; Bassani JWM et al., 1994). However, it has also been shown in this cell type that Ca2+ stored in mitochondria (corresponding to >15% of total cell Ca2+, Kessar and Crompton, 1983) may redistribute to the sarcoplasmic reticulum (Bassani JWM et al., 1993), which is the main source of contraction-activating Ca2+. Considering that in neonatal rat ventricle sarcoplasmic reticulum appears to be functionally underdeveloped (Nakanishi and Jarmakani, 1984; Kaufman et al., 1990; Wibo et al., 1991; Vetter et al., 1997) and that Ca2+ buffering by passive binding is lower than in adults (Bassani RA et al., 1998), the higher rate of mitochondrial Ca2+ uptake might result in a greater role of mitochondria in overall cell Ca2+ regulation in cardiac cells of young animals, possibly with increased contribution to cell relaxation and Ca2+ buffering than in adult cells. This possibility is under current investigation in our laboratory.
Acknowledgements This work was supported by FAPESP (Proc. N. 95/ 0355-3) and PADCT (Proc. N. 620123/94.0). We are indebted to Dr Nivaldo Baccan (Institute of Chemistry/UNICAMP) for determination of Ca2+ concentrations by atomic absorption spectrometry. We are also grateful to Mr Gilson B. Maia Jr, Mr Luis Henrique G. Ribeiro, Ms Karen A. Silva, Ms Caroline S. Sukushima and Mr Mateus Vercesi for technical assistance.
References ˚ KEO, 1978. Charge transfer during vaA linomycin-induced Ca2+ uptake in rat liver mitochondria. FEBS Lett 93: 293–296. ˚ KEO, W¨ MKF, 1976. Safranin as a A probe of the mitochondrial membrane potential. FEBS Lett 68: 191–197. A A, S J, M A, 1984. Development of enzymes of energy metabolism in rat heart. Biol Neonate 45: 78–85. B JWM, B RA, B DM, 1993. Ca2+ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes. J Physiol 460: 603–621.
B JWM, B RA, B DM, 1994. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol 476: 279– 293. B RA, B JWM, B DM, 1992. Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes. J Physiol 453: 591–608. B RA, S TS, B DM, 1998. Passive Ca2+ binding in ventricular myocardium of neonatal and adult rats. Cell Calcium 23: 433–442. B M, P T, A GF, 1979. Kinetics of Ca2+ carrier in rat liver mitochondria. Biochemistry 18: 5972–5978. C M, 1990. The role of Ca2+ in the function and dysfunction of heart mitochondria. Langer GA (ed.). Calcium and the Heart. New York: Raven Press, 167– 198. C M, C M, C E, 1976, The sodiuminduced efflux of calcium from heart mitochondria. A possible mechanism for the regulation of mitochondrial calcium. Eur J Biochem 69: 453–462. D RM, MC JG, 1990. Ca2+ as a second messenger within mitochondria of the heart and other tissues. Annu Rev Physiol 52: 451–466. F G, R B, L AL, 1979. The electric charge stoichiometry of respiration-dependent Ca2+ uptake by mitochondria. J Biol Chem 254: 6288– 6295. F WF, 1970. The intrinsic physiologic properties of the developing heart. Progr Cardiovasc Dis 15: 87– 111. G EJ, S MD, S HS, 1997. Measurement of mitochondrial calcium in single living cardiomyocytes by selective removal of cytosolic indo-1. Am J Physiol 273 (Cell Physiol 42): C37–C44. G TE, P DL, 1990. Mechanisms by which mitochondria transport calcium. Am J Physiol 258 (Cell Physiol 27): C755–C786. G TE, G KK, S S-S, G CE, 1994. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol 267 (Cell Physiol 36): C313–C339. H DA, D AM, 1991. Control of mitochondrial ATP synthesis in the heart. Biochem J 280: 561–573. J DW, D MH, B GP, 1988. Estimates of pH gradient and Donnan potential in de-energized heart mitochondria. Arch Biochem Biophys 263: 19–28. K TM, H JW, W DJ, M L, 1990. Age-related changes in myocardial relaxation and sarcoplasmic reticulum function. Am J Physiol 259 (Heart Circ Physiol 28): H309–H316. K P, C M, 1983. The sequestration of Ca2+ by mitochondria in rat heart cells. Cell Calcium 4: 295–305. L N K, N WJ, W JR, 1970. Control of citric acid cycle activity in rat heart mitochondria. J Biol Chem 245: 102–111. L JR, G LW, S DA, S RC J, 1993. Regulation of cardiac mitochondrial calcium by average extramitochondrial calcium. Am J Physiol 265 (Heart Circ Physiol 34): H1203–H1208. MC JG, D RM, 1979. The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem J 180: 533–544.
Cardiac Mitochondria Ca2+ Uptake and Development M V, B R, N Z, M A, M R, B V, K B, B GC, 1996. Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria. Biochem J 320: 329–334. M-S´ R, H BA, H RG, 1990. Influence of NAD-linked dehydrogenase activity on flux through oxidative phosphorylation. Biochem J 268: 421–428. N T, J JM, 1984. Developmental changes in myocardial mechanical function and subcellular organelles. Am J Physiol 246 (Heart Circ Physiol 15): H615–H625. O G, A P, L AV, 1980. 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 46: 503–512. R KC, B FL, 1974. The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red. Biochem J 140: 143–155. R H, 1979. The measurement of membrane potential and DpH in cells, organelles and vesicles. Meth Enzymol 55: 547–569. S¨ H, N H, H W, B U, J G, 1995. Cytochrome-c oxidase in developing rat heart. Enzymic properties and aminoterminal sequences suggest identity of the fetal heart and the adult liver isoforms. Eur J Biochem 230: 235–241. S JHM, F VM, M M, S DM,
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B AJ, C SA, 1996. Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: effects of high extracellular Ca2+. Biophys J 70: 2571–2580. S¨ P, S L, B R, 1996. Expression of the ADP/ATP carrier and expansion of the mitochondrial (ATP+ADP) pool contribute to postnatal maturation of the rat heart. Eur J Biochem 241: 895– 900. V R, S R, R H, K´ F, O´ ´ I, D H, 1997. Reciprocal changes in post-natal expression of the sarcolemmal Na+–Ca2+-exchanger and SERCA2 in rat heart. J Mol Cell Cardiol 27: 1689– 1701. V M, 1992. Force-frequency relationship, contraction duration and recirculating fraction of calcium in post-natally developing rat heart ventricles: correlation with heart rate. Acta Physiol Scand 145: 311– 321. W JC, W V, M J, S HG, 1982. Perinatal changes in mitochondrial respiration of the rabbit heart. Biol Neonate 42: 208–216. W M, B G, G T, 1991. 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 68: 662–673. W WJ, R KA, G E, S LA, 1991. Postnatal changes in heart mitochondrial calcium and energy metabolism. Am J Physiol 261 (Heart Circ Physiol 30): H1–H8.
Changes in Calcium Uptake Rate by Rat Cardiac Mitochondria during Postnatal Development Rosana A. Bassani, Marcia M. Fagian1, Jose´ W. M. Bassani and Anibal E. Vercesi1 Centro de Engenharia Biome´dica and Departmento de Engenharia Biome´dica, Faculdade de Engenharia Ele´trica e de Computac¸a˜o; 1Departamento de Patologia Clinica, Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil (Received 9 February 1998, accepted in revised form 24 June 1998) R. A. B, M. M. F, J. W. M. B A. E. V. Changes in Calcium Uptake Rate by Rat Cardiac Mitochondria during Postnatal Development. Journal of Molecular and Cellular Cardiology (1998) 30, 2013–2023. Ca2+ uptake, transmembrane electrical potential (Dwm) and oxygen consumption were measured in isolated ventricular mitochondria of rats from 3 days to 5 months of age. Estimated values of ruthenium redsensitive, succinate-supported maximal rate of Ca2+ uptake (Vmax, expressed as nmol Ca2+/min/mg protein) were higher in neonates and gradually fell during postnatal development (from 435±24 at 3–6 days, to 156±10 in adults, P<0.001), whereas K0.5 values (>10 l) were not significantly affected by age. Under similar conditions, mitochondria from adults (5 months old) and neonates (4–6 days old) showed comparable state 4 (succinate and a-ketoglutarate as substrates) and state 3ADP (a-ketoglutarate-supported) respiration rates, as well as Dwm values (>−150 mV). Respiration-indpendent Dwm and Ca2+ uptake, supported by valinomycin-induced K+ efflux were also investigated at these ages. A transient Dwm (>−30 mV) was evoked by valinomycin in both neonatal and adult mitochondria. Respiration-independent Ca2+ uptake was also transient, but its initial rate was significantly higher in neonates than in adults (49.4±10.0 v 28.0±5.7 mmol Ca2+/min/mg protein, P<0.01). These results indicate that Ca2+ uptake capacity of rat cardiac mitochondria is remarkably high just after birth and declines over the first weeks of postnatal life, without change in apparent affinity of the transporter. Increased mitochondrial Ca2+ uptake rate in neonates appears to be related to the uniporter itself, rather than 1998 Academic Press to modification of the driving force of the transport. K W: Mitochondria; Ca2+ uptake; Ca2+ uniporter; Electrical potential; Respiration; Ontogeny; Postnatal development.
Introduction Cardiac mitochondria can accumulate significant amounts of Ca2+ in physiological conditions. It is generally accepted that Ca2+ influx into mitochondria is mediated by a Ca2+ uniporter, driven by the electrochemical gradient of the ion. This gradient is mainly accounted for by the establishment of an electrical potential (Dwm) across the mitochondrial inner membrane (>−150 mV),
due to H+ extrusion from the matrix through the electron transport system or via ATP hydrolysis by the F1-ATP synthase. Ca2+ uptake by the uniporter does not appear to be coupled to transport of other ions or to require direct energy expenditure (for review, see e.g. Crompton 1990; Gunter and Pfeiffer, 1990; Gunter et al., 1994). Ca2+ may be extruded from mitochondria via a Na+-dependent pathway and a Na+-independent mechanism. In cardiac mitochondria, the former predominates, with a
Please address all correspondence to: Dr Rosana A. Bassani, Centro de Engenharia Biomedica, Universidade Estadual de Campinas, Caixa Postal 6040, 13083-970 Campinas, SP, Brazil.
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maximal velocity of Ca2+ transport (Vmax) of approximately one tenth of that of the Ca2+ uniporter (Crompton et al., 1976). After electrical activation of cardiac cells, cytosolic Ca2+ concentration ([Ca2+]i) oscillates between 0.1 and 1 l over a few hundred milliseconds. Although the Ca2+ uniporter appears to be kinetically incompatible with the generation of intra-mitochondrial Ca2+ transients on a beat-tobeat basis, steady-state intra-mitochondrial [Ca2+] ([Ca2+]m) would be expected to rise in response to increased time-averaged [Ca2+]i (Crompton, 1990). This might result from the increase in the amplitude and/or frequency of the [Ca2+]i transients, which usually happens, for instance, during adrenergic stimulation of the heart. Indeed, these predictions have been experimentally confirmed in isolated rat ventricular mitochondria (Leisey et al., 1993), myocytes (Griffiths et al., 1997) and whole hearts (Scheur et al., 1996). Increase in [Ca2+]m would ultimately lead to an increase in mitochondrial ATP production, since the activity of several intramitochondrial dehydrogenases (i.e. pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and aketoglutarate dehydrogenase) is stimulated by Ca2+ (McCormack and Denton, 1979; Denton and McCormack, 1990; Moreno-Sa´nchez et al., 1990). It has been proposed that F1-ATP synthase and adenine nucleotide translocase are also targets for Ca2+ stimulatory effects (Harris and Das, 1991; Gunter et al., 1994), and there is evidence that Ca2+ can stimulate directly the mitochondrial phosphorylation system (Mildaziene et al., 1996). Thus, increase in time-averaged [Ca2+]i might act, via Ca2+ uptake by the mitochondrial uniporter, as a signal for enhanced respiration and ATP synthesis, to match elevation of ATP consumption (e.g. by myofilament and sarcoplasmic reticulum Ca2+ATPases) during increased cardiac work. Several changes have been described in cardiac mitochondria during postnatal maturation both in the structural (e.g. increase in the number and volume of mitochondria, see Olivetti et al., 1980) and biochemical aspects (e.g. changes in respiration rate, as well as in protein and lipid composition of the inner membrane, see La Noue et al., 1970; Werner et al., 1982; Andres et al., 1984; Wolf et al., 1991; Scha¨gger et al., 1995; Scho¨nfeld et al., 1996). Increased respiration rate has been reported in cardiac mitochondria from newborn rabbits (Werner et al., 1982; Wolf et al., 1991), paralleled by increase in Ca2+ uptake rate (Wolf et al., 1991). However, in the rat, it was observed that oxygen consumption during the perinatal period is lower than in adults (Scho¨nfeld et al., 1996), whereas
information regarding the rate of Ca2+ uptake in this species is scarce in literature. The aims of the present study were: (a) to compare the ability of isolated cardiac mitochondria of developing and adult rats to take up Ca2+; and (b) to investigate whether possible changes are due to age-dependent alteration of mitochondrial respiration.
Materials and Methods Mitochondria isolation Mitochondria were isolated from hearts of developing (3- to 21-day-old) and adult (5-month-old) Wistar rats by differential centrifugation. Briefly, hearts were dissected and immersed in ice-cold isolation buffer with the following composition: 210 m mannitol, 75 m sucrose, 10 m N-2hydroxyethylpiperazine-N′-2 ethanosulfonic acid (HEPES), 1 m ethylene-glycol-bis-(b-aminoethylether)-N-N-N′-N′-tetraacetic acid (EGTA) and 0.1% bovine serum albumin (BSA), pH 7.2 at 4°C. After atria and major blood vessels were discarded, ventricles were washed in ice-cold medium, finely minced and homogenized (Polytron, 1 second at setting 4; Van Potter, 3 strokes) in the same buffer. The homogenate was centrifuged (4°C) at 800×g for 10 min. The supernatant was retrieved and centrifuged at 7000×g for 10 min. The resulting pellet was then resuspended in the buffer above lacking EGTA and recentrifuged at 7000× g for 10 min. The final pellet was resuspended in the EGTA-free buffer to a protein concentration of 30–50 mg/ml and kept on ice. Protein concentration was determined by the biuret method using BSA as standard.
Respiration-dependent measurements
Respiration rates Maximal state 4 respiration rate was estimated by the rate of oxygen consumption at 25°C using a Clark-type electrode in an incubation medium containing 130 m KCl, 15 m HEPES, 2 l rotenone, 2 m Pi and 10 m succinate or 5 m aketoglutarate (pH 7.2). Respiration was started by mitochondria addition (0.5–1 mg protein/ml). aketoglutarate-supported maximal state 3ADP respiration rate was measured, in the absence and presence of 0.5 m EGTA (free [Ca2+] >3.5–4.5 l
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and <5 n, respectively), after addition of 245 nmol ADP per mg protein. Succinate-dependent uncoupled respiration rate was measured after addition of 0.5 l carbonyl-cyanide p-(tri-fluoromethoxy)-phenylhydrazone (FCCP).
Transmembrane electrical potential (Dwm) Changes in Dwm were monitored at 25°C with ˚ ckerman and Wickstro¨m, 1976) using safranin O (A a spectrophotometer (Hitachi, mod. F-4010). Excitation and emission wavelengths were 495 and 586 nm, respectively. Mitochondria (final concentration 0.25–0.5 mg protein/ml) were added to a cuvette containing control incubation medium with the following composition: 130 m KCl, 15 m HEPES, 0.2 m Pi, and 2 l rotenone (pH 7.2), to which 5 l safranin O were added. Development of Dwm was initiated by addition of 10 m potassium succinate. After stabilization of Dwm, the FCCP-sensitive signal was identified by addition of the uncoupler (1 l). Calibration of safranin O signals was based on K+ equilibration across the mitochondrial membrane after addition of valinomycin (Rottenberg, 1979). Mitochondria were incubated in a K+-free medium containing 250 m sucrose, 10 m tris-(hydroxymethyl) aminomethane (pH 7.2 with HCl), 2 l rotenone, 5 l safranin O and 10 m sodium succinate. After stabilization of succinate-dependent Dwm, valinomycin (0.5 l) was applied, followed by several KCl additions (0.5–2 lmol each). Finally, the generated potential was collapsed with 0.12 m nigericin. The value of K+ equilibrium potential (EK) at each extra-mitochondrial [K+] was calculated according to Nernst’s equation, assuming: (a) constant intramitochondrial [K+] of 120 m; (b) similar K+ activity coefficient in intra- and extramitochondrial compartments; and (c) negligible passive K+ binding to mitochondria. Experimental points obtained in adult and neonatal preparations were satisfactorily fitted by Nernst’s equation (r>0.99, P<0.001).
Ca2+ uptake Succinate-supported Ca2+ uptake rates were measured at 25 and 30°C using the metallochromic Ca2+ indicator arsenazo III. Extramitochondrial [Ca2+] was estimated by arsenazo III differential absorbance at 675 and 685 nm (SLM-Aminco spectrophotometer, mod. DW2000). Mitochondrial suspension aliquots (final concentration 0.5 mg protein/ml) were added to a cuvette containing
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control incubation medium with 40 l arsenazo III (pH 7.2). In some experiments, K+-free medium was used (250 m sucrose, 10 mm Tris-HCl and 2 l rotenone, pH 7.2), to which 1 m Pi and oligomycin (2 lg/ml) were added. Ca2+ uptake was started by addition of 10 m succinate. Preliminary experiments showed that comparable uptake rates were obtained with 5–20 m succinate, in neonatal and adult mitochondria. After the arsenazo III signal attained a stable level, Ca2+ release from the mitochondria was induced by addition of FCCP (1 l). Experiments in which the absorbance signal after FCCP was different from that before succinate addition were discarded. Calibration of the arsenazo III signal was achieved by addition of CaCl2 before mitochondria energization, which not only allowed the increase in extramitochondrial [Ca2+] to the desired level, but also provided internal calibration signals in the range of [Ca2+] attained during uptake. Concentration of contaminating Ca2+ in the incubation media (typically 3.5–5.5 l), as well as in the CaCl2 stock solutions, was measured by atomic absorption spectrometry. Maximal rates of Ca2+ uptake (Vmax) and Ca2+ concentrations at which uptake rate is 50% of Vmax (K0.5) were estimated from the uptake rates (V) at different extramitochondrial Ca2+ concentrations ([Ca2+]) using the second-order Hill equation (Gunter and Pfeiffer, 1990): V=(Vmax · [Ca2+]2) / {(K0.5)2+[Ca2+]2} In all cases, the equation above fitted satisfactorily the experimental points of the V–[Ca2+] relationship (P<0.01). Ruthenium red ability to inhibit Ca2+ uptake was assessed in adult and neonatal mitochondria (0.5 mg/ml) at 25°C in separate experiments (extramitochondrial [Ca2+] of >8–9 l). In this case, ruthenium red (1–500 n) was added 10–20 s after mitochondria energization with 10 m succinate. Absence of significant ruthenium red contamination of glass and plastic material was checked with interpolated control assays (in the absence of ruthenium red). Total Ca2+ uptake (i.e. after stabilization of the indicator signal) was expressed as the percentage of control and fitted as a sigmoid function of ruthenium red molar concentration (r>0.98, P<0.01), for estimation of the ruthenium red concentration necessary to inhibit Ca2+ uptake by 50% (IC50). Triplicate assays were performed at each ruthenium red concentration. Ruthenium red solutions were prepared daily.
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Respiration-independent measurements
SUC
In these experiments, Dwm and Ca uptake supported by valinomycin-induced K+ efflux were assessed in de-energized mitochondria incubated in ˚ ckerman, 1978) at 25°C. Besides K+-free medium (A + rotenone, the K -free incubation medium also contained 1 m Pi, 0.5 l antimycin A and 2 lg/ml oligomycin. For Dwm measurements, 5 l safranin O, 0.1 m EGTA and 1 m MgCl2 were included in the medium. For Ca2+-uptake measurements, the medium contained 40 l arsenazo III. Mitochondria (0.5 mg protein/ml) were incubated in this medium for 5 min, after which respirationindependent Dwm was generated by addition of 30 n valinomycin. Valinomycin-dependent Dwm and Ca2+ uptake were compared to those supported by succinate (in which case antimycin A was omitted) or 0.2 m tetramethylenephenylene diamine (TMPD) plus 0.5 m ascorbate in a similar medium. All chemicals were purchased from Sigma Co. (St Louis, MO, USA). 2+
Statistical analysis Data were compared by one- and two-way analysis of variance, followed by Student–Newmann–Keuls test for multiple comparisons. Values are presented as mean±... or accompanied by the 95% confidence interval. P values Ζ0.05 were considered as indicative of statistical significance.
Results Ca2+ uptake during postnatal development The rate of succinate-supported Ca2+ uptake at 30°C was markedly enhanced in cardiac mitochondria from newborn rats, compared with those from adult animals, as seen in Figure 1, which shows traces recorded from mitochondrial suspensions from neonatal and adult rats containing the same amount of Ca2+ (>17 l) and mitochondrial protein (0.4 mg). Estimation of Ca2+ uptake parameters revealed different effects of age on the maximal uptake rate (Vmax) and the Ca2+ concentration for half-maximal uptake rate (K0.5). The analysis of variance showed that K0.5 was not significantly altered by development (3–6 days, 9.3±0.5 l, n=13; 7–10 days, 7.8±0.4 l, n=3; 14–15 days, 12.3±1.1 l, n=
–3
∆Abs (× 10 )
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Figure 1 Succinate-suppported Ca2+ uptake by cardiac mitochondria from adult and neonatal (3–6-day-old) rats. On the ordinates arsenazo III differential absorption (in arbitrary units). Experiments were carried out at 30°C. After mitochondria addition (MITO), extramitochondrial [Ca2+] was increased in 2.5-l steps. Uptake was started by succinate (SUC) addition and intramitochondrial Ca2+ release was induced by FCCP application. Observe the markedly higher uptake rate in neonatal mitochondria, although the content of mitochondrial protein was the same.
11; 19–21 days, 10.0±1.4 l, n=5; and adult, 9.0±1.0 l, n=19). However, Vmax was markedly influenced by age (P<0.001), attaining its highest values just after birth, then declining gradually during maturation (see Fig. 2). Vmax values estimated at these ages were: 3–6 days, 435±24 nmol Ca2+/min/mg protein; 7–10 days, 365±10 nmol Ca2+/min/mg protein; 14–15 days, 305±25 nmol Ca2+/min/mg protein; 19–21 days, 217±15 nmol Ca2+/min/mg protein; and adult, 156±10 nmol Ca2+/min/mg protein. Interestingly, even after 3 weeks of postnatal life, estimated Vmax values were still significantly higher than in adult rats (P<0.05). In both adult and neonatal (5–7-day-old) mitochondria, Ca2+ uptake was sensitive to ruthenium red in the nanomolar range [see Figs 3(a) and (b)]. Complete uptake inhibition was achieved at 300–500 n ruthenium red. Concentration– inhibition curves obtained in neonatal and adult mitochondria were superimposable [Fig. 3(b)] and estimated ruthenium red IC50 values were not statistically different (neonate, 17 n 95% confidence interval from 12–23 n, DF=4; adult, 21 n, 95% confidence interval from 17–25 n, DF=4). In a few experiments, 0.5 l ruthenium red and 10 m NaCl (to block the Ca2+ uniporter and induce Na+/Ca2+ exchange-mediated Ca2+ efflux, respectively) were added to mitochondrial suspensions after Ca2+ accumulation had reached a
Cardiac Mitochondria Ca2+ Uptake and Development (a)
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Figure 2 Vmax and K0.5 values of succinate-supported Ca2+ uptake by isolated cardiac mitochondria from rats during post-natal development (ages in days are indicated). Values are mean±... ∗ indicates statistical difference with relation to the adult group.
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Figure 3 (a) Succinate-supported Ca2+ uptake by cardiac mitochondria from adult rats in the absence (control) and presence of ruthenium red (RR). (b) Relationship between ruthenium red concentration and inhibition of Ca2+ uptake in adult (Χ) and neonatal (Β) mitochondria. Points are means and bars represent ... values (n=3 for each point).
steady level. This caused a slow release of the accumulated Ca2+, as inferred by rise of the arsenazo III signal. Although interference in signal level after NaCl addition (probably due to Na+ binding to the indicator) precluded estimation of Ca2+ efflux, no gross differences in the rate of change of the arsenazo III signal were observed in adult and neonatal mitochondria (data not shown).
Adult v neonate: respiration-dependent and independent Dwm and Ca2+ uptake In this series of experiments, we compared cardiac mitochondria from adult and neonatal (5–7-dayold) ventricles in assays carried out at 25°C, to slow down the time-course of valinomycin-supported Dwm and Ca2+ uptake.
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R. A. Bassani et al. Table 1 Respiration in cardiac mitochondria from adult and neonatal rats. Oxygen consumption rates (nmol O2/min/mg protein) measured in cardiac mitochondria from adult (5 months old) and neonatal (4–6 days old) rats (25°C, 0.5 mg protein/ml). Data are presented as mean±... (3–4 determination). For further details, see Materials and Methods Substrate State State State State
4a 4a 3ADPa 3ADP+EGTAb
Succinate a-Ketoglutarate a-Ketoglutarate a-Ketoglutarate
Neonate
Adult
60.3±6.9 28.0±0.7 107.2±6.4∗ 77.8±9.8†
51.8±3.4 30.1±2.9 100.8±11.8∗ 60.5±6.2†
a Extramitochondrial [Ca2+]≈3.5–4.5 l; b extramitochondrial [Ca2+]<5 n. ∗ Significantly different (P<0.01) from state 4 rate in respective age group; † significantly different (P<0.01) from state 3ADP rate in the absence of EGTA in respective age group.
Figure 4 Transmembrane electrical potential (Dwm) in neonatal (a) and adult (b) cardiac mitochondria. Mitochondria were incubated in K+-free medium at 25°C. The arrow indicates addition of 10 m succinate (SUC) or 30 n valinomycin (VAL). Note that succinate-dependent Dwm is sustained (reverted by FCCP addition), while respiration-independent Dwm developed after valinomycin attains a lower maximum and then dissipates.
Succinate- and a-ketoglutarate-dependent oxygen consumption rates at resting state were not significantly different in neonatal and adult mitochondria (see Table 1). FCCP addition significantly enhanced succinate-driven respiration in both age groups (P<0.01), but no statistical difference was observed in the uncoupled state respiration rates between ages (112.3±15.4 and 98.0±4.8 nmol O2/min/mg protein in neonates and adults, respectively). ADP addition significantly (P<0.01) enhanced a-ketoglutarate-supported respiration, but
state 3ADP respiration rates were still similar at both ages (Table 1). It is interesting to note that incubation with 0.5 m EGTA caused a significant (P<0.01) decrease in state 3ADP respiration rates, which was not statistically different between ages. Succinate-dependent steady-state Dwm was in the range of −150 to −160 mV in both neonatal and adult cardiac mitochondria (see Fig. 4). Comparable magnitudes of FCCP-sensitive Dwm were also observed after addition of TMPD plus ascorbate in K+-free medium containing rotenone, oligomycin
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Figure 5 Respiration-dependent and -independent Ca2+ uptake in isolated cardiac mitochondria. Preparations were incubated in K+-free medium at 25°C (see Materials and Methods). (a) Ca2+ uptake was started by succinate (SUC) or valinomycin (VAL) addition in adult mitochondria. Notice that, in contrast to succinate-supported Ca2+ accumulation, valinomycin-dependent Ca2+ accumulation is not FCCP-sensitive. (b) Initial phase of respiration-dependent Ca2+ uptake in neonatal (N) and adult (A) mitochondria. (c) Initial phase of respiration-independent Ca2+ uptake in both preparations.
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and antimycin A (data not shown). We have also induced respiration-independent Dwm by valinomycin addition to de-energized mitochondria incubated in K+-free medium containing respiration inhibitors and oligomycin. This Dwm was transient, attaining a much lower amplitude (peak around −30 to −35 mV), which was similar in neonatal and adult mitochondria. The time-course of respirationindependent Dwm was also comparable at the two ages, with time to peak of 10–15 s and half-time of decay of approximately 15 s (see Fig. 4). Although not sustained, the Dwm generated by valinomycin was able to support transiently Ca2+ uptake by cardiac mitochondria. Since the amount of Ca2+ taken up was too small at either very low and high extra-mitochondrial [Ca2+], we have chosen to measure the peak rate of Ca2+ uptake (Vp) within the first 5 s after valinomycin addition at 8–10 l extramitochondrial [Ca2+]. Respirationdependent Vp values were obtained after succinate addition in the same medium (in the absence of antimycin A). Figure 5(a) shows traces from experiments in which valinomycin- and succinatesupported Ca2+ uptake was induced in adult preparations. In contrast with the sustained succinatesupported uptake, respiration-independent Ca2+ uptake was transient (with a time-course similar to the respiration-independent Dwm), attaining a maximal uptake of >3 nmol Ca2+, followed by a slow FCCP-insensitive release of the Ca2+ previously taken up. The same pattern of response was observed in neonatal mitochondria. At both ages, respiration-dependent Vp values were significantly greater (P<0.001) than those obtained after valinomycin addition. However, in either condition, Ca2+ uptake was faster in neonatal mitochondria [Figs 5(b) and 5(c). Both succinateand valinomycin-dependent Vp values were significantly higher (P<0.01) in neonatal (91.9±10.3 and 49.4±10.0 nmol Ca2+/min/mg protein after succinate and valinomycin, respectively, n=5) than in adult mitochondria (48.5±2.9 and 28.0±5.7 nmol Ca2+/min/mg protein after succinate and valinomycin, respectively, n=5). The effects of the experimental protocol (i.e. whether in the presence or absence of respiration) and age were independent, as indicated by the absence of statistically-significant interaction between these factors (P>0.25).
Discussion In this study, we observed that Ca2+ uptake rate was markedly increased in cardiac mitochondria of
neonatal rats and progressively declined as maturation proceeded. Enhancement of Ca2+ uptake by cardiac mitochondria from rabbits up to 14 days old has been reported by Wolf et al. (1991). The present results show that this phenomenon does not seem to be species-dependent and that even in hearts from 3-week-old rats, in which most of cell mechanisms involved in [Ca2+]i regulation have practically reached the adult pattern (Wibo et al., 1991; Vonanen, 1992; Vetter et al., 1997), mitochondrial Ca2+ uptake rate was still significantly higher than in adults. The increase in Ca2+ uptake rate by mitochondria of young rats was paralleled by an increase in the estimated Vmax of the transport, without apparent changes in K0.5. This result suggests that enhanced capacity of transport, rather than increased affinity of the transporter, might be responsible for the agedependent changes in Ca2+ uptake rates. Ruthenium red inhibited Ca2+ uptake in a concentrationdependent fashion. At high concentrations (300–500 n), ruthenium red was able to completely block Ca2+ uptake in neonatal and adult mitochondria, which indicates that uptake was mediated via the Ca2+ uniporter, without significant contribution of other influx mechanisms. The similar potency of ruthenium red at inhibiting Ca2+ uptake at both ages (IC50 ranging from 12–25 n; compare with Ki of >30 n found in liver mitochondria by Reed and Bygrave, 1974), in addition to similar K0.5 values for Ca2+ uptake, suggests absence of marked changes in the uniporter conformation during postnatal development. Increased Vmax with unchanged K0.5 was also reported by Wolf et al. (1991) in neonatal rabbit cardiac mitochondria. Additionally, these authors observed increased phosphorylating respiration rate in these preparations, as previously observed by Werner et al. (1982), who also described augmented resting-state respiration rates in rabbit aged 1 and 10 days. In rat cardiac mitochondria, however, Scho¨enfeld et al. (1996) reported lower oxygen utilization in both phosphorylating and resting states just after birth, with attainment of adult values by the 5th day. At this age, we have also observed state 4 respiration rates similar to those of adult mitochondria, albeit, under the same conditions (i.e. using succinate as substrate in the absence of ADP), Ca2+ uptake rates were higher in the neonate. Comparable rates at both age groups were also observed when a-ketoglutarate was used as substrate, either at state 4 or state 3ADP. Respiration supported by a-ketoglutarate relies on NAD reduction by a-ketoglutarate dehydrogenase, a Ca2+-stimulated enzyme. When state 3ADP
Cardiac Mitochondria Ca2+ Uptake and Development
respiration rates were determined in the presence of 0.5 m EGTA (which was estimated to produce a drop in extramitochondrial [Ca2+] from 3.5–4.5 l to <5 n), a decrease of 30–40% in oxygen utilization rate in both preparations took place. This observation is in agreement with those of McCormack and Denton (1979) and MorenoSa´nchez et al. (1990), which indicate decline in enzyme activity, NAD reduction and state 3ADP respiration after decrease of extramitochondrial Ca2+ availability, and indicates that the enzyme appears to be Ca2+-sensitive also in neonatal mitochondria. In addition to comparable succinate-driven respiration, succinate-supported Dwm was similar at both ages. Although Dwm estimation with safranin O may not be highly accurage (Jung et al., 1988), the values obtained here are in agreement with previous reports (see Gunter and Pfeiffer, 1990) and allowed to discard gross differences in Dwm which might be present at different ages. To investigate whether respiration might in some way be involved in the age-dependent differences in Ca2+ uptake rates, we attempted to induce respiration-independent Ca2+ uptake. The generation of Dwm by valinomycin addition to de-energized mitochondria in K+-free medium was previously shown to support Ca2+ uptake in liver mitochondria ˚ ckerman, 1978; Bragadin et al., 1979; Fiskum (A et al., 1979). Here, we show that respiration-independent Ca2+ uptake can also be induced in cardiac mitochondria. In our conditions, the Dwm developed after valinomycin addition was transient, as expected for transient K+ efflux from mitochondria, which declines as transmembrane [K+] gradient dissipates. Accordingly, valinomycin-dependent Ca2+ uptake was also transient. However, the initial rate of Ca2+ uptake was significantly higher in neonatal mitochondria, despite the similar magnitude and time-course of the Dwm generated with this procedure. This indicates that the driving force for Ca2+ uptake does not appear to be the factor responsible for the differences in Ca2+ transport presently observed during post-natal maturation. One possibility would be that higher net Ca2+ uptake rates measured in young animals might be due to diminished mitochondrial Ca2+ extrusion by mitochondrial Na+/Ca2+ exchange, rather than enhanced Ca2+ influx via the Ca2+ uniporter. However, this seems to be unlikely, since our experiments were conducted in the absence of extramitochondrial Na2+. Moreover, addition of 10 m Na+ to Ca2+-loaded, ruthenium red-exposed mitochondria resulted in apparently similar rates of Ca2+ release in neonatal and adult preparations. Wolf et al. (1991) also failed to observe changes in
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Na+-evoked Ca2+ efflux in cardiac mitochondria free from neonatal rabbits which showed enhanced Ca2+ uptake rates. Although the Na+-independent mechanism mediating mitochondrial Ca2+ efflux has not been investigated in this study, the increase in Ca2+ uptake rates in young animals is too large (up to 2.7-fold) to be accounted for by a mechanism which typically transports Ca2+ at a rate less than 3 orders of magnitude lower than that of the Ca2+ uniporter (Gunter and Pfeiffer, 1990). Thus, a more likely explanation might be the occurrence of agedependent changes in the Ca2+ uniporter capacity to transport Ca2+, perhaps due to modification of lipid composition (Wolf et al., 1991) and/or of density of the uniporter molecules in the inner mitochondrial membrane. During the first weeks after birth, the mammalian heart undergoes dramatic structural development. Left-ventricular wall thickness increases up to fivefold and myocytes undergo marked hypertrophy, with a three- to five-fold increase in sarcoplasmic reticulum and myofibril relative volumes (Olivetti et al., 1980). These changes are accompanied by increase in sarcoplasmic reticulum and myofibrillar. ATPase activity, as well as in muscle contractility (Friedman, 1972; Nakanishi and Jarmakani, 1984; Kaufman et al., 1990). Both growth and enhanced contractile function represent a significant increase in energy demand. However, oxidative phosphorylation does not seem to be completely developed in rat heart at birth and there is evidence that at this age respiration control is exerted mainly (>80%) by the adenine nucleotide translocase and the F1-ATP synthase (Scho¨nfeld et al., 1996). During the first postnatal week, the role of the translocase in the respiration control decreases markedly, while expression and activity of the adenine nucleotide translocase increase with a longer time-course (Scho¨enfeld et al., 1996). Other enzymes also undergo postnatal development in rat heart, such as cytochromes and citric acid cycle enzymes (La Noue et al., 1970; Andres et al., 1984; Scha¨gger et al., 1995). It would be tempting to speculate whether increased mitochondrial Ca2+ uptake in the neonatal period might be important to assure adequate ATP production, via stimulation of Ca2+-sensitive mitochondrial enzymes (i.e. matrix dehydrogenases, F1-ATP synthase, adenine nucleotide translocase) by elevated [Ca2+]m. It seems reasonable to suppose the enhanced mitochondrial Ca2+ uptake in the first days after birth might play a role in metabolic compensation, since cardiac myocytes are rather poor in mitochondria (mitochondrial volume of 20% of cell volume in 1-day-old rat v 30% in adult rat, Olivetti
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et al., 1980). However, from the 10th day on, when relative mitochondrial volume is comparable to that in adults, but Ca2+ uptake rate is still higher, mitochondria might play a greater role in cell Ca2+ homeostasis. It has been estimated that mitochondrial Ca2+ uptake accounts for less than 1–2% of the Ca2+ fluxes during [Ca2+]i decline associated with cell relaxation in adult mammalian cardiac myocytes (Bassani RA et al., 1992; Bassani JWM et al., 1994). However, it has also been shown in this cell type that Ca2+ stored in mitochondria (corresponding to >15% of total cell Ca2+, Kessar and Crompton, 1983) may redistribute to the sarcoplasmic reticulum (Bassani JWM et al., 1993), which is the main source of contraction-activating Ca2+. Considering that in neonatal rat ventricle sarcoplasmic reticulum appears to be functionally underdeveloped (Nakanishi and Jarmakani, 1984; Kaufman et al., 1990; Wibo et al., 1991; Vetter et al., 1997) and that Ca2+ buffering by passive binding is lower than in adults (Bassani RA et al., 1998), the higher rate of mitochondrial Ca2+ uptake might result in a greater role of mitochondria in overall cell Ca2+ regulation in cardiac cells of young animals, possibly with increased contribution to cell relaxation and Ca2+ buffering than in adult cells. This possibility is under current investigation in our laboratory.
Acknowledgements This work was supported by FAPESP (Proc. N. 95/ 0355-3) and PADCT (Proc. N. 620123/94.0). We are indebted to Dr Nivaldo Baccan (Institute of Chemistry/UNICAMP) for determination of Ca2+ concentrations by atomic absorption spectrometry. We are also grateful to Mr Gilson B. Maia Jr, Mr Luis Henrique G. Ribeiro, Ms Karen A. Silva, Ms Caroline S. Sukushima and Mr Mateus Vercesi for technical assistance.
References ˚ KEO, 1978. Charge transfer during vaA linomycin-induced Ca2+ uptake in rat liver mitochondria. FEBS Lett 93: 293–296. ˚ KEO, W¨ MKF, 1976. Safranin as a A probe of the mitochondrial membrane potential. FEBS Lett 68: 191–197. A A, S J, M A, 1984. Development of enzymes of energy metabolism in rat heart. Biol Neonate 45: 78–85. B JWM, B RA, B DM, 1993. Ca2+ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes. J Physiol 460: 603–621.
B JWM, B RA, B DM, 1994. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol 476: 279– 293. B RA, B JWM, B DM, 1992. Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes. J Physiol 453: 591–608. B RA, S TS, B DM, 1998. Passive Ca2+ binding in ventricular myocardium of neonatal and adult rats. Cell Calcium 23: 433–442. B M, P T, A GF, 1979. Kinetics of Ca2+ carrier in rat liver mitochondria. Biochemistry 18: 5972–5978. C M, 1990. The role of Ca2+ in the function and dysfunction of heart mitochondria. Langer GA (ed.). Calcium and the Heart. New York: Raven Press, 167– 198. C M, C M, C E, 1976, The sodiuminduced efflux of calcium from heart mitochondria. A possible mechanism for the regulation of mitochondrial calcium. Eur J Biochem 69: 453–462. D RM, MC JG, 1990. Ca2+ as a second messenger within mitochondria of the heart and other tissues. Annu Rev Physiol 52: 451–466. F G, R B, L AL, 1979. The electric charge stoichiometry of respiration-dependent Ca2+ uptake by mitochondria. J Biol Chem 254: 6288– 6295. F WF, 1970. The intrinsic physiologic properties of the developing heart. Progr Cardiovasc Dis 15: 87– 111. G EJ, S MD, S HS, 1997. Measurement of mitochondrial calcium in single living cardiomyocytes by selective removal of cytosolic indo-1. Am J Physiol 273 (Cell Physiol 42): C37–C44. G TE, P DL, 1990. Mechanisms by which mitochondria transport calcium. Am J Physiol 258 (Cell Physiol 27): C755–C786. G TE, G KK, S S-S, G CE, 1994. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol 267 (Cell Physiol 36): C313–C339. H DA, D AM, 1991. Control of mitochondrial ATP synthesis in the heart. Biochem J 280: 561–573. J DW, D MH, B GP, 1988. Estimates of pH gradient and Donnan potential in de-energized heart mitochondria. Arch Biochem Biophys 263: 19–28. K TM, H JW, W DJ, M L, 1990. Age-related changes in myocardial relaxation and sarcoplasmic reticulum function. Am J Physiol 259 (Heart Circ Physiol 28): H309–H316. K P, C M, 1983. The sequestration of Ca2+ by mitochondria in rat heart cells. Cell Calcium 4: 295–305. L N K, N WJ, W JR, 1970. Control of citric acid cycle activity in rat heart mitochondria. J Biol Chem 245: 102–111. L JR, G LW, S DA, S RC J, 1993. Regulation of cardiac mitochondrial calcium by average extramitochondrial calcium. Am J Physiol 265 (Heart Circ Physiol 34): H1203–H1208. MC JG, D RM, 1979. The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem J 180: 533–544.
Cardiac Mitochondria Ca2+ Uptake and Development M V, B R, N Z, M A, M R, B V, K B, B GC, 1996. Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria. Biochem J 320: 329–334. M-S´ R, H BA, H RG, 1990. Influence of NAD-linked dehydrogenase activity on flux through oxidative phosphorylation. Biochem J 268: 421–428. N T, J JM, 1984. Developmental changes in myocardial mechanical function and subcellular organelles. Am J Physiol 246 (Heart Circ Physiol 15): H615–H625. O G, A P, L AV, 1980. 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 46: 503–512. R KC, B FL, 1974. The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red. Biochem J 140: 143–155. R H, 1979. The measurement of membrane potential and DpH in cells, organelles and vesicles. Meth Enzymol 55: 547–569. S¨ H, N H, H W, B U, J G, 1995. Cytochrome-c oxidase in developing rat heart. Enzymic properties and aminoterminal sequences suggest identity of the fetal heart and the adult liver isoforms. Eur J Biochem 230: 235–241. S JHM, F VM, M M, S DM,
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B AJ, C SA, 1996. Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: effects of high extracellular Ca2+. Biophys J 70: 2571–2580. S¨ P, S L, B R, 1996. Expression of the ADP/ATP carrier and expansion of the mitochondrial (ATP+ADP) pool contribute to postnatal maturation of the rat heart. Eur J Biochem 241: 895– 900. V R, S R, R H, K´ F, O´ ´ I, D H, 1997. Reciprocal changes in post-natal expression of the sarcolemmal Na+–Ca2+-exchanger and SERCA2 in rat heart. J Mol Cell Cardiol 27: 1689– 1701. V M, 1992. Force-frequency relationship, contraction duration and recirculating fraction of calcium in post-natally developing rat heart ventricles: correlation with heart rate. Acta Physiol Scand 145: 311– 321. W JC, W V, M J, S HG, 1982. Perinatal changes in mitochondrial respiration of the rabbit heart. Biol Neonate 42: 208–216. W M, B G, G T, 1991. 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 68: 662–673. W WJ, R KA, G E, S LA, 1991. Postnatal changes in heart mitochondrial calcium and energy metabolism. Am J Physiol 261 (Heart Circ Physiol 30): H1–H8.