Calcium signal transmission between ryanodine receptors and mitochondria.

Control of energy metabolism by increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) may represent a fundamental mechanism to meet the ATP demand imposed by heart contractions, but the machinery underlying propagation of [Ca(2+)] signals from ryanodine receptor Ca(2+) release channels (RyR) to the mitochondria remains elusive. Using permeabilized cardiac (H9c2) cells we investigated the cytosolic [Ca(2+)] ([Ca(2+)](c)) and [Ca(2+)](m) signals elicited by activation of RyR. Caffeine, Ca(2+), and ryanodine evoked [Ca(2+)](c) spikes that often appeared as frequency-modulated [Ca(2+)](c) oscillations in these permeabilized cells. Rapid increases in [Ca(2+)](m) and activation of the Ca(2+)-sensitive mitochondrial dehydrogenases were synchronized to the rising phase of the [Ca(2+)](c) spikes. The RyR-mediated elevations of global [Ca(2+)](c) were in the submicromolar range, but the rate of [Ca(2+)](m) increases was as large as it was in the presence of 30 microm global [Ca(2+)](c). Furthermore, RyR-dependent increases of [Ca(2+)](m) were relatively insensitive to buffering of [Ca(2+)](c) by EGTA. Therefore, RyR-driven rises of [Ca(2+)](m) appear to result from large and rapid increases of perimitochondrial [Ca(2+)]. The falling phase of [Ca(2+)](c) spikes was followed by a rapid decay of [Ca(2+)](m). CGP37157 slowed down relaxation of [Ca(2+)](m) spikes, whereas cyclosporin A had no effect, suggesting that activation of the mitochondrial Ca(2+) exchangers accounts for rapid reversal of the [Ca(2+)](m) response with little contribution from the permeability transition pore. Thus, rapid activation of Ca(2+) uptake sites and Ca(2+) exchangers evoked by RyR-mediated local [Ca(2+)](c) signals allow mitochondria to respond rapidly to single [Ca(2+)](c) spikes in cardiac cells.

Excitation-contraction coupling involves local interactions between dihydropyridine receptors (DHPR) 1 located in the plasma membrane and ryanodine receptor Ca 2ϩ release chan-nels (RyR) located in the sarcoplasmic reticulum Ca 2ϩ stores. The mechanism for excitation-contraction coupling differs between skeletal and cardiac muscle. Conformational coupling between DHPR and RyR is utilized in skeletal muscle, whereas RyR respond to local [Ca 2ϩ ] signals established by Ca 2ϩ entering through DHPR in cardiac muscle. RyR-mediated [Ca 2ϩ ] signals lead to activation of the contractile machinery (for review see Ref. 1) and may also be utilized to activate mitochondrial energy metabolism to meet the ATP requirements of muscle contraction (2)(3)(4). This regulatory pathway depends on propagation of [Ca 2ϩ ] c signals to the mitochondria and, in turn, activation of the Ca 2ϩ -sensitive mitochondrial dehydrogenases. However, significant mitochondrial Ca 2ϩ uptake does not occur until [Ca 2ϩ ] c exceeds 1 M in studies carried out with permeabilized cardiac myocytes (5) and the threshold [Ca 2ϩ ] c for mitochondrial Ca 2ϩ uptake is 300 -500 nM in intact myocytes (6). Moreover, the rate of activation of mitochondrial Ca 2ϩ uptake is slow at submicromolar [Ca 2ϩ ] levels (7,8). Thus, RyR-mediated increases of global [Ca 2ϩ ] c that peak in the submicromolar range are not expected to yield rapid rises of [Ca 2ϩ ] m . Importantly, [Ca 2ϩ ] peaks at substantially higher levels in the vicinity of activated RyR than the peak of the global [Ca 2ϩ ] c rise (9,10). Therefore, if mitochondria are located in sufficient proximity to experience the local [Ca 2ϩ ] gradients, propagation of RyR-mediated [Ca 2ϩ ] signals to the mitochondria may result from local coupling between these sites. Recent data on the distance between mitochondria and adjacent Ca 2ϩ release units in cardiac myocytes are in support of the idea that RyR and mitochondria are sufficiently close to each other to establish local [Ca 2ϩ ] communication (11). Significantly, local [Ca 2ϩ ] signaling between IP 3 receptor Ca 2ϩ release channels and mitochondria has also been shown in many cell types (12)(13)(14)(15)(16).
Most of the studies carried out in recent years to investigate propagation of RyR-mediated [Ca 2ϩ ] c responses to the mitochondria used fluorescence measurements of [Ca 2ϩ ] m in intact cardiac cells. Some reports showed that changes of [Ca 2ϩ ] m appear in association with changes in the frequency of [Ca 2ϩ ] c spiking (6,(17)(18)(19), whereas other studies showed beat-to-beat control of [Ca 2ϩ ] m in myocytes (20 -23). In smooth muscle cells, [Ca 2ϩ ] m was also demonstrated to closely follow the kinetics of [Ca 2ϩ ] c rise evoked by activation of RyR (24). Though strategies have been developed for loading of the mitochondria with Ca 2ϩsensitive probes, it is difficult to eliminate the cytosolic tracer component in many cell types, particularly in cardiac myocytes. Thus, the concern was raised that the cytosolic contribution from Ca 2ϩ -sensitive dyes could contribute to the beat-to-beat fluctuations reported in [Ca 2ϩ ] m (e.g. Ref. 6). Studies that evaluated mitochondrial Ca 2ϩ uptake by investigating the effect of mitochondrial inhibitors on RyR-linked [Ca 2ϩ ] c signals also provided conflicting data on the contribution of mitochondria to single [Ca 2ϩ ] c transients (Refs. 6, 17 versus 25). Electron probe microanalysis studies carried out to determine the changes of total mitochondrial calcium showed the presence (26) as well as the absence of changes (27) during the contractile cycle. Nevertheless, membrane potential (⌬⌿ m ) and mitochondrial NAD(P)H responses, which depended on mitochondrial Ca 2ϩ uptake, have been reported during RyR-mediated [Ca 2ϩ ] c spikes (28,29). This suggests that propagation of [Ca 2ϩ ] c increases to the mitochondria can regulate Ca 2ϩ -dependent mitochondrial functions.
The present study was undertaken to determine whether a privileged Ca 2ϩ transfer is involved in the Ca 2ϩ coupling between RyR and mitochondria and to address how mitochondrial Ca 2ϩ transport is controlled during RyR-mediated [

EXPERIMENTAL PROCEDURES
Cell Culture-H9c2 cells (obtained from ATCC) were cultured in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 1 mM pyruvate in humidified air (CO 2 5%) at 37°C. For imaging experiments cells were plated onto poly-D-lysine-coated glass coverslips, and for PCR assays cells were cultured in 75-cm 2 flasks. Cells were grown for 3-5 days for studies with myoblasts (subconfluent cultures), whereas cells were grown to reach confluency (1 week on average) and subsequently for an additional 3-7 days to allow differentiation for studies with myotubes.
Fluorescence Imaging Measurements-Prior to loading with fluorescent dyes, the cells were preincubated for 30 min in an extracellular medium (2% bovine serum albumin/ extracellular medium) consisting of 121 mM NaCl, 5 mM NaHCO 3 , 10 mM Na-HEPES, 4.7 mM KCl, 1. For permeabilized cell measurements the dye-loaded cells were washed three times with a Ca 2ϩ -free extracellular buffer, containing 120 mM NaCl, 5 mM KCl, 1 mM KH 2 PO 4 , 0.2 mM MgCl 2 , 0.1 mM EGTA, and 20 mM Hepes/NaOH, pH 7.4, and then permeabilized with 20 g/ml digitonin for 4 min in an intracellular medium composed of 120 mM KCl, 10 mM NaCl, 1 mM KH 2 PO4, 20 mM Hepes/Tris, pH 7.2, supplemented with 2 mM MgATP, 20 M EGTA/Tris, and 1 g/ml each of antipain, leupeptin, pepstatin. The intracellular medium was passed through a Chelex column prior to the addition of protease inhibitors to reduce ambient Ca 2ϩ . To detect perimembrane [Ca 2ϩ ] ([Ca 2ϩ ] pm ), cells were labeled with 0.5-4 M fura-C18 during the permeabilization. After permeabilization the cells were washed with the same buffer without digitonin. Measurements were performed in intracellular medium that contained ϳ300 nM [Ca 2ϩ ] after the addition of 2 mM ATP, 2 mM succinate, and protease inhibitors. During measurements of ⌬⌿ m , 20 nM TMRE was present in the buffer to maintain the fluorescence labeling achieved during the loading with TMRE.
Images were acquired using an Olympus IX70 inverted microscope (40ϫ, UApo340, NA 1.35 oil immersion objective) fitted with a cooled CCD camera (PXL, Photometrics) under computer control. The computer also controlled a scanning monochromator (DeltaRAM, PTI) to select the excitation wavelength. Excitation at 340 and 380 nm was used for fura2 and fura-C18, 380 nm was used for mitoGFP, 360 nm was used for NAD(P)H, and 545 nm was used for rhod2 and TMRE. Using multiwavelength beamsplitter/emission filter combinations (Chroma Technology Corp., Brattleboro, VT) simultaneous detection of fura-C18 and rhod2 fluorescence or NADH and rhod2 fluorescence was achieved.
Experiments were carried out with at least three different cell preparations, and 20 -60 cells were monitored in each experiment. Traces represent single cell responses unless indicated otherwise.
Reverse Transcriptase-PCR Amplification and Sequencing-Total RNA was prepared from H9c2 cells using the RNeasy Mini Kit (Qiagen). First strand cDNA was synthesized from 0.5-1 g of RNA using MuLV reverse transcriptase (Perkin-Elmer). cDNAs were amplified using Am-pliTaq DNA polymerase (Perkin-Elmer). PCR reactions (100 l) contained first strand cDNA template, 150 nM of each primer, 0.2 mM of each dNTP, 2 mM MgCl 2 , 1ϫ PCR buffer II, and 2.5 units of AmpliTaq DNA polymerase. Reactions were carried out in a Perkin-Elmer Gene-Amp PCR System 9600 for 35 cycles, after an initial 105 s at 95°C, melting for 15 s at 95°C, and anneal-extending for 30 s at the appropriate temperature (50 -60°C). This was followed by a final extension for 7 min at 72°C. Primers to distinguish between rat cardiac and skeletal muscle ␣ 1 subunits were selected by (GCG Prime program) and corresponded to a region in the fourth domain. The cardiac forward primer (3955-3973) was as follows: 5Ј-TCCTCATCCTGCTCAACAC-3Ј. The cardiac reverse primer (4403-4420) was as follows: 5Ј-AATACCT-GCATCCCAATC-3Ј. The skeletal forward primer (1297-1314) was as follows: 5Ј-ATGAACCACATATCGGAC-3Ј. The skeletal reverse primer (1665-1682) was as follows: 5Ј-ATCAGCAAAGCCACATAC-3Ј.
Degenerate primers were used to detect the three RyR isoforms and corresponded to a region in the membrane-spanning domains M3 and M4 (30) 100 l of PCR product was fractionated on a 1-2% low melting temperature agarose gel (Life Technologies, Inc.), and individual bands were purified using a QIAquick gel extraction kit (Qiagen). The purified PCR product was sequenced by the Molecular Core Facility at UMDNJ.

RESULTS AND DISCUSSION
Calcium Signaling in H9c2 Myoblasts and Myotubes-In subconfluent cultures, H9c2 myoblasts displayed a spindle or polygonal shape, and multinucleated myotubes were not present ( Fig. 1, upper row of images). Depolarization by high [K ϩ ] (60 mM KCl) or activation of RyR with caffeine did not elicit a [Ca 2ϩ ] c signal, whereas an IP 3 -linked agonist, vasopressin (VP), induced large [Ca 2ϩ ] c spikes in most of the cells (Fig. 1, i-v). Myoblast cultures grown to reach confluency and subsequently for an additional 3-7 days to allow differentiation contained a number of long, multinucleated myotubes ( Fig. 1, lower row of images). Depolarization resulted in a rise in [Ca 2ϩ ] c in all of the cells in these differentiated cultures (Fig. 1, vii). This [Ca 2ϩ ] c rise was prevented by the dihydropyridine Ca 2ϩ channel inhibitor, nifedipine (10 M, not shown). The addition of caffeine also caused a [Ca 2ϩ ] c rise, which was particularly large in the multinucleated cells (Fig. 1, viii). The rise in [Ca 2ϩ ] c evoked by VP added after caffeine was relatively small in differentiated cells (Fig. 1, iv-v versus ix-x). In these experiments, depletion of intracellular Ca 2ϩ stores evoked by caffeine before the addition of VP might explain the decreased VP-dependent [Ca 2ϩ ] c response in the differentiated cells. However, the relatively small effect of VP was also apparent when the hormone was added to naive cells (data not shown).
To identify the Ca 2ϩ channels that were involved in the depolarization-and caffeine-induced [Ca 2ϩ ] c responses in myotubes, the expression of voltage-operated L-type Ca 2ϩ channels and RyR were examined by reverse transcriptase-PCR. Isotype-specific regions of the ␣ 1 subunit of L-type Ca 2ϩ channel mRNA were amplified by reverse transcriptase-PCR. Fig. 2 shows that differentiation of the myoblasts was associated with substantial increases in the mRNA encoding both cardiac and skeletal isoforms of the L-type channels. This is consistent with previous studies (31)(32)(33). RyR mRNA was not detected in myoblasts but it appeared after differentiation (Fig. 2). The reverse transcriptase-PCR results suggest the presence of one isotype that shows 85, 76, and 80% sequence identity to human RyR1, RyR2, and RyR3, respectively. Taken together, these data provide evidence for expression of the Ca 2ϩ channels that are hallmarks of the myotube phenotype in differentiated H9c2 cells and also suggest that up-regulation of the expression of these Ca 2ϩ channels occurring during differentiation accounts for the appearance of [Ca 2ϩ ] c signals in response to depolarization and to RyR activators in differentiated cells.
Propagation of RyR-mediated [Ca 2ϩ ] Signals to the Mitochondria in Permeabilized Myotubes-To study whether the Ca 2ϩ signals mediated by the activation of RyR are relayed to the mitochondria, [Ca 2ϩ ] m was studied in rhod2-loaded, permeabilized H9c2 cells. Compartmentalization of rhod2 in the mitochondria is facilitated by the net positive charge of the dye, and the cytosolic dye component is eliminated during cell permeabilization. In myoblasts, ryanodine or caffeine caused no rise in [Ca 2ϩ ] rhod2 , whereas the addition of IP 3 yielded large responses in most of the cells (Fig. 3A, upper row). By contrast, myotubes displayed large increases of [Ca 2ϩ ] rhod2 in response to ryanodine (Fig. 3A, v) or caffeine (viii). Images obtained at higher spatial resolution revealed a pattern of [Ca 2ϩ ] rhod2 similar to the distribution of GFP targeted to the mitochondria (Fig. 3, B versus C). Moreover, the decrease of [Ca 2ϩ ] rhod2 resulting from uncoupler-induced dissipation of the mitochondrial membrane potential was also associated with the structures that displayed the caffeine-induced [Ca 2ϩ ] rhod2 rise (Fig.  3B, iii versus iv). These data suggest that activation of RyR in the myotubes brings about increases of [Ca 2ϩ ] m .
Several intramitochondrial dehydrogenases are activated by elevated [Ca 2ϩ ] m , and this activation can be monitored fluorometrically through changes in the pyridine nucleotide redox state. Caffeine-induced increases of [Ca 2ϩ ] rhod2 were associated with an increase in NAD(P)H fluorescence, reflecting the activation of Ca 2ϩ -sensitive mitochondrial dehydrogenases (Fig.  4A). The increase in NAD(P)H fluorescence was transient, but the addition of Ca 2ϩ to the permeabilized cells elicited a further rise of [Ca 2ϩ ] rhod2 and a second transient in NAD(P)H. The fluorescence increase associated with maximal reduction of pyridine nucleotides was obtained by the addition of rotenone to block oxidation of NADH by the respiratory chain. These data provide evidence that the rise of [Ca 2ϩ ] m coupled to activation of RyR exerts control over the Ca 2ϩ -sensitive steps of mitochondrial metabolism in permeabilized myotubes.
To further investigate the signal transmission machinery between RyR and mitochondria, rhod2-loaded permeabilized cells were exposed to fura-C18, a dye that allows measurements of [Ca 2ϩ ] immediately adjacent to cellular membranes (16,34,35 (Fig. 4B). Relaxation of the FIG. 2. PCR products from cardiac myoblasts and myotubes. RNA was isolated from myoblasts (non-diff) and differentiated H9c2 cells (diff) as described under "Experimental Procedures." PCR products generated by primers designed to amplify sequences of the specific genetic messages for cardiac and skeletal muscle L-type Ca 2ϩ channels and RyR were fractionated on agarose gel. Negative controls (no RNA but with reverse primer) were performed and showed no bands. [Ca 2ϩ ] m spikes was slower than the fall of [Ca 2ϩ ] pm , and in some cells an effectively sustained rise of [Ca 2ϩ ] m was coupled to a baseline-spike pattern of the caffeine-induced [Ca 2ϩ ] pm oscillations ( Fig. 4D and presumably, C). Treatment with supramaximal caffeine caused spikes in [Ca 2ϩ ] pm and [Ca 2ϩ ] m displaying a prolonged decay phase (Fig. 4, B and D). The addition of mitochondrial uncoupler caused a small increase in [Ca 2ϩ ] pm but induced a rapid decay of the caffeine-induced elevation of [Ca 2ϩ ] m (Fig. 4B). Rapid reversal of the caffeineinduced rise of [Ca 2ϩ ] m was also evoked by Ru360, a drug reported to inhibit mitochondrial Ca 2ϩ uptake without inhibition of RyR (36). Consistent with this report, the Ru360-induced decay of [Ca 2ϩ ] m was observed in the absence of inhibition of RyR-mediated [Ca 2ϩ ] pm oscillations (Fig. 4D). Furthermore, Ru360 added before supramaximal caffeine did not affect the magnitude of the [Ca 2ϩ ] pm increase but abolished the rise in [Ca 2ϩ ] m (Fig. 4, E and F). Coupled oscillations of [Ca 2ϩ ] pm and [Ca 2ϩ ] m also occurred in permeabilized myotubes exposed to other activators of the RyR, such as Ca 2ϩ and ryanodine (Fig. 5, A and B). Taken together, these data show that activation of RyR in permeabilized myotubes yields oscillatory Ca 2ϩ release and reuptake that is associated with rapid activation of mitochondrial Ca 2ϩ uptake to yield [Ca 2ϩ ] m signals.
Local  were observed in the absence of any caffeine-induced [Ca 2ϩ ] pm increase. After washout of the Ca 2ϩ buffer, the caffeine-induced Ca 2ϩ release brought about a [Ca 2ϩ ] pm rise and an augmented [Ca 2ϩ ] m increase (Fig. 6) response by a slow Ca 2ϩ buffer like EGTA suggests that the spatial separation between RyR and mitochondrial Ca 2ϩ uptake sites is probably in the range of 100 nm rather than Ͻ20 nm (10,16). This conclusion is in agreement with recent measurements of the distances between Ca 2ϩ release units and mitochondria in ventricular myocardium (11).
In an effort to assess the magnitude of the local [Ca 2ϩ ] increases to which the mitochondrial Ca 2ϩ uptake sites are exposed during RyR-mediated Ca 2ϩ release, rates of mitochondrial Ca 2ϩ uptake were measured with varying concentrations of added medium Ca 2ϩ and compared with the rate of Ca 2ϩ uptake obtained during caffeine-induced Ca 2ϩ release. Fig. 7 shows that the addition of Ca 2ϩ led to dose-dependent increases in mitochondrial Ca 2ϩ uptake rates. Half-maximal stimulation was attained at Ϸ20 M [Ca 2ϩ ] c , and maximal activation appeared to require at least 50 M [Ca 2ϩ ] c . Importantly, mitochondrial Ca 2ϩ uptake was not limited by the mitochondrial membrane potential (⌬⌿ m ) under the substrate conditions used in these experiments (2 mM succinate, 2 mM MgATP), because only very small depolarizations were evoked by the addition of large pulses of Ca 2ϩ (Fig. 7, left panel). When caffeine-induced mitochondrial Ca 2ϩ uptake was studied under ] m changes were recorded simultaneously in a permeabilized myotube. Cells were exposed to submaximal and maximal concentrations of caffeine and then to uncoupler (unc; 1 M carbonyl cyanide p-trifluoromethoxyphenylhydrazone ϩ 2.5 g/ml oligomycin). C, time courses of [Ca 2ϩ ] m changes were recorded in a permeabilized myotube exposed to caffeine and Ru360 (Calbiochem, 2 M). D, time courses of [Ca 2ϩ ] pm and [Ca 2ϩ ] m changes were recorded simultaneously in a permeabilized myotube. Cells were exposed to caffeine and Ru360 (2 M). E and F, mean increases of [Ca 2ϩ ] pm and [Ca 2ϩ ] m in naive and Ru360-pretreated myotubes challenged with supramaximal caffeine. Myotubes were exposed to Ru360 (10 M for 3 min) or solvent and then to caffeine (20 mM). The mean traces for [Ca 2ϩ ] pm and [Ca 2ϩ ] m shown in E are from separate runs using cells from the same culture. All myotubes in the field were included for calculation of the mean responses. Calculated magnitude of the [Ca 2ϩ ] pm and [Ca 2ϩ ] m responses shown in F are mean Ϯ S.E. of values from 19 (control) and 16 (Ru360-pretreated) cells from two different cell cultures. Fluorescence of NAD(P)H and compartmentalized rhod2 are shown as fluorescence arbitrary units. the same conditions in permeabilized myotubes, the Ca 2ϩ uptake rate was similar to that achieved with 30 M added free [Ca 2ϩ ] in the bulk medium (Fig. 7). Thus, the local [Ca 2ϩ ] c rise sensed by the mitochondrial Ca 2ϩ uptake sites appears to be in the range of 30 M.
Although the demonstration of efficient Ca 2ϩ transfer from RyR to the mitochondrial matrix in H9c2 myotubes does not prove that the same functional organization occurs in myocytes of the beating heart, there are a number of observations that suggest that mitochondria are exposed to local [Ca 2ϩ ] gradients generated by RyR in cardiac myocytes. The distance between mitochondria and RyR estimated from our [Ca 2ϩ ] flux studies in the presence of EGTA/Ca 2ϩ buffer as well as the large magnitude of perimitochondrial [Ca 2ϩ ] rises are consistent with the data from morphological measurements in cardiac myocytes showing proximity of mitochondria and calcium release units (11). Moreover, a recent study demonstrated large [Ca 2ϩ ] m elevations in association with caffeine-induced [Ca 2ϩ ] c spikes in skinned myocytes. 3 Therefore, it is likely that a privileged pathway of calcium signaling also exists between RyR and mitochondria in cardiac myocytes.  (Fig. 8A). At higher caffeine concentrations, the interspike period decreased (Fig. 8, B  and F), and the fraction of cells showing more prolonged [Ca 2ϩ ] pm transients increased (Fig. 8, C and E). The prolonged responses at high levels of caffeine presumably reflect sustained activation of RyR, and the decay phase was because of depletion of the caffeine-sensitive Ca 2ϩ store. The gradual decrease in the amplitude of spikes during [Ca 2ϩ ] pm oscillations observed in a few cells (e.g. Figs. 8B and 9A) may also reflect depletion of the Ca 2ϩ store. Moreover, bleaching of the fluorescent probe or dissociation from the membranes that occurred during the measurements may also contribute to the decrease in spike amplitudes during [Ca 2ϩ ] pm oscillations. In most cells, spikes of [Ca 2ϩ ] pm were associated with transients of [Ca 2ϩ ] m that displayed a cell-specific pattern of the decay phase. Increases in the oscillation frequency established by successive caffeine additions did not affect the amplitude and upstroke kinetics of the individual spikes (Fig. 8D). Thus, the [Ca 2ϩ ] pm and [Ca 2ϩ ] m spiking triggered by activators of the RyR display the fundamental features of the frequency modulation. Interestingly, in a few cells the [Ca 2ϩ ] m oscillations associated with uniform [Ca 2ϩ ] pm oscillations were found to begin with a relatively small initial [Ca 2ϩ ] m spike (e.g. Fig. 9B; spike at 200 s versus spikes at 250 and 300 s). This result suggests that the Ca 2ϩ coupling between RyR and mitochondrial Ca 2ϩ uptake sites was optimized during the first spikes, and this process may include a decrease in the distance between RyR and mitochondria or sensitization of the mitochondrial Ca 2ϩ uptake sites.
Mechanisms Underlying Rapid Ca 2ϩ Efflux from the Mitochondria-The temporal pattern of the [Ca 2ϩ ] m spikes recorded in individual cells displaying similar [Ca 2ϩ ] pm spikes was heterogeneous. In particular, the decay phase of the [Ca 2ϩ ] m spikes showed cell-to-cell variability. To identify the mechanisms that are responsible for the decay phase, the effect of inhibitors of the two main mitochondrial Ca 2ϩ efflux pathways (Ca 2ϩ exchanger, permeability transition pore) (37) on [Ca 2ϩ ] m oscillations was studied (Fig. 9). Both pathways have 3 S.-S. Sheu, personal communication. been suggested to play a role in physiological Ca 2ϩ regulation in cardiac myocytes (38,39). An inhibitor of the Ca 2ϩ exchanger, CGP37157, did not affect the shape of the [Ca 2ϩ ] pm spikes or the frequency of [Ca 2ϩ ] pm and [Ca 2ϩ ] m spikes but slowed the decay phase of [Ca 2ϩ ] m spikes (Fig. 9, A and C). In contrast, an inhibitor of the permeability pore, cyclosporin-A, failed to affect the decay phase of the [Ca 2ϩ ] m spikes (Fig. 9, B  and C)  each heartbeat is associated with a [Ca 2ϩ ] m rise, complete reversal between consecutive [Ca 2ϩ ] c spikes would require that the relaxation of [Ca 2ϩ ] m be at least an order of magnitude faster than that which occurs in caffeine-stimulated permeabilized H9c2 myotubes. Interestingly, the relaxation phases shown in our study display cell-specific differences and increases in the oscillation frequency are not associated with major changes in the relaxation rate of [Ca 2ϩ ] m spikes. Although inhibition of mitochondrial Ca 2ϩ efflux did not result in any major changes in the pattern of RyR-driven [Ca 2ϩ ] c spiking, a plethora of data suggests that mitochondrial uptake and release of Ca 2ϩ is important in IP 3 -linked cytosolic Ca 2ϩ signaling (35, 40 -47). As such, rapid extrusion of Ca 2ϩ from mitochondria may contribute to the control of cytosolic effector systems and may also contribute to the refilling of the RyRsensitive Ca 2ϩ store. Thus, translation of RyR-driven [Ca 2ϩ ] c signals into brief [Ca 2ϩ ] m transients may have multiple roles in mitochondrial and cytosolic [Ca 2ϩ ] regulation.
Conclusions-This study provides direct evidence that calcium signal propagation from RyR to the mitochondria may occur without significant changes in global [Ca 2ϩ ] c . Thus, it appears that this signaling pathway, which is sufficient to yield an essentially maximal activation of mitochondrial Ca 2ϩ uptake, is established by local communication between the RyR and mitochondrial uptake sites. Remarkably, the physiological regulatory cascade initiated by plasma membrane depolarization in the heart includes privileged communication between DHPR and RyR as well as between RyR and mitochondrial Ca 2ϩ uptake sites, albeit with lesser proximity in the latter case. One function of this pathway is to enhance mitochondrial oxidative metabolism to increase ATP formation, as demonstrated by our finding that the RyR-mediated [Ca 2ϩ ] m signal yields activation of Ca 2ϩ -sensitive mitochondrial dehydrogenases. It appears to be characteristic to the mitochondrial calcium signaling displayed by heart cells that the RyR-driven large spikes of [Ca 2ϩ ] m can be reversed very rapidly owing to activation of the mitochondrial Ca 2ϩ exchanger. The rapid mitochondrial turnover of Ca 2ϩ may protect against sequestration of large amounts of Ca 2ϩ in the mitochondria under physiological conditions and may also contribute to cytosolic calcium signaling on a beat-to-beat basis. All these properties of the signal detection and processing by the mitochondria suggest that the mitochondrial calcium signaling pathway may have an important role in adjusting the activity of energy metabolism to the needs of the contractile machinery.