- Email: [email protected]
Selective Loading of Rhod 2 into Mitochondria Shows Mitochondrial Ca2+Transients during the Contractile Cycle in Adult Rabbit Cardiac Myocytes
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 738–742 (1997)
RC977042
Selective Loading of Rhod 2 into Mitochondria Shows Mitochondrial Ca2/ Transients during the Contractile Cycle in Adult Rabbit Cardiac Myocytes Donna R. Trollinger, Wayne E. Cascio,* and John J. Lemasters Department of Cell Biology and Anatomy, and *Department of Medicine, University of North Carolina, Chapel Hill, North Carolina
Received June 16, 1997
A strategy of cold loading of the Ca2/-indicating fluorophore Rhod 2-AM followed by warm incubation was developed to selectively label mitochondria of adult rabbit cardiac myocytes. After electrical stimulation, mitochondrial Rhod 2 fluorescence observed by confocal microscopy increased and then rapidly decayed to baseline. In regions between mitochondria, the fluorescent transients were small or absent. Subsequent addition of calcium ionophore increased mitochondrial but not cytosolic fluorescence, confirming the mitochondrial localization of Rhod 2. These experiments directly demonstrate rapid mitochondrial free Ca2/ transients during the contractile cycle in rabbit cardiac myocytes. q 1997 Academic Press
Mitochondrial Ca2/ is widely proposed to regulate mitochondrial metabolism and to match mitochondrial ATP production to cellular ATP demand in heart and other tissues (1-3). However, investigations addressing how rapidly mitochondrial Ca2/ responds to changes of energy demand have yielded conflicting results. In Mn2/-quenched Indo 1-loaded rat cardiac myocytes, Miyata et al. (4) observed a gradual rise of mitochondrial Ca2/ over 60 seconds as pacing frequency increased. Similarly, Leisey et al. (5) concluded that mitochondrial free Ca2/ changes relatively slowly in isolated rat heart mitochondria in response to changes of extramitochondrial free Ca2/. Although Moravec and Bond (6) failed to show by electron probe microanalysis that changes of total mitochondrial Ca2/ occur during the contractile cycle in hamster cardiac myocytes, Abbreviations: AM, acetoxymethyl ester; KRH, Krebs-RingersHEPES buffer containing in mM: 110 NaCl, 5.0 KCl, 1.25 CaCl2 , 0.5 Na2HPO4 , 0.5 KH2PO4 , 1.0 MgSO4 , 10 glucose, 1.0 octanoic acid and 20 HEPES; HEPES, N-2-hydroxyethylpiperazine-N*-2-ethanesulfuronic acid; TMRM, tetramethylrhodamine methyl ester; CCCP, carbonyl cyanide m-chlorophenylhydrazone. 0006-291X/97 $25.00
Wendt-Gallitelli et al. (7) reported a 260% increase of total mitochondrial Ca2/ 40 ms after stimulation of isolated guinea pig cardiac myocytes. Additionally, Rizzuto et al. (8) and Jou et al. (9) reported rapid changes of mitochondrial Ca2/ in response to agonist stimulation. Moreover, Sparagna and co-workers (10) recently described in isolated rat liver mitochondria a rapid phase of mitochondrial Ca2/ uptake in response to physiological pulses of Ca2/. Recently using laser scanning confocal microscopy, we observed rapid mitochondrial free Ca2/ transients accompanying each contractile cycle in Fluo 3 and Indo 1-loaded adult rabbit cardiac myocytes (11,12). An assumption of these confocal experiments was that mitochondrial and cytosolic fluorescence were measured independently without spill-over of signal between the cytosol and mitochondrial compartments. Accordingly to exclude the possibility of spill-over of mitochondrial and cytosolic signals, the aim of the present work was to develop a technique to load Ca2/-indicating fluorophores exclusively into mitochondria without significant cytosolic labeling. Here, we report a procedure of cold loading followed by warm incubation that achieves virtually exclusive loading of the Ca2/-indicating fluorophore Rhod 2 into the mitochondria of adult rabbit cardiac myocytes. Rapid fluorescence transients were then observed after electrical stimulation, which directly demonstrated rapid mitochondrial Ca2/ transients during the excitation-contraction cycle. MATERIALS AND METHODS Myocyte isolation. Adult cardiac myocytes were isolated from New Zealand white rabbits (3-4 kg) by collagenase digestion, as previously described (13), and attached to laminin-coated coverslips (10 mg/cm2) for 2 hours in an air/5% CO2 incubator at 377C in nutrient medium (1:1 mixture of Joklik’s medium and medium 199 supplemented with 0.05 U/ml insulin, 1 mM creatine, 1 mM octanoic acid, 1 mM taurine, 10 U/ml penicillin and 10 mg/ml streptomycin). Rhod 2-AM loading. Myocytes were loaded with Rhod 2-AM1 (10 mM) for 1 hour at 47C in HEPES-buffered nutrient medium (20 mM
738
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HEPES) containing 10% fetal calf serum at pH 7.4. After cold loading, some cells were further incubated for 3-5 h at 377C in nutrient medium without serum. Prior to mounting on the microscope, cells were washed twice with KRH. Electrical field stimulations. Myocytes in KRH were field-depolarized in the presence of 1 mM isoproterenol at twice threshold voltage in 5 msec pulses using a Grass Model SD9 stimulator (Quincy, MA). Collection of Rhod 2 images. Fluorescent images of Rhod 2-labeled myocytes were collected with a Zeiss 410 laser scanning confocal microscope using a Zeiss 63X N.A. 1.4 planapochromat objective lens. Rhod 2 was excited with a 568 nm argon-krypton laser line and fluorescence was collected through a 590 long pass barrier filter. A pinhole setting of 15 was used that corresponded to an airy unit of approximately 0.9. All experiments were performed at room temperature. Materials. Br-A23187 was obtained from Sigma (St. Louis, MO). Rhod 2-AM and TMRM were obtained from Molecular Probes (Eugene, OR). All other reagents were of analytical grade and obtained from the usual commercial sources.
RESULTS AND DISCUSSION Rhod-2 fluorescence after cold-loading. Previously, the Ca2/ sensitive fluorophore, Rhod-2, was used to measure changes in mitochondrial Ca2/ in living cells (14-17). Rhod 2-AM, a derivative of rhodamine 123, contains one net positive charge and accumulates into mitochondria electrophoretically. Once inside the matrix, mitochondrial esterases cleave the AM ester to liberate Rhod 2 free acid. Previous work from our laboratory indicated that ester loading of fluorophores into mitochondria is temperature dependent (11,12,18). A warm loading temperature (377C) favors cytosolic loading, whereas cold temperature (47C) favors mitochondrial loading in addition to cytosolic loading. Presumably at the warm temperature, cytosolic esterases are so active that the AM esters are first cleaved before they can even enter mitochondria. At cold loading temperatures when enzymatic activity is slowed, the fluorophore esters can reach mitochondria before being hydrolyzed, allowing both cytosolic and mitochondrial loading to occur. Accordingly, we loaded Rhod 2-AM at 47C to enhance mitochondrial loading in freshly isolated myocytes. Confocal imaging of Rhod 2 fluorescence immediately after cold loading however did not show an obvious mitochondrial pattern (Fig. 1, unstimulated). Some bright spots of fluorescence were evident (double arrows in Fig. 1), which presumably correspond to fluorophore entrapped in the endosomal/lysosomal compartment, as observed previously with Fluo 3 (13). Moreover, during field stimulations, Rhod 2 fluorescence transients occurred throughout the cytoplasm without an apparent compartmental pattern. (Fig. 1). Cold loading followed by warm incubation. Previously, Weider and coworkers (19) noted that fluorophores loaded into myocytes gradually leak from the cytosol during warm incubation. Accordingly, we tested the hypothesis that warm incubation of myocytes would cause selective loss of cytosolic Rhod 2 with the
result that only mitochondrial Rhod 2 remained in the myocytes. After cold loading followed by warm incubation, Rhod 2 fluorescence in unstimulated cells showed a distinctly mitochondrial pattern (Fig. 2, unstimulated), comparable to that observed when myocytes are loaded with the mitochondrial dyes TMRM and rhodamine 123 (11,13). Subsequently, when these myocytes were electrically stimulated at pacing frequencies of 1 Hz and 2 Hz, strong fluorescence transients again occurred (Fig. 2). The largest fluorescence transients occurred in the bright regions corresponding to mitochondria. Fluorescence in areas between mitochondria either remained dark or increased moderately after electrical stimulation. To verify Rhod 2 localization, we added the calcium ionophore Br-A23187. CCCP, a powerful protonophoric uncoupler, was also added to depolarize mitochondria and block cycling of free Ca2/ after ionophore treatment. Glucose and oligomycin were also added. Oligomycin is an inhibitor of the mitochondrial H/-translocating ATPase that in the presence of a glycolytic substrate prevents ATP depletion due to activation of the uncoupler-stimulated mitochondrial ATPase (20-22). Additionally, butanedione monoxime (BDM) was added prior to Br-A23187 to prevent myocyte contracture (23). After Br-A23187 treatment in this way, Rhod 2 fluorescence increased maximally and continued to show a pattern virtually indistinguishable from that obtained with specific mitochondrial markers such as TMRM and Rhodamine 123 (Fig. 2, far right panel; compare with 11-13). By contrast, cytosolic areas between mitochondria remained dark. Occasional toxic blebs formed after Br-A23187 treatment. The contents of these blebs are an extension of the cytosol (13, 24). These blebs in Br-A23187-treated myocytes remained dark, indicating the virtual absence of cytosolic Rhod 2 loading. In other experiments, we added Br-A23187 in the absence of butanedione monoxime. Under such conditions, the myocytes hypercontracted, but Rhod 2 fluorescence remained confined to mitochondria (data not shown). Toxic blebs were again very weakly fluorescent, indicating that Rhod-2 was nearly absent in the cytosol. Quantification of Rhod 2 fluorescence. We used Image PC (Scion Corp., Frederick, MD) to quantify Rhod 2 fluorescence transients during electrical excitation. A confocal image was collected in a single 16 sec scan, as shown in Figure 2. Each horizontal line in the image corresponded to a 32 ms time interval. A rectangular area was then selected within the myocyte, as shown in Fig. 2. Average pixel values for each horizontal line in the selected area were then plotted against time. This analysis showed that intracellular Rhod-2 fluorescence after electrical stimulation was quite rapid and was followed by a slower recovery to baseline (Fig. 3A). To improve signal-to-noise, we averaged 8 consecutive
739
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC
740
AID
BBRC 7042
/
6933$$7042
07-17-97 08:54:30
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Plot analysis of Rhod 2 fluorescence in warm incubated myocytes during field stimulation at 2 Hz. The selected area in Fig. 2 was analyzed using Image PC. In A, average pixel intensity of each horizontal line was plotted against Time. In B, fluorescent transients during 8 consecutive stimulations were averaged. Fluorescence was normalized with 100% representing the diastolic intracellular fluorescence. Error bars are S.E.M.
transients. The averaged transient showed a half rise time of about 65 ms with a total of 300 ms to rise from baseline to peak fluorescence after electrical stimulation (Fig. 3B). Recovery of mitochondrial Ca2/, by contrast, was slower with a half decay time of 160 ms. Interpretation of fluorescence transients. Previously, we used a dual-labeling approach to identify mitochondrial and cytosolic Ca2/ transients in beating cardiac myocytes (11,12). Myocytes were co-loaded with the green-fluorescing Ca2/-indicator, Fluo 3, which labeled both cytosolic and mitochondrial compartments, and
the red fluorescing cationic dye, TMRM, which accumulated electrophoretically into mitochondria in response to their highly negative membrane potential (13,25). During electrical stimulations, transients of Fluo 3 fluorescence occurred both in regions corresponding to TMRM-labeled mitochondria and in unlabeled non-mitochondrial regions, which indicated the presence of both mitochondrial and cytosolic Ca2/ transients. In the present study, we extended this approach using myocytes in which labeling with Ca2/-indicating fluorophore was almost completely inside mitochondria. Using cold loading of Rhod 2-AM followed by warm incubation, Rhod 2 fluorescence was confined almost exclusively to mitochondria. During electrical stimulation, Rhod 2 fluorescence increased sharply in the bright regions corresponding to mitochondria. However, areas between mitochondria sometimes appeared to increase moderately in fluorescence, an apparent contradiction of our conclusion that Rhod 2 was confined exclusively to mitochondria. This inconsistency is explained by the fact that the confocal slice thickness is finite, in the range of 0.8-0.9 mm (26). Cardiac mitochondria are approximately 1 mm in diameter and many are only partially sectioned within the confocal slice. Areas of confocal images containing partially sectioned mitochondria will be darker than areas with fully sectioned mitochondria. However, fluorescence will nonetheless increase as Ca2/ increases even in those regions containing partially sectioned mitochondria. Previously, we excluded the contribution of partially sectioned mitochondria on the basis of the distribution of the mitochondrial marker, TMRM. Only pixels with the brightest and lowest TMRM fluorescence were analyzed in comparison to the fluorescence of the Ca2/ indicator Fluo-3. Pixels of intermediate TMRM fluorescence were ignored to exclude partially sectioned mitochondria. An advantage of the present technique using cold loading followed by warm incubation is that all pixels can be used for analysis, which increases signal strength and decreases signal-to-noise. Mitochondrial Ca2/ transients and metabolic regulation. In vivo in working hearts, it is estimated that ATP consumption is 23 mmol ATP/s/g dry weight (27).
FIG. 1. Ca2/ transients in freshly loaded myocytes. A myocyte was imaged immediately after loading with Rhod 2 at 47C. Note that Rhod 2 fluorescence increases across the entire cell after each electrical stimulation at 1 Hz (arrows). Bright spots of fluorescence (double arrows) presumably correspond to fluorophore entrapped in endosomal/lysosomal compartments. The 16-sec confocal scans proceed from top to bottom. The images are pseudocolored using the black body look-up table of Photoshop (Adobe Systems, San Jose, CA). The inset is a linear scale of pixel intensities from 0 to 255. FIG. 2. Mitochondrial transients after cold loading followed by warm incubation. A cardiac myocyte was cold loaded with Rhod 2-AM and then incubated at 377C for 3 hours. Note the mitochondrial pattern of Rhod 2 fluorescence in the unstimulated cell. After stimulation at 1 Hz (arrows) and then 2 Hz, Rhod 2 fluorescence rapidly increased in the bright mitochondrial regions and then returned to baseline. Oligomycin (10 mM), CCCP (10 mM), butanedione monoxime (20 mM), and then Br-A23187 (20 mM) were added to increase free Ca2/ in all compartments and saturate Rhod 2. Note that Rhod 2 fluorescence was confined to mitochondria and that cytosolic areas, such as in blebs (arrow), remained only faintly fluorescent. Each 16-sec confocal scan proceeds from top to bottom. The images are pseudocolored using the black body look-up table of Photoshop, as described in Fig. 1. The white box in the third panel represents the selected area analyzed in Fig. 3. 741
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Since ATP and phosphocreatine (PCr) content are about 20 mmol/g dry wt. and 40 mmol/g, respectively (28), an equivalent of all cellular ATP and PCr must turn over every 3 seconds. With exercise, cardiac work changes rapidly by up to 10 fold. Thus, mitochondrial ATP production must change rapidly to maintain constant ATP and PCr levels as cardiac output changes. Changes of mitochondrial Ca2/ are widely proposed to be responsible for matching mitochondrial ATP supply to cellular ATP demand (1-3). Here, we show that transients of mitochondrial free Ca2/ during the contractile cycle are indeed kinetically competent to drive the tight kinetic coupling of ATP production to ATP demand in adult rabbit cardiac myocytes. ACKNOWLEDGMENTS This work was supported, in part, by Grant N00014-96-0283 from the Office of Naval Research. Portions of this work were presented at FOCUS ON MULTI-DIMENSIONAL MICROSCOPY ’97, Buffalo, New York, April 28–30, 1997.
REFERENCES 1. Denton, R. M., and McCormack, J. G. (1980) FEBS Lett. 119, 1– 8. 2. Denton, R. M., and McCormack, J. G. (1985) Am. J. Physiol. 249, E543–E554. 3. Hansford, R. G. (1985) Rev. Physiol. Biochem. Pharmacol. 102, 1–72. 4. Miyata, H., Silverman, H. S., Sollott, S. J., Lakatta, E. G., Stern, M. D., and Hansford, R. G. (1991) Am. J. Physiol. 261, H1123– H1134. 5. Leisey, J. R., Grotyohann, L. W., Scott, D. A., and Scaduto, R. C., Jr. (1993) Am. J. Physiol. 265, H1203–H1208. 6. Moravec, C. S., and Bond, M. (1991) Am. J. Physiol. 260, H989– H997. 7. Wendt-Gallitelli, M. F., and Isenberg, G. (1991) J. Physiol. 435, 349–372. 8. Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325–327.
9. Jou, M. J., Peng, T. I., and Sheu, S-S. (1996) J. Physiol. 497, 299–308. 10. Sparagna, G. C., Gunter, K. K., Sheu, S-S., and Gunter, T. E. (1995) J. Biol. Chem. 270, 27510–27515. 11. Chacon, E., Ohata, H., Harper, I. S., Trollinger, D. R., Herman, B., and Lemasters, J. J. (1996) FEBS Lett. 382, 31–36. 12. Ohata, H., Trollinger, D. R., Chacon. E., Tesfai, S. A., Harper, I. S., Herman, B., Cascio, W. E., and Lemasters, J. J. (1997) [Submitted for publication] 13. Chacon, E., Reece, J. M., Nieminen, A-L., Zahrabelski, G., Herman, B., and Lemasters, J. J. (1994) Biophys. J. 66, 942–952. 14. Burnier, M., Centeno, G., Burki, E., and Brunner, H. R. (1994) Am. J. Physiol. 266, C1118–C1127. 15. Jou, M-L., and Sheu, S-S. (1994) Biophys. J. 66, A94. 16. Hajno´czky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995) Cell 82, 415–425. 17. Bouchard, R. A., Clark, R. B., and Giles, W. R. (1995) Circ. Res. 76, 790–801. 18. Nieminen, A-L., Saylor, A. K., Tesfai, S. A., Herman, B., and Lemasters, J. J. (1995) Biochem. J. 307, 99–106. 19. Wieder, E. D., Hang, H., and Fox, M. H. (1993) Cytometry 14, 916–921. 20. Nieminen, A-L., Dawson, T. L., Gores, G. J., Kawanishi, T., Herman, B., and Lemasters, J. J. (1990) Biochem. Biophys. Res. Commun. 167, 600–606. 21. Nieminen, A-L., Saylor, A. K., Herman, B., and Lemasters, J. J. (1994) Am. J. Physiol. 267, C67–C74. 22. Bers, D. M., Bassani, J. W., and Bassani, R. A. (1993) Cardiovasc. Res. 27, 1772–1777. 23. Armstrong, S. C., and Ganote, C. E. (1991) J. Mol. Cell Cardiol. 23, 1001–1014. 24. Lemasters, J. J., Ji, S., and Thurman, R. G. (1981) Science 213, 661–663. 25. Minta, A., Kao, J. P., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171–8178. 26. Lemasters, J. J., Chacon, E., Zehrebelski, G., Reece, J. M., and Nieminen, A-L. (1991) in Optical Microscopy: New Technologies and Applications (Herman, B., and Lemasters, J. J., Eds.), pp. 339–354. Academic Press, San Diego, CA. 27. Taegtmeyer, H. (1988) in Myocardial Energy Metabolism (De Jong, J. W., Ed.), pp. 17–34. Kluwer Academic, Dordrecht/Boston/Lancaster. 28. Achterberg, P. W. (1988) in Myocardial Energy Metabolism (De Jong, J. W., Ed.), pp. 45–52. Kluwer Academic, Dordrecht/Boston/Lancaster.
742
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC
236, 738–742 (1997)
RC977042
Selective Loading of Rhod 2 into Mitochondria Shows Mitochondrial Ca2/ Transients during the Contractile Cycle in Adult Rabbit Cardiac Myocytes Donna R. Trollinger, Wayne E. Cascio,* and John J. Lemasters Department of Cell Biology and Anatomy, and *Department of Medicine, University of North Carolina, Chapel Hill, North Carolina
Received June 16, 1997
A strategy of cold loading of the Ca2/-indicating fluorophore Rhod 2-AM followed by warm incubation was developed to selectively label mitochondria of adult rabbit cardiac myocytes. After electrical stimulation, mitochondrial Rhod 2 fluorescence observed by confocal microscopy increased and then rapidly decayed to baseline. In regions between mitochondria, the fluorescent transients were small or absent. Subsequent addition of calcium ionophore increased mitochondrial but not cytosolic fluorescence, confirming the mitochondrial localization of Rhod 2. These experiments directly demonstrate rapid mitochondrial free Ca2/ transients during the contractile cycle in rabbit cardiac myocytes. q 1997 Academic Press
Mitochondrial Ca2/ is widely proposed to regulate mitochondrial metabolism and to match mitochondrial ATP production to cellular ATP demand in heart and other tissues (1-3). However, investigations addressing how rapidly mitochondrial Ca2/ responds to changes of energy demand have yielded conflicting results. In Mn2/-quenched Indo 1-loaded rat cardiac myocytes, Miyata et al. (4) observed a gradual rise of mitochondrial Ca2/ over 60 seconds as pacing frequency increased. Similarly, Leisey et al. (5) concluded that mitochondrial free Ca2/ changes relatively slowly in isolated rat heart mitochondria in response to changes of extramitochondrial free Ca2/. Although Moravec and Bond (6) failed to show by electron probe microanalysis that changes of total mitochondrial Ca2/ occur during the contractile cycle in hamster cardiac myocytes, Abbreviations: AM, acetoxymethyl ester; KRH, Krebs-RingersHEPES buffer containing in mM: 110 NaCl, 5.0 KCl, 1.25 CaCl2 , 0.5 Na2HPO4 , 0.5 KH2PO4 , 1.0 MgSO4 , 10 glucose, 1.0 octanoic acid and 20 HEPES; HEPES, N-2-hydroxyethylpiperazine-N*-2-ethanesulfuronic acid; TMRM, tetramethylrhodamine methyl ester; CCCP, carbonyl cyanide m-chlorophenylhydrazone. 0006-291X/97 $25.00
Wendt-Gallitelli et al. (7) reported a 260% increase of total mitochondrial Ca2/ 40 ms after stimulation of isolated guinea pig cardiac myocytes. Additionally, Rizzuto et al. (8) and Jou et al. (9) reported rapid changes of mitochondrial Ca2/ in response to agonist stimulation. Moreover, Sparagna and co-workers (10) recently described in isolated rat liver mitochondria a rapid phase of mitochondrial Ca2/ uptake in response to physiological pulses of Ca2/. Recently using laser scanning confocal microscopy, we observed rapid mitochondrial free Ca2/ transients accompanying each contractile cycle in Fluo 3 and Indo 1-loaded adult rabbit cardiac myocytes (11,12). An assumption of these confocal experiments was that mitochondrial and cytosolic fluorescence were measured independently without spill-over of signal between the cytosol and mitochondrial compartments. Accordingly to exclude the possibility of spill-over of mitochondrial and cytosolic signals, the aim of the present work was to develop a technique to load Ca2/-indicating fluorophores exclusively into mitochondria without significant cytosolic labeling. Here, we report a procedure of cold loading followed by warm incubation that achieves virtually exclusive loading of the Ca2/-indicating fluorophore Rhod 2 into the mitochondria of adult rabbit cardiac myocytes. Rapid fluorescence transients were then observed after electrical stimulation, which directly demonstrated rapid mitochondrial Ca2/ transients during the excitation-contraction cycle. MATERIALS AND METHODS Myocyte isolation. Adult cardiac myocytes were isolated from New Zealand white rabbits (3-4 kg) by collagenase digestion, as previously described (13), and attached to laminin-coated coverslips (10 mg/cm2) for 2 hours in an air/5% CO2 incubator at 377C in nutrient medium (1:1 mixture of Joklik’s medium and medium 199 supplemented with 0.05 U/ml insulin, 1 mM creatine, 1 mM octanoic acid, 1 mM taurine, 10 U/ml penicillin and 10 mg/ml streptomycin). Rhod 2-AM loading. Myocytes were loaded with Rhod 2-AM1 (10 mM) for 1 hour at 47C in HEPES-buffered nutrient medium (20 mM
738
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HEPES) containing 10% fetal calf serum at pH 7.4. After cold loading, some cells were further incubated for 3-5 h at 377C in nutrient medium without serum. Prior to mounting on the microscope, cells were washed twice with KRH. Electrical field stimulations. Myocytes in KRH were field-depolarized in the presence of 1 mM isoproterenol at twice threshold voltage in 5 msec pulses using a Grass Model SD9 stimulator (Quincy, MA). Collection of Rhod 2 images. Fluorescent images of Rhod 2-labeled myocytes were collected with a Zeiss 410 laser scanning confocal microscope using a Zeiss 63X N.A. 1.4 planapochromat objective lens. Rhod 2 was excited with a 568 nm argon-krypton laser line and fluorescence was collected through a 590 long pass barrier filter. A pinhole setting of 15 was used that corresponded to an airy unit of approximately 0.9. All experiments were performed at room temperature. Materials. Br-A23187 was obtained from Sigma (St. Louis, MO). Rhod 2-AM and TMRM were obtained from Molecular Probes (Eugene, OR). All other reagents were of analytical grade and obtained from the usual commercial sources.
RESULTS AND DISCUSSION Rhod-2 fluorescence after cold-loading. Previously, the Ca2/ sensitive fluorophore, Rhod-2, was used to measure changes in mitochondrial Ca2/ in living cells (14-17). Rhod 2-AM, a derivative of rhodamine 123, contains one net positive charge and accumulates into mitochondria electrophoretically. Once inside the matrix, mitochondrial esterases cleave the AM ester to liberate Rhod 2 free acid. Previous work from our laboratory indicated that ester loading of fluorophores into mitochondria is temperature dependent (11,12,18). A warm loading temperature (377C) favors cytosolic loading, whereas cold temperature (47C) favors mitochondrial loading in addition to cytosolic loading. Presumably at the warm temperature, cytosolic esterases are so active that the AM esters are first cleaved before they can even enter mitochondria. At cold loading temperatures when enzymatic activity is slowed, the fluorophore esters can reach mitochondria before being hydrolyzed, allowing both cytosolic and mitochondrial loading to occur. Accordingly, we loaded Rhod 2-AM at 47C to enhance mitochondrial loading in freshly isolated myocytes. Confocal imaging of Rhod 2 fluorescence immediately after cold loading however did not show an obvious mitochondrial pattern (Fig. 1, unstimulated). Some bright spots of fluorescence were evident (double arrows in Fig. 1), which presumably correspond to fluorophore entrapped in the endosomal/lysosomal compartment, as observed previously with Fluo 3 (13). Moreover, during field stimulations, Rhod 2 fluorescence transients occurred throughout the cytoplasm without an apparent compartmental pattern. (Fig. 1). Cold loading followed by warm incubation. Previously, Weider and coworkers (19) noted that fluorophores loaded into myocytes gradually leak from the cytosol during warm incubation. Accordingly, we tested the hypothesis that warm incubation of myocytes would cause selective loss of cytosolic Rhod 2 with the
result that only mitochondrial Rhod 2 remained in the myocytes. After cold loading followed by warm incubation, Rhod 2 fluorescence in unstimulated cells showed a distinctly mitochondrial pattern (Fig. 2, unstimulated), comparable to that observed when myocytes are loaded with the mitochondrial dyes TMRM and rhodamine 123 (11,13). Subsequently, when these myocytes were electrically stimulated at pacing frequencies of 1 Hz and 2 Hz, strong fluorescence transients again occurred (Fig. 2). The largest fluorescence transients occurred in the bright regions corresponding to mitochondria. Fluorescence in areas between mitochondria either remained dark or increased moderately after electrical stimulation. To verify Rhod 2 localization, we added the calcium ionophore Br-A23187. CCCP, a powerful protonophoric uncoupler, was also added to depolarize mitochondria and block cycling of free Ca2/ after ionophore treatment. Glucose and oligomycin were also added. Oligomycin is an inhibitor of the mitochondrial H/-translocating ATPase that in the presence of a glycolytic substrate prevents ATP depletion due to activation of the uncoupler-stimulated mitochondrial ATPase (20-22). Additionally, butanedione monoxime (BDM) was added prior to Br-A23187 to prevent myocyte contracture (23). After Br-A23187 treatment in this way, Rhod 2 fluorescence increased maximally and continued to show a pattern virtually indistinguishable from that obtained with specific mitochondrial markers such as TMRM and Rhodamine 123 (Fig. 2, far right panel; compare with 11-13). By contrast, cytosolic areas between mitochondria remained dark. Occasional toxic blebs formed after Br-A23187 treatment. The contents of these blebs are an extension of the cytosol (13, 24). These blebs in Br-A23187-treated myocytes remained dark, indicating the virtual absence of cytosolic Rhod 2 loading. In other experiments, we added Br-A23187 in the absence of butanedione monoxime. Under such conditions, the myocytes hypercontracted, but Rhod 2 fluorescence remained confined to mitochondria (data not shown). Toxic blebs were again very weakly fluorescent, indicating that Rhod-2 was nearly absent in the cytosol. Quantification of Rhod 2 fluorescence. We used Image PC (Scion Corp., Frederick, MD) to quantify Rhod 2 fluorescence transients during electrical excitation. A confocal image was collected in a single 16 sec scan, as shown in Figure 2. Each horizontal line in the image corresponded to a 32 ms time interval. A rectangular area was then selected within the myocyte, as shown in Fig. 2. Average pixel values for each horizontal line in the selected area were then plotted against time. This analysis showed that intracellular Rhod-2 fluorescence after electrical stimulation was quite rapid and was followed by a slower recovery to baseline (Fig. 3A). To improve signal-to-noise, we averaged 8 consecutive
739
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC
740
AID
BBRC 7042
/
6933$$7042
07-17-97 08:54:30
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Plot analysis of Rhod 2 fluorescence in warm incubated myocytes during field stimulation at 2 Hz. The selected area in Fig. 2 was analyzed using Image PC. In A, average pixel intensity of each horizontal line was plotted against Time. In B, fluorescent transients during 8 consecutive stimulations were averaged. Fluorescence was normalized with 100% representing the diastolic intracellular fluorescence. Error bars are S.E.M.
transients. The averaged transient showed a half rise time of about 65 ms with a total of 300 ms to rise from baseline to peak fluorescence after electrical stimulation (Fig. 3B). Recovery of mitochondrial Ca2/, by contrast, was slower with a half decay time of 160 ms. Interpretation of fluorescence transients. Previously, we used a dual-labeling approach to identify mitochondrial and cytosolic Ca2/ transients in beating cardiac myocytes (11,12). Myocytes were co-loaded with the green-fluorescing Ca2/-indicator, Fluo 3, which labeled both cytosolic and mitochondrial compartments, and
the red fluorescing cationic dye, TMRM, which accumulated electrophoretically into mitochondria in response to their highly negative membrane potential (13,25). During electrical stimulations, transients of Fluo 3 fluorescence occurred both in regions corresponding to TMRM-labeled mitochondria and in unlabeled non-mitochondrial regions, which indicated the presence of both mitochondrial and cytosolic Ca2/ transients. In the present study, we extended this approach using myocytes in which labeling with Ca2/-indicating fluorophore was almost completely inside mitochondria. Using cold loading of Rhod 2-AM followed by warm incubation, Rhod 2 fluorescence was confined almost exclusively to mitochondria. During electrical stimulation, Rhod 2 fluorescence increased sharply in the bright regions corresponding to mitochondria. However, areas between mitochondria sometimes appeared to increase moderately in fluorescence, an apparent contradiction of our conclusion that Rhod 2 was confined exclusively to mitochondria. This inconsistency is explained by the fact that the confocal slice thickness is finite, in the range of 0.8-0.9 mm (26). Cardiac mitochondria are approximately 1 mm in diameter and many are only partially sectioned within the confocal slice. Areas of confocal images containing partially sectioned mitochondria will be darker than areas with fully sectioned mitochondria. However, fluorescence will nonetheless increase as Ca2/ increases even in those regions containing partially sectioned mitochondria. Previously, we excluded the contribution of partially sectioned mitochondria on the basis of the distribution of the mitochondrial marker, TMRM. Only pixels with the brightest and lowest TMRM fluorescence were analyzed in comparison to the fluorescence of the Ca2/ indicator Fluo-3. Pixels of intermediate TMRM fluorescence were ignored to exclude partially sectioned mitochondria. An advantage of the present technique using cold loading followed by warm incubation is that all pixels can be used for analysis, which increases signal strength and decreases signal-to-noise. Mitochondrial Ca2/ transients and metabolic regulation. In vivo in working hearts, it is estimated that ATP consumption is 23 mmol ATP/s/g dry weight (27).
FIG. 1. Ca2/ transients in freshly loaded myocytes. A myocyte was imaged immediately after loading with Rhod 2 at 47C. Note that Rhod 2 fluorescence increases across the entire cell after each electrical stimulation at 1 Hz (arrows). Bright spots of fluorescence (double arrows) presumably correspond to fluorophore entrapped in endosomal/lysosomal compartments. The 16-sec confocal scans proceed from top to bottom. The images are pseudocolored using the black body look-up table of Photoshop (Adobe Systems, San Jose, CA). The inset is a linear scale of pixel intensities from 0 to 255. FIG. 2. Mitochondrial transients after cold loading followed by warm incubation. A cardiac myocyte was cold loaded with Rhod 2-AM and then incubated at 377C for 3 hours. Note the mitochondrial pattern of Rhod 2 fluorescence in the unstimulated cell. After stimulation at 1 Hz (arrows) and then 2 Hz, Rhod 2 fluorescence rapidly increased in the bright mitochondrial regions and then returned to baseline. Oligomycin (10 mM), CCCP (10 mM), butanedione monoxime (20 mM), and then Br-A23187 (20 mM) were added to increase free Ca2/ in all compartments and saturate Rhod 2. Note that Rhod 2 fluorescence was confined to mitochondria and that cytosolic areas, such as in blebs (arrow), remained only faintly fluorescent. Each 16-sec confocal scan proceeds from top to bottom. The images are pseudocolored using the black body look-up table of Photoshop, as described in Fig. 1. The white box in the third panel represents the selected area analyzed in Fig. 3. 741
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Since ATP and phosphocreatine (PCr) content are about 20 mmol/g dry wt. and 40 mmol/g, respectively (28), an equivalent of all cellular ATP and PCr must turn over every 3 seconds. With exercise, cardiac work changes rapidly by up to 10 fold. Thus, mitochondrial ATP production must change rapidly to maintain constant ATP and PCr levels as cardiac output changes. Changes of mitochondrial Ca2/ are widely proposed to be responsible for matching mitochondrial ATP supply to cellular ATP demand (1-3). Here, we show that transients of mitochondrial free Ca2/ during the contractile cycle are indeed kinetically competent to drive the tight kinetic coupling of ATP production to ATP demand in adult rabbit cardiac myocytes. ACKNOWLEDGMENTS This work was supported, in part, by Grant N00014-96-0283 from the Office of Naval Research. Portions of this work were presented at FOCUS ON MULTI-DIMENSIONAL MICROSCOPY ’97, Buffalo, New York, April 28–30, 1997.
REFERENCES 1. Denton, R. M., and McCormack, J. G. (1980) FEBS Lett. 119, 1– 8. 2. Denton, R. M., and McCormack, J. G. (1985) Am. J. Physiol. 249, E543–E554. 3. Hansford, R. G. (1985) Rev. Physiol. Biochem. Pharmacol. 102, 1–72. 4. Miyata, H., Silverman, H. S., Sollott, S. J., Lakatta, E. G., Stern, M. D., and Hansford, R. G. (1991) Am. J. Physiol. 261, H1123– H1134. 5. Leisey, J. R., Grotyohann, L. W., Scott, D. A., and Scaduto, R. C., Jr. (1993) Am. J. Physiol. 265, H1203–H1208. 6. Moravec, C. S., and Bond, M. (1991) Am. J. Physiol. 260, H989– H997. 7. Wendt-Gallitelli, M. F., and Isenberg, G. (1991) J. Physiol. 435, 349–372. 8. Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325–327.
9. Jou, M. J., Peng, T. I., and Sheu, S-S. (1996) J. Physiol. 497, 299–308. 10. Sparagna, G. C., Gunter, K. K., Sheu, S-S., and Gunter, T. E. (1995) J. Biol. Chem. 270, 27510–27515. 11. Chacon, E., Ohata, H., Harper, I. S., Trollinger, D. R., Herman, B., and Lemasters, J. J. (1996) FEBS Lett. 382, 31–36. 12. Ohata, H., Trollinger, D. R., Chacon. E., Tesfai, S. A., Harper, I. S., Herman, B., Cascio, W. E., and Lemasters, J. J. (1997) [Submitted for publication] 13. Chacon, E., Reece, J. M., Nieminen, A-L., Zahrabelski, G., Herman, B., and Lemasters, J. J. (1994) Biophys. J. 66, 942–952. 14. Burnier, M., Centeno, G., Burki, E., and Brunner, H. R. (1994) Am. J. Physiol. 266, C1118–C1127. 15. Jou, M-L., and Sheu, S-S. (1994) Biophys. J. 66, A94. 16. Hajno´czky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995) Cell 82, 415–425. 17. Bouchard, R. A., Clark, R. B., and Giles, W. R. (1995) Circ. Res. 76, 790–801. 18. Nieminen, A-L., Saylor, A. K., Tesfai, S. A., Herman, B., and Lemasters, J. J. (1995) Biochem. J. 307, 99–106. 19. Wieder, E. D., Hang, H., and Fox, M. H. (1993) Cytometry 14, 916–921. 20. Nieminen, A-L., Dawson, T. L., Gores, G. J., Kawanishi, T., Herman, B., and Lemasters, J. J. (1990) Biochem. Biophys. Res. Commun. 167, 600–606. 21. Nieminen, A-L., Saylor, A. K., Herman, B., and Lemasters, J. J. (1994) Am. J. Physiol. 267, C67–C74. 22. Bers, D. M., Bassani, J. W., and Bassani, R. A. (1993) Cardiovasc. Res. 27, 1772–1777. 23. Armstrong, S. C., and Ganote, C. E. (1991) J. Mol. Cell Cardiol. 23, 1001–1014. 24. Lemasters, J. J., Ji, S., and Thurman, R. G. (1981) Science 213, 661–663. 25. Minta, A., Kao, J. P., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171–8178. 26. Lemasters, J. J., Chacon, E., Zehrebelski, G., Reece, J. M., and Nieminen, A-L. (1991) in Optical Microscopy: New Technologies and Applications (Herman, B., and Lemasters, J. J., Eds.), pp. 339–354. Academic Press, San Diego, CA. 27. Taegtmeyer, H. (1988) in Myocardial Energy Metabolism (De Jong, J. W., Ed.), pp. 17–34. Kluwer Academic, Dordrecht/Boston/Lancaster. 28. Achterberg, P. W. (1988) in Myocardial Energy Metabolism (De Jong, J. W., Ed.), pp. 45–52. Kluwer Academic, Dordrecht/Boston/Lancaster.
742
AID
BBRC 7042
/
6933$$1021
07-17-97 08:54:30
bbrcg
AP: BBRC