ATP Regulation in Adult Rat Cardiomyocytes
2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês
10.1074/jbc.m604540200
ISSN1083-351X
AutoresChristopher J. Bell, Nicholas A. Bright, Guy A. Rutter, Elinor J. Griffiths,
Tópico(s)Advanced MRI Techniques and Applications
ResumoThe mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca2+ in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP]m) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP]c) were much smaller (<5%), indicating that [ATP]c was effectively buffered in this compartment. Although mitochondrial [Ca2+] ([Ca2+]m) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca2+ transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP]m occurred over the same time scale as average [Ca2+]m was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca2+]m were observed at 0.9 or 1.3 μm, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca2+ transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand. The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca2+ in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP]m) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP]c) were much smaller (<5%), indicating that [ATP]c was effectively buffered in this compartment. Although mitochondrial [Ca2+] ([Ca2+]m) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca2+ transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP]m occurred over the same time scale as average [Ca2+]m was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca2+]m were observed at 0.9 or 1.3 μm, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca2+ transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand. Aerobic energy production in the heart is essential for the maintenance of normal contractility, but the mechanisms through which ATP homeostasis is achieved are incompletely understood. Early studies on isolated mitochondria showed that the main regulator of ATP production was likely to be ADP (1Chance B. Williams G.R. J. Biol. Chem. 1956; 221: 477-489Abstract Full Text PDF PubMed Google Scholar), so it seemed probable that this parameter had to increase, and ATP levels fall, before any stimulation of respiration occurred (reviewed by Brown (2Brown G.C. Biochem. J. 1992; 284: 1-13Crossref PubMed Scopus (504) Google Scholar)). However, subsequent studies using 31P NMR in whole hearts showed that ATP levels were maintained during increased workload (3Katz L.A. Swain J.A. Portman M.A. Balaban R.S. Am. J. Physiol. 1989; 256: H265-H274Crossref PubMed Google Scholar), implicating alternative regulatory mechanisms in the control of mitochondrial respiration. One such mechanism is provided by Ca2+; in many mammalian cell types, changes in the free Ca2+ concentration in the mitochondrial matrix ([Ca2+]m), 3The abbreviations used are: [Ca2+]m, mitochondrial [Ca2+]; [Ca2+]c, cytosolic free [Ca2+]; [ATP]m, mitochondrial [ATP]; [ATP]c, cytosolic [ATP]; SR, sarcoplasmic reticulum; CaUP, Ca2+ uniporter; mNCX, Na+/Ca2+ exchanger; cAq, cytosolically targeted aequorin; mAq, mitochondrially targeted aequorin; GFP, green fluorescent protein. by agonists that increase cytosolic free [Ca2+] ([Ca2+]c), stimulate ATP production by activating three mitochondrial dehydrogenases (4McCormack J.G. Halestrap A.P. Denton R.M. Physiol. Rev. 1990; 70: 391-425Crossref PubMed Scopus (1162) Google Scholar) and also possibly by activating the F0F1-ATPase (5Das A.M. Harris D.A. Cardiovasc. Res. 1990; 24: 411-417Crossref PubMed Scopus (95) Google Scholar, 6Territo P.R. Mootha V.K. French S.A. Balaban R.S. Am. J. Physiol. 2000; 278: C423-C435Crossref PubMed Google Scholar). However, the ability of [Ca2+]m to increase ATP on a rapid time scale, as would have to occur in the heart to meet sudden changes in energy demand, has not been investigated, and exactly how ATP supply and demand are so well matched in the heart remains controversial even after over 40 years of research (7Balaban R.S. J. Mol. Cell. Cardiol. 2002; 34: 1259-1271Abstract Full Text PDF PubMed Google Scholar). Another problem is that the mitochondrial Ca2+ transporters would have to respond very rapidly to increases in [Ca2+]c in order to maintain constant ATP levels. Previous work on isolated mitochondria suggested that the mitochondrial Ca2+ uniporter (CaUP) was a relatively slow Ca2+-uptake pathway, and the efflux pathway, the Na+/Ca2+ exchanger (mNCX), was even slower (8Gunter T.E. Pfeiffer D.R. Am. J. Physiol. 1990; 258: C755-C786Crossref PubMed Google Scholar, 9Nicholls D.G. Crompton M. FEBS. Lett. 1980; 111: 261-268Crossref PubMed Scopus (206) Google Scholar). So these pathways could certainly not respond quickly enough to the very rapid (millisecond) changes in [Ca2+]c that occur during excitation-contraction coupling to achieve beat-to-beat regulation of [Ca2+]m. However, more recent work using isolated myocytes has suggested that mitochondrial Ca2+ transients do occur during excitation-contraction coupling (10Trollinger D.R. Cascio W.E. Lemasters J.J. Biochem. Biophys. Res. Commun. 1997; 236: 738-742Crossref PubMed Scopus (170) Google Scholar), although there are conflicting data on this in the literature, because other studies reported that mitochondrial Ca2+ accumulation occurred over tens of seconds (11Miyata H. Silverman H.S. Sollott S.J. Lakatta E.G. Stern M.D. Hansford R.G. Am. J. Physiol. 1991; 261: H1123-H1134Crossref PubMed Google Scholar, 12Griffiths E.J. Stern M.D. Silverman H.S. Am. J. Physiol. 1997; 273: C37-C44Crossref PubMed Google Scholar, 13Zhou Z. Matlib M.A. Bers D.M. J. Physiol. (Lond.). 1998; 507: 379-403Crossref Scopus (95) Google Scholar). Such controversy, together with a resurgence of interest in the role of intramitochondrial Ca2+ in both cell signaling and the regulation of energy metabolism, has highlighted the need for methods that can be used to measure [Ca2+]m dynamically and specifically in living cells. Most previous work attempting to measure [Ca2+]m has used fluorescent indicators, and an evident problem with such studies is that the dyes may not localize exclusively to the mitochondrial compartment. Similarly, although ATP has been measured in whole hearts, and now in animals and humans using noninvasive 31P NMR (7Balaban R.S. J. Mol. Cell. Cardiol. 2002; 34: 1259-1271Abstract Full Text PDF PubMed Google Scholar), again only relatively slow responses were measured, and so it could not be determined whether ATP was varying beat-to-beat or during the time taken for mitochondria to accumulate Ca2+ to a level sufficient to activate the dehydrogenases. Although microinjection of luciferase into single cardiomyocytes has been reported (14Bowers K.C. Allshire A.P. Cobbold P.H. J. Mol. Cell. Cardiol. 1992; 24: 213-218Abstract Full Text PDF PubMed Scopus (64) Google Scholar), we did not find it possible to microinject our cells without damaging them. This study reported rapid depletion of (cytosolic) ATP levels in response to an uncoupler but did not report changes in response to physiological conditions or in response to normal cell contraction. Furthermore, free ATP levels have not been directly measured in different compartments of the living cardiomyocyte, and the relationship between mitochondrial [ATP] ([ATPm]) and cytosolic ATP ([ATP]c) is not known. Recently, Robert et al. (15Robert V. Gurlini P. Tosello V. Nagai T. Miyawaki A. Di Lisa F. Pozzan T. EMBO J. 2001; 20: 4998-5007Crossref PubMed Scopus (194) Google Scholar) expressed a mitochondrially targeted aequorin (16Rizzuto R. Simpson A.W. Brini M. Pozzan T. Nature. 1992; 358: 325-327Crossref PubMed Scopus (787) Google Scholar) in neonatal cardiomyocytes using the "FuGENE" transfection reagent and observed beat-to-beat mitochondrial Ca2+ transients with kinetics that were broadly similar to those observed in the cytosol. However, these findings have not been extended to adult cardiomyocytes because the latter are not amenable to transfection by conventional procedures. To investigate the relationship between ATP and Ca2+ in both mitochondrial and cytosolic compartments, we generated adenoviruses containing luciferase or aequorin targeted to either cytosol or mitochondria (17Jouaville L.S. Pinton P. Bastianutto C. Rutter G.A. Rizzuto R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13807-13812Crossref PubMed Scopus (647) Google Scholar, 18Kennedy H.J. Pouli A.E. Ainscow E.K. Jouaville L.S. Rizzuto R. Rutter G.A. J. Biol. Chem. 1999; 274: 13281-13291Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 19Ainscow E.K. Rutter G.A. Biochem. J. 2001; 353: 175-180Crossref PubMed Scopus (78) Google Scholar). Parallel experiments were performed to determine whether ATP levels changed under conditions where [Ca2+]m increased. Our results indicate that [ATP]c is extremely well buffered in myocytes, even under conditions where the cells were stimulated to beat rapidly in the presence of an adrenergic agonist to further increase ATP demand by the cell. However, an initial drop in [ATP]m was observed before it recovered to higher than resting values. We also observed beat-to-beat changes in [Ca2+]m, the amplitude of which was increased in the presence of an adrenergic agonist. Furthermore, although the peak amplitude of the transient increased immediately upon rapid stimulation, there was an underlying level of [Ca2+]m that increased more slowly. Together, these results suggest that changes in [Ca2+]m may be translated time-dependently into steady-state alterations in free mitochondrial and, subsequently, cytosolic [ATP]. Materials—All materials were from Sigma or BDH (distributed by VWR International Ltd., Poole, UK) unless stated otherwise. Myocyte Isolation and Culture—Adult male Wistar rats (250 g) were humanely killed by a blow on the head followed by cervical dislocation, and the heart was excised and placed into ice-cold modified Krebs Buffer (MKB) (in mm: 4.2 Hepes, pH 7.6, 130 NaCl, 5.4 KCl, 1.4 MgCl2, 0.4 NaH2PO4, 10 glucose, 20 taurine, 10 creatine) containing 0.75 mm CaCl2. Ca2+-tolerant adult myocytes were then isolated by a Langendorf perfusion method (20Mitcheson J.S. Hancox J.C. Levi A.J. Cardiovasc. Res. 1998; 39: 280-300Crossref PubMed Scopus (211) Google Scholar) and were cultured on laminin-coated (5 μg/ml) dishes in Medium 199 containing penicillin (100 units/ml) and streptomycin (100 μg/ml). The cells were allowed to adhere for 4 h at 37 °C in an atmosphere of 5% CO2, and then fresh medium was added. Measurement of Mitochondrial and Cytosolic [ATP] Using Targeted Luciferase—Myocytes were cultured for 24 h in the presence of adenoviruses encoding either cytosolically or mitochondrially targeted luciferase at an multiplicity of infection of 50–100 (17Jouaville L.S. Pinton P. Bastianutto C. Rutter G.A. Rizzuto R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13807-13812Crossref PubMed Scopus (647) Google Scholar). This time provided optimum expression of luciferase, since longer periods in culture with the luciferase viruses led to clear decreases in cell viability. One hour prior to recording, the cells were transferred into MKB, and 1 mm luciferin was added 1–2 min before recording. Culture dishes were placed on the stage of an Olympus IX-70 inverted microscope, and experiments were performed at 37 °C. Light emission from luciferase was detected in time-resolved imaging mode (60 frames s–1) using a triply intensified charge coupled camera (ICCD218; Photek, Lewes, East Sussex, UK) (21Rutter G.A. Burnett P. Rizzuto R. Brini M. Murgia M. Pozzan T. Tavare J.M. Denton R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5489-5494Crossref PubMed Scopus (220) Google Scholar). Light was detected from an entire field of view (typically 50–100 cells), using a –10 objective. Smoothing of traces was performed off-line using Microsoft Excel (18Kennedy H.J. Pouli A.E. Ainscow E.K. Jouaville L.S. Rizzuto R. Rutter G.A. J. Biol. Chem. 1999; 274: 13281-13291Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 19Ainscow E.K. Rutter G.A. Biochem. J. 2001; 353: 175-180Crossref PubMed Scopus (78) Google Scholar), and images generated over an appropriate integration period. Measurement of Mitochondrial and Cytosolic [Ca2+] Using Targeted Aequorin—Myocytes were cultured as above for 48 h in the presence of adenoviruses encoding either cytosolically or mitochondrially targeted aequorin (cAq and mAq, respectively), at a multiplicity of infection of 50–100 (19Ainscow E.K. Rutter G.A. Biochem. J. 2001; 353: 175-180Crossref PubMed Scopus (78) Google Scholar). This period in culture was necessary for sufficient expression of aequorin and did not result in loss of cell viability, unlike the luciferase viruses. The adenoviruses also encoded untargeted green fluorescent protein (GFP). 1 h before experiments, myocytes were transferred to Ca2+-free MKB containing 5 μm coelenterazine to reconstitute aequorin. Adult cells were stimulated electrically, and light emission from aequorin was detected at 37 °C, as above. Free [Ca2+] was calculated using the ratio of the counts at a specific time point to the total number of counts detected. This was determined by permeabilization of the cells to consume unused aequorin in the presence of digitonin (2–5 mg/ml) and 10 mm CaCl2 (21Rutter G.A. Burnett P. Rizzuto R. Brini M. Murgia M. Pozzan T. Tavare J.M. Denton R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5489-5494Crossref PubMed Scopus (220) Google Scholar). Dying cells, identified by sudden bursts of light because of essentially instantaneous aequorin consumption, were eliminated from further analysis. Immunocytochemistry—Immunocytochemistry was performed as described (22Saghir A.N. Tuxworth Jr., W.J. Hagedorn C.H. McDermott P.J. Biochem. J. 2001; 356: 557-566Crossref PubMed Scopus (37) Google Scholar). Briefly, infected cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Goat polyclonal anti-luciferase (Promega, Madison, WI) or mouse monoclonal anti-hemagglutinin tag antibody (Covance Research Products, Berkeley, CA) was used at 1:50 in phosphate-buffered saline containing 1% bovine serum albumin and revealed with an Alexa 568-conjugated secondary antibody (Molecular Probes, 1:500 dilution) using a Leica TCS-NT confocal microscope (×63 objective). Electron Microscopy—Immunoelectron microscopy was performed as described (23Searle S. Bright N.A. Roach T.I. Atkinson P.G. Barton C.H. Meloen R.H. Blackwell J.M. J. Cell Sci. 1998; 111: 2855-2866Crossref PubMed Google Scholar). Briefly, cells were washed in phosphate-buffered saline and fixed in 8% paraformaldehyde, 250 mm Hepes, pH 7.2, for 1 h at room temperature prior to embedding in gelatin and infusion with sucrose. Sections were cut on a Reichert Ultracut S ultramicrotome with a cryochamber attachment (Leica, Milton Keynes, UK) and observed on a Philips CM 100 transmission electron microscope (Philips Electron Optics, Cambridge, UK). Ultrathin cryosections were indirectly labeled as described (23Searle S. Bright N.A. Roach T.I. Atkinson P.G. Barton C.H. Meloen R.H. Blackwell J.M. J. Cell Sci. 1998; 111: 2855-2866Crossref PubMed Google Scholar). Grids were incubated with primary antibody (1:100 mouse monoclonal anti HA) for 1 h at room temperature before detection with goat anti-mouse IgG 10 nm gold conjugate. Statistics—Statistical significance was calculated using paired or unpaired Student's t tests, as appropriate. Measurement of Mitochondrial and Cytosolic ATP Using Luciferase in Adult Myocytes—Targeted luciferases, introduced into myocytes by means of an adenoviral expression system, were used to monitor changes in [ATP] in both mitochondria and cytosol in living cells. The correct localization of the luciferase enzymes was confirmed by immunofluorescence studies using antibodies directed against luciferase (Fig. 1, A and B). Mitochondrially targeted luciferase (modified to include the amino-terminal leader sequence to cytochrome c oxidase subunit VIII) showed a restricted distribution (Fig. 1A), with no fluorescence in the cell nuclei and enhanced fluorescence around the periphery of the nuclei, consistent with a mitochondrial localization as expected (17Jouaville L.S. Pinton P. Bastianutto C. Rutter G.A. Rizzuto R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13807-13812Crossref PubMed Scopus (647) Google Scholar, 18Kennedy H.J. Pouli A.E. Ainscow E.K. Jouaville L.S. Rizzuto R. Rutter G.A. J. Biol. Chem. 1999; 274: 13281-13291Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). In contrast, untargeted (cytosolic) luciferase was found throughout the cell, including the cell nuclei (Fig. 1B). Characteristic cytosolic or mitochondrial localization patterns were also seen in neonatal myocytes (see Supplemental Material). To investigate whether there are any changes in ATP in either cytosol or mitochondria upon changes in ATP demand by the cell, adult myocytes were infected with adenoviruses encoding cytosolically or mitochondrially targeted luciferase; cells were then paced to beat by electrical stimulation at 2 Hz, before being subjected to an increase in workload induced by addition of the β-adrenergic agonist isoproterenol. No significant changes in apparent free [ATP] (other than those that could be reproduced by mock additions and were thus considered artifacts), either in cytosol or mitochondria, were observed upon addition of isoproterenol (Fig. 1, C and D) or upon transition to either an increased extracellular [Ca2+] or increased stimulation rate (data not shown). In addition, no changes in [ATP]m were observed beat-to-beat (Fig. 1, E and F). [ATP]m did, however, change when myocytes were stimulated to beat from a resting state in the presence of isoproterenol, giving a large increase in workload. Under these conditions [ATP]m dropped initially by ∼10% over 30–60 s. This drop was followed by a slower recovery to give a sustained rise in [ATP]m (Fig. 2, A and C). [ATP]m then returned to initial levels when the electrical stimulus was removed. This pattern was apparent when either glucose (Fig. 2A) or the mitochondrial substrates pyruvate and lactate (Fig. 2C) were used as fuels; the latter were used to enhance the likely contribution of mitochondrial ATP synthesis to overall changes in cellular [ATP]. No significant difference in magnitude of either the initial drop or the sustained rise between the two fuels was observed (Table 1). [ATP]c showed much smaller changes in response to increased work rate with either fuel (Fig. 2, B and D; Table 1).TABLE 1Percentage change in ATP observed in different compartments upon rapid stimulation of myocytes from restmLuccLucGlucosePyr and LacGlucosePyr and LacATP fall9.2% ± 1.6 (9)13.9% ± 2.4 (7)2.0% ± 0.9 (7)*4.0% ± 2.3 (3)*ATP rise19.7% ± 4.8 (9)28.4% ± 5.0 (7)8.9% ± 3.3 (7)*5.0% ± 2.5 (3)* Open table in a new tab Measurement of [Ca2+] Changes—Because [Ca2+]m is considered to be an important signal regulating ATP production in mitochondria (4McCormack J.G. Halestrap A.P. Denton R.M. Physiol. Rev. 1990; 70: 391-425Crossref PubMed Scopus (1162) Google Scholar, 24Rutter G.A. Rizzuto R. Trends Biochem. Sci. 2000; 25: 215-221Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), targeted aequorin molecules were used to monitor mitochondrial and cytosolic [Ca2+] under similar conditions of large increases in workload. The localization of aequorin was confirmed by immunofluorescence and immunoelectron microscopy techniques using antibodies against the HA tag present on the aequorin molecule. Because the aequorin virus constructs also contained GFP expressed from a separate cytomegalovirus promoter (thus being untargeted) the aequorin distribution could also be compared with the GFP distribution. Mitochondrially targeted aequorin showed a similar distribution to mitochondrially targeted luciferase (Fig. 3A), with no fluorescence in the nuclei and enhanced fluorescence in the perinuclear regions. This distribution was in marked contrast to both GFP and untargeted (cytosolic) aequorin (Fig. 3, B, E, and F), which was distributed throughout the cell, including the nuclei. GFP was also absent from the perinuclear regions (Fig. 3B), suggesting exclusion from the mitochondria. In addition, immuno-EM studies showed ∼90% localization to the mitochondria (Fig. 3, C and D). Immunofluorescence studies of mAq and cAq in neonatal myocytes also showed characteristic distributions (see Supplemental Material). Given that even a limited mislocalization of mAq to a nonmitochondrial site might significantly interfere with calculations of [Ca2+] in this compartment, we also determined the fraction of mistargeted aequorin by additional, more quantitative methods. First, we determined the fraction of aequorin released upon selective permeabilization of cells with digitonin, saponin, or α-hemolysin (α-toxin) in "intracellular buffer." The mitochondrial Ca2+ uniporter inhibitor Ru360 was added to prevent mitochondrial Ca2+ uptake, along with 10 μm Ca2+ to fully activate any nonmitochondrial aequorin. Initial experiments using mAq-infected cells indicated that 24.4 ± 3.4% (n = 20) of the total light output was emitted at low concentrations of digitonin or 15 ± 1.08% (n = 11) in the presence of α-toxin. To determine whether mAq light output correlated with release of a mitochondrial marker enzyme (citrate synthase), we permeabilized cells with a low concentration of saponin (1 μg/ml). Even this concentration caused some release of citrate synthase, confirming that low concentrations of either saponin or digitonin are likely to damage intracellular membranes and contribute to the slightly higher estimates for mitochondrial mistargeting obtained using the detergents compared with α-toxin. However, by comparing the amount of citrate synthase activity released by saponin with mAq light output, it could be calculated that 82 ± 2% (n = 4) of aequorin was localized to mitochondria, in line with the value of 85% obtained using α-toxin. Full details of these experiments and their analysis are given in the Supplemental Material. When cells were stimulated from rest in the presence of isoproterenol, large increases in [Ca2+] were observed in both cytosol and mitochondria (Fig. 2, E and F), peaking at ∼40 s. [Ca2+] then decreased gradually to a lower steady-state level after ∼60–90 s, the time scale of the increase in [Ca2+] therefore paralleling the decrease in [ATP] in both compartments. [Ca2+]m and [Ca2+]c both returned to base line when electrical stimulation ceased. Detection of Beat-to-Beat Ca2+ Transients in Adult Cardiomyocytes—Beat-to-beat mitochondrial calcium transients have been observed previously in populations of neonatal rat myocytes (15Robert V. Gurlini P. Tosello V. Nagai T. Miyawaki A. Di Lisa F. Pozzan T. EMBO J. 2001; 20: 4998-5007Crossref PubMed Scopus (194) Google Scholar) using targeted aequorin, and so it was interesting to determine whether these could also be detected in adult myocytes. Having used the adenovirally expressed aequorin system to achieve similar results to those reported previously in neonatal myocytes (see Supplemental Material), we monitored changes in [Ca2+] using the same system in adult ventricular myocytes. In cAq-expressing cells, beat-to-beat changes in free [Ca2+]c could easily be visualized (Fig. 4) in a cell population (10–100 cells), although they were close to the limit of detection at the single cell level. However, the amount of light at each systolic peak was much smaller than that observed in neonatal myocytes, as was the total amount of light released on addition of high concentrations of digitonin (not shown) indicating that expression was lower in these cells. No Ca2+ transients were ever observed in resting cells (in over 20 fields of cells studied (not shown)). In mAq-expressing cells, the total light output (assessed as described above) was very small compared with that in experiments using cAq. This limitation made it difficult to observe changes in [Ca2+] at normal (1–2 mm) extracellular [Ca2+], because even systolic [Ca2+]m peaks gave a minimal increase in photon production and hence were close to the limit of detection. The reasons for the less efficient expression of mAq versus cAq (also observed in neonatal myocytes) are unclear; however, beat-to-beat Ca2+ transients were clearly observed at higher extracellular [Ca2+] (4 mm) or in the presence of isoproterenol (Fig. 5). Again, no Ca2+ transients were ever observed in resting cells (15 separate experiments). The time course for mitochondrial and cytosolic transients is compared in Fig. 5D, whereas the initial phase of the upstroke of the mitochondrial transient is qualitatively very similar to that of the cytosolic, and the down stroke appears to lag behind, indicating a slower mitochondrial Ca2+ efflux. The recording time scale possible with aequorin is unfortunately not fast enough to allow accurate quantification of these kinetics. Although diastolic [Ca2+] could not be accurately determined in either compartment because of very low light output at these low [Ca2+] (<0.2 μm), peak systolic [Ca2+]m and [Ca2+]c could be measured and were similar at ∼0.9 μm, a value that rose upon a further increase of extracellular calcium or the addition of isoproterenol (Table 2 and Figs. 4 and 5). The effect of isoproterenol was more pronounced in adult cells than in neonatal cells with peak systolic concentrations of ∼1.6 and 1.2 μm, respectively (Table 2). The diastolic concentration of both [Ca2+]m and [Ca2+]c also appeared to increase upon addition of isoproterenol (Figs. 4 and 5), although this could not be accurately quantified (see above).TABLE 2Peak systolic [Ca2+]m and [Ca2+]c in adult and neonatal cardiomyocytesAdultNeonateExternal [CaCl2]cAqmAqExternal [CaCl2]cAqmAqmmμmmmμm20.89 ± 0.04 (36)0.90 ± 0.09 (11)20.84 ± 0.07 (13)0.83 ± 0.07 (9)41.14 ± 0.10 (14)*1.00 ± 0.09 (14)*40.94 ± 0.07 (11)*1.09 ± 0.06 (5)*2 + ISO1.65 ± 0.07 (19)#1.23 ± 0.11 (10)#2 + ISO0.98 ± 0.10 (3)#0.92 ± 0.10 (3)#4 + ISO1.46 ± 0.11 (9)#1.35 ± 0.06 (15)#4 + ISO1.23 ± 0.09 (8)#1.28 ± 0.16 (3)# Open table in a new tab We are confident that the mitochondrial Ca2+ transients observed do in fact originate largely from the mitochondria; if all of the systolic light output were due to mAq in the cytosol, then the [Ca2+] values we observed would have required a 60% mistargeting of mAq, greatly in excess of our actual mistargeting of 15%. Full details of this theoretical calculation are given in the Supplemental Material. Using a similar calculation (not shown), we estimate that the mistargeting of mAq to the cytosol leads to only a small (≤7%) underestimate of the [Ca2+]m spike amplitude. Inhibition of Mitochondrial Ca2+ Transport—To investigate whether inhibition of mitochondrial Ca2+ transport might affect cytosolic Ca2+ signaling, we attempted to inhibit these pathways in living cells. Clonazepam is an inhibitor of the mNCX, the main mitochondrial Ca2+-efflux pathway in the heart, and does not affect L-type Ca2+ channels, Ca2+ transporters of the sarcoplasmic reticulum (SR) (25Cox D.A. Conforti L. Sperelakis N. Matlib M.A. J. Cardiovasc. Pharmacol. 1993; 21: 595-599Crossref PubMed Scopus (166) Google Scholar, 26Matlib M.A. Schwartz A. Life Sci. 1983; 32: 2837-2842Crossref PubMed Scopus (42) Google Scholar, 27Matlib M.A. Doane J.D. Sperelakis N. Riccippo-Neto F. Biochem. Biophys. Res. Commun. 1985; 128: 290-296Crossref PubMed Scopus (25) Google Scholar), or cell contraction (28Griffiths E.J. Wei S.K. Haigney M.C. Ocampo C.J. Stern M.D. Silverman H.S. Cell Calcium. 1997; 21: 321-329Crossref PubMed Scopus (45) Google Scholar, 29Brandes R. Bers D.M. Biophys. J. 2002; 83: 587-604Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). We have previously attempted to use CGP37147, which inhibits the mNCX in isolated mitochondria, but we found that it could not be used effectively in living cardiomyocytes, as it did not consistently inhibit the mNCX (28Griffiths E.J. Wei S.K. Haigney M.C. Ocampo C.J. Stern M.D. Silverman H.S. Cell Calcium. 1997; 21: 321-329Crossref PubMed Scopus (45) Google Scholar). Here, clonazepam eliminated mitochondrial Ca2+ transients but produced
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