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General Medical Education Center and Department of Physiology, Yamagata University School of Medicine, Yamagata, Japan
Submitted 15 February 2008 ; accepted in final form 14 April 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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m) and ATP levels at the anoxic cell core were relatively sustained. In contrast, treatment with 0.5 mM iodoacetamide (IA) to inhibit creatine kinase (CK) resulted in depletion of both m and ATP at the anoxic cell core. Even at normal extracellular PO2, actively respiring cardiomyocytes developed rigor contracture followed by necrotic cell death. Furthermore, such rigor was remarkably accelerated by IA, whereas cell injury was perfectly rescued by mitochondrial F1Fo inhibition by oligomycin. These results suggest that increases in radial gradients of O2 potentially promote cell death through the reverse action of F1Fo in mitochondria located at the anoxic cell core. However, in the intact cardiomyocyte, the CK-mediated energy flux from the subsarcolemmal space may sustain m at the cell core, thus avoiding uncontrolled consumption of ATP that can lead to necrotic cell death. Mitochondria at the anoxic core can cause necrotic cell death in cardiomyocytes at physiological extracellular PO2.
necrosis; mitochondria; hypoxia; creatine kinase; energy shuttle
Spectrophotometry, at a submicrometer spatial resolution, has demonstrated radial gradients of fractional O2 saturation of myoglobin (SMb) in isolated single ventricular myocytes in which mitochondrial O2 consumption has been elevated at physiological extracellular PO2 (25, 28). At elevated mitochondrial O2 demands, such intracellular gradients of O2 supply have been demonstrated to restrict oxidative metabolism in mitochondria, particularly those located at the center of cardiomyocytes (25, 27). Thus, an anoxic cell core may be produced in actively respiring cardiomyocytes despite normal extracellular O2. In the in vivo heart, interruption of the capillary O2 supply for a prolonged time results in cell death due to the suppression of oxidative phosphorylation. How then does such regional anoxia within a cell (i.e., the anoxic core) affect the overall functions of cardiomyocytes at normal extracellular O2?
Here, I demonstrate that intracellular radial O2 concentration gradients may promote necrotic cell death in single ventricular myocytes isolated from rats, even when cells are exposed to physiological O2 concentrations (2–5%). It is also demonstrated that such cell injury is significantly delayed by an endogenous intracellular energy transfer system, the phosphocreatine (PCr)-creatine kinase (CK) system. Finally, a new physiological function for this enzyme system is proposed.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Isolation of Single Rat Cardiomyocytes
Prior approval for all experiments was obtained from the Animal Research Committee of Yamagata University School of Medicine. Single cardiomyocytes were isolated from Sprague-Dawley rats and suspended in HEPES-Tyrode solution as previously described (26). Briefly, after an injection of pentobarbital sodium (50 mg/kg ip), the heart was excised, and a Langendorff perfusion was conducted. Single ventricular myocytes were isolated from the heart using collagenase (type 2, Worthington, Lakewood, NJ) and suspended in a HEPES-Tyrode solution containing 130 mM NaCl, 6 mM HEPES, 10 mM glucose, 5.4 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 1.2 mM CaCl2 supplemented with 2.5 mM Na-pyruvate and 10 mM taurin (pH 7.4 at 37°C). Cells were quiescent, and contractions could be induced by electrical stimulations. All experiments were conducted at 37°C.
Increasing O2 Flux to the Mitochondria
In isolated single cardiomyocytes, intracellular radial O2 gradients were augmented by elevating mitochondrial O2 consumption. This was achieved using an oxidative phosphorylation uncoupler (1 µM CCCP) rather than electrically pacing the cell to avoid movement artifacts in optical measurements. As previously reported from our laboratory (4), the O2 consumption rate in the presence of 1 mM Ca2+ was 130 ± 32 nmol O2·min–1·10–6 rod cells at 37°C (mean ± SD; n = 7), whereas this rate increased to 790 ± 173 nmol O2·min–1·10–6 rod cells after an incubation with 1 µM CCCP. Because the normal heart can increase steady-state O2 utilization up to 20-fold from basal levels (31), a 6-fold increase in O2 consumption in 1 µM CCCP-treated cardiomyocytes represents a level that is well below the maximal O2 consumption in vivo.
Cell Survival Experiments
An aliquot (8 µl) of the cell suspension (
106 cells/ml) was placed in an airtight cuvette with gas inlet/outlet ports on the stage of an inverted microscope (IX71, Olympus). The suspension was superfused with humidified gas (2 ml/min) with O2 concentrations ranging from <0.001% to 10%. To facilitate a PO2 equilibrium between gas and liquid phases, the thickness of the medium was reduced to 230 µm. The transmitted light image at 435 nm was captured by a charge-coupled device (CCD) camera (SV512, PixelVision, Tigard, OR) every 15 s (unless otherwise noted), and data were stored in the computer. Drugs [CCCP, iodoacetamide (IA), or vehicle (DMSO)] were added to the suspension immediately before measurements. Rigor development was defined as >30% shortening of the cell length. The fraction of cardiomyocytes that developed rigor contracture is indicated in the Kaplan-Meier plot.
Using phosphorescence quenching techniques, Rumsey et al. (19) generated a high-resolution O2 map in the beating heart in the anesthetized piglet ventilated with room air. Intravascular PO2, presumably representing that of capillaries and venules, ranged 18–26 mmHg. In the in vivo heart, PO2 at the sarcolemma should be lower than the capillary PO2 of 20 mmHg (19, 31). In the present study, superfusion of the medium in the measuring cuvette with 10% (71 mmHg), 5% (36 mmHg), 2% (14 mmHg), and <0.001% (<0.07 mmHg) O2 gas was designated as hyperoxia, normoxia, hypoxia, and anoxia, respectively. Because the PO2 drop between the superfusion gas and sarcolemma was not determined, "hypoxia" merely means that the cell is less oxygenated compared with the "normoxia."
Measurements of Intracellular Ca2+ Concentration
Changes in intracellular Ca2+ concentration ([Ca2+]i) were assessed by calculating the fluorescence ratio at 340 and 380 nm (F340/F380) of the Ca2+ indicator dye fura-2 (Invitrogen/Molecular Probes, Eugene, OR). Cardiomyocytes were incubated with 5 µM fura-2 AM for 30 min at 35°C. Fluorescence images of cells at 510 nm were serially captured by a CCD camera for excitation at both 340 nm (F340) and 380 nm (F380). After the background fluorescence had been subtracted, F340/F380 was calculated using image-processing software (IPLab, Scanalytics/BD Biosciences, Rockville, MD). Transmitted cell images at 435 nm were captured simultaneously.
Imaging of SMb
SMb was imaged at a subcellular spatial resolution from light absorption of individual cells at 435 nm (an absorption peak of deoxy-Mb). Transmitted light images of cells at 435 nm were captured by a CCD camera every 10 s. In a separate experiment using a Mb solution (with the addition of Na2S2O4), oxy-Mb was found to be converted to the deoxy-Mb form within 150 s in the present experimental setup (data not shown). A rectangular region of interest (ROI) was arbitrary defined perpendicular to the long axis of the cell image (see Fig. 5A). Following low-pass filtering, optical density (OD) was calculated along the arrow (see Fig. 5A) using IPLab software. Finally, SMb was determined using the following formula: SMb = (OD – ODanaerobic)/(ODaerobic – ODanaerobic). Detailed methods have been published elsewhere (25, 28).
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Mitochondrial oxidative metabolism was assessed by autofluorescence of mitochondrial NADH at a subcellular spatial resolution. Single cardiomyocytes were first superfused with 18% O2. Fluorescence images were captured using a CCD camera (excitation at 365 nm and emission at 450 nm). Subsequently, the O2 concentration of the superfusion gas was reduced to 2%. After 5 min, subsequent fluorescence images were taken. Changes in NADH fluorescence were determined, after subtraction of the nonspecific background fluorescence, by dividing the second image by the first image. Image processing was conducted using IPLab software. Data are represented in pseudocolors. Detailed methods have been published elsewhere (27).
Imaging of Mitochondrial Membrane Potential
The fluorescent dyes JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, Invitrogen/Molecular Probes) or tetramethylrhodamine methyl ester (TMRM; Invitrogen/Molecular Probes) were used to assess changes in mitochondrial membrane potential (m) in isolated single cardiomyocytes.
Cells were incubated with 2 µM JC-1 for 30 min at 35°C and washed twice with HEPES-Tyrode buffer. m was assessed from the ratio of the J-aggregate fluorescence (red fluorescence, excitation at 525 nm and emission at 595 nm) and monomer fluorescence (green fluorescence, excitation at 490 nm and emission at 535 nm). A CCD camera captured fluorescence images. Intensities of the fluorescence images were averaged over the cells, and the fluorescence ratios (red/green fluorescence) were calculated.
Single cardiomyocytes were incubated with 0.5 µM TMRM for 15 min at 35°C and washed twice with HEPES-Tyrode solution. TMRM was excited at 525 nm, and fluorescence images at 595 nm were captured by a CCD camera every 7.5 s. ROIs of 20 pixel width were defined near the periphery and at the center of the fluorescence images. Average fluorescence intensities in the respective ROIs were calculated. Radial gradients of TMRM fluorescence were determined by dividing the average fluorescence at the center by that at the periphery.
Indirect Imaging of ATP Changes
There is no convenient technique currently available that allows for repeated measurements of ATP at a subcellular spatial resolution in living cells. In the present study, ATP heterogeneities were indirectly assessed by imaging the intracellular free Mg2+ concentration ([Mg2+]i) (14). This technique is based on the assumption that, at a physiological pH, Mg2+ is liberated upon hydrolysis of ATP and taken up during ADP phosphorylation. Thus, transient reductions of ATP may be reflected by increases in [Mg2+]i. [Mg2+]i was imaged at a subcellular spatial resolution using the Mg2+-sensitive ratiometric fluorescent dye mag-fura-2 (Invitrogen/Molecular Probes). The affinity of mag-fura-2 to Ca2+ is low (Kd = 25 µM at 22°C) (11) compared with resting [Ca2+]i in rat ventricular myocytes (0.09 µM) (23). Mag-fura-2 fluorescence has been demonstrated to behave independently of [Ca2+]i under a variety of physiological and pathophysiological conditions (14).
Cells were incubated with 5 µM mag-fura-2 for 30 min at 35°C. Fluorescence images of cells at 510 nm were serially captured by a CCD camera for excitation at both 340 nm (F340) and 380 nm (F380). After the background fluorescence had been subtracted, the fluorescence ratio (F340/F380) was calculated.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Cell Survival Experiments
Cell survival experiment 1: definition of the end point. In isolated single cardiomyocytes treated with 1 µM CCCP, cell death at lowered extracellular O2 concentrations followed a typical time course (Fig. 1) : 1) in the early phase, there was the development of rigor contracture (represented by a longitudinal shortening) with slight increases in [Ca2+]i; 2) over the following 2 h, there were continuing increases in [Ca2+]i without significant changes in cellular morphology; and 3) finally, there was the formation of blebs and eventual disruption of the sarcolemma, reflected by an almost complete disappearance of fura-2 fluorescence in the cell. Reoxygenation (10% O2) of the cell did not restore cell morphology once rigor contracture had developed; the eventual disruption of the cell membrane was observed. Thus, in the following cell survival experiments, the development of rigor contracture was taken as the hallmark of irreversible cell injury.
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Next, the effects of an increased magnitude of radial O2 gradients on cellular viability were tested. In quiescent cardiomyocytes, intracellular radial O2 gradients were augmented by elevating O2 flux to the mitochondria using an uncoupler of oxidative phosphorylation. CCCP (1 µM) produced a sixfold increase in O2 consumption in quiescent single cardiomyocytes (see MATERIALS AND METHODS). Figure 2 (thick curves) shows the development of rigor contracture under conditions of reduced O2 concentration. In both hyperoxia (10% O2) and normoxia (5% O2), most cells retained a normal morphology at the end of the 20-min observation period. In contrast, under conditions of both hypoxia (2% O2) and anoxia (<0.001% O2), cells deteriorated quickly. The fraction of cells in which rigor development was detected at the end of the observation period was 0% (n = 18, superfused 10% O2), 7.1% (n = 42, superfused 5% O2), 45.7% (n = 35, superfused 2% O2), and 100% (n = 17, superfused <0.001% O2).
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IA was used to nonspecifically inhibit CK (5, 9, 10, 29). Strikingly, as shown in Fig. 2 (thin curves), 0.5 mM IA considerably accelerated cell injury in both normoxic (5% O2) and hypoxic (2% O2) cardiomyocytes, whereas hyperoxic (10% O2) cardiomyocyte viability was unaffected. Treatment with 0.5 mM IA in normoxic (5% O2) cardiomyocytes with low O2 flux (i.e., without CCCP treatment) did not affect viability (0% rigor contracture at 20 min, n = 11; data not shown).
Cell survival experiment 4: effects of IA on survival of energy-depleted cardiomyocytes. First, the role of glycolytic ATP in the survival of cardiomyocytes was determined. Oxidative ATP production in mitochondria was inhibited by anoxia (<0.001% O2) along with inhibition of mitochondrial ATP synthase by oligomycin (15 µg/ml). Cardiomyocytes were not treated with the uncoupler. Thus, mitochondrial respiration was predicted to be slow and radial O2 gradients negligible (28).
Cardiomyocytes incubated with a normal concentration of glucose successfully survived without oxidative ATP for at least for 40 min [99% survival; Fig. 3, IA(–)/glucose]. In contrast, cardiomyocytes in which glycolysis was inhibited (in addition to oxidative ATP inhibition) by 2-deoxyglucose (2-DG; 10 mM) deteriorated quickly [49% survival at 20 min and 19% survival at 40 min; Fig. 3, IA(–)/2-DG]. These results may indicate that glycolytic ATP alone is sufficient for maintaining basic (noncontracting) cellular functions in cardiomyocytes.
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In cardiomyocytes with arrested ATP productions, inhibition of CK by 0.5 mM IA did not significantly affect rigor contracture at 20 min [log-rank test; Fig. 3; comparisons between IA(–)/2-DG and IA(+)/2-DG], indicating that PCr, as a source of high-energy phosphate, does not appear to contribute to cardiomyocyte survival in this time frame.
Cell survival experiment 5: role of cyclosporin A-sensitive permeability transition pore in cell injury at elevated O2 flux. In cardiomyocytes, cell death may be preceded by opening of the mitochondrial permeability transition pore (PTP). The role of cyclosporin A (CsA)-sensitive mitochondrial permeability transition (mPT) in necrotic cell death in actively respiring cardiomyocytes was examined. As shown in Fig. 4, 0.5 µM CsA significantly reduced rigor development in the early phase in cardiomyocytes treated with IA. However, CsA treatment did not preclude eventual rigor development. These findings suggest an additional mechanism for rigor development that is independent of CsA-sensitive PTP opening.
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Imaging experiment 1: intracellular gradients of O2 and NADH oxidation at elevated O2 flux. Previously, we have reported that in 1 µM CCCP-treated cardiomyocytes, significant radial gradients of SMb have been demonstrated at a constant extracellular O2 ranging from 2% to 4% (see Fig. 7 in Ref. 28 and Figs. 2 and 4 in Ref. 25). Also, we have demonstrated similar radial SMb gradients in electrically paced (CCCP untreated) cardiomyocytes (see Figs. 3 and 4 in Ref. 25). In addition to these steady-state measurements, dynamic changes in SMb heterogeneities during a transition from aerobic to anaerobic conditions were demonstrated in the present study (Fig. 5B). Thus, radial gradients of SMb in actively respiring single cardiomyocytes were demonstrated in both steady and transient states.
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m gradients at elevated O2 flux.
Suppression of mitochondrial NADH oxidation in the O2-deficient cell core (Fig. 5C) should affect m. Depolarization of the mitochondrial inner membrane may affect cell fate in two distinct ways: 1) suppression of oxidative ATP production and 2) opening of the mitochondrial PTP (22).
To seek a possible linkage between the formation of an anoxic core and accelerated cell injury in IA-treated cardiomyocytes, changes in m were imaged at a subcellular spatial resolution. As shown in Fig. 6A, top, a decrease in the extracellular O2 concentration from 10% to 2% in 1 µM CCCP-treated cardiomyocytes mildly reduced TMRM fluorescence, indicating a depolarization of the mitochondria. This reduction in fluorescence appeared relatively uniform in these control cardiomyocytes. These findings appear inconsistent with the suppression of NADH oxidation due to a lack of O2 at the anoxic cell core (Fig. 5C).
In contrast, significant heterogeneities in TMRM fluorescence were demonstrated in 0.5 mM IA-treated cardiomyocytes with significant reductions in fluorescence specifically at the cell core (Fig. 6A, bottom). Figure 6B shows heterogeneities in TMRM fluorescence in individual cardiomyocytes. After levels of superfused O2 declined from 10% to 2%, TMRM fluorescence in the cell core was lower than that at the periphery. In IA-treated cardiomyocytes (red), such heterogeneities were significantly larger, and rigor developed in all cells within 12 min.
Imaging experiment 3: intracellular ATP gradients at elevated O2 flux. At the anoxic core, it is reasonable to assume the existence of regional depletions of ATP since intracellular diffusion of ATP in muscles (0.5 x 10–9 m2/s) (3) is much slower than that of O2 (2.4 x 10–9 m2/s) (1). It is important, therefore, to clarify the role, if any, of the ATP-depleted cell core in the overall cell fate.
Intracellular ATP heterogeneities were indirectly assessed by imaging [Mg2+]i. Figure 7A shows serial imaging of [Mg2+]i in 1 µM CCCP-treated cardiomyocytes. The percentage of superfused gas was lowered from 10% O2 to <0.001% O2. The mag-fura-2 fluorescence ratio (F340/F380) increased over time, presumably reflecting gradual ATP decreases, whereas radial gradients of F340/F380 were not observed (Fig. 7C). The effects of 0.5 mM IA were then examined. As shown in Fig. 7B, as the concentration of O2 gradually decreased, F340/F380 increased first in the cell core, producing a radial gradient of F340/F380 (Fig. 7D). These findings were consistent with the m imaging experiments shown in Fig. 6. In cardiomyocytes with moderately elevated O2 flux, IA-sensitive mechanisms may sustain m and ATP levels in the cell core where the mitochondrial O2 supply is limited.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Recently, we (25, 28) visualized radial SMb gradients in isolated single cardiomyocytes with elevated O2 consumptions. In separate experiments, we (27) demonstrated radial gradients of NADH oxidation that were consistent with the SMb gradients. These results indicate that the intracellular diffusion of O2 may be an important factor that regulates oxidative metabolism in mitochondria, particularly those located in the center of the cell.
The question then arises as to whether the heterogeneities of intracellular O2 and mitochondrial oxidative metabolism, particularly the anoxic cell core, affect the overall cell fate. To answer this question, imaging of predominant parameters of O2 metabolism (SMb, mitochondrial NADH oxidation, m, and Mg2+/ATP), along with cell survival experiments, was carried out in single cardiomyocytes with elevated O2 flux.
Increased O2 flux induced irreversible cell injury at physiological extracellular O2 levels. Importantly, cell injury developed at O2 concentrations (for example, 2% O2 or 14 mmHg) that are substantially higher than the apparent Km for cytochrome c oxidase determined in isolated mitochondria (0.1 mmHg) (6). Augmented intracellular radial O2 gradients in actively respiring cardiomyocytes may, in fact, decrease PO2 averaged over the cell. However, despite such O2 concentration gradients, most mitochondria, particularly those located in the subsarcolemmal space, should still enjoy abundant O2 supply due to their very high affinity for O2. Therefore, a decrease in diffusional O2 supply to the mitochondria does not simply explain such cell injuries. Another important finding is that the administration of 0.5 mM IA, aimed at nonspecifically inhibiting CK, significantly accelerated the cell injury. In cardiomyocytes, contrary to expectations, significant heterogeneity was not found in either ATP levels or m despite the radial gradients of SMb and mitochondrial NADH oxidation. In contrast, IA treatment in these cells revealed ATP- and m-depleted cell cores. How could the findings regarding cell survival and intracellular gradients of O2 metabolism be connected?
Pharmacological Interventions
In the present study, two drugs, CCCP and IA, were used to increase mitochondrial O2 consumption and inhibit CK, respectively. Before the present findings are interpreted, the effects of these drugs should be carefully examined.
Cell injuries after treatment with the uncoupler are not surprising, at least qualitatively. Oxidative phosphorylation uncouplers, such as CCCP, are H+ ionophores and reduce H+ gradients across the mitochondrial inner membrane. Thus, these uncouplers decrease m. It is possible that 1 µM CCCP might have eliminated m, the driving force for ATP synthase, which would lead to the depletion of cellular ATP stores. Then, to determine whether 1 µM CCCP completely abolishes m, a semiquantitative measurement of m was carried out in single cardiomyocytes loaded with JC-1 (Fig. 8). At 5% O2, the JC-1 fluorescence ratio (red/green fluorescence) was 86% of controls, whereas the ratio declined to 53% at <0.001% O2 (n = 5). These results are consistent with the hypothesis that the magnitude of m is a function of both H+ leaks across the mitochondrial inner membrane and the rate of electron transport in the respiratory chain, the latter being limited by O2 availability. It is concluded that in the present CCCP-treated cardiomyocytes, m was not abolished, at least under conditions of normal extracellular O2 (5%), whereas the equilibrium between the H+ leak and the electron transport-coupled H+ pumping was shifted.
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Reductions of m in the anoxic core after the administration of IA would be consistent with the inhibition of glycolysis by this alkylating agent, if m in the anoxic core is sustained exclusively by glycolytically produced ATP through the reverse action of F1Fo (see below). However, the results of the cell survival experiments are not fully compatible with this hypothesis. In the present study, it was demonstrated that survival of oligomycin-treated cardiomyocytes (with a slow mitochondrial respiration) relies on glycolytic ATP [Fig. 3, IA(–)/2-DG vs. IA(–)/glucose]. Furthermore, the finding that 0.5 mM IA induced cell death at 40 min [Fig. 3, IA(–)/glucose vs. IA(+)/glucose] certainly suggests an effect of this drug on glycolytic ATP production. However, at 20 min, the effect of IA on rigor development was not significant (Fig. 3, dashed curves). Additionally, in 1 µM CCCP-treated cardiomyocytes in which oxidative phosphorylation was suppressed by anoxic superfusion, rigor development was identical in control and IA-treated cardiomyocytes (Fig. 2, bottom right). This result is inconsistent to the hypothesis that the inhibition of glycolysis by IA causes accelerated cell injury. Thus, in the time frame in which cell survival experiments were conducted, inhibition of glycolysis by IA did not fully account for the accelerated rigor development in cardiomyocytes with elevated O2 consumption (Fig. 2, thin curves). Why, then, does IA treatment accelerate rigor development in cardiomyocytes with elevated intracellular O2 flux?
Mechanisms for Accelerated Cell Death in CCCP-Treated Cardiomyocytes Without Functional CK
It is assumed that complex V (F1Fo-ATP synthase/ATPase) in mitochondria located at the anoxic core might be the causative factor. At the anoxic core, corruption of m due to a lack of O2 may turn mitochondrial F1Fo-ATP synthase to a potent ATPase, thus partially restoring m through the reverse mode action of this enzyme (18, 30). At the same time, rapid and uncontrolled ATP consumption by F1Fo in mitochondria at the anoxic core may produce a flux of ATP from the cytoplasm to the mitochondrial matrix with a reverse action of adenine nucleotide translocase in the inner membrane. Finally, if intracellular diffusion of ATP from the O2-abundant subsarcolemmal space is not fast enough to replenish the anoxic core with ATP, cellular ATP levels would reduce at normal extracellular O2 levels. A decline in ATP in myofibrils results in rigor development (12). If cellular ATP levels continue to decline, ionic homeostasis, particularly that of Ca2+, is lost. Plasma membrane integrity is disturbed, the final step in necrotic cell death (16, 32).
Additionally, at the moment when ATP (including that produced by anaerobic glycolysis) is completely consumed at the anoxic core, complex V no longer sustains m. Dissipation of m at the anoxic core could open the PTP (22). This would be followed by a massive release of Ca2+ from the mitochondrial matrix to the cytosol under pathophysiological conditions. In the present study, mPT is likely to partially account for cell injury (Fig. 4). These distinct mechanisms with different time courses would result in Ca2+ overload and eventual necrotic cell death. Thus, mitochondria at the anoxic core can lead to overall cell injury in cardiomyocytes exposed to normal extracellular PO2.
In the intact cardiomyocyte, there may be an intrinsic mechanism by which such cell injuries at increased O2 consumption is avoided. In cardiac muscle, intracellular transfer of energy predominantly relies on CK-mediated PCr/Cr flux rather than direct diffusion of adenine nucleotides (3, 21). Therefore, it is likely that CK-mediated energy flux from the O2-abundant subsarcolemmal space to the O2-depleted cell core might stabilize m at the anoxic core by substantially accelerating diffusional ATP transport to F1Fo. Thus, CK-mediated flux of energy to the anoxic core delays overall cell death.
Stabilization of m Blocks Cell Injury in Cardiomyocytes With Elevated O2 Flux
Untreated cardiomyocytes can survive without oxidatively produced ATP for a prolonged period of time (Fig. 3). As outlined above, cell injury in actively respiring cardiomyocytes may be caused by the extra consumption of ATP through the reverse operation of F1Fo following m dissipations (18, 30) in the anoxic core. Theoretically, such catastrophic wasting of ATP may be interrupted by stabilizing m through the supply of ATP to F1Fo in the anoxic core or by inhibiting this enzyme.
Based on these assumptions, the ability of inhibition of this enzyme to prevent irreversible injury in rapidly respiring cardiomyocytes lacking a CK-mediated ATP supply to the anoxic core was examined. As shown in Fig. 9, within the 20-min observation period, rigor developed in 94% of actively respiring IA-treated cells (presumably due to ATP wasting in the anoxic cell core). This was perfectly reversed by the inhibition of mitochondrial F1Fo by 15 µg/ml oligomycin.
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Expansion of the PCr Energy Shuttle Theory
In cardiomyocytes, the PCr-CK system is the predominant energy transfer system (the energy shuttle) between the site of production and those of consumptions (21). Intermyofibrillar mitochondria in the mammalian ventricular myocyte are uniformly distributed, with a highly ordered pattern such that strands of mitochondria run along the corresponding myofibrils, in which the average distance between adjacent mitochondria is 1.8 µm (2). Thus, it is tempting to assume that the transfer of energy between mitochondria and myofibril ATPases is confined to a small, spatially restricted compartment. Saks et al. (20) proposed the intracellular energy unit (ICEU), in which mitochondria form a functional complex with adjacent myofibrils and sarcoplasmic reticulum ATPases. However, if the energy shuttling takes place predominantly within the ICEU, the benefit of the CK-mediated and -facilitated energy transfer may be somewhat discounted because both mitochondria and myofibrils in the ICEU are arranged closely. The direct diffusion of adenine nucleotides may suffice for energy transfer over such a short diffusion length (<2 µm) (3).
The present findings appear to expand the energy shuttle theory of the PCr-CK system. Here, the CK-mediated energy shuttling along the radial axis of the cell, with a maximum diffusion distance of 10 µm in the rat ventricular myocyte, is proposed to provide an important step in the intracellular energy transport. Consequently, at elevated O2 flux, not only intra-ICEU energy transfer but also inter-ICEU energy transfer may be supported by the PCr-CK system. A radial flux of energy may be required for the equilibration of the supply of energy under inherent heterogeneities of intracellular O2 supply within actively respiring cardiomyocytes.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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