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REVIEW

Ion Transport and Energetics During Cell Death and Protection

Elizabeth Murphy and Charles Steenbergen

National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland; and Department of Pathology, Johns Hopkins Medical Institute, Baltimore, Maryland, murphy1{at}mail.nih.gov

	Abstract

 
During ischemia, ATP and phosphocreatine (PCr) decline, whereas intracellular hydrogen ion, intracellular sodium (Na⁺), calcium (Ca²⁺), and magnesium (Mg²⁺) concentrations all rise. If the ischemia is relatively short and there is little irreversible injury (cell death), PCr, pH, Na⁺, Mg²⁺, and Ca²⁺ all recovery quickly on reperfusion. ATP recovery can take up to 24 h because of loss of adenine base from the cell and the need for de novo synthesis. There are correlative data showing that a sustained rise in Ca²⁺ during ischemia and/or lack of recovery during reperfusion is associated with irreversible cell injury. Interventions that reduce the rise in Ca²⁺ during ischemia and reperfusion have been shown to reduce cell death. Therefore, a better understanding of the mechanisms responsible for the rise in Ca²⁺ during ischemia and early reperfusion could have important therapeutic implications. This review will discuss mechanisms involved in alterations in ions and high energy phosphate metabolites in perfused or intact heart during ischemia and reperfusion.

	High-Energy Phosphates

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 
ATP levels have been measured in snap-frozen glucose perfused hearts by enzymatic methods or by luciferase (49). Under aerobic conditions, ATP levels in heart are normally about 20–25 µmol/g tissue dry wt (7, 49, 71); this can be converted to mM by dividing by 2.5, based on 2.5 µl intracellular water/g dry wt tissue. ATP content can be continuously measured in the same heart without freezing and extraction using nuclear magnetic resonance (NMR) spectroscopy. Content can be converted to concentration using standards or by measuring ATP content by biochemical methods at the end of the study. These different methods generally agree and report ATP concentrations in heart of 8–10 mM. Values for phosphocreatine (PCr) concentrations measured using similar methods are 10–25 mM (7, 49) and are more variable because this parameter is more sensitive to work state and substrate than ATP. PCr is easily degraded during extraction and tissue grinding, and this may account for some of the lower levels. Inorganic phosphate is measured in the range of 2.5 mM (7). ADP measured in snap-frozen hearts is typically ~2.5–3 µmol/g dry wt, corresponding to ~1.2 mM ADP (49). However, using NMR spectroscopy, ADP levels were undetectable. ADP calculated using the creatine kinase equilibrium is typically in the range of 0.05–0.08 mM, considerably lower than the values measured in extracts from frozen hearts (7, 19, 71). This difference is usually attributed to a bound ADP pool that is measured in extracts but is not measured by NMR (because the line-width of bound ADP is broadened, making it undetectable), and this bound ADP does not contribute to the ATP phosphorylation potential. Table 1 shows baseline levels of high-energy phosphates measured in the glucose-perfused rat heart. Values are similar in other species.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Representative changes in Na, Ca, Mg, ATP, CrP, and pH during ischemia

	Intracellular pH

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 
Intracellular pH has been measured, at baseline and during ischemia, by ³¹P-NMR spectroscopy using the shift difference between creatine phosphate and inorganic phosphate:

(Eq. 1)

where _max, _min and _x are the maximum, minimum, and measured shift difference between PCr and inorganic phosphate, and pK is the pK for inorganic phosphate. Creatine phosphate is pH insensitive in the physiological range, whereas inorganic phosphate has a pK_a of ~6.9. The position of inorganic phosphate shifts depending on the pH (the degree of protonation). Therefore, using a rearrangement of the Henderson-Hasselbach equation (Eq. 1), intracellular pH can be calculated from the shift difference between creatine phosphate and inorganic phosphate. Basal pH in heart is in the range of 7.05–7.20 (16, 20, 45). These values agree with older literature in which intracellular pH was measured using the equilibrium of a weak acid usually with a radioactive label such as ¹⁴C (58). During total ischemia in the rodent heart, intra-cellular pH rapidly declines, reaching about 6.0 after 15 min of ischemia, and remains at this level during ischemia (see FIGURE 1) (45).

	Intracellular Na+

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 
Intracellular Na⁺ has been measured in beating perfused hearts by ²³Na-NMR, using a shift reagent to shift the extracellular Na⁺ so that it can be separated from the resonance peak for the intracellular Na⁺ (57). By measuring the area under the intracellular Na⁺ resonance peak, ²³Na-NMR measures the amount of intra-cellular Na⁺. The amount of Na⁺ can be converted to an intracellular Na⁺ concentration with information about intracellular volumes. Investigators using ²³Na-NMR have reported intracellular Na⁺ values in the range of 7–15 mM (6, 45, 51, 66). These values agree well with those measured in isolated cardiac myocytes using ion-selective electrodes or fluorescent indicators (5). As illustrated in FIGURE 1, ²³Na-NMR data indicate that intracellular Na⁺ rises about three- to fourfold during ischemia to a level in the range of 25–40 mM (1, 45, 51).

	Cytosolic Ionized Ca2+

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 
Intracellular Ca²⁺ has been measured in cardiac myocytes using fluorescent calcium indicators by many investigators (4, 15, 69). Diastolic Ca²⁺ levels have been reported to be ~100 nM with systolic levels reported in the range of 0.6–2 µM (4, 15, 69). It has been more difficult to measure Ca²⁺ with fluorescent indicators in a beating heart because of motion artifacts. Some measurements of surface fluorescence have been made, and they support the measurements obtained in isolated myocytes (39). Ca²⁺-sensitive indicators based on BAPTA have also been labeled with fluorine to allow measurement of Ca²⁺ via ¹⁹F-NMR spectroscopy (38, 45). A limitation of this NMR method is that the low sensitivity of NMR (relative to fluorescence) requires loading with high levels of the indicator, which results in buffering of Ca²⁺; however, gated Ca²⁺ measurements agree well with measurements obtained in isolated myocytes (37). Ischemia is defined as the lack of blood flow and can therefore only be studied in an intact organ, although measurements have been made of Ca²⁺ during metabolic inhibition in isolated myocytes. Studies have been done in perfused hearts using fluorescent Ca²⁺ indicators (39) or NMR measurements using ¹⁹F-BAPTA (38, 59). Surface fluorescent measurements of Indo-1-loaded hearts show a rise in Ca²⁺ during ischemia (39). ¹⁹F-NMR measurements of Ca²⁺ using FBAPTA have reported a rise in Ca²⁺ to 3 µM by 20 min of global ischemia (38, 59).

	Cytosolic Ionized Mg2+

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 
Cytosolic free Mg²⁺ has also been measured using fluorescent and fluorine-labeled NMR-sensitive indicators (44, 47). Using a ¹⁹F-labeled Mg²⁺ chelators APTRA, which undergoes an NMR chemical shift on binding Mg²⁺, intracellular Mg²⁺ was determined to be 0.8 mM in a beating perfused heart (47). Similar levels for free Mg²⁺ were measured using fluorescent Mg²⁺ indicators loaded into cardiomyocytes (44). During ischemia, ¹⁹F-NMR was used to measure Mg²⁺, and it was reported that the rise in Mg²⁺ during ischemia tracked the decline in ATP during ischemia. At 20 min of ischemia, Mg²⁺ was measured at 2.1 mM in the rat heart (47).

	Mechanisms Responsible for Ionic Changes During Ischemia and Reperfusion

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 
pH during ischemia
pH during ischemia is determined by the cellular production of acid and the acid extrusion tranporters. The initial decline in pH during ischemia is usually attributed to anerobic glycolysis and release of protons from ATP breakdown (56). In a global Langendorff rat heart model, glycolysis slows markedly after about 15 min of ischemia. The virtual cessation of production of protons during ischemia has usually been attributed to inhibition of glycolysis (due to increased NADH and low pH-mediated inhibition of GAPDH) before complete glycogen depletion (54), whereas the rate of fall and the final extent of intracellular acidification can be modulated by interventions that reduce ATP breakdown during ischemia (reduce metabolic demand). Interestingly, the fall in pH_i during ischemia is reduced (less acidification) with many cardioprotective interventions (30, 46, 61). It is also interesting that oligomycin, an inhibitor of the F₁F₀-ATPase, which has been shown to consume ATP during ischemia by running in reverse, also reduced ischemic acidification (12). These data suggest that the rate of fall and final pH reached during ischemia is also influenced by factors other than direct inhibition of glycolysis.

The protons generated during ischemia are removed from the cell by extrusion of weak acids such as lactic acid and other proton extrusion mechanisms such as Na⁺-H⁺ exchange (NHE), resulting in extracellular acidification. This eventually inhibits further acid efflux from the cell during ischemia in intact myocardium where the extracellular volume is limited. Concomitant measurements of intra- and extracellular pH during ischemia demonstrated final values of 5.9 and 5.5, respectively (18). Lactate efflux and proton extrusion via NHE or Na⁺-bicarbonate cotransporters have been suggested to be the primary transporters responsible for proton extrusion during ischemia. However, the role of NHE in extruding protons during ischemia is debated because NHE is inhibited by low extracellular pH that occurs during ischemia. However, the fall in extracellular pH does not occur immediately, so it is likely that inhibition of proton extrusion mechanisms does not occur immediately on ischemia, and extracellular pH is lower than intracellular pH, and this cannot be achieved by efflux of weak acids alone. Also, the fall in pH can be accounted for based on metabolism without invoking inhibition of proton extrusion mechanisms (56).

pH during reperfusion
On reperfusion, extracellular pH returns to normal (pH ~7.4), allowing extrusion of intracellular protons via NHE and Na⁺-dependent bicarbonate exchange. These acid-extruding mechanisms, such as NHE, return the intracellular pH to normal within a few minutes of reperfusion (28, 29, 52). A number of studies have shown that NHE inhibition causes a slight slowing of the rate of recovery of pH on reperfusion (52, 62). Interestingly, even with NHE inhibition, pH still recovers rapidly during reperfusion, demonstrating that other acid extrusion mechanisms can regulate pH if NHE is inhibited.

Na⁺ during ischemia
Na⁺ has been shown to rise during ischemia (36, 45, 51). This increase in Na⁺ could be due to an increase in Na⁺ influx, a decrease in Na⁺ extrusion, or a combination of both (see FIGURE 2). The Na⁺-K⁺-ATPase extrudes Na⁺ from the cell and thereby sets the inwardly directed Na⁺ gradient that provides the driving force for many other exchangers. Generally, a modest increase in a Na⁺ influx pathway does not increase intracellular Na⁺, because of an increase in activity of the Na⁺-K⁺-ATPase. Because intracellular Na⁺ rises during ischemia, it is generally assumed that the activity of the Na⁺-K⁺-ATPase is reduced during ischemia (11). However, data suggest that the pump is active during the first few minutes of ischemia (1, 24). The reasons for the eventual inhibition of the Na⁺-K⁺-ATPase are not completely clear. Clearly, a fall in ATP will result in inhibition of the Na⁺-K⁺-ATPase; however, it is not clear whether the pump becomes inhibited before ATP levels decline to concentrations that would result in inhibition of the Na⁺ pump (24). Once the pump is active, it is reported that it remains active even with ATP levels of <0.2 mM and ADP levels as high as 2 mM (24). There are also data suggesting that the Na⁺ pump might be inhibited by posttranslational modifications that occur during ischemia (17). With the Na⁺ pump inhibited, Na⁺ will rise because of Na⁺ entry via Na⁺ influx pathways. Regarding the Na⁺ influx mechanism, the relative role of NHE vs. persistent (non-inactivating) Na⁺ channels has been debated (43, 70), and it is likely that both contribute. If Na⁺-K⁺-ATPase activity is reduced during ischemia but not completely eliminated, reducing Na⁺ influx through either NHE or non-inactivating sodium channels could be sufficient to prevent sodium accumulation. Studies have shown that addition of NHE inhibitors significantly attenuates the rise in Na⁺ during ischemia, suggesting a role for NHE in the rise in Na⁺ during ischemia (9, 13, 23, 45, 52). This mechanism has been questioned for several reasons. First, NHE inhibitors do not increase the fall in pH during ischemia as might be expected. However, there are other pathways that regulate pH, and, if one is inhibited, other pathways can regulate pH. Inhibition of NHE will alter the kinetics of pH regulation but not necessarily the intracellular pH set point. Also, reducing Ca²⁺ overload by reducing sodium influx can reduce ATP utilization and slow ischemic metabolism and thereby reduce the generation of protons. Second, it has been shown that NHE is inhibited by low extracellular pH that occurs during ischemia (67). However, as discussed above, the fall in extracellular pH does not occur immediately, so it is likely that inhibition of NHE does not occur immediately during ischemia. Furthermore, although the rate of NHE activity is reduced by low extracellular pH, some low level of activity can still occur (67), which may be sufficient to extrude the intracellular acid that is being generated slowly after the first minutes of ischemia. Also, the fall in pH can be accounted for without inhibition of proton extrusion mechanisms (56). Thirdly, the early NHE inhibitors were shown to also inhibit the persistent Na⁺ channels (70). It is clear that the marked inhibition of the rise in Na⁺ during ischemia that occurs with amiloride and other nonselective NHE inhibitors is due in part to inhibition of persistent Na⁺ channels. However, a role for Na⁺ channels in Na⁺ entry does not preclude a role for NHE. Indeed, recent studies with newer, more specific NHE inhibitors find that these more specific inhibitors also reduce the rise in Na⁺ during ischemia (9, 23, 65), although the attenuation of the rise in Na⁺ appears to be less than with nonspecific inhibitors such as amiloride. Further support for a role for NHE comes from studies using mice lacking NHE. NHE-null mice were shown to be resistant to ischemia/reperfusion injury compared with wild-type, with better preserved ATP during ischemia and a reduction in the degree of contracture during ischemia (68).

View larger version (69K): [in this window] [in a new window]   FIGURE 2. Illustration of ion channels and transporters involved in cardiac ion homeostasis as described in the text

The ²³Na-NMR and SBFI surface fluorescence studies both suggest a role for persistent Na⁺ channels in the rise in Na⁺ during ischemia, although they differ somewhat as to whether this is the sole Na⁺ entry mechanism. The preponderance of data obtained using inhibitors as well as NHE knockout mice suggest that NHE is at least partially responsible for the rise in Na⁺ during ischemia. Taken together, the data support a role for both NHE and persistent Na⁺ channels in the rise in Na⁺ during ischemia, and the relative contribution of each may depend on the metabolic activity of the cell. Although it is controversial whether there are restricted or fuzzy space Na⁺ gradients, this would also have implications for the interpretation of Na⁺ gradients (2).

Na⁺ during reperfusion
In contrast to the debate over the mechanism responsible for the rise in Na⁺ during ischemia, there appears to be agreement that NHE is primarily responsible for the rise in Na⁺ at the start of reperfusion. On reperfusion when extracellular pH is restored, the protons accumulated in the cytosol are extruded via NHE in exchange for Na⁺. There is some disagreement regarding whether the Na⁺ that enters on reperfusion results in a measurable increase in Na⁺ or whether the Na⁺ entering is rapidly extruded via the Na⁺-pump and reverse mode NCX resulting in only a slight and very transient spike in Na⁺. Most of the ²³Na-NMR studies find little or no measurable rise in Na⁺ during reperfusion, unless the Na⁺-K⁺-ATPase is inhibited (27, 66). These data suggest that, on reperfusion, the Na⁺-K⁺-ATPase is reactivated and can extrude the increased Na⁺ that enters via NHE. Therefore, on reperfusion, inhibitors of NHE appear to slightly delay the recovery of pH and slightly reduce the very transient rise in Na⁺. In contrast to the ²³Na-NMR studies, Williams et al. found a large rise in Na⁺ (to ~40 mM) on reperfusion (70), which remained at this high level for ~10 min. The reason for this difference is unclear. The larger and more persistent rise in Na⁺ on reperfusion, which was measured in hearts loaded with SBFI, suggest that either ATP recovery and/or Na⁺-K⁺-ATPase recovery is slower in this study in SBFI-loaded hearts or that the rise in Na⁺ is larger in this model and overwhelms the pump. A small amount of leakage of the indicator with slow washout should also be considered.

Ca²⁺ during ischemia
As shown in FIGURE 2, intracellular Ca²⁺ is normally maintained several orders of magnitude below extracellular Ca²⁺ by sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA), the sarcolemmal Ca²⁺ ATPase, and the sarcolemmal Na⁺-Ca²⁺ exchanger (NCX), which uses the energy of the Na⁺ gradient to extrude Ca²⁺ from the cell. As discussed, during ischemia, intracellular Na⁺ rises, and the inwardly directed Na⁺ gradient is reduced allowing Ca²⁺ to rise via NCX. The decline in ATP or posttranslational modification of the Ca²⁺ ATPase results in inhibition of the Ca²⁺ ATPase. However, for a rise in Ca²⁺ to occur, Ca²⁺ must enter via some mechanism. The mechanism responsible for the rise in Ca²⁺ during ischemia is debated (50). There are data showing that the rise in Ca²⁺ is linked to the rise in Na⁺. Blocking the rise in Na⁺ during ischemia has been shown to delay and attenuate the rise in Ca²⁺. However, it is not fully agreed whether the Na⁺-dependent rise in Ca²⁺ is because of Ca²⁺ entry via reverse mode of the NCX or whether Ca²⁺ enters via another mechanism, but Ca²⁺ rises because Ca²⁺ extrusion via NCX is inhibited because of the reduced Na⁺ gradient. This issue could have clinical implications because inhibitors of NCX have been suggested to reduce the rise in Ca²⁺ during ischemia, and these inhibitors would only be beneficial if Ca²⁺ rises because of Ca²⁺ entry via reverse-mode NCX. NCX exchanges 3 Na⁺ for 1 Ca²⁺ and is therefore electrogenic. NCX is generally close to equilibrium, and it depends on membrane potential and the Na⁺ and Ca²⁺ gradients, as indicated in Eq. 2

(Eq. 2)

Noble had questioned the level of Ca²⁺ measured during ischemia based on modeling of the equilibrium using assumptions about the Na⁺ gradient and the membrane potential (50). However, the discrepancy between the model and the data appear to be due to the value chosen for the extracellular Na⁺; in a global ischemia model, the extracellular Na⁺ falls to levels approaching 120 mM due to sodium influx and the relatively smaller extracellular volume compared with intracellular volume during ischemia in intact myocardium. If an extracellular [Na⁺] of 120 mM is assumed, then the mathematic model used by Noble shows that NCX is in equilibrium with a cytosolic Ca²⁺ of ~3 µM (50).

Because NCX is inhibited by low pH, it has been suggested that NCX will not be active during ischemia when pH falls to 6.0 (34). Although NCX activity will be reduced by the low pH, it may not be totally inhibited. Furthermore, the increase in [Ca²⁺]_i during ischemia is modulated by the level of Na⁺ consistent with NCX activity (45). Also consistent with the concept that Ca²⁺ entry via reverse mode of NCX has a role in Ca²⁺ entry during ischemia and enhances ischemic injury, studies have shown that mice lacking cardiac NCX (29) or inhibitors of NCX (25, 31, 35) reduce ischemic injury. Imahashi et al. (29) found that mice with cardiac-specific ablation of the plasma membrane NCX had a slower decline in ATP during ischemia, a slower onset of ischemic contracture, a reduced maximum contracture, and less of a rise in Na⁺ during ischemia. It is interesting that NCX-KO hearts had a reduced rise in Na⁺ during ischemia (29). If NCX runs in reverse mode during ischemia, it might be expected that inhibition of NCX would increase the rise in [Na⁺]_i during ischemia.

However, the reduced rise in Ca²⁺ during ischemia, which would occur due to inhibition of reverse-mode NCX, would result in better preservation of ATP, which in turn might reduce metabolic generation of protons and reduce Na⁺ entry via NHE. Taken together these data suggest that NCX is active during ischemia, although the level of activity might be reduced. Thus there appears to be reasonable agreement that NCX is a major mechanism responsible for the rise in Ca²⁺ during ischemia and that attenuation of NCX during ischemia would be beneficial. However, blocking the rise in Na⁺ during ischemia does not completely block the rise in Ca²⁺ (45), so there are also likely to be Na⁺-independent mechanisms for Ca²⁺ entry. Ca²⁺ entry via the L-type Ca²⁺ channel appears to play a role in the rise in Ca²⁺ during ischemia (8, 64). Inhibition of the L-type Ca²⁺ channel via S-nitrosylation has been shown to reduce ischemic injury (63, 64). A rise in cytosolic Ca²⁺ could also occur due to release of Ca²⁺ from intracellular organelles such as the SR or the mitochondria. Ca²⁺ uptake into the SR is mediated by the SERCA, which uses the energy from ATP hydrolysis to transport Ca²⁺ into the SR against a concentration gradient. Under normoxic conditions, Ca²⁺ in the SR is reported to be near 1 mM (7); thus the Ca²⁺ gradient across the SR is approximately four orders of magnitude, a value close to the thermodynamic potential based on the G for ATP. During ischemia, when ATP falls, it might be expected that Ca²⁺ would be released from the SR. However, measurements of SR Ca²⁺ during ischemia show no measurable change in SR Ca²⁺. The lack of decline in SR Ca²⁺ is consistent with the rise in cytosolic Ca²⁺; calculations show that, during ischemia even with the reduced G for ATP (calculations show it falls from –60 to –49 kJ/mol), there is still sufficient energy to maintain a SR Ca²⁺ of ~1 mM with a cytosolic Ca²⁺ of 3 µM (see Ref. 7 for details). Because SR Ca²⁺ does not change during ischemia, it does not appear that release of SR Ca²⁺ during ischemia is a significant source of the rise in cytosolic Ca²⁺.

The role of mitochondria in regulating cytosolic Ca²⁺ has long been debated (26). Currently there is controversy as to whether mitochondrial matrix Ca²⁺ follows cytosolic Ca²⁺ transients or whether the change in matrix Ca²⁺ reflects a more time-averaged change in cytosolic [Ca²⁺]. Several studies (3, 53) report that mitochondrial Ca²⁺ cycles on a beat-to-beat basis from ~0.2 to 0.9 µM. Others such as Miyata et al. (41) suggest that mitochondrial Ca²⁺ is in the range of 0.1–0.2 µM and increases to a higher steady state as cytosolic Ca²⁺ is increased by increasing beating frequency or by addition of isoproterenol. One key issue is whether a mitochondrial release mechanism exists with sufficient time resolution to extrude Ca²⁺ from the mitochondria on a beat-to-beat basis. The contribution of mitochondrial Ca²⁺ to changes in cytosolic Ca²⁺ during ischemia is even less clear. Ca²⁺ is taken up into the mitochondria by the Ca²⁺ uniporter, which uses the energy of the mitochondrial p (protomotive force) as a driving force. The mitochondrial p and thus the driving force for Ca²⁺ uptake into the mitochondrial falls during ischemia. Ca²⁺ can exit cardiac mitochondria via a NCX. The matrix Na⁺ level has been reported to be regulated by mitochondrial NHE and thus by the inwardly directed pH gradient across the inner mitochondrial membrane. In energized mitochondria, the Na⁺ gradient is reported to be outwardly directed, and Na⁺ is reported to be as much as eightfold lower in the matrix (14, 32).

There are very few studies measuring mitochondrial Ca²⁺ in a perfused heart during true ischemia. Miyamae et al. (39) measured mitochondrial Ca²⁺ in perfused hearts with Indo1 and surface fluorescence, using Mn²⁺ to quench cytosolic Ca²⁺. They found a rise in mitochondrial Ca²⁺ during ischemia and an inverse correlation between mitochondrial Ca²⁺ during ischemia and recovery of LVDP on reperfusion. A number of studies have measured mitochondrial Ca²⁺ during simulated ischemia, and most studies suggest that there is a small rise in mitochondrial Ca²⁺ (21, 40, 42, 48, 55). Interestingly, Griffiths et al. observed that the rise in mitochondrial Ca²⁺ during ischemia was inhibited by clonazepam (an inhibitor of mitochondrial NCX), thus suggesting a role for mitochondrial NCX operating in the reverse mode to increase mitochondrial matrix Ca²⁺ (21). It is suggested that during ischemia the mitochondrial Na⁺ gradient decreases as a result of the decrease in mitochondrial pH gradient, and this will reduce the inwardly directed Na⁺ gradient (21). The decrease in Na⁺ gradient along with the rise in cytosolic Ca²⁺ may contribute to Ca²⁺ entry into the mitochondria by mitochondrial NCX. Taken together, the data suggest that mitochondrial Ca²⁺ efflux does not contribute to the rise in cytosolic Ca²⁺ during ischemia. In fact, the data suggest the contrary, that the rise in cytosolic Ca²⁺ during ischemia contributes to the rise in mitochondrial Ca²⁺. In summary, Ca²⁺ entry via NCX and the L-type Ca²⁺ channel appear to be the primary mechanisms responsible for the rise in cytosolic Ca²⁺ during ischemia.

Ca²⁺ during reperfusion
During the first few minutes of reperfusion, before the Na⁺ gradient is restored to normal and during the time of increased Na⁺ entry by NHE (stimulated by the pH gradient that occurs with the return of the normal extracellular pH), a rise in cytosolic Ca²⁺ can occur due to reverse-mode NCX. Within a few minutes of reperfusion after 20–30 min of global ischemia, the Na⁺ gradient is restored (by the Na⁺-K⁺ ATPase and the return of the normal pH gradient which reduces intra-cellular Na⁺ loading) to its normal inwardly directed gradient allowing Ca²⁺ extrusion via NCX. ATP is also resynthesized allowing operation of Ca²⁺-ATPases. Ca²⁺ typically returns to preischemic levels within a few minutes of reperfusion after 20–30 min of global ischemia, resulting in more or less normal Ca²⁺ transients. On reperfusion, the mitochondrial p is also restored providing a large driving force for Ca²⁺ uptake into the mitochondria. If the Na⁺-K⁺-ATPase returns to normal function, it will extrude Na⁺, sparing the cell from a large Ca²⁺ overload. If a rise in Ca²⁺ on reperfusion is sustained, this can lead to mitochondrial Ca⁺² uptake, which dissipates p, thus reducing mitochondrial ATP generation (see Ref. 46). Accumulation of mitochondrial Ca²⁺ is also reported to activate a poorly defined mitochondrial permeability transition pore, which will totally dissipate the mitochondrial p and is suggested to result in immediate cell death.

	Consequence of Ionic Alterations

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 
The rise in cytosolic Ca²⁺ during ischemia may lead to activation of enzymes such as calpains that are involved in initiating apoptosis and necrosis. Data seem to suggest that the rise in Ca²⁺ at the start of reperfusion is a major factor in the development of irreversible injury. This rise in Ca²⁺ can lead to increased mitochondrial Ca²⁺ uptake, which will use the energy from electron transport for Ca²⁺ influx rather than to make ATP, and if mitochondrial Ca²⁺ increases above a threshold amount, it can activate the mitochondrial permeability transition pore that triggers cell death (46). It should be noted, however, that there are some data suggesting that a rise in Ca²⁺ on reperfusion is not an important trigger for the MPT (33). It should be noted also that there are several cell death pathways (necrosis, apoptosis, and autophagy), and they might be regulated differently by ions.

What relationship (if any) is there between altered ion gradients during ischemia and the rise in Ca²⁺ at the start of reperfusion? Another way of phrasing this question is: Is the rise in Ca²⁺ during ischemia a predictor of the rise in Ca²⁺ on reperfusion? Correlative data suggest that irreversible injury correlates with the rise in cytosolic and/or mitochondrial Ca²⁺ during ischemia (39, 60). However, this increase in Ca²⁺ may just indicate an increase in severity of injury in general, and it may not be the increase in [Ca²⁺] per se that is injurious. In support of a causal role for Ca²⁺, interventions that reduce the rise in Ca²⁺ during ischemia also reduce the amount of injury (60). Furthermore, in mice with cardiac-specific ablation of plasma membrane NCX or NHE, there was a reduced rate of fall in ATP during ischemia, suggesting a beneficial effect of reducing Ca²⁺ during ischemia (29, 68). It is also possible that the rise in Ca²⁺ during ischemia primes the mitochondria for opening of the MPT when pH is restored to normal on reperfusion, thus removing the inhibition of the MPT by low pH. The increase in cytosolic Ca²⁺ is also a factor in maintaining SR Ca²⁺ during ischemia, and this might contribute to SR Ca²⁺ oscillations on reperfusion.

The fall in pH during ischemia has a number of effects. The low pH inhibits contractility, which will help to conserve ATP. The low pH during ischemia also contributes to Na⁺ and Ca²⁺ loading of the cell and organelles. Furthermore, the low pH during ischemia inhibits the MPT. On reperfusion, intracellular pH is rapidly restored, allowing contractility to resume, but also allowing activation of MPT. Indeed, it has been suggested that postconditioning protects in part by slowing the recovery of intracellular pH (10).

In summary, during ischemia, ATP and PCr decline, whereas intracellular hydrogen ion concentration, Na⁺, Ca²⁺, and Mg²⁺ all rise. If the ischemia is relatively short and there is little irreversible injury (cell death), PCr, pH, Na⁺, Ca²⁺, and Mg²⁺ all recover quickly on reperfusion. ATP recovery can take up to 24 h because of loss of adenine base and the need for de novo synthesis. Interventions that reduce the rise in Ca²⁺ during ischemia and reperfusion have been shown to reduce ischemia-reperfusion-mediated cell death. Therefore, a better understanding of the mechanisms responsible for the rise in Ca²⁺ during ischemia and early reperfusion could have important therapeutic implications. A rise in Na⁺ occurs during ischemia due to NHE and persistent Na⁺ channel activity; this rise in Na⁺ leads to an increase in Ca²⁺ via NCX. Ca²⁺ entry via the L-type Ca²⁺ channel may also contribute to the rise in Ca²⁺ during ischemia. On reperfusion, there is additional Na⁺ entry via NHE, but if PCr recovers and ATP phosphorylation potential is restored, there is little or no rise in bulk Na⁺ because of extrusion via the Na⁺-K⁺-ATPase and NCX. However, Na⁺ extrusion via NCX results in increased Ca²⁺ entry into the cell, which can increase mitochondrial Ca²⁺ loading, activating the mitochondrial permeability pore, resulting in cell death. Maintaining low pH for a few minutes at the start of reperfusion appears to reduce injury, perhaps by inhibition of the MPT. The increase in Ca²⁺ can also lead to Ca²⁺ cycling across the SR, which impairs contractility and leads to futile cycling. The relative role of the rise in Ca²⁺ during ischemia vs. reperfusion has been debated. It appears the Ca²⁺ entry during ischemia and reperfusion both contribute to irreversible cell death, and reducing Ca²⁺ entry during both phases would be worthwhile therapeutic targets. The difficulties with administering cardioprotective agents (such as NHE inhibitors) before the onset of ischemia is likely to have been a major factor in the failure of these clinical trials. Interventions initiated at the start of reperfusion are more clinically relevant; however, it is important that the interventions be applied during the first few seconds of reperfusion. Thus strategies to reduce Ca²⁺ during ischemia and reperfusion are worth developing and testing.

	References

Top High-Energy Phosphates Intracellular pH Intracellular Na+ Cytosolic Ionized Ca2+ Cytosolic Ionized Mg2+ Mechanisms Responsible for Ionic... Consequence of Ionic Alterations References

 

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