INTRODUCTION

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REPERFUSION AFTER CARDIAC ISCHEMIA causes mechanical dysfunction because of cell injury. This has been demonstrated in isolated myocyte models with simulated ischemia and in intact isolated hearts and whole animal models. Depending upon the duration and/or magnitude of the ischemic insult, the cellular injury may be reversible (stunned) or irreversible (infarcted) (14). Various interrelated mechanisms have been implicated in reperfusion cellular injury. Among these are 1) cytosolic Na⁺ (17, 26, 28) and Ca²⁺ (1) overload resulting in myofibrillar hypercontracture, cytoskeletal damage, and cell disruption during reperfusion; 2) mitochondrial Ca²⁺ overload (3, 13, 24) causing inefficient ATP synthesis and utilization as a result of NADH accumulation, 3) reduced maximal Ca²⁺ activated force and/or sensitivity of myofilaments to Ca²⁺; and 4) impaired myocardial vascular perfusion.

Of these several mechanisms, cytosolic and/or mitochondrial Ca²⁺ overload, along with free radical damage, ultimately accounts for much of the cellular damage during reperfusion after ischemia. Free radicals damage sarcolemmal and intracellular membranes and impair ATP-dependent Na⁺ and Ca²⁺ reuptake mechanisms. Intracellular acidosis promotes Na⁺/H⁺ exchange, and this enhances reverse Na⁺/Ca²⁺ exchange (28, 37) during reperfusion (21), as suggested by Ca²⁺ loading on reperfusion (22). Myofilament Ca²⁺ sensitivity may also be reduced because of free radical damage and Ca²⁺ overload (14).

Time-dependent interrelationships of intracellular free Na⁺ and compartmental Ca²⁺ concentrations, reduction-oxidation potential, cardiac metabolism, contractility, and relaxation during ischemia and reperfusion have not been examined in the beating, intact heart. It is critically important to know whether Na⁺ overloading occurs simultaneously with cytosolic Ca²⁺ overloading, whether the mitochondria also accumulate Ca²⁺, and whether NADH/NAD⁺, primarily a measure of mitochondrial energy state, is restored on reperfusion. The hypothesis tested was that NADH increases incrementally during ischemia and that Na⁺ and compartmental Ca²⁺ rise together early during reperfusion and remain elevated during later reperfusion with continued myocardial dysfunction. To test this, we measured sequential changes in intracellular Na⁺, NADH, and cytosolic and mitochondrial Ca²⁺ with myocardial contractility, relaxation, metabolism, and coronary perfusion. The aim was to gain a more complete understanding of the dissociation among these variables at distinct time points during global ischemia and reperfusion. From this knowledge, treatments directed toward improving ionic homeostasis and function during reperfusion injury can be rationally designed and applied.

	METHODS

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Langendorff Isolated Heart Preparation and Measurements

Left ventricular (LV) pressure (LVP) was measured isovolumetrically with a transducer connected to a thin, saline-filled latex balloon inserted into the LV through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain a diastolic LVP (LVP_dia) of 0 mmHg during the initial control period, so that any increase in LVP_dia reflected an increase in LV wall stiffness or diastolic contracture. Pairs of bipolar electrodes were placed in the right atrial appendage and right ventricular free wall to monitor spontaneous heart rate and atrial-ventricular conduction time. Coronary flow (aortic inflow) was measured at constant temperature and constant perfusion pressure (55 mmHg) by a self-calibrating, in-line, ultrasonic flowmeter (Transonic T106X, Ithaca, NY) placed directly into the aortic inflow line.

Coronary inflow and coronary effluent Na⁺, K⁺, Ca²⁺, PO₂, PCO₂ , and pH were measured off-line with an intermittently self-calibrating analyzer system (Radiometer Copenhagen ABL 505, Copenhagen, Denmark). Coronary sinus effluent was collected by placing a small catheter into the right ventricle through the pulmonary artery after ligating both venae cavae. Coronary sinus venous PO₂ tension was also measured continuously on-line with an O₂ Clark-type electrode (model 203B, Instech, Plymouth Meeting, PA). The percent O₂ extraction was calculated as 100 × arterial PO₂  (venous PO₂/arterial PO₂); myocardial O₂ consumption (MO₂) was calculated as (coronary flow/g) × (arterial PO₂  venous PO₂) × 24 µl O₂/ml at 760 mmHg; and cardiac work efficiency was calculated as LVP systolic-diastolic (LVP_sys-dia) × heart rate/MO₂.

Measurement of Cytosolic and Noncytosolic Free Ca²+ in Intact Hearts

Calculation of dissociation constant. Eight hearts (1.5 ± 0.1 g) were perfused with standard KR perfusate (see Langendorff Isolated Heart Preparation and Measurements) for 20 min to wash out the blood. Hearts were removed from the perfusion apparatus, immersed in 15 ml of a mixture of 5 mM HEPES buffer (pH adjusted to 7.0) with 20 mM NaCl and 115 mM KCl, and homogenized with the use of a polytron blender. The soluble protein fraction was collected after centrifugation at 5,000 g for 20 min and ultracentrifugation at 100,000 g for 1 h at 25°C. With all liquid retained, final soluble heart protein concentration was 0.4 g/ml. Fluorescence (F; subscripted numbers indicate fluorescence wavelength) was measured with a modified luminescence spectrophotometer (SLM Aminco-Bowman II, Spectronic Instruments, Urbana, IL). Peak fluorescence activity of paired test homogenates, 75 µM free indo 1 and an indo 1 free blank, was measured in a quartz cuvette at 37°C for a ratio (R) of Ca²⁺ fluorescence (F) at 456 and 385 nm for minimal Ca²⁺ (R_min, 20 mM EGTA to chelate Ca²⁺) and from 0.7 to 160 µM (R_max).

Loading fluorescent probe indo 1 and recording Ca²+ transients. Experiments were carried out in a light-shielded Faraday cage. The heart was partially immobilized by hanging it from the aortic cannula, the pulmonary artery catheter, and the LV balloon catheter. The heart was immersed in a bath. The distal end of a trifurcated fiber silica fiber-optic cable (optical surface area 3.85 mm²) was placed against the LV epicardial surface through a hole in the bath, and a rubber O ring was placed between the ferrule and the heart to reduce cardiac motion at the contact point of the fiber-optic tip. Background autofluorescence was determined for each heart after initial perfusion and equilibration at 37°C. Thereafter, hearts were loaded with indo 1-acetoxymethyl ester (AM) at room temperature (25 ± 0.6°C) for 20-30 min with 165 ml of a recirculated, modified KR solution containing 6 µM indo 1-AM (Sigma Chemical, St. Louis, MO). Indo 1-AM was initially dissolved in 1 ml of dimethyl sulfoxide containing 16% (wt/vol) pluronic I-127 (Sigma Chemical) and diluted to 165 ml with the modified KR solution. Loading was stopped when the F₃₈₅ intensity was increased 10-fold. Residual indo 1-AM was washed out by perfusing the heart with standard perfusate for at least another 20 min, and each heart was then rewarmed to 37.5 ± 0.2°C before initiating the study. The perfusate contained probenecid (100 µM) to retard leakage of indo 1. Loading and washout of indo 1 in its vehicle reduced LVP from 96 ± 3 mmHg before loading to 72 ± 4 mmHg after washout. In the hearts given the vehicle alone to assess changes in tissue autofluorescence, LVP decreased from 92 ± 2 mmHg prevehicle to 70 ± 2 mmHg postvehicle; this showed that reduced contractility is mostly due to the vehicle rather than to intracellular Ca²⁺ buffering by indo 1 per se. Emission F₃₈₅ and F₄₅₆ declined over time after washout of extracellular indo 1-AM but remained at least fivefold greater than background for at least 3 h at 37°C; however, the F₃₈₅-to-F₄₅₆ ratio did not change over time.

Calculation of compartmental Ca²+ concentration from Ca²⁺ transients. Background fluorescence, but not indo 1 dye fluorescence, is influenced by the tissue oxygenation state at these two isobestic wavelengths (6, 7). Thus it was necessary to measure any change in tissue fluorescence over the course of the study, especially during ischemia and reperfusion, both with and without infusion of MnCl₂ to quench indo 1 in the cytosol. In eight hearts, only the indo 1 vehicle was washed in and out, after which autofluorescence was measured during the time course of ischemia and reperfusion; in eight additional hearts, this protocol was repeated in the absence of ischemia and reperfusion (cytosolic Ca²⁺ time control). In nine hearts, the vehicle was washed out just before continuous infusion of MnCl₂ and followed by ischemia and reperfusion; in six hearts, this protocol was repeated in the absence of ischemia and reperfusion (noncytosolic Ca²⁺ time control). In all experiments, the mean F₃₈₅ and F₄₅₆ background values were subtracted from the corresponding indo 1 F₃₈₅ and F₄₅₆ values at the same time point and for the same experimental condition.

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Measurement of Mitochondrial (Noncytosolic) Ca²+ in Intact Hearts

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Measurement of Intracellular NADH in Intact Hearts

Measurement of Intracellular Free Na+ in Intact Hearts

Loading of sodium benzofuran isophthalate fluorescent indicator. The Na⁺ binding fluorescent dye sodium benzofuran isophthalate (SBFI) was used to follow changes in myocyte Na⁺ concentration in the isolated heart (28, 38). The ratiometric technique for Na⁺ is similar to that for indo 1 except that the fluorescence ratio is obtained by alternating the excitation wave length from 340 to 380 nm and collecting and dividing the emitted light at 530 nm (10, 18, 20, 28, 38). The same trifurcated fiber-optic system was used as for indo 1, but Na⁺ and Ca²⁺ measurements were done in separate hearts because a change in filters and software was necessary. Each of the eight hearts were loaded for ~30 min with 6 µM SBFI at 25°C until the emitted signal after loading was between 6 to 10 times the baseline signal. SBFI fluorescence gradually declined over time but the F₃₄₀-to-F₃₈₀ ratio remained relatively stable. To assure that the fluorescence ratio was being calculated only from the component of heart fluorescence produced by SBFI, in 11 additional hearts only the SBFI vehicle was loaded and washed out before initiating the same ischemia-reperfusion protocol. Corrections were made for the average basal autofluorescence (AF), measured before SBFI loading, and for the change in autofluorescence that occurred during ischemia and reperfusion at each measurement point [time (t)]. The ratio (R) was calculated from the formula

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Calibration of SBFI in isolated hearts. Cardiac cell surface membranes were made permeable to Na⁺ by adding the Na⁺ ionophores gramicidin D and monensin to clamp [Na⁺] (and strophanthidin to poison Na⁺-K⁺-ATPase) and by removing divalent ions (primarily Ca²⁺ and Mg²⁺) from the perfusate solution (38). The pore-forming antibiotics allow extracellular Na⁺ to pass through the sarcolemmal L-type Ca²⁺ channels to equilibrate with the internal milieu. Under these conditions, intracellular [Na⁺] ([Na⁺]_i) approaches extracellular [Na⁺] ([Na⁺]_e); however, because SBFI is also sensitive to [K⁺], it was necessary to calibrate at a constant [Na⁺] and [K⁺]. Hence, calibration solutions were made that contained unequal proportions (0, 10, 70, and 140 mM) of NaCl or 140 mM KCl in a solution containing 10 mM HEPES, 1 mM EGTA 1, 10 mM glucose, 0.2 µg/ml gramicidin D, and 40 µM monensin; pH was then adjusted to 7.4 through either NaOH or KOH. In additional experiments, 5 and 70 mM [Na⁺]_e were also used. From the calibration curves of six hearts, the K_d for SBFI was calculated as 9.1 ± 0.9 mM at 37°C.

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Protocol

Data Presentation and Interpretation

Cytosolic-systolic Ca²+ and diastolic Ca²⁺. F₃₈₅, F₄₅₆, and F₃₈₅/F₄₅₆ Ca²⁺ transient signals, LVP, and the first derivative of LVP (LV dP/dt) were displayed simultaneously on a computer screen and stored digitally using proprietary software on an IBM OS/2 system. After correcting for tissue autofluorescence over time, with or without ischemia and reperfusion, and quenching of the cytosolic Ca²⁺ compartment to assess the mitochondrial Ca²⁺ compartment, we calibrated the signals to nanomolar [Ca²⁺] with the use of algorithms developed by our group. LVP and raw metabolic data were recorded (MacLab, AD Instruments, Castle Hills, Australia) and, together with the Ca²⁺ transient data, were later analyzed together (Excel Microsoft). Various characteristics of [Ca²⁺]_cyto were analyzed: peak systolic, peak diastolic, and phasic systolic-diastolic [Ca²⁺], i.e., released [Ca²⁺]; [Ca²⁺]_mito is not phasic. The characteristics of isovolumetric LVP analyzed were the following: LVP_sys, LVP_dia, LVP_sys-dia, the rise in LV dP/dt (LV dP/dt_max), and the fall in LV dP/dt (LV dP/dt_min).

Statistical Analysis

	RESULTS

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Figures 1A-5A show data obtained from the cytosolic Ca²⁺ group; Figs. 5B and 6B show data from the mitochondrial Ca²⁺ group; Fig. 7A shows data from the NADH group; and Fig. 7B shows data from the Na⁺ group. Mechanical and metabolic data obtained before ischemia and after reperfusion were similar among these groups; these intergroup comparisons were not conducted. Variables unchanged from before ischemia to 60-min reperfusion, respectively, for all groups were heart rate (252 ± 4 and 252 ± 3 beats/min), atrial-ventricular conduction time (76 ± 3 and 72 ± 2 ms), and percent O₂ extraction (75 ± 2 and 73 ± 3%).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   A: beat-to-beat changes in cytosolic [Ca2+] and left ventricular (LV) pressure (LVP) before global ischemia and after 2- and 60-min reperfusion in 1 heart. Note the peaked Ca2+ transient before the rise in LVP transient, the elevated systolic and diastolic [Ca2+] during early reperfusion, the depressed systolic LVP, and the elevated diastolic LVP during early reperfusion. B: individual coordinates of LVP and cytosolic [Ca2+] over 7 cardiac cycles before and after ischemia in 1 heart. Again, note that [Ca2+] peaks before LVP rises and that the coordinates are shifted rightward during reperfusion.

Figure 1A displays calibrated Ca²⁺ transients and LVP from one experiment before ischemia and during reperfusion. During early reperfusion, both [Ca²⁺]_sys and [Ca²⁺]_dia rose, whereas LVP_dia increased and LVP_sys decreased; during late reperfusion, [Ca²⁺]_sys and [Ca²⁺]_dia approached the preischemia controls, whereas LVP_dia remained elevated, and LVP_sys remained depressed. Figure 1B shows three 2.5-s recordings of 250 LVP/[Ca²⁺] coordinates at the same time periods shown in Fig. 1A. The relationship shifted downward and to the right during early reperfusion and leftward during late reperfusion, whereas (developed) LVP_sys-dia remained depressed.

Figure 2A shows [Ca²⁺]_sys plotted against LVP_sys and LV dP/dt_max during 60-min reperfusion after global ischemia. Compared with before ischemia, the relationship was shifted downward and far to the right at 1-min reperfusion and gradually shifted upward and to the left during later reperfusion. At 60-min reperfusion, the relationship remained shifted downward. Figure 2B shows [Ca²⁺]_dia plotted against LVP_dia and LV dP/dt_min during 60-min reperfusion after global ischemia. Compared with before ischemia, the relationship was shifted upward and far to the right at 1-min reperfusion and gradually shifted downward and to the left during later reperfusion. At 60-min reperfusion, the relationship remained shifted upward.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Relationship between systolic [Ca2+] and the rise in the first derivative of LVP (LV dP/dtmax) and between diastolic [Ca2+] and the fall in the first derivative of LVP (LV dP/dtmin) before ischemia and during 1- to 60-min reperfusion in 12 hearts. A: note that systolic [Ca2+] is highest at 1-min reperfusion but that systolic [Ca2+] returns toward control over 60 min (P > 0.1), whereas LV dP/dtmax (contractility) only slowly and incompletely increases during reperfusion. B: note that diastolic [Ca2+] is highest at 1-min reperfusion but that diastolic [Ca2+] returns toward control over 60 min, whereas LV dP/dtmin (relaxation) only slowly and incompletely decreases during reperfusion.

Figure 3A shows that [Ca²⁺]_sys fell from 564 ± 63 to 365 ± 22 nM at 30-min ischemia and increased twofold to 1,236 ± 114 nM at 1-min reperfusion; [Ca²⁺]_sys returned to control levels by 10-min reperfusion. At 60-min reperfusion, [Ca²⁺]_sys was 598 ± 75 nM, which was not different from the preischemia value. Both LVP_sys and LV dP/dt_max remained depressed throughout 60-min reperfusion. Figure 3B shows that [Ca²⁺]_dia progressively increased during global ischemia from 152 ± 14 nM before ischemia to 290 ± 16 nM at 30-min ischemia; [Ca²⁺]_dia peaked at 394 ± 44 nM at 1-min reperfusion and was 175 ± 18 nM at 60-min reperfusion, which was not different from the preischemia value. [Ca²⁺]_dia was associated with a marked increase in LVP_dia and a marked decrease in LV dP/dt_min throughout 60-min reperfusion.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   A: time course of systolic [Ca2+], LVP, and LV dP/dtmax before, during, and after global ischemia. B: time course of diastolic [Ca2+], LVP, and LV dP/dtmax before, during, and after global ischemia. During global ischemia, diastolic [Ca2+] rose, whereas the indexes of contractility and relaxation were absent, except for diastolic LVP, which increased during the last 5 min of ischemia. During initial reperfusion, systolic and diastolic [Ca2+] peaked almost twofold higher than before ischemia at 1 min but returned to control values by 60-min reperfusion. Indexes of contractility and relaxation did not return to preischemia values by 60-min reperfusion. Systolic LVP and LV dP/dtmax gradually increased during reperfusion but remained lower than control values after 60-min reperfusion.

Figure 4A displays LV dP/dt_max as a function of [Ca²⁺]_sys-dia during incremental increases in [Ca²⁺]_e from 0.39 ± 0.03 to 4.81 ± 0.21 mM. These changes were conducted 30 min before and 30 min after ischemia and were repeated in the time control group, which was not exposed to ischemia. The peak value for LV dP/dt_max was decreased by 56 ± 3% after ischemia; there was no significant change in this relationship in the time control group. Figure 4B displays the same data after all values for LV dP/dt_max were normalized to 100%. There were no significant differences in the slopes, indicating no apparent change in contractile sensitivity to Ca²⁺. In data not displayed, peak LV dP/dt_max and dP/dt_min as a function of maximal [Ca²⁺]_dia during the change in [Ca²⁺]_e was markedly reduced after ischemia, and there were significant rightward shifts in the 100% normalized LV dP/ dt_max and dP/dt_min slopes after ischemia of 30 ± 9 and 36 ± 7 nM [Ca²⁺], respectively, compared with before ischemia.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Contractility as a function of increasing systolic-diastolic [Ca2+] resulting from incremental increases in perfusate extracellular [Ca2+] ([Ca2+]e) before and after ischemia compared with a nonischemia, time control group. A: note the decrease in peak LV dP/dtmax at peak systolic-diastolic [Ca2+] in the ischemia-reperfusion group. B: when normalized to 100%, there was neither a shift in the curve nor a change in the slope after ischemia. See text for the diastolic [Ca2+] -LV dP/dtmin relationship.

Figure 5A shows that phasic [Ca²⁺] gradually fell from a preischemia value of 412 ± 53 to <25 ± 10 nM at 30-min ischemia. On reperfusion, phasic [Ca²⁺] increased twofold to 842 ± 110 nM at 1 min and then declined to within the preischemia value, 446 ± 66 nM, at 10-min reperfusion. As shown, MO₂, coronary flow, and cardiac efficiency remained depressed throughout reperfusion, but cardiac efficiency rose from 5 ± 1 to 14 ± 1 mmHg · beat · 0.1 µl · O₂¹ · g between 1- and 60-min reperfusion. For data not displayed, percent O₂ extraction was 81 ± 2, 63 ± 3, 61 ± 2, 62 ± 2, 61 ± 2, and 63 ± 3% at 10 min before ischemia and at 1-, 5-, 10-, 30-, and 60-min reperfusion, respectively. Figure 5B shows that noncytosolic, primarily [Ca²⁺]_mito increased during ischemia from a preischemia value of 234 ± 30 to 427 ± 74 nM at 30-min ischemia; on initial reperfusion, [Ca²⁺]_mito increased nearly 2.5-fold and remained significantly elevated at 60-min reperfusion. LVP_sys remained depressed, and LVP_dia remained elevated, throughout 60-min reperfusion.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   A: time course of phasic (systolic-diastolic) [Ca2+], myocardial O2 consumption (MO2), coronary flow, and cardiac efficiency (in mmHg · beat · 0.1 µl · O21 · g) before, during, and after global ischemia. During initial reperfusion, phasic [Ca2+] doubled, whereas the indexes of MO2, perfusion, and efficiency were depressed. Note that efficiency improved up to 60-min reperfusion but only phasic [Ca2+] returned to control levels. B: time course of mitochondrial [Ca2+] during ischemia and reperfusion in 9 hearts treated with MnCl2 after indo 1 loading and washout to quench cytosolic [Ca2+]. Note that mitochondrial [Ca2+] more than doubled during early reperfusion and remained elevated at 60-min reperfusion. The decrease in systolic LVP and the increase in diastolic LVP were similar to that of the cytosolic [Ca2+] group (1A-5A).

Figure 6A displays LV dP/dt_max as a function of [Ca²⁺]_mito during the induced 0.3 to 4.8 mM increase in [Ca²⁺]_e conducted 30 min before and 30 min after reperfusion. The peak value for LV dP/dt_max was decreased by 32 ± 4% after reperfusion. [Ca²⁺]_mito did not rise significantly to an increase in [Ca²⁺]_e alone before or after ischemia and reperfusion. Figure 6B displays the same data after LV dP/dt_max was normalized to 100%. There was a marked rightward shift of 125 ± 33 nM in the curve but no significant difference between the slopes, indicating similar contractile responses with elevated [Ca²⁺]_mito despite Ca²⁺ loading after reperfusion. Results were nearly identical for 100%-normalized LV dP/dt_min (data not displayed). In the time control group (n = 5), there was no shift in LV dP/dt_max and dP/dt_min as function of [Ca²⁺]_mito.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Contractility as a function of mitochondrial [Ca2+] resulting from incremental increases in perfusate [Ca2+]e before and after ischemia in 9 hearts. A: note the very small increase in mitochondrial [Ca2+] by [Ca2+]e before or after ischemia, the decrease in peak LV dP/dtmax, and the large shift to the right after 30-min reperfusion. B: when normalized to 100%, the right shift in the curve persisted after reperfusion.

Figure 7A shows that NADH (in arbitrary units) increased up to 2.5-fold during ischemia but that there was a tendency for NADH to decrease during the last 10 min of ischemia. On reperfusion, NADH immediately decreased to the preischemia value, indicating rapid removal of the excess protons. Figure 7B shows a small, but significant, increase in [Na⁺] from a control of 9.7 ± 0.6 to 12.2 ± 0.9 mM at 30-min ischemia. On reperfusion, [Na⁺] increased over twofold and peaked at 24.5 ± 2.3 mM; this was followed by a slow decline to 12.2 ± 1.1 mM at 60 min that was higher than the preischemia value.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   A: time course of NADH [in arbitrary fluorescence units (F, where the subscripted number indicates the wavelength in nm)], systolic LVP, and diastolic LVP before, during, and after global ischemia in 8 hearts. Note the nearly threefold increase in NADH during ischemia, which returned to near-baseline values immediately on reperfusion. B: time course of intracellular [Na+], systolic LVP, and diastolic LVP before, during, and after ischemia in 8 other hearts loaded with sodium benzofuran isophthalate. Note that [Na+] increased significantly during ischemia, peaked rapidly on initial reperfusion, and did not attain the preischemic control level by 60-min reperfusion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cardiac ischemia and reperfusion cause a multitude of derangements in cardiac function and metabolism. This is the first report in which the time course and interrelationship of a number of ionic and metabolic factors that underlie contractile, relaxant, and metabolic dysfunction were measured repetitively during global ischemia and early and late reperfusion in intact hearts. We found that NADH rapidly accumulated on initial ischemia and persisted during ischemia; this was accompanied by slow increases in myoplasmic [Ca²⁺]_dia and especially [Ca²⁺]_mito but little change in [Na⁺]_i. Phasic Ca²⁺ transients remained, but phasic contractility was absent during initial ischemia. On initial reperfusion, there was a more than doubling of [Ca²⁺]_dia, [Ca²⁺]_sys, [Ca²⁺]_mito, and [Na⁺]_i. Each of these variables peaked at 1- to 2-min initial reperfusion; however, NADH returned toward preischemia values within the first minute of reperfusion. Moreover, by 10-min reperfusion, [Ca²⁺]_dia, [Ca²⁺]_sys, and phasic [Ca²⁺], like NADH, had returned to the preischemia levels, but [Na⁺]_i and [Ca²⁺]_mito remained elevated for up to 60-min reperfusion.

These ionic changes were associated with a depression of developed LVP, LV dP/dt_max, LV dP/dt_min, coronary flow, MO₂, and cardiac efficiency for up to 60 min; much of this results from a reduction of viable cells (about a 45% LV infarct size with this protocol). The loop of LVP as a function of transient Ca²⁺ over the cardiac cycle at normal [Ca²⁺]_e was shifted downward and right at 2-min reperfusion; by 60-min reperfusion, this loop remained truncated, indicating depressed contractility and relaxation at a given [Ca²⁺]_cyto. As a function of external Ca²⁺-induced increases in phasic [Ca²⁺]_cyto, peak LV dP/dt_max at 30-min reperfusion was markedly reduced, but when peak LV dP/dt_max was normalized to 100% of the preischemia value, the curve was neither significantly shifted nor the slope altered. Thus, over the range of external Ca²⁺, functional myocardial sensitivity to cytosolic Ca²⁺ was not significantly altered after reperfusion. In contrast to [Ca²⁺]_cyto, [Ca²⁺]_mito remained persistently elevated during reperfusion but was insensitive to external Ca²⁺. However, LV dP/dt_max and dP/dt_min as a function of [Ca²⁺]_mito or [Ca²⁺]_dia was markedly right shifted after 30-min reperfusion. These experiments demonstrate interesting links between excess intracellular Na⁺, cytosolic Ca²⁺, mitochondrial Ca²⁺, and NADH. The data support the hypothesis that Na⁺ overload is an essential cause of Ca²⁺ overload and cardiac dysfunction during early reperfusion and suggest that the rapid return of NADH is related to the elevation in [Ca²⁺]_mito. A limitation of this study is that the contribution of oxygen-reactive species to reperfusion contractile dysfunction was not assessed. Moreover, the intracellular measurements apply only to cells surviving reperfusion because nonviable cells are permeable to large molecules.

There are many reports in a variety of models showing that Ca²⁺ is elevated on reperfusion, but there are no known studies in which beat-to-beat changes in diastolic, systolic, and mitochondrial Ca²⁺ were measured repetitively in intact hearts during the critical periods of ischemia-reperfusion injury. We found that cytosolic Ca²⁺ transients were not immediately abolished on ischemia so energy consumption during the anaerobic period might contribute to impaired relaxation and contractility on reperfusion. Moreover, cytosolic Ca²⁺ overload was reversed after 10-min reperfusion, whereas mitochondrial Ca²⁺ remained elevated over 60-min reperfusion. Dissociation between mitochondrial Ca²⁺ and contractility and relaxation were also evidenced by decreased maximal contractile effort as a function of maximum [Ca²⁺] and by the rightward shift in mitochondrial Ca²⁺ contractility and relaxation curves after reperfusion. Excess cytosolic Ca²⁺ inhibits breaking of the troponin C-Ca²⁺ complex so that relaxation, and consequently contraction, becomes impaired. Improved cardiac efficiency during reperfusion may reflect cellular respiration increasingly directed toward contractile function.

Activation of contraction by Ca²⁺ ultimately derives from increased availability of myosin-binding sites on actin. The number of sites relates to the experimental determination of maximal Ca²⁺-activated force (11, 23). The equivalent of this force, contractility, for a given [Ca²⁺], was depressed by more than 30% after ischemia in isolated hearts; a large part of this effect must be due to nonviable tissue no longer contributing to contractile function. Ca²⁺ also regulates actin-myosin interactions via changes in myofilament Ca²⁺ sensitivity, which can be induced theoretically by a change in the Ca²⁺ affinity of troponin C or by a change in cross-bridge kinetics. Inotropic mechanisms can be defined as "upstream" if they primarily affect the amplitude or time course of the Ca²⁺ transient, "central" if they change the Ca²⁺ binding to regulatory proteins such as troponin C, and "downstream" if they change the response of myofilaments, per se, to a given level of occupancy of troponin C by Ca²⁺ (4, 23, 41). Although central and downstream Ca²⁺ sensitivity in terms of phasic cytosolic Ca²⁺ was not affected after reperfusion, central Ca²⁺ sensitivity was affected in terms of diastolic or mitochondrial Ca²⁺, so this suggests diastolic and or mitochondrial Ca²⁺ loading does contribute in some way to the Ca²⁺-myofilament interaction.

Normal functions of myocyte mitochondria are the generation of ATP via the proton pump and Ca²⁺ homeostasis. Both processes are driven by the proton gradient (µH) generated by electron transport in the inner mitochondrial membrane. A small increase in [Ca²⁺]_mito during increased work demand activates pyruvate and other dehydrogenases to produce NADH from NAD⁺ (5, 8, 16, 40). Excess NADH inhibits these dehydrogenases, so entry of pyruvate into the tricarboxylic acid cycle is blocked and oxidative phosphorylation ceases (40). As proton pumps fail, matrix pH decreases as H⁺ is increased. During ischemia, cytosolic ATP is generated by glycolysis and may be used in reverse fashion by F₀F₁-ATPase to attempt to maintain µH (11). A very large acute increase in [Ca²⁺]_mito is thought to be a marker for irreversible cellular injury (33). Indeed, persistently elevated [Ca²⁺]_mito, rather than phasic [Ca²⁺]_cyto, may be associated with nonreversible myocyte injury (24). Reperfusion after prolonged ischemia may result in mitochondrial Ca²⁺ overload severe enough to decrease µH and the proton force for ATP synthesis (11) so that damage to mitochondrial membranes leads to cell death (1, 3).

ATP synthesis is dependent on the proton motive force generated by NADH, so mitochondrial NADH/NAD⁺ is a measure of mitochondrial energy state. Reduced NADH is generated from NAD⁺ in the cytosol and matrix from substrates via pyruvate and fatty acids (31). Energy stored by the electrochemical µH in the inner membrane drives ATP synthesis by the F₀F₁-ATP synthetic complex. The electron transfer from NADH (and FADH₂) to O₂ via cytochrome oxidase generates the motive force µH to drive H⁺ through the complex to power ATP synthesis (9). With adequate substrate and O₂, this system is in equilibrium to maintain µH at about 200 mV at a pH of ~8.2. Thus oxidation of NADH and phosphorylation are normally matched. During ischemia, as the supply of [1/2]O₂ to accept electrons from NADH diminishes, electron flux through the electron transport chain falters and NADH accumulates (12). Normalization of NADH early during reperfusion may reflect energy expenditure away from myofilament relaxation via actin-myosin-ATPase, resulting in systolic contractile dysfunction and toward noncontractile repair processes, such as restoration of transmembrane ion transport.

Excess mitochondrial Ca²⁺ can impair cellular respiration and reduce ATP synthesis (25, 29). ATP is required to break the actin-myosin bond, reaccumulate Ca²⁺ into the sarcoplasmic reticulum, extrude Ca²⁺ from the myocyte, and reestablish the sarcolemmal membrane potential, primarily by restoring Na⁺ equilibrium. But it is unknown if a moderate increase in mitochondrial Ca²⁺ directly contributes to contractile dysfunction. On the contrary, because NADH rapidly returned to normal in the present study, ATP levels may have also been restored. The elevation in mitochondrial Ca²⁺ might actually help to reestablish NADH during reperfusion. In this way, the very quickly renormalized mitochondrial electrochemical gradient (µH  NADH/NAD⁺) could restore synthesis of ATP, which is hydrolyzed by Ca²⁺-ATPase and Na⁺-K⁺-ATPase to reestablish ion pump activities, perhaps at the expense of actin-myosin-ATPase required for relaxation contraction cycling. It is assumed that only viable myocytes contribute to the measure of NADH autofluorescence, as suggested by the small decline in NADH during late ischemia.

A major cause of myocardial damage during reperfusion is likely excess Na⁺ accumulation during reperfusion leading to Ca²⁺ loading. During ischemia, metabolic acidosis is believed to cause excessive Na⁺ entry via Na⁺/H⁺ exchange, which in turn raises intracellular pH. The resting membrane potential becomes depolarized during ischemia as Na⁺-K⁺-ATPase pump activity decreases; this increase in [Na⁺] shifts the reversal potential for Na⁺/Ca²⁺ exchange toward more negative membrane potentials. This, in turn, promotes a greater Ca²⁺ influx by the exchanger to increase [Ca²⁺], i.e., by enhanced "reversed" mode operation (18, 37). Because repolarization is slowed during reperfusion, probably because the Na⁺-K⁺-ATPase pump is incapable of rapidly reversing the Na⁺ load (19), the increase in Na⁺ entry, via Na⁺/H⁺ exchange, increases [Ca²⁺] as a result of reduced Ca²⁺ efflux or increased Ca²⁺ influx via reversed Na⁺/Ca²⁺ exchange (30). Consistent with this idea, we observed a small, but significant, increase in [Na⁺] during ischemia but a more than doubling of [Na⁺] immediately on reperfusion. Na⁺ remained elevated along with mitochondrial Ca²⁺ during later reperfusion, so it is possible that cytosolic Ca²⁺ was removed at the expense of excess cytosolic Na⁺ to reduce excess mitochondrial Ca²⁺. The above mechanism is supported by a preliminary study in the same model. We found that a Na⁺/H⁺ exchange inhibitor, BIIB-513, given for 10 min before 30-min global ischemia, improved return of cardiac function (2); importantly, this drug normalized [Na⁺] during ischemia and at 60-min reperfusion and reduced peak [Na⁺] on initial reperfusion by 50%.

In summary, Na⁺ accumulation, during ischemia and particularly on reperfusion, likely accounts for the initial increase in cytosolic Ca²⁺ that leads to mitochondrial Ca²⁺ excess and cardiac dysfunction. Elevated mitochondrial Ca²⁺ may help to normalize NADH early on reperfusion while buffering excess cytosolic Ca²⁺ during later reperfusion. Therapeutic interventions that reduce Na⁺ and Ca²⁺ loading, e.g., Na⁺/H⁺ exchange inhibitors, should be quite beneficial to attenuate ischemia-reperfusion injury, particularly if effective immediately on reperfusion. Lastly, it is important to recognize that other factors, such as free radical formation, leukocyte migration, and complement activation, are also involved in reperfusion injury. The role of the endothelium and vasculature in mediating reperfusion injury must also be acknowledged.