INTRODUCTION

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DURING ISCHEMIA, lactate and P_i accumulate in the cytosol as a result of anaerobic glycolysis and the breakdown of ATP and phosphocreatine (PCr), thereby increasing the intracellular osmolarity of the tissue (1, 15, 34). As water enters the myocytes with the increase in osmotic gradient, the cells swell (3, 17, 34). Such water movement should be particularly evident at the start of reperfusion, when the return of flow exposes the heart to a much lower extracellular osmolarity (34). It has been postulated that cell swelling exacerbates tissue damage during ischemia (11, 14), alters the energetic state of the myocardium, and modulates ion transport (7, 25, 29). An understanding of the mechanisms by which cardiac myocytes regulate their volume is therefore critical to the development of ways to lessen the contribution of volume changes to ischemic injury.

Studies of isolated myocytes have shown the effects of anisosmotic solutions on various sarcolemmal ion transporters (20, 28). For example, exposure to hyposmotic buffers causes a decrease in activity of the Na⁺/Ca²⁺ exchanger (41), activation of the Cl (12, 13, 32, 33, 35, 42) nonselective cation (15) and K⁺ channels (27, 30, 36), and an increase in Na⁺-K⁺-ATPase activity (30, 40). Amino acids are extruded on cell swelling, which suggests that they act as osmolytes in volume regulation (5, 25, 26). Na⁺-K⁺-ATPase activity is inhibited by hyperosmotic solutions (40), whereas Na⁺-K⁺-2Cl cotransport (17, 18, 25), Na⁺/H⁺ exchange (17, 25, 29), Cl/HCO₃ exchange (17, 25, 29) and Na⁺/Ca²⁺ exchange (41) are activated. Swelling and shrinkage have been shown to alter the intracellular pH (pH_i) of trabecular tissue (38, 39), suggesting that the transporters involved in pH regulation are also affected by changes in cell volume.

However, most studies have determined the effects of swelling and shrinkage on isolated myocytes, rather than on the whole heart, and on ion transporters and channels, rather than as a cause of myocardial damage. Correlations with cardiac performance are often inferred (39), but little experimental evidence directly links cardiac function with the changes in cell volume.

To determine how swelling and shrinkage may injure the heart, we have perfused isolated guinea pig hearts with hyposmotic and hyperosmotic buffers under normal and ischemic conditions. We determined the effects on pH_i and phosphorus metabolite levels using ³¹P NMR spectroscopy while measuring cardiac contractile function.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Heart Perfusions

Hearts were placed into a 20-mm-diameter NMR tube. Mannitol buffer (10 mM sodium HEPES and 265 mM mannitol, pH 7.4) was pumped past the heart at a rate of 30 ml/min to minimize NMR signals originating from coronary effluent in the bath surrounding the heart. The osmolarity of the mannitol buffer was matched to that of the perfusion buffer and changed simultaneously with the change in perfusion buffer. Coronary effluent and mannitol buffer were removed through an overflow outlet above the heart. Coronary flow was calculated from the effluent flow rate minus that of the mannitol. A constant temperature of 37°C at the heart was maintained using water-jacketed perfusion buffer reservoirs and lines and a variable-temperature unit attached to the NMR spectrometer.

Changes in tissue phosphorus metabolite concentrations and pH were measured from ³¹P NMR spectra obtained using a Varian Inova Unity spectrometer attached to a 9.4-T, vertical-bore, superconducting magnet, producing a phosphorus resonance frequency of 161.92 MHz. Each spectrum consisted of 112 summed transients of 60° pulses with an interpulse delay of 1.97 s, giving a total acquisition time of 4 min. Peak resolution was enhanced by shimming the water proton signal to a line width of ~30 Hz to reduce magnetic field inhomogeneities. Before Fourier transformation, the signal-to-noise ratio of the ³¹P NMR signal was increased by multiplying the free induction decays by an exponential function that generated a line broadening of 20 Hz.

To determine absolute tissue concentrations of intracellular metabolites, an external standard containing 20 µl of 500 mM methylphosphonic acid solution was sealed in polythene tubing and placed adjacent to the heart during acquisition of the ³¹P NMR spectra. Each spectral peak was fitted to a Lorentzian line shape using the NMR1 line-fitting software program (Tripos, St. Louis, MO), and the amount of each metabolite was expressed relative to the standard. The pH_i was determined from the difference in chemical shift between the P_i and PCr peaks by using the previously established titration curve (9)

Experimental Protocols

Standard protocol. Once in the spectrometer the hearts were perfused with Krebs-Henseleit buffer for 20 min, then they were perfused for 40 min with isosmotic (300 mosM), hyposmotic (220 mosM), or hyperosmotic (380 mosM) low-Na⁺ Krebs-Henseleit buffer. The NaCl concentration was lowered by 40 mM in the buffers to maintain a constant buffer Na⁺ concentration during osmotic shock, with 80 or 160 mM mannitol added to increase the osmolarity. After an osmotic challenge the hearts were perfused for 20 min with Krebs-Henseleit buffer to allow recovery. Cardiac function was monitored continuously throughout each 80-min experiment. ³¹P NMR spectra were recorded continuously from 12 min into the equilibration period until the end of the experiment.

Na⁺/H⁺ exchange. To determine the contribution of Na⁺/H⁺ exchange to the alkalosis caused by the hyperosmotic buffer, hearts were perfused in the presence or absence of the Na⁺/H⁺ exchange inhibitor HOE-642 (31). HOE-642 was introduced into the perfusion buffer via an outlet just above the cannulated aorta at a rate calculated to give a concentration of 1 µM at the heart. Addition of HOE-642 was begun 2 min before the hyperosmotic period of perfusion.

Bicarbonate transport. To investigate the contribution of bicarbonate-dependent ion transport to the alkalosis caused by hyperosmotic buffer, hearts were perfused with isosmotic or hyperosmotic HEPES buffers. The HEPES buffers were the same as the Krebs-Henseleit buffers, except 25 mM NaHCO₃ was replaced by 20 mM HEPES (Na⁺ salt), pH 7.4, with or without 80 mM mannitol. Total Na⁺ concentration was made up to 145 mM with NaCl, and the buffers were gassed with 100% O₂ at 37°C.

Lactate-H⁺ cotransport. To determine whether the pH changes during osmotic challenge were via lactate-H⁺ cotransport, the lactate content in the coronary effluent from another set of hearts, perfused with hyposmotic or hyperosmotic buffer, was determined using a spectrophotometric lactate assay (6).

Tissue water content. Intra- and extracellular water content were determined by perfusing hearts with hyposmotic or hyperosmotic buffer containing 0.1 µCi/ml ³H₂O and 0.005 µCi/ml [¹⁴C]inulin for 5 min before freeze clamping (19). Heart tissue was extracted in 5.6% perchloric acid solution, and total ³H₂O and extracellular water ([¹⁴C]inulin) space were estimated from the ³H and ¹⁴C counts in 0.5 ml of neutralized perchloric acid extract and 0.2 ml of perfusate. ³H and ¹⁴C were counted simultaneously using a scintillation counter. Tissue water spaces were determined as follows The intracellular water space was taken as the difference between the ³H₂O space and the [¹⁴C]inulin space. The total water content and wet-to-dry weight ratios were also determined by drying 100-mg tissue samples for 48 h at 80°C.

Ischemia protocol. After 8 min of equilibration with standard Krebs-Henseleit buffer, hearts (n = 18) were perfused for 8 min with standard Krebs-Henseleit or isosmotic, hyposmotic, or hyperosmotic low-Na⁺ buffers before being subjected to 20 min of total global ischemia by clamping the buffer line to the aorta. Flow of the appropriate mannitol buffer was stopped during the ischemic period. After 20 min the hearts were reperfused with the appropriate preischemic buffer. ³¹P NMR spectra were acquired throughout the protocol.

Expression of Results

	RESULTS

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Isosmotic Low-Na⁺ Buffer

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Developed pressure and intracellular pH changes (pHi, n = 4-8/group) in isolated guinea pig hearts perfused with hyperosmotic (380 mosM), isosmotic (300 mosM), or hyposmotic (220 mosM) low-Na+ Krebs-Henseleit (K-H) buffers. * P < 0.05 compared with isosmotic perfusion.

Hyposmotic Low-Na⁺ Buffer

Hyperosmotic Low-Na⁺ Buffer

The effects of the buffers (Fig. 1) were due to a combination of the decreased Na⁺ concentration and the different osmolarities. To emphasize the effects due to the change in buffer osmolarity alone, Fig. 2 shows the changes in developed pressure and pH_i plotted as the difference between the isosmotic buffer and the hyposmotic and hyperosmotic buffers. Decreasing the osmolarity of the perfusion buffer from 300 to 220 mosM caused an average decrease in developed pressure of 46% accompanied by an acidosis of 0.04 unit. When the buffer osmolarity was increased to 380 mosM the developed pressure increased on average by 19% with an intracellular alkalosis of 0.14 unit. Similar increases in developed pressure and pH_i, although of smaller magnitude, have been observed during perfusion of hearts with standard Krebs-Henseleit buffer (140 mM Na⁺) made hyperosmolar with the addition of 80 mM mannitol (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Developed pressure and pHi changes in heart due to osmotic challenge alone plotted as difference between isosmotic buffer and hyperosmotic or hyposmotic buffer. Raw data are shown in Fig. 1.

Tissue Water Content

Lactate-H⁺ Cotransport

Na⁺/H⁺ Exchange

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Changes in myocardial developed pressure and pHi during perfusion with isosmotic (300 mosM), hyposmotic (220 mosM), and hyperosmotic (380 mosM) low-Na+ buffers (A) (n = 4-8/group), perfusion with isosmotic or hyperosmotic (Hyper) buffer ± 1 µM HOE-642 (B) (n = 4), or perfusion with K-H buffer, bicarbonate-free HEPES buffer, and hyperosmotic HEPES buffer (C) (n = 4). * P < 0.05 compared with isosmotic;  P < 0.05 compared with K-H buffer.

Bicarbonate-Free Perfusions

Ischemia

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Changes in developed pressure and end-diastolic pressure in isolated guinea pig hearts perfused with K-H buffer or isosmotic (Iso), hyposmotic (Hypo), or hyperosmotic (Hyper) low-Na+ buffers and subjected to 20 min of total global ischemia and 20 min of reperfusion (n = 3-6/group).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Phosphocreatine, ATP, and pHi during preischemic osmotic shock and 20 min of total global ischemia and reperfusion with K-H buffer or isosmotic, hyposmotic, or hyperosmotic low-Na+ buffers (n = 3-6/group).

	DISCUSSION

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We have shown in the perfused heart that buffer osmolarity and extracellular Na⁺ concentrations modulated myocardial developed pressure and pH_i but did not alter heart rate or energetics. Lowering buffer Na⁺ concentration alone caused a transient increase in developed pressure. The alkalinization and the developed pressure increase observed during perfusion with hyperosmotic buffer occurred in the presence and absence of Na⁺/H⁺ exchange inhibition and without extrusion of lactate, suggesting that Na⁺/H⁺ exchange and the lactate-H⁺ symport were not involved in proton extrusion. Bicarbonate-free buffers caused an increase in developed pressure and pH_i. Increasing the osmolarity of the buffer to 380 mosM did not further increase developed pressure or pH. This suggests that alkalosis may have been via bicarbonate-dependent transport mechanisms.

There were no significant differences in the recovery of cardiac function or energetics between hearts perfused with Krebs-Henseleit buffer and those perfused with isosmotic low-Na⁺ buffer during ischemia or reperfusion. Altering the buffer osmolarity by ±80 mosM did not significantly alter the myocardial changes during ischemia, apart from the rate of ATP depletion in hearts perfused with hyperosmotic buffer. During reperfusion, hearts perfused with hyposmotic buffer had faster recoveries and increased contractile function, accompanied by greater recoveries of PCr and ATP. Hearts perfused with hyperosmotic buffer did not recover from 20 min of total global ischemia and had decreased recoveries of PCr and ATP.

Our hypothesis is that changes in myocardial cell volume, caused by hyposmotic and hyperosmotic buffers, alter the intracellular concentrations of all ions. The effect on HCO₃, H⁺, and Ca²⁺ concentrations perturb cardiac function, in addition or in opposition to the positive inotropic effect caused by the low buffer Na⁺ concentration. The low extracellular Na⁺ concentration decreased the transsarcolemmal Na⁺ electrochemical gradient and, thereby, Na⁺/Ca²⁺ exchange, causing increased intracellular Ca²⁺ concentrations and developed pressure (1). Over the 40 min of exposure to the isosmotic buffer, the hearts equilibrated to the new Na⁺ concentration gradient, and contraction returned toward control levels.

Intracellular ionic strength may be altered by changing buffer ionic strength or by changing buffer osmolarity, leading to cell swelling or shrinkage. Cardiac contraction is influenced by intracellular ionic strength, such that an increase in buffer ionic strength slows Ca²⁺-activated tension development and decreases tension in a variety of muscle types (2, 22, 23, 37). However, we found that hyperosmotic buffer increased developed pressure in isolated hearts, the opposite effect to that expected due to increased intracellular ionic strength after cell shrinkage, indicating an overriding effect of osmolarity on contractile function.

Hyperosmolar buffer caused the cardiac myocytes to shrink, which may have increased the intracellular concentrations of Ca²⁺ and HCO₃. Therefore, there was an additional direct positive inotropic effect due to the intracellular Ca²⁺ concentration increase and an indirect effect, whereby the H⁺-buffering capacity of the cells was increased, leading to alkalinization and decreased competition with Ca²⁺ for troponin C-binding sites (10, 21, 24). Osmolarity was the only variable between the isosmotic and hyperosmotic buffers; thus the increased osmolarity must have increased pH_i (Fig. 2). Interventions that modify pH_i of cardiac tissue usually exhibit pH regulation within ~20 min (1, 38), but notably during our experiments the change in pH_i persisted throughout the 40 min of osmotic shock (Fig. 1).

Conversely, hyposmotic buffer caused cell swelling, decreasing intracellular Ca²⁺ concentration and pH, both of which invoked negative inotropy exceeding the positive inotropic effect due to the lowered Na⁺ concentration of the buffer and lowered the developed pressure. This hyposmolar pH effect has been observed in single cells (10) and isolated muscle fibers (24), but not in trabecular tissue (38).

The changes in pH and contractile function were rapid after introduction of hyperosmotic or hyposmotic buffers. Diffusion of water may have been the only process that occurred fast enough to alter the intracellular concentration of HCO₃ and, therefore, H⁺. This was supported by the lack of response during bicarbonate-free perfusions (Fig. 3), which not only modulated the activity of the bicarbonate-dependent transporter HCO₃/Cl exchange and HCO₃-Na⁺ cotransport (16) but altered the intracellular buffering capacity. The lack of effect of HOE-642 (Fig. 3) indicated that Na⁺/H⁺ exchange did not mediate the changes in pH and developed pressure during hyperosmotic perfusions. In trabecular tissue, hyposmolar acidosis took >5 min to reach a maximum that, the authors suggested, was mediated by Na⁺/H⁺ exchange (38).

From our observations that cytosolic energetics were unaffected by changes in osmolarity and the pH and functional changes were reversible, we conclude that osmotic shocks of ±80 mosM were not, per se, deleterious to the perfused heart. In contrast, buffer osmolarity had a profound effect on cardiac recovery from an ischemic episode. If ischemic cell swelling intensifies reperfusion injury, then it would be expected that swelling before ischemia should hinder recovery even further, and shrinking should increase recovery on reperfusion. However, our experimental results show the opposite, implying that cell size does not necessarily modulate ischemic damage but that intracellular osmolarity or ionic strength causes the beneficial or detrimental effects.

The damaging effect of perfusion with hyperosmotic buffer may have occurred by two distinct mechanisms. First, hyperosmolar buffer may have increased the intracellular osmolarity of the tissue and added to the osmolar load during ischemia. This would have resulted in increased swelling during ischemia and reperfusion and, therefore, increased damage. Second, perfusion with hyperosmolar buffer may have increased the intracellular concentration of Na⁺, either due to cell shrinkage or via the accumulation of ions during a regulatory volume increase. The increased Na⁺ concentration would increase Na⁺-K⁺-ATPase activity and ATP consumption. Normally, perfused hearts would be able to increase ATP production to match the increased Na⁺-K⁺-ATPase activity, but during total global ischemia, when ATP production is solely glycolytic, the increased demand for ATP would lead to faster ATP depletion. This hypothesis is supported by the faster initial ATP loss during ischemia in hearts exposed to hyperosmotic buffer.

In summary, we have shown that changing the osmolarity of the perfusion buffer between 220 and 380 mosM altered cardiac pH_i and developed pressure, but such osmolar changes were not, per se, deleterious to the perfused heart. The increase in pH_i on perfusion with hyperosmolar buffer was not due to increased Na⁺/H⁺ exchange or lactate-H⁺ symport activity but may have been via bicarbonate-dependent transport mechanisms. During 20 min of total global ischemia and reperfusion, hearts perfused with hyposmotic buffer had faster and greater recovery of contractile function, accompanied by increased recoveries of PCr and ATP. Hearts perfused with hyperosmotic buffer had faster ATP loss during ischemia and did not recover. Thus buffer osmolarity significantly alters myocardial ischemic injury.