Time course of myocardial sodium accumulation after burn trauma: a 31P- and 23Na-NMR study
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《应用生理学杂志》
Departments of 1 Surgery and 2 Radiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9160
ABSTRACT
In this study, 23Na- and 31P- nuclear magnetic resonance (NMR) spectra were examined in perfused rat hearts harvested 1, 2, 4, and 24 h after 40% total body surface area burn trauma and lactated Ringer resuscitation, 4 ml · kg1 · %1 burn. 23Na-NMR spectroscopy monitored myocardial intracellular Na+ using the paramagnetic shift reagent thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid). Left ventricular function, cardiac high-energy phosphates (ATP/PCr), and myocyte intracellular pH were studied by using 31P NMR spectroscopy to examine the hypothesis that burn-mediated acidification of cardiomyocytes contributes to subsequent Na+ accumulation by this cell population. Intracellular Na+ accumulation was confirmed by sodium-binding benzofuran isophthalate loading and fluorescence spectroscopy in cardiomyocytes isolated 1, 2, 4, 8, 12, 18, and 24 h postburn. This myocyte Na+ accumulation as early as 2 h postburn occurred despite no changes in cardiac ATP/PCr and intracellular pH. Left ventricular function progressively decreased after burn trauma. Cardiomyocyte Na+ accumulation paralleled cardiac contractile dysfunction, suggesting that myocardial Na+ overload contributes, in part, to the progressive postburn decrease in ventricular performance.
keywords:nuclear magnetic resonance spectroscopy; cardiomyocyte loading of the fluorescent indicator sodium-binding benzofuran isophthalate; ventricular contraction-relaxation assessed by Langendorff perfusion; myocardial acidosis (intracellular pH); myocardial high-energy phosphate stores
INTRODUCTION
MAJOR TRAUMA SUCH AS THERMAL INJURY produces a rise in intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i) concentrations, and this cation dyshomeostasis may contribute to the myocardial cell injury and contractile dysfunction that have been described after burn injury (13, 16, 21, 22, 24, 30, 32). The mechanisms by which a cutaneous burn injury alters myocardial Na+ homeostasis remains unclear. However, studies using models of ischemia and reperfusion have shown that myocyte acidification early in the ischemic period is a precipitating event in both myocardial Na+ and Ca2+ accumulation and cardiac contractile dysfunction (6, 7, 10, 25, 28). The negative inotropic effects of intracellular acidosis may be related to competition of hydrogen ions with Ca2+ for binding sites on the contractile proteins, impairing actin-myosin interaction and reducing tension development (26). However, intracellular acidosis may also alter left ventricular function by mediating Na+/H+ exchange, and the resulting accumulation of Na+ within the myocardium may increase the [Ca2+]i via Na+/Ca2+ countertransport (9, 11, 29). Alternatively, a rise in myocyte [Na+]i may modulate the effects of the sympathetic nervous system on the heart, contributing to contractile dysfunction (8).
Few studies of traumatic injury have examined the direct effects on myocardial acidification an intracellular-extracellular Na+ dyshomeostasis or cellular function. Studies from our laboratory examined myocardial Na+ late in the postburn period (24 h postburn), but those previous studies ignored the potential of myocardial acidosis in the early postburn period (1-12 h). Furthermore, it is unclear whether Na+ accumulation within cardiac myocytes promotes cellular injury and dysfunction or whether the myocyte Na+ accumulation described late in the postburn occurred secondary to burn-induced cardiac contraction and relaxation defects. The availability of 23Na-nuclear magnetic resonance (NMR) spectroscopy as well as 23Na-NMR shift reagents allow frequency resolution of intra- and extracellular Na+ resonances in the isolated perfused heart. Resolution of [Na+]i is compatible with acquisition of the 31P spectra, allowing simultaneous measurements of [Na+]i, intracellular pH (pHi), high-energy phosphate stores, and cardiac mechanical performance.
In this present study, subsets of rats were killed at several times after burn trauma, and hearts were perfused to determine 1) the time course of myocardial Na+ accumulation after burn trauma, 2) the contribution of intracellular acidosis to myocardial Na+ accumulation, and 3) whether burn-mediated myocardial Na+ accumulation occurred before or as a consequence of myocardial contractile abnormalities. These studies were designed to examine the hypothesis that burn-related acidification of cardiomyocytes contributes to Na+ accumulation by this cell population; Na+ dyshomeostasis in turn contributes to myocardial contractile abnormalities. In addition, we compared ventricular Na+ concentrations measured by 23Na-NMR spectroscopy in perfused hearts with [Na+]i measured by fluorescent dye sodium-binding benzofuran isophthalate (SBFI) loading of isolated ventricular myocytes. Our data showed that burn trauma promoted a rise in myocardial [Na+]i as early as 2 h after burn injury and achieved maximal myocardial Na+ levels 4 h postburn. Thus burn-mediated myocardial Na+ accumulation preceded the development of contractile defects. In our study, the burn-mediated increase in [Na+]i was not related to intracellular acidification as indicated by a stable pHi at all times after burn trauma. Our data suggest that postburn myocardial Na+ accumulation is not related solely to Na+/H+ exchange mechanisms, but it is likely that the influx of Na+ ions occurs via other pathways such as Na+-K+-ATPase or Na+/Ca2+ exchange systems.
MATERIALS AND METHODS
Experimental animals and burn procedure. The experimental protocol was reviewed and approved by the Institutional Animal Care and Research Advisory Committee of The University of Texas Southwestern Medical Center at Dallas. Adult male Sprague-Dawley rats, weighing 280-320 g each, were randomly divided into burn and sham-burn groups. Burn injury was produced using a template model as previously described (1, 15). Briefly, rats were deeply anesthetized with methoxyflurane, shaved, and secured in a template device. A full-thickness scald burn over 40% of the total body surface area was produced by immersing the skin on the back and sides exposed through the template in 100°C water for 12 s. The animals were quickly dried after immersion to avoid additional injury. The rats evidenced no postburn pain as indicated by their eating, drinking, moving freely about the cage, and responding to external stimuli; in addition, the animals evidenced no discomfort with handling by the investigators. Despite the presence of third-degree burns and destruction of underlying neural tissue, all rats received analgesics (Buprenex, 0.05 mg/kg) every 8 h postburn. Fluid resuscitation was initiated after trauma (lactated Ringer solution, 4 ml · kg1 · %1 burn) with one-half of the calculated volume given intraperitoneally immediately after the burn procedure and before recovery from anesthesia. After recovery from inhalation anesthesia, the animals were placed in separate cages and given standard rat chow and water ad libitum until death. The remaining volume of lactated Ringer solution was given 8 h postburn intraperitoneally. Sham-burn rats were anesthetized and handled in an identical manner except they were exposed to room-temperature water.
Isolated coronary perfused hearts. At the designated time after burn or sham burn (1, 2, 4, and 24 h), rats were anesthetized with methoxyflurane, and the heart was excised through a midline thoracotomy and placed in ice-cold Krebs-Henseleit buffer. A cannula placed in the aorta was connected to a reservoir located 95 cm above the heart for perfusion of the coronary arteries by the Langendorff method with a modified phosphate-free Krebs-Henseleit bicarbonate buffer (in mM: 118 NaCl, 25 NaHCO3, 6 KCl, 1.2 MgSO4, 1.25 CaCl2, and 10 glucose; pH 7.4, temperature 37°C and bubbled continuously with 95% O2-5% CO2). The all-glass water-jacketed perfusate reservoir, which also serves as the oxygenation chamber, was located in the bore of the magnet. Coronary effluent was removed from the magnetic resonance spectroscopy (MRS) tube, filtered through 5-μm filters and recirculated. An intraventricular balloon attached to a catheter (0.8 mm OD) was placed in the left ventricle (via left atrium) for monitoring spontaneous heart rate, left ventricular pressure, and coronary flow rate. The balloon cannula was connected to a Gould P23 pressure transducer, and functional parameters were measured by using a physiological monitor (Coulbourn, Lehigh Valley, PA) and recorded on a chart recorder (WR 3101, Western Graphtec, Irvine, CA). The heart was suspended in a 20-mm-OD MRS tube with a microtube containing 50 μl of 1.3 M NaCl and 3.3 mM Dy(DOTP)5 (as a Na+ external standard).
Shift reagent. Thulium 1,4,7,10-tetraazacylododecane-1,4,7,10-tetra(methylenephosphonic acid) (Na+ salt; TmDOTP4), characterized by favorable paramagnetic and Na+ binding properties, has been used to separate intra- and extracellular 23Na signals both in isolated organ studies and in vivo (5, 31, 32). Shift reagents such as TmDOTP4 are membrane impermeable and interact with extracellular Na+, causing its magnetic resonance signal to be shifted away from the intracellular signal. Compared with other shift reagents (such as DyTTHA3), TmDOTP4 has two advantages: a larger 23Na chemical shift per unit concentration and narrower line widths due, in part, to a lower bulk magnetic susceptibility. Because TmDOTP4 alters Ca2+ availability, the concentration of Ca2+ was adjusted to 3.9 mM so that the free Ca2+ in the perfusate was maintained at 1.0 mM as measured with Ca2+-sensitive electrode.
NMR spectroscopy. Hearts were allowed to equilibrate for 15 min before MRS spectra as well as functional parameters were obtained. The perfusate was switched to an identical solution containing TmDOTP4 (3.5 mM) for 31P- and 23Na- MR spectroscopy. The spectroscopy was performed on Varian INOVA 300 spectrometer equipped with an Oxford 4.2-T vertical-bore superconducting magnet. A Bruker 20-mm broadband probe was used, tunable to either 23Na or 31P; the magnetic field homogeneity was adjusted by using the 23Na-free induction decay. A 23Na line width of 9-10 Hz was typical, and 23Na spectra were acquired by using a ±6,000-Hz spectral width, 2,000 data points, a 65-μs excitation pulse, 72-1,280 acquisitions, and a 0.40-s interpulse delay over a period of 1 min. 31P spectra were acquired over a ±6,000-Hz spectral width by using 2,000 data points with a 38-μs excitation pulse, 56-520 acquisitions, and a 1.1- or 2.0-s interpulse delay.
MRS data analysis. The spectra were zero-filled to 4,096 points before Fourier transformation. The resonance areas in both 23Na- and 31P-NMR spectra were determined using the NMR utility transform software (two-dimensional version; Acorn, NMR, 1998) curve analysis program. Phosphocreatine (PCr), ATP, and Pi were assumed proportional to resonance areas, and the relative contents of intracellular Na+, PCr, ATP, and Pi were determined from the respective resonance area values corrected for differential saturation as noted. Myocardial energy metabolism was evaluated by determining the ratios PCr/ATP and Pi/ATP. Intracellular pH was determined from the relationship
where v is the chemical shift difference between inorganic phosphate and the PCr resonance (5, 31).
Effects of intracellular acidosis in the perfused rat heart. To determine whether controlled acidification could alter Na+ homeostasis as well as contractile function in the perfused rat heart, a technique was used to manipulate pHi with minimal alterations in perfusate pH, oxygenation, or flow rate, as previously described by Jeffrey and colleagues (17). This technique included the introduction and subsequent washout of ammonium chloride (NH4Cl) and allowed us to examine the effects of acidification on ionic movement across the cell membrane (28). Hearts isolated from naive control rats (n = 10) were perfused with a modified Krebs-Henseleit bicarbonate buffer as described under Isolated coronary perfused hearts. For these experiments, two perfusate-oxygenating columns were used, with one containing 10 mM NH4Cl in the buffer. After stabilization of the heart with the Krebs-Henseleit bicarbonate buffer perfusion contained in the first column, the introduction of NH4Cl was produced by switching to the second column. The change to NH4Cl perfusion was completed in <20 s. The heart was perfused with the NH4Cl solution for 15 min, and ventricular performance, pHi, and high-energy phosphate metabolites were measured at 1- to 2-min intervals during the NH4Cl perfusion. Sixteen minutes after the NH4Cl perfusion began, the perfusate was switched back to the original Krebs-Henseleit bicarbonate buffer, and ventricular function, pHi, high-energy phosphates, and [Na+]i were again measured at 1- to 2-min intervals during and after the washout of NH4Cl.
Cardiac myocyte isolation. To further examine the time course of burn-mediated myocardial Na+ accumulation as measured by NMR spectroscopy, hearts (n = 3-4 hearts/group per time period) were collected at several times postburn (1, 2, 4, 8, 12, 18, and 24 h); time-matched sham burns (n = 3 hearts/group per time period) were included to provide appropriate controls. Hearts were harvested and placed in ice-cold physiological Ca2+-free HEPES buffer (pH 7.4) containing (in mM) 136 NaCl, 5.4 KCl, 0.6 MgCl2, 0.3 NaH2PO4, 10 glucose, and 10 HEPES. The hearts were then cannulated and perfused with the Ca2+-free HEPES buffer in a nonrecirculating mode. Enzymatic digestion was initiated by perfusing the hearts with 0.5 mg/ml collagenase and 0.05 mg/ml of protease in a recirculating mode at a flow rate of 12 ml/min for 8-10 min. The temperature of the buffer was kept constant at 37°C, and the pH was maintained at 7.4 by bubbling perfusate with 95% O2 and 5% CO2. At the end of the enzymatic digestion, the ventricles were removed and mechanically dissociated in the HEPES buffer containing 100 μM CaCl2 by mincing with fine scissors. The tissue homogenate was filtered through a mesh filter into a conical tube and allowed to pellet for 10 min. The cells were washed and pelleted further in the physiological HEPES buffer with three increasing increments of Ca2+ up to 1.8 mM (14) and 1% each of MEM-amino acid solution (Life Technologies, Rockville, MD) and MEM (Life Technologies).
Use of SBFI to measure cardiomyocyte [Na+]i. [Na+]i was measured by quantitative fluorescence microscopy using the InCyt Im2 fluorescence imaging system (Intracellular Imaging, Cincinnati, OH). Cardiomyocytes were loaded with 10 μmol/l acetoxymethyl ester of SBFI plus pluronic acid (Molecular Probes, Eugene, OR) for 2 h at room temperature. Cells were then washed to remove extracellular dye and were placed in a chamber on the stage of a Nikon inverted microscope. The microscope was interfaced with Grooney optics for epi-illumination, a triocular head, phase optics, and ×10 phase-contrast objective and mechanical stage; the excitation illumination source (300-W compact xenon arc illuminator) was equipped with a power supply. In addition, this fluorescence imaging system included an imaging workstation and Intel Pentium Pro200 MHZ-based personal computer. The computer-controlled filter changer allowed alternation between the 340 and 380 excitation wavelengths, and fluorescence was measured at 510 nm. Images were captured by monochrome charge-coupled device camera equipped with a TV relay lens. InCyt Im2 Image software allowed measurement of [Na+]i from the ratio of the two fluorescent signals generated from the two excitation wavelengths (340 and 380 nm); autofluorescence of myocytes that had not been loaded with SBFI was measured with each experiment and subtracted. The calibration procedure included measuring fluorescence ratio with different Na+ concentration buffers. At each wavelength, the fluorescence emissions were collected for 1-min intervals, and the time between data collection was 1-2 min. Cellular Na+ concentration was calculated as previously described (3, 23).
Statistical analysis. The left ventricular data and the Na+ peak area ratio data were analyzed via a two-factor ANOVA. In the case of significant interaction, a simple means analysis contrasting sham and burn was performed at each time point. Also, for the ratio data, each treatment-time combination was examined by a one-way ANOVA followed by Dunnett's test comparing all combinations to the control. The pH and internal Na+ peak observations were analyzed by using a single-factor repeated-measures mixed model. A first-order autoregressive covariance structure was assumed. Post hoc testing was conducted contrasting the baseline time period to successive time points in the washout segment. Observed differences P < 0.05 were considered to be statistically significant in post hoc testing.
RESULTS
Time course of burn-mediated changes in mechanical performance. Left ventricular developed pressure (LVDP) was significantly lower 1 h after burn trauma compared with LVDP measured in sham-burn animals; however, LVDP was similar in sham-burn animals and burn animals 2 h after burn trauma. This initial myocardial contractile depression was likely related to the deep anesthesia required for burn trauma over 40% of the body surface; although this level of anesthesia transiently impairs myocardial function, it ensured humane care for the rats. Myocardial function progressively fell 4-24 h after trauma (Table 1). Compared with values measured in sham-burn hearts, hearts collected 24 h postburn generated significantly lower LVDP (sham 110 ± 10 vs. burn 61 ± 5 mmHg, P < 0.05). Heart rate and coronary flow rate were similar in sham (312 ± 12 beats/min and 15 ± 1 ml/min) and in burned rats (304 ± 14 beats/min and 16 ± 2 ml/min, P > 0.05) 24 h after burn trauma. The rise in LVDP 4 and 24 h after sham burn was attributed to recovery from anesthesia in this control group.
Myocardial pHi and energy availability in sham and burned hearts. A representative tracing of the 23Na and 31P spectra is shown in Fig. 1. Constant infusion of the shift reagent successfully achieved separation of intracellular and extracellular Na+; the shift reagent did not alter any aspect of the 31P spectra or any aspect of ventricular performance (before shift reagent: LVDP, 102 ± 11 mmHg; heart rate, 306 ± 6 beats/min; after shift reagent: LVDP, 102 ± 10 mmHg; heart rate, 313 ± 12 beats/min). Indexes of energy metabolism are shown in Table 2. ATP and PCr values as well as PCr/ATP and Pi/ATP ratios measured in hearts from burn resuscitated rats were not significantly different from those values measured in sham-burn animals. In addition, myocardial pHi was similar in burn and sham-burned groups at all times during the experimental period (Fig. 2).
23Na NMR measurement of myocardial [Na+]i. Myocardial Na+ content measured in both experimental groups is shown in Fig. 3. The ratio of Na+ signal from the intracellular compartment (i.e., [Na+]i) compared with external standards increased significantly 2 h after burn trauma (+33% increase), achieving a 59% increase 4 h after burn trauma. Twenty-four hours postburn, the ratio of Na+ signal from the intracellular compartment to external standard was increased 58% above that measured in time-matched sham-burned hearts as determined by NMR spectroscopy. Because cardiac mass was the same in all experimental groups, this change is consistent with an increase in intracellular Na+ content.
Time course of myocardial Na+ accumulation (fluorescence spectroscopy). To confirm the substantial rise in myocardial [Na+]i measured by 23Na NMR spectroscopy, ventricular myocytes were isolated at several times postburn, loaded with SBFI, and examined by fluorescence spectroscopy. These studies confirmed significant Na+ accumulation 4 h postburn (Fig. 4). These data support the 23Na-NMR spectroscopy data and confirm that burn trauma promotes Na+ accumulation by ventricular myocytes. There was no significant difference in [Na+]i measured in cardiomyocytes prepared from sham-burn animals at several experimental time points (Fig. 4).
Influence of intracellular acidosis on myocardial Na+ and on cardiac contractile function. NH4Cl perfusion of the heart produced an initial alkalosis followed by a recovery to near control pH values after 15 min of continuous NH4Cl perfusion. Washout of the NH4Cl solution produced transient myocardial acidosis, which persisted for <5 min (Fig. 5A). NH4Cl-induced myocardial acidosis was associated with a significant rise in [Na+]i as measured by 23Na-NMR spectroscopy (Fig. 5, B and C). In addition, myocardial acidification was paralleled by a significant fall in LVDP, whereas high-energy phosphate levels remained unchanged (Table 3).
DISCUSSION
Previous studies from our laboratory have shown that major burn trauma promotes significant Na+ accumulation in several cell types, including hepatocytes and cardiomyocytes; however, these burn-mediated changes in intracellular Na+ levels were documented 24 h postburn (13, 16, 24, 31, 32). The mechanisms by which ionic disturbances occur after burn trauma as well as the functional consequences remain unclear, but it is likely that alterations in myocardial ion homeostasis contribute to postburn cardiac arrhythmias and progressive cardiac cellular injury. Evidence has accumulated that, within the myocardium, [Na+]i and [Ca2+]i and pHi are intimately linked. Katz and Hecht (19) proposed that protons exert a negative inotropic effect by competing with Ca2+ for the binding sites on the contractile proteins, confirming the adverse affects of intracellular acidosis on systolic and diastolic function and diastolic compliance (21). This present study examined the hypothesis that burn trauma promoted myocardial intracellular acidification during the early postburn period, which secondarily promoted Na+ accumulation within the myocardium, contributing ultimately to myocardial contractile dysfunction. Although our NMR data showed that burn trauma promoted a significant rise in myocardial intracellular Na+ levels as early as 2 h after trauma, these burn-related ionic derangements occurred in the absence of myocardial acidosis and with no change in myocardial high-energy phosphate stores.
Normally, transport of Na+ across the cell membrane is determined by both passive diffusion as well as active transport. The steady-state low level of intracellular Na+ is maintained by active extrusion via the Na+ pump/Na+-K+-ATPase in the membrane (3, 19, 21). When the rate of Na+ extrusion from the cell is less than the diffusion rate of Na+ down the electrochemical gradient into the cell, then intracellular Na+ accumulation occurs. Our initial concern in this study was that Na+ accumulation occurred as a result of altered ATP production and decreased cellular energy. The energy that is needed to maintain pump function and contractile activity of the heart comes from hydrolysis of ATP by ATPases; a decrease in the rate of ATP production after burn trauma would lessen the energy available for membrane pumps and impair the effective removal of Na+ from the intracellular compartment. In models of ischemia-reperfusion, cellular Na+ and Ca2+ accumulation have been linked to a fall in ATP and PCr (27), and these previous studies suggested that compromised energy availability may limit myocyte Na+ efflux after major burn trauma. In our study, measures of energy availability did not decrease during the postburn period, suggesting that the rise in myocardial [Na+]i and the decreased contractile performance could not be attributed to inadequate ATP production to support ion-pump function or to sustain contractile activity. The rise in myocardial ATP levels 4 and 24 h after burn trauma paralleled the fall in LVDP at these times and likely reflected decreased energy utilization.
Because burn-mediated changes in energy availability were eliminated as a mechanism for ion dyshomeostasis after burn trauma, we considered the role of myocardial intracellular acidification and Na+/H+ exchange as a mechanism for postburn myocardial Na+ overload. Our consideration of burn-mediated intracellular acidosis as a causative factor was based on previous ischemia-reperfusion studies that showed that myocardial cation derangement was unequivocally linked to myocardial acidosis (2, 18, 20, 27, 29). To first determine whether our NMR technology had sufficient sensitivity to detect acidosis-mediated effects on cellular Na+ levels, we used a model of moderate intracellular acidosis in perfused hearts that has been described previously to maintain normal external pH and optimal oxygen delivery (17). In our study, NH4Cl perfusion of the isolated heart indeed produced transient myocardial acidification, and, as expected, acidosis was associated with a fall in ventricular performance. More importantly, this moderate NH4Cl-induced acidosis produced a substantial rise in [Na+]i, addressing our concern that Na+/H+ exchange could be activated in the rat heart with even a moderate myocardial acidosis. The NH4Cl-washout technique reduced pHi from 7.10 to 6.86 and was associated with a 34% increase in [Na+]i, similar to the percent increase in [Na+]i observed after burn trauma.
One explanation for the absence of myocardial acidosis after burn trauma could be our use of a bicarbonate buffer in the perfused hearts and perhaps rapid in vitro correction of a previous in vivo acidosis. Previous studies have described a Na+/HCO/Cl/H+ exchanger that has been shown to be active at a physiological pH; this exchanger promotes HCO influx and H+ efflux. It is possible that perfusing the isolated heart with a bicarbonate-based buffer during NMR studies may have promoted H+ efflux, masking burn-mediated myocardial acidosis. However, our finding of an absence of myocardial acidosis after burn trauma contrasts with the significant acidification reported in models of myocardial ischemia; and these data further suggest that burn-related ischemia is not the primary mechanism underlying myocardial Na+ dyshomeostasis.
An alternative mechanism by which burn trauma may promote Na+ loading of cardiomyocytes is myocyte accumulation of Ca2+ and decreased efficiency of the Na+/Ca2+ exchanger (4). In this regard, our laboratory has shown previously that a major cutaneous burn injury promotes significant Ca2+ accumulation by cardiomyocytes (13, 16). It is attractive to hypothesize that the oxidant stress and lipid peroxidation that are recognized to occur with burn trauma directly modify the Na+/Ca2+ exchanger protein. For example, sulfhydryl alterations may modify the affinity of the Na+/Ca2+ exchanger for Ca2+, promoting cytosolic Ca2+ and Na+ accumulation. Alternatively, burn-mediated lipid peroxidation of membranes may alter some aspect of sarcolemma phospholipid asymmetry, promoting subsequent inhibition of Na+/Ca2+ exchanger (12).
Although NMR spectroscopy provides a powerful tool to examine the ionic status of perfused organs, this technical approach also measures ion concentrations of several nonmyocyte cell populations within the perfused heart. For example, 23Na-NMR spectroscopy in the isolated heart measures Na+ levels in cardiomyocytes as well as endothelial cells and perhaps emigrated neutrophils. Therefore, SBFI loading of isolated ventricular myocytes and fluorescence spectroscopy were included to examine the effects of burn trauma on Na+ accumulation specifically by the cardiomyocyte cell population. Although the major advantages of multinuclear magnetic resonance spectroscopy include the ability to simultaneously measure ion homeostasis, energy change, and functional performance in the isolated heart, such simultaneous measures in isolated ventricular myocytes are, at best, problematic. Additional limitations of fluorescent techniques applied to isolated ventricular myocytes include the need for collagenase perfusion of the isolated heart and the possibility of collagenase-related changes in intracellular homeostasis. In this study, use of NMR technology to measure intracellular Na+ rapidly and noninvasively in the perfused heart, paralleled by the fluorescent indicator loading of isolated cardiomyocytes and fluorescent spectroscopy, provided an ideal experimental approach to examine the time course of burn-mediated cardiomyocyte Na+ dyshomeostasis.
In summary, 31P- and 23Na-NMR spectroscopy were used to assess the time course of burn-mediated changes in pHi, energy availability, [Na+]i, and ventricular function in the isolated perfused heart. Myocardial Na+ accumulation paralleled the development of contractile dysfunction after burn trauma, but Na+ accumulation occurred despite no change in postburn myocardial pHi or high-energy phosphates. Our in vitro studies showed that an NH4Cl-washout technique produced moderate acidosis in the perfused heart, and this acidosis promoted significant Na+ accumulation, confirming acidosis-mediated changes in cellular ion status. Although these in vitro studies confirmed that myocardial acidification can activate Na+/H+ exchange in the rodent heart, the absence of a burn-mediated myocardial acidosis suggests that active Na+/H+ exchange is not the primary mechanism by which the myocardium accumulates Na+ after burn trauma.
ACKNOWLEDGEMENTS
We thank Dr. William Frawley, University of Texas Southwestern Medical Center, Academic Computing Services, for assistance in the statistical analysis.
FOOTNOTES
This study was supported by National Institute of General Medical Sciences Grant GM-57054-01A1.
Address for reprint requests and other correspondence: J. W. Horton, Dept. of Surgery, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9160 (E-mail: jureta.horton@utsouthwestern.edu).
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.
Received 3 May 2001; accepted in final form 27 July 2001.
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ABSTRACT
In this study, 23Na- and 31P- nuclear magnetic resonance (NMR) spectra were examined in perfused rat hearts harvested 1, 2, 4, and 24 h after 40% total body surface area burn trauma and lactated Ringer resuscitation, 4 ml · kg1 · %1 burn. 23Na-NMR spectroscopy monitored myocardial intracellular Na+ using the paramagnetic shift reagent thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid). Left ventricular function, cardiac high-energy phosphates (ATP/PCr), and myocyte intracellular pH were studied by using 31P NMR spectroscopy to examine the hypothesis that burn-mediated acidification of cardiomyocytes contributes to subsequent Na+ accumulation by this cell population. Intracellular Na+ accumulation was confirmed by sodium-binding benzofuran isophthalate loading and fluorescence spectroscopy in cardiomyocytes isolated 1, 2, 4, 8, 12, 18, and 24 h postburn. This myocyte Na+ accumulation as early as 2 h postburn occurred despite no changes in cardiac ATP/PCr and intracellular pH. Left ventricular function progressively decreased after burn trauma. Cardiomyocyte Na+ accumulation paralleled cardiac contractile dysfunction, suggesting that myocardial Na+ overload contributes, in part, to the progressive postburn decrease in ventricular performance.
keywords:nuclear magnetic resonance spectroscopy; cardiomyocyte loading of the fluorescent indicator sodium-binding benzofuran isophthalate; ventricular contraction-relaxation assessed by Langendorff perfusion; myocardial acidosis (intracellular pH); myocardial high-energy phosphate stores
INTRODUCTION
MAJOR TRAUMA SUCH AS THERMAL INJURY produces a rise in intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i) concentrations, and this cation dyshomeostasis may contribute to the myocardial cell injury and contractile dysfunction that have been described after burn injury (13, 16, 21, 22, 24, 30, 32). The mechanisms by which a cutaneous burn injury alters myocardial Na+ homeostasis remains unclear. However, studies using models of ischemia and reperfusion have shown that myocyte acidification early in the ischemic period is a precipitating event in both myocardial Na+ and Ca2+ accumulation and cardiac contractile dysfunction (6, 7, 10, 25, 28). The negative inotropic effects of intracellular acidosis may be related to competition of hydrogen ions with Ca2+ for binding sites on the contractile proteins, impairing actin-myosin interaction and reducing tension development (26). However, intracellular acidosis may also alter left ventricular function by mediating Na+/H+ exchange, and the resulting accumulation of Na+ within the myocardium may increase the [Ca2+]i via Na+/Ca2+ countertransport (9, 11, 29). Alternatively, a rise in myocyte [Na+]i may modulate the effects of the sympathetic nervous system on the heart, contributing to contractile dysfunction (8).
Few studies of traumatic injury have examined the direct effects on myocardial acidification an intracellular-extracellular Na+ dyshomeostasis or cellular function. Studies from our laboratory examined myocardial Na+ late in the postburn period (24 h postburn), but those previous studies ignored the potential of myocardial acidosis in the early postburn period (1-12 h). Furthermore, it is unclear whether Na+ accumulation within cardiac myocytes promotes cellular injury and dysfunction or whether the myocyte Na+ accumulation described late in the postburn occurred secondary to burn-induced cardiac contraction and relaxation defects. The availability of 23Na-nuclear magnetic resonance (NMR) spectroscopy as well as 23Na-NMR shift reagents allow frequency resolution of intra- and extracellular Na+ resonances in the isolated perfused heart. Resolution of [Na+]i is compatible with acquisition of the 31P spectra, allowing simultaneous measurements of [Na+]i, intracellular pH (pHi), high-energy phosphate stores, and cardiac mechanical performance.
In this present study, subsets of rats were killed at several times after burn trauma, and hearts were perfused to determine 1) the time course of myocardial Na+ accumulation after burn trauma, 2) the contribution of intracellular acidosis to myocardial Na+ accumulation, and 3) whether burn-mediated myocardial Na+ accumulation occurred before or as a consequence of myocardial contractile abnormalities. These studies were designed to examine the hypothesis that burn-related acidification of cardiomyocytes contributes to Na+ accumulation by this cell population; Na+ dyshomeostasis in turn contributes to myocardial contractile abnormalities. In addition, we compared ventricular Na+ concentrations measured by 23Na-NMR spectroscopy in perfused hearts with [Na+]i measured by fluorescent dye sodium-binding benzofuran isophthalate (SBFI) loading of isolated ventricular myocytes. Our data showed that burn trauma promoted a rise in myocardial [Na+]i as early as 2 h after burn injury and achieved maximal myocardial Na+ levels 4 h postburn. Thus burn-mediated myocardial Na+ accumulation preceded the development of contractile defects. In our study, the burn-mediated increase in [Na+]i was not related to intracellular acidification as indicated by a stable pHi at all times after burn trauma. Our data suggest that postburn myocardial Na+ accumulation is not related solely to Na+/H+ exchange mechanisms, but it is likely that the influx of Na+ ions occurs via other pathways such as Na+-K+-ATPase or Na+/Ca2+ exchange systems.
MATERIALS AND METHODS
Experimental animals and burn procedure. The experimental protocol was reviewed and approved by the Institutional Animal Care and Research Advisory Committee of The University of Texas Southwestern Medical Center at Dallas. Adult male Sprague-Dawley rats, weighing 280-320 g each, were randomly divided into burn and sham-burn groups. Burn injury was produced using a template model as previously described (1, 15). Briefly, rats were deeply anesthetized with methoxyflurane, shaved, and secured in a template device. A full-thickness scald burn over 40% of the total body surface area was produced by immersing the skin on the back and sides exposed through the template in 100°C water for 12 s. The animals were quickly dried after immersion to avoid additional injury. The rats evidenced no postburn pain as indicated by their eating, drinking, moving freely about the cage, and responding to external stimuli; in addition, the animals evidenced no discomfort with handling by the investigators. Despite the presence of third-degree burns and destruction of underlying neural tissue, all rats received analgesics (Buprenex, 0.05 mg/kg) every 8 h postburn. Fluid resuscitation was initiated after trauma (lactated Ringer solution, 4 ml · kg1 · %1 burn) with one-half of the calculated volume given intraperitoneally immediately after the burn procedure and before recovery from anesthesia. After recovery from inhalation anesthesia, the animals were placed in separate cages and given standard rat chow and water ad libitum until death. The remaining volume of lactated Ringer solution was given 8 h postburn intraperitoneally. Sham-burn rats were anesthetized and handled in an identical manner except they were exposed to room-temperature water.
Isolated coronary perfused hearts. At the designated time after burn or sham burn (1, 2, 4, and 24 h), rats were anesthetized with methoxyflurane, and the heart was excised through a midline thoracotomy and placed in ice-cold Krebs-Henseleit buffer. A cannula placed in the aorta was connected to a reservoir located 95 cm above the heart for perfusion of the coronary arteries by the Langendorff method with a modified phosphate-free Krebs-Henseleit bicarbonate buffer (in mM: 118 NaCl, 25 NaHCO3, 6 KCl, 1.2 MgSO4, 1.25 CaCl2, and 10 glucose; pH 7.4, temperature 37°C and bubbled continuously with 95% O2-5% CO2). The all-glass water-jacketed perfusate reservoir, which also serves as the oxygenation chamber, was located in the bore of the magnet. Coronary effluent was removed from the magnetic resonance spectroscopy (MRS) tube, filtered through 5-μm filters and recirculated. An intraventricular balloon attached to a catheter (0.8 mm OD) was placed in the left ventricle (via left atrium) for monitoring spontaneous heart rate, left ventricular pressure, and coronary flow rate. The balloon cannula was connected to a Gould P23 pressure transducer, and functional parameters were measured by using a physiological monitor (Coulbourn, Lehigh Valley, PA) and recorded on a chart recorder (WR 3101, Western Graphtec, Irvine, CA). The heart was suspended in a 20-mm-OD MRS tube with a microtube containing 50 μl of 1.3 M NaCl and 3.3 mM Dy(DOTP)5 (as a Na+ external standard).
Shift reagent. Thulium 1,4,7,10-tetraazacylododecane-1,4,7,10-tetra(methylenephosphonic acid) (Na+ salt; TmDOTP4), characterized by favorable paramagnetic and Na+ binding properties, has been used to separate intra- and extracellular 23Na signals both in isolated organ studies and in vivo (5, 31, 32). Shift reagents such as TmDOTP4 are membrane impermeable and interact with extracellular Na+, causing its magnetic resonance signal to be shifted away from the intracellular signal. Compared with other shift reagents (such as DyTTHA3), TmDOTP4 has two advantages: a larger 23Na chemical shift per unit concentration and narrower line widths due, in part, to a lower bulk magnetic susceptibility. Because TmDOTP4 alters Ca2+ availability, the concentration of Ca2+ was adjusted to 3.9 mM so that the free Ca2+ in the perfusate was maintained at 1.0 mM as measured with Ca2+-sensitive electrode.
NMR spectroscopy. Hearts were allowed to equilibrate for 15 min before MRS spectra as well as functional parameters were obtained. The perfusate was switched to an identical solution containing TmDOTP4 (3.5 mM) for 31P- and 23Na- MR spectroscopy. The spectroscopy was performed on Varian INOVA 300 spectrometer equipped with an Oxford 4.2-T vertical-bore superconducting magnet. A Bruker 20-mm broadband probe was used, tunable to either 23Na or 31P; the magnetic field homogeneity was adjusted by using the 23Na-free induction decay. A 23Na line width of 9-10 Hz was typical, and 23Na spectra were acquired by using a ±6,000-Hz spectral width, 2,000 data points, a 65-μs excitation pulse, 72-1,280 acquisitions, and a 0.40-s interpulse delay over a period of 1 min. 31P spectra were acquired over a ±6,000-Hz spectral width by using 2,000 data points with a 38-μs excitation pulse, 56-520 acquisitions, and a 1.1- or 2.0-s interpulse delay.
MRS data analysis. The spectra were zero-filled to 4,096 points before Fourier transformation. The resonance areas in both 23Na- and 31P-NMR spectra were determined using the NMR utility transform software (two-dimensional version; Acorn, NMR, 1998) curve analysis program. Phosphocreatine (PCr), ATP, and Pi were assumed proportional to resonance areas, and the relative contents of intracellular Na+, PCr, ATP, and Pi were determined from the respective resonance area values corrected for differential saturation as noted. Myocardial energy metabolism was evaluated by determining the ratios PCr/ATP and Pi/ATP. Intracellular pH was determined from the relationship
where v is the chemical shift difference between inorganic phosphate and the PCr resonance (5, 31).
Effects of intracellular acidosis in the perfused rat heart. To determine whether controlled acidification could alter Na+ homeostasis as well as contractile function in the perfused rat heart, a technique was used to manipulate pHi with minimal alterations in perfusate pH, oxygenation, or flow rate, as previously described by Jeffrey and colleagues (17). This technique included the introduction and subsequent washout of ammonium chloride (NH4Cl) and allowed us to examine the effects of acidification on ionic movement across the cell membrane (28). Hearts isolated from naive control rats (n = 10) were perfused with a modified Krebs-Henseleit bicarbonate buffer as described under Isolated coronary perfused hearts. For these experiments, two perfusate-oxygenating columns were used, with one containing 10 mM NH4Cl in the buffer. After stabilization of the heart with the Krebs-Henseleit bicarbonate buffer perfusion contained in the first column, the introduction of NH4Cl was produced by switching to the second column. The change to NH4Cl perfusion was completed in <20 s. The heart was perfused with the NH4Cl solution for 15 min, and ventricular performance, pHi, and high-energy phosphate metabolites were measured at 1- to 2-min intervals during the NH4Cl perfusion. Sixteen minutes after the NH4Cl perfusion began, the perfusate was switched back to the original Krebs-Henseleit bicarbonate buffer, and ventricular function, pHi, high-energy phosphates, and [Na+]i were again measured at 1- to 2-min intervals during and after the washout of NH4Cl.
Cardiac myocyte isolation. To further examine the time course of burn-mediated myocardial Na+ accumulation as measured by NMR spectroscopy, hearts (n = 3-4 hearts/group per time period) were collected at several times postburn (1, 2, 4, 8, 12, 18, and 24 h); time-matched sham burns (n = 3 hearts/group per time period) were included to provide appropriate controls. Hearts were harvested and placed in ice-cold physiological Ca2+-free HEPES buffer (pH 7.4) containing (in mM) 136 NaCl, 5.4 KCl, 0.6 MgCl2, 0.3 NaH2PO4, 10 glucose, and 10 HEPES. The hearts were then cannulated and perfused with the Ca2+-free HEPES buffer in a nonrecirculating mode. Enzymatic digestion was initiated by perfusing the hearts with 0.5 mg/ml collagenase and 0.05 mg/ml of protease in a recirculating mode at a flow rate of 12 ml/min for 8-10 min. The temperature of the buffer was kept constant at 37°C, and the pH was maintained at 7.4 by bubbling perfusate with 95% O2 and 5% CO2. At the end of the enzymatic digestion, the ventricles were removed and mechanically dissociated in the HEPES buffer containing 100 μM CaCl2 by mincing with fine scissors. The tissue homogenate was filtered through a mesh filter into a conical tube and allowed to pellet for 10 min. The cells were washed and pelleted further in the physiological HEPES buffer with three increasing increments of Ca2+ up to 1.8 mM (14) and 1% each of MEM-amino acid solution (Life Technologies, Rockville, MD) and MEM (Life Technologies).
Use of SBFI to measure cardiomyocyte [Na+]i. [Na+]i was measured by quantitative fluorescence microscopy using the InCyt Im2 fluorescence imaging system (Intracellular Imaging, Cincinnati, OH). Cardiomyocytes were loaded with 10 μmol/l acetoxymethyl ester of SBFI plus pluronic acid (Molecular Probes, Eugene, OR) for 2 h at room temperature. Cells were then washed to remove extracellular dye and were placed in a chamber on the stage of a Nikon inverted microscope. The microscope was interfaced with Grooney optics for epi-illumination, a triocular head, phase optics, and ×10 phase-contrast objective and mechanical stage; the excitation illumination source (300-W compact xenon arc illuminator) was equipped with a power supply. In addition, this fluorescence imaging system included an imaging workstation and Intel Pentium Pro200 MHZ-based personal computer. The computer-controlled filter changer allowed alternation between the 340 and 380 excitation wavelengths, and fluorescence was measured at 510 nm. Images were captured by monochrome charge-coupled device camera equipped with a TV relay lens. InCyt Im2 Image software allowed measurement of [Na+]i from the ratio of the two fluorescent signals generated from the two excitation wavelengths (340 and 380 nm); autofluorescence of myocytes that had not been loaded with SBFI was measured with each experiment and subtracted. The calibration procedure included measuring fluorescence ratio with different Na+ concentration buffers. At each wavelength, the fluorescence emissions were collected for 1-min intervals, and the time between data collection was 1-2 min. Cellular Na+ concentration was calculated as previously described (3, 23).
Statistical analysis. The left ventricular data and the Na+ peak area ratio data were analyzed via a two-factor ANOVA. In the case of significant interaction, a simple means analysis contrasting sham and burn was performed at each time point. Also, for the ratio data, each treatment-time combination was examined by a one-way ANOVA followed by Dunnett's test comparing all combinations to the control. The pH and internal Na+ peak observations were analyzed by using a single-factor repeated-measures mixed model. A first-order autoregressive covariance structure was assumed. Post hoc testing was conducted contrasting the baseline time period to successive time points in the washout segment. Observed differences P < 0.05 were considered to be statistically significant in post hoc testing.
RESULTS
Time course of burn-mediated changes in mechanical performance. Left ventricular developed pressure (LVDP) was significantly lower 1 h after burn trauma compared with LVDP measured in sham-burn animals; however, LVDP was similar in sham-burn animals and burn animals 2 h after burn trauma. This initial myocardial contractile depression was likely related to the deep anesthesia required for burn trauma over 40% of the body surface; although this level of anesthesia transiently impairs myocardial function, it ensured humane care for the rats. Myocardial function progressively fell 4-24 h after trauma (Table 1). Compared with values measured in sham-burn hearts, hearts collected 24 h postburn generated significantly lower LVDP (sham 110 ± 10 vs. burn 61 ± 5 mmHg, P < 0.05). Heart rate and coronary flow rate were similar in sham (312 ± 12 beats/min and 15 ± 1 ml/min) and in burned rats (304 ± 14 beats/min and 16 ± 2 ml/min, P > 0.05) 24 h after burn trauma. The rise in LVDP 4 and 24 h after sham burn was attributed to recovery from anesthesia in this control group.
Myocardial pHi and energy availability in sham and burned hearts. A representative tracing of the 23Na and 31P spectra is shown in Fig. 1. Constant infusion of the shift reagent successfully achieved separation of intracellular and extracellular Na+; the shift reagent did not alter any aspect of the 31P spectra or any aspect of ventricular performance (before shift reagent: LVDP, 102 ± 11 mmHg; heart rate, 306 ± 6 beats/min; after shift reagent: LVDP, 102 ± 10 mmHg; heart rate, 313 ± 12 beats/min). Indexes of energy metabolism are shown in Table 2. ATP and PCr values as well as PCr/ATP and Pi/ATP ratios measured in hearts from burn resuscitated rats were not significantly different from those values measured in sham-burn animals. In addition, myocardial pHi was similar in burn and sham-burned groups at all times during the experimental period (Fig. 2).
23Na NMR measurement of myocardial [Na+]i. Myocardial Na+ content measured in both experimental groups is shown in Fig. 3. The ratio of Na+ signal from the intracellular compartment (i.e., [Na+]i) compared with external standards increased significantly 2 h after burn trauma (+33% increase), achieving a 59% increase 4 h after burn trauma. Twenty-four hours postburn, the ratio of Na+ signal from the intracellular compartment to external standard was increased 58% above that measured in time-matched sham-burned hearts as determined by NMR spectroscopy. Because cardiac mass was the same in all experimental groups, this change is consistent with an increase in intracellular Na+ content.
Time course of myocardial Na+ accumulation (fluorescence spectroscopy). To confirm the substantial rise in myocardial [Na+]i measured by 23Na NMR spectroscopy, ventricular myocytes were isolated at several times postburn, loaded with SBFI, and examined by fluorescence spectroscopy. These studies confirmed significant Na+ accumulation 4 h postburn (Fig. 4). These data support the 23Na-NMR spectroscopy data and confirm that burn trauma promotes Na+ accumulation by ventricular myocytes. There was no significant difference in [Na+]i measured in cardiomyocytes prepared from sham-burn animals at several experimental time points (Fig. 4).
Influence of intracellular acidosis on myocardial Na+ and on cardiac contractile function. NH4Cl perfusion of the heart produced an initial alkalosis followed by a recovery to near control pH values after 15 min of continuous NH4Cl perfusion. Washout of the NH4Cl solution produced transient myocardial acidosis, which persisted for <5 min (Fig. 5A). NH4Cl-induced myocardial acidosis was associated with a significant rise in [Na+]i as measured by 23Na-NMR spectroscopy (Fig. 5, B and C). In addition, myocardial acidification was paralleled by a significant fall in LVDP, whereas high-energy phosphate levels remained unchanged (Table 3).
DISCUSSION
Previous studies from our laboratory have shown that major burn trauma promotes significant Na+ accumulation in several cell types, including hepatocytes and cardiomyocytes; however, these burn-mediated changes in intracellular Na+ levels were documented 24 h postburn (13, 16, 24, 31, 32). The mechanisms by which ionic disturbances occur after burn trauma as well as the functional consequences remain unclear, but it is likely that alterations in myocardial ion homeostasis contribute to postburn cardiac arrhythmias and progressive cardiac cellular injury. Evidence has accumulated that, within the myocardium, [Na+]i and [Ca2+]i and pHi are intimately linked. Katz and Hecht (19) proposed that protons exert a negative inotropic effect by competing with Ca2+ for the binding sites on the contractile proteins, confirming the adverse affects of intracellular acidosis on systolic and diastolic function and diastolic compliance (21). This present study examined the hypothesis that burn trauma promoted myocardial intracellular acidification during the early postburn period, which secondarily promoted Na+ accumulation within the myocardium, contributing ultimately to myocardial contractile dysfunction. Although our NMR data showed that burn trauma promoted a significant rise in myocardial intracellular Na+ levels as early as 2 h after trauma, these burn-related ionic derangements occurred in the absence of myocardial acidosis and with no change in myocardial high-energy phosphate stores.
Normally, transport of Na+ across the cell membrane is determined by both passive diffusion as well as active transport. The steady-state low level of intracellular Na+ is maintained by active extrusion via the Na+ pump/Na+-K+-ATPase in the membrane (3, 19, 21). When the rate of Na+ extrusion from the cell is less than the diffusion rate of Na+ down the electrochemical gradient into the cell, then intracellular Na+ accumulation occurs. Our initial concern in this study was that Na+ accumulation occurred as a result of altered ATP production and decreased cellular energy. The energy that is needed to maintain pump function and contractile activity of the heart comes from hydrolysis of ATP by ATPases; a decrease in the rate of ATP production after burn trauma would lessen the energy available for membrane pumps and impair the effective removal of Na+ from the intracellular compartment. In models of ischemia-reperfusion, cellular Na+ and Ca2+ accumulation have been linked to a fall in ATP and PCr (27), and these previous studies suggested that compromised energy availability may limit myocyte Na+ efflux after major burn trauma. In our study, measures of energy availability did not decrease during the postburn period, suggesting that the rise in myocardial [Na+]i and the decreased contractile performance could not be attributed to inadequate ATP production to support ion-pump function or to sustain contractile activity. The rise in myocardial ATP levels 4 and 24 h after burn trauma paralleled the fall in LVDP at these times and likely reflected decreased energy utilization.
Because burn-mediated changes in energy availability were eliminated as a mechanism for ion dyshomeostasis after burn trauma, we considered the role of myocardial intracellular acidification and Na+/H+ exchange as a mechanism for postburn myocardial Na+ overload. Our consideration of burn-mediated intracellular acidosis as a causative factor was based on previous ischemia-reperfusion studies that showed that myocardial cation derangement was unequivocally linked to myocardial acidosis (2, 18, 20, 27, 29). To first determine whether our NMR technology had sufficient sensitivity to detect acidosis-mediated effects on cellular Na+ levels, we used a model of moderate intracellular acidosis in perfused hearts that has been described previously to maintain normal external pH and optimal oxygen delivery (17). In our study, NH4Cl perfusion of the isolated heart indeed produced transient myocardial acidification, and, as expected, acidosis was associated with a fall in ventricular performance. More importantly, this moderate NH4Cl-induced acidosis produced a substantial rise in [Na+]i, addressing our concern that Na+/H+ exchange could be activated in the rat heart with even a moderate myocardial acidosis. The NH4Cl-washout technique reduced pHi from 7.10 to 6.86 and was associated with a 34% increase in [Na+]i, similar to the percent increase in [Na+]i observed after burn trauma.
One explanation for the absence of myocardial acidosis after burn trauma could be our use of a bicarbonate buffer in the perfused hearts and perhaps rapid in vitro correction of a previous in vivo acidosis. Previous studies have described a Na+/HCO/Cl/H+ exchanger that has been shown to be active at a physiological pH; this exchanger promotes HCO influx and H+ efflux. It is possible that perfusing the isolated heart with a bicarbonate-based buffer during NMR studies may have promoted H+ efflux, masking burn-mediated myocardial acidosis. However, our finding of an absence of myocardial acidosis after burn trauma contrasts with the significant acidification reported in models of myocardial ischemia; and these data further suggest that burn-related ischemia is not the primary mechanism underlying myocardial Na+ dyshomeostasis.
An alternative mechanism by which burn trauma may promote Na+ loading of cardiomyocytes is myocyte accumulation of Ca2+ and decreased efficiency of the Na+/Ca2+ exchanger (4). In this regard, our laboratory has shown previously that a major cutaneous burn injury promotes significant Ca2+ accumulation by cardiomyocytes (13, 16). It is attractive to hypothesize that the oxidant stress and lipid peroxidation that are recognized to occur with burn trauma directly modify the Na+/Ca2+ exchanger protein. For example, sulfhydryl alterations may modify the affinity of the Na+/Ca2+ exchanger for Ca2+, promoting cytosolic Ca2+ and Na+ accumulation. Alternatively, burn-mediated lipid peroxidation of membranes may alter some aspect of sarcolemma phospholipid asymmetry, promoting subsequent inhibition of Na+/Ca2+ exchanger (12).
Although NMR spectroscopy provides a powerful tool to examine the ionic status of perfused organs, this technical approach also measures ion concentrations of several nonmyocyte cell populations within the perfused heart. For example, 23Na-NMR spectroscopy in the isolated heart measures Na+ levels in cardiomyocytes as well as endothelial cells and perhaps emigrated neutrophils. Therefore, SBFI loading of isolated ventricular myocytes and fluorescence spectroscopy were included to examine the effects of burn trauma on Na+ accumulation specifically by the cardiomyocyte cell population. Although the major advantages of multinuclear magnetic resonance spectroscopy include the ability to simultaneously measure ion homeostasis, energy change, and functional performance in the isolated heart, such simultaneous measures in isolated ventricular myocytes are, at best, problematic. Additional limitations of fluorescent techniques applied to isolated ventricular myocytes include the need for collagenase perfusion of the isolated heart and the possibility of collagenase-related changes in intracellular homeostasis. In this study, use of NMR technology to measure intracellular Na+ rapidly and noninvasively in the perfused heart, paralleled by the fluorescent indicator loading of isolated cardiomyocytes and fluorescent spectroscopy, provided an ideal experimental approach to examine the time course of burn-mediated cardiomyocyte Na+ dyshomeostasis.
In summary, 31P- and 23Na-NMR spectroscopy were used to assess the time course of burn-mediated changes in pHi, energy availability, [Na+]i, and ventricular function in the isolated perfused heart. Myocardial Na+ accumulation paralleled the development of contractile dysfunction after burn trauma, but Na+ accumulation occurred despite no change in postburn myocardial pHi or high-energy phosphates. Our in vitro studies showed that an NH4Cl-washout technique produced moderate acidosis in the perfused heart, and this acidosis promoted significant Na+ accumulation, confirming acidosis-mediated changes in cellular ion status. Although these in vitro studies confirmed that myocardial acidification can activate Na+/H+ exchange in the rodent heart, the absence of a burn-mediated myocardial acidosis suggests that active Na+/H+ exchange is not the primary mechanism by which the myocardium accumulates Na+ after burn trauma.
ACKNOWLEDGEMENTS
We thank Dr. William Frawley, University of Texas Southwestern Medical Center, Academic Computing Services, for assistance in the statistical analysis.
FOOTNOTES
This study was supported by National Institute of General Medical Sciences Grant GM-57054-01A1.
Address for reprint requests and other correspondence: J. W. Horton, Dept. of Surgery, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9160 (E-mail: jureta.horton@utsouthwestern.edu).
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.
Received 3 May 2001; accepted in final form 27 July 2001.
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