Cardiomyocyte sodium accumulation after burn injury precedes the development of myocardial contractile dysfunction. The present study examined the effects of burn injury on Na-K-ATPase activity in adult rat hearts after major burn injury and explored the hypothesis that burn-related changes in myocardial Na-K-ATPase activity are PKC dependent. A third-degree burn injury (or sham burn) was given over 40% total body surface area, and rats received lactated Ringer solution (4 ml·kg−1·% burn−1). Subgroups of rats were killed 2, 4, or 24 h after burn (n = 6 rats/time period), hearts were homogenized, and Na-K-ATPase activity was determined from ouabain-sensitive phosphate generation from ATP by cardiac sarcolemmal vesicles. Additional groups of rats were studied at several times after burn to determine the time course of myocyte sodium loading and the time course of myocardial dysfunction. Additional groups of sham burn-injured and burn-injured rats were given calphostin, an inhibitor of PKC, and Na-K-ATPase activity, cell Na+, and myocardial function were measured. Burn injury caused a progressive rise in cardiomyocyte Na+, and myocardial Na-K-ATPase activity progressively decreased after burn, while PKC activity progressively rose. Administration of calphostin to inhibit PKC activity prevented both the burn-related decrease in myocardial Na-K-ATPase and the rise in intracellular Na+ and improved postburn myocardial contractile performance. We conclude that burn-related inhibition of Na-K-ATPase likely contributes to the cardiomyocyte accumulation of intracellular Na+. Since intracellular Na+ is one determinant of electrical-mechanical recovery after insults such as burn injury, burn-related inhibition of Na-K-ATPase may be critical in postburn recovery of myocardial contractile function.
- myocyte sodium
- ventricular function
- protein kinase C inhibitor
- rat burn injury model
- protein kinase C activity
the myocardial na-k-atpase ion exchange protein functions to transport Na+ out of and K+ into the cardiac myocyte. The Na-K-ATPase transporter plays a major role in electrical-mechanical activity of cardiac muscle, establishing a transarcolemmal Na+ gradient that, in turn, generates the rapid rise of the action potential and contributes to numerous ion transport functions that maintain cell homeostasis (19, 36). Na-K-ATPase function is dependent on ATP availability and is regulated, in part, by phosphorylation by protein kinase C (PKC) (29).
Myocardial contractile dysfunction has been linked to defects in the functional activity of several ion exchange proteins, including Na-K-ATPase (5, 14, 15, 31). In this regard, myocardial ischemia and dysfunction that occur in several injury and disease states are associated with a rise in intracellular Na+ that, in turn, promotes Na+/Ca2+ exchange, a rise in cellular Ca2+, and cell injury (21, 29, 34, 47, 58). Pharmacological interventions that limit cellular accumulation of Na+ (for example, amiloride inhibits the Na+/H+ exchanger and correction of intracellular acidosis eliminates the stimulus for Na+/H+ exchange) have been shown to improve cardiac performance (26, 40, 42, 43). Recent data have suggested that activity of the Na-K-ATPase during injury and disease is regulated by PKC, which produces phosphorylation of serine and threonine residues on the Na-K-ATPase, reducing ion transport function (6, 11, 16, 59). Since major burn injury increases myocardial PKC activity, increases cardiac myocyte Na+ levels, and impairs myocardial contraction and relaxation (21, 48, 58), we postulated that postburn inhibition of PKC would alter burn-related changes in myocardial Na-K-ATPase activity, reducing myocyte Na+ accumulation and improving myocardial contractile performance. To examine this hypothesis, we used a rat model of burn injury over 40% of the total body surface area (TBSA) and inhibited PKC activity with a specific inhibitor, calphostin. Na-K-ATPase activity, PKC activity, myocyte Na+, and left ventricular performance were measured in hearts collected at several time points after burn injury.
MATERIALS AND METHODS
Adult Sprague-Dawley male rats (320–350 g) were obtained from Harlan Laboratories (Houston, TX) and housed in the animal care facility. Commercial rat chow and tap water were available ad libitum, and rats were maintained at a constant temperature with a 12:12-h light-dark cycle. All work was performed under a protocol that was approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory Committee. The work conformed to the guidelines outlined in the “Guiding Principles in the Care and Use of Animals” of the American Physiological Society and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Rats were deeply anesthetized with isoflurane and secured in a constructed template device as described previously (1). The skin exposed through the template was immersed in 100°C water for 12 s on each side to produce a full-thickness dermal burn over 40% TBSA. This burn technique produces complete destruction of the underlying neural tissue. After immersion the rats were immediately dried, and each animal was placed in an individual cage. All burn-injured animals received standard fluid resuscitation consisting of 4 ml·kg−1·% burn−1 lactated Ringer solution, with one-half of this calculated volume given intraperitoneally immediately after the burn injury was completed and the remaining volume given 8 h after burn injury. Buprenorphine (0.05–0.1 mg/kg) was given every 12 h during the postburn period. Burn-injured rats did not display discomfort or pain, moved freely about the cage, and consumed food and water within 15 min after recovering from isoflurane anesthesia. Sham burn-injured rats also received identical regimens of analgesics (buprenorphine) throughout the study period.
Protein extraction of sarcolemmal membranes from rat heart tissue fractionation was carried out as described by Fuller et al. (19). All procedures were performed at 4°C. Rat heart tissue was homogenized with a glass homogenizer in buffer containing (in mM) 20 HEPES, 250 sucrose, 2 EDTA, and 1 MgCl2, pH 7.4. To isolate a purified sarcolemmal/particulate (SLP) fraction, the resulting homogenate was centrifuged at 10,000 rpm for 20 min. The supernatant containing partially purified SLP fraction was centrifuged again for 30 min at 20,000 rpm, and the resulting pellet was suspended in lysis buffer. This SLP fraction contained sarcolemmal membranes as well as other subcellular components that do not contain Na-K-ATPase, including sarcoplasmic reticulum, nuclei, residual myofilaments, and mitochondria.
Measurement of PKC activity.
PKC activity was measured in myocardial membrane with a PKC assay kit (Upstate Biotechnology, Lake Placid, NY). Following the manufacturer's protocol, [γ-32P]ATP (6,000 Ci/mmol stock solution) was used in this study. The reaction mixture included 2.5 μl of 10× reaction buffer, 2.5 μl of PKC substrate, 2.5 μl of inhibitor cocktail, 2.5 μl of freshly sonicated PKC lipid activator, and 50 μg of sample protein. The reaction was initiated by adding 7 μl of the Mg2+-ATP cocktail containing [γ-32P]ATP, followed by incubation. The reaction was stopped by transferring 25 μl of the reaction mixture onto the center of a P81 paper square (provided in the assay kit from Upstate Biotechnology). The assay squares were then washed three times for 5 min each with 0.75% phosphoric acid. Finally, the assay squares were washed once with acetone for 3 min and then transferred into scintillation vials; 5 ml of scintillation cocktail was added, and samples were counted with a scintillation counter. PKC activity was calculated as picomoles of phosphate per microgram of protein according to the manufacturer's suggested protocol.
Na-K-ATPase activity was measured in triplicate by the inorganic phosphate (Pi) released from ATP as described by Tsakiris (52). Cardiac sarcolemmal membrane protein (50 μg) was used to measure total ATPase activity (Na,K,Mg-dependent ATPase and Mg2+-dependent ATPase activity). The incubation medium contained (in mM) 120 NaCl, 20 KCl, 1 K-EDTA, 240 sucrose, 4 MgCl2, and 50 Tris·HCl, pH 7.4, in a final volume of 200 μl. The reaction was initiated by addition of ATP to a final concentration of 3.0 mM. Ouabain-insensitive Mg-ATPase was assayed under the same conditions after addition of 2 mM ouabain and without NaCl and KCl. The reaction was initiated by adding ATP at 37°C and stopped after an incubation period of 15 min by addition of 60 μl of an ice-cold mixture of 1% ammonium molybdate in 0.9 M H2SO4 (7, 52, 53). Released Pi was colorimetrically measured at 390 nm by the method of Chen and colleagues (10). ATPase activity of these preparations ranged from 0.8 to 1.5 μmol Pi·mg protein−1·min−1. Enzyme activity was determined after a 20-min preincubation with various concentrations of each myocardial protein sample. Each sample was assayed in triplicate, and Na-K-ATPase activity was calculated by the difference in values measured with and without ouabain. Specific activities of the enzymes were expressed as micromoles of Pi released per minute per milligram of protein.
To examine the time course of burn-related changes in myocardial PKC and Na-K-ATPase activities, burn-injured rats were killed at several times after burn (2, 4, and 24 h; n = 5 or 6 rats/time period); hearts were collected and freeze clamped for in vitro study. Sham burn-injured rats were included for controls. To examine the time course of changes in cardiac contractile performance after burn injury and to correlate these changes in left ventricular performance with changes in myocyte Na+ levels, additional groups of burn-injured rats were killed 2, 4, 8, 12, 24, 48, or 72 h or 8 days after burn over 40% TBSA. Hearts were either perfused in vitro (Langendorff) to assess contraction and relaxation defects (n = 7–9 rats per group per time) or perfused to isolate cardiac myocytes to measure intracellular sodium (n = 4 or 5 rats per group per time).
The effects of PKC inhibition on myocardial PKC and Na-K-ATPase activities, myocyte Na+ levels, and cardiac function were also examined in additional groups of rats treated with either vehicle (saline) or calphostin (0.1 mg/kg injected iv over 5 min, 30 min, 6 h, and 21 h after burn injury). The vehicle- and calphostin-treated groups were randomized and killed at baseline (control), 2, 4, or 24 h after burn injury to examine the effects of PKC inhibition on myocardial PKC and Na-K-ATPase activity (n = 4 rats per experimental groups per time period). The effects of PKC inhibition on left ventricular function (n = 7–9 rats per group) and myocyte sodium accumulation (n = 4 rats/group) were studied 24 h after burn injury (a time when burn-related myocardial contractile deficits were maximal); groups included group 1, sham burn + vehicle; group 2, sham burn + calphostin; group 3, burn injury + vehicle; group 4, burn + calphostin (n = 7–9 rats/experimental group). The experimental protocol and times for tissue harvest are summarized in Fig. 1.
Isolated heart perfusion.
To examine the time course of cardiac contraction and relaxation responses to burn injury in the absence of calphostin therapy, subgroups of rats were anticoagulated with sodium heparin (200 U; Elkins-Sinn, Cherry Hill, NJ) and decapitated 2, 4, 8, 12, 24, 48, or 72 h or 8 days after burn over 40% TBSA (n = 7–9 rats/time period). To examine the effects of PKC inhibition on myocardial performance, additional burn-injured rats (and sham-injured rats) treated with calphostin or vehicle after injury were anticoagulated and killed 24 h after burn injury, a time when myocardial defects were maximal after burn injury. The heart from each experimental animal was rapidly removed and placed in a petri dish containing ice-cold (4°C) Krebs-Henseleit bicarbonate-buffered solution (in mM: 118 NaCl, 4.7 KCl, 21 NaHCO3, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11 glucose). All solutions were prepared each day with demineralized, deionized water and bubbled with 95% O2-5% CO2 (pH 7.4, Po2 550 mmHg, Pco2 38 mmHg). A cannula placed in the ascending aorta was connected via glass tubing to a buffer-filled reservoir for perfusion of the coronary circulation at a constant flow rate. Hearts were suspended in a temperature-controlled chamber maintained at 38°C, and a constant flow pump (Ismatec, model 7335-30, Cole-Parmer Instrument, Chicago, IL) was used to maintain perfusion of the coronary artery (ml/min) by retrograde perfusion of the aortic stump cannula. Coronary perfusion pressure was measured, and effluent was collected to confirm coronary flow rate. Contractile function was assessed by measuring intraventricular pressure with a water-filled latex balloon attached to a polyethylene tube and threaded through the apex of the left ventricular chamber. Peak systolic left ventricular pressure (LVP) was measured with a Statham pressure transducer (model P23ID, Gould Instruments, Oxnard, CA) attached to the balloon cannula, and the rates of LVP rise (+dP/dt) and fall (−dP/dt) were obtained with an electronic differentiator (model 7P20C, Grass Instruments, Quincy, MA) and recorded (Grass model 7DWL8P). Left ventricular developed pressure was calculated from peak systolic LVP and left ventricular end-diastolic pressure. Data from the Grass recorder were input into a Dell Pentium computer, and a Grass PolyVIEW Data Acquisition System was used to convert acquired data into digital form.
Cardiac myocyte isolation.
After burn injury (or sham burn), animals from each experimental group (n = 4–6 rats/group) were heparinized, blood samples were collected, and rats were decapitated; hearts were harvested and placed in a petri dish containing ice-cold heart medium [in mM: 113 NaCl, 4.7 KCl, 0.6 KH2PO4, 0.6 Na2HPO4, 1.2 MgSO4, 12 NaHCO3, 10 KHCO3, 20 d-glucose, 10 HEPES; 30 taurine, 2.0 carnitine, and 2.0 creatine with 0.5× minimum essential medium (MEM) and amino acids (50×, GIBCO/BRL 11130-051)]. Hearts were cannulated via the aorta and perfused with heart medium at a rate of 12 ml/min for a total of 5 min in a nonrecirculating mode. Enzymatic digestion was initiated by perfusing the heart with digestion solution, which contained 34.5 ml of the heart medium described above plus 50 mg of collagenase II (Worthington 4177, lot no. MOB3771), 50 mg of bovine serum albumin (BSA, endotoxin free), fraction V (GIBCO/BRL 11018-025), 0.5 ml of trypsin (2.5%, 10×, GIBCO/BRL 15090-046), and 15 μM CaCl2. Enzymatic digestion was accomplished by recirculating this solution through the heart at a flow rate of 12 ml/min for 20 min. All solutions perfusing the heart were maintained at a constant temperature of 37°C. At the end of the enzymatic digestion, the ventricles were removed and mechanically disassociated in 6 ml of enzymatic digestion solution containing a 6-ml aliquot of 2× BSA solution (2 mg BSA fraction V to 100 ml of heart medium). After mechanical dissociation with fine forceps, the tissue homogenate was filtered through a mesh filter into a conical tube. The cells adhering to the filter were collected by washing with an additional 10-ml aliquot of 1× BSA solution (100 ml of heart medium described above and 1 g of BSA fraction V). Cells were then allowed to pellet in the conical tube for 10 min. The supernatant was removed, and the pellet was resuspended in 10 ml of 1× BSA. The cells were washed and pelleted further in BSA buffer with increasing increments of calcium (100, 200, 500 μM, to a final concentration of 1,000 μM). After the final pelleting step the supernatant was removed, and the pellet was resuspended in MEM [prepared by adding 10.8 g of 1× MEM (Sigma M-1018), 11.9 mM NaHCO3, 10 mM HEPES, and 10 ml penicillin-streptomycin, 100×, GIBCO/BRL 1540-122 with 950 ml of MilliQ water]; total volume was adjusted to 1 liter. At the time of MEM preparation, the medium was bubbled with 95% O2-5% CO2 for 15 min and the pH was adjusted to 7.1 with 1 M NaOH. The solution was then filter sterilized and stored at 4°C until use. At the final concentration of calcium, the cardiomyocyte cell number was calculated and myocyte viability was determined.
Measurement of myocyte-derived cytokines.
Myocytes were pipetted into microtiter plates at 5 × 104 cells·ml−1·well−1 (12-well cell culture cluster, Corning, Corning NY) and incubated for 18 h (CO2 incubator at 37°C). Supernatants were collected to measure myocyte-secreted TNF-α, IL-1β, IL-6, and IL-10 (rat ELISA, Endogen, Woburn, MA). We previously examined (22) the contribution of contaminating cells (nonmyocytes) in our cardiomyocyte preparations with flow cytometry, cell staining (hematoxylin and eosin), and light microscopy. We confirmed that <2% of the total cell number in a myocyte preparation was noncardiomyocytes. Since immediately after myocyte isolation our preparations are 98% cardiomyocytes, we concluded that a majority of the inflammatory cytokines measured in the cardiomyocyte supernatant were indeed cardiomyocyte derived (22). Myocyte viability after 24-h incubation was 96–97%.
Cardiac myocyte Na+ levels.
Additional aliquots of cardiac myocytes were loaded with sodium-binding benzofuran isophthalate (SBFI) for 1 h at room temperature in the dark. Myocytes were then suspended in 1.0 mM calcium-containing MEM and washed to remove extracellular dye; myocytes were placed on a glass slide on the stage of a Nikon inverted microscope. The microscope was interfaced with Grooney optics for epi-illumination, a triocular head, phase optics, and ×30 phase-contrast objective and mechanical stage. This InCyt Im2 Fluorescence Imaging System (Intracellular Imaging, Cincinnati, OH) included an imaging workstation and computer. The computer-controlled filter changer allowed alternation between the 340- and 380-nm excitation wavelengths, and images were captured by monochrome charge-coupled device camera equipped with a TV relay lens. InCyt Im2 Image software allowed measurement of intracellular sodium concentrations from the ratio of the two fluorescent signals generated from the two excitation wavelengths (340 nm/380 nm); background was removed by the InCyt IM2 software. The calibration procedure included measuring fluorescence ratio with buffers containing different concentrations of sodium. At each wavelength the fluorescence emissions were collected for 1-min intervals, and the time between data collection was 1–2 min. Since quiescent or noncontracting myocytes were used in these studies, the Na+ levels measured reflect diastolic levels.
All values are expressed as means ± SE. Analysis of variance (ANOVA) was used to assess an overall difference among the groups for each of the variables. Cardiac function determined by the Langendorff preparation (including stabilization data) is expressed as the mean ± SE, and separate analysis were performed for each LVP, maximum +dP/dt (+dP/dtmax), and maximum −dP/dt (−dP/dtmax) as a function of treatment group and coronary flow rate with a repeated-measures ANOVA. Levene's test for equality of variance was used to suggest the multiple comparison procedure to be used. If equality of variance among the four groups was suggested, multiple-comparison procedures were performed (Bonferroni). Probability values <0.05 were considered statistically significant (analysis was performed with SPSS for Windows, version 7.5.1).
Time course of myocyte sodium accumulation and time course of myocardial contractile dysfunction.
Burn injury caused a progressive rise in cardiac myocyte Na+ over the first 24 h after burn as measured by the fluorescent indicator SBFI. As shown in Table 1, cardiomyocyte Na+ levels were significantly elevated as early as 12 h after burn over values measured in sham burn-injured animals (P < 0.05), and a maximal increase in cardiac myocyte Na+ levels was evident 24 h after burn. Myocyte Na+ levels measured 72 h after burn returned to values that were comparable to those measured in sham burn-injured rats and remained at this basal level 8 days after burn injury (Table 1).
Our time course studies examining cardiac contractile function showed significant burn-mediated cardiac contraction and relaxation deficits that were evident 12 h after burn injury, a time consistent with significant myocyte Na+ loading (Table 1). LVP and ±dP/dtmax progressively fell after burn, achieving a nadir 24 h after burn. Left ventricular performance improved 48 h after burn compared with values measured 24 h after burn, but LVP measured 8 days after burn failed to return to values measured in sham burn-injured rats (P < 0.05).
Myocardial PKC activity and Na-K-ATPase activity.
In this study, vehicle-treated burn injury caused a progressive rise in myocardial PKC activity in tissue harvested 2, 4, or 24 h after burn over 40% TBSA (Fig. 2). This progressive change in PKC activity was paralleled by a progressive decrease in Na-K-ATPase activity 2, 4, and 24 h after burn (Fig. 3).
Administration of calphostin, a specific inhibitor of PKC, after burn injury attenuated the burn-related increases in myocardial PKC (Fig. 2) and blunted the burn-related decrease in Na-K-ATPase activity (Fig. 3). Recovery of Na-K-ATPase activity during the postburn period also attenuated the burn-related accumulation of Na+ by cardiomyocytes.
Effects of burn injury on hemodynamic and metabolic function.
All animals survived the experimental period. Twenty-four hours after burn injury, mean arterial blood pressure was lower in group 3 (burn injury + vehicle; 112 ± 8 mmHg) compared with that measured in sham burn-injured rats (group 1, 151 ± 5 mmHg; P < 0.002). Metabolic acidosis occurred after vehicle-treated burn injury, as indicated by the increase in whole blood lactate (3.3 ± 0.4 mM) compared with values measured in sham burn-injured rats (2.2 ± 0.3 mM; P < 0.04). Hematocrit levels fell 24 h after burn injury, likely because of the aggressive fluid resuscitation (Table 2). Plasma cytokines were significantly elevated 24 h after vehicle-treated burn injury (group 3) compared with values measured in sham burn-injured rats (group 1; P < 0.05). While administration of calphostin in burns (group 4) improved mean arterial blood pressure and improved metabolic acidosis compared with values measured in vehicle-treated burns (group 3), these values did not return to the levels measured in sham burn-injured rats. Calphostin administration after burn injury lowered plasma cytokine levels compared with values that were measured in vehicle-treated burns (P < 0.05), but plasma cytokine levels in calphostin-treated burns remained above those measured in sham burns (P < 0.05; Table 2).
Effects of burn injury and calphostin on cardiac inflammation.
Burn injury produced significant pro- and anti-inflammatory responses in the myocardium. as indicated by increased secretion of TNF-α, IL-1β, IL-6, and IL-10. Calphostin therapy after burn injury attenuated cardiac myocyte secretion of the proinflammatory cytokines TNF-α (Fig. 4A), IL-1β (Fig. 4B), and IL-6 (Fig. 4C), as well as the anti-inflammatory cytokine IL-10 (Fig. 4D).
Effects of calphostin treatment on cardiac myocyte Na+ levels and myocardial function.
As shown in Fig. 5, the burn-related rise in myocyte Na+ levels measured 24 h after vehicle-treated burn injury was significantly attenuated by calphostin treatment of burns (P < 0.05). The attenuation of cardiomyocyte Na+ overload and restoration of intracellular Na+ homeostasis in calphostin-treated burns was associated with improved myocardial contraction and relaxation in group 4 compared with left ventricular performance measured in vehicle-treated burn (group 3; P < 0.05). Left ventricular developed pressure (LVP) and ±dP/dt responses (Table 3) were measured in all four experimental groups as the hearts were perfused at constant preload, constant heart rate, and constant coronary flow rate. Additional studies included in vitro perfusion of the heart 24 h after burn injury (Langendorff approach), providing further evidence of cardiac contractile depression as indicated by the reduced LVP and ±dP/dt responses to incremental increases in either preload (Fig. 6A) or increases in perfusate Ca2+ (Fig. 6B). Hearts harvested from calphostin-treated burn-injured rats generated significantly greater levels of LVP and ±dP/dt at all levels of left ventricular preload (Fig. 6A) and better left ventricular performance in response to incremental increases in perfusate Ca2+ (Fig. 6B) compared with values generated by hearts harvested from vehicle-treated burn-injured rats (P < 0.05).
The present study confirmed that administration of a specific PKC inhibitor, calphostin, attenuated the burn-related increase in myocardial PKC activity. Furthermore, calphostin attenuated burn-related myocardial contraction and relaxation defects, and this improved myocardial performance was associated with increased Na-K-ATPase activity and significant attenuation of the burn-related increase in cardiomyocyte intracellular Na+ loading. These data are consistent with our hypothesis that PKC activation in burn injury decreases Na-K-ATPase activity, interrupting Na+ efflux during the postburn course and contributing to cardiomyocyte Na+ overload. These factors contribute, in part, to burn-related myocardial contraction and relaxation defects.
In the present study, intracellular Na+ rose after burn injury, likely because of an increase in anaerobic metabolism, a decrease in intracellular pH, and activation of the Na+/H+ exchange mechanism (2, 18, 30, 41, 44, 57). A rise in intracellular Na+ has been shown to activate the Na-K-ATPase enzyme, and the Na+ gradient regulates the Na+/Ca2+ exchanger, an ion transport system that couples cellular Na+ and Ca2+ transport (11, 16, 20, 30, 41, 44, 50, 54). Aggressive fluid resuscitation from burn injury likely reactivates Na+/H+ exchange, correcting intracellular acidosis but exacerbating cellular Na+/Ca2+ overload. Altered Na+ and Ca2+ handling by the myocyte have been shown to play a pivotal role in myocardial injury and dysfunction in a number of experimental models (21, 44, 45, 47, 49, 51, 58). Further support for the role of myocyte Na+ accumulation in postburn myocardial dysfunction was provided by the time course data in the present study. There was no cardiac contractile depression 2 h after burn, when there was little change in myocyte intracellular Na+ levels. However, progressive Na+ accumulation was followed by progressive myocardial contraction and relaxation defects. These data suggest that Na+ accumulation by the cardiac myocyte likely initiated a cellular signaling cascade that perhaps includes increased myocyte inflammation, as indicated by increased myocyte secretion of TNF-α, IL-1β, and IL-6, culminating in myocardial contractile depression. Pharmacological interventions that limit the rise in intracellular Na+ have been shown to reduce myocardial injury and improve myocardial performance in several experimental models (12, 26, 37, 43). Since the Na-K-ATPase exchanger is the primary regulator of sodium efflux from the cell, a pharmacological agent that increases Na-K-ATPase activity should promote Na+ efflux from the cell, limit injury-related Na+ accumulation, and provide organ protection (13). Data from the present study support this hypothesis with increased myocardial Na-K-ATPase activity after calphostin treatment of burn injury paralleled by reduced myocyte Na+ levels, reduced myocardial inflammation, and improved cardiac contractile performance.
While the Na-K-ATPase transporter plays a pivotal role in regulating cellular ionic homeostasis through its transmembrane transport of Na+ and K+, Na-K-ATPase function is modulated in several cell types by phosphorylation by either PKA or PKC (3, 8, 17, 27, 32). In this regard, PKC activity has been shown to inhibit Na-K-ATPase activity in noncardiac tissue by phosphorylating the enzyme, decreasing affinity of the Na-K-ATPase for sodium, and producing a decrease in activity of the enzyme (4, 6, 28, 59). However, the present study is the first, to our knowledge, to suggest that burn-related changes in myocardial Na-K-ATPase activity are PKC dependent. In our study, the PKC inhibitor calphostin increased myocardial Na-K-ATPase after burn injury, and these data are consistent with studies by Lundmark and colleagues (29), who described that chelerythrine increased Na-K-ATPase activity after ischemic injury in isolated rat hearts. While increased PKC activity after burn injury in our study was paralleled by a fall in Na-K-ATPase activity and impaired cardiac function, increased PKC activity has been associated with both protective and detrimental effects. While PKC translocation from the cytosol to the cell membrane has been shown to play a role in ischemic preconditioning (33, 46), PKC inhibition has been shown to lessen myocardial injury and dysfunction, and these beneficial effects were linked to downregulation of proinflammatory responses (23, 25). Similarly, PKC inhibition has been shown to afford cardioprotection in models of coronary occlusion (25).
While burn-related PKC activation likely contributed to the progressive decrease in myocardial Na-K-ATPase in our study, other factors should be considered. Alterations in membrane lipid content clearly alter the function of membrane-bound enzymes (38). In this regard, Pierce and Dhalla (39) described an association between increased cholesterol content of the sarcolemmal membranes and a decrease in a sarcolemmal Na-K-ATPase activity. Similarly, feeding a cholesterol-enriched diet increased sarcolemmal cholesterol content and depressed Na-K-ATPase activity in adult rats (35). The rise in plasma cholesterol and triglycerides that occurs after burn injury may provide another mechanism by which burn injury alters Na-K-ATPase activity (9, 24, 56). Alternatively, increased proinflammatory activities after burn injury as indicated in our study by a rise in plasma and tissue TNF-α, IL-1β, and IL-6 levels may exert direct inhibitory effects on Na-K-ATPase enzymatic activity, promoting myocyte accumulation of sodium and cellular injury. We showed previously (44) that increased myocyte secretion of TNF-α and IL-1β parallel a rise in myocyte sodium.
Several limitations of the present study must be considered. The changes in myocardial Na-K-ATPase, cardiac myocyte Na+ levels, myocyte cytokine secretion, and left ventricular function parallel one another, but do not indicate causality. We proposed that burn injury initiates a sequence of events that include increased PKC activity, which in turn impaired Na-K-ATPase expression and activity, promoting a subsequent rise in myocyte Na+. The rise in cellular Na+ likely triggered local inflammatory responses, culminating in impaired left ventricular function. Our finding that calphostin (a PKC inhibitor) improved Na-K-ATPase expression and pump activity, lowered myocyte Na+, and improved left ventricular function supports this proposal. However, calphostin may have directly altered cytokine secretion by myocytes, improving cardiac function regardless of calphostin's effects on either PKC or Na-K-ATPase activity or myocyte Na+ levels. Other limitations include the fact that calphostin may have altered several aspects of cellular function in other organs as well as in the heart. While the administration of calphostin in vivo after major burn injury allowed us to examine several aspects of the burn-related sequelae (cell signaling, inflammation, as well as organ or heart function), this in vivo approach was complicated by the potential effects of calphostin on many cell types and organ systems.
In summary, burn injury over 40% of the total body surface area in adult rats increased myocardial PKC activity and decreased Na-K-ATPase activity. These biochemical changes were paralleled by cardiac myocyte accumulation of sodium and myocardial contraction and relaxation deficits that were evident by 12 h after burn. Administration of calphostin, a specific PKC inhibitor, increased postburn myocardial Na-K-ATPase activity, decreased burn-related myocardial sodium overload, and improved myocardial performance. These data suggest that burn-related changes in Na-K-ATPase activity promoted sodium accumulation by the myocyte, which likely initiated a cellular signaling cascade that culminated in myocardial contractile depression. Our data further suggest that inhibition of PKC activity after burn injury provides cardiac protection by preventing myocyte sodium loading that occurs via the PKC-related decrease in Na-K-ATPase activity.
This work was supported by National Institute of General Medical Sciences Grant R01 GM-57054.
↵† Aug. 20, 2007.
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