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J Appl Physiol 94: 1169-1176, 2003. First published November 27, 2002; doi:10.1152/japplphysiol.00778.2002
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Vol. 94, Issue 3, 1169-1176, March 2003

Effect of myocardial volume overload and heart failure on lactate transport into isolated cardiac myocytes

Ronald K. Evans1, Dean D. Schwartz2, and L. Bruce Gladden3

1 School of Human Performance and Recreation, The University of Southern Mississippi, Hattiesburg, Mississippi 39406; and 2 Departments of Anatomy, Physiology, and Pharmacology, and 3 Health and Human Performance, Auburn University, Auburn, Alabama 36849


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to determine lactate transport kinetics in single isolated rat ventricular cardiac myocytes after 1) 8 wk of myocardial volume overload (MVO) and 2) congestive heart failure (CHF). Twenty male Sprague-Dawley rats were assigned to one of four groups: myocardial hypertrophy (MH), MH sham (MHS), CHF, or CHF sham (CHFS). A chronic MVO was induced in the MH and CHF groups by an infrarenal arteriovenous fistula. Postdeath heart and lung weights were significantly greater (P < 0.05) for the MH and CHF groups compared with controls. Isolated cardiac myocytes were loaded with BCECF to determine intracellular pH (pHi) changes after the addition of lactate to the extracellular superfusate. Alterations in pHi with the addition of varied lactate concentrations were attenuated 72-89% by 5.0 mM alpha -cyano-4-hydroxycinnamate. Significant differences (P < 0.05) were found in estimated maximal lactate transport rates between the experimental and sham groups (MH = 19.4 ± 1.1 nmol · µl-1 · min-1 vs. MHS = 15.1 ± 1.1 nmol · µl-1 · min-1; CHF = 20.2 ± 2.0 nmol · µl-1 · min-1 vs. CHFS = 14.0 ± 0.9 nmol · µl-1 · min-1). Western blot analysis confirmed a 270% increase in monocarboxylate symport protein 1 (MCT1) protein content in CHF compared with CHFS rats. The results of this study suggest that MH and CHF induced by MVO engender a greater maximal lactate transport capacity across the cardiac myocyte sarcolemma along with an increase in MCT1 protein content. These alterations would likely benefit the cell by attenuating intracellular acidification during a period of increased myocardial load.

monocarboxylate; monocarboxylate symport protein 1; membrane transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UNDER NORMAL CONDITIONS, lactic acid, an important respiratory substrate for the heart, is transported into the cardiac myocyte and oxidized. In contrast, lactic acid must be transported out of the cell to prevent intracellular acidification during hypoxia or any other condition that increases lactic acid production or diminishes intracellular oxidation of the molecule (2, 6, 23). In either situation, the importance of effective lactic acid transport across the sarcolemmal membrane is evident.

Sarcolemmal lactate transport has been studied in several cardiac preparations, including the intact heart, isolated sarcolemmal membrane vesicles, and isolated cardiac myocytes (5, 18, 20, 24). At physiological pH values, lactic acid is ~99% dissociated into a lactate anion and a proton (H+) and has been shown to traverse cell membranes by three separate pathways. These pathways include 1) free diffusion of the undissociated acid, 2) exchange of the lactate anion for another anion (e.g., Cl- or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) on the band 3 protein, and 3) a H+-monocarboxylate symport protein (MCT) (19). The MCT pathway has been determined to be the major pathway for the transport of lactate across the sarcolemmal membrane (18-20, 25-27) and evidence suggests the presence of at least two separate MCT isoforms in cardiac tissue (13). Studies of carrier-mediated lactate transport kinetics in isolated rat cardiac myocytes have been performed by using BCECF, a fluorescent intracellular pH (pHi) indicator (25, 26). These studies confirmed the 1:1 lactate-to-proton stoichiometry of the transporter and the earlier hypothesis that two transporter isoforms existed in a single rat cardiac myocyte and further revealed differences in substrate and inhibitor specificities of the two transporters (25, 26).

Recently, Jòhannsson et al. (14) investigated the lactate-transport effects of an induced myocardial infarction (MI) and subsequent congestive heart failure (CHF) in rats. Six weeks after the MI, left ventricular end-diastolic pressure was >15 mmHg. At the same time, maximum lactate influx into isolated cardiomyocytes was increased by 250% above that of sham-operated animals. In addition, the MCT1 protein level was increased by 260%. Because glucose transporters were not similarly upregulated, the authors (14) suggested that lactate was an important substrate in these failing hearts.

In the present study, we sought to extend these observations to a different model of CHF, chronic myocardial volume overload (MVO), such as would be present with aortic or mitral valve insufficiency, aortocaval fistula, or aortopulmonary shunt. We chose the infrarenal arteriovenous (A-V) fistula model because it has been shown to be an effective and reproducible model for the study of volume overload myocardial hypertrophy (MH) in the rat (10). After the creation of an A-V fistula, the heart undergoes sustained ventricular volume overload that results in an early and progressive dilatation of the ventricles and eccentric MH (7). MVO also gives rise to several alterations in substrate utilization in the heart, including increased lactate uptake and oxidation. Gratama et al. (11) found a greater net myocardial lactate uptake in lambs with chronic MVO and suggested that the myocardium preferred lactate to free fatty acids during a chronic load. Beaufort-Krol et al. (6) have shown an increase in myocardial lactate oxidation and glycogen breakdown in aortopulmonary shunt lambs at rest and during exercise, primarily due to decreased fatty acid oxidation. The decreased fatty acid oxidation has been related to a decrease in carnitine acetyltransferase activity (1, 6). The ability of a cardiac myocyte to maintain normal function in the presence of a chronic MVO will depend on the adaptations made in response to the previously mentioned alterations in structure and substrate utilization. An increased lactate influx capacity subsequent to a decrease in carnitine acetyltransferase activity and beta -oxidation may enhance respiratory substrate supply during a chronic myocardial load.

We hypothesized that, similar to the results of Jòhannsson et al. (14) for MI-induced CHF, lactate transport capacity across the cardiac myocyte sarcolemmal membrane will be increased after a period of chronic MVO and subsequent CHF. We further hypothesized that the increased lactate influx capacity will be due to an increase in the carrier-mediated component (MCT). Accordingly, the purpose of this study was to determine the lactate transport kinetics across the sarcolemmal membrane of ventricular cardiac myocytes isolated from chronic MVO and sham-operated rats. The chronic MVO group was further divided into two groups: 1) an 8-wk MH group and 2) a CHF group. Furthermore, carrier-mediated lactate transport was differentiated in all groups by utilizing the competitive inhibitor, alpha -cyano-4-hydroxycinnamate (CHC).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Twenty male Sprague-Dawley rats weighing ~150 g before surgical intervention were randomly assigned to one of four groups: MH, MH sham (MHS), CHF, and CHF sham (CHFS). All groups were fed ad libitum and maintained on a 12:12-h light-dark cycle. The Auburn University Institutional Animal Care and Use Committee approved all experimental procedures.

Surgical intervention. A MVO was induced in the MH and CHF groups by opening an infrarenal A-V fistula, as described by Garcia and Diebold (10) with modifications. Briefly, the rat was anesthetized with an injection of pentobarbital sodium (50 mg/kg ip), and a ventral abdominal laparotomy was performed to expose a 20.0-mm portion of the aorta and inferior vena cava at a level ~3.0 mm below the renal arteries. Both vessels were occluded with finger pressure above and below the fistula site, and an 18-gauge short-bevel needle (Becton-Dickenson, 4-1514) was passed through the exposed abdominal aorta and advanced into the vena cava. The needle was withdrawn, and the puncture site was sealed with cyanoacrylate (3M). Age-matched rats (MHS and CHFS) underwent a sham operation consisting of a ventral abdominal laparotomy and exposure of the aorta and inferior vena cava without subsequent fistula procedures. After an 8-wk period of volume overload, the MH group and corresponding sham-operated group were killed in a pair-wise manner (one MH and one MHS) on consecutive days. The CHF group was allowed to progress into CHF and killed in a pair-wise manner (one CHF and one CHFS) on consecutive days. Rapid weight gain (>20 g/day), peripheral edema, and labored breathing were used as death criteria for the CHF animals.

Isolation of cardiac myocytes. Ventricular myocytes were isolated as detailed by Rodrigues and Severson (21) with modifications designed to increase myocyte yield and viability. After application of anesthesia, a ventral laparotomy and thoracotomy were performed, and the hepatic portal vein was cannulated and perfused for 5 min with calcium-free perfusion buffer (in mM: 142.0 NaCl, 13.4 KCl, 20.0 HEPES, pH 7.4, gassed with 100% O2) at a flow rate of 15.0 ml/min. This method was very effective for removing blood from the coronary circulation. After the initial hepatic portal vein perfusion, the heart was excised and weighed with ~5.0 mm of aorta attached, and the aorta was cannulated for retrograde perfusion. A portion of left ventricle (~100 mg) from CHF and CHFS animals was removed and immediately frozen in liquid nitrogen. The tissue was stored at -80°C for subsequent Western blot analysis. The calcium-free perfusion buffer was replaced with isolation solution [Joklik minimal essential medium (in mM): 10.0 HEPES, 1.2 NaHCO3, 1.2 MgSO4, 1.0 DL-carnitine, pH 7.4, gassed with 100% O2] containing collagenase (Worthington type II, 0.5 mg/ml). Approximately 100 ml of collagenase solution was perfused in a single pass at a flow rate of 6.0-7.0 ml/min. Dissociated ventricular cells were sequentially subjected to isolation solution containing increasing CaCl2 (25.0 µM, 250.0 µM, 500.0 µM, and 1.0 mM) in a 37.0°C shaking water bath and allowed to settle for 10-15 min at 37.0°C under 100% O2. During the myocyte isolation procedures, the lungs were removed, blotted dry, and weighed.

BCECF loading. The isolated cardiac myocytes were incubated for 20 min with BCECF-AM (B-1150, Molecular Probes) at a final concentration of 5.0 µM. After the BCECF incubation, the cells were rinsed several times in a Tyrode solution (in mM: 140.0 NaCl, 4.0 KCl, 1.0 CaCl2, 10.0 HEPES, 10.0 glucose, pH 7.4) to remove any exogenous BCECF from the cell suspension. The cells were also allowed a 20-min unloading period in Tyrode solution to remove any uncleaved BCECF-AM from the cell before pHi measurement. The isolated cells were stored at room temperature under oxygenated conditions until use.

Determination of pHi changes. At least three elongated, quiescent cells with a nongranular appearance were selected from each heart for study. pHi changes, after the application of varied concentrations of lactic acid, were assessed as described by Wang et al. (25, 26) with modifications. The isolated BCECF-loaded cardiac myocytes were placed in a small (~22 µl total volume) superfusion chamber (Model RFS-30, Wagner Instruments, Hamden, CT) that allowed a complete chamber fluid change within ~3.0 s at a flow rate of 2.0 ml/min. The chamber was placed on the stage of a microscope (Nikon Diaphot 300) integrated with a fluorescence ratio imaging system (MD 1220, Photon Technologies, Lawrenceville, NJ). A single BCECF-loaded cell was alternately excited at two wavelengths (440 and 500 nm), and the emitted fluorescence was passed through a 535-nm emission filter. pHi changes were calculated on the basis of changes in the ratio of emitted fluorescence at the two excitation wavelengths. All fluorescence measurements were performed at room temperature in Tyrode solution with varied concentrations of lactate added to the superfusate as sodium salts and readjusted to a pH of 7.4. The influx of lactate was determined by superfusing a BCECF-loaded myocyte with Tyrode solution containing extracellular concentrations of lactate (2.0, 5.0, 10.0, and 20.0 mM) while subsequently measuring changes in the 440:500-nm ratio. The initial rates of lactate influx were calculated based on the 440:500-nm ratio change that occurred during the first 20 s after the application of lactate.

Determination of pHi changes in the presence of MCT inhibitor. With the addition of the known inhibitor of proton-linked monocarboxylate transport (CHC), lactate transport via the MCT protein was separated from total lactate transport at each lactate concentration. Initially, the cardiac myocyte was perfused with 5.0 mM CHC in Tyrode solution containing no lactate. The perfusion solution was then changed to Tyrode solution containing the inhibitor and different concentrations of lactate (5.0, 10.0, 20.0 mM). The initial rate of pHi change in the presence of CHC represented the diffusive component because CHC blocks the carrier-mediated (MCT) and the band-3 protein components. The initial rate of fluorescence change attributable to carrier-mediated lactate transport was determined by subtracting the initial rate of pHi change in the presence of CHC from the initial rate of pHi change in the absence of CHC.

Calibration of the fluorescence signal. The change in the 440:500-nm ratio after the addition of lactic acid to the perfusion solution represents a specific change in pHi. The ratio was converted to pH units to allow for quantification of lactate transport. The calibration of the 440:500 ratio was accomplished by superfusing the cell with 7.0 µM nigericin in a physiological solution containing a high KCl concentration: (140.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 8.0 mM dextrose, 10.0 mM HEPES). This solution was titrated to four different pH standard values (pH 6.9, 7.2, 7.4, and 7.9). Nigericin is a H+-K+ ionophore, which equalizes intracellular and extracellular proton concentration so that pHi equals pHo. The change in the fluorescence ratio units analogous to a specific change in pHi units was determined by linear regression analysis by plotting the 440:500-nm ratio against the pH value of the corresponding pH solution.

Determination of intracellular buffering capacity. To allow accurate calculation of lactate transport as a result of changes in pHi, the intracellular buffering capacity of the cardiac myocytes was determined. Intracellular buffering capacity refers to the cell's ability to resist fluctuations in pHi and was determined by the method of Wang et al. (25). Briefly, 2.0 mM butyric acid was added to the extracellular medium perfusing a cardiac myocyte. Butyrate, which has a pK of 4.8 and is lipid soluble, enters the cells rapidly by diffusion and results in pHi changes similar to an equal concentration of lactate, which mostly enters by the MCT pathway. The intracellular butyrate concentration was calculated as follows
[Butyr]<SUB>in</SUB> = [Butyr]<SUB>out</SUB> · Cal<SUB>pH</SUB><SUP>(Y−7.4)</SUP>
where [Butyr]in and [Butyr]out are the intracellular and extracellular concentrations of butyrate, respectively; CalpH is the number of pH units per fluorescence ratio unit, which is equal to the slope of the linear relationship between the 440:500-nm ratio and the change in pH after addition of each pH standard as described above; Y is the new pHi after the application of butyrate; and 7.4 is the pH of the extracellular superfusion medium. Because butyrate is taken up by free diffusion of the undissociated acid, a single proton must accompany each butyrate anion (25), and the buffering capacity (BC) was determined by
BC = [Butyr]<SUB>in</SUB>/&Dgr;pH<SUB>i</SUB>
where Delta pHi indicates the pH units of intracellular acidification caused by the addition of 2.0 mM butyrate to the extracellular medium and the resulting increase in intracellular butyrate concentration.

Calculation of lactate transport. Lactate transport velocity (Vlac) into the cardiac myocyte was calculated on the basis of the following equation (25)
V<SUB>lac</SUB> = Cal<SUB>pH</SUB> (&Dgr;R) · BC
where CalpH is the pH change per unit change in the fluorescence ratio, Delta R is the measured change in the fluorescence ratio per minute for a given lactate concentration, BC is given in millimoles per liter, and Vlac is in nanomoles of lactate per microliter per minute. Where appropriate, nonlinear, least-squares regression analysis (KaleidaGraph, Synergy Software) was used to fit data to the Michaelis-Menten equation. From this fit, the maximal Vlac (Vmax) and the lactate concentration at one-half Vmax (Km) were determined.

Electrophoresis and Western blotting for MCT1. Approximately 100 mg of frozen ventricle were homogenized in 2.0 ml of buffer containing (in mM) 210.0 sucrose, 30.0 HEPES, 2.0 EGTA, 5.0 EDTA, 40.0 NaCl, 2.0 phenylmethylsulfonyl fluoride, pH 7.4, by using a Polytron homogenizer (2 × 15 s at setting 10). The homogenate was mixed with an equal volume of a KCl (500.0 mM)-sodium pyrophosphate (25.0 mM) buffer and incubated on ice for 15 min. Homogenates were centrifuged at 200,000 g for 75 min at 4°C. Pellets were resuspended and homogenized in 0.2 ml of buffer (10.0 mM Tris base, 1.0 mM EDTA, pH 7.4). Protein was determined by the Bio-Rad assay using BSA as a standard.

For immunoblotting, 25.0 µg of ventricular protein from sham and CHF rats were separated on 10% Tris-glycine PAGEr Gold precast gels (Biowhittaker Molecular Applications, Rockland, ME) and transferred to nitrocellulose membranes (trans-blot, BioRad, Hercules, CA). Membranes were stained with Ponceau stain for visualization of protein lanes and quantification of transfer efficiency. Immunoblotting for membrane fractions was performed by using chicken anti-MCT1 polyclonal antibodies (AB1286; Chemicon International, Temecula, CA) and detected by using enhanced chemiluminescence plus (Amersham). Immunoblots were analyzed by using a Bio-Rad FluorS multiimager system and Quantity One software. The density of MCT1 bands in the sham hearts was determined, and the mean was used to compare the band intensity for MCT1 in CHF hearts on the same blot. The intensity for MCT1 in CHF rats was expressed as a percentage of the intensity in the sham-operated rat hearts.

Statistical analysis. A two groups (experimental vs. sham)-by-four lactate concentrations (2.0, 5.0, 10.0, 20.0 mM) split-plot ANOVA was performed for the 8-wk MH (MH vs. MHS) and the CHF (CHF vs. CHFS) groups with repeated measures on the lactate concentration factor. Vmax and Km values, as determined by nonlinear, least-squares regression analysis fit to the Michaelis-Menten equation, were analyzed by independent t-tests. The significance level was set at P < 0.05 for all analyses. All statistical procedures were performed by utilizing SPSS for Windows, release 10.0.5 (SPSS).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Initial experiments were performed in isolated ventricular myocytes from control rats to establish baseline parameters. Isolated myocytes were loaded with BCECF and superfused with Tyrode solution, and the 440:500-nm fluorescence was measured. The addition of lactate (2.0, 5.0, 10.0, and 20.0 mM) to the extracellular superfusion medium resulted in an increase in the measured 440:500-nm fluorescence ratio in isolated ventricular cardiac myocytes (Fig. 1). The increase in fluorescence ratio represents a decrease in pHi. Furthermore, when the superfusion solution was replaced with one containing a lactate concentration of 0.0 mM, the fluorescence ratio returned to baseline, which represented a return to a baseline pHi. The fluorescence ratio increase was found to be linear during the first 20 s after the application of lactate-containing superfusate (Fig. 2). This initial (20 s) increase in fluorescence ratio was utilized to calculate lactate transport.


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  		Fig. 1.   Representative changes in fluorescence ratio (440:500 nm) in response to increasing lactate concentration in the extracellular superfusion medium (Tyrode solution). The superfusion protocol consisted of a 60-s superfusion with 0.0 mM lactate, followed by 60-s superfusion periods with Tyrode solution containing lactate at 20.0, 10.0, 5.0, or 2.0 mM. The chamber was rinsed with Tyrode solution containing 0.0 mM lactate between each lactate concentration, which returned intracellular lactate concentration and intracellular pH (pHi) to baseline levels.



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  		Fig. 2.   Representative figure showing initial (20 s) period of fluorescence ratio change after the application of 20.0 mM lactate. Data were fit by linear regression.

The relationship between the 440:500-nm ratio and the change in pHi (Delta pH) was found to be linear with a slope of 6.3 ± 0.3 pH units per fluorescence ratio unit (Fig. 3). In addition, the pHi of the cardiac myocytes (n = 5) with intact membranes in Tyrode solution (pH 7.4) was determined to be 7.44 ± 0.01, which is in the range of previously reported values (25). After the addition of 2.0 mM butyrate to the extracellular superfusion medium, the intracellular buffering capacity (n = 12) was determined to be 22.5 ± 0.9 mmol · l-1 · pH unit-1. Buffering capacity data from experimental and control animals were not significantly different and were therefore combined.


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  		Fig. 3.   Data depict the linear relationship between pHi and fluorescence ratio. Data (n = 5) were obtained with the addition of 7.0 µM nigericin to the extracellular perfusate titrated to the 4 different pH values (7.6, 7.4, 7.2, 6.9). The slope of this relationship (6.3 ± 0.9 pH units per fluorescence ratio unit) was utilized to calculate lactate transport values.

Lactate transport in the absence of CHC, the known inhibitor of monocarboxylate transport, represents total lactate transport [carrier-mediated component (MCT) and diffusive component] (Fig. 4). The addition of extracellular superfusion solutions containing different lactate concentrations (0.0, 5.0, 10.0, and 20.0 mM) along with 5.0 mM CHC resulted in a linear increase in the initial rate of fluorescence change that represents the diffusive component of lactate transport (Fig. 5). There were no significant differences in the diffusive component of lactate transport in cells isolated from experimental and control animals; therefore, the data for diffusion were subsequently combined. After subtracting the diffusive component from total lactate transport at each lactate concentration, carrier-mediated transport at each lactate concentration was plotted and fit by nonlinear, least-squares regression analysis to the Michaelis-Menten equation (Fig. 5).


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  		Fig. 4.   Total lactate transport [carrier-mediated monocarboxylate symport protein (MCT) component and diffusive component] into cardiac myocytes isolated from congestive heart failure (CHF; ), CHF-sham (CHFS; ), myocardial hypertrophy (MH; ), and MH-sham (MHS; open circle ) animals (n = 5 for each group) at different lactate concentrations. Data were fitted by nonlinear, least-squares regression analysis to the Michaelis-Menten equation plus a linear function.



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  		Fig. 5.   Initial rate (20 s) of fluorescence ratio change of single BCECF-loaded rat cardiac myocytes after the addition of different lactate concentrations in the absence (open circle ) and presence () of 5.0 mM CHC. Lactate transport in the presence of alpha -cyano-4-hydroxycinnamate (CHC) is taken to represent diffusion of the undissociated acid and was fit by linear regression analysis. Subtraction of lactate transport in the presence of CHC from transport in the absence of CHC represents lactate transport via the specific MCT (). Calculated carrier-mediated transport () was fit by nonlinear, least-squares regression analysis to the Michaelis-Menten equation. Because lactate transport in the absence of CHC (open circle ) contains a linear portion (diffusion), the Michaelis-Menten equation plus a linear function was used when fitting those data.

Experimental groups. No significant differences in body weight occurred during the treatment period; however, heart weight, wet lung weight, heart weight-to-body weight ratio, and lung weight-to-body weight ratio were significantly greater in the experimental groups (MH and CHF) compared with their corresponding sham-operated groups (MHS and CHFS) (Table 1). The progressive nature of myocardial changes associated with chronic MVO is illustrated by the fact that the CHF animals had significantly higher heart weight, wet lung weight, heart weight-to-body weight ratio, and lung weight-to-body weight ratio values compared with the MH group (Table 1).

                              
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  		Table 1.   Body and tissue weights in experimental groups

In isolated ventricular myocytes loaded with BCECF, the estimated Vmax across the sarcolemmal membrane was significantly increased by 28% (P < 0.05) in the MH group compared with the MHS group (Fig. 6). This same effect was seen in the CHF myocytes in that there was a 44% increase (P < 0.05) in Vmax in the CHF group compared with the CHFS group (Fig. 7). However, there were no significant differences in the lactate transport values between control and treatment groups at any individual lactate concentration. Furthermore, no differences were seen in the Km values obtained from experimental and control rats.


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  		Fig. 6.   Lactate transport via the MCT pathway into cardiac myocytes isolated from MH (; n = 5) and MHS (open circle ; n = 5) animals at different lactate concentrations. Data were fitted by nonlinear, least-squares regression analysis to the Michaelis-Menten equation to determine maximal lactate transport velocity (Vmax; in nmol · µl-1 · min-1) and Km (in mM) values. * P < 0.05, MH vs. MHS



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  		Fig. 7.   Lactate transport via the MCT pathway into cardiac myocytes isolated from CHF (; n = 5) and CHFS (open circle ; n = 5) animals at different lactate concentrations. Data were fitted by nonlinear, least-squares regression analysis to the Michaelis-Menten equation to determine Vmax (in nmol · µl-1 · min-1) and Km (in mM) values. * P < 0.05, CHF vs. CHFS

To determine whether the increase in Vmax in the CHF group was due to an increase in MCT1 protein expression, immunoblots were performed on membranes from hearts of CHFS (n = 3) and CHF (n = 3) rats. Because of storage problems, tissues from two rats in each group were lost. The MCT1 antibody detected a single band of ~40 kDa in the rat heart membrane (Fig. 8A). MCT1 protein levels were increased 270 ± 37% in the CHF compared with the CHFS hearts (Fig. 8B).


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  		Fig. 8.   Representative Western blot (top) and summary data for MCT1 protein expression (bottom) in CHFS (n = 3) and CHF rats (n = 3). Western blots were repeated 3 times. Values are means ± SE of the 3 experiments. * Significantly different from CHFS (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, chronic MVO increased maximal lactate transport capacity across the ventricular cardiac myocyte sarcolemma under both compensated and decompensated conditions. Furthermore, the increased lactate transport capacity was shown to be the result of an increase in the carrier-mediated component (MCT). Although lactate transport into cardiac myocytes isolated from normal, healthy animals has been well characterized (18, 20, 25-27), this is only the second study to reveal alterations in maximal lactate transport capacity after a period of abnormal heart function. This is the first study, however, to evaluate the alterations in lactate transport during the progression from a compensated to a decompensated state as a result of a chronic MVO induced by A-V fistula.

Volume overload model. The A-V fistula model is a common and reproducible technique for inducing a chronic MVO in the rat (1, 2, 7, 9, 10). MH resulting from a chronic MVO has been shown to result in several alterations in cardiac myocyte structure, function, and substrate utilization (1-4, 6-8, 11, 12, 15-17, 22). In the present study, the increases in heart weight, lung weight, heart weight-to-body weight ratio, and lung weight-to-body weight ratio in the MH and CHF groups were consistent with previous studies (1, 2, 7). Furthermore, the progressive nature of the ventricular dilatation and MH was evident in our study in the form of significant increases in the heart weights and lung weights of the CHF group beyond the 8-wk MH group.

We expected that enhancements in lactate transport capacity would allow the hypertrophied myocardium to meet the increased energy requirements during a period of MVO. Previous investigations of alterations in the hypertrophied myocardium have revealed impairments in long-chain fatty acid uptake and oxidation (2, 6). Furthermore, chronic MVO has also been shown to increase net myocardial lactate uptake and oxidation (6, 11). Possible explanations for the shifting from fatty acid to carbohydrate metabolism include a decreased tissue carnitine content and/or an increase in the expression of fetal myocardial protein isoforms in the hypertrophied myocardium (1-3, 6).

Lactate transport changes. Despite the previous findings of increased lactate uptake and decreased fatty acid oxidation by the hypertrophied myocardium (2, 6, 11), our results do not support the notion of a role for enhanced lactate transport in these metabolic adaptations. If lactate transport changes were important in this scenario, one would expect to see transport differences at low, even resting, lactate concentrations. In the present study, repeated-measures ANOVA did not reveal any significant differences among lactate influx values at the different lactate concentrations. However, when we chose to perform independent sample t-test analysis to compare the mean lactate transport values at each lactate concentration, significant increases (P < 0.05) were revealed in lactate transport at lactate concentrations of 10.0 mM in the MH group and at 20.0 mM in both MH and CHF groups compared with the sham groups. This finding further supports the idea that there were no changes in lactate transport at low lactate concentrations but that there was a trend for significantly faster transport rates at high lactate concentrations.

What is the physiological benefit of an increased maximal lactate transport capacity across the cardiac myocyte sarcolemma? Under certain conditions (e.g., myocardial ischemia, hypoxia, or high myocardial energy demand), glycolysis is stimulated in an attempt to provide adequate tissue concentrations of ATP (13). The increased lactic acid production and subsequent decreased pHi associated with an increase in glycolytic metabolism can inhibit glycolysis and diminish the strength of myocardial contraction (13, 25). Halestrap's group (13, 20, 25) has suggested that the lactate transporter may be working at a level close to its maximal transport capacity in the normal working heart. During conditions such as hypoxia or ischemia, the transporter might actually become saturated and limit the rate of lactate efflux from the cell. During these situations, an enhanced capacity for the transport of lactic acid out of the cell (efflux) at high lactate concentrations may provide a means of attenuating intracellular acidification.

As previously mentioned, this study is the second to demonstrate a functional increase in maximal lactate transport capacity via the MCT pathway after a period of abnormal heart function. Jòhannsson et al. (14) induced a MI by left coronary artery ligation in a group of male Wistar rats. Six weeks after the initial surgery, lactate transport rates were determined by using the BCECF technique. They found significant increases in Vmax values (107 and 42 mmol · l-1 · min1 in infarcted and sham-operated animals, respectively), with no change in Km values. Furthermore, MCT1 protein levels increased by 260% compared with sham-operated rats. In contrast, using the MVO model, we found a 28-44% increase in Vmax, no change in Km, and a 270% increase in MCT1 content in CHF rats. Why the increase in MCT1 content was similar in the two studies, whereas the Vmax increases were much smaller in the present study, is unclear. One possible explanation may be the units used to express the lactate influx. In both our study and that of Jòhannsson et al. (14), lactate influx is normalized to cell volume. It would have been preferable to normalize to cell surface area, especially since it is likely that the cardiomyocytes in both studies were larger after MI or MVO, respectively. For example, because larger cells have a larger volume-to-surface area ratio, the influx rates corrected to surface area for the MH and CHF cardiomyocytes in our study would likely be elevated at each lactate concentration, and the calculated Vmax values would likely be larger as well. We do not know whether cardiomyocyte size would be increased more after volume overload vs. infarction. More importantly, however, the overall results of our study and that of Jòhannsson et al. (14) are qualitatively similar, although there are quantitative differences.

Possible limitations. Although we found higher Vmax values for influx of lactate into cardiac myocytes isolated from volume overload animals, we cannot be absolutely certain that this finding correlates with a similar increase in Vmax for lactate efflux. Utilizing BCECF, Wang et al. (25) examined Vmax and Km values for carrier-mediated lactate efflux and found threefold higher Km values and twofold higher Vmax values for lactate efflux compared with lactate influx, which suggests that the lactate transport via the MCT protein is asymmetrical. However, even if there were no increase in lactate efflux properties, the enhancement of lactate influx could operate to improve cell-to-cell transfer of lactate, such as might occur between an ischemic cardiomyocyte and an adjacent oxygenated cardiomyocyte.

Considerable variability exists in previously reported Vmax and Km values for lactate transport via the MCT pathway in cardiac myocytes. Vmax and Km values have been reported in the range of 2.27-42.0 nmol · min-1 · µl-1 and 2.15-7.1 mM, respectively (14, 20, 25-27). Comparisons of previous results are confounded when species, superfusion solutions, experimental temperatures, and methodologies are considered. In the present study, all experiments were performed by utilizing BCECF at room temperature (23°C) in a Tyrode solution. The only other studies (14, 25) utilizing similar experimental conditions reported Vmax values of 5.21 and 42.0 nmol · min-1 · µl-1 and Km values of 2.74 and 7.1 mM, respectively, in control animals. Vmax and Km values obtained for control animals in the present study are approximately threefold higher than the results of one study (25) and approximately threefold lower than the values of the other (14). Although all studies evaluated cardiac myocytes isolated from rats, the present study utilized male Sprague-Dawley rats, whereas male Wistar rats were used in the previous studies (14) or the strain was not reported (25).

Because total lactate transport includes a diffusive component, a commonly utilized (18-20, 25-27) MCT pathway inhibitor, CHC, was employed in the present study to differentiate carrier-mediated lactate transport from total lactate transport. The MCT pathway was shown to account for 72-84% of the total lactate transport across the cell membrane. CHC is a substituted aromatic monocarboxylate and has been shown to inhibit, to varying degrees, both monocarboxylate transport isoforms in the heart (25-27). One MCT isoform in the rat heart is thought to be MCT1, whereas some uncertainty still exists concerning the other isoform. Prior investigations (26, 27) have determined that one isoform is sensitive to stilbene disulphonates, whereas the other isoform is not. Wang et al. (25) have suggested that CHC is not equally effective at inhibiting both MCT isoforms in heart tissue, especially at physiological temperatures and in the hearts of species that contain greater amounts of the stilbene disulphonate-insensitive MCT isoform. The ability of CHC to block lactate transport via the MCT pathway has been shown to be decreased as the pH gradient increases or as extracellular substrate concentration increases (19, 26). Despite these possible pitfalls, we believe that our results are valid. This belief is supported by the fact that the portion of total lactate transport remaining after CHC inhibition was linear across the range of lactate concentrations employed (see Fig. 5). This linearity suggests a diffusive component and argues against any significant contribution of MCT activity that could not be blocked by CHC. Furthermore, total lactate transport values that are not corrected for a diffusive component clearly demonstrate a trend toward an increase in lactate influx at higher lactate concentrations in the experimental animals compared with controls (Fig. 4). This, along with the increased MCT1 protein content in the experimental animals, supports the suggestion that the increase in Vmax for lactate transport seen after the experimental period is due to increases in the carrier-mediated component (MCT).

In summary, this study suggests that maximal lactate transport capacity across the rat cardiac myocyte sarcolemma is increased after an 8-wk period of MVO. A similar increase in Vmax is apparent after the appearance of signs of CHF. Km values, which are considered an indication of the affinity of the transport protein for the transport substrate, were not significantly increased in the treatment groups compared with the control groups. The higher Vmax values appear to be the result of an increase in the number of MCT1 proteins in the heart, as previously reported for a different model of heart failure (14). The enhanced maximal lactate influx could improve cell-to-cell transfer of lactate from an ischemic cell into an adjacent, oxygenated cardiomyocyte. Furthermore, if our results also imply an increased capacity for lactate efflux, this would allow the hypertrophied myocardium to attenuate intracellular acidification. Further studies to determine lactate efflux kinetics during periods of increased myocardial demand, hypoxia, or ischemia are warranted.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants 1R01-AR-40342 and HL-66956.


    FOOTNOTES

Address for reprint requests and other correspondence: R. K. Evans, School of Human Performance and Recreation, Univ. of Southern Mississippi, 2609 W. 4th St., Box 5142, Hattiesburg, MS 39406 (E-Mail: Ronnie.Evans{at}USM.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.

First published November 27, 2002;10.1152/japplphysiol.00778.2002

Received 26 August 2002; accepted in final form 20 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alaoui-Talibi, ZE, Guendouz A, Moravec M, and Moravec J. Control of oxidative metabolism in volume-overloaded rat hearts: effect of propionyl-L-carnitine. Am J Physiol Heart Circ Physiol 272: H1615-H1624, 1997[Abstract/Free Full Text].

2.   Alaoui-Talibi, ZE, Landormy S, Loireau A, and Moravec J. Fatty acid oxidation and mechanical performance of volume-overloaded rat hearts. Am J Physiol Heart Circ Physiol 262: H1068-H1074, 1992[Abstract/Free Full Text].

3.   Allard, MF, Schönekess BO, Henning SL, English DR, and Lopaschuk GD. The contribution of oxidative metabolism and glycolysis to ATP production in hypertrophied heart. Am J Physiol Heart Circ Physiol 267: H742-H750, 1994[Abstract/Free Full Text].

4.   Alyono, D, Ring WS, Anderson MR, and Anderson RW. Left ventricular adaptation to volume overload from large aortocaval fistula. Surgery 96: 360-367, 1984[Web of Science][Medline].

5.   Baker, SK, McCullagh KJA, and Bonen A. Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle. J Appl Physiol 83: 987-994, 1998.

6.   Beaufort-Krol, GCM, Takens J, Molenkamp MC, Smid GB, Meuzelaar KJ, Zijlstra WG, and Kuipers JRG Increased myocardial lactate oxidation in lambs with aortopulmonary shunts at rest and during exercise. Am J Physiol Heart Circ Physiol 275: H1503-H1512, 1998[Abstract/Free Full Text].

7.   Brower, GL, Henegar JR, and Janicki JS. Temporal evaluation of left ventricular remodeling and function in rats with chronic volume overload. Am J Physiol Heart Circ Physiol 271: H2071-H2078, 1996[Abstract/Free Full Text].

8.   Ferrans, VJ. Morphology of the heart in hypertrophy. Hosp Pract (Off Ed) 18: 67-78, 1983.

9.   Flaim, SF, Minteer WJ, Nellis SH, and Clark DP. Chronic arteriovenous shunt: evaluation of a model for heart failure in rat. Am J Physiol Heart Circ Physiol 236: H698-H704, 1979[Abstract/Free Full Text].

10.   Garcia, R, and Diebold S. Simple, rapid, and effective method of producing aortocaval shunts in the rat. Cardiovasc Res 24: 430-432, 1990[Abstract/Free Full Text].

11.   Gratama, JW, Dalinghaus M, Meuzelaar JJ, Gerding AM, Koers JH, Muskiet FA, and Kuipers JR. Myocardial carbohydrate, ketone, and fatty acid uptake in conscious lambs with aortopulmonary shunts. Pediatr Res 32: 27-32, 1992[Web of Science][Medline].

12.   Grossman, W, Jones D, and McLaurin LP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest 56: 56-64, 1975[Web of Science][Medline].

13.   Halestrap, AP, Wang X, Poole RC, Jackson VN, and Price NT. Lactate transport in heart in relation to myocardial ischemia. Am J Cardiol 80: 17-25, 1997.

14.   Jòhannsson, E, Lunde PK, Heddle C, Sjaastad I, Thomas M, Bergersen L, Halestrap A, Blackstad T, Ottersen OP, and Sejersted O. Upregulation of the cardiac monocarboxylate transporter MCT1 in a rat model of congestive heart failure. Circulation 104: 729-734, 2001[Abstract/Free Full Text].

15.   Katz, AM. Cardiomyopathy of overload. N Engl J Med 322: 100-110, 1990[Web of Science][Medline].

16.   Katz, AM. The cardiomyopathy of overload: an unnatural growth response in the hypertrophied heart. Ann Intern Med 121: 363-371, 1994[Abstract/Free Full Text].

17.   Legault, F, Rouleau JL, Juneau C, Rose C, and Rakusan K. Functional and morphological characteristics of compensated and decompensated cardiac hypertrophy in dogs with chronic infrarenal aorto-caval fistulas. Circ Res 66: 846-859, 1990[Abstract/Free Full Text].

18.   Poole, RC, Cranmer SL, Halestrap AP, and Levi AJ. Substrate and inhibitor specificity of monocarboxylate transport into heart cells and erythrocytes. Biochem J 269: 827-829, 1990[Web of Science][Medline].

19.   Poole, RC, and Halestrap AP. Transport of lactate and other monocarboxylates across mammalian plasma membranes. Am J Physiol Cell Physiol 264: C761-C782, 1993[Abstract/Free Full Text].

20.   Poole, RC, Halestrap AP, Price SJ, and Levi AJ. The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Biochem J 264: 409-418, 1989[Web of Science][Medline].

21.   Rodrigues, B, and Severson DL. Preparation of cardiac myocytes. In: Biochemical Techniques in the Heart, edited by McNeill JH.. New York: CRC Press, 1997, p. 101-115.

22.   Schönekess, BO, Allard MF, and Lopaschuk GD. Proprionyl L-carnitine improvement of hypertrophied heart function is accompanied by an increase in carbohydrate oxidation. Circ Res 77: 726-734, 1995[Abstract/Free Full Text].

23.   Stanley, WC. Myocardial lactate metabolism during exercise. Med Sci Sports Exerc 23: 920-924, 1991[Web of Science][Medline].

24.   Trosper, TL, and Philipson KD. Lactate transport by cardiac sarcolemmal vesicles. Am J Physiol Cell Physiol 252: C483-C489, 1987[Abstract/Free Full Text].

25.   Wang, X, Levi AJ, and Halestrap AP. Kinetics of the sarcolemmal lactate carrier in single heart cells using BCECF to measure pHi. Am J Physiol Heart Circ Physiol 267: H1759-H1769, 1994[Abstract/Free Full Text].

26.   Wang, X, Levi AJ, and Halestrap AP. Substrate and inhibitor specificities of the monocarboxylate transporters of single rat heart cells. Am J Physiol Heart Circ Physiol 270: H476-H484, 1996[Abstract/Free Full Text].

27.   Wang, X, Poole RC, Halestrap AP, and Levi AJ. Characterization of the inhibition by stilbene disulphonates and phloretin of lactate and pyruvate transport into rat and guinea-pig cardiac myocytes suggests the presence of two kinetically distinct carriers in heart cells. Biochem J 290: 249-258, 1993.

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