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Am J Physiol Heart Circ Physiol 277: H1435-H1440, 1999;
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Vol. 277, Issue 4, H1435-H1440, October 1999

Alteration of gene expression for glycolytic enzymes in aerobic and ischemic myocardium

A. James Liedtke and Matthew L. Lynch

University of Wisconsin Hospital and Clinics, Madison, Wisconsin 53792-3248


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this report was to describe mRNA abundance for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and pyruvate dehydrogenase in ischemic and adjacent aerobic myocardium. Mechanical, metabolic, and mRNA data were acquired in a pig model of regulated coronary flow using extracorporeal perfusion. Trials of coronary hypoperfusion included sustained and intermittent exposures of acute ischemia with or without reperfusion. These were compared with a chronic 4-day model of partial coronary stenosis. In ischemic tissues, levels of mRNA, normalized by mRNA for beta -actin, were increased over control values for GAPDH (range 2.7- to 4.6-fold), pyruvate kinase (2.9-fold), and pyruvate dehydrogenase (2.1-fold). It is of interest that increases in mRNA levels over control values were also observed in adjacent aerobic heart muscle from intervention hearts, including 3.6- to 4.5-fold elevations in message for GAPDH and a 2.1-fold increase in signal for pyruvate dehydrogenase. Augmentation in mRNA abundance occurred in as short a time as 40 min of ischemia and was maintained for as long as 4 days in partial coronary stenosis. Whether the former time was of an interval sufficient to affect protein production is problematic, but the latter time was ample to influence enzyme concentration, which may in turn have regulated glycolysis in this condition.

acute ischemia; reperfusion; myocardial hibernation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MYOCARDIAL ISCHEMIA, sometimes in association with postischemic reperfusion, is a potent stimulus to a variety of gene signals for protein production in response to stress. mRNA was reportedly induced or increased for such proteins as beta -adrenergic receptor kinase 1, which inactivates beta -receptors (33); beta 1-adrenergic receptors per se (12); heat shock proteins 70 (16, 24, 35), 72 (29), and 90 (27), which are most intensely expressed in reperfusion, perhaps as a result of secondary oxidative injury; vascular endothelial growth factor and endothelin-1 (1, 9, 32); interleukin-6 and intracellular adhesion molecule (15, 36); insulin-like growth factor II (13); and the Na+/H+ exchanger (4). Other reports have described the appearance of two new heart transcripts and three new stress-inducible genes in response to ischemia (14) and the suppression of mRNA message regulating Gs and Gi proteins, which may explain the observed disruption to the G protein-adenylate cyclase system (28).

We have expanded this survey in a recent report to include an evaluation of the effects of myocardial ischemia to influence the levels of mRNA regulating the proteins of glucose metabolism (5). In animals preselected for increased glucose utilization in myocardium rendered ischemic in several protocols with or without reperfusion, it was noted that gene signal was increased for GLUT-1, GLUT-4, hexokinase, phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase. Gene signal for several of these proteins was upregulated in as short a time as 40 min after ischemic exposure and was maintained for as long as 4 days in a model of partial coronary stenosis. The initial study focused on characterizing the proximal enzymes in the glycolytic pathway as well as the glucose transporters and was a descriptive review surveying mRNA abundance for a select animal in each of the interventional conditions. The purpose of the present study was to describe mRNA levels for the mid-to-distal enzymes in the glycolytic cascade (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and pyruvate dehydrogenase) and included a larger sampling of representative hearts (n = 3 hearts/intervention). Gene signal for these enzymes was normalized by mRNA levels of beta -actin also acquired in aerobic and ischemic myocardium. mRNA data in hearts were collected and contrasted between tissue perfused aerobically in the left circumflex (LCF) circulation and that rendered ischemic with and without reperfusion in the left anterior descending (LAD) circulation. The same interventional states that were previously reported (5) will be reviewed. Our test hypothesis is also the same, i.e., inducibility of gene signals for various glycolytic enzymes occurs as a consequence of exposures to acute ischemic syndromes and/or to coronary perfusion states simulating myocardial hibernation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental conditions. The surgical preparation and protocols are described elsewhere (5). Frozen tissues of myocardium for RNA isolation were obtained from hearts acquired in several previous studies conducted in the laboratory. An intact, working pig heart preparation was employed in which regional coronary flows were controlled and regulated by extracorporeal perfusion. Physiological and metabolic information for the several interventional groups were previously reported (7, 8, 18, 19, 21). Mechanical and metabolic data from the subset of animals representing each of the interventional groups to be reported in this study are listed in RESULTS. Conditions included 1) an aerobic "control" state in which regional coronary flow was maintained at aerobic levels for 60 min (for further details, see Ref. 21); 2) three conditions of acute ischemic syndromes, and 3) two experimental protocols designed to simulate conditions of ischemic myocardial hibernation. The acute ischemic syndromes included two protocols of sustained hypoperfusion (60% reduction from aerobic levels) to the LAD-perfused myocardium for 40 min either with or without accompanying reperfusion (for further description, see Refs. 18 and 19), designated respectively as "ischemia" and "ischemia-reflow." A third protocol of acute intermittent LAD ischemia, termed "intermittent ischemia-reflow," was also employed (7) in which LAD flow was cyclically reduced by 60% for 5 min, reperfused aerobically between cycles for 15 min, rendered ischemic for a total of 4 cycles, and terminally reperfused for 20 min. The two conditions of ischemic myocardial hibernation included an acute protocol of mild regional ischemia (40% decrease in LAD flow) modified after the experiments of Schulz et al. (30, 31) (for further details, see Ref. 8), designated "mild ischemia," and a chronic 4-day model of partial coronary stenosis (21), termed "chronic coronary stenosis." From the original groupings, three hearts were selected from each experimental condition to be surveyed for mRNA abundance in this study.

RNA isolation. Tissue samples (~2 g) from LAD and LCF perfusion beds were quickly excised, freeze-clamped in liquid nitrogen, and stored at -70°C. With the use of TRIzol reagent (Life Technologies, Grand Island, NY) and an improved single-step RNA isolation method, based on Chomczynski and Sacchi (2), total RNA was obtained. The total RNA was quantitated spectrophotometrically at 260 nm. The ratio of absorption at 260 nm to that at 280 nm was >1.6 for all samples.

Electrophoresis and transfer. Equal amounts of total RNA (20 µg) were fractionated by electrophoresis through a denaturing 1% agarose gel with 0.6 M formaldehyde in 1× MOPS. The RNA was transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH) using the Turboblotter system and protocol (Schleicher and Schuell). The transfer was done for 3 h with 20× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). The RNA was fixed to the membrane by ultraviolet cross-linking at 302 nm.

Probe generation. To generate the probes for beta -actin and pyruvate kinase, total RNA from porcine myocardium was reverse transcribed, and the corresponding cDNA was amplified by PCR to obtain short sequences for the NH2-terminal regions of each gene. AMV (avian myeloblastosis virus) Reverse Transcriptase, Tfl DNA polymerase, and the primers for beta -actin were purchased from Promega (Madison, WI). The primers for pyruvate kinase were synthesized by the University of Wisconsin Biotechnology Center (Madison, WI).

The reverse transcription and amplification were carried out using the Access RT-PCR system (Promega). Total RNA (1 µg) was reverse transcribed and amplified in a 50-µl reaction containing 1× AMV/Tfl reaction buffer, 200 µM dNTP, 1 µM primers, 0.1 U/µl AMV Reverse Transcriptase, and 0.l U/µl Tfl DNA polymerase. The MgSO4 concentration was 1 mM for the pyruvate kinase reaction and 0.5 mM for the beta -actin reaction. The reverse transcription and subsequent amplification were performed in a Techne Cyclogene HL-1 thermal cycler. The reactions were incubated at 48°C for 45 min for reverse transcription. Amplification by PCR was followed immediately with 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and elongation at 72°C for 2 min. A final elongation at 72°C for 10 min was added to allow for complete extension of the products.

The PCR products were gel purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The purified PCR products were used directly as probes for Northern analysis. Also, the purified products were cloned into the pCR2.1 vector from Invitrogen (Carlsbad, CA) by using their Original TA Cloning Kit for sequencing and future use. To ensure the fidelity of the PCR products, sequencing of the two inserts was performed by the University of Wisconsin Biotechnology Center (Madison, WI). Sequencing confirmed the identity of the inserts as beta -actin and pyruvate kinase.

The probe for rat glyceraldehyde-3-phosphate dehydrogenase was a generous gift from Dr. J. M. Aiken (University of Wisconsin, Madison, WI). The probe for human pyruvate dehydrogenase E1-alpha -subunit was a generous gift from Dr. Henrik Dahl (Royal Children's Hospital, Melbourne, Australia). The sequences for these nonporcine probes were compared with the sequences for their porcine counterparts, and the sequence similarity was found to be >90%.

Hybridization and detection. The Renaissance Random Primer Fluorescein Labeling Kit from NEN (Boston, MA) was used to generate labeled probes using random hexamers, fluorescein-N6-dATP, and other nucleotides. The labeled probes were denatured at 95°C for 5 min, placed on ice for 5 min, and added to hybridization tubes with the membranes. The hybridization was carried out for 18 h in Church buffer (3) and 50 µg/ml sheared, sonicated herring sperm DNA. The hybridization was performed in a Hybaid hybridization oven at 62°C.

After hybridization, the membranes were washed once at 62°C in 125 mM Na2HPO4, pH 7.2, 0.05 mM EDTA, and 2.5% SDS for 10 min and twice for 10 min each at room temperature in 25 mM Na2HPO4, pH 7.2, 0.05 mM EDTA, and 0.5% SDS. All subsequent washes and incubations were carried out at room temperature. The membranes were then vigorously washed for 5 min in 0.1 M Tris · HCl, pH 7.5, and 0.15 M NaCl. The membranes were blocked for 1 h with gentle agitation in 0.1 M Tris · HCl, pH 7.5, 0.15 M NaCl, and 0.5% blocking reagent. After blocking, the membranes were incubated for 1 h with gentle agitation in 0.1 M Tris · HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent, and 0.1% antifluorescein-alkaline phosphatase conjugate. After incubation, the membranes were vigorously washed four times for 5 min each in 0.1 M Tris · HCl, pH 7.5, and 0.15 M NaCl. The membranes were given two final washes for 5 min each in 0.1 M Tris · HCl, pH 9.5, and 0.1 M NaCl. From the final wash, the membranes were incubated for 5 min with the chemiluminescent substrate CDP-Star. The excess CDP-Star was blotted from the membranes, and the membranes were wrapped in plastic and exposed to film.

Analysis of mRNA data. Analysis of the mRNA data was accomplished with the Bio-Rad model GS-700 Imaging Densitometer and Bio-Rad Molecular Analyst Software (version 1.4.1; Hercules, CA). The signals were quantified within the linear range of the film's sensitivity. The densitometric values were normalized to a signal obtained with the beta -actin probe to account for minor differences in RNA integrity. To ensure that equal amounts of RNA were loaded onto different gels, visual inspection of the gels after they were stained with ethidium bromide was routinely performed. Representative gels that show this loading for beta -actin and glyceraldehyde-3-phosphate dehydrogenase are included in Fig. 1.


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  		Fig. 1.   Ethidium bromide-stained gels before blotting. Total RNA (20 µg) was fractionated by electrophoresis through a denaturing 1% agarose gel with 0.6 M formaldehyde in 1× MOPS. RNA from left anterior descending (LAD) beds for intermittent ischemia (animals 71-13, 71-23, and 71-29), ischemia-reflow (animals 64-3, 64-8, and 64-13), and ischemia (animals 48-14, 48-16, and 48-22) interventions are shown. The gels were transferred and fixed to Nytran membranes and probed for beta -actin (A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; B).

Statistics. The data are expressed as means ± SE. Despite the large variation inherent and anticipated with a small data set of animals per intervention, statistical comparisons for mRNA abundance were made between and among experimental conditions in an effort to identify appreciable and meaningful differences between and among groups. For select comparisons of intervention groups with control hearts, nonpaired Student t-tests were employed. For generic comparisons among all experimental groups, ANOVA was used. Statistical significance was defined for P values <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Performance data for the animal subsets in control and intervention groups are listed in Table 1. Reductions in LAD flow conscribed to the protocol specifics given in METHODS. Average aerobic flow values ranged from 4.4 to 6.1 ml · min-1 · g dry wt-1 (average 5.5 ml · min-1 · g dry wt-1). Coronary perfusion in the chronic coronary stenosis group, which was keyed to in situ resting flow rates after 4 days of partial coronary stenosis, did not appear as an appreciable outlier and was only 10% lower than the average for all groups. Systolic shortenings during ischemia were appreciably compromised from their respective aerobic values in all intervention groups (range -9 to 60% of initial values) and were followed by evidence of mechanical stunning in those hearts that were reperfused. Errors in some subsets were large because of small sample size and, in the case of the mild ischemia group, no doubt reflected the modest nature of the stress. Myocardial oxygen consumption (MVO2) fell by 50% (range 44-55%) in hearts exposed to a 60% reduction in LAD flow and by 10% in the mild ischemia group. In the chronic coronary stenosis group, MVO2 appeared to be maintained at aerobic levels. Exogenous glucose utilization was calculated from data acquired from steady-state infusions of [5-3H]glucose into the LAD arterial perfusate (for further details, see Refs. 7, 8, 18, 20, 21). Direct measurements of glucose metabolism were not obtained in the ischemia (without reflow) group, which involved experiments to describe changes in the radioactive pool size of acetate in moderate myocardial ischemia (19). However, it may be reasonably assumed that glycolysis was increased in this group because, as demonstrated by either indirect or direct estimates of glucose utilization (18, 20), this was the case in other studies using this protocol. Furthermore, in the animals from that group in this study, other demonstrable effects of ischemia were noted (Table 1).

                              
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  		Table 1.   Performance values for mechanical and metabolic functions in hearts surveyed for mRNA abundance encoding messages for glycolytic enzymes

mRNA data are presented in Table 2. Gene signals for glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and pyruvate dehydrogenase are listed for both LAD- and LCF-perfused myocardium. All data were normalized by mRNA data for beta -actin, also measured directly from the same LAD- and LCF-perfused myocardium. This latter signal for cytoskeletal protein is used routinely for calibration of nucleic acid gels. In comparing the ethidium bromide-stained gels with those showing Northern hybridization for beta -actin, we could find no evidence that this signal was altered because of ischemia per se (see examples in Figs. 1 and 2). To facilitate comparisons and increase sample size in Table 2, aerobic values of mRNA in control hearts were combined from LAD- and LCF-perfused tissue to yield a sample size of n = 5 (for technical concerns, one LCF sample was deleted). Nonpaired Student t-test analysis was used here as a surveying tool to evaluate shifts in mRNA abundance against control values. It was noted that in no instance in any of the interventions was there an appreciable decrease in mRNA signal for any of the enzymes during acute or chronic ischemia. Conversely, in several conditions appreciable increases were observed. In LAD-perfused myocardium, these included a 2.7- and 4.6-fold increase in mRNA abundance for glyceraldehyde-3-phosphate dehydrogenase in the mild ischemia and chronic coronary stenosis groups, respectively, a 2.9-fold increase in mRNA levels for pyruvate kinase in the chronic coronary stenosis group, and a 2.1-fold increase in mRNA signal for pyruvate dehydrogenase in the ischemia (without reflow) group. With the exception of the mild ischemia group, all of these increases achieved statistical significance by nonpaired Student t-test analysis but not by ANOVA. Because of the possible influence of LAD ischemia and its effects on adjacent aerobic myocardium, we also interrogated LCF-perfused myocardium for possible shifts in glycolytic mRNA. Surprisingly, mRNA increases were noted here as well. These included 4.5-, 4.3-, and 3.6-fold increases in mRNA for glyceraldehyde-3-phosphate dehydrogenase for the ischemia, mild ischemia, and chronic coronary stenosis groups, respectively, and a 2.1-fold increase in mRNA abundance for pyruvate dehydrogenase in the ischemia (without reflow) group. The shifts in signal for glyceraldehyde-3-phosphate dehydrogenase in the ischemia and mild ischemia conditions over control values met statistical significance by ANOVA. An increase in mRNA signal from either LAD- or LCF-perfused myocardium in select groups occurred with as little as 40 min of ischemic exposure and was maintained for as long as 4 days of altered coronary reserve. The exact mechanism responsible for triggering this gene message remains unknown at this time.

                              
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  		Table 2.   mRNA abundance for three glycolytic enzymes normalized to mRNA levels of beta -actin



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  		Fig. 2.   Northern hybridization of beta -actin and GAPDH from LAD beds for intermittent ischemia (animals 71-13, 71-23, and 71-29), ischemia-reflow (animals 64-3, 64-8, and 64-13), and ischemia (animals 48-14, 48-16, and 48-22) interventions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this report was to evaluate the effect of myocardial ischemia as a stimulus to trigger alterations in gene signal responsible for the glycolytic enzymes located in the mid-to-distal portion of the glycolytic cascade. Glucose metabolism was chosen as a study area because glucose utilization, given a sufficient level of residual coronary washout (26), is almost invariably increased during episodes of myocardial ischemia in both clinical and animal studies. In this report, and in that of a previous study bearing on the same general topic (5), mRNA levels for glycolytic enzymes and glucose transporters were never appreciably reduced from aerobic control values. Conversely, increases in mRNA abundance, sometimes reaching values severalfold higher than control levels, were noted at several conditions of ischemia. In select instances these were accompanied by increases in gene signal in adjacent aerobic myocardium. The changes in mRNA abundance in either perfusion bed were noted most frequently for glyceraldehyde-3-phosphate dehydrogenase and to a lesser extent for pyruvate kinase and pyruvate dehydrogenase. The potency of this ischemic stimulus was reflected by the abrupt onset of the mRNA increase (40 min) and the prolonged nature of its augmentation (4 days).

Critique of the methods. All mRNA data in this study were expressed as a ratio by using the RNA signal for beta -actin as a normalizing factor. beta -Actin is a cytoskeleton protein found ubiquitously in several cell lines and is distributed regionally within cells, possibly as part of a signal transduction pathway for cell motility (11). Certain cytoskeletal proteins in heart muscle (alpha -actinin, desmin, myomesin, spectrin, and tubulin) are reportedly altered in settings of myocardial ischemia (10, 23), but we could find no evidence in the literature that beta -actin is affected. Furthermore, in our study, comparison of ethidium bromide-stained gels (Fig. 1) with Northern hybridization gels for beta -actin (Fig. 2) suggested no shift in beta -actin signal as a function of ischemia per se.

The relationship between gene expression and protein production for the enzymes reported in this study must remain circumspect at this juncture not only because of the short time intervals used for some of the protocols in the acute ischemic groups but also because enzyme concentrations and kinetics were not directly measured. Moreover, the increased transcript signal may indicate an increase in either gene expression or RNA stability. However, by inference, activities in one or more protein systems must have been increased to account for the accelerated glycolysis noted in all intervention groups. It does seem reasonable, given the duration of the protocol in the chronic coronary stenosis model, that augmented protein production secondary to the elevated mRNA levels was plausible and likely in the 4-day model of myocardial hibernation.

The data are expressed as means ± SE. The SEs for certain mechanical, metabolic, and gene data are sometimes large, consistent with the small sample size of the data subsets. Data trends for gene signal are presented as multifold shifts over control values. Nonpaired Student t-test analyses were used only to qualitatively survey comparisons in this report, and, given the small sample size, their absolute significance values should not be overemphasized. The ANOVA by Tukey's test for the LCF comparisons are, however, valid absolute markers.

The method of measuring mRNA for glycolytic proteins varied from that of a previous report, i.e., RT-PCR amplification (5) vs. Northern blot analysis. The previous report characterized the glucose transporters and proximal enzymes in the glucose utilization pathway, whereas the present study focused on the mid-to-distal enzymes in the cascade. Both studies measured mRNA for glyceraldehyde-3-phosphate dehydrogenase, and it was possible to make comparisons between methods. Results varied, showing greater increases in mRNA signal for hearts from the acute ischemic syndromes in the previous study and greater increases in tissue from the hibernation models in this study. Such variation may have resulted from differences in sample size and strategies of normalizing the data. It was also possible to describe variation for individual animals in these two studies because identical hearts were employed in both studies. To facilitate comparisons between studies, in the present report mRNA from LAD tissue in intervention groups was referenced to control tissue as was done previously (5). With the use of Northern blot analysis, mRNA signal appeared more amplified than with the use of RT-PCR (average 5.6:1 vs. 1.5:1 for all intervention groups). Correlation between measuring methods was directionally identical for the ischemia, ischemia-reflow, and intermittent ischemia-reflow groups but not for the mild ischemia and chronic coronary stenosis groups, in which signal increase was noted by Northern blot but not by RT-PCR. Variations here may have been intrinsic to the methods employed and to possible differences in the quality of the extracted RNA.

In general, the present study adds to the information currently being developed about the potent affects of ischemia in evoking elevations of gene message to a variety of protein systems. In the introduction we listed increased gene expression or induction for beta -adrenergic receptor kinase 1, beta 1-adrenergic receptors, heat shock proteins, vascular endothelial growth factor, endothelin-1, interleukin-6, intracellular adhesion molecule, insulin-like growth factor II, and the Na+/H+ exchanger. To this list may now be added myocardial glucose transporters and several enzymes of glycolysis. Webster et al. (34) previously described mRNA increases for glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase in hypoxic skeletal muscle myogenic cultures, but we are unaware of any previous information on ischemic myocardium. Signal increases included those for hexokinase, phosphofructokinase (5), and, in this study, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and pyruvate dehydrogenase. Together with the glucose transporters, these enzymes regulate the key reactions of carbohydrate utilization in well-oxygenated hearts and, with respect to glyceraldehyde-3-phosphate dehydrogenase, the key site of flux inhibition during myocardial ischemia (25). Possible interpretations of the present and previous data from our laboratory are that the mRNA increases represent a random response to some ischemic stimulus, perhaps mechanical or chemical in nature, or rather a purposeful response to upregulate glycolysis in an attempt to preserve residual energy production. It is of added interest that this stimulus was not confined to the hypoperfused LAD circulation but involved the adjacent aerobic myocardium as well. Mäki et al. (22) noted brisk increases in myocardial glucose uptake in normal segments from patients with chronically dysfunctional (hibernating) myocardium. We had previously reported (17) a greater preference for glucose utilization in aerobic myocardium adjacent to ischemic, restricted tissue. It has been postulated that, during ischemia, tethering and other influences place an added burden on aerobic myocardium to compensate mechanically for the dysfunction occurring in adjacent stressed tissue. The increase in gene signal noted in the LCF bed may be part of a regulatory mechanism to achieve energy balance for the increased energy demands that occur during this stress period.


    ACKNOWLEDGEMENTS

This research was supported in part by the Oscar and Rosalie Mayer Cardiovascular Research Fund, the Oscar G. and Elsa S. Mayer Charitable Trust, and the Rennebohm Foundation of Wisconsin as well as National Institutes of Health and American Heart Association grants.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. J. Liedtke, Univ. of Wisconsin Hospital and Clinics, Cardiology Section H6/349, 600 Highland Ave., Madison, WI 53792-3248 (E-mail: ajl{at}medicine.wisc.edu).

Received 20 November 1998; accepted in final form 27 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 277(4):H1435-H1440
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society



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