Eur J Cardiothorac Surg 2003;24:493-501
© 2003 Elsevier Science NL
Restoration of sarcoplasmic reticulum protein level by thyroid hormone contributes to partial improvement of myocardial function, but not to glucose metabolism in an early failing heart
Masahito Minakawa,
Koh Takeuchi,
Kazuo Ito,
Takao Tsushima,
Kozo Fukui,
Shunichi Takaya,
Ikuo Fukuda*
First Department of Surgery, Hirosaki University School of Medicine, Aomori Prefecture, Hirosaki, Japan
Received 8 August 2002;
received in revised form 1 June 2003;
accepted 16 June 2003.
* Corresponding author
e-mail: ikuofuku{at}cc.hirosaki-u.ac.jp
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Abstract
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Objective: In heart failure, sarcoplasmic reticulum Ca2+-ATPase (SERCA2) activity is decreased, resulting in abnormal Ca2+ handling and contractile dysfunction. We have previously reported that impaired glucose transporter (GLUT4) activity was an early indicator of progression of heart failure in pressure overload hypertrophied heart. This study was aimed to examine the contribution of both SERCA2 and glucose metabolism in pressure overload hypertrophied heart. Thyroid hormone, which is known to restore GLUT4 and/or SERCA2 function, was also tested. Methods: Hypertrophied rat heart was created by abdominal aortic banding for 16 and 26 weeks. Then 20–40 µg/kg of 3,5,3'-triiodo-L-thyronine (T3) was administered subcutaneously daily for the last 4 weeks. Hypertrophied myocytes were created by the stimulation of H9c2(2-1) rat heart myoblasts with 2 µmol/L of isoproterenol for 3, 7 and 10 days. Left ventricle function of the hypertrophied rat hearts were measured in Langendorff perfusion. Myocardial protein levels of GLUT4 and SERCA2 in two models were analyzed by Western immunoblotting. Glucose and lactate concentration of cultured medium of myocytes were measured enzymatically to determine the efficacy of glycolysis. Results: Diastolic function (tau) was significantly deteriorated in 16-week heart with significantly lower SERCA2 protein (89.3%) than control. In 26-week heart, both systolic and diastolic function (+dP/dt max, -dP/dt max and tau) was significantly deteriorated. This was associated with significant decrease in both GLUT4 and SERCA2 protein (84.8 and 91.6%, respectively). In cultured hypertrophied myocytes, glycolysis was shifted from aerobic to anaerobic during progression of hypertrophy. GLUT4 protein was significantly decreased at day 7 (45.6% of control). This led to a down-regulation of SERCA2 protein at day 10 (51.8% of control). Although there was no impact of T3 treatment on GLUT4, SERCA2 protein level was almost reversed with partial improvement of myocardial function. Conclusions: We conclude that impairment of both glucose metabolism and SERCA2 function were seen in an early heart failure. Thyroid hormone partially improved myocardial function with successful improvement of SERCA2 protein but no impact on GLUT4 protein expression in hypertrophied rat heart. Restoration of glucose metabolism is a critical step to avoid further progression of heart failure.
Key Words: Myocardial hypertrophy • Glucose transporter • SR Ca2+-ATPase • Glycolysis • Thyroid hormone
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1. Introduction
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Myocardial hypertrophy and heart failure are important risk factors of cardiac surgery. Several events during adaptive remodeling in pressure-overload hypertrophy include an increase in ventricular mass and a switch to immature isoforms of contractile proteins [1]. Metabolic adaptation can occur such as decreased mitochondrial ß-oxidation increased glucose use for energy production, and increased glycolytic enzyme activity [2]. In hypertrophied-failing heart, further derangements related to contractile dysfunction have been reported; these include ineffective intracellular calcium handling [3], impaired excitation–contraction coupling, and a reduced sensitivity of contractile protein to calcium. The derangement of SERCA2 protein in the failing heart has been reported [2]. A more recent report showed that SERCA2 protein level was depressed in an early heart failure model and this defective SR calcium loading contributed to the onset of contractile failure in animals with chronic pressure overload [4].
Glucose that becomes a major substrate in hypertrophied myocytes is not freely permeable across the lipid membrane and requires a specific sarcolemmal membrane protein to facilitate its entrance into the myocytes. Two types of glucose transporters are known to work in myocytes, namely, GLUT1 and GLUT4. GLUT1 is constitutively present in the sarcolemma and mediates basal glucose transport. GLUT4 [5] is mainly located in myocytes, stored in the intracellular pool and translocated from cytosol to membrane in response to insulin stimulation. Glucose transported into the myocytes would be phosphorylated by hexokinase and then incorporated into the glycolytic pathway. Only 2 mol of ATP can be produced by glycolysis from 1 mol of glucose, but this small amount of high energy phosphates is believed to play a pivotal role in maintaining intracellular homeostasis by supplying energy to various ionic channels including SR Ca2+-ATPase (SERCA2) [6] and K+-ATPase, which prevent contracture during ischemia–reperfusion insult [7]. Due to the increased dependence of the hypertrophied heart on exogenous glucose, this impairment may lead to contractile dysfunction through the subsequent dysfunction of calcium regulation. In fact, accumulated data suggest a reduced glucose uptake-phosphorylation [8] in pressure overload hypertrophied heart model and a reduced GLUT4 mRNA content in experimental hypertensive heart. Pharmacological inhibition of glycolysis is known to lead to diastolic dysfunction of the left ventricle in normal heart. We hypothesize that during the development of myocardial hypertrophy, glucose becomes the key substrate for energy production; and in the transition from compensated to decompensated myocardium in a pressure overloaded hypertrophied heart, glucose metabolism would be impaired, and this might thereby lead to contractile dysfunction due to the malfunction of SERCA2. To examine the contribution of both SERCA2 and glucose metabolism to pressure overload hypertrophied heart we used two different hypertrophy models, i.e. aortic banding model (16 week, 26 week) and isoproterenol (2 µmol/L) stimulated cultured myocytes (up to 10 days) to study glucose metabolism including GLUT4 protein expression, SERCA2 protein expression, as well as myocardial function. Thyroid hormone, which is known to restore GLUT4 and/or SERCA2 function, was also tested in the aortic banding models.
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2. Materials and methods
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2.1. Hypertrophied rat heart preparation
All animals received humane care in compliance with the European Convention on Animal Care and investigations were conducted in accordance with the institutional Guidelines for Animal Experiment. Using Wister rats weighing 60–70 g (4–5 weeks old), suprarenal aortic banding was carried out under intraperitoneal (i.p.) pentobarbital sodium (30 mg/kg) anesthesia to create left ventricular hypertrophy. A 0.035-inch guide wire was placed parallel to the suprarenal abdominal aorta, and encircled with a 3-0 silk suture. After the abdominal aorta was ligated with the suture, the guide wire was removed. Mild aortic coarctation was created with the same size, which then creates relatively severer stenosis with animal growth. Aortic banding group was subdivided into two groups: one group was 16 week aortic banding (HT-16wk, n=10), while another was 26 week aortic banding (HT-26wk, n=7). These groups were compared to normal heart (20 weeks old, n=7). In another three groups prepared in the same manner as described above, 3,5,3'-triiodo-L-thyronine (T3) (20–40 µg/kg, Sigma, St. Louis, MO) was administered subcutaneously daily for the last 4 weeks before the animals were sacrificed, grouped as follows: control group (C-T3, n=7), HT-16wk group (HT-T3 16wk, n=6), and HT-26wk group (HT-T3 26wk, n=7). The degree of left ventricular (LV) hypertrophy was assessed by LV/body weight (LV/BW) ratio. Serum T3 concentration was measured by radioimmunoassay.
2.2. Cell culture
Single cell cultures were prepared from subculture of H9c2(2-1) rat heart myoblast (Dainippon Pharm. Co. Osaka, Japan). H9c2(2-1) is a subclone of the original clonal cell line derived from embryonic BD1X rat heart [9]. On arrival from the company, cells were subcultured 2 times on 100-mm diameter culture dishes in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum, 75 unit/ml penicillin G, 75 µg/ml streptomycin and 0.63 µg/ml amphotericin B in a 5% CO2 in air incubator at 37 °C. At the time when the culture dishes became confluent in the second subculture, cells in culture medium of DMEM containing 10% fetal bovine serum and 10% DMSO were stored in liquid nitrogen according to the manufacturer's instructions until the next experimental protocol.
2.3. Hypertrophied myocytes
Cells were rapidly thawed and seeded on the 100-mm culture dishes for Western immunoblotting, and on the 60-mm culture dishes for the glucose and lactate determination. Cells were suspended occupying 60–70% confluence in the dishes initially, and the protocol was started when they reached the state of 90% confluence. Medium was exchanged to medium 199 containing 5% fetal bovine serum, 1.5 µM vitamin B12, 75 units/ml penicillin G, 75 µg/ml streptomycin and 0.63 µg/ml amphotericin B. All cultures were kept at 37 °C in humidified air with sufficient CO2 (about 1%) to maintain pH 7.3. The protocol of stimulation with isoproterenol has been described previously [10]. Medium was not allowed to be alkaline, since catecholamines are unstable at high pH. Isoproterenol was freshly prepared for each experiment as 10 mM stock solution in 1 mM HCl and was diluted to 0.1 mM by medium 199 containing 100 µM ascorbic acid when added to the cultures. Isoproterenol concentration of the medium was maintained at around 2 µM. Cultured myocytes were stimulated with isoproterenol for 3 days (Iso-day3), 7 days (Iso-day7) or 10 days (Iso-day10). These were compared with control myocytes that had been cultured for 3 days without isoproterenol stimulation.
2.4. Isolated heart preparation
Rats prepared for hypertrophied heart model were anesthetized with pentobarbital sodium (30 mg/kg i.p.). After blood sample was collected from inferior vena cava for the analysis of serum T3 concentration and the injection of 1,000U heparin into the inferior vena cava, hearts were rapidly excised and arrested in ice-cold modified Krebs–Henseleit solution, and perfused in the Langendorff mode. The modified Krebs–Henseleit solution consisted of the following (in mM): NaCl 118, NaHCO3 25, glucose 11.1, KCl 4.9, CaCl2 2.7, MgSO4 1.2, KH2PO4 1.2, insulin 10 units/L equilibrated with 95% O2–5% CO2. The whole system was maintained at 37 °C. Isolated hearts were perfused retrogradely via the ascending aorta at a constant pressure of 75–80 cm H2O. The heart was suspended in a sealed chamber from which the coronary effluent was collected. A latex balloon connected to a pressure transducer was inserted through the mitral valve into the left ventricle. LV pressure curve was recorded on polygraph (RTA-1200M, Nihon Kohden Inc., Tokyo, Japan) connected to the pressure transducer. Pacing electrodes were attached to RA and RV of the heart.
After the 15 min of equilibration period, sequential measurements were made with LV end-diastolic pressure (LVEDP) at 5, 10, 20, 30 and 40 mmHg. The maximal rate of contraction (+dP/dt max) and left ventricular developed pressure (LVDP) were used as measures of LV systolic function. The maximal rate of relaxation (-dP/dt max) and isovolumic relaxation time (
: tau) were used as measures of diastolic function. Tau was calculated using a monoexponential curve fitting according to the following formula:
where P0 is the pressure at -dP/dt max, is the calculated time constant, t is the time from P0 to P. All parameters were derived from the pressure tracings. Myocardial oxygen consumption (MVO2) was calculated as follows: MVO2=CF (CaO2-CcsO2)/100/LV g wet weight, where CF is coronary flow, CaO2 is oxygen-content of the perfusate just before heart, and CcsO2 is oxygen-content of coronary effluent. LVDP/MVO2 was used as an index of mechanical efficiency of myocardium.
Thapsigargin was added to the control rat heart with the modified Krebs–Henseleit solution to observe the function in the state in which SERCA2 was specially inhibited. Thapsigargin was dissolved in 100% dimethyl sulfoxide (DMSO) diluted to a final DMSO concentration of 0.01% with the modified Krebs–Henseleit solution. During the stabilization time for 15 min with the LVEDP at 5 mmHg, this solution was perfused by the infusion pump from the side connector just above the aortic cannula at a constant rate of 7.7x10-3 µmol/min. This group was designated Control-TG (n=7), which was an additional control group.
2.5. Western immunoblotting analysis
Myocardial protein level of GLUT4 and SERCA2 were analyzed by Western immunoblotting. From the hypertrophied heart model, LV tissue was pulverized under liquid nitrogen and then homogenized on ice in buffer containing the following (in mM): Tris–HCl (pH 7.5) 20, sucrose 330, EDTA 2, EGTA 0.5, phenylmetylsulfonyl fluoride (PMSF) 1.0, and leupeptin 25 µg/ml. The homogenate was centrifuged to remove particulate debris (1000g at 4 °C for 10 min). The supernatant was diluted with electrophoresis sample buffer containing the following: sodium dodecyl sulfate (SDS) 10% (wt/vol), Tris–HCl 100 mM (pH 8.0), dithiothreitol 0.7% (wt/vol), EDTA 3 mM, and speck of bromophenol blue as a tracking dye.
In the cultured hypertrophied myocytes model, sample was prepared as follows. The cells cultured on 100 mm dish were washed in PBS and lysed in a buffer containing the following: Tris 50 mM (pH 7.4), EDTA 1 mM, EGTA 1 mM, NaF 50 mM, Na orthovanadate 1 mM, Triton X-100 1%, PMSF 1 mM, leupeptin 20 µg/ml, pepstatin 20 µg/ml. This lysate was centrifuged at 15,000g for 20 min at 4 °C, and the supernatant was used as total cell lysate. Total protein content was determined by using the DC protein assay reagent (Bio-Rad, Hercules, CA) with bovine serum albumin for standard.
Equal amounts of sample protein (20 µg/lane) were separated on 10% SDS-polyacrylamide gels. Electrophoretically separated proteins were transferred to nitrocellulose membranes by electroblotting. Blots were blocked for non-specific binding by incubation in 5% non-fat milk for 40 min at room temperature, and then incubated for 3 h with monoclonal mouse anti-rat GLUT4 (1:1000 dilution, Genzyme, Cambridge, MA) or monoclonal mouse anti-SERCA2 (1:2500 dilution, Affinity Bioreagents, Golden, CO). Primary antibody binding was detected with horseradish peroxidase-coupled anti-mouse secondary antibody (1:25 000 dilution, Chemicon International, Temecula, CA) using the enhanced chemiluminescence method (ECL, Amersham, Buckinghamshire, England) according to the manufacturer's instructions. Band intensity was quantified densitometrically using the NIH imaging system (National Institute of Health, Bethesda, Maryland).
2.6. Determination of glucose and lactate level in culture medium
Glucose level in the culture medium was quantified by coupled enzymatic reactions of hexokinase and glucose-6-phosphate dehydrogenase [11]. During these reactions, added nicotinamide adenine dinucleotide (NAD) was reduced to NADH in an amount equimolar to glucose. An increase in absorbance at 340 nm, which is proportional to glucose concentration, was monitored. All values of glucose and lactate were correlated with the total protein content.
2.7. Data analysis
All results were expressed as mean ± standard errors of mean. One-way analysis of variance (ANOVA) followed by Bonferroni test was used for multiple group comparisons. Repeated measure ANOVA followed by Bonferroni test was used to compare the groups in the determination of LV functions. Differences were considered significant at P<0.05. Analyses were performed using commercially available software Statview 5.0 (Abacus Concepts, Berkeley, CA).
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3. Results
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3.1. Hypertrophied heart model
Left ventricle to body weight (LV/BW) ratio became significantly higher in HT-16wk, and this continued to be significantly higher in HT-26wk than Control (Table 1). The administration of T3 did not increase whole heart weight or LV wet weight, but LV/ BW ratio was increased due mostly to the loss of body weight in C-T3. In T3 treated hypertrophied hearts, LV/BW ratio in HT-T3 16wk was not significantly higher than HT-16wk. This became significantly increased in HT-T3 26wk due to both increase of heart weight (developing further hypertrophy) and loss of body weight. Serum T3 concentration was significantly higher in the T3 treated groups, but there were no significant differences among the T3 treated groups.
3.2. Left ventricular function
LV function was assessed in isolated perfused hearts. All data were recorded at a condition of sequential increase in LVEDP from 5 to 40 mmHg (Table 2, Fig. 1
). Although the +dP/dt max in HT-16wk was decreased only when the LVEDP was high at 30–40 mmHg (P<0.05 vs. Control), that in HT-26wk was significantly lower in any pre-load condition (LVEDP; 5–40 mmHg, P<0.05 vs. Control). The LVDP in HT-26wk was significantly lower than Control, but it was not significant in HT-16wk (Fig. 1). In the condition in which SERCA2 was selectively inhibited by thapsigargin, the +dP/dt max and LVDP in Control-TG were significantly worse than control hearts and were similar to that seen in HT-26wk heart. The effect of T3 on LVDP was insignificant in both HT-16wk and HT-26wk (data were not shown).
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Fig. 1. Left ventricular developed pressure (LVDP) was measured at 5, 10, 20, 30 and 40 mmHg of left ventricular end-diastolic pressure (LVEDP). Thapsigargin was administered to the Langendorff perfused normal hearts to observe the functional response when SR Ca2+-ATPase was specially inhibited. LVDP was measured in HT-16wk (closed circle), HT-26wk (open circle), control- thapsigargin (TG) (open triangle) and control (closed triangle).
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The -dP/dt max was not improved by T3 administration. The diastolic isovolumic relaxation (tau, Table 2) was slower in HT-16wk at higher LVEDP. In HT-26wk, tau was significantly slower than Control with significant stiffness of the LV myocardium. The tau was significantly improved by T3 in HT-T3 16wk, suggesting an improvement of diastolic relaxation of LV myocardium, while that was not significant in HT-26wk. The -dP/dt max was not reduced significantly in HT-16wk, and further deterioration was seen in HT-26wk (Table 2). Thapsigargin caused significant diastolic dysfunction (both tau and -dP/dt max) in non-banded rat heart (Table 2).
The work state to energy requirement (LVDP/MVO2) was significantly higher in HT-16wk than Control, suggesting a compensated hypertrophied heart (Table 2). In HT-26wk, however, this index was lower than HT-16wk, suggesting a decompensated hypertrophied heart. Interestingly, T3 could change this mechanical efficiency towards the baseline in HT-T3 16wk, but not in HT-T3 26wk (Table 2). This might be related to the dosage of triiodothyronine: decrease of body weight, increase of metabolism (i.e. increased MVO2).
3.3. Myocardial GLUT4 and SERCA2 content in hypertrophied rat model
GLUT4 protein content decreased gradually with a progression of cardiac hypertrophy from 16 to 26 weeks (Fig. 2A
). On the other hand, the change of SERCA2 protein content was earlier than that of GLUT4 (significant decrease in HT-16wk) (Fig. 2B), and this was associated with mild impairment of LV function, particularly in diastolic function. There was no difference in SERCA2 protein content between HT-16wk and HT-26wk.
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Fig. 2. Western immunoblotting of GLUT4 (A) and SR Ca2+-ATPase (SERCA2) (B) in pressure overload hypertrophied heart. Samples from Control, HT-16 wk, HT-26 wk hearts were loaded into each well on 10% SDS-polyacrylamide gel for the electrophoresis, then the gel was transferred to nitrocellulose paper. Monoclonal mouse anti-rat GLUT4 or SERCA2 were used as primary antibody. Band intensities (bar graphs) were quantified densitometrically using the NIH imaging system. Data are mean ±SE. *P<0.05 vs. control.
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3.4. Glucose and lactate level in culture medium
Glucose concentration in the culture medium was reduced, but lactate release was not changed in Iso-day3 compared with Control, suggesting enhanced glucose uptake (Fig. 3
). In Iso-day7, both glucose level and lactate were significantly higher than control. This may suggest restrictive glucose uptake and promoting anaerobic glycolysis for energy production in the course of developing hypertrophy. In Iso-day10, glucose uptake was further deteriorated (high glucose level in culture media) with an impairment of anaerobic glycolysis (decrease of lactate release).
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Fig. 3. Glucose and lactate concentration of the culture medium. Glucose (solid line) was measured by coupled enzymatic reactions by hexokinase and glucose-6-phosphate dehydrogenase. Lactate (broken line) was measured by enzymatic assay using lactate dehydrogenase converting lactate to pyruvate. Glucose and lactate levels were normalized by total protein. Control (no isoproterenol stimulation), Iso-day3 (isoproterenol stimulation for 3 days), Iso-day7 (isoproterenol stimulation for 7 days) and Iso-day10 (isoproterenol stimulation for 10 days). Data are mean±SE. *P<0.05 vs. Control,  P<0.05 vs. Iso-day3.
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3.5. GLUT4 and SERCA2 content in cultured hypertrophied myocyte model
GLUT4 content (Fig. 4A
) was not altered in Iso-day3 in which aerobic glycolysis was augmented, but then this was significantly reduced at Iso-day7 when anaerobic glycolysis became prominent. On the other hand, SERCA2 content gradually decreased with a progression of hypertrophy over the isoproterenol stimulation (Fig. 4B). Then, it was significantly decreased at Iso-day10, when both aerobic and anaerobic glycolysis was impaired.
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Fig. 4. Western immunoblotting of GLUT4 (A), and SR Ca2+-ATPase (SERCA2) (B) in isoproterenol stimulated cultured myocytes. The cultured myocytes were lysed in an extraction buffer and centrifuged, and then the supernatant was used as total cell lysate. Equal amounts of total cell lysate (20 µg) were loaded into each well on 10% SDS-polyacrylamide gel. Band intensities (bar graphs) were quantified densitometrically. Control (no isoproterenol stimulation), Iso-day3 (isoproterenol stimulation for 3 days), Iso-day7 (isoproterenol stimulation for 7 days) and Iso-day10 (isoproterenol stimulation for 10 days). Data are mean±SE. *P<0.05 vs. Iso-day3, P<0.05 vs. Control.
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3.6. The effect of T3 on the expression of GLUT4 and SERCA2 protein
There was no impact of T3 treatment on GLUT4 level in HT-T3 16wk or HT-T3 26wk (Table 3). However, T3 treatment attained significant improvement of SERCA2 protein levels, which were down-regulated in HT-16wk and HT-26wk. This recovery attained by T3 treatment reached almost normal SERCA2 protein level.
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4. Discussion
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The important finding of this study is that an impairment of glucose metabolism and down-regulation of SERCA2 occurs as early events during the transition of compensated to decompensated hypertrophied heart. Thyroid hormone restored the SR Ca2+-ATPase protein level with partial restoration of myocardial function, but had no impact on GLUT4 protein level.
Abdominal aortic banding created the LV hypertrophy model used in this experiment, and LV hypertrophy had been made chronic so that acute ischemic change or acute hormonal change was not an influence on the process of hypertrophy. The degree of hypertrophy was assessed with LV/BW ratio, and was measured to be 25–40% increased. It is known that in the process of transition to heart failure, diastolic dysfunction occurs first and then finally leads to systolic dysfunction[12]. In this HT-16wk model, diastolic function was significantly impaired, as revealed in the result of -dP/dt max and tau, though systolic functions stayed at the normal level. Systolic functions were significantly deteriorated in HT-26wk model. So, the model used in this experiment was considered to be a transition from compensated hypertrophied heart (HT-16wk) to failing heart (HT-26wk) or early heart failure.
GLUT4 is specifically located in myocytes and mediates the glucose transport from extracellular space to intracellular space (cytosol). This protein is known to be sensitive to insulin stimulation (activation of GLUT4) [5]. Because of the shift in substrate preference from fatty acid to glucose, decreased mitochondrial fatty acid beta-oxidation, increased glycolytic enzyme [2], as a main glucose transporter in myocytes, GLUT4 is considered to be an important molecule for hypertrophied heart to maintain energy production. This is one of the major reasons why hypertrophied myocardium has less tolerance to ischemic insult, and why hypertrophied myocardium is a risk factor for cardiac surgery. In this experiment, we found a mild decrease of GLUT4 protein level in 16-week model and a significant decrease in 26-week model. Down regulation of GLUT4 protein level has been reported previously and it has been an accepted phenomenon, but it remains controversial in the early heart failure model. In a previous report, GLUT4 protein level remained unchanged in an early heart failure model caused by pressure overload, but the glucose uptake and phosphorylation activity (2-deoxy-D glucose 6 phosphates) measured by 31P-NMR spectroscopy were reduced [8]. This functional down-regulation of glucose uptake and phosphorylation was improved by treatment with vanadyl sulfate, which is probably related to a post-transcriptional effect such as an inhibition of phosphotyrosin phosphatase [8]. GLUT4 mRNA content is decreased and glucose uptake ratio in the presence of insulin is also reduced in hypertensive rat heart. A more recent paper suggested that impaired glucose transporter activity was an early indicator of progression of heart failure in pressure overload hypertrophied heart [13]. ATP from glycolysis is important to maintain various protein functions on the sarcolemma such as ATP-sensitive K+ channel [7] and SERAC2 [6], and the impairment of these proteins may cause ionic imbalances. In hypertrophied hearts, increased glycolytic potential may be an adaptive response to relatively compromised coronary flow and oxygen delivery, because glucose oxidation yields more ATP per mole of oxygen than do other substrates. Chemical inhibition of glycolysis with resultant ADP accumulation causes significant impairment of diastolic function in hypertrophied rat hearts. Clearly, many of these phenomena are potentially affected by events occurring during a progression of heart failure. A particular sensitivity of the heart to decreased ATP in hypertrophied heart, where the energy substrate preference is fairly shifted to glucose, probably relates in large part to the contractile apparatus. It is likely that the source of ATP for the myofibrils may be more important than total cellular ATP content. Glycolysis may be most important in this regard [7].
Calcium is an important molecule for myocardial contraction. Several findings of intracellular calcium change in hypertrophied-failing heart have been reported in association with contractile dysfunction, such as impaired calcium handling, and less sensitivity of contractile protein to calcium. The major storage site of intracellular calcium is believed to be the sarcoplasmic reticulum (SR), where both release channel (Ryanodine receptor) and uptake channel (SR Ca2+-ATPase; SERCA2) play a role in maintaining the cytosolic calcium level. Down-regulation of SERCA2 [14] or decrease in activity [15] have been found in decompensated failing heart and might be associated with contractile dysfunction. These alterations due to depressed SERCA2 function could cause enhanced cytosolic calcium extrusion via Na+/Ca2+ exchange [16] as a compensating mechanism in end-stage failing heart. Because it has been shown that sustained elevation of resting Ca2+ leads to activation of serine–threonine phosphatase, including calcineurin, inducing hypertrophy and cell death [17]. Therefore, a decrease in diastolic Ca2+ may in effect reduce the proapoptotic and prohypertrophy signaling. Preventing an increase in intracellular calcium prevents the induction of triggered activity. Over expression of SERCA2 attained by adenovirus gene transfection in failing hearts improved the contractile function and phosphocreatine/ATP ratio [18]. On the other hand, only a partial effect by transgenic expression of SERCA2 is reported in early heart failure model [4]. Glycolytic ATP is very important to maintain SERCA2 function [4,19,20] as well as other ionic channels including K+-ATPase. In the normal heart, although the majority of the energy consumption is due to cross-bridge cycling, relaxation requires an energy expenditure of 15% to remove Ca2+ from cytoplasm. This high level of energy required by SERCA2 reaction is directly related to the magnitude of the Ca2+ gradient across the SR [21]. Increased ADP concentration through the inhibition of glycolysis contributes to diastolic dysfunction in hypertrophied rat hearts [22]. Functional coupling of glycolytic ATP and SR Ca2+-ATPase have been described in normal heart by Xu et al. [6]. We previously showed that in normal heart, inhibition of glycolysis by 2-deoxy-glucose or SERCA2 by thapsigargin caused an increase of intracellular calcium and slower diastolic calcium removal, which were similar to those seen in hypertrophied failing heart. Pyruvate completely preserved myocardial function and intracellular calcium handling during glycolytic inhibition [23]. This is consistent with our data in cultured myocytes in which glucose metabolism was temporally up-regulated (increased aerobic glycolysis), then impaired (anaerobic glycolysis) in combination with down-regulation of SR Ca2+-ATPase protein during the progression of hypertrophy. Although GLUT4 protein level did not change in the aortic banding model, this may not correctly explain the protein activity including glucose uptake and phosphorylation, as we mentioned previously [8]. A temporal decrease of glucose utilization may be enough to affect the SERCA2 activity resulting in sustained elevations of resting calcium, the induction of which may trigger activities such as further myocardial hypertrophy.
The primary effect of thyroid hormone is on gene expression rather than on enzyme activities or transporter processes, and the primary site of action is in the cell nucleus rather than on the plasma membrane. The receptors for these hormones are DNA-binding proteins that possess metal-binding fingers. The binding of thyroid hormone to its receptor converts it into a transcriptional enhancer; specific genes are then expressed. Numerous actions of thyroid hormone have been reported previously such as SERCA2 protein synthesis [14], GLUT4 protein synthesis [24] and improvement of myocardial function [25] as well. We could find an effect of thyroid hormone on SERCA2 protein synthesis, which might be related to the delay in functional deterioration of myocardium in pressure overload hypertrophied heart. However no significant effect was seen on glucose metabolism (consistent decrease of GLUT4 protein). One potential explanation for this is related to the dosage of the thyroid hormone, although we decided this dosage from the previous publication. Because in the thyroid hormone treated heart, body weight was decreased and body metabolism might be increased. This might effectively augment the SERCA2 function, or at least SERCA2 protein expression, but would not augment GLUT4 protein expression.
We conclude that there is functional derangement of both glucose metabolism and SR Ca2+-ATPase in an early failing heart of pressure overload hypertrophied heart. Thyroid hormone restores SR Ca2+-ATPase protein level with no impact on glucose metabolism and incomplete recovery of myocardial function. Utilizing strategies known to restore GLUT4 function and/or glucose metabolism might be useful to slow the progression from compensated hypertrophy to decompensated hypertrophy.
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Acknowledgments
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We thank Dr Mamoru Munakata (Hirosaki, Japan) and Dr Paul Hollister (Misawa, Japan) for helpful discussion and preparation for the manuscript. This study was partly funded by Grants-In-Aid for the Scientific Research (No. 12671290) from the Ministry of Education, Science, and Culture.
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Abstract
1. Introduction
