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Relatively little is known concerning the regulation of uncoupling proteins (UCPs) in the heart. We investigated in the adult rodent heart 1) whether changes in ...
Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor ␣ in the adult rodent heart MARTIN E. YOUNG, SARITA PATIL, JUN YING, CHRISTOPHE DEPRE, HARLEEN SINGH AHUJA,* GREGORY L. SHIPLEY,* STANISLAW M. STEPKOWSKI,† PETER J. A. DAVIES,* AND HEINRICH TAEGTMEYER1 Division of Cardiology, *Department of Integrative Biology, and the †Division of Organ Transplantation, University of Texas Houston Medical Center, Houston, Texas 77030, USA Relatively little is known concerning the regulation of uncoupling proteins (UCPs) in the heart. We investigated in the adult rodent heart 1) whether changes in workload, substrate supply, or cytokine (TNF-␣) administration affect UCP-2 and UCP-3 expression, and 2) whether peroxisome proliferator-activated receptor ␣ (PPAR␣) regulates the expression of either UCP-2 or UCP-3. Direct comparisons were made between cardiac and skeletal muscle. UCP-2, UCP-3, and PPAR␣ expression were reduced when cardiac workload was either increased (pressure overload by aortic constriction) or decreased (mechanical unloading by heterotopic transplantation). Similar results were observed during cytokine administration. Reduced dietary fatty acid availability resulted in decreased expression of both cardiac UCP-2 and UCP-3. However, when fatty acid (the natural ligand for PPAR␣) supply was increased (high-fat feeding, fasting, and STZ-induced diabetes), cardiac UCP-3 but not UCP-2 expression increased. Comparable results were observed in rats treated with the specific PPAR␣ agonist WY-14,643. The level of cardiac UCP-3 but not UCP-2 expression was severely reduced (20-fold) in PPAR␣ⴚ/ⴚ mice compared to wild-type mice. These results suggest that in the adult rodent heart, UCP-3 expression is regulated by PPAR␣. In contrast, cardiac UCP-2 expression is regulated in part by a fatty acid-dependent, PPAR␣independent mechanism.—Young, M. E., Patil, S., Ying, J., Depre, C., Ahuja, H. S., Shipley, G. L., Stepkowski, S. M., Davies, P. J. A., Taegtmeyer H. Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor ␣ in the adult rodent heart. FASEB J. 15, 833– 845 (2001) ABSTRACT

Key Words: diabetes 䡠 fasting 䡠 fatty acids 䡠 hypertrophy 䡠 unloading Very little is known about either the function or the regulation of uncoupling proteins (UCPs) in the adult heart. There are three known members of the family of UCPs. Thermogenin, also known as UCP-1, was first discovered in brown adipose tissue as the principal mediator of nonshivering thermogenesis (1). UCP-1 is 0892-6638/01/0015-0833 © FASEB

a transmembrane protein found in the inner mitochondrial membrane, where it uncouples ATP synthesis and mitochondrial electron transport, dissipating the energy as heat (2). More recently, two genes have been described with high sequence homology to UCP-1— UCP-2 and UCP-3 (3–5)— both of which have been shown to possess uncoupling activity in cells (6). UCP-2 is ubiquitously expressed whereas UCP-3 is highly expressed in skeletal muscle, adipose tissue, and, to a lesser extent, the heart. Although much research has focussed on UCPs in skeletal muscle, the mechanism(s) by which cardiac UCP expression alter(s) require elucidation. There are four primary hypotheses concerning the physiological roles of UCP-2 and UCP-3 (7): thermogenesis, regulation of fatty acid oxidation, regulation of ATP synthesis, and reduction of mitochondrial reactive oxygen species (ROS) generation. Whether UCPs in the heart play a role in one or several of the above mentioned functions is unknown. The roles of UCPs in tissues, such as skeletal muscle, as well as white and brown adipocytes have been investigated by determining the changes in gene expression of the UCPs in response to various stimuli. For example, increases in plasma free fatty acid levels result in increased skeletal muscle UCP expression, suggesting a role in fatty acid utilization and/or prevention of lipotoxicity and insulin resistance (7–9). Thus, determining UCP expression in the heart in response to altered workload (in which ATP demand, substrate utilization, and ROS generation are altered) and/or substrate availability might aid an understanding of the role of cardiac UCPs. In addition, tumor necrosis factor ␣ (TNF-␣) has been shown to increase skeletal muscle UCP expression, suggesting a role in cytokine-induced thermogenesis (10). TNF-␣ levels are known to increase in the failing heart(11), which has also been described as ‘energy starved’ (12). Whether increased TNF-␣ levels result in increased UCP expression and whether the 1 Correspondence: Division of Cardiology,, University of Texas Houston Medical School, 6431 Fannin, MSB 1.246, Houston, TX 77030, USA. E-mail: [email protected]

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latter affects the efficiency of the failing heart are unknown. The purpose of the present study was twofold. First, we wanted to investigate both physiological and pathophysiologic situations under which cardiac UCP-2 and UCP-3 expression might be altered. These included increased and decreased workload and altered substrate availability (e.g., fasting/refeeding, altered dietary fatty acid composition, streptozotocin-induced diabetes), as well as cytokine (TNF-␣) challenge. Second, we were curious about the potential role of peroxisome proliferator-activated receptor ␣ (PPAR␣, a nuclear transcription factor that has been suggested to play a role in skeletal muscle and adipocyte UCP expression) in the changes observed in UCP expression. We 1) measured PPAR␣ expression under conditions in which UCP expression altered; 2) determined the effects of specific activation of PPAR␣ in vivo, with WY-14,643, on UCP expression; and 3) measured the level of UCP expression in PPAR␣⫺/⫺ mice. Where possible, we made direct comparisons between cardiac and skeletal muscle to determine the similarities and differences of UCP control in these two tissues. The results show that during increased availability of fatty acids, the heart increases the expression of UCP-3 only, with no effect on cardiac UCP-2 expression. In contrast, in skeletal muscle, the expression of both UCP-2 and UCP-3 is increased with PPAR␣ activation. UCP-3, but not UCP-2, expression was severely reduced in the heart of PPAR␣⫺/⫺ mice compared to wild-type mice. Together, these findings strongly implicate PPAR␣ as the major regulator of UCP-3, but not UCP-2, expression in the adult rodent heart. During mechanical unloading and pressure overloading of the heart, UCP-2, UCP-3, and PPAR␣ expression all decrease. The results provide new hypotheses for the roles of UCPs in the adult heart.

MATERIALS AND METHODS Animals Male Sprague-Dawley rats (200 –225 g initial weight) were kept in the Animal Care Center of the University of Texas Houston Medical School under controlled conditions (23⫾1°C; 12 h light/12 h dark cycle), and received standard laboratory chow and water ad libitum unless otherwise stated. Changes in workload In the first series of experiments, cardiac unloading was induced by heterotopic transplantation of a rat heart into the abdomen of a recipient rat, as described earlier (13, 14). After 2 wk, the animals were anesthetized and both donor (unloaded) and recipient (control) hearts were removed, freeze clamped, and stored at ⫺80°C prior to RNA extraction. In the second series of experiments, cardiac pressure overload was induced by banding the ascending aorta, with a 20-gauge needle, as described previously (14, 15). In control animals, sham operations were performed without banding of the aorta. Either 7 or 9 days after aortic constriction (to be 834

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specified), the animals were anesthetized, hearts were removed, freeze-clamped, and stored at ⫺80°C. Changes in substrate availability In the first set of experiments, rats were fed either a highcarbohydrate/low-fat (HC/LF) diet or a low-carbohydrate/ high-fat (LC/HF) diet (Purina Mills). These isocaloric diets varied only in the proportion of energy obtained from carbohydrate and fat. The contribution of carbohydrate, fat and protein to total energy available were 71%, 6% and 23% for the HC/LF diet, and 24%, 53% and 23% for the LC/HF diet, respectively. The source of carbohydrate was a combination of sucrose and dextrin, while the source of fat was a combination of corn oil and lard. Nonnutritive fiber was also increased in the LC/HF diet. The length of time in which the rats were fed the special diets is specified for individual experiments. In selected experiments, soleus (skeletal) muscle was removed in addition to the heart. The effects of fasting and refeeding on cardiac gene expression were investigated. Rats were fasted for either 1 or 2 days, after which the heart and soleus muscles were isolated. A subset of fasted rats were refed with the HC/LF diet for an additional 4 days. Diabetes was induced through a single injection of streptozotocin [STZ; 55 mg/kg intravenous (i.v.)]. Control animals were administered with buffer (Hank’s buffer; Life Technologies, Inc., Grand Island, N.Y.) only. Five, 7, 14, or 182 days (6 months) after the initial injection, the animals were anesthetized, hearts were removed, freeze-clamped, and stored at ⫺80°C. Animals were considered diabetic if their blood glucose level was greater than 300 mg per deciliter. Pharmacological interventions To test the effects of specific PPAR␣ activation, WY-14,643 was added to standard powdered Purina rodent chow at a concentration of 0.01% (w/w). Rats were fed a WY-14,643containing diet for 4 days. Control animals received powered rodent chow only. In a separate set of experiments, rats received a single i.v. (tail vein) injection with TNF-␣ (human recombinant, 30 ␮g/kg body weight in 1 ml 0.9% NaCl); controls were injected with NaCl only. Twelve hours later, heart and soleus muscles were removed. As cytokine administration decreases rodent food intake (which can potentially affect gene expression), rats were injected at 07.00 h. During the following 12 h period in the light, rodent ingestion is minimal. To ensure standardized experimental conditions, food was withdrawn from all animals at the time of injection. PPAR␣ⴚ/ⴚ mice Isolated RNA from hearts of age matched wild-type and PPAR␣⫺/⫺ mice was a kind gift from Dr L. Nagy (The Salk Institute for Biological Studies, Gene Expression Laboratory, La Jolla, Calif.). RNA extraction and quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) RNA extraction and quantitative RT-PCR of samples was performed using previously described methods well established in our laboratory (14, 16 –18). Specific quantitative assays were designed from the rat sequences available in GenBank (Table 1). Primers and probes were designed from unconserved sequences of the genes (allowing for isoform

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TABLE 1. Primer and probe sequences used in the quantitative PCR for UCP-2, UCP-3, PPAR␣, PDK-2, PDK-4, muscle CPT-I, iNOS, and cyclophilin Gene

Primer/probe

Sequence

Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe

5⬘-TCATCAAAGATACTCTCCTGAAAGC-3⬘ 5⬘-TGACGGTGGTGCAGAAGC-3⬘ 5⬘-FAM-TGACAGACGACCTCCCTTGCCACT-TAMRA-3⬘ 5⬘-GTGACCTATGACATCATCAAGGA-3⬘ 5⬘-GCTCCAAAGGCAGAGACAAAG-3⬘ 5⬘-FAM-CTGGACTCTCACCTGTTCACTGACAACTTCC-TAMRA-3⬘ 5⬘-TGCTGAGATGGTGACCTACGA-3⬘ 5⬘-CCAAAGGCAGAGACAAAGTGA-3⬘ 5⬘-FAM-AAGTTGTCAGTAAACAGGTGAGACTCCAGCAA-TAMRA-3⬘ 5⬘-ACTACGGAGTTCACGCATGTG-3⬘ 5⬘-TTGTCGTACACCAGCTTCAGC-3⬘ 5⬘-FAM-AGGCTGTAAGGGCTTCTTTCGGCG-TAMRA-3⬘ 5⬘-TCAGCAAGTTCTCCCCGTC-3⬘ 5⬘-ATGAAGTTTTCTCGCAGGCA-3⬘ 5⬘-FAM-TGCTGGATCCGAAGTCTAGAAACTGCTTCAT-TAMRA-3⬘ 5⬘-TTCACACCTTCACCACATGC-3⬘ 5⬘-AAAGGGCGGTTTTCTTGATG-3⬘ 5⬘-FAM-CGTGGCCCTCATGGCATTCTTG-TAMRA-3⬘ 5⬘-ATCATGTATCGCCGCAAACT-3⬘ 5⬘-ACCCATGTGCTCCTACCAGAT-3⬘ 5⬘-FAM-TCAAGCCGGTAATGGCACTGGG-TAMRA-3⬘ 5⬘-GAGAAGCTGAGGCCCAGG-3⬘ 5⬘-ACCTTCCGCATTAGCACAGA-3⬘ 5⬘-FAM-CAGTCTTGGTGAAAGCGGTGTTCTTTG-TAMRA-3⬘ 5⬘-CTGATGGCGAGCCCTTG-3⬘ 5⬘-TCTGCTGTCTTTGGAACTTTGTC-3⬘ 5⬘-FAM-CGCGTCTGCTTCGAGCTGTTTGCA-TAMRA-3⬘

r/m UCP-2a r UCP-3 m UCP-3 r PPAR␣ r PDK-2 r PDK-4 r muscle CPT-I r iNOS r/m Cyclophilin

a

r denotes a rat-specific assay; m denotes a mouse-specific assay; r/m denotes a rat- and mouse-compatible assay.

specificity). The correlation between Ct (the number of PCR cycles required for the fluorescent signal to reach a detection threshold) and the amount of standard was linear over a 5 log range of RNA for all assays (Fig. 1 illustrates the values for rat/mouse UCP-2 and rat UCP-3). The level of transcripts for the constitutive housekeeping gene product cyclophilin was quantitatively measured in each sample to control for sampleto-sample differences in RNA concentration. PCR data are reported as the number of transcripts per number of cyclophilin molecules. Statistical analysis Data are presented as the mean ⫾ se. Statistically significant differences between groups were calculated by the Student’s t test. A value of P⬍0.05 was considered significant.

RESULTS Mechanical unloading and pressure overload both decrease cardiac UCP expression Decreased workload results in significant atrophy over the course of 2 wk, at which time there is a 55% decrease in the heart weight-to-body weight ratio (14). The expression of UCP-2 and UCP-3 were decreased in unloaded hearts (Fig. 2A, B). Pressure overload resulted in cardiac hypertrophy (after 1 wk the heart weight to body weight ratios were 3.42⫾0.06 vs. 2.98⫾0.05 in the experimental and control groups; P⬍0.05). Hypertrophy also decreased the expression of UCP-2 and UCP-3 (Fig. 2C, D). In all hearts, levels of UCP-2 expression were 100-fold higher compared to UCP-3 (Fig. 2). Mechanical unloading decreases cardiac PPAR␣ expression

Figure 1. Sensitivity of the quantitative assays for UCP-2 and UCP-3. Each graph shows the Ct obtained with various amounts of standard RNA molecules. The relation between the number of standard molecules and the Ct was linear over the range investigated (from ⬃103 to ⬃107). CARDIAC UCP GENE EXPRESSION

A possible mechanism by which reduced cardiac workload results in decreased UCP expression is a decrease in the level of PPAR␣ expression. Unloading reduced cardiac PPAR␣ expression by half (0.137⫾0.027 vs. 0.282⫾0.018 in experimental and control groups; P⬍0.05). 835

their specific diets for 9 days after surgery, then the hearts were isolated. Aortic banding resulted in similar degrees of cardiac hypertrophy in both groups (Table 2). Hearts isolated from sham-operated rats fed the LC/HF diet compared to sham-operated rats fed the HC/LF diet possessed significantly higher levels of both UCP-2 and UCP-3 expression (Table 2). However, the fold induction of UCP-3 was far greater than that of UCP-2. As observed during standard laboratory chow feeding, banding significantly reduced the expression of both UCP-2 and UCP-3 when rats were fed the LC/HF diet (Table 2). In contrast, pressure overload did not decrease the cardiac expression of either UCP-2 or UCP-3 for rats fed the HC/LF diet (Table 2). Banding resulted in a significant decrease in PPAR␣ expression for rats fed either diet (Table 2). Furthermore, rats fed the HC/LF diet showed significantly higher cardiac PPAR␣ expression compared to rats fed the LC/HF diet (Table 2). Figure 2. UCP-2 and UCP-3 expression during cardiac unloading and overloading. Altered expression of UCP-2 and UCP-3 in unloaded (A, B) and overloaded (C, D) hearts. Values are shown as the mean ⫾ se for 9 or 10 separate observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P ⬍ 0.05 vs. control.

Pressure overload induced changes in UCP expression are dependent on dietary fatty acids The involvement of PPAR␣ in decreased UCP expression during pressure overload was investigated in two ways. First, we measured the expression of PPAR␣ during overloading. Second, we determined whether the availability of the natural ligand for PPAR␣ (fatty acids) affected pressure overload induced alterations in UCP expression. Therefore, rats were fed one of two diets: either a high-carbohydrate/low-fat (HC/LF) diet or a low-carbohydrate/high-fat (LC/HF) diet. On day 7 after the initiation of the special diets, half of the rats underwent aortic banding, whereas the other half were operated without banding. Rats were maintained on

Increased dietary fat increases cardiac UCP-3, but not UCP-2, expression; heart vs. skeletal muscle To investigate the time course over which cardiac UCP expression changes, rats were fed the LC/HF diet for various lengths of time (1, 2, 4, or 8 days), after which the expression of cardiac UCP-2 and –3 as well as PPAR␣ were measured (Fig. 3). At all time points, feeding with the LC/HF diet had no effect on cardiac UCP-2 expression (Fig. 3A). In contrast, UCP-3 expression was rapidly induced by the LC/HF diet (within 24 h), and this induction was maintained (Fig. 3B). In the case of PPAR␣, the LC/HF diet repressed expression, an effect that was partially normalized after 8 days of continuous feeding (Fig. 3C). We also measured UCP and PPAR␣ expression in soleus muscle (Fig. 3D–F). Feeding of rats with the LC/HF diet resulted in a rapid increase in soleus muscle UCP-2 and UCP-3 expression (Fig. 3D, E). This increase was maintained for the full duration of the feeding study. PPAR␣ expression was not affected by the LC/HF diet (Fig. 3F).

TABLE 2. Effects of banding and altered dietary fatty acid content on body weight (BW), heart weight (HW), hypertrophy (HW/BW ratio), and expression of UCP-2, UCP-3, and PPAR␣

Body weight (g) Heart weight (g) HW/BWa UCP-2/cyclo UCP-3/cyclob PPAR␣/cyclo

HC/LF sham

HC/LF banded

LC/HF sham

LC/HF banded

255 ⫾ 7 0.81 ⫾ 0.03 3.16 ⫾ 0.08 0.278 ⫾ 0.024 0.214 ⫾ 0.041 0.146 ⫾ 0.007

257 ⫾ 6 0.99 ⫾ 0.09* 3.91 ⫾ 0.24* 0.306 ⫾ 0.021 0.345 ⫾ 0.061 0.106 ⫾ 0.014*

267 ⫾ 6 0.85 ⫾ 0.02 3.17 ⫾ 0.07 0.360 ⫾ 0.018* 1.147 ⫾ 0.215*** 0.092 ⫾ 0.006**

262 ⫾ 6 1.02 ⫾ 0.07$ 3.90 ⫾ 0.13$ 0.285 ⫾ 0.019$ 0.719 ⫾ 0.115$ 0.074 ⫾ 0.004***$

a

HW/BW ratio is multiplied by a factor of 1000. UCP-3/cyclo ratio is multiplied by a factor of 100. The table shows the body weight (BW), heart weight (BW), and heart weight to body weight ratio (HW/BW; a marker of cardiac hypertrophy) for sham operated and banded animals fed either a high-carbohydrate/low-fat (HC/LF) diet or a low-carbohydrate/high-fat (LC/HF) diet, as well as the expression of UCP-2, UCP-3, and PPAR␣. Duration of diet feeding was a total of 16 days (7 days before and 9 days after surgery). Values are shown as the mean ⫾ se for 5–10 observations. All gene expression values are normalized against the expression of the housekeeping gene cyclophilin (cyclo). $ * P ⬍ 0.05, ** P ⬍ 0.01 and *** P ⬍ 0.001 vs. HC/LF control. P ⬍ 0.05 vs. LC/HF control. b

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Figure 3. Time course for altered cardiac and soleus muscle UCP-2, UCP-3, and PPAR␣ expression during increased dietary fat content. The time course for altered UCP-2, UCP-3, and PPAR␣ expression in cardiac (A—C) and soleus (D—F) muscle in response to feeding on the low-carbohydrate/high-fat (LC/HF) diet. Values are shown as the mean ⫾ se for five separate observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P⬍0.05, **P⬍0.01, and ***P⬍0.001 vs. control (day zero).

Fasting and refeeding modulates cardiac UCP-3, but not UCP-2, expression; heart vs. skeletal muscle

STZ-induced diabetes increases cardiac UCP-3 expression

Rats were fasted for either 1 or 2 days, after which a subset was refed for an additional 4 days on the HC/LF diet. Fasting had no effect on cardiac UCP-2 expression, although refeeding with the HC/LF diet resulted in a significant decrease in expression (Fig. 4A. In contrast, fasting significantly increased UCP-3 expression by threefold within 24 h (Fig. 4B). This increased UCP-3 expression was severely blunted when fasting continued for 2 days (Fig. 4B). Refeeding returned cardiac UCP-3 expression to normal levels (Fig. 4B). Fasting for either 1 or 2 days significantly lowered the expression of cardiac PPAR␣, an effect that was reversed on refeeding (Fig. 4C). To investigate further differences in UCP regulation between cardiac and skeletal muscle, we also determined the effects of fasting and refeeding on soleus muscle UCP expression. Fasting resulted in significant increases in UCP-2 and -3 expression (Fig. 4D, E, respectively). In the case of UCP-3, this increase observed after 1 day was severely reduced at the second day of fasting, a result similar to that observed in the heart (Fig. 4E, B). Refeeding normalized soleus muscle UCP (both -2 and -3) expression (Fig. 4D, E, respectively). Soleus muscle PPAR␣ expression was not affected by either fasting or refeeding (Fig. 4F).

STZ-induction of diabetes through pancreatic ␤-cell destruction resulted in significant elevations in plasma glucose levels at 5 (3.12-fold; P⬍0.001), 7 (4.45-fold; P⬍0.001), 14 (3.18-fold; P⬍0.001), and 182 (5.00-fold; P⬍0.001) days after STZ injection (compared to agematched controls). UCP-2 expression in the heart was not affected by STZ-induced diabetes (Fig. 5A). However, cardiac UCP-3 expression increased rapidly in STZ-induced diabetes and remained elevated at all time points investigated (Fig. 5B). The highest fold induction in cardiac UCP-3 expression occurred 14 days after the initial STZ injection (6.4-fold). Despite a maintenance in the induction of UCP-3 at day 182 (4.6-fold), there was a substantial decrease in the absolute level of cardiac UCP-3 expression in both control and STZ diabetic animals (Fig. 5B). Although there was no significant difference in cardiac UCP-2 expression with respect to time, there was a trend for older animals to show reduced expression (Fig. 5A). Thus, cardiac UCP expression declined with age. We also investigated the effects of STZ-induced diabetes on cardiac PPAR␣ gene expression. After either 5 or 7 days of diabetes, there is no effect on PPAR␣ expression (Fig. 5C). After 14 days of diabetes there is a decrease in PPAR␣ expression, although this effect is

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Figure 4. Effects of fasting and refeeding on cardiac and soleus muscle UCP-2, UCP-3, and PPAR␣ expression. The effects of fasting (either 1 or 2 days) and refeeding on UCP-2, UCP-3, and PPAR␣ expression in cardiac (A—C) and soleus (D—F) muscle. Animals were refed the high-carbohydrate/low-fat (HC/LF) diet. Values are shown as the mean ⫾ se for four or five separate observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P⬍0.05, **P⬍0.01, and ***P⬍0.001 vs. control (day zero).

not significant (Fig. 5C). However, there is a significant decrease in PPAR␣ expression at 182 days after the induction of diabetes (Fig. 5C). Age alone had no effect on cardiac PPAR␣ expression (Fig. 5C). The PPAR␣ agonist WY-14,643 alters heart and soleus muscle gene expression Rats were fed either control diet or diet containing the PPAR␣ agonist WY-14,643 (0.01% w/w) for 4 days, after which cardiac and soleus muscle UCP-2, UCP-3, PPAR␣, PDK-2, PDK-4, and muscle CPT-I expression were measured. WY-14,643 caused an increase in soleus muscle UCP-2 expression, with no effect on cardiac UCP-2 expression (Fig. 6A). Both cardiac and soleus muscle UCP-3 expression was increased by WY-14,643 (3.5- and 13.4-fold, respectively; Fig. 6B). PPAR␣ expression was not affected by WY-14,643 in either heart or soleus muscle (Fig. 6C). To determine the effectiveness of WY-14,643 feeding, we measured pyruvate dehydrogenase kinase-4 (PDK-4) and muscle-specific carnitine palmitoyltransferase-I (CPT-I) expression, genes 838

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known to be induced by PPAR␣ activation (19, 20). PDK-4 and muscle CPT-I expression increased in response to WY-14,643 feeding in both heart and soleus muscle (Fig. 6C, F). PDK-2, whose expression is not believed to be regulated by PPAR␣ (19), was not affected by WY-14,643 (Fig. 6D). TNF-␣ decreases cardiac UCP-2, UCP-3, and PPAR␣ expression We investigated whether cytokine exposure alters cardiac gene expression by injecting rats with TNF-␣. TNF-␣ administration (30 ␮g/kg; i.v.) resulted in a significant decrease in cardiac PPAR␣ expression after 12 h (Fig. 7C). Reduced PPAR␣ expression in response to TNF-␣ was accompanied by decreased cardiac UCP-2 and UCP-3 expression (Figs. 7A, B). Similarly, TNF-␣ administration resulted in decreased soleus muscle UCP-3 and PPAR␣ expression (Fig. 7B, C). In contrast, soleus muscle UCP-2 expression increased (Fig. 7A). To determine the effectiveness of TNF-␣ administration, cardiac and soleus muscle inducible nitric oxide syn-

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Figure 5. Time course for altered cardiac UCP-2, UCP-3, and PPAR␣ expression by streptozotocin-induced diabetes. The effects of streptozotocin (STZ; 55 mg/kg i.v.) -induced diabetes on cardiac UCP-2 (A), UCP-3 (B), and PPAR␣ (C) expression either 5, 7, 14, or 182 days after the initial injection. Values are shown as the mean ⫾ se for five separate observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. age-matched controls.

thase (iNOS) expression was also measured. TNF-␣ increased both cardiac and soleus muscle iNOS expression to similar extents (Fig. 7D). Genetic mutation of the PPAR␣ gene decreases expression of cardiac UCP-3, but not UCP-2 We investigated whether PPAR␣ was essential for the expression of cardiac UCP-2 and UCP-3 by comparing wild-type and PPAR␣⫺/⫺ mouse hearts. The level of cardiac UCP-2 and UCP-3 in wild-type mice were relatively similar (Table 3). There was a 20-fold lower level of cardiac UCP-3 expression in PPAR␣⫺/⫺ mice compared to wild-type hearts (Table 3). No differences were observed in the level of UCP-2 expression in hearts isolated from PPAR␣⫺/⫺ mice compared to those isolated from wild-type mice (Table 3). DISCUSSION We show that cardiac UCP-2 and UCP-3 expression changes in response to altered physiological and pathoCARDIAC UCP GENE EXPRESSION

physiologic states; in the case of UCP-3, these changes are regulated by PPAR␣. In contrast to soleus muscle, cardiac UCP-2 expression does not significantly change in response to high-fat feeding, fasting, diabetes, or WY-14,643 treatment, all of which lead to PPAR␣ activation. However, UCP-3 expression increases in cardiac and soleus muscle under the same conditions. Expression of both UCP-3 and PPAR␣ decrease during cardiac mechanical unloading and pressure overloading. Mutation of the PPAR␣ gene results in a near complete inhibition of cardiac UCP-3 expression, with no effect on UCP-2 expression. These results suggest that in the adult rodent heart, UCP-3 expression is regulated by PPAR␣ whereas UCP-2 expression is not. Cardiac UCP-2 expression decreased during mechanical unloading and pressure overloading. Furthermore, decreased UCP-2 expression during pressure overload was dependent on the presence of dietary fatty acids. Decreased dietary fatty acid intake alone reduced cardiac UCP-2 and UCP-3 expression. These results suggest that UCP-2 expression in the heart is regulated, at least in part, by a fatty acid-dependent, PPAR␣-indepen839

Figure 6. Effects of the specific PPAR␣ agonist WY-14,643, on cardiac and soleus muscle UCP-2, UCP-3, PPAR␣, PDK-2, PDK-4, and muscle CPT-I expression. The effects of feeding animals with a diet containing WY-14,643 (0.01% w/w) on cardiac and soleus muscle UCP-2 (A), UCP-3 (B), PPAR␣ (C), PDK-2 (D), PDK-4 (E), and muscle CPT-I (F) expression. Values are shown as the mean ⫾ se for six separate observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. tissue-matched control.

dent mechanism. Last, we show that TNF-␣ reduces the expression of both UCP-2 and UCP-3 in the heart, suggesting that TNF-␣ induced UCP expression in the failing heart is not involved in energetic dysfunction. The results of the present study must be interpreted in a wider context. The uncoupling of mitochondrial electron transport from ADP phosphorylation causes a collapse of the proton gradient across the inner mitochondrial membrane and thereby limits ATP generation through oxidative phosphorylation. Instead, the potential energy ‘stored’ by the gradient is liberated as heat, an essential process in nonshivering thermogenesis (1, 2). Such a proton leak is catalyzed by UCPs. Potential roles for UCP-2 and UCP-3 have been suggested for skeletal muscle and adipose tissue, primarily from studies investigating regulation of their expression in response to different stimuli. For example, cold exposure, thyroid hormone, TNF-␣, elevated dietary fat composition, insulin-dependent diabetes mellitus, and specific PPAR agonists have all been shown to increase skeletal muscle UCP expression (8 –10, 21–23). In contrast, exercise training lowers skeletal muscle UCP expression (23). These results, plus consideration of the sheer mass of skeletal muscle in the body, have implicated skeletal muscle UCPs in the processes of heat generation, obesity, and perhaps maintenance of 840

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insulin sensitivity (9, 24, 25). However, the role(s) of UCPs in the adult heart have not been addressed and are more difficult to rationalize, which gives rise to the following considerations. Altered cardiac workload affects both UCP-2 and UCP-3 expression In response to increased or decreased workload, the heart increases glucose utilization and decreases fatty acid utilization (26 –28). This change is mirrored by changes in the expression of several genes encoding metabolic enzymes, including decreases in the expression of PDK-4 (M. E. Young et al., unpublished observation), muscle-specific CPT-I, and medium chain acyl CoA dehydrogenase (MCAD) in the heart (14, 29). All three genes are regulated by PPAR␣ in the heart (19, 20, 30, 31). The present study has found that PPAR␣ expression decreases during both unloading and overloading (see Results section and Table 2), which is responsible for the observed changes in PDK-4, CPT-I, and MCAD. Likewise, cardiac expression of both UCP-2 and UCP-3 decreased during unloading and overloading (Fig. 2). This decrease in UCP expression depended on the presence of fatty acids, as pressure overloaded hearts isolated from animals

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feeding and fasting caused induction of both UCP-2 and UCP-3 in soleus muscle (Figs. 3 and 4); previously reported studies have shown both UCP-2 and UCP-3 expression to increase in skeletal muscle during STZinduced diabetes (21). The fold induction of UCP-3 in soleus muscle was greater than that of the heart during high-fat feeding and fasting. Specific activation of PPAR␣ through feeding with WY-14,643 resulted in a similar pattern of UCP expression observed with elevation of plasma fatty acid levels: increased UCP-3 expression in both heart and soleus muscles, with the largest induction in the latter, whereas UCP-2 was induced only in soleus, not heart, muscle (Fig. 6). Cardiac UCP-3, but not UCP-2, expression is dependent on PPAR␣ signaling in the adult rodent heart

Figure 7. Effects of TNF-␣ administration on cardiac and soleus muscle UCP-2, UCP-3, PPAR␣ and iNOS expression. The effects of TNF-␣ injection (30 ␮g/kg; i.v.) on cardiac and soleus muscle UCP-2 (A), UCP-3 (B), PPAR␣ (C), and iNOS (D) expression. Values are shown as the mean ⫾ se for six separate observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P ⬍ 0.05 and **P ⬍ 0.01 vs. tissue-matched control.

fed a low-fat diet (HC/LF) did not possess decreased UCP expression compared to diet-matched controls, despite a decrease in PPAR␣ expression (Table 2). These results initially suggested to us that PPAR␣ acts in the decreased UCP expression during hypertrophy only in the presence of its ligand (fatty acids). Similarly, the decreased PPAR␣ expression during unloading could potentially result in decreased UCP expression. Consistent with the decrease observed in UCP expression during pressure overload, exercise training, which is associated with cardiac hypertrophy, reduces UCP expression in the heart (as well as in skeletal muscle) (23). Fatty acid availability influences both cardiac and skeletal muscle UCP expression High-fat feeding, fasting, and insulin-dependent diabetes mellitus would be expected to activate PPAR␣ (30, 32, 33). In certain experiments, a direct comparison was made between expression of UCPs in heart and a slow-twitch skeletal muscle (soleus) that is relatively similar to the heart (34, 35). Still, there were differences between the muscles. High-fat feeding, fasting, and diabetes all increased the expression of cardiac UCP-3 expression but had no effect on UCP-2 expression (Figs. 3–5). These results agree with a recent study that investigated cardiac UCP expression during diabetes and fasting (36). In contrast to the heart, high-fat CARDIAC UCP GENE EXPRESSION

Table 4 summarizes the relationship between PPAR␣ and the expression of cardiac UCP-2 and UCP-3, as observed in the present study. Decreased PPAR␣ expression during cardiac unloading and overloading occurs in concert with decreased expression of UCP-2 and UCP-3. Furthermore, the decreased UCP expression during hypertrophy is dependent on the presence of fatty acids in the diet, providing evidence for the hypothesis that this decrease is related to the reduced PPAR␣ expression. Similarly, on low-fat feeding (either postfasting or comparing the HC/LF and LC/HF feeding effects), when PPAR␣ activation should be reduced, UCP-2 and UCP-3 expression both decrease (Fig. 4 and Table 2). However, situations in which PPAR␣ is stimulated (high-fat feeding, fasting, diabetes, and WY14,643 feeding) increase the expression of cardiac UCP-3, with no effect on cardiac UCP-2 expression. These observations are consistent with the hypothesis that UCP-3 expression is regulated by PPAR␣. One possible explanation for these observations was that in the adult rat heart, UCP-2, but not UCP-3, expression is maximal through PPAR␣ signaling. Thus, a further stimulation of PPAR␣ would have no effect on UCP-2 expression, yet reduced PPAR␣ signaling would reduce UCP-2 expression. To investigate this hypothesis further, we determined the level of expression of cardiac UCP-2 and UCP-3 in PPAR␣⫺/⫺ mice. In this model, expression of various PPAR␣-regulated genes is decreased (37). Because we find reduced expression of TABLE 3. Expression of cardiac UCP-2 and UCP-3 in PPAR␣⫺/⫺ and wild-type mice

UCP-2/cyclo UCP-3/cyclo

Wild-type

PPAR␣⫺/⫺

0.248 ⫾ 0.034 0.193 ⫾ 0.074

0.259 ⫾ 0.076 0.010 ⫾ 0.002*

a Expression of UCP-2 and UCP-3 in wild-type and PPAR␣⫺/⫺ hearts. Values are shown as the mean ⫾ se for four (wild-type) or five (PPAR␣⫺/⫺) separate observations. All values are normalized against the expression of the housekeeping gene cyclophilin (cyclo). * P ⬍ 0.05 vs wild-type.

841

TABLE 4. Relationship between PPAR␣ and the expression of UCP-2 and UCP-3 in the hearta PPAR␣ (expression/ligand UCP-2 availability) expression

Model

Unloading Pressure overload TNF␣ administration PPAR␣⫺/⫺ mouse High-fat feeding Fasting STZ-induced diabetes WY-14,643 treatment

UCP-3 expression

2Expression 2 Expression 2 Expression 2 Expression 1 Ligand 1 Ligand

2 2 2 ⫽ ⫽ ⫽

2 2 2 2 1 1

1 Ligand 1 Ligand

⫽ ⫽

1 1

a 1 represents an increase, 2 represents a decrease, and ⫽ represents no change.

UCP-3, but not UCP-2, in PPAR⫺/⫺ mice, these results provide evidence that UCP-2 expression in the adult rodent heart is not regulated by PPAR␣. Instead, there must be a fatty acid-dependent, PPAR␣-independent mechanism modulating cardiac UCP-2 expression, which is maximally activated under normal physiological circumstances. PPAR␥, another member of the family of PPAR transcription factors, does not appear to regulate UCP-3 expression in the same way as PPAR␣ in the heart. Treatment of adult rats with the PPAR␥ agonist troglitazone (0.1% w/w in the diet for 4 days), has no effect on UCP-3 (nor UCP-2) expression in the heart (M. E. Young et al., unpublished observation). This lack of an effect by troglitazone treatment (which did induce known PPAR␥-regulated genes in the skeletal muscle) on cardiac UCP gene expression is most likely due to the very low abundance of PPAR␥ in the heart, as compared with both adipose tissue and skeletal muscle. TNF-␣ reduces cardiac UCP expression TNF-␣ is known to cause contractile dysfunction (38), and the expression of TNF-␣ increases in the failing heart (11). Furthermore, TNF-␣ antagonism appears to have beneficial effects in subjects with heart failure (39). TNF-␣ has been shown to induce UCP expression in skeletal muscle (10). As overexpression of UCPs might limit mitochondrial ATP production, we hypothesized that TNF-␣-induced cardiac UCP expression could result in the contractile dysfunction observed previously. However, administration of TNF-␣ to adult rats caused a significant reduction in cardiac UCP expression (Fig. 7). Whether reduced cardiac UCP expression in response to TNF-␣ administration was due to a direct effect of TNF-␣ on the cardiomyocyte or to a systemic effect cannot be determined in the present study. It is also still possible that local TNF-␣ generation by the cardiomyocyte, as observed in failing myocardium, induces UCP expression. 842

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Complexity of cardiac and skeletal muscle UCP gene expression regulation There are obvious differences in the regulation of UCP-2 and UCP-3 expression, some of which are summarized in Table 4. Under physiological conditions, cardiac UCP-2 expression is ⬃100-fold greater than that of UCP-3. Expression of UCP-3 is regulated by PPAR␣ in the adult rodent heart, whereas UCP-2 is not. On the second day of exposure to elevated fatty acid levels (for both the high-fat feeding and fasting experiments), both cardiac and soleus muscle UCP-3 expression fall transiently (Figs. 3 and 4). As fatty acid exposure continues, UCP-3 expression increases again (Fig. 3). Increased age was associated with decreased cardiac UCP expression. In addition, TNF-␣ increases soleus muscle UCP-2 expression while decreasing cardiac UCP-2 expression (Fig. 7). Thus, multiple mechanisms for the regulation of UCP expression must operate in heart and skeletal muscle. These might include altered expression and/or activity in PPAR␣ (as observed in the heart for the present study), PPAR␣ dimerization partners (e.g., RXR) or coactivators (e.g., PGC-1), which confer specificity between UCP-2 and UCP-3 promoter regions, or even as yet unidentified PPAR␣independent mechanisms (40 – 42). A study published during the preparation of this manuscript suggests that cardiac UCP-2 expression is regulated by PPAR␣ (43). In this study, neonatal cardiomyocytes were cultured in the presence of triiodothyronine, fatty acids or WY14,643, resulting in increased UCP-2 expression. From these observations, it was concluded that increased cardiac UCP-2 expression on birth was due to combined stimulation of the thyroid hormone receptor and PPAR␣. However, at birth there is a substantial increase in mitochondrial biogenesis, which might explain the increased expression of a mitochondrial protein such as UCP-2 (44). It should be noted that the results observed in the present study are not consistent with altered mitochondrial biosynthesis (e.g., acute pressure overload results in increased mitochondrial biogenesis whereas UCP expression decreases; 45). It is possible that neonatal cardiomyocytes in culture possess a specific factor (e.g., a PPAR␣ dimerization partner or coactivator) that is not present in the adult heart. Increased fatty acid availability reduces cardiac PPAR␣ expression The present results suggest that conditions associated with increased cardiac fatty acid utilization (high-fat feeding, fasting, and diabetes) result in decreased PPAR␣ expression, a mechanism not acutely observed in skeletal muscle (Figs. 3–5). Two hypotheses can be drawn regarding the potential mechanism by which this phenomenon occurs. First, increased signaling through PPAR␣ might cause the altered expression of a protein that affects the expression of PPAR␣. This autoregulation mechanism would therefore be PPAR␣-dependent. A second, PPAR␣-independent mechanism can

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be postulated wherein fatty acids activate a specific pathway that regulates the expression of PPAR␣. Thus, the first mechanism is fatty acid independent and PPAR␣ dependent whereas the latter mechanism is fatty acid dependent and PPAR␣ independent. To investigate which mechanism was responsible for the observed results, we used a specific PPAR␣ agonist (WY-14,643) that would lead to PPAR␣ activation in the absence of elevated fatty acid levels. WY-14,643 increased cardiac and skeletal muscle UCP-3, PDK-4, and muscle CPT-I expression, suggesting PPAR␣ was activated. In contrast to fatty acids, WY-14,643 feeding had no effect on cardiac PPAR␣ expression (or soleus muscle PPAR␣ expression). The results are consistent with the hypothesis that a fatty acid-dependent mechanism is responsible for the observed down-regulation of cardiac PPAR␣. Further evidence toward this hypothesis is the observation that hearts isolated from the obese Zucker rat, a model of insulin resistance in which plasma fatty acid levels are elevated, have severely reduced PPAR␣ expression (46). It could be hypothesized that repression of PPAR␣ expression by fatty acids serves as a mechanism to prevent excessive fluctuations in PPAR␣ signaling in this tissue during altered fatty acid availability. Potential roles of UCPs in the adult rodent heart The present study has focused on uncovering the mechanisms by which gene expression of the uncoupling proteins is regulated in the adult heart during both physiological and pathophysiological conditions. Determination of UCP protein levels (requiring antibody generation) and altered mitochondrial function is beyond the scope of the present study. It is believed that elucidating the mechanisms by which the heart alters the expression of the UCPs might aid in the understanding of their function. Possible major functions for UCPs in the heart include thermogenesis, regulation of fatty acid oxidation, regulation of ATP synthesis, and reduction of ROS formation. Due to its size (compared with the body), the heart is unlikely to play a role in global thermogenesis. UCP-2 expression is relatively insensitive to substrate availability, suggesting it plays little function in cardiac fatty acid utilization (which increases, for example, in diabetes). However, UCP-3 is highly responsive to fatty acids. Whether UCP-3 induction helps prevent lipotoxicity when fatty acid levels are high or acts in an antioxidant capacity when oxidative metabolism increases (fatty acids cannot be metabolized anaerobically) are distinct possibilities. When the heart requires increased efficiency, for example, during increased or decreased workload or substrate limitation (as observed during low dietary fatty acid availability, as fatty acids are the primary fuel for the heart under physiological conditions), cardiac UCP expression decreases. If cardiac UCPs affect mitochondrial ATP synthesis, decreased UCP expression would be expected to increase mitochondrial efficiency. Last, evidence suggests that uncoupling proCARDIAC UCP GENE EXPRESSION

teins can act as antioxidants (47, 48). The heart is a continuously contracting organ, with high oxidative metabolism. The observation that cardiac UCP-2 expression is relatively high (even higher than skeletal muscle) and relatively constant during various conditions suggests that it has an essential, constitutive role in the heart, such as prevention of ROS formation. Indirect evidence for this hypothesis comes from the observations that TNF-␣ administration, which causes a rapid decrease in cardiac UCP-2 and UCP-3 expression, is known to increase oxidative stress (49). Limitations of the study Whether changes in UCP gene expression result in changes in either UCP protein or activity or ultimately lead to altered cardiac function has not been determined. Future studies are required to address these issues. For example, the expression of UCP-2 in the heart is ⬃100-fold greater than that of UCP-3. Therefore, if both UCP-2 and UCP-3 possess uncoupling activity, what is the physiological significance of UCP-3 induction during increased fatty acid availability? It is possible that UCP-2 and UCP-3 possess different intrinsic activities, are differentially regulated post-transcriptionally, or are located within different regions of the inner mitochondrial membrane, akin to the light harvesting complexes and photosystems of chloroplasts. Whether allosteric factors other than guanosine nucleotides and fatty acids differentially affect the activity of UCP-2 and UCP-3 is unknown. Another concern is that each intervention investigated in the present study could potentially affect multiple factors in these complex in vivo models. For example, nervous activity, workload, and multiple growth factor and cytokine signaling cascades are all altered in both the unloaded and pressure overloaded heart. In addition, various hormonal alterations occur during nutritional manipulation and diabetes. Even in knockout mice, compensatory mechanisms become activated, allowing for adaptation in the absence of a specific gene. All these factors could potentially affect cardiac UCP gene expression. However, one common factor that links cardiac UCP-3 (but not UCP-2) expression with these diverse animal models is the transcription factor PPAR␣ (see Table 4). Although the present study has not directly measured PPAR␣ protein or activity, previous studies have shown that increased fatty acid availability results in PPAR␣ activation (30, 32, 33) and that reduced PPAR␣ transcription—for example, during cardiac hypertrophy—is associated with reduced activity and expression of PPAR␣-regulated genes (50).

CONCLUSIONS In the adult rodent heart, expression of UCP-3 but not UCP-2 is regulated by PPAR␣. In contrast to skeletal muscle, in which UCP expression alters dramatically in response to substrate availability, heart UCP expression 843

does not fluctuate very much. The expression of UCP-2, the major UCP isoform in the rat heart (100-fold higher expression compared to UCP-3), changes relatively little in response to dramatic alterations in substrate availability. Uncoupling of skeletal muscle mitochondria may be important for the utilization of fatty acids, whereas the uncoupling of cardiac mitochondria might play more of a role in an antioxidant capacity, preventing over-reduction of the electron transport chain. In situations in which increased cardiac efficiency is required, such as unloading, overloading, and decreased fatty acid availability, cardiac UCP expression is decreased. Whether other factors are able to significantly increase the expression of uncoupling proteins in the adult heart is unknown.

12. 13. 14.

15.

16. 17. 18.

We wish to thank Patrick H. Guthrie and Wenhao Chen for technical assistance. This work was supported in part by grants from the NIH (HL-43133 and HL-61483) and from the American Heart Association National Center. C.D. was a Daland Fellow of the American Philosophical Society.

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