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or high-fat feeding in a rat infarct model of heart failure
1Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio; 2Department of Pediatrics, Baylor College of Medicine, Houston, Texas; 3Veteran Affairs Medical Research Center, Cleveland, Ohio; and 4Department of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio
Submitted 23 September 2005 ; accepted in final form 6 December 2005
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ABSTRACT |
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(PPAR) increases intramyocellular ceramide and left ventricular (LV) dysfunction. We tested the hypothesis that activation of fatty acid metabolism with fat feeding or a PPAR agonist increases myocardial triglyceride and/or ceramide and exacerbates LV dysfunction in HF. Rats with infarct-induced HF (n = 38) or sham-operated rats (n = 10) were either untreated (INF, n = 10), fed a high-fat diet (45% kcal fat, INF + Fat, n = 15), or fed the PPAR agonist fenofibrate (150 mg·kg–1·day–1, INF + Feno, n = 13) for 12 wk. LV ejection fraction was significantly reduced with HF (49 ± 6%) compared with sham operated (86 ± 2%) with no significant differences in ejection fraction (or other functional or hemodynamic measures) among the three infarcted groups. Treatment with the PPAR agonist resulted in LV hypertrophy (24% increase in LV/body mass ratio) and induced mRNAs encoding for PPAR-regulated genes, as well as protein expression and activity of medium chain acyl-CoA dehydrogenase (compared with INF and INF + Fat groups). Myocardial ceramide content was elevated in the INF group compared with sham-operated rats, with no further change in the INF + Fat or INF + Feno groups. Myocardial triglyceride was unaffected by infarction but increased in the INF + Fat group. In conclusion, LV dysfunction and dilation are not worsened despite upregulation of the fatty acid metabolic pathway and LV hypertrophy or accumulation of myocardial triglyceride in the rat infarct model of HF.
cardiac; left ventricular dysfunction; ceramide; triglycerides; fatty acids
(PPAR) is a key modulator of the expression of genes encoding proteins controlling fatty acid uptake and metabolism (10). Transgenic mice with cardiac overexpression of PPAR have increased myocardial fatty acid uptake, TG, and ceramide accumulation, cardiac hypertrophy, and left ventricular (LV) dilation that is exacerbated by high-fat feeding (8, 9). Upregulation of myocardial fatty acid uptake and esterification in transgenic mice have clear adverse effects on cardiac function and structure (3, 4, 9, 25); however, it is unclear if these findings can be translated to clinical conditions like heart failure (26).
There is clear evidence that upregulation of myocardial fatty acid uptake through genetic manipulations can result in "cardiac lipotoxicity" (3, 4, 8, 9, 31). From a clinical perspective, however, it is important to know if relevant dietary and pharmacology manipulations (e.g., high-fat feeding or activation of PPAR) have adverse effects on LV function and remodeling in established heart failure. The present study tested the hypothesis that activation of fatty acid metabolism by high-fat feeding would increase myocardial TG and/or ceramide content and exacerbate LV dysfunction and remodeling in established heart failure. In addition, the effect of upregulation of proteins involved in myocardial fatty acid metabolism by chronic treatment with a PPAR agonist in heart failure was evaluated. Studies were conducted in the rat infarct model of heart failure (24), and treatment was initiated 8 wk after myocardial infarction and continued for 12 additional weeks. Because cardiac dysfunction has been associated with myocardial ceramide accumulation (4, 35), we also assessed cardiac ceramide content.
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METHODS |
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Heart failure was induced by coronary artery ligation as previously described in detail (1). Eight-week-old male Wistar-Kyoto rats (
250 g) were anesthetized with isoflurane (1.5–2.0%), intubated, and ventilated. A myocardial infarction was induced by left coronary artery ligation, and sham-operated animals underwent a similar surgical procedure without arterial ligation. Eight weeks after surgery, LV function was evaluated by echocardiography (described below), and the infarcted rats were assigned to either no treatment (INF, n = 10), treatment with a high-fat diet [Research Diets, 45% kcal from fat (37% saturated and 46% mono- and 19% polyunsaturated fatty acids); INF + Fat, n = 15] or treatment with the PPAR agonist fenofibrate (150 mg·kg–1·day–1, INF + Feno, n = 13, milled into the food based on previously recorded average daily food consumption). The INF and INF + Feno groups were fed normal chow with 10% of calories from fat. Sham-operated animals were left untreated (n = 10). [Comparisons between data from 8 and 20 wk of sham-operated and untreated INF groups have been reported separately (21).] Animals were assigned so that the initial level of LV dysfunction [determined by the wall motion score index as previously described (22)] was the same in each group. After 12 wk of treatment with either high-fat diet or fenofibrate (20 wk postsurgery), echocardiography was again performed, and LV pressures were measured as described below. After LV cannulation, blood samples were drawn from the inferior vena cava, and myocardial tissue samples were harvested and quick-frozen immediately after obtaining LV, right ventricular (RV), and scar mass by gravimetric measurements.
Echocardiography. LV function was evaluated by echocardiography using a Sequoia C256 System (Siemens Medical) with a 15-MHz linear array transducer as previously described (22). Briefly, rats were anesthetized with 1.5–2.0% isoflurane by mask, the chest was shaved, the animal was situated in the supine position on a warming pad, and ECG limb electrodes were placed. Two-dimensional (2-D), 2-D-guided M-mode, and Doppler echocardiographic studies of aortic and transmitral flows were performed from parasternal and foreshortened apical windows. End-diastolic and end-systolic dimensions (EDD and ESD) were measured by using software resident on the ultrasonograph, and fractional shortening (FS), myocardial performance index (MPI), and cardiac index (CI) were calculated as previously described (22).
Hemodynamic measurements.
After 12 wk of treatment (20 wk postsurgery), rats were anesthetized (1.5–2.0% isoflurane), intubated, and ventilated as previously described (22). A microtip pressure transducer catheter (3.5 Fr, Millar Instruments) was introduced via the right carotid artery into the LV. Measurements of heart rate, maximum LV pressure, peak end-diastolic pressure (EDP), peak positive (+dP/dt) and negative first derivative of LV pressure (–dP/dt), and tau, the time constant of isovolumic LV relaxation [previously described (21)] were recorded using a Digi-Med Heart Performance Analyzer-
over a 30-s period.
Metabolic products and enzyme activity. Plasma free fatty acids (FFA) and TGs were measured by using a commercially available enzymatic spectrophotometric kit (Wako Chemicals, Richmond, VA). Myocardial activities of medium chain acyl-CoA dehydrogenase (MCAD) and citrate synthase (CS) and tissue TG content were measured from homogenate extracts by using enzymatic spectrophotometer methods as previously described (18, 23). C16-ceramide content in the LV was measured by a capillary gas chromatographic procedure with a flame ionization detector using C17-ceramide as an internal standard, as previously described by Tserng and Griffin (29).
RNA extraction and quantitative RT-PCR.
RNA extraction and quantitative RT-PCR were performed on frozen powdered LV tissue by using previously described methods (5, 11, 14). Specific quantitative assays were designed from rat sequences available in GenBank for expression of PPAR, retinoid X receptor- (RXR), CS, uncoupling protein 2 (UCP2), atrial natriuretic factor (ANF), and genes that are known to be regulated by PPAR: MCAD, carnitine palmitoyltransferase I (CPT-I), pyruvate dehydrogenase kinase 4 (PDK-4), uncoupling protein 3 (UCP3), and mitochondrial thioesterase 1 (MTE-1) (14, 27, 28, 30, 33, 34). Standard RNA was made for all assays by the T7 polymerase method (Ambion), by using total RNA isolated from rat hearts. The correlation between the Ct (the number of PCR cycles required for the fluorescent signal to reach a detection threshold), and the amount of standard was linear over at least a 5-log range of RNA for all assays. To control for sample-to-sample differences in RNA concentration and to adjust for possible changes in overall expression with heart failure, the mRNA level for 18S was quantitatively measured in each sample. The number of 18S molecules was not different among the experimental groups (data not shown); therefore, the PCR data are normalized to 18S and expressed as a percentage of the sham-operated value.
Western immunoblot analysis.
Protein was extracted from frozen powdered LV tissue as previously described (23). Either 50 or 75 µg of total protein was separated by electrophoresis in 10% SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane. Membranes were incubated with specific antibodies to PPAR, RXR (1:100; Santa Cruz Biotechnology), or MCAD (1:2,000; Caymen Chemical). After conjugation with the secondary antibody, the membranes were developed in a chemiluminescence substrate solution (Santa Cruz Biotechnology), and bands were quantified by using commercially available software. All samples were run in duplicate and normalized to a standard sample loaded on each gel.
Statistical analysis. Differences among the sham-operated, INF, INF + Fat, and INF + Feno groups were determined by using one-way ANOVA followed by a Bonferroni t-test for multiple comparisons. Data are expressed as group means ± SE. For all statistical analyses, significance was accepted at P < 0.05.
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RESULTS |
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(MCAD, PDK-4, MTE-1, and UCP3) were significantly decreased in the INF group compared with the sham-operated group (Fig. 3, bottom). There were no differences in any of the mRNA levels between the INF and the INF + Fat groups. Treatment with fenofibrate increased the expression of MCAD, CPT-I, PDK-4, and MTE-1 compared with the INF and INF + Fat groups (Fig. 3, bottom). UCP3 expression was significantly increased in the INF + Feno group compared with the INF group alone.
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and RXR was not different among groups; however, fenofibrate administration significantly increased MCAD protein expression relative to the sham-operated INF and INF + Fat groups (Table 4). CS activity was not different between groups, but MCAD activity (either normalized to gram wet weight tissue or CS activity) was significantly increased by fenofibrate treatment compared with the sham-operated, INF, and INF + Fat groups (Table 4).
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DISCUSSION |
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agonist increases fatty acid oxidation capacity and causes LV hypertrophy in rats with heart failure but does not affect myocardial TG or ceramide levels or exacerbate LV dysfunction and remodeling. In contrast, high-fat feeding significantly increases cardiac TG stores but again does not exacerbate LV dysfunction. Thus, in a model of established LV dysfunction and dilation, neither increased fatty acid oxidation capacity nor accumulation of myocardial TG alone exacerbates heart failure.
The lack of a functional consequence of PPAR activation on LV contractile function contrasts with previous work that showed impaired cardiac function in cardiac-specific PPAR-overexpressing mice and in rats fed a PPAR agonist during the initial development of cardiac hypertrophy induced by aortic banding (8, 9, 32). In the current investigation, fenofibrate treatment increased MCAD mRNA and protein expression and enzyme activity in rats with established heart failure but did not worsen LV dysfunction or dilation. It is important to note in this study that PPAR activation did not result in an increase in myocardial TG or ceramide content. On the other hand, we observed that treatment with PPAR agonist caused LV hypertrophy (24% increase in LV mass/body mass ratio) without any evidence of further contractile dysfunction or LV remodeling. Marked cardiac hypertrophy has been reported in mice with cardiac-restricted overexpression of PPAR (9) and PPAR
/
(2). Furthermore, treatment of Fischer 344 rats with the specific PPAR agonist WY-14643 for 26 wk resulted in a 23% increase in cardiomyocyte diameter and a greater heart mass/body mass ratio (13). The mechanism for this effect is unclear, but perhaps activation of PPAR stimulates hypertrophy through signaling mechanisms that are not maladaptive (6). Jamshidi et al. (16) found that a G/C polymorphism in intron 7 of the PPAR gene in young men predicted greater LV growth in response to exercise training and hypertension. It is not yet known if this mutation activates or inhibits PPAR activation of gene expression, but it nevertheless demonstrates a link between PPAR and LV hypertrophy (16).
In contrast to the fenofibrate-induced upregulation of the fatty acid metabolic pathway, administration of a high-fat diet did not significantly increase the transcription of PPAR-regulated genes in the rat infarct model of heart failure. Although PPAR can recognize a broad array of ligands, including long-chain polyunsaturated fatty acids and eicosanoids, the response to long chain saturated and monounsaturated fatty acids is markedly reduced (10). Administration of long-chain saturated and monounsaturated fatty acids did result in moderate increases in the mRNA expression of several PPAR-regulated genes; however, this was seen only in isolated cardiomyocytes (12). Similarly, increasing the long-chain but not medium-chain fatty acid supply upregulates UCP3 (a PPAR-regulated gene) mRNA and protein levels in both skeletal and cardiac muscles, with the greatest effect being seen in cardiac muscle (15). The lack of activation we observed may result from the fatty acid composition of our study's diet. The high-fat diet used in this study contained primarily saturated and monounsaturated fatty acids of varying chain lengths, which may have been inadequate for optimal activation of PPAR.
Previous work has shown that elevated myocardial ceramide and TG levels are associated with cardiac dilatation and reduced contractility and that the reduction of these lipid intermediates is associated with improved cardiac function (3, 9, 35). In this study, we hypothesized that increased delivery of fatty acids to the myocardium would result in a mismatch between fatty acid import and utilization, resulting in an accumulation of myocardial ceramide and TG and an exacerbation of LV dysfunction and dilation in heart failure. Although we found ceramide levels to be increased in the heart failure animals compared with sham-operated animals (21), increasing fatty acid delivery to the heart via a high-fat diet did not further increase cardiac ceramide content or exacerbate LV dysfunction or dilation observed in this study. On the other hand, high-fat fed animals had significantly higher myocardial TG content compared with the infarcted rats fed normal chow with no increase in LV dysfunction or dilation. These results are consistent with previous studies using isolated cells or transgenic mice where the ability to synthesize cardiac TGs has been suggested to play a critical role in protection from "lipotoxicity" by diverting excess fatty acids from cytotoxic pathways (19, 20). The data further suggest that myocardial ceramide accumulation is not dependent on the quantity of fatty acids delivered to the myocardium and that myocardial ceramide content is not affected by cardiac TG accumulation in heart failure.
There are several limitations of the present study that need to be addressed. First, although fenofibrate administration upregulated the fatty acid metabolic pathway, we did not measure myocardial fatty acid uptake or oxidation directly. These measurements require ex vivo perfusion, which would have precluded the biochemical measurements made in the present study. Second, the lack of effect of high-fat feeding on the progression of heart failure and activation of PPAR-regulated genes may be due to the lipid composition and duration of treatment with the high-fat diet. Finally, it is possible that despite elevations in myocardial TGs, the duration of treatment was insufficient to upregulate the fatty acid metabolic pathway or accelerate the progression of heart failure. Future studies should consider increasing both the content of the long-chain fatty acid moieties and the length of treatment with high-fat feeding.
In summary, prolonged administration of a PPAR agonist upregulated the fatty acid metabolic pathway and caused LV hypertrophy but did not affect LV dysfunction in a rat model of infarct-induced heart failure. In addition, high-fat feeding significantly increased cardiac TG stores but also did not exacerbate LV dysfunction or remodeling. Thus LV dysfunction and dilation are not worsened despite upregulation of the fatty acid metabolic pathway or accumulation of myocardial TG in the rat infarct model of heart failure.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy. Nat Med 10: 1245–1250, 2004.[CrossRef][ISI][Medline]-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: modulation by dietary fat content. Proc Natl Acad Sci USA 100: 1226–1231, 2003. overexpression mimics that caused by diabetes mellitus. J Clin Invest 109: 121–130, 2002.[CrossRef][ISI][Medline] and -. Proc Natl Acad Sci USA 94: 4312–4317, 1997. and PPAR/, but not PPAR
, modulate the expression of genes involved in cardiac lipid metabolism. Circ Res 92: 518–524, 2003. (PPAR-) agonist, WY-14,643, increased transcription of myosin light chain-2 in cardiomyocytes. J Toxicol Sci 26: 275–284, 2001.[CrossRef][Medline] gene regulates left ventricular growth in response to exercise and hypertension. Circulation 105: 950–955, 2002. in pacing-induced heart failure. Circulation 106: 606–612, 2002.-regulated gene in cardiac and skeletal muscle. Am J Physiol Endocrinol Metab 287: E888–E895, 2004. is associated with contractile dysfunction in hypertrophied rat heart. J Biol Chem 276: 44390–44395, 2001. in the adult rodent heart. FASEB J 15: 833–845, 2001.This article has been cited by other articles:
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  H. Yamashita, K. G. Bharadwaj, S. Ikeda, T.-S. Park, and I. J. Goldberg Cardiac metabolic compensation to hypertension requires lipoprotein lipase Am J Physiol Endocrinol Metab, September 1, 2008; 295(3): E705 - E713. [Abstract] [Full Text] [PDF]
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 C. X. Fang, F. Dong, D. P. Thomas, H. Ma, L. He, and J. Ren Hypertrophic cardiomyopathy in high-fat diet-induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1206 - H1215. [Abstract] [Full Text] [PDF]
|
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|
|
 |
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 J. H. Rennison, T. A. McElfresh, I. C. Okere, H. V. Patel, A. B. Foster, K. K. Patel, M. S. Stoll, P. E. Minkler, H. Fujioka, B. D. Hoit, et al. Enhanced acyl-CoA dehydrogenase activity is associated with improved mitochondrial and contractile function in heart failure Cardiovasc Res, July 15, 2008; 79(2): 331 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
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 E. D. Abel, S. E. Litwin, and G. Sweeney Cardiac Remodeling in Obesity Physiol Rev, April 1, 2008; 88(2): 389 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Rodriguez-Calvo, L. Serrano, E. Barroso, T. Coll, X. Palomer, A. Camins, R. M. Sanchez, M. Alegret, M. Merlos, M. Pallas, et al. Peroxisome Proliferator-Activated Receptor {alpha} Down-Regulation Is Associated With Enhanced Ceramide Levels in Age-Associated Cardiac Hypertrophy J. Gerontol. A Biol. Sci. Med. Sci., December 1, 2007; 62(12): 1326 - 1336. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Lopaschuk, C. D.L. Folmes, and W. C. Stanley Cardiac Energy Metabolism in Obesity Circ. Res., August 17, 2007; 101(4): 335 - 347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Blanco-Rivero, I. Marquez-Rodas, F. E. Xavier, R. Aras-Lopez, I. Arroyo-Villa, M. Ferrer, and G. Balfagon Long-term fenofibrate treatment impairs endothelium-dependent dilation to acetylcholine by altering the cyclooxygenase pathway Cardiovasc Res, July 15, 2007; 75(2): 398 - 407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. King, M. E. Young, J. Kerner, H. Huang, K. M. O'Shea, S. E. H. Alexson, C. L. Hoppel, and W. C. Stanley Diabetes or peroxisome proliferator-activated receptor {alpha} agonist increases mitochondrial thioesterase I activity in heart J. Lipid Res., July 1, 2007; 48(7): 1511 - 1517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-A. S. Duhaney, L. Cui, M. K. Rude, N. K. Lebrasseur, S. Ngoy, D. S. De Silva, D. A. Siwik, R. Liao, and F. Sam Peroxisome Proliferator-Activated Receptor {alpha}-Independent Actions of Fenofibrate Exacerbates Left Ventricular Dilation and Fibrosis in Chronic Pressure Overload Hypertension, May 1, 2007; 49(5): 1084 - 1094. [Abstract] [Full Text] [PDF]
|
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N. Sharma, I. C. Okere, M. K. Duda, D. J. Chess, K. M. O'Shea, and W. C. Stanley Potential impact of carbohydrate and fat intake on pathological left ventricular hypertrophy Cardiovasc Res, January 15, 2007; 73(2): 257 - 268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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B. N. Finck The PPAR regulatory system in cardiac physiology and disease Cardiovasc Res, January 15, 2007; 73(2): 269 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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I. C. Okere, M. E. Young, T. A. McElfresh, D. J. Chess, V. G. Sharov, H. N. Sabbah, B. D. Hoit, P. Ernsberger, M. P. Chandler, and W. C. Stanley Low Carbohydrate/High-Fat Diet Attenuates Cardiac Hypertrophy, Remodeling, and Altered Gene Expression in Hypertension Hypertension, December 1, 2006; 48(6): 1116 - 1123. [Abstract] [Full Text] [PDF]
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P < 0.05, INF + Feno compared with INF and INF + Fat.
P < 0.05 compared with INF only. ANF, atrial natriuretic factor; RXR