Suppression of Cardiac Myocyte Hypertrophy by Conjugated Linoleic Acid

Conjugated linoleic acid (CLA) refers to a naturally occurring mixture of positional and geometric isomers of linoleic acid. Evidence suggests that CLA is a dietary constituent and nutraceutical with anti-cancer, insulin-sensitizing, immunomodulatory, weight-partitioning, and cardioprotective properties. The aim of this study was to evaluate the effects of intervention with CLA on cardiac hypertrophy. In vitro, CLA prevented indicators of cardiomyocyte hypertrophy elicited by endothelin-1, including cell size augmentation, protein synthesis, and fetal gene activation. Similar anti-hypertrophic effects of CLA were observed in hypertrophy induced by angiotensin II, fibroblast growth factor, and mechanical strain. CLA may inhibit hypertrophy through activation of peroxisome proliferator-activated receptors (PPARs). CLA stimulated PPAR activity in cardiomyocytes, and the anti-hypertrophic effects of CLA were blocked by genetic and pharmacological inhibitors of PPAR isoforms α and γ. CLA may disrupt hypertrophic signaling by stimulating diacylglycerol kinase ζ, which decreases availability of diacylglycerol and thereby inhibits the protein kinase Cϵ pathway. In vivo, dietary CLA supplementation significantly reduced blood pressure and cardiac hypertrophy in spontaneously hypertensive heart failure rats. These data suggest that dietary supplementation with CLA may be a viable strategy to prevent pathological cardiac hypertrophy, a major risk factor for heart failure.

Cardiac hypertrophy is the increase in myocardial mass provoked by hemodynamic stress or myocardial injury and is a convergence point for many risk factors leading to heart failure. For more than a century, hypertrophy was viewed as a compensatory response that preserves ventricular performance (1,2), but prolonged hypertrophy is maladaptive and leads to cardiac arrest and/or failure (3,4). Thus, attenuation of hypertrophy is a promising therapeutic target to prevent heart failure.
Hypertrophy is characterized at the cardiomyocyte level by increases in cell size, protein synthesis, sarcomeric reassembly, and changes in gene expression (5). Re-induction of fetal genes such as brain natriuretic peptide (BNP) 2 is one of the most consistent markers of hypertrophy. Hence, BNP expression and BNP promoter-reporter constructs are used as experimental indicators of hypertrophy, since virtually every hypertrophic stimulus activates the BNP gene (6).
Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid (LA), an 18-carbon polyunsaturated fatty acid with cis double bonds at carbons 9 and 12. In CLA double bonds are conjugated and may be cis or trans. Most biological actions have been ascribed to cis-9, trans-11-, and trans-10, cis-12-CLA (7). Humans acquire CLA through diet from dairy and meat products from ruminant animals because monogastric intestinal bacteria do not make CLA (8). Currently, although CLA is receiving considerable attention for its many salutary properties (9), its effects on the heart are largely unknown, although CLA may be protective through anti-arrhythmic actions (10).
Previous studies have reported that some of the non-cardiac benefits of CLA involve activation of peroxisome proliferatoractivated receptors (PPARs) (11)(12)(13). PPARs constitute a nuclear receptor family of transcription factors that regulate fatty acid and triglyceride metabolism (14). The crystal structure of PPAR ligand binding domains reveals a large binding pocket that may confer promiscuity for several ligands, including fatty acids (15). There are three isoforms: ␣, ␦, and ␥. In the heart all PPAR isoforms are expressed in myocytes, and bona fide PPAR agonists prevent hypertrophy in vitro (16 -20) and in vivo (17,21).
We determined the effect of CLA on hypertrophy stimulated in neonatal rat ventricular cardiomyocytes. Because CLA activates PPARs and PPAR agonists prevent hypertrophy, we considered whether PPAR activation might underlie anti-hypertrophic effects of CLA. Then we sought to elucidate the mechanism by which CLA attenuates hypertrophic signaling. Finally, we verified the anti-hypertrophic effect of CLA in vivo in the spontaneously hypertensive heart failure (SHHF) rat. * This work was supported by the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Manitoba, Dairy Farmers of Canada, and the Manitoba Medical Service Foundation and a fellowship from the Manitoba Health Research Council (to C. P. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
Mechanical Strain-Cells were cultured on collagen I-coated Flex plates (Flexcell International Corp.). After transfection, cells were serum-deprived for 24 h, then subjected to cyclical strain (60 Hz) on the FX3000 unit (Flexcell International Corp.) to promote a calculated increment in surface area of ϳ20% at the point of maximal distension on the culture surface (23). Cell Size-Myocytes were cultured on glass coverslips (2 ϫ 10 6 cells/coverslip), serum-deprived for 24 h, pretreated with vehicle or CLA (30 M; 1 h), then stimulated by addition of ET-1 (0.1 M; 24 h). Myocyte size was assessed by immunofluorescence, fluorescence microscopy, and computer-assisted planimetry as previously described (24). Pixel values were converted to surface area (m 2 ) by multiplying by scale factors of the x and y axes.
Transfection and Luciferase Assay-Myocytes were cultured in 12-well plates (1 ϫ 10 6 cells/well) then co-transfected with Renilla luciferase and other plasmids with Lipofectin according to the manufacturer's protocol. Myocytes were maintained in DMEM, 10% cosmic calf serum for 24 h, then serum-deprived for 24 h. After treatments, luciferase activity was measured from lysates using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to Renilla luciferase activity.
PKC⑀ Translocation-Myocytes were cultured in 10-cm plates (15 ϫ 10 6 cells/plate), serum-deprived for 24 h, pretreated with vehicle or CLA (30 M; 1 h), then stimulated by the addition of ET-1 (0.1 M; 5 min). Cell lysates were prepared in radioimmune precipitation assay buffer and clarified by centrifugation. Supernatants were centrifuged at 100 000 ϫ g at 4°C to separate soluble and particulate fractions. Particulate fractions were resuspended in radioimmune precipitation assay buffer by sonication. PKC⑀ was detected each in soluble and particulate fractions using conventional Western blotting.
DGK Immunoblotting-Myocytes were cultured in 10-cm plates (15 ϫ 10 6 cells/plate), serum-deprived for 24 h, then treated with vehicle or CLA (30 M; 1-4 h). Cell lysates were prepared in radioimmune precipitation assay buffer and clarified by centrifugation, and DGK was detected by conventional Western blotting. Membranes were stripped and reprobed with ␤-actin antibody to account for loading variations among lanes.
Labeling of Phospholipids with ortho-[ 32 P]Phosphate and Measurement of PA Formation-Myocytes cultured in 6-well plates (2 ϫ 10 6 cells/well) were deprived of serum in phosphate-free DMEM for 24 h. Cells were incubated with 40 Ci/ml ortho-[ 32 P]phosphate in phosphate-free DMEM for 3 h. Excess [ 32 P] was washed out, cells were stimulated with vehicle or CLA in phosphate-free DMEM, and reactions were terminated. Cells were scraped into PBS, and lipids were extracted and separated by thin layer chromatography as previously described (25). [ 32 P]PA in each sample was normalized by total [ 32 P]phospholipid.
In Vivo Experiments-7-Week-old male SD (control) and SHHF rats were obtained from Charles River Canada. Animals were maintained at 20°C, 50% humidity and a 12-h light-dark cycle and allowed free access to water and food. After 2 weeks of acclimatization, a standard diet or one supplemented with 0.5% CLA was offered. Total fat percentage was controlled in diets by adjusting soybean oil content. After 8 weeks, rats were weighed, blood pressure and echocardiographic measurements were performed, and then rats were sacrificed by pentobarbital overdose.
Animals-The study was conducted according to recommendations from the Animal Care Committee of the University of Manitoba and the Canadian Council of Animal Care.
Statistics-All data except for body weights in Fig. 7 were subjected to 1-way analysis of variance followed by a Newman-Keuls multiple comparison test to detect all between-group differences. Body weight data were analyzed using a 2-way repeated measures analysis of variance. p Ͻ 0.05 was considered significant.
We also examined changes in myocyte size, which was measured directly by computer-assisted planimetry. We next determined whether the effect of CLA on hypertrophy is specific or an artifactual effect of fatty acids by examining the effect of LA on ET-1-induced BNP promoter activity. LA differs from CLA in that the double bonds are unconjugated (i.e. separated by two, rather than one, single bonds). Before stimulation with ET-1 (0.1 M; 24 h), myocytes were treated with vehicle or LA (30 M, 1 h). In myocytes, pretreatment with LA did not have an inhibitory effect on ET-1-induced BNP promoter activity (298 Ϯ 62% versus control, p Ͻ 0.05; Fig. 1E).
CLA Inhibits BNP Promoter Activation Induced by Multiple Hypertrophic Stimuli-Several other hypertrophic stimuli activated the BNP promoter (Fig. 2 PPAR␣ and PPAR␥ Mediate the Anti-hypertrophic Effects of CLA-Activated PPARs bind to peroxisome-proliferator response elements (PPREs) to modulate target gene expression. To measure PPAR activation, myocytes were transfected with PPRE 3 -TK-LUC, a reporter construct containing three copies of the acyl-CoA oxidase PPRE upstream of the thymidine kinase promoter driving luciferase expression (29). As shown in Fig. 3, CLA (30 M, 48 h) stimulated significant increases in  We next determined whether PPAR isoforms participate in the anti-hypertrophic actions of CLA. Dominant negative PPAR␣ (Fig. 4B) and dominant negative PPAR␥ (Fig.  4D) abolished the inhibitory effect of CLA on ET-1-stimulated BNP promoter activity, whereas dominant negative PPAR␦ had no significant effect (Fig. 4C). This finding was confirmed pharmacologically using MK886 and GW9662, which selectively antagonize PPARs ␣ (30) and ␥, respectively. Both MK886 (Fig. 4E) and GW9662 (Fig. 4F) abolished the anti-hypertrophic actions of CLA. None of the dominant negative constructs or pharmacological inhibitors affected ET-1-induced BNP promoter activity alone.
CLA Inhibits ET-1-induced Hypertrophic Signaling-Production of diacylglycerol (DAG) and subsequent translocation and activation of PKC isoforms, especially ⑀, are key early signaling events stimulated through the ET A receptor (31,32). Therefore, we examined the effect of CLA on ET-1-induced translocation of PKC⑀. ET-1 increased the ratio of particulate to total PKC⑀ (0.1 M, 5 min; 247 Ϯ 58% versus control, p Ͻ 0.01), and this translocation of PKC⑀ to the particulate fraction was abolished by CLA (98 Ϯ 17% versus control; Fig. 5).
DGK may have a role in the regulation of cardiac hypertrophy (33). DGK phosphorylates DAG to produce PA. The resultant decrease in availability of DAG attenuates translocation and activation of PKC. Thus, we sought to determine whether CLA activates DGK. We observed that CLA (30 M, 1 h) increases protein expression of DGK compared with vehicle-treated controls (304.6 Ϯ 43.8 versus control, p Ͻ 0.05; Fig. 6A), an event that was sustained over 2-and 4-h time points. Additionally, MK886 (1 M, 1 h) or GW9662 (1 M, 1 h) each blocked the ability of CLA to increase DGK protein expression, suggesting a role for PPAR␣ and/or -␥ in the activation of DGK by CLA (Figs. 6, B and C). We also measured [ 32 P]PA formation in 32 P-labeled myocytes in the presence of CLA. We were unable to detect a difference in [ 32 P]PA formation between myocytes treated with vehicle or CLA for 1 h (Fig. 6D). However, treatment with CLA for 2 h induced a significant increase in [ 32 P]PA formation, which indicates that CLA stimulates DGK activity (Fig. 6D).
CLA Attenuates Hypertrophy in Vivo-M-mode echocardiography showed early cardiac hypertrophy in SHHF rats (Table 1). Hypertrophic parameters including LVPWd, LVPWs, LV mass, and relative wall thickness were augmented in SHHF rats compared with age-matched SD rats. Fractional shortening in SD and SHHF rats were not significantly different, demonstrating that at 17 weeks of age, SHHF rats do not show signs of declining left ventricular function. Experimental control or CLA diets were palatable to the rats and well tolerated, with no incidents of ill health, diarrhea, weight loss, lethargy, or aggression. Dietary CLA (0.5%, 8 weeks) had no effect on body weight in SD (Fig. 7A) or SHHF rats (Fig. 7B) yet had improved cardiac geometry by attenuating thickening of LVPWd and LVPWs, reducing LV mass, and decreasing relative wall thickness. CLA had no effect on cardiac geometry in SD rats (Table 1). SBP (187 Ϯ 11 versus 114 Ϯ 4 mm Hg in SD rats, p Ͻ 0.001), DBP (92 Ϯ 6 versus 55 Ϯ 2 mm Hg in SD rats, p Ͻ 0.001), and mean blood pressure (123 Ϯ 7 versus 74 Ϯ 3 mm Hg in SD rats, p Ͻ 0.001) were elevated in SHHF rats compared with SD rats. 8-Week treatment with CLA had no effect on blood pressure in SD rats but partially attenuated the development of hypertension in SHHF rats (Table 2). Notably, the anti-hypertrophic effect of CLA reached statistical significance at 3 weeks in terms of reduced thickening of LVPWd and LVPWs before its reducing effect on blood pressure (data not shown).

DISCUSSION
This study shows that CLA suppresses cardiomyocyte hypertrophy through activation of PPAR isoforms ␣ and ␥. This action of CLA is applicable to a number of models of hypertrophy, since CLA attenuated BNP promoter activation triggered by mechanical strain, AngII, and FGF. To our knowledge this is the first report that supplementation with a dietary polyunsaturated fatty acid can prevent cardiac hypertrophy in vivo. CLA supplementation to SHHF rats, which would normally exhibit hypertrophic cardiac growth at this age, resulted in reductions in thickening of the left ventricular wall, left ventricular mass, and relative wall thickness as well as blood pressure. It should be noted that in our experiments the anti-hypertrophic effect of CLA in SHHF rats was evident after only 3 weeks of CLA treatment, whereas BP was not yet significantly reduced. Thus, the ability of CLA to prevent cardiac hypertrophy is attributable at least in part to direct actions on the heart rather than improved hypertension, which is consistent with our in vitro data. Thus, in addition to its other cardioprotective attributes such as antiarrhythmic (10, 34), anti-atherogenic (35)(36)(37), and anti-hypertensive (38 -40) effects, our study suggests that CLA also protects against the development of cardiac hypertrophy.
In humans, basal plasma levels of CLA are in the M range (ϳ7 M) (41). Daily supplementation with CLA in healthy male (3.0 g/day) and female (3.9 g/d) volunteers resulted in 3-4-fold increases in plasma CLA (42). Given the 3-4-fold increase of plasma CLA with supplementation (41)(42)(43) and the fact that local tissue concentrations easily reach levels 10-fold greater than plasma concentrations (44,45), our experimental concentration of CLA (30 M) should be physiologically attainable and relevant.
Several lines of evidence indicate that CLA inhibited cardiomyocyte hypertrophy by activating PPARs. Previous studies have reported that specific PPAR agonists such as fibrates or thiazolidinediones inhibit hypertrophy in vivo (21, 46 -48) and in vitro (18 -21, 49) and that CLA activates all three PPAR isoforms in non-cardiac tissues (11,(51)(52)(53)(54)(55). We found that CLA activated all PPAR isoforms in myocytes in the presence of overexpressed PPARs. Similarly, the PPAR␣, -␦, and -␥-specific agonists, fenofibrate, GW501516, and troglitazone, also required overexpression of PPARs to induce PPAR activation. CLA activated PPAR␣ and -␥ to similar extents as fenofibrate and troglitazone, respectively. In contrast, CLA weakly stimulated PPAR␦ compared with GW501516. Weak activation of PPAR␦ by CLA in these experiments does not preclude an antihypertrophic action of activated PPAR␦. However, it may explain why CLA prevented hypertrophy in response to ET-1, AngII, FGF, and mechanical strain but failed to block PE-induced hypertrophy. Lack of CLA effect on PE-induced growth may be due to insufficient activation of PPAR␦ by CLA, since the selective PPAR␦ agonist, L-165041, inhibits PE-induced hypertrophy (18).
Dominant negative (dn) mutant PPAR␦ failed to abrogate the growth inhibitory action of CLA. We did not determine expression levels of endogenous wild type (wt) PPAR␦ nor dn PPAR␦ mutant, so we cannot comment on the stoichiometric ratio of wt:dnPPAR␦. However, we did test a range of dnPPAR␦ con- centrations (0.01 (not shown) Ϫ 0.1 (Fig. 4C) g/well); none of these blocked CLA. In contrast, dn mutants of PPAR␣ or -␥ abolished the inhibitory effect of CLA on ET-1-induced hypertrophy, as did pharmacologically antagonizing PPAR␣ or -␥. Therefore, we provide direct evidence that prevention of hypertrophy by CLA is mediated by PPAR␣ or ␥, but probably not ␦.
In this study we suggest that CLA attenuates cardiomyocyte hypertrophy via activation of DGK, leading to inhibition of PKC⑀ translocation and activation (illustrated in Fig. 8). PKC activation is an early event of ET-1 signaling. Binding of ET-1 to the ET A receptor triggers activation of G␣ q /␣ 11 , resulting in DAG production and accumulation in the sarcolemmal membrane. In cardiomyocytes, PKC⑀ and to a much lesser degree, PKC␦, rapidly bind to DAG in the sarcolemma and are activated (31,32,56 (58). We report here that CLA attenuated ET-1-induced PKC⑀ translocation to the particulate or membrane fraction. We also found that CLA induced an increase in the protein expression of DGK and stimulated the production of PA, which suggests increased activity of DGK. Despite observing an  increase in DGK protein expression in myocytes treated with CLA for 1 h, we did not detect DGK activity (increase in PA formation) until 2 h of treatment with CLA. It is entirely possi-ble that small changes in DGK activity that elude our detection at 1 h would be enough to disrupt hypertrophic signaling. Additionally, at 2 h of CLA treatment, PA may have accumulated to a level that is measurable by our assay. We also observed that increased expression of DGK is dependent on PPAR␣ or -␥. A role for PPAR in the activation of DGK and inhibition of PKC signaling has also been observed in endothelial cells. PPAR␥ agonists, specifically ciglitazone, troglitazone, and 15d-PGJ2, were each shown to inhibit endothelial cell activation by upregulating DGK␣ and disrupting translocation of PKC␤ (59). It should be noted that since we employed a mixed (albeit pure) preparation of CLA, the question arises whether its antihypertrophic activity is supported by one or more of the isomers that constitute the preparation. Individual CLA isomers may suffice to prevent hypertrophy. Alternatively, a synergistic interaction(s) between isomers may contribute to certain biological effects of CLA. Further studies using pure CLA isomers are required to fully elucidate the specificity of different isomers in hypertrophy.
In summary, this study shows that activation of PPAR␣ and ␥ by CLA prevents cardiac hypertrophy through activation of DGK and subsequent inhibition of the PKC⑀ pathway. These findings provide novel support for the role of polyunsaturated fatty acids as cardioprotective dietary elements. In a time when prevention of hypertrophy is viewed as a promising therapeutic target (60) and ϳ44% of heart failure patients resort to nutrition-based therapies (50), this study may have important implications for effective nutritional intervention toward the prevention of cardiac disease. FIGURE 8. Proposed mechanism by which CLA abrogates ET-1-induced hypertrophic signaling. ET-1 induces accumulation of DAG in the plasma membrane, which causes translocation and activation of PKC⑀, an early signaling mediator of hypertrophy. CLA may interrupt hypertrophic signaling by activating PPAR␣ and/or ␥ isoforms, which stimulates DGK to phosphorylate DAG, producing PA. The resulting decreased availability of DAG inhibits PKC⑀ recruitment to the plasma membrane.

TABLE 2 Blood pressure
Effect of dietary CLA on blood pressure in the SHHF rat is shown. 9-Week old rats were fed control (0.5% soybean oil) or CLA (0.5% CLA) diets ad libitum. Experimental diets were maintained for 8 weeks. Blood pressure was measured using tail-cuff methodology.