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. 2015 Sep 18;290(38):23371-84.
doi: 10.1074/jbc.M115.682195. Epub 2015 Aug 3.

Excess Linoleic Acid Increases Collagen I/III Ratio and "Stiffens" the Heart Muscle Following High Fat Diets

Affiliations

Excess Linoleic Acid Increases Collagen I/III Ratio and "Stiffens" the Heart Muscle Following High Fat Diets

Julianne Beam et al. J Biol Chem. .

Abstract

Controversy exists on the benefits versus harms of n-6 polyunsaturated fatty acids (n-6 PUFA). Although n-6 PUFA demonstrates anti-atherosclerotic properties, survival following cardiac remodeling may be compromised. We hypothesized that n-6 PUFA like linoleic acid (LA) or other downstream PUFAs like γ-linolenic acid or arachidonic acid alter the transforming growth factor-β (TGFβ)-collagen axis in the heart. Excess dietary LA increased the collagen I/III ratio in the mouse myocardium, leading to cardiac "stiffening" characterized by impaired transmitral flow indicative of early diastolic dysfunction within 5 weeks. In vitro, LA under TGFβ1 stimulation increased collagen I and lysyl oxidase (LOX), the enzyme that cross-links soluble collagen resulting in deposited collagen. Overexpression of fatty acid desaturase 2 (fads2), which metabolizes LA to downstream PUFAs, reduced collagen deposits, LOX maturation, and activity with LA, whereas overexpressing fads1, unrelated to LA desaturation, did not. Furthermore, fads2 knockdown by RNAi elevated LOX activity and collagen deposits in fibroblasts with LA but not oleic acid, implying a buildup of LA for aggravating such pro-fibrotic effects. As direct incubation with γ-linolenic acid or arachidonic acid also attenuated collagen deposits and LOX activity, we concluded that LA itself, independent of other downstream PUFAs, promotes the pro-fibrotic effects of n-6 PUFA. Overall, these results attempt to reconcile opposing views of n-6 PUFA on the cardiovascular system and present evidence supporting a cardiac muscle-specific effect of n-6 PUFAs. Therefore, aggravation of the collagen I/III ratio and cardiac stiffening by excess n-6 PUFA represent a novel pathway of cardiac lipotoxicity caused by high n-6 PUFA diets.

Keywords: collagen; fatty acid metabolism; heart failure; lysyl oxidase; polyunsaturated fatty acid (PUFA).

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Figures

FIGURE 1.
FIGURE 1.
Various diets lead to similar weight gains and no overt fibrotic changes over 5 weeks. a, fatty acid composition of high fat diet groups. Oleic acid (C18:1) and linoleic acid (C18:2n-6) were significantly higher in olive oil and linoleic acid groups, respectively. b, average daily food intake over 5 weeks. Mice on high fat diets consumed less food on a per g basis than chow diets. c, body weight of mice over 5 weeks of diet regimens. Body weight was not significantly different among any group of mice. Bottom panel, Masson trichrome stains of LV depicting perivascular and interstitial regions of LV at ×400 and 200 magnifications, respectively. No difference in total collagen staining (blue) in either areas were observed with any diet. Data were analyzed using one-way ANOVA with Tukey post hoc tests (n = 6 mice). *, p < 0.05 versus the chow-fed group.
FIGURE 2.
FIGURE 2.
Corn oil alters collagen subtypes in the mouse heart. a, distribution and localization of collagen I and III in the heart across diet groups via double immunolabeling. Chow mouse hearts depicted low collagen I (fine arrow) with negligible collagen III positivity. OO increased diffuse collagen III across the myocardium (block arrow) and reduced punctate collagen I stains. CO hearts demonstrated the highest collagen I (fine arrows) with negligible collagen III stains. Magnification ×200. b, cardiac mRNA expression of tgfβ1 and tgfβ3 across diet groups. c, cardiac mRNA expression of col1α1 and col3α1. d, representative bands from Western blots for collagen (Col) I and III with total protein depicted by Ponceau stain. e, Western blot analysis of collagen I and III in mouse hearts normalized to total protein under various diets. Data were analyzed using one-way ANOVA with Tukey post hoc tests (n = 6 mice). *, p < 0.05 versus chow fed group. †, p < 0.05 versus corresponding olive oil fed group. A.U., arbitrary units; tgfβ, gene of tissue growth factor β.
FIGURE 3.
FIGURE 3.
Linoleic acid induces specific TGFβ and collagen isoforms in fibroblasts. a, col1a 1 and col3a1 expression following 24 h of treatment with 0.1 mm fatty acids. b, ratio of col1a1/col3a1 mRNA expression in fibroblasts with 0.1 mm fatty acids over 24 h. c, deposited collagen over 24 h following incubations with 0.1 mm fatty acids with or without 10 ng/ml TGFβ1 and TGFβ3. LA treatment increased collagen deposition following TGFβ1 but not TGFβ3 treatment. Conversely, TGFβ3 reduced collagen deposition with LA but increased with OA. RAU, relative absorbance units. d, representative micrographs of fibroblasts demonstrating collagen deposits with 0.1 mm fatty acids and 10 ng/ml TGFβ1. Upper panel magnification, ×200; lower panel magnification, ×400. LA increased collagen in fibroblasts under TGFβ1 stimulation. Experiments were repeated at least twice in triplicate. Data were analyzed using two-way ANOVA with Tukey post hoc tests, p < 0.05. *, p < 0.05 versus control cells. †, p < 0.05 versus corresponding treatments with OA. A.U., arbitrary units.
FIGURE 4.
FIGURE 4.
Overexpression of fads2 does not alter 16:1n-10 in fibroblasts. a, characterization of gas chromatographic separations for 16:1n9 and 16:1n10. Panel i, representative chromatogram of a spiked fibroblast cell extract with 1 μg of 16:1n9. Panel ii, representative chromatogram of a spiked fibroblast cell extract with 1 μg of 16:1n10. Palmitate (C16:0) is also depicted in panels i and ii for positional reference. Representative chromatograms of C16:0, C16:1n9, and C16:1n10 in untransfected NIH/3T3 cell extracts treated with BSA (b), OA (d) and LA (f). Impact of fads2 overexpression in NIH/3T3 cells treated with BSA (c), OA (e) and LA (g). Overexpression of fads2 did not affect either C16:0, C16:1n9, or 16:1n10 content in any group. Experiments were repeated at least twice in triplicate. Table 6 depicts numerical values for these data. fads2, fatty acid desaturase 2.
FIGURE 5.
FIGURE 5.
Overexpression of fads2 but not fads1 reduces fibroblast collagen deposition with LA. a, impact of fads2 overexpression in fatty acid-treated cells with or without 10 ng/ml TGFβ1 on collagen deposition. fads2 overexpression lowers collagen deposition with and without TGFβ1 with LA but not OA. b, impact of fads2 overexpression in NIH/3T3 cells with or without 10 ng/ml TGFβ3 on collagen deposition. fads2 overexpression lowers collagen deposition with both OA and LA, with or without TGFβ3. c, impact of fads2 overexpression in NIH/3T3 cells with or without 10 ng/ml TGFβ1 on collagen deposition. Unlike fads2, overexpression of fads1 does not affect collagen deposition in cells treated with either OA or LA, with or without TGFβ1. Experiments were repeated at least twice in triplicate. Data are presented relative to untreated control. Data were analyzed using two-way ANOVA with Tukey post hoc tests, p < 0.05. *, p < 0.05 versus control cells. †, p < 0.05 versus corresponding OA-treated cells. RAU, relative absorbance units; fads1, fatty acid desaturase 1; fads2, fatty acid desaturase 2.
FIGURE 6.
FIGURE 6.
LOX activity is increased with LA both in vivo and in vitro. a, LOX activity in hearts of mice fed various diets. Corn oil increased whereas olive oil decreased LOX activity compared with chow-fed mice. b, mRNA expression of lox and loxl1 in hearts of mice. No change in either gene was noted with any diet. c, protein expression of prolysyl oxidase and mature LOX from Western blot of LOX in vivo. Inset, representative bands of prolysyl oxidase (50 kDa) and mature LOX (30 kDa). Corn oil-fed hearts demonstrated higher mature 30-kDa fraction of LOX compared with other groups. d, soluble collagen after 24-h incubations with 0.1 mm fatty acids with or without 10 ng/ml TGFβ1 and TGFβ3. Soluble collagen was increased with TGFβ1 in both LA- and OA-treated cells, whereas TGFβ3 only increased soluble collagen in the presence of OA. e, LOX activity in fibroblasts treated with 0.1 mm fatty acids for 24 h with or without TGFβ1. LOX activity was the highest in LA-treated fibroblasts with TGFβ1. In vitro experiments were repeated at least twice in triplicate. Data were analyzed using two-way ANOVA with Tukey post hoc tests, p < 0.05. *, p < 0.05 versus corresponding control cells. †, p < 0.05 versus corresponding OA-treated cells. lox, gene for LOX; loxl1, gene for lysyl oxidase like 1; RFU, relative fluorescence units; fads2, fatty acid desaturase 2.
FIGURE 7.
FIGURE 7.
Effect of fads2 overexpression on LOX activity in fibroblasts. Control or stably fads2 transfected fibroblasts were treated with 0.05, 0.1, or 0.2 mm of OA (a) and LA (b) for 24 h. Experiments were repeated at least twice in triplicate. Overexpression of fads2 abolished LOX activity in LA-treated fibroblasts. c, representative Western blots for LOX across 0.2 mm fatty acid treatments. Mature 30-kDa fraction of LOX was profoundly inhibited by fads2 overexpression with LA. Data were analyzed using two-way ANOVA with Tukey post hoc tests, p < 0.05. *, p < 0.05 versus corresponding untransfected cells. †, p < 0.05 versus corresponding 0.05 mm cells.††, p < 0.05 versus corresponding 0.1 mm cells. RFU, relative fluorescence units; fads2, fatty acid desaturase 2.
FIGURE 8.
FIGURE 8.
Effect of fads2 RNAi on collagen deposition and LOX activity in fibroblasts. a, fads2 gene levels after incubation with scrambled or fads2 RNAi in fibroblasts. b, deposited collagen in fibroblasts treated with incremental doses of OA (b) or LA (c) in the presence of either scrambled or fads2 RNAi after 24 h. No effect of fads2 RNAi was noted with OA, but it increased collagen deposition with both 0.1 and 0.2 mm LA. LOX activity in fibroblasts treated with incremental doses of OA (d) or LA (e) in the presence of either scrambled or fads2 RNAi after 24 h. OA demonstrated increased LOX activity at 0.05 mm, whereas LA demonstrated increased LOX activity at 0.1 and 0.2 mm under fads2 RNAi. f, Western blots for LOX from fibroblasts with fads2 RNAi with 0.2 mm fatty acids. fads2 RNAi induced significant increase in mature/prolysyl oxidase levels with LA but not OA. Experiments were repeated at least twice in triplicate. Data were analyzed using two-way ANOVA with Tukey post hoc tests, p < 0.05. *, p < 0.05 versus corresponding scrambled controls. RFU, relative fluorescence units; fads2, fatty acid desaturase 2.
FIGURE 9.
FIGURE 9.
n-6 PUFAs, GLA, and AA does not induce collagen deposition in fibroblasts. a, deposited collagen with 0.1 mm fatty acids with or without TGFβ1 for 24 h. b, deposited collagen in fads2 overexpressing fibroblasts with 0.1 mm fatty acids with or without TGFβ1 for 24 h. c, LOX activity in normal and fads2 overexpressing fibroblasts with 0.1 mm fatty acids and TGFβ1 for 24 h. Experiments were repeated at least twice in triplicate. Data are expressed as a percentage of control. Data were analyzed by two-way ANOVA with Tukey post hoc tests, p < 0.05. *, p < 0.05 versus corresponding LA treated cells. †, p < 0.05 versus corresponding unstimulated cells. RAU, relative absorbance units, RFU, relative fluorescence units, fads2, fatty acid desaturase 2.
FIGURE 10.
FIGURE 10.
Proposed schematic of the opposing roles of linoleic and oleic acids on TGFβ-collagen axis. The parent n-6 polyunsaturated fatty acid, linoleic acid (LA, blue arrows), activates TGFβ1 and induces collagen I and promotes LOX maturation and activity. Together, LOX forms insoluble collagen fibers and induces a stiff myocardium that may induce abnormal transmitral flow, indicative of early diastolic dysfunction. Downstream PUFAs of LA, generated by fads2 or fads1 action, were not found to have significant roles in this axis. Oleic acid (blue arrow), a monounsaturated fatty acid, induces TGFβ3 and collagen III as a direct effect. However, OA can also block LA action (red lines) by directly inhibiting TGFβ1, collagen I, and LOX activity, thereby promoting an elastic myocardial phenotype.

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