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. 2022 Mar 16;118(4):1061-1073.
doi: 10.1093/cvr/cvab111.

Free fatty acid receptor 4 responds to endogenous fatty acids to protect the heart from pressure overload

Katherine A Murphy  1 , Brian A Harsch  2 , Chastity L Healy  1 , Sonal S Joshi  1 , Shue Huang  2 , Rachel E Walker  2 , Brandon M Wagner  1 , Katherine M Ernste  1 , Wei Huang  3 , Robert C Block  4 , Casey D Wright  5 , Nathan Tintle  6 , Brian C Jensen  3 , Quinn S Wells  7 , Gregory C Shearer  2 , Timothy D O'Connell  1
Affiliations

Free fatty acid receptor 4 responds to endogenous fatty acids to protect the heart from pressure overload

Katherine A Murphy et al. Cardiovasc Res. .

Abstract

Aims: Free fatty acid receptor 4 (Ffar4) is a G-protein-coupled receptor for endogenous medium-/long-chain fatty acids that attenuates metabolic disease and inflammation. However, the function of Ffar4 in the heart is unclear. Given its putative beneficial role, we hypothesized that Ffar4 would protect the heart from pathologic stress.

Methods and results: In mice lacking Ffar4 (Ffar4KO), we found that Ffar4 is required for an adaptive response to pressure overload induced by transverse aortic constriction (TAC), identifying a novel cardioprotective function for Ffar4. Following TAC, remodelling was worsened in Ffar4KO hearts, with greater hypertrophy and contractile dysfunction. Transcriptome analysis 3-day post-TAC identified transcriptional deficits in genes associated with cytoplasmic phospholipase A2α signalling and oxylipin synthesis and the reduction of oxidative stress in Ffar4KO myocytes. In cultured adult cardiac myocytes, Ffar4 induced the production of the eicosapentaenoic acid (EPA)-derived, pro-resolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE). Furthermore, the activation of Ffar4 attenuated cardiac myocyte death from oxidative stress, while 18-HEPE rescued Ffar4KO myocytes. Systemically, Ffar4 maintained pro-resolving oxylipins and attenuated autoxidation basally, and increased pro-inflammatory and pro-resolving oxylipins, including 18-HEPE, in high-density lipoproteins post-TAC. In humans, Ffar4 expression decreased in heart failure, while the signalling-deficient Ffar4 R270H polymorphism correlated with eccentric remodelling in a large clinical cohort paralleling changes observed in Ffar4KO mice post-TAC.

Conclusion: Our data indicate that Ffar4 in cardiac myocytes responds to endogenous fatty acids, reducing oxidative injury, and protecting the heart from pathologic stress, with significant translational implications for targeting Ffar4 in cardiovascular disease.

Keywords: 18-hydroxyeicosapentaenoic acid (18-HEPE); Cytoplasmic phospholipase A2α (cPLA2α); Eicosapentaenoic acid (EPA); Free fatty acid receptor 4 (Ffar4); GPR120; Heart failure.

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Figures

Figure 1
Figure 1
Four weeks following TAC, mice were euthanized and hearts were collected. (A) Heart weight (HW) and (B) heart weight-to-body weight ratio (HW/BW) of male WT and Ffar4KO mice. (C) Representative images of ventricular fibrosis quantified from ventricular cross sections from male WT and Ffar4KO mice stained with Sirius red/Fast Green. (D) Ventricular fibrosis quantified by fibrotic area (Sirius red)/total ventricular area (Fast green) from male WT and Ffar4KO mice. (E) Lung weight of male WT and Ffar4KO mice. Four weeks following TAC, cardiac function measured by echocardiography in male WT and Ffar4KO mice. (F) Ejection fraction (EF, %); (G) E/A ratio. Data were compared by a Welch’s two sample t test. Error bars represent the mean with SD.
Figure 2
Figure 2
Transcriptome analysis (RNA-seq) was performed on cardiac myocytes isolated from male WT and Ffar4KO mice 3-days post-TAC or sham surgery. (A) Principal component analysis of RNA transcriptomes from WT and Ffar4KO cardiac myocytes. (B) Venn diagram indicating genes differentially expressed ≥ 1.7-fold uniquely in WT cardiac myocytes post-TAC (1380 genes), upregulated ≥ 1.7-fold uniquely in Ffar4KO cardiac myocytes post-TAC (247 genes), or upregulated ≥ 1.7-fold in both (1409). (C) Differentially expressed genes identified in B were sorted based on gene ontology (GO) terms for biological function. Graphical representation of the number of genes up- or down-regulated in each category that were unique to the WT TAC (relative to sham, green), unique to Ffar4KO TAC (relative to sham, blue), and shared between WT and Ffar4KO (grey). (D, E) Data sets of gene expression from WT (D) and Ffar4KO (E) cardiac myocytes were analysed using Ingenuity Pathway Analysis Software (version 01-16). A custom pathway for known Ffar4 signalling pathways was generated, and expression data for WT and Ffar4KO were analysed (WT, green map, Ffar4KO blue map, as indicated). Genes upregulated (red) or down-regulated (green) for each group are indicated. (F) Induction of gene cardiac gene expression by TUG-891 (35 mg/kg/d, IP, for 3 days) in male WT mice. Each gene is indicated at the top of the graph. Data are mean ± SEM, n = 4 mice per group, data were analysed by Student’s t-test.
Figure 3
Figure 3
(A–H) Cultured adult cardiac myocytes from WT (green) and Ffar4KO (blue) male mice were treated with the Ffar4 agonist, TUG-891 (50 µM) for 0, 15, 30, and 60 min. Oxylipins were detected by mass spectrometry from cardiac myocyte membranes (esterified) or cytosolic fractions [non-esterified, or from the culture medium in lipoproteins (esterified) or free (non-esterified)]. Probability plots for oxylipins detected from cardiac myocytes in the (A) esterified or (B) unesterified fractions after 60 min, or in the culture medium in the (C) esterified or (D) unesterifed fractions, or time course of 18-HEPE production in myocytes in the (E) esterified or (F) unesterifed fractions, or in the medium in the (G) esterified or (H) unesterified fractions (dashed lines represent the 95% CI). (I) Cytoplasmic phospholipase A2α (cPLA2α) mediated cleavage of EPA from membrane phospholipids and production of 18-HEPE by CYPhydroxylase. 18-HEPE is re-acylated into membrane phospholipids (sequestration), remains free in the cell and is further metabolized (potentially E-Resolvins), or exported, esterified in a lipoprotein. (J) EPA, DHA, and AA concentration in cardiac myocyte nuclear membrane (NM) or sarcolemma/plasma membrane (PM) fractions. Data are mean ± SD, n = 3 separate preparations, fold differences indicated.
Figure 4
Figure 4
(A) Genes identified by the oxidation–reduction gene ontology sorting that could specifically affect the redox state of cardiac myocytes post-TAC. Data are n = 4–6, as in Figure 3, and fold change in expression for WT (green) and Ffar4KO (blue) are shown. (B) Cultured adult cardiac myocytes from WT mice were treated for 24 h with vehicle or 10 µM TUG-891, and for the final 2 h, myocytes were treated with vehicle or 10 µM H2O2 to induce oxidative damage and cell death. For all panels, images were captured using a ×10 objective, the entire field is shown, and cell death was indicated by round-shaped myocytes. The number of rod-shaped/round myocytes is in the inset. (C) Cell morphology was recorded, with at least 150 cells from 5 different fields measured per condition. Data are mean ± SEM, n = 4 separate cultures, data were analysed by two-way ANOVA, with Sidek post-test. (D) Cultured adult cardiac myocytes from WT (top) and Ffar4KO (bottom) mice were treated for 4 h with vehicle or 100 nM 18-HEPE and for the final 2 h, myocytes were treated with vehicle or 10 µM H2O2. The number of rod-shaped/round myocytes is in the inset. (E) Cell morphology was assessed as above. Data are mean ± SEM, n = 4 separate cultures, data were analysed by three-way ANOVA, with Sidek post-test. In this case, the primary interaction was not significant, but the post-test indicated a significant effect of both 18-HEPE and H2O2, indicating no difference between WT and Ffar4KO.
Figure 5
Figure 5
Plasma was collected 4 weeks following TAC or sham surgery, and oxylipins in HDL were detected by liquid chromatography/mass spectrometry. To discriminate changes in oxylipin content between WT and Ffar4KO in sham and TAC operated mice, wide principal component analysis was performed (A). Scores plot of group locations by PC Scores 1 (x-axis) and PC Scores 2 (y-axis). (B) PCA loadings with the identification and location of selected oxylipins. To exemplify changes, five oxylipins selected according to total variance explained in the first three-principle components were selected along with 9-HETE, an autoxidative marker. Important fold-differences (95%CIs) are summarized in each graph. (C) 18-HEPE, EPA metabolite of CYPhydroxylase. (D) 15-HETE, AA-metabolite of 12/15-LOX. (E) 12-HETE, AA-metabolite of 12/15-LOX. (F) 14(15)-EpETE, EPA metabolite of CYPepoxygenase. (G) 8(9)-EpETrE, AA metabolite of CYPepoxygenase and (H) 9-HETE, auto-oxidatively derived from AA.
Figure 6
Figure 6
RT-PCR to detect the expression of human, (A) total Ffar4 (both short and long isoforms) (B) Ffar4L only, and (C) Ffar1 in cardiac tissue obtained from non-failing (NF) and failing (F) human hearts. RT-PCR to detect the expression of mouse (D) Ffar4 and (E) Ffar1 in wild-type (WT) and Ffar4KO (KO) mouse heart.
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