FFAR4 regulates cardiac oxylipin balance to promote inflammation resolution in HFpEF secondary to metabolic syndrome

Abstract

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome, but a predominant subset of HFpEF patients has metabolic syndrome (MetS). Mechanistically, systemic, nonresolving inflammation associated with MetS might drive HFpEF remodeling. Free fatty acid receptor 4 (Ffar4) is a GPCR for long-chain fatty acids that attenuates metabolic dysfunction and resolves inflammation. Therefore, we hypothesized that Ffar4 would attenuate remodeling in HFpEF secondary to MetS (HFpEF-MetS). To test this hypothesis, mice with systemic deletion of Ffar4 (Ffar4KO) were fed a high-fat/high-sucrose diet with L-NAME in their water to induce HFpEF-MetS. In male Ffar4KO mice, this HFpEF-MetS diet induced similar metabolic deficits but worsened diastolic function and microvascular rarefaction relative to WT mice. Conversely, in female Ffar4KO mice, the diet produced greater obesity but no worsened ventricular remodeling relative to WT mice. In Ffar4KO males, MetS altered the balance of inflammatory oxylipins systemically in HDL and in the heart, decreasing the eicosapentaenoic acid-derived, proresolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE), while increasing the arachidonic acid-derived, proinflammatory oxylipin 12-hydroxyeicosatetraenoic acid (12-HETE). This increased 12-HETE/18-HEPE ratio reflected a more proinflammatory state both systemically and in the heart in male Ffar4KO mice and was associated with increased macrophage numbers in the heart, which in turn correlated with worsened ventricular remodeling. In summary, our data suggest that Ffar4 controls the proinflammatory/proresolving oxylipin balance systemically and in the heart to resolve inflammation and attenuate HFpEF remodeling.

Keywords: 18-hydroxyeicosapentaenoic acid; free fatty acid receptor 4 (Ffar4); heart failure preserved ejection fraction (HFpEF); inflammation; lipidomics; metabolic syndrome; obesity; omega-3 fatty acids; phospholipase a2.

Fig. 1

Male WT (green) and Ffar4KO (KO, blue) mice were fed a control diet (Cont., open symbols) or the combination of a high-fat (42%)/high-sucrose (30%) diet and L-NAME (1 mg/ml) in the drinking water (HFpEF, closed symbols) for 20 weeks. After 20 weeks; (A) Body weight was recorded, and body composition, including: (B) Fat Mass and (C) Lean Mass were determined by EchoMRI; and (D) Adiposity Index (fat mass/lean mass) was calculated. (E) Intraperitoneal glucose tolerance test (IPGTT) and (F) calculation of AUC. (G) Mean Arterial Pressure (MAP) measured by tail-cuff. (H) Triglyceride (TG) levels and (I) HDL-C levels were measured from plasma. Data (A–I) are presented as mean ± 95% confidence interval and were analyzed by two-way ANOVA with Tukey's multiple comparison test. Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction; L-NAME, L-nitroarginine methyl ester.

Fig. 2

Cardiac function was measured by echocardiography in male WT (green) and Ffar4KO (KO, blue) mice after 20 weeks on the control diet (Cont., open symbols) or HFpEF diet (HFpEF, closed symbols). (A) E/e’ ratio. (B) E/A ratio. (C) Ejection fraction (EF, %). (D) Ventricular fibrosis quantified hydroxyproline content (μg/mg ventricular wet weight). (E) Ventricular myocardial capillary density quantified by Isolectin-B4. Data (A–E) are presented as mean ± 95% CI and were analyzed by two-way ANOVA with Tukey's multiple comparison test. ∗Primary interaction: P = 0.0779, Welch’s t test for WT HFpEF versus Ffar4KO HFpEF, employed due to unequal variance between Control and HFpEF diet groups: P = 0.0438. Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction.

Fig. 3

Cardiac function was measured by echocardiographic strain analysis (A, B) or using Langendorff perfused hearts (C–G) from male WT (green) and Ffar4KO (KO, blue) mice after 20 weeks on the control diet (Cont., open symbols) or HFpEF diet (HFpEF, closed symbols). (A) Global longitudinal strain (GLS, %). (B) Reverse longitudinal peak strain rate (1/s). (C) dP/dt max. (D) dP/dt min. (E) O₂ extraction normalized to heart weight (%). (F) Cardiac efficiency normalized to heart weight (mmHg/μmol/mg). (G) Change in dP/dt from baseline following isoproterenol administration. Data (A–G) are presented as mean ± 95% CI and were analyzed by two-way ANOVA with Tukey's multiple comparison test. Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction.

Fig. 4

Female WT (orange) and Ffar4KO (KO, purple) mice were fed a control diet (Cont., open symbols) or the combination of a high-fat (42%)/high-sucrose (30%) diet and L-NAME (1 mg/ml) in the drinking water (HFpEF, closed symbols) for 20 weeks. After 20 weeks; (A) body weight was recorded and (B) Adiposity Index (fat mass/lean mass, determined by EchoMRI) was calculated. (C) Mean Arterial Pressure (MAP) measured by tail-cuff. (D) Intraperitoneal glucose tolerance test (IPGTT). (E) calculation of AUC. (F) Triglyceride levels and (G) HDL-C levels were measure from plasma. Cardiac function was measured by echocardiography. (H) E/e’ ratio. (I) E/A ratio. (J) ejection fraction (EF, %). Data (A–I) are presented as mean ± 95% CI and were analyzed by two-way ANOVA with Tukey's multiple comparison test. Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction; L-NAME, L-nitroarginine methyl ester.

Fig. 5

After 20 weeks on diet (Cont., open symbols, HFpEF, closed symbols), plasma was collected from both male and female, WT and Ffar4KO mice (Male: WT, green; KO, blue, Female: WT, orange; KO, purple) and HDL oxylipin content was detected by liquid chromatography/mass spectrometry. (A, C) Levels of the 18-HEPE, an EPA-derived, proresolving oxylipin and 12-HETE, an AA-derived, proinflammatory oxylipin in HDL from males. (B, D) Levels of 18-HEPE and 12-HETE in HDL from females. (E) 12-HETE/18-HEPE ratio in HDL from males and females. Data are presented as mean ± 95% CI and were analyzed by two-way ANOVA with Tukey's multiple comparison test. 18-HEPE, 18-hydroxyeicosapentaenoic acid; 12-HETE, 12-hydroxyeicosatetraenoic acid; Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction.

Fig. 6

After 20 weeks on diet (Cont., open symbols, HFpEF, closed symbols), hearts were harvested from both male, WT (WT, green) and Ffar4KO (KO, blue) mice, and heart oxylipin content in the esterified and nonesterified (NEOx) fractions was detected by liquid chromatography/mass spectrometry. (A, D) 18-HEPE, esterified, and non-esterified. (B, E): 12-HETE, esterified, and nonesterified. (C, F) 12-HETE/18-HEPE ratio, esterified, and nonesterified. Data are presented as mean ± 95% CI and were analyzed by two-way ANOVA with Tukey's multiple comparison test. 18-HEPE, 18-hydroxyeicosapentaenoic acid; 12-HETE, 12-hydroxyeicosatetraenoic acid; Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction.

Fig. 7

A–C: After 20 weeks on diet (Cont., open symbols, HFpEF, closed symbols), RNA was isolated from male WT (WT, green) and Ffar4KO (KO, blue) hearts, and (A) Ffar4 (Ffar4), (B) ChemR23 (Cmklr1) , and (C) GPR31 (Gpr31) mRNA levels were quantified by qRT-PCR. Data are presented as mean ± 95% CI and were analyzed by two-way ANOVA with Tukey's multiple comparison test. Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction.

Fig. 8

(A, B) After 20 weeks on diet (Cont., open symbols, HFpEF, closed symbols), ventricular sections from male WT (WT, green) and Ffar4KO (KO, blue) hearts were stained CD64 to detect the total macrophage population (A) and total CD64⁺ cells were counted (B). Data (B) are presented as mean ± 95% CI and were analyzed by two-way ANOVA with Tukey's multiple comparison test. (C–E) Total CD64⁺ macrophages were correlated with markers of HFpEF remodeling: (C) E/e’ ratio; (D) E/A ratio; and (E) Capillary density. Ffar4, free fatty acid receptor 4; HFpEF, heart failure with preserved ejection fraction.