A Novel Endocrine Role for the BAT-Released Lipokine 12,13-diHOME to Mediate Cardiac Function

Abstract

Background: Brown adipose tissue (BAT) is an important tissue for thermogenesis, making it a potential target to decrease the risks of obesity, type 2 diabetes, and cardiovascular disease, and recent studies have also identified BAT as an endocrine organ. Although BAT has been implicated to be protective in cardiovascular disease, to this point there are no studies that identify a direct role for BAT to mediate cardiac function.

Methods: To determine the role of BAT on cardiac function, we utilized a model of BAT transplantation. We then performed lipidomics and identified an increase in the lipokine 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME). We utilized a mouse model with sustained overexpression of 12,13-diHOME and investigated the role of 12,13-diHOME in a nitric oxide synthase type 1 deficient (NOS1^-/-) mouse and in isolated cardiomyocytes to determine effects on function and respiration. We also investigated 12,13-diHOME in a cohort of human patients with heart disease.

Results: Here, we determined that transplantation of BAT (+BAT) improves cardiac function via the release of the lipokine 12,13-diHOME. Sustained overexpression of 12,13-diHOME using tissue nanotransfection negated the deleterious effects of a high-fat diet on cardiac function and remodeling, and acute injection of 12,13-diHOME increased cardiac hemodynamics via direct effects on the cardiomyocyte. Furthermore, incubation of cardiomyocytes with 12,13-diHOME increased mitochondrial respiration. The effects of 12,13-diHOME were absent in NOS1^-/- mice and cardiomyocytes. We also provide the first evidence that 12,13-diHOME is decreased in human patients with heart disease.

Conclusions: Our results identify an endocrine role for BAT to enhance cardiac function that is mediated by regulation of calcium cycling via 12,13-diHOME and NOS1.

Keywords: 12; 13-diHOME; NOS1; brown adipose tissue (BAT); cardiac function; exercise; lipokines.

Figure 1.. Exercise or increasing BAT mass by transplantation improves cardiac function and structure in mice.

(A) Systolic function and (B) diastolic function measured by in vivo cardiac hemodynamics (n=4-6/group). Cardiac function and structure measured by (C) ejection fraction, (D) end diastolic volume, (E) left ventricular mass, and (F) diastolic diameter. Data are mean ± S.E.M (n=6/group). Asterisks represent difference vs. Sham-Sedentary (*P<0.05). Repeated measures two-way ANOVA was used for A and B with Tukey’s multiple comparisons tests; one-way ANOVA was used for C, D, E, and F with Tukey’s multiple comparisons tests.

Figure 2.. Exercise or transplantation of BAT increases circulating 12,13-diHOME, 9,10-diHOME, and 9-HODE.

(A) Heat map and (B,C) volcano plot representing 88 lipids comparing the fold induction of Sham-Sedentary to the p value; 12,13-diHOME is circled in red. Data are mean ± S.E.M (n=6/group). Plasma concentrations of (D) 12,13-diHOME, (E) 9,10-diHOME, and (F) 9-HODE. Data are mean ± S.E.M (n=6/group). Asterisks represent difference vs. Sham-Sedentary (*P<0.05; **P<0.01; ***P<0.001). One-way ANOVA was used for C, D, and E with Tukey’s multiple comparisons tests.

Figure 3.. 12,13-diHOME improves in vivo cardiac function and structure.

(A) Systolic and (B) diastolic function in mice acutely injected with saline (n=3), 12,13-diHOME (n=8), 9-HODE (n=5), or 9,10-diHOME (n=3). Data are mean ± S.E.M Asterisks represent difference compared to mice injected with saline, 9-HODE, or 9,10-diHOME (*P<0.05; **P<0.01). (C) Systolic and (D) diastolic function measured by in vivo cardiac hemodynamics in Sham or +BAT mice fed the sEH inhibitor AUDA. (E) Systolic and (F) diastolic function measured by in vivo cardiac hemodynamics in Sham or +BAT mice fed the sEH inhibitor t-AUCB. Data are mean ± S.E.M (n=6/group). (G) 12,13-diHOME in Baseline (n=8), Sham (n=5), TNT-Ephx1/2 (n=7), or TNT-Ucp1 (n=5) mice. Data are mean ± S.E.M Asterisks represent difference compared to Sham (*P<0.05). Cardiac function and structure measured by (H) ejection fraction, (I) posterior wall thickness, (J) left ventricular mass, (K) diastolic diameter, and (L) end diastolic volume (EDV) was measured in Sham (n=5), TNT-Ephx1/2 (n=7), or TNT-Ucp1 (n=5). Data are mean ± S.E.M Asterisks represent differences compared to baseline cohort (*P<0.05; **P<0.01). (M) Systolic function and (N) diastolic function measured by in vivo cardiac hemodynamics. Data are mean ± S.E.M Asterisks represent difference in TNT-Ephx1/2 compared to all other groups (*P<0.05; **P<0.01; ***P<0.001). Repeated measures two-way ANOVA was used for C, D, E, F, M, and N with Tukey’s multiple comparisons tests. One-way ANOVA was used for G, H, I, J, K, and L with Tukey’s multiple comparisons tests. Kruskal-Wallis test was used for A and B.

Figure 4.. 12,13-diHOME increases function and respiration in isolated cardiomyocytes.

(A) Peak shortening, (B) Ca2+ transient, (C) maximal velocity of shortening, and (D) maximal velocity of relengthening in isolated cardiomyocytes ±12,13-diHOME. Data are mean ± S.E.M (n=4/group; 10-12 myocytes per mouse). Asterisks represent difference compared to vehicle (**P<0.01; ***P<0.001). (E) Fatty acid uptake in cardiomyocytes constitutively expressing firefly luciferase that were treated with either 12,13-diHOME or vehicle, as measured by luciferase activity using 10 uM FFA-SS-Luc. Data are mean ± S.E.M (n=9 technical replicate wells per group. Asterisks represent difference compared to vehicle (**P<0.01). (F) Bioenergetic profile of cardiomyocytes treated with 12,13-diHOME or vehicle. (G) Basal OCR, (H) maximal respiration, and (I) non-mitochondrial respiration were measured. Data are mean ± S.E.M (n=5/group). Asterisks represent differences compared to vehicle (*P<0.05; ***P<0.001). (J) Bioenergetic profile of cardiomyocytes treated with 12,13-diHOME or vehicle with or without BDM. (K) Basal OCR, (L) maximal respiration, and (M) non-mitochondrial respiration were measured. Data are mean ± S.E.M (n=5/group). Asterisks represent differences compared to vehicle (*P<0.05; ***P<0.001), compared to vehicle + BDM (##P<0.01; ###P<0.001), or compared to 12,13-diHOME + BDM ($$$P<0.001). Unpaired two-tailed Student’s t-test was used for A, B, C, D, G, H, and I. Two-way ANOVA was used for E, F and J with Tukey’s multiple comparisons tests; one-way ANOVA was used for K, L, and M with Tukey’s multiple comparisons tests.

Figure 5.. 12,13-diHOME improves cardiac function and respiration via NOS1 and RyR.

(A) Systolic and (B) diastolic function in wild-type (WT) (n=6) or NOS1^−/− mice (n=6). (C) Peak shortening, (D) Ca2+ transient, (E) maximal velocity of shortening, and (F) maximal velocity of relengthening in isolated cardiomyocytes from NOS1^−/− mice treated with PBS or 12,13-diHOME. Data are mean ± S.E.M (n=5/group; 5-16 myocytes per mouse). (G) Bioenergetic profile of NOS1^−/− cardiomyocytes treated with 12,13-diHOME or vehicle. (H) Basal OCR, (I) maximal respiration, and (J) non-mitochondrial respiration were measured. Data are mean ± S.E.M (n=5/group). (K) Bioenergetic profile of cardiomyocytes treated with 12,13-diHOME or vehicle with or without tetracaine. (L) Basal OCR, (M) maximal respiration, and (N) non-mitochondrial respiration were measured. Data are mean ± S.E.M (n=5/group). Asterisks represent differences compared to vehicle (*P<0.05; ***P<0.001), compared to vehicle + tetracaine (#P<0.05; ###P<0.001), or compared to 12,13-diHOME + tetracaine ($$$P<0.001). (O) Proposed model for 12,13-diHOME to regulate cardiac function via NOS1. Unpaired two-tailed Student’s t-test was used for A, B, C, D, E, F, H, I, and J. Two-way ANOVA was used for G and K with Tukey’s multiple comparisons tests. One-way ANOVA was used for L, M, and N with Tukey’s multiple comparisons tests.

Figure 6.. 12,13-diHOME is decreased in human patients with heart disease.

(A) Plasma concentrations of 12,13-diHOME of human subjects (healthy and heart disease). Data are mean ± S.E.M (healthy males n=25; healthy female n=26; males with heart disease n=17; females with heart disease n=7). Asterisks represent differences compared to healthy controls of same gender (*P<0.05; **P<0.01), or an overall effect of heart disease (#P<0.05). (B) BMI among groups and (C) correlation among BMI and 12,13-diHOME. (D) Age among groups and (E) correlation among age and 12,13-diHOME. Asterisks represent differences among healthy male and female subjects (*P<0.05). In a subset of patients with heart disease, (F) ejection fraction and (G) fractional shortening correlated to 12,13-diHOME in plasma. One-way ANOVA was used for A, B, and D with Tukey’s multiple comparisons tests. Spearman’s correlation was used for C and E. Linear regression analyses were used for F and G.