Lipid exposure leads to metabolic dysfunction in fetal sheep cardiomyocytes

Abstract

Fetal circulating lipids are low but rise precipitously following birth. It is unknown how prematurely elevated lipids affect the fetal heart, which primarily uses carbohydrates for energy. Fetal sheep were surgically instrumented and received Intralipid 20® or Lactated Ringer's Solution intravenously. After 8 days, myocardial biopsies were taken, and cardiomyocytes were dispersed. Lipid uptake was assessed by labeled saturated long-chain fatty acids (LCFA) and very long-chain fatty acids (VLCFA) incorporation. Maximal oxygen consumption rates (OCR) were measured. Gene and protein expression levels were measured by quantitative PCR and Western blotting. Intralipid treatment increased LCFA (p < 0.001) and VLCFA (p < 0.001) lipid droplet number, and LCFA (males p = 0.002) and VLCFA (p = 0.018) droplet size. Fetal Intralipid treatment reduced maximal OCR in basal media (p = 0.005). Palmitic acid decreased maximal OCR regardless of fetal treatment or length of in vitro exposure (p = 0.006). Fetal Intralipid upregulated genes included CD36 (p = 0.001), CPT1A (p < 0.001), CPT1B (p < 0.001), VLCAD (p < 0.001), and PDK4 (p < 0.001), with no differences in protein expression. There were no effects on ER stress, apoptosis, or autophagy markers. Extended elevated lipid levels in the fetus increased lipid uptake and may have shifted substrate preference towards lipids, but all lipid exposure depressed fetal cardiac metabolism. Prematurely elevated lipids mature but suppress oxidative metabolism.

Keywords: cardiomyocyte; fetus; metabolism; parenteral nutrition.

FIGURE 1

Lipid uptake and droplet formation in isolated cardiomyocytes following in vivo Intralipid administration in fetal sheep. Left panels: Representative images of BODIPY™‐labeled fatty acid incorporation into cellular lipid droplets (green; magnification 630×). Right panels: Summarized data of labeled long‐chain fatty acid (LCFA; upper panels) or very long‐chain fatty acid (VLCFA; lower panels) lipid droplet number per cell area and droplet size in left ventricular cardiomyocytes from Intralipid‐infused fetuses compared to vehicle‐infused fetuses. Scale bars = 25 μm. Raw data with mean. Number for Control (C) female (F) = 7, male (M) = 4; Intralipid female = 4, male = 6. Data were assessed by 2‐way ANOVA. Significance determined at α = 0.05 and shown for treatment main effects unless otherwise noted. Simple main effects tested following significant interaction using Bonferroni correction for multiple comparisons; family‐wise α = 0.025.

FIGURE 2

Maximal cellular respiration in cardiomyocytes provided palmitic acid following in vivo Intralipid administration in fetal sheep. (a) Oxygen consumption rates (OCR) were measured in cultured fetal cardiomyocytes from Intralipid or vehicle‐treated fetal sheep exposed only to standard cardiomyocyte media without palmitic acid using the Seahorse Extracellular Flux Analyzer. Data are expressed normalized to baseline OCR. Data were assessed by unpaired t‐test. (b) Palmitic acid must be conjugated at a 6:1 molar ratio with bovine serum albumin (BSA) for in vitro treatment. To assess the effects of this vehicle, cardiomyocytes were given fatty acid‐free BSA at a range of doses either acutely during the metabolic analysis, or for 24 h or 48 h prior to measurement. (c) Cardiomyocytes from Intralipid or vehicle‐treated fetal sheep were given palmitic acid (PA) at a range of doses either acutely at the time of measurement, for 24 h, or for 48 h prior to measurement. Data are expressed normalized to the appropriate dose of the bovine serum albumin vehicle. Data for (b) and (c) were assessed by mixed measures 3‐way ANOVA (four levels of repeated measures for in vitro treatment, three levels for in vitro treatment duration) with the Greenhouse–Geisser correction for sphericity (α = 0.05; details in Table 4). As there were no significant interactions analysis by in vivo treatment or duration of in vitro PA exposure, data are grouped only by in vitro treatment in the graph; p values for multiple comparisons using Bonferroni correction following significant main effects are shown. Numbers for Control = 6 (female = 3, male = 3), Intralipid = 6 (female = 1, male = 5), except for acute in vitro treatment Control = 5 (female = 3, male = 2), Intralipid = 5 (female n = 1, male = 4). All data raw with mean.

FIGURE 3

Cardiac expression levels of metabolism‐related genes and proteins following in vivo Intralipid administration in fetal sheep. (a) Fatty acid transporters CD36, CPT1A, and CPT1B mRNA and protein. (b) Esterification and lipid droplet enzymes DGAT, GPAT, and PLPP1 mRNA. (c) Beta‐oxidation enzymes HADH, LCAD, and VLCAD mRNA. (d) Metabolic and TCA cycle enzymes IDH and PDK4 mRNA. (e) Signaling molecules PPARA mRNA and protein, and VLDLR mRNA. (f) Representative images of Western blots. Number for Control female (F) = 7, male (M) = 4; Intralipid female = 4, male = 7. Data were assessed by 2‐way ANOVA. Significant p values (p < 0.05) shown for differences by treatment; there were no differences by sex.

FIGURE 4

Cardiac expression levels of ER stress, apoptosis, and autophagy‐related genes and proteins following in vivo Intralipid administration in fetal sheep. (a) ATF6, DDIT3 (CHOP), and HSPA5 mRNA, and phospho‐EIF2A protein. (b) Cleaved caspase 3 protein. (c) BECN1 mRNA. Number for Control female (F) = 7, male (M) = 4; Intralipid female = 4, male = 7. Data were assessed by 2‐way ANOVA. Significant p values (p < 0.05) shown for differences by sex; there were no differences by treatment.