Thyroid hormone increases fatty acid use in fetal ovine cardiac myocytes

Abstract

Cardiac metabolic substrate preference shifts at parturition from carbohydrates to fatty acids. We hypothesized that thyroid hormone (T₃ ) and palmitic acid (PA) stimulate fetal cardiomyocyte oxidative metabolism capacity. T₃ was infused into fetal sheep to a target of 1.5 nM. Dispersed cardiomyocytes were assessed for lipid uptake and droplet formation with BODIPY-labeled fatty acids. Myocardial expression levels were assessed PCR. Cardiomyocytes from naïve fetuses were exposed to T₃ and PA, and oxygen consumption was measured with the Seahorse Bioanalyzer. Cardiomyocytes (130-day gestational age) exposed to elevated T₃ in utero accumulated 42% more long-chain fatty acid droplets than did cells from vehicle-infused fetuses. In utero T₃ increased myocardial mRNA levels of CD36, CPT1A, CPT1B, LCAD, VLCAD, HADH, IDH, PDK4, and caspase 9. In vitro exposure to T₃ increased maximal oxygen consumption rate in cultured cardiomyocytes in the absence of fatty acids, and when PA was provided as an acute (30 min) supply of cellular energy. Longer-term exposure (24 and 48 h) to PA abrogated increased oxygen consumption rates stimulated by elevated levels of T₃ in cultured cardiomyocytes. T₃ contributes to metabolic maturation of fetal cardiomyocytes. Prolonged exposure of fetal cardiomyocytes to PA, however, may impair oxidative capacity.

Keywords: fetus; heart; heart metabolism; lipid metabolism.

FIGURE 1

Lipid uptake and droplet formation in isolated cardiomyocytes. On the left are representative images of BODIPY™‐labeled fatty acid incorporation into cellular lipid droplets (green; magnification 630×). Summarized data are shown on the right. There are a greater number of labeled long‐chain fatty acid (LCFA) lipid droplets per cell area (upper panels) in left ventricular cardiomyocytes from T₃‐infused fetuses compared to vehicle‐infused fetuses. There are no differences in lipid droplet density for very long‐chain (VLCFA; lower panels). Scale bars = 25 μm. n = 7 fetuses per group. Mean ± SD. Treatments compared by Student's unpaired t‐test.

FIGURE 2

Left ventricular metabolic gene mRNA expression levels following 5 days of exposure to elevated T₃ in utero. Expression levels of mRNA were measured in left ventricular myocardium from fetuses that had received an intravenous infusion of T₃ (54 μg d⁻¹; n = 8) or vehicle (n = 7) for 5 days. Following T₃ infusion there were increases in mRNA levels for genes involved in (a) fatty acid transporter, (b) beta oxidation, and (c) the citric acid cycle, but not (d) fatty acid esterification and lipid droplet formation. Mean ± SD. Treatments compared by Student's unpaired t‐test.

FIGURE 3

Left ventricular regulatory gene mRNA expression levels following 5 days of exposure to elevated T₃ in utero. Expression levels of mRNA were measured in left ventricular myocardium from fetuses that had received an intravenous infusion of T₃ (54 μg d⁻¹; n = 8) or vehicle (n = 7) for 5 days. Following T₃ infusion (a) the metabolic regulator PDK for was elevated by T₃ exposure, but the transcription factor PPARα was not. (b) Apoptosis regulators were not affected, but caspase 3 was elevated. Mean ± SD. Treatments compared by Student's unpaired t‐test.

FIGURE 4

The effects of T₃ and PA on oxygen consumption rates in fetal cardiomyocytes. Oxygen consumption rates (OCR) were measured in cultured fetal cardiomyocytes using the Seahorse Extracellular Flux Analyzer. (a) Treatment with T₃ (24 h; all doses) increased maximal OCR. A threshold rather than a dose‐dependent effect was observed. (b) Adding palmitic acid (PA) as an energetic substrate at the time of analysis yielded a similar result, but shifted the T₃ threshold (0.75 nM to 1.5 nM). In contrast, 24 h treatment of cells with (c) 100 μM PA or (d) 200 μM PA eliminated the T₃‐stimulated increase in maximal OCR, suggesting suppression of oxygen consumption by PA. n = 9 fetuses per group (Panels a–c), n = 6 (Panel D, except n = 5 for T₃ 0.75 nM). Groups were compared by 1‐way ANOVA followed, if warranted, by Tukey's multiple comparisons test. Mean ± SD.

FIGURE 5

The role of palmitic acid on maximal oxygen consumption rate in the presence of T₃. Maximal oxygen consumption rate (OCR) was compared in the presence of varying doses of T₃ across all exposures to palmitic acid (PA). (a) Acute PA (100 μM) suppressed maximum OCR compared to control cells that only received 0.75 nM T₃. However, maximal OCR following 24 h exposure to PA (100 or 200 μM) was similar to that in control cells, suggesting cellular adaptation to lipid as a substrate at this low level of T₃. At (b) 1.5 nM T₃, and (c) 10 nM T₃ there were PA dose‐dependent depressions in maximal OCR with chronic PA exposure. n = 9 fetuses per group, except n = 6 for the groups treated with 200 μM PA and n = 5 for 200 μM PA + T₃ 0.75 nM. Groups were compared by 1‐way ANOVA followed by Tukey's multiple comparisons test. Mean ± SD.