Role of pyruvate dehydrogenase inhibition in the development of hypertrophy in the hyperthyroid rat heart: a combined magnetic resonance imaging and hyperpolarized magnetic resonance spectroscopy study

Abstract

Background: Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy.

Methods and results: Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg x kg(-1) x d(-1)). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022 ± 0.0002 versus 0.0055 ± 0.0005 second(-1); P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-(13)C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100 ± 20 versus 200 ± 30 mg; P=0.04) despite no change in the increase observed in cardiac output.

Conclusions: This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand.

Figure 1

(A) Single representative MR cardiac spectrum of a control rat at day 0 following infusion of [1-¹³C]pyruvate, recorded at t = 10 s. Pyruvate (and its equilibrium product pyruvate hydrate), as well as lactate, alanine and bicarbonate, metabolic products of pyruvate, are annotated. (B) The rate of exchange of ¹³C label from [1-¹³C]pyruvate in to [1-¹³C]bicarbonate in each group assessed using hyperpolarized ¹³C-MRS. (C) The activity of the active PDH fraction (PDH_a) assessed ex vivo using a spectrophotometric approach. (D) Relative expression of PDK4 as assessed by Western blotting. (Key: ‘Control’ - combined day 0 data (n=18); ‘T3’ - hyperthyroid rats (n=7); ‘T3 + DCA Infusion’ - received an acute infusion of DCA immediately prior to MRS analysis (n=6), ‘T3 + DCA Treatment’ received DCA treatment throughout the 7 day T3 administration period (n=5); * P < 0.05 vs. control, ^‡ P < 0.05 vs. T3.).

Figure 2

(A) The maximum [1-¹³C]alanine / maximum [1-¹³C]pyruvate ratio and (B) The maximum [1-¹³C]lactate / maximum [1-¹³C]pyruvate ratio in each group. (*P<0.05 vs. control, ^‡P<0.05 vs. T3) (C) An example high-resolution 500 MHz ¹H NMR spectrum of an extract of cardiac tissue. Peak 1: valine/leucine/isoleucine, peak 2: β-hydroxybutyrate, peak 3: lactate, peak 4: alanine, peak 5: acetate, peak 6: glutamate, peak 7: glutamate and glutamine, peak 8: glutamine, peak 9: succinate, peak 10: malate, peak 11: citrate, peak 12: aspartate, peak 13: creatine, peak 14: choline, peak 15: phosphocholine / glycerophosphocholine, peak 16: taurine, peak 17: glycine, peak 18: glucose, peak 19: glycerol backbone. (D) Metabolic perturbations in the hyperthyroid heart measured using ex vivo ¹H-NMR spectroscopy. Changes are expressed as percentages of the measured control values and were all significant at the P<0.05 level.

Figure 3

(A) MR cardiac spectrum of a T3 rat at day 8 following infusion of [2-¹³C]pyruvate. Ten individual spectra were summed to generate this spectrum (t = 4-13 s). [2-¹³C]pyruvate and its metabolic products are annotated (unlabelled peaks are impurities in the [2-¹³C]pyruvate preparation). (B) The [3-¹³C]citrate/[2-¹³C]pyruvate ratio in each group assessed using hyperpolarized ¹³C-MRS. (Key: Control- combined day 0 data (n=12); T3-hyperthyroid rats (n=7); ‘T3 + DCA Treatment’ received DCA treatment throughout the 7 day T3 administration period (n=5); * P<0.05 vs. control).

Figure 4

Cardiac mass and cardiac function (cardiac output) assessed using MRI, in the hyperthyroid rat heart with and without DCA treatment at Day 0 (Black) and Day 8 (White) (*P<0.05).