The cycling of acetyl-coenzyme A through acetylcarnitine buffers cardiac substrate supply: a hyperpolarized 13C magnetic resonance study

Abstract

Background: Carnitine acetyltransferase catalyzes the reversible conversion of acetyl-coenzyme A (CoA) into acetylcarnitine. The aim of this study was to use the metabolic tracer hyperpolarized [2-(13)C]pyruvate with magnetic resonance spectroscopy to determine whether carnitine acetyltransferase facilitates carbohydrate oxidation in the heart.

Methods and results: Ex vivo, following hyperpolarized [2-(13)C]pyruvate infusion, the [1-(13)C]acetylcarnitine resonance was saturated with a radiofrequency pulse, and the effect of this saturation on [1-(13)C]citrate and [5-(13)C]glutamate was observed. In vivo, [2-(13)C]pyruvate was infused into 3 groups of fed male Wistar rats: (1) controls, (2) rats in which dichloroacetate enhanced pyruvate dehydrogenase flux, and (3) rats in which dobutamine elevated cardiac workload. In the perfused heart, [1-(13)C]acetylcarnitine saturation reduced the [1-(13)C]citrate and [5-(13)C]glutamate resonances by 63% and 51%, respectively, indicating a rapid exchange between pyruvate-derived acetyl-CoA and the acetylcarnitine pool. In vivo, dichloroacetate increased the rate of [1-(13)C]acetylcarnitine production by 35% and increased the overall acetylcarnitine pool size by 33%. Dobutamine decreased the rate of [1-(13)C]acetylcarnitine production by 37% and decreased the acetylcarnitine pool size by 40%.

Conclusions: Hyperpolarized (13)C magnetic resonance spectroscopy has revealed that acetylcarnitine provides a route of disposal for excess acetyl-CoA and a means to replenish acetyl-CoA when cardiac workload is increased. Cycling of acetyl-CoA through acetylcarnitine appears key to matching instantaneous acetyl-CoA supply with metabolic demand, thereby helping to balance myocardial substrate supply and contractile function.

Figure 1

The involvement of acetylcarnitine in cardiac carbohydrate metabolism. Acetyl-CoA formed from pyruvate can be incorporated into the Krebs cycle via citrate synthase or can be incorporated into the larger acetylcarnitine ‘overflow pool’ via CAT. CAT liberates free CoA and allows for cytosolic disposal of acetyl moieties in excess of demand. Abbreviations: PDH, pyruvate dehydrogenase; MPC, mitochondrial pyruvate carrier; CAT, carnitine acetyltransferase; CPT, carnitine palmitoyltransferase; CS, citrate synthase.

Figure 2

Design and results of acetylcarnitine saturation experiments (n = 5). A) Control spectra were acquired to identify the exact frequency of the acetylcarnitine peak, as shown. Next, a saturation pulse (gray box: 160 Hz bandwidth) was applied continuously to the acetylcarnitine resonance, which was 730 Hz downstream of citrate. The same pulse was applied 730 Hz upstream of citrate as a control to ensure that citrate was not affected by RF saturation. B) Saturation crushed all acetylcarnitine ¹³C signal, and significantly reduced the citrate and glutamate peak areas. The control-saturation experiment confirmed that acetylcarnitine saturation had no effect on citrate. Glutamate, which was 330 Hz nearer to the control saturation pulse than citrate, was significantly reduced by RF effects. All data are ± SEM and statistical significance was taken at p<0.05. *p<0.05 compared with controls (in which no saturation was applied).

Figure 3

Representative spectrum acquired from the heart in vivo, after hyperpolarised [2-¹³C]pyruvate was infused into a rat. A) For display purposes, 3 s of data were summed and a line broadening of 15 Hz was applied. Modified from Schroeder MA, Clarke K, Neubauer S, Tyler DJ. Hyperpolarized magnetic resonance: a novel technique for the in vivo assessment of cardiovascular disease. Circulation. 2011; 124:1580-1594. B) Metabolic products of [2-¹³C]pyruvate acquired in control rat hearts (n = 8). Data are mean ± SEM.

Figure 4

The in vivo effects of infusing DCA (n = 6) and dobutamine (n = 8) on the production of [1-¹³C]citrate, [5-¹³C]glutamate, and [1-¹³C]acetylcarnitine following infusion of [2-¹³C]pyruvate. Data are ± SEM and statistical significance was taken at p<0.05.

Figure 5

A) The effect of infusing 0.25 mmol/kg pyruvate into a living rat (n = 4 vs. n = 7), as per our in vivo hyperpolarised ¹³C MRS experiments, on myocardial acetylcarnitine levels. B) The effects of DCA (n = 4) and dobutamine (n = 4) on myocardial acetylcarnitine levels. All data are ± SEM and statistical significance was taken at p<0.05. §p<0.05 compared with tissue not exposed to pyruvate, *p<0.05 compared with controls (that were exposed to 80 μmol pyruvate).

Figure 6

A) In the presence of dichloroacetate (DCA), PDH flux is increased, in turn increasing mitochondrial acetyl-CoA levels. Under these conditions excess acetyl-CoA is converted into acetylcarnitine by CAT, to liberate CoA. B) In the presence of dobutamine, cardiac workload and thus Krebs cycle demand are increased. Under these conditions, mitochondrial acetylcarnitine acts as an extra source of energetic substrate. C) This study has revealed that acetyl-CoA rapidly cycles through the acetylcarnitine pool. It is likely that acetyl-CoA/acetylcarnitine cycling enables myocytes to ‘fine-tune’ the supply of mitochondrial acetyl-CoA, to ensure constant provision of energetic substrate without potentially inhibitory acetyl-CoA accumulation. Abbreviations: PDH, pyruvate dehydrogenase; MPC, mitochondrial pyruvate carrier; CAT, carnitine acetyltransferase; CS, citrate synthase; OAA, oxaloacetate.