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AICAR prevents heart failure in a rat model of doxorubicin-induced cardiotoxicity

Kerstin N Timm¹, Vicky Ball¹, Benjamin Thackray¹, Michael P Murphy², Lisa C Heather¹, and Damian J Tyler¹
¹Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, ²MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom

Synopsis

Doxorubicin (DOX) is a commonly used chemotherapeutic agent for the treatment of many cancers. However, DOX has serious cardiotoxic side effects culminating in congestive heart failure. We have previously shown in a clinically-relevant rat model of DOX-induced heart failure (DOX-HF), that this is due to loss and dysfunction of mitochondria. We show here that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, can prevent heart failure in DOX-treated rats. This cardioprotective effect appears to be, at least in part, achieved through improved fatty acid oxidation in cardiac mitochondria which can be indirectly assessed with hyperpolarized [2-¹³C]pyruvate MRS.

Introduction

Doxorubicin (DOX) is a commonly used anthracycline chemotherapeutic agent for the treatment of many cancers. However, DOX is cardiotoxic, causing heart failure in ~5% of patients¹. Different mechanisms have been proposed to cause this cardiotoxicity, including mitochondrial impairment². Hyperpolarized MRS by dissolution dynamic nuclear polarization of ¹³C-labelled substrates³ has revolutionized metabolic flux measurements in preclinical models⁴ and in the human heart^5,6. We have previously shown in a clinically-relevant rat model of doxorubicin-induced heart failure (DOX-HF), that cardiac metabolic fluxes, assessed by hyperpolarized ¹³C magnetic resonance spectroscopy (MRS), are decreased in the hearts of DOX-treated rats (abstract 3605 ISMRM 2017) and that this is not due to oxidative stress but due to a loss and dysfunction of mitochondria (abstract 0786 ISMRM 2019). We now set out to establish whether 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates AMPK⁷, increasing mitochondrial biogenesis⁸ and fatty acid oxidation⁹, can prevent heart failure in DOX-treated rats.

Methods

Male Wistar rats were weight-matched and divided into 3 groups (Fig 1): a saline control group (n=6) receiving weekly iv injections of saline for 5 weeks, a DOX+saline group (n=9) receiving weekly iv injections of DOX (3 mg/kg) for 5 weeks and daily sc injections of saline and a DOX+AICAR group (n=9) receiving weekly iv injections of DOX (3 mg/kg) for 5 weeks and daily sc injections of AICAR (500 mg/kg). Body weight was monitored weekly. At weeks 1, 3 and 6 of the study, cardiac function was assessed with a CINE-FLASH sequence using a 72-mm dual-tuned ¹H/¹³C birdcage volume transmit and a four-channel ¹H-receive coil (Rapid Biomedical), acquiring 8-10 cardiac-gated short axis slices (1.6 mm thick) of the heart. Images were analyzed using the free hand drawing function in ImageJ. [2-¹³C]pyruvate was hyperpolarized on a prototype hyperpolarizer (Oxford Instruments) for 45 min. One mL of 80 mM hyperpolarized [2-¹³C]pyruvate was injected into the tail vain over 10s and ¹³C MR spectra were acquired over the heart using a ¹³C two-channel surface receive coil (Rapid Biomedical) every second for 60s (10 mm slab, hard pulse, 13° flip angle, bandwidth 17kHz). Multi-coil spectra were added in phase, and the first 30s of spectra from the initial appearance of the pyruvate peak were summed and quantified with AMARES/jMRUI¹⁰. Blood samples were taken at all 3 time points and the plasma was analyzed for non-esterified fatty acid (NEFA) content using a spectrophotometric assay kit (Randox). At week 6, rats were sacrificed, hearts excised and subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated for mitochondrial oxygen consumption measurements with a Clarke-type oxygen electrode as previously described¹¹.

Results

We hypothesized that AICAR could achieve cardioprotection in DOX-HF by increasing mitochondrial biogenesis and/or increasing mitochondrial fatty acid oxidation (Fig 2a). Both DOX+saline rats and DOX+AICAR rats gained significantly less weight over the study compared to saline control rats. However, DOX+AICAR rats showed significantly increased body weight gain compared to DOX+saline rats (Fig 2b). Epididymal fat pad weights in rats at the end of the study were reduced in both DOX+saline and DOX+AICAR rats compared to saline control rats (Fig 2c). AICAR normalized plasma NEFA levels in DOX-treated rats at week 6 of the study (Fig 2d). DOX+saline rats had a significantly reduced ejection fraction (EF) compared to saline treated control rats at week 6 and this decrease in EF was prevented in DOX+AICAR rats (Figure 3a). The heart weight:tibia length ratio, indicative of cardiac atrophy, was decreased in both DOX groups compared to saline controls at week 6 but was significantly higher in DOX+AICAR rats than DOX+saline rats (Fig 3b). We next assessed cardiac metabolic fluxes with hyperpolarized [2-¹³C]pyruvate MRS (Fig 4a). There was a marked decrease of tricarboxylic acid (TCA)-cycle derived glutamate labelling at week 6 in DOX+saline treated rats (Fig 4b) and of acetyl-carnitine labelling at weeks 3 and 6 (Fig 4c). Interestingly, both glutamate and acetyl-carnitine labeling were decreased in DOX+AICAR rats already at week 1 (Fig 4b-c). Mitochondrial number was decreased in both DOX+saline and DOX+AICAR rat compared to saline-treated controls (Fig 5a). SSM isolated from high dose DOX hearts showed a decreased oxygen consumption rate with all substrates (Fig 5b), while IFM had a reduced oxygen consumption rate only with glutamate (Fig 5c). Both SSM and IFM from DOX+AICAR rats showed reduced oxygen consumption rates only with glutamate. However, oxygen consumption was significantly improved in IFM with palmitoyl-CoA+carnitine (Fig 5c), suggesting that increased fatty acid oxidation may be the mechanism of AICAR-cardioprotection in this model of DOX-HF (Fig 5d).

Discussion and conclusion

We have shown here that AICAR can prevent DOX-HF in rats. AICAR did not increase mitochondrial number in DOX-treated hearts, however, AICAR improved fatty acid oxidation in cardiac mitochondria from DOX-treated rats, which may overall allow improved cardiac energetics despite low mitochondrial number, as fatty acids are more energy efficient, producing more ATP per mole than glucose. Hyperpolarized [2-¹³C]pyruvate showed an early decrease in carbohydrate-derived TCA cycle flux in DOX+AICAR rats, which may reflect increased fatty acid oxidation due to reciprocal inhibition from the Randle cycle¹². AICAR is already being used in clinical trials for other pathologies⁴ and its use in cancer patients receiving DOX is therefore feasible in the future to prevent DOX-HF.

Acknowledgements

This work was supported by a British Heart Foundation Immediate Postdoctoral Basic Science Research Fellowship (FS/16/7/31843).

References

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Figures

Figure 1: Study outline depicting treatment scheme and timepoints for MRI and further experiments.

Figure 2: AICAR prevents body weight loss and decreases plasma fatty acids in DOX-treated rats. a) AICAR-cardioprotection in DOX-HF could be due to increased mitochondrial biogenesis and/or improved fatty acid (FA) oxidation. b) Body weight change over time. c) Epididymal fat pad weights at week 6. d) Plasma non-esterified fatty acid (NEFA) levels. Mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to saline control; ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 compared to DOX+saline.

Figure 3: AICAR prevents cardiac dysfunction and ameliorates cardiac atrophy in DOX-treated rats. a) Left ventricular ejection fraction assessed by CINE MRI. b) Heart weight:tibia length ratio indicative of cardiac atrophy. Mean±SEM. *p<0.05, ***p<0.001 compared to saline control; ^$p<0.05, ^$$p<0.01 compared to DOX+saline.

Figure 4: AICAR decreases cardiac carbohydrate-derived TCA-cycle flux in DOX-treated rats. a) Labeling pattern of downstream metabolites in cardiac ¹³C MR spectra after injection of hyperpolarized [2-¹³C]pyruvate. Cardiac b) glutamate:pyruvate and c) acetyl-carnitine:pyruvate ratio as markers of tricarboxylic acid cycle flux. Mean±SEM. *p<0.05, **p<0.01, ****p<0.0001 compared to saline control; ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 compared to DOX+saline.

Figure 5: AICAR does not increase cardiac mitochondrial number but improves cardiac mitochondrial fatty acid oxidation. a) Mitochondrial number assessed by qPCR of a mitochondrial gene (cytB) compared to a nuclear gene (GAPDH). State 3 respiration in b) subsarcolemmal (SSM) and c) interfibrillar (IFM) mitochondria isolated from rat hearts. d) Working hypothesis of AICAR-cardioprotection in DOX-HF. Mean±SEM. *p<0.05, **p<0.01, ***p<0.001 compared to saline control; ^$$p<0.01 compared to DOX+saline.

Proc. Intl. Soc. Mag. Reson. Med. 28 (2020)

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