Assessment of Necrosis in the Ischemic Heart using Hyperpolarized [1,4-13C2]Fumarate
Damian J Tyler1, Angus Lau1, Ferdia Gallagher2, and Marie A Schroeder1

1DPAG, University of Oxford, Oxford, United Kingdom, 2Radiology, University of Cambridge, Cambridge, United Kingdom

Synopsis

The aim of this study was to evaluate the potential of hyperpolarised [1, 4-13C2]fumarate, coupled with MRS, to measure cardiac necrosis during ischemia and reperfusion. Hyperpolarised [1, 4-13C2]fumarate was infused into rat hearts at three time points, corresponding with the healthy heart, early reperfusion after a 20 min ischemic period, and late reperfusion. The amount of [1, 4-13C2]malate production was measured using MRS and quantified to reflect degree of cardiomyocyte necrosis. We observed a 3.8-fold increase in [1,4-13C2]malate during the late reperfusion period but no change in early reperfusion, suggesting that necrotic cell death takes place during reperfusion only. This technique shows potential to evaluate therapies targeting necrosis to prevent cardiac remodeling into failure.

Target Audience

Clinicians and basic scientists with an interest in cardiovascular disease and metabolic imaging/spectroscopy.

Purpose

The discovery that necrosis may be programmed and thus represents a novel therapeutic target has renewed interest in the role of cell death during ischemia and reperfusion.1 Dissecting out the relative occurrences of necrosis and apoptosis remains challenging, but it is now evident that conventional markers of apoptosis are not specific for apoptosis, and that there is considerable overlap between death signalling pathways. Furthermore, the timeline and contribution of necrotic cardiomyocyte death in ischemic heart disease is not yet known,2 but blocking programmed necrosis improves outcome in the isolated perfused heart3 and in vivo.4 Understanding when necrosis takes place could lead to therapies that reduce cell death following a myocardial infarction (MI), thus precluding the cycle of cell death and remodelling that ultimately results in heart failure and patient mortality. To develop new therapies that inhibit necrotic signalling, a method to measure necrosis non-invasively will be required. Therefore, the aim of this study was to evaluate the potential of hyperpolarised [1, 4-13C2]fumarate, coupled with 13C magnetic resonance spectroscopy (MRS),5 to measure cardiac necrosis during ischemia and reperfusion.

Methods

Perfused heart experiments

Rat hearts (n = 5) were perfused in the Langendorff mode and placed in an 11.7 T vertical bore MR scanner. Krebs-Henseleit buffer was supplemented with 11 mM glucose, and hearts were perfused with a constant flow rate of 20 ml/min. Hyperpolarized [1,4-13C2]fumarate (1.25 mM) was infused for 2 min to monitor baseline necrosis, while MR spectra were acquired every 1 s using a flip angle of 30 degrees. 31P MRS assessed energetics, and a pressure transducer measured left ventricular (LV) function. Next, total global ischemia was applied to each heart for 20 minutes, taking care to maintain a constant temperature of 37°C, followed by 45 min of reperfusion. A second dose of hyperpolarized [1,4-13C2]fumarate was infused into the heart immediately after reperfusion to measure necrosis incurred directly from ischemia. After reperfusion, a third dose of [1,4-13C2]fumarate was infused into the heart and LV function and energetics were assessed. A control experiment (n=1), where the heart was not subjected to global ischemia, was undertaken to ensure that there were no detrimental effects on the heart due to the perfusion protocol over the timeframe of the experiment.

Data analysis

All cardiac 13C spectra were analysed using the AMARES algorithm in the jMRUI software package.6 Malate production from hyperpolarized [1,4-13C2]fumarate was quantified based on 60 s of summed spectra, and reported as a ratio: the percentage of malate signal relative to fumarate. Statistical significance was considered at P < 0.05.

Results

Figure 1 shows example 13C spectra acquired from the perfused heart upon initial preparation, immediately following reperfusion and 45 min after reperfusion. The infused [1,4-13C2]fumarate is clearly visible in all spectra along with a small but detectable [1,4-13C2]malate resonance pair in the healthy heart, and immediately following reperfusion. The [1,4-13C2]malate resonance becomes clearly visible at the late reperfusion time point, on average 3.8-fold higher than at baseline, indicating the occurrence of necrosis over this 45 min time frame. Figure 2 shows a quantitative analysis of the % malate signal ratio at each time point and demonstrates a significant increase in the conversion of the injected [1,4-13C2]fumarate to [1,4-13C2]malate after 45 min of reperfusion. The control experiment showed no effect of the perfusion protocol on the generation of hyperpolarized malate in the absence of the 20-minute period of global ischemia.

Discussion and Conclusions

The results presented here demonstrate that the use of hyperpolarized [1,4-13C2]fumarate shows promise as a positive contrast agent to detect cardiac necrosis following a myocardial infarction (MI). The conversion of hyperpolarized [1,4-13C2]fumarate into [1,4-13C2]malate has been shown to be a marker of cellular necrosis because the loss of cell membrane integrity provides access to the intracellular enzyme, fumarase, that is responsible for this conversion.5 The observation of a significant increase in [1,4-13C2]malate during the late reperfusion period but not immediately following reperfusion suggests that necrotic cell death takes place to a substantial degree during reperfusion following MI, but not during the ischemic period itself. Further work is warranted to translate this technique in vivo, and to evaluate putative therapies that target cardiac cell death to prevent cardiac remodeling into failure.

Acknowledgements

This study was supported by the British Heart Foundation, the Oxford-BHF Centre for Research Excellence, and GE Healthcare.

References

1. Zhang et al, Science, 2009.

2. Kung et al, Circ Res, 2011.

3. Lim et al, Cardiovasc Drugs Ther, 2007.

4. Luedde et al, Cardiovasc Res, 2014.

5. Gallagher et al, PNAS, 2009.

6. Naressi, Computers in Biology and Medicine, 2001.

Figures

Representative MR spectra obtained following infusion of hyperpolarized [1,4-13C2]fumarate into the isolated perfused heart at three time points: at baseline into the healthy heart, during reperfusion immediately following 20 min of global ischemia (early reperfusion), and after 45 min of reperfusion (late reperfusion).

Quantitative assessment of the malate/fumarate ratio, expressed as a percentage of fumarate signal, at the three time points studied. A significant increase in the production of [1,4-13C2]malate from the infused [1,4-13C2]fumarate was observed at the late reperfusion time point, indicating the occurrence of necrosis over the 45 min time frame of reperfusion. ** p<0.01 compared to control and to healthy heart.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
0596