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
heart
3 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.