Clinical Implications of Cardiac Hyperpolarized Magnetic Resonance Imaging

The basis of magnetic resonance imaging lies in the interaction between the static magnetic field of the MRI system and the molecules of the body. When placed in the magnetic field, the molecules act like small bar magnets, aligning themselves in one of two orientations, either in the same direction as the field or opposed to it. The signal generated by the MRI system is then proportional to the difference in the number of molecules aligned in the two orientations, referred to as the polarization. At normal clinical magnetic field strengths and room temperature, the polarization is very small (e.g. at 3 T, the polarization is in the order of 0.001%). The aim of hyperpolarization techniques is to artificially increase the number of molecules in one orientation and thus the polarization level. This then results in an increase in the signal that can be generated by a given sample. Currently, there are four main approaches used to generate hyperpolarized compounds. These are brute force polarization [17], optical pumping of noble gases [18], parahydrogen-induced polarization (PHIP) [19] and finally, dynamic nuclear polarization (DNP), which will form the focus of the rest of this article [20].

The majority of cardiac DNP investigations to date have used ¹³C-pyruvate to interrogate myocardial metabolism. The rationale for this lies in the fact that pyruvate sits at a key intersection of multiple metabolic pathways and plays an integral part in cellular energy homeostasis. For hyperpolarized ¹³C studies of cardiac metabolism, pyruvate has been labelled in the one-carbon [1-¹³C] and two-carbon [2-¹³C] positions. Depending on which carbon position is labelled with ¹³C, interrogation of different metabolic pathways can be achieved (Figure 2). As the fate of pyruvate; namely conversion to alanine (via Alanine Aminotransferase, ALT), lactate (via Lactate Dehydrogenase, LDH) and acetyl-CoA/CO₂ (via Pyruvate Dehydrogenase, PDH), is dependent on prevailing metabolic conditions, this provides a window on several important metabolic processes that are essential to cardiac function, and which vary during differing disease processes. If pyruvate is ¹³C labeled in the third carbon position ([3-¹³C]-pyruvate) the methyl carbon group results in a T₁ relaxation time that is short, making it an unattractive target for in vivo hyperpolarization studies. In contrast, the T₁ relaxation times of [1-¹³C] and [2-¹³C]-pyruvate are sufficiently long to make them good targets for hyperpolarization.

The initial hyperpolarized ¹³C MRS measurements of in vivo substrate selection were validated against analogous data collected in vitro and ex vivo. It was first demonstrated in the isolated perfused rat heart that infusion of hyperpolarized [1-¹³C]pyruvate, and MRS detection of total carbonic acid (¹³CO₂ plus ¹³C-bicarbonate), measured flux through the PDH enzyme complex [22]. Subsequent in vivo work rapidly showed that if hyperpolarized [1-¹³C]Pyruvate was metabolized, the resulting signal was transferred to lactate, CO_2, bicarbonate and alanine allowing the assessment of 3 separate enzyme reactions, namely 1) LDH flux (pyruvate-lactate conversion), 2) PDH flux (pyruvate-CO₂/bicarbonate conversion) and 3) ALT flux (pyruvate-alanine conversion, Figure 2) [15, 23, 24].

In disease models, alterations in the flux of hyperpolarized [1-¹³C]pyruvate through myocardial pyruvate dehydrogenase (PDH), as assessed by the production of ¹³C-bicarbonate, has not only been shown to be 65% lower than normal in the type 1diabetic heart but also to correlate with disease severity [15]. In addition, hyperpolarized [1-¹³C]pyruvate spectroscopy has been performed in models of cardiac hypertrophy allowing a greater understanding of the variation in substrate switching that occurs in different models. For example, in contrast to the spontaneously hypertensive rat, where a move away from predominantly fatty acid oxidative metabolism towards an increased reliance on glucose oxidation has been observed [25], left ventricular hypertrophy in the setting of hyperthyroidism was shown to be related to a reduced PDH flux [26]. Hyperpolarized [1-¹³C]pyruvate spectroscopy was also able to demonstrate that this inhibition of glucose oxidation in the hyperthyroid heart was mediated by Pyruvate Dehydrogenase Kinase (PDK). Treatment with dichloroacetic acid (DCA), a potent inhibitor of PDK, restored the metabolic flexibility of the hyperthyroid heart and the level of cardiac hypertrophy was significantly reduced [26]. The ability of hyperpolarized [1-¹³C] pyruvate spectroscopy to discriminate distinct patterns of metabolic dysregulation in different causes of cardiac hypertrophy is unparalleled and, therefore, has the potential to allow targeted therapeutics to prevent/treat different aetiologies of cardiac hypertrophy.

Myocardial oxygen consumption has been shown to be sensitive to cardiac substrate selection, with fatty acid utilization increasing oxygen consumption [27]. Whilst this is considered to be unimportant to physiology in the heart under conditions of normal oxygen supply, in the setting of ischemia or ischemia–reperfusion, increased metabolism of fatty acids impairs contractility and recovery [28] and multiple studies have proved that increased oxidation of carbohydrates relative to fatty acids improves the outcome after myocardial ischemia [29, 30].

The effects of transient global ischaemia (10mins) followed by reperfusion on cardiac substrate selection have been investigated in a perfused heart model using hyperpolarized [1-¹³C]pyruvate. Using this method, it has been shown that in the early reperfusion period PDH flux was essentially zero and coupled with an increased appearance of [1-¹³C]lactate (via cytosolic LDH). This was followed later, within 20 minutes of reperfusion, by recovery of PDH flux observed through the reappearance of the products of PDH, ¹³CO₂ and ¹³C-bicarbonate [31].

In addition to these spectral data acquisitions, pre-clinical experiments in both rodents and pigs have also demonstrated that metabolic maps of the spatial distribution of the downstream metabolites of hyperpolarized [1-¹³C]pyruvate; namely bicarbonate, lactate and alanine, can provide a sensitive marker of ischaemia, with myocardial lactate production during coronary occlusion providing a direct visualisation of ischaemia [24, 32, 33]. When oxygen is present, pyruvate is converted to acetyl-CoA, by Pyruvate Dehydrogenase (PDH), supplying substrate for the tricarboxylic acid (TCA) cycle. However, under anaerobic conditions, pyruvate is converted to lactate (via Lactate Dehydrogenase) with resulting NAD + production that allows glycolysis to continue to produce ATP in the absence of oxygen (the usual terminal electron acceptor during mitochondrial oxidative metabolism).

As current clinical imaging techniques rely on indirect measures of ischaemia (either perfusion abnormalities or changes in wall motion during stress), it is hoped that direct visual assessment of ischaemia in the form of lactate imaging will aid guided revascularisation. Given the evidence that only if revascularisation is targeted to the presence of significant myocardial ischaemic burden (>10%) [34] does it improve outcome, this potential for downstream metabolites of hyperpolarized [1-¹³C]pyruvate imaging to localise and grade the extent of myocardial ischaemia is potentially of great clinical importance.

Identifying areas of myocardium that are hibernating and would benefit from revascularization, and discriminating them from areas that are non-viable and would not recover after revascularization is another clinically important question to which hyperpolarized [1-¹³C]pyruvate imaging may be able to contribute. Hibernating myocardium is in essence a state of persistently impaired myocardial function at rest due to chronically reduced coronary blood flow, which can be partially or completely restored to normal either by improving blood flow or by reducing oxygen demand [35]. In viable myocardium, cell membrane integrity is typically retained, and there is some mitochondrial activity, together with an active glucose metabolism, existence of coronary flow, and the presence of contractile reserve [36]. As imaging of hyperpolarized [1-¹³C]pyruvate metabolism can produce localised metabolite maps of lactate, alanine and bicarbonate, the viability of myocardial segments could be evaluated on the basis of these datasets [24].

Using an interleaved-frequency, time-resolved volumetric pulse sequence, robust and reliable three-dimensional measurements of cardiac metabolic signals have been obtained (Figure 3) [33, 37]. These “single-shot” pulse sequences selectively produce images of metabolites in a very rapid time frame (~100 milliseconds per image). In large animal models of ischaemia-reperfusion, transient coronary occlusion resulted in regional hypokinesia with a reduced bicarbonate signal and an increased lactate signal consistent with acute infarction [33]. However, restoration of flow at 45 minutes was accompanied by restoration of function at 1 week and increased bicarbonate signal. This pattern of metabolites with normalisation of the bicarbonate signal, in the absence of late gadolinium enhancement, is consistent with viable myocardium. However, in a perfused heart model of chronic infarction, using ¹³C-hyperpolarized metabolite maps, the combination of reduced perfusion and significant reductions in both bicarbonate and lactate signals after prolonged coronary artery occlusion has been shown to reflect a loss of normal glycolytic metabolism indicative of cell membrane integrity disruption and non-viability [32].

In the majority of cases suitability for coronary revascularisation in the presence of depressed myocardial systolic function is based on the detection of regional myocardial viability as assessed by myocardial perfusion scanning (MPS) and stress echocardiography. However, recent results from the STICH trial, albeit using global measures of viability, did not show an advantage to viability assessment pre coronary artery bypass grafting, disrupting these traditionally accepted methods of assessing suitability for revascularisation [38]. It is now clear that better detection of viable myocardium is needed and the ability of ¹³C hyperpolarized imaging to detect and localise specific patterns of myocardial metabolism associated with ischaemia and viability promises to be an exciting advance in this area of cardiac imaging.

If pyruvate is enriched with ¹³C on the second carbon atom, the hyperpolarized label is not lost in the cleavage of pyruvate into acetyl-CoA and carbon dioxide (¹³CO₂). Instead the ¹³C label is carried through acetyl-CoA and into the TCA cycle (Figure 2) allowing for the observation of various TCA cycle intermediates in real-time [46]. This has allowed TCA flux to be investigated in multiple cardiac disease models.

The use of hyperpolarized CMR at multiple time-points following a myocardial infarction induced by ligation of the left anterior descending coronary artery has shown, in vivo, that tricarboxylic acid (TCA) cycle flux (as indicated by the reduced production of citrate and glutamate from [2-¹³C]pyruvate) is significantly reduced from six weeks after infarction in the infarcted heart [47]. Also, using metabolic mapping in a pacing induced porcine model of dilated cardiomyopathy (DCM) it has been shown that, despite early impairment of cardiac energetics (reduced PCr/ATP ratio) and changes in [2-¹³C]pyruvate incorporation into the TCA cycle (reduced ¹³C glutamate production), pyruvate oxidation was maintained until overt DCM developed, when the heart’s capacity to oxidize both pyruvate and fats was reduced (Figure 4) [48]. As a result, hyperpolarized [2-¹³C]pyruvate imaging may be important to characterize metabolic changes that occur during heart failure progression and provide potential treatment targets.

Pyruvate itself has been reported as an attractive treatment for heart failure and has been the subject of multiple clinical studies [52‐54]. Supra-physiological levels of pyruvate (150 mmol/L infused at up to 740 ml/hr) have been infused into the coronary arteries invasively at angiography and have been shown in small studies to be well tolerated and result in increased cardiac output, decreased pulmonary capillary-wedge pressure and decreased heart rate in patients with dilated cardiomyopathy and heart failure [54]. Despite the safety and tolerability of slow infusions in the above heart failure study and high doses in chronic liver disease studies (up to 82.4 g/day for 10 days) [55], owing to the rapid loss of hyperpolarization and thus the short imaging window it will provide, pyruvate injection must be administered as a bolus injection at a rate of ~ 5 ml/second, resulting in potentiality supra-physiological doses.

The first application of hyperpolarized magnetic resonance in humans (using bolus injection) has now been performed at the University of California in San Francisco (UCSF) [56, 57]. In this “first in man” study, the potential for hyperpolarized magnetic resonance to stage prostate cancer and assess response to treatment has been investigated. Although no studies of human cardiac metabolism have been performed to date, the pyruvate concentrations used in the UCSF clinical trial of prostate cancer are likely to be identical to initial cardiac studies.

In order to be a useful metabolic probe, any potential tracer molecule needs to have the following physical properties; 1) the tracer needs to maintain its polarization (i.e. have a sufficiently long T₁ relaxation time) for a period of time necessary for the study to be carried out, 2) the compound needs to be enriched with non-zero nuclear spin nuclei, 3) when the mixture is frozen it needs to form a glass rather than a crystallized solid (as successful polarization levels are generally achieved by DNP with glass formation), and finally 4) that the tracer is rapidly incorporated into a metabolic pathway.

In addition to pyruvate and bicarbonate, several other potential hyperpolarized probes have been proposed. For example, distinct regions of the TCA cycle have been assessed using hyperpolarized [1-¹³C]glutamate and [1,4-¹³C₂]fumarate, the respective conversions of which to [1-¹³C]α-ketoglutarate and [1,4-¹³C₂]malate have been demonstrated in vivo[58, 59]. Hyperpolarized butyrate has been used as a marker of short chain fatty acid metabolism [60] and hyperpolarized [1-¹³C]acetate has also been used in preclinical models as an assay for intracellular CoA levels [61]. Hyperpolarized glucose, vitamin C, lactate and alanine, amongst many others, have also been demonstrated to be useful in vivo[62‐66]. However, the extent to which these, or any other, tracers can be used in clinical applications has yet to be determined.