Probing the cardiac malate-aspartate shuttle non-invasively using hyperpolarized [1,2-¹³ C₂ ]pyruvate

Previous studies have demonstrated that using hyperpolarized [2-¹³ C]pyruvate as a contrast agent can reveal ¹³ C signals from metabolites associated with the tricarboxylic acid (TCA) cycle. However, the metabolites detectable from TCA cycle-mediated oxidation of [2-¹³ C]pyruvate are the result of several metabolic steps. In the instance of the [5-¹³ C]glutamate signal, the amplitude can be modulated by changes to the rates of pyruvate dehydrogenase (PDH) flux, TCA cycle flux and metabolite pool size. Also key is the malate-aspartate shuttle, which facilitates the transport of cytosolic reducing equivalents into the mitochondria for oxidation via the malate-α-ketoglutarate transporter, a process coupled to the exchange of cytosolic malate for mitochondrial α-ketoglutarate. In this study, we investigated the mechanism driving the observed changes to hyperpolarized [2-¹³ C]pyruvate metabolism. Using hyperpolarized [1,2-¹³ C]pyruvate with magnetic resonance spectroscopy (MRS) in the porcine heart with different workloads, it was possible to probe ¹³ C-glutamate labeling relative to rates of cytosolic metabolism, PDH flux and TCA cycle turnover in a single experiment non-invasively. Via the [1-¹³ C]pyruvate label, we observed more than a five-fold increase in the cytosolic conversion of pyruvate to [1-¹³ C]lactate and [1-¹³ C]alanine with higher workload. ¹³ C-Bicarbonate production by PDH was increased by a factor of 2.2. Cardiac cine imaging measured a two-fold increase in cardiac output, which is known to couple to TCA cycle turnover. Via the [2-¹³ C]pyruvate label, we observed that ¹³ C-acetylcarnitine production increased 2.5-fold in proportion to the ¹³ C-bicarbonate signal, whereas the ¹³ C-glutamate metabolic flux remained constant on adrenergic activation. Thus, the ¹³ C-glutamate signal relative to the amount of ¹³ C-labeled acetyl-coenzyme A (acetyl-CoA) entering the TCA cycle was decreased by 40%. The data strongly suggest that NADH (reduced form of nicotinamide adenine dinucleotide) shuttling from the cytosol to the mitochondria via the malate-aspartate shuttle is limited on adrenergic activation. Changes in [5-¹³ C]glutamate production from [2-¹³ C]pyruvate may play an important future role in non-invasive myocardial assessment in patients with cardiovascular diseases, but careful interpretation of the results is required.