Impairment of Myocardial Glutamine Homeostasis Induced By Suppression of the Amino Acid Carrier SLC1A5 in Failing Myocardium

The normal heart derives ATP primarily from fatty acids by way of oxidative metabolism and the tricarboxylic acid cycle. In heart failure, this is altered, with an increase of anaerobic glycolysis and an impairment of fatty acid and glucose oxidation and mitochondrial activity. Only little is known about amino acid (AA) metabolism in heart failure (HF), the third source of ATP production. Glutamine, the most abundant AA, has pleiotropic functions, and the phenotype of several hereditary glutamine disorders such as glutamine synthetase deficiency suggests a pivotal role of glutamine metabolism for cardiac homeostasis. AAs are transported into the cell via carriers of the solute carrier family SLC. Of those, SLC1A5 is crucial for cellular glutamine homeostasis.¹ We hypothesized that myocardial glutamine uptake is decreased in the failing heart due to derangements of myocardial SLC.

We compared myocardial and serum AA concentrations of HF patients (n=10) to healthy controls and to patients supported with left ventricular assist devices. We performed quantitative polymerase chain reaction and western blot analyses of genes and proteins with pivotal function in AA metabolism using human cardiac samples (control, n=5; HF, n=8) and an AC 16 cell culture model for mechanistic analyses. AA was measured by high-pressure liquid chromatography with UV detector (Alliance). The study was approved by the IRB at Columbia University Medical Center. One-way ANOVA with Tukey post hoc analysis or the Student t test was performed where applicable. P<0.05 was considered to be statistically significant. P<0.05, 0.01, and 0.001 were indicated as *, **, and ***, respectively. Mean and the SEM is reported unless stated otherwise.

Of the 19 AAs analyzed, 9 showed lower levels in failing myocardium compared with controls (Figure [A]). Mechanical unloading (left ventricular assist device) increased AA concentrations of Arg, Gln, Ser, Ala, and Pro (Figure [A]). In contrast to myocardial AA concentrations, circulating levels were mostly not significantly altered by left ventricular assist device implantation in comparison to HF subjects. (Figure [B]). Of note, circulating glutamate levels were increased in the HF cohort compared with controls.

SLC1A5 is a neutral AA transporter crucial for cellular glutamine homeostasis. SLC1A5 mRNA levels were suppressed in human HF samples compared with healthy controls (Figure [C]). SLC1A5 protein expression was also lower in human failing myocardium (Figure [D]). The expression of other SLC carriers was not significantly changed (Figure [C]).

Gene expression levels of rate-limiting enzymes of glutamine metabolism (glutaminase, glutamic-oxaloacetic transaminase1, glutamic-oxaloacetic transaminase2, and malate dehydrogenase) assessed by quantitative polymerase chain reaction showed no significant changes, suggesting that changes in SLC expression is a main cause for derangements in glutamine levels and homeostasis. As proline metabolism is closely linked to glutamine metabolism, we also tested mRNA levels of proline-dehydrogenase, the key enzyme of proline degradation. This analysis revealed suppression of proline-dehydrogenase in failing myocardium, which suggests a proline storage preserving state (Figure [C]).

HF is associated with a systemic pro-inflammatory state with increased levels of circulating cytokines such as tumor necrosis factor alpha.² To mimic this milieu, we incubated AC16 cells with TNF-α (100 ng/mL; 24 and 48 hours). TNF-α decreased SLC1A5 levels (Figure [E]). Confocal microscopy quantification showed a decrease of SLC1A5 levels by 21±10% after incubation with TNF-α (Figure [F]). To assess for effects of TNF-α on SLC1A5 expression, we measured 3H-glutamine uptake in AC16 cells (Figure [G]). TNF-α also decreased cellular glutamine uptake. In the presence of TNF-α and insulin (100 nmol/L), glutamine uptake remained decreased, suggesting that TNF-α has an inhibitory effect on insulin-mediated glutamine uptake (Figure [G]). To quantify the contribution of SLC1A5 to cellular glutamine uptake, we blocked SLC1A5-dependent glutamine uptake using L-glutamic acid p-nitroanilide hydrochloride (L-γ-Glutamyl-p-nitroanilide, 300 umol/L), a specific SLC1A5 inhibitor.³ L-γ-Glutamyl-p-nitroanilide reduced 3H-glutamine uptake by 33±9% (Figure [G]). In combination with insulin (+L-γ-Glutamyl-p-nitroanilide), 3H-glutamine levels remained unchanged.

Energy production in HF is characterized by decreased oxidative metabolism and increased glycolysis. Anaplerotic pathways, meaning that certain AAs can function as substrates to replenish the tricarboxylic acid cycle, are induced in HF, most likely to compensate for loss of tricarboxylic acid cycle substrates due to a decrease in FA oxidation. Our study reveals derangements in myocardial glutamine storage in HF with decreased expression of SLC1A5. Glutamine is an anaplerotic glucogenic AA. In this context, the decrease of myocardial SLC1A5 expression appears to be detrimental to the energetic status in HF, impairing cellular glutamine homeostasis by decreasing cellular glutamine uptake.

Loss of SLC1A5 function has been shown to activate autophagy.⁴ Oka et al⁵ revealed a mechanistic link between systemic inflammation with increased circulating pro-inflammatory cytokines such as TNF-α in HF and the myocardial autophagy complex. This suggests a vicious cycle in which systemic inflammation with cytokine induction decreases myocardial SLC1A5 expression, leading to induction of the autophagy complex, which in return further aggravates systemic inflammation through processes such as mitochondrial DNA release. In our model, TNF-α reduced SLC1A5 expression and decreased cellular glutamine uptake which provides a link between SCL1A5 loss in HF and autophagy.⁴

To summarize, this study reveals novel insights into the derangement of myocardial AAs and in particular glutamine homeostasis in HF. Chronic HF has profound effects on myocardial AA homeostasis. SLC1A5, an important carrier for glutamine and other neutral AAs, is decreased in HF and is influenced by inflammatory signals. This study reveals a new pathophysiological mechanism that may contribute to myocardial energy derangements in the HF.