Extended Bloch-McConnell equations for mechanistic analysis of hyperpolarized 13C magnetic resonance experiments on enzyme systems

Abstract. We describe an approach to formulating the kinetic master equations of the time evolution of NMR signals in reacting (bio)chemical systems. Special focus is given to studies that employ signal enhancement (hyperpolarization) methods such as dissolution dynamic nuclear polarization (dDNP) and involving nuclear spin-bearing solutes that undergo reactions mediated by enzymes and membrane transport proteins. We extend the work given in a recent presentation on this topic to now include enzymes with two or more substrates and various enzyme reaction mechanisms as classified by Cleland. Using this approach, we can address some pressing questions in the field from a theoretical standpoint. For example, why does binding of a hyperpolarized substrate to an enzyme not cause an appreciable loss of the signal from the substrate or product? Why does the concentration of an unlabelled pool of substrate, for example 12C lactate, cause an increase in the rate of exchange of the 13C labelled pool? To what extent is the equilibrium position of the reaction perturbed during administration of the substrate? The formalism gives a full mechanistic understanding of the time courses derived and is of relevance to ongoing clinical trials using these techniques. </jats:p

SGLT2 inhibitors and the cardiac Na⁺/H⁺ exchanger-1:the plot thickens

No abstract available

Off-target effects of SGLT2 blockers: empagliflozin does not inhibit Na+/H+ exchanger-1 or lower [Na+]i in the heart

Aims: Empagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose cotransporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. Methods and results: The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10 or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i-rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively. Conclusions Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+]. Translational Perspective: Heart failure remains a huge clinical burden. Clinical trials of SGLT2 inhibitors in patients with diabetes and heart failure have reported highly significant cardiovascular benefit that appears independent of improved glycaemic control. As SGLT2 is not expressed in the heart, the mechanism by which SGLT2 inhibitors are cardioprotective remains unknown. Understanding this mechanism is clearly essential as the use of SGLT2 inhibitors in non-diabetics is increasing and a better understanding may allow refinement of therapeutic approaches in both HFpEF and HFrEF. One suggested mechanism that has received significant attention, inhibition of cardiac Na+/H+ exchanger, is investigated here

Developing hyperpolarized 13C spectroscopy and imaging for metabolic studies in the isolated perfused rat heart

Hyperpolarized 13C magnetic resonance is a powerful tool for the study of cardiac metabolism. In this work, we have implemented protocols for the real-time hyperpolarized 13C investigation of Langendorff-perfused rat hearts using both non-selective non-localized spectroscopy and fast spectroscopic imaging. Following [1-13C] pyruvate infusion, we observed both catabolic and anaplerotic metabolic processes resulting in a number of metabolites, including bicarbonate, carbon dioxide, lactate, alanine and aspartate. Employing fast spectroscopic imaging, we were able to observe regional variations in pyruvate perfusion as well as in lactate and bicarbonate production

Multiple quantum filtered ²³Na NMR in the Langendorff perfused mouse heart:Ratio of triple/double quantum filtered signals correlate with [Na]_i

AbstractWe investigate the potential of multiple quantum filtered (MQF) 23Na NMR to probe intracellular [Na]i in the Langendorff perfused mouse heart. In the presence of Tm(DOTP) shift reagent the triple quantum filtered (TQF) signal originated largely from the intracellular sodium pool with a 32±6% contribution of the total TQF signal arising from extracellular sodium, whilst the rank 2 double-quantum filtered signal (DQF), acquired with a 54.7° flip-angle pulse, originated exclusively from the extracellular sodium pool. Given the different cellular origins of the 23Na MQF signals we propose that the TQF/DQF ratio can be used as a semi-quantitative measure of [Na]i in the mouse heart. We demonstrate a good correlation of this ratio with [Na]i measured with shift reagent at baseline and under conditions of elevated [Na]i. We compare the measurements of [Na]i using both shift reagent and TQF/DQF ratio in a cohort of wild type mouse hearts and in a transgenic PLM3SA mouse expressing a non-phosphorylatable form of phospholemman, showing a modest but measurable elevation of baseline [Na]i. MQF filtered 23Na NMR is a potentially useful tool for studying normal and pathophysiological changes in [Na]i, particularly in transgenic mouse models with altered Na regulation

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial