Intracellular sodium elevation reprograms cardiac metabolism.

Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 µM ouabain 100 nM blebbistatin) or chronic myocardial Nai load (PLM3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLMWT), transgenic (PLM3SA), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23Na, 31P, 13C NMR followed by 1H-NMR metabolomic profiling. Elevated Nai leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or inhibition of Na/Camito may be a new approach to ameliorate metabolic dysregulation in heart failure.

Is there a causal link between intracellular Na elevation and metabolic remodelling in cardiac hypertrophy?

Alterations in excitation-contraction coupling and elevated intracellular sodium (Nai) are hallmarks of pathological cardiac remodelling that underline contractile dysfunction. In addition, changes in cardiac metabolism are observed in cardiac hypertrophy and heart failure (HF) that lead to a mismatch in ATP supply and demand, contributing to poor prognosis. A link between Nai and altered metabolism has been proposed but is not well understood. Many mitochondrial enzymes are stimulated by mitochondrial calcium (Camito) during contraction, thereby sustaining production of reducing equivalents to maintain ATP supply. This stimulation is thought to be perturbed when cytosolic Nai is high due to increased Camito efflux, potentially compromising ATPmito production and leading to metabolic dysregulation. Increased Nai has been previously shown to affect Camito; however, whether Nai elevation plays a causative role in energetic mismatching in the hypertrophied and failing heart remains unknown. In this review, we discuss the relationship between elevated Nai, NaK ATPase dysregulation and the metabolic phenotype in the contexts of pathological hypertrophy and HF and their link to metabolic flexibility, capacity (reserve) and efficiency that are governed by intracellular ion homeostasis. The development of non-invasive analytical techniques using nuclear magnetic resonance able to probe metabolism in situ in the functioning heart will enable a better understanding of the underlying mechanisms of Nai overload in cardiac pathophysiology. They will lead to novel insights that help to explain the metabolic contribution towards these diseases, the incomplete rescue observed with current therapies and a rationale for future energy-targeted therapies.

Identification of Flavin-Containing Monooxygenase 5 (FMO5) as a Regulator of Glucose Homeostasis and a Potential Sensor of Gut Bacteria.

We have previously identified flavin-containing monooxygenase 5 (FMO5) as a regulator of metabolic aging. The aim of the present study was to investigate the role of FMO5 in glucose homeostasis and the impact of diet and gut flora on the phenotype of mice in which the Fmo5 gene has been disrupted (Fmo5-/- mice). In comparison with wild-type (WT) counterparts, Fmo5-/- mice are resistant to age-related changes in glucose homeostasis and maintain the higher glucose tolerance and insulin sensitivity characteristic of young animals. When fed a high-fat diet, they are protected against weight gain and reduction of insulin sensitivity. The phenotype of Fmo5-/- mice is independent of diet and the gut microbiome and is determined solely by the host genotype. Fmo5-/- mice have metabolic characteristics similar to those of germ-free mice, indicating that FMO5 plays a role in sensing or responding to gut bacteria. In WT mice, FMO5 is present in the mucosal epithelium of the gastrointestinal tract where it is induced in response to a high-fat diet. In comparison with WT mice, Fmo5-/- mice have fewer colonic goblet cells, and they differ in the production of the colonic hormone resistin-like molecule ßFmo5-/- mice have lower concentrations of tumor necrosis factor α in plasma and of complement component 3 in epididymal white adipose tissue, indicative of improved inflammatory tone. Our results implicate FMO5 as a regulator of body weight and of glucose disposal and insulin sensitivity and, thus, identify FMO5 as a potential novel therapeutic target for obesity and insulin resistance.

Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms.

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65-85; 100-135; 160-250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC-MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose (r² = 0.45; P = 0.002), acetyl-carnitine (r² = 0.50; P = 0.001), glutamine (r² = 0.59; P = 0.0002); and negative correlations with taurine (r² = 0.74; P < 0.0001), fumarate (r² = 0.45; P = 0.003), aspartate (r² = 0.59; P = 0.0002), alanine (r² = 0.66; P < 0.0001) and phosphocholine (r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.

Contractile function assessment by intraventricular balloon alters the ability of regional ischaemia to evoke ventricular fibrillation.

BACKGROUND AND PURPOSE: In drug research using the rat Langendorff heart preparation, it is possible to study left ventricular (LV) contractility using an intraventricular balloon (IVB), and arrhythmogenesis during coronary ligation-induced regional ischaemia. Assessing both concurrently would halve animal requirements. We aimed to test the validity of this approach. EXPERIMENTAL APPROACH: The electrocardiogram (ECG) and LV function (IVB) were recorded during regional ischaemia of different extents in a randomized and blinded study. KEY RESULTS: IVB-induced proarrhythmia was anticipated, but in hearts with an ischaemic zone (IZ) made deliberately small, an inflated IVB reduced ischaemia-induced ventricular fibrillation (VF) incidence as a trend. Repeating studies in hearts with large IZs revealed the effect to be significant. There were no changes in QT interval or other variables that might explain the effect. Insertion of an IVB that was minimally inflated had no effect on any variable compared with 'no IVB' controls. The antiarrhythmic effect of verapamil (a positive control drug) was unaffected by IVB inflation. Removal of an inflated (but not a non-inflated) IVB caused a release of lactate commensurate with reperfusion of an endocardial/subendocardial layer of IVB-induced ischaemia. This was confirmed by intracellular (31) phosphorus ((31) P) nuclear magnetic resonance (NMR) spectroscopy. CONCLUSIONS AND IMPLICATIONS: IVB inflation does not inhibit VF suppression by a standard drug, but it has profound antiarrhythmic effects of its own, likely to be due to inflation-induced localized ischaemia. This means rhythm and contractility cannot be assessed concurrently by this approach, with implications for drug discovery and safety assessment.