Extending the cardiac metabolism toolkit: Bringing extracellular flux analyses to the living cardiac tissue slice.

In this issue of Acta Physiologica, Bottermann et al,1 describe a novel tool to examine cardiac substrate metabolism in the mouse myocardium, offering a valuable addition to the existing toolkit for studying cardiac metabolic reprogramming in the diseased heart. The heart is a high energy-demanding organ that requires a continuous supply of ATP to sustain its contractile function.2 To meet its high-energy requirements, the heart can utilize a variety of substrates such as fatty acids, glucose, amino acids, lactate, and ketone bodies.3 In normal physiological conditions, the adult heart primarily depends on fatty acids (40%–70%) and glucose (20%–30%) as energy sources, with lesser contributions from lactate (5%–20%) and ketone bodies (5%–15%).4 Importantly, changes in substrate utilization can drive the progression of pathological conditions like ischaemia-reperfusion injury and heart failure. For example, heart failure is generally associated with a shift in cardiac energy substrate preference, leading to reduced fatty acid oxidation and increased glucose utilization.2-4 This metabolic remodeling is linked to impaired mitochondrial function and reduced ATP production, contributing to the energy deficiency observed in failing hearts.2 Metabolic modulation holds great promise as a novel therapeutic approach to treating heart failure and improving cardiac function, therefore understanding the interplay between altered substrate metabolism, mitochondrial dysfunction and heart failure is crucial. The current state-of-the-art techniques to gain insights into cardiac metabolism encompass a diverse often complementary array of cutting-edge methodologies.5 Advanced imaging techniques like positron emission tomography (PET) and magnetic resonance spectroscopy (MRS) enable non-invasive assessment of cardiac metabolism in vivo using radiotracers. Metabolomics and stable isotope-resolved metabolomics (fluxomics) are widely used techniques.5 Metabolomics provides a comprehensive snapshot of metabolite abundances in a biological system (e.g., isolated cardiac cells or whole hearts), while fluxomics allows the analysis of metabolic flux by measuring the incorporation rate of stable isotope-labeled substrates, such as glucose and fatty acids, as a read-out of metabolic pathway activity.5 Recently, extracellular flux (XF) analysis has emerged as a powerful and widely used tool for studying cellular metabolism and bioenergetics.6 Seahorse XF Analysers enable continuous and simultaneous monitoring of O2 consumption and extracellular acidification rates (OCR and ECAR), as readouts of mitochondrial respiration and glycolysis respectively, in living cells. The technique is non-destructive and non-invasive, meaning cells can be continuously monitored throughout the assay without the need to lyse or disrupt them. It also has the advantage of analyzing multiple samples simultaneously (24 or 96 depending on the instrument type), and it is suitable for various cell types, including adherent7 and non-adherent cells,8 organoids,9 and intact tissues.10 However, when studying cardiac metabolism, XF analysis has primarily been conducted in isolated cardiomyocytes.11 Isolated cardiomyocytes offer precise control and manipulation of the cellular environment, eliminating potential confounding factors from other cardiac cell types. Nonetheless, drawbacks include the possibility of metabolic alterations during enzymatic digestion and cell culture, as well as the loss of tissue architecture and intercellular interactions, which might not fully recapitulate in vivo conditions. To overcome these limitations, Bottermann et al,1 have developed novel protocols for measuring extracellular flux analysis in intact murine cardiac tissue slices.1 Myocardial slices are 100- to 400-μm-thick slices of living adult myocardium that can be obtained, using a high-precision vibratome, from various small and large mammals, including mice, rats, pigs, and human tissue biopsies. Cardiac tissue slices provide a powerful platform for cardiovascular research as they maintain essential features of the native multicellular structure, tissue architecture, and physiological characteristics found in adult cardiac tissue.12 Previous studies on porcine cardiac slices have demonstrated their suitability as models for metabolic measurements.13, 14 However, these studies were limited to measuring glucose utilization using radio-labelled tracers,13 or baseline oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)14 to examine metabolic alterations over time in culture. In this study, Bottermann et al1 have set up and optimized the conditions to perform mitochondrial stress tests in murine cardiac slices, demonstrating that they are a suitable model for assessing myocardial tissue metabolism and bioenergetics. Notably, they tested a range of myocardial slice thicknesses revealing a linear correlation between thickness and baseline OCR, and indicating that oxygen diffusion is likely not a limiting factor in murine cardiac slices up to 200 μm thick. The authors also examined substrate preference by exposing the myocardial slices to a complete media, including BSA-conjugated palmitate, glucose, and glutamine, along with injections of substrate specific inhibitors. The results indicate a preference for fatty acids as the primary energy source in murine myocardial slices. To further validate the method and to test its sensitivity, the authors also measured substrate preference in cardiac tissue slices obtained from the remote non-ischemic area of mice subjected to myocardial ischemia and reperfusion injury (I/R) or sham-operation. Remarkably, the extracellular flux analysis results showed a decrease in oxygen consumption rate derived from fatty acids utilization and increased reliance on glucose/glutamine in the myocardial slices from the I/R mice, in line with previous findings. The use of cardiac slices offers a unique opportunity to study regional differences in heart metabolism, such as in remote areas I/R, which is challenging to investigate using other approaches such as in vivo or ex vivo metabolomics. Future studies may be designed to explore regional differences in cardiac metabolism in other models of heart disease and under physiological conditions. In this regard, when designing experiments using cardiac slices, it is important to carefully consider and standardize the location from which the slices are obtained to minimize variation and increase data reproducibility. Furthermore, it is essential to validate the functional integrity of the cardiac tissue slices, as described by Bottermann et al,1 in order to obtain meaningful and consistent data. In conclusion, this study1 reveals that XF analysis of myocardial slices represent a novel and valuable approach to measure cardiac metabolism and bioenergetics. Integrating this new tool with existing methodologies will advance research aimed at understanding the relationship between cardiac metabolism and heart disease, and at developing novel therapies targeting metabolism. I do not have any conflict of interest to declare.