A New Method to Analyse Myocardial Substrate Metabolism and Mitochondrial Function in Intact Cardiac Tissue Slices.

The progression of cardiac diseases is often accompanied by disturbances in substrate metabolism. Extracellular flux analysis has become a standard tool to investigate metabolic alterations in cell lines. However, the enzymatic digestion of the heart to isolate adult cardiomyocytes as well as the cultivation procedure that is required for cell attachment to the cell culture plates might affect metabolism. Therefore, we developed a flux analyser-based method to measure substrate metabolism of intact cardiac tissue slices. Furthermore, we tested this method in a proof-of-principle approach in remote myocardium after myocardial infarction. To yield cardiac tissue pieces of comparable size, mouse cardiac tissue was sliced (150 µm) using a vibratome, and tissue pieces (diameter 1.9 mm) of these slices were punched out. Using "islet capture plates" in a Seahorse XFe 24 analyser, oxygen consumption rates (OCR) were measured at baseline and after FCCP-induced uncoupling in palmitate, glucose (Glc) and glutamine (Gln) enriched medium. To determine long-chain fatty acid metabolism, CPT1 was inhibited by etomoxir, and Glc/Gln metabolism by inhibition of mitochondrial pyruvate carrier (MPC) and glutaminase (Gls) with UK5099/BPTES. Optical mapping of membrane potential was used to assess action potentials in tissue slices as indicator of cellular integrity. Finally, the developed method was used to analyse substrate metabolism in the remote myocardium at day 3 after myocardial ischemia and reperfusion (n=7) in comparison to sham mice (n=8). Data are mean±SD; unpaired two-sample t-test. Basal OCR was 53±8 pmol/min, and FCCP increased OCR to 92±18 pmol/min. Both etomoxir and UK5099/BPTES reduced OCR indicating that both palmitate and Glc/Gln are metabolised. Optical mapping of tissue slices showed regular action potential characteristics and propagation. After myocardial infarction, CPT1 inhibition caused a smaller reduction of uncoupled mitochondrial OCR in I/R animals compared to sham (40±13 vs. 52±4%, p<0.05). This effect was caused by an increased metabolism of Glc/Glu (37±13 vs. 24±4 pmol/min, p<0.05), whilst the effect of CPT1 was not different. Here, we describe a new method to analyse cardiac metabolism using cardiac tissue slices that metabolise fatty acids as well as glucose, and show high functional integrity. Therefore, this method has the potential to expand the methodological alternatives to investigate cardiac substrate metabolism. In a proof-of-principle approach, the analysis of cardiac substrate metabolism of the remote myocardium after I/R showed an augmented glucose/glutamine metabolism.