Background:
Alterations of cardiac substrate metabolism and mitochondrial energetics are a hallmark of many cardiac diseases. A standard tool to investigate metabolic alterations in cell lines is the extracellular flux analysis using the Seahorse XFe system. However, the enzymatic digestion of the heart to isolate adult cardiomyocytes and the cultivation procedure required for cell attachment to the cell culture plates might affect metabolism and impact study outcome. Therefore, we aimed to develop a Seahorse XFe 24 flux analyser-based method for the cellular and mitochondrial substrate metabolism of intact cardiac tissue slices. Furthermore, we tested this method in a proof-of-principle approach in remote myocardium after myocardial infarction.

Methods:
Mouse cardiac tissue was sliced using a vibratome. Tissue pieces of these slices were punched out to yield comparable-sized samples, and transferred to “islet capture plates”. Oxygen consumption rates (OCR) were measured at baseline and after FCCP-stimulation in palmitate, glucose (Glc) and glutamine (Gln) enriched medium. To determine long-chain fatty acid (LCFA) 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 an indicator of cellular integrity. Finally, the developed method was used to analyse substrate metabolism in the remote myocardium at day 3 after myocardial ischemia (45 min LAD-occlusion, n=7) in comparison to sham mice (n=8). Data are mean±SD; unpaired two-sample t-test.

Results:
Basal mitochondrial OCR was 40±8 pmol/min, and non-mitochondrial OCR 14±3 pmol/min. Uncoupling by FCCP increased mitochondrial OCR to 79±18 pmol/min. Etomoxir reduced this uncoupled OCR by 54%; and UK5099/BPTES by 28% indicating that both palmitate and Glc/Gln are metabolised. Optical mapping of tissue slices showed typical action potential characteristics and propagation at different stimulation frequencies.

After myocardial infarction, basal and uncoupled OCR of remote myocardium from I/R animals was higher compared to sham animals. CPT1 inhibition caused a smaller reduction of uncoupled 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 pmol/min vs. 24±4 pmol/min, p<0.05), whereas the effect of CPT1 inhibition was not different between groups.

Conclusions:
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.

(Funding: DFG SFB1116, A06/A01)