Bioenergetic analysis

Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used to detect oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), representing oxidative phosphorylation and glycolysis, respectively, as previously described^54,55. The general scheme of the mitochondrial stress test is shown in Fig. 5a. Oligomycin (1.5 μM), FCCP (2 μM), rotenone/antimycin A (0.5 μM) were sequentially introduced to measure basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. Maximal respiratory capacity was estimated by inducing maximal OCR via chemical dissipation of the mitochondrial membrane potential with the protonophore FCCP on the background of oligomycin (used to prevent the ATP-consuming reverse activity of ATP synthase, which may lead to cellular metabolic dysfunction and death). Maximal respiratory capacity is a measure of the maximal ability of the ETC to produce energy. Spare respiratory capacity is derived from the difference between maximal OCR and basal respiration. A cell with a larger spare respiratory capacity can produce more ATP to maintain adequate levels of energetic molecules and overcome more stress.

The general scheme of glycolysis stress test is shown in Fig. 5b. Sequential injections of 3 μM rotenone (to block complex I, thereby eliminating mitochondrial respiration and force cells to rely on glycolysis), 10 mM glucose, and 100 mM 2-deoxyglucose (2-DG; glucose analog and inhibitor of glycolytic ATP production) were used to measure glycolysis, glycolytic capacity and allow estimation of glycolytic reserve and non-glycolytic acidification.

H9c2 cells (40,000 cells/well) were seeded on the XFp cell culture mini plates (Seahorse Bioscience, Billerica MA, USA) and subjected to the H/R challenge (Fig. 1). At the end of the first hour of reoxygenation, the Tyrode’s solution was replaced with 500 μl/well of XF24 running media. The plates were pre-incubated at 37 °C for 20 min in the XF Prep Station incubator (Seahorse Bioscience, Billerica MA, USA) in the absence of CO₂ and then run on the XF24 analyzer to obtain OCR and ECAR.

OCR and ECAR were recorded during specified programmed time periods (three readings each) as the average numbers between the injections of inhibitors mentioned above. The final data calculation was performed after the readings had been normalized for total protein/well.