Background

Mitochondrial dysfunction is a core feature of acute pancreatitis (AP), a debilitating and potentially fatal disease for which there is currently no specific therapy (Criddle, 2016; Habtezion et al., 2019). The elucidation of pivotal pathological mechanisms which underlie mitochondrial damage in pancreatic acinar cells (PACs) is paramount for the development of new therapies. Previous evaluations of mitochondrial bioenergetics in isolated PACs have been mostly performed using confocal microscopy, including assessments of mitochondrial membrane potential (tetramethyl rhodamine methyl ester: TMRM), NADH/FAD⁺ autofluorescence and ATP (Magnesium Green), coupled with patch-clamp electrophysiology (Voronina et al., 2002; Criddle et al., 2004 and 2006). Such studies have pinpointed a reduction of ATP in response to precipitants of AP, via the opening of the mitochondrial permeability transition pore (MPTP), as a critical event that leads to necrotic cell death (Criddle et al., 2006; Mukherjee et al., 2016); supplementation with intracellular ATP ameliorated damage. Furthermore, luciferase measurements in PACs have provided details of changes of both mitochondrial and cytosolic ATP concentrations induced by pathophysiological stimulation (Voronina et al., 2010). Such experimental approaches in single cells, however, are technically difficult and time-consuming. In contrast, population-based assays provide important information about mitochondrial dysfunction and cell death using a convenient plate-reader format (Armstrong et al., 2019). The use of Seahorse Flux analysis allows a detailed evaluation of bioenergetics changes to be performed in PACs, measuring rates of oxygen consumption (OCR) and extracellular acidification (ECAR); these inform about cellular respiration and glycolysis, respectively. A respiratory function “stress” test can further be applied in which pharmacological manipulation of the electron transport chain (ETC) is used to derive parameters such as the Maximal Respiration, Spare Respiratory Capacity, ATP-linked Turnover and Non-mitochondrial Respiration. Such detailed bioenergetics information can be coupled with parallel studies of apoptosis and necrosis to inform the influence of mitochondrial dysfunction on cell death patterns (Armstrong et al., 2018).