The role of mitochondrial Ca2+ uniporter in initiation and development of acute pancreatitis

Acute pancreatitis is a frequent, debilitating and life threatening inflammatory disease. The incidence of this disease is increasing rapidly whilst specific therapy is unavailable. This highlights the importance of research into the mechanisms of acute pancreatitis and coordination between basic and translational researchers working in this area.
Pancreatic acinar cells store and secrete digestive enzymes and precursors of digestive enzymes. The damage to pancreatic acinar cells is an important early event in the development of acute pancreatitis. Such damage can be triggered by the inducers of acute pancreatitis like bile acids and ethanol metabolites and is manifested by formation of large intracellular vacuoles, intracellular activation of digestive enzymes and, importantly for this study, mitochondrial damage.
Ca2+ is an important second messenger regulating pancreatic secretion. However excessive Ca2+ accumulation is toxic for the cells. We hypothesise that the mitochondrial damage in pancreatic acinar cells, exposed to the inducers of acute pancreatitis, develops as a result of excessive Ca2+ entry into mitochondria via the Mitochondrial Ca2+ Uniporter (MCU) and that such MCU-dependent Ca2+ overload could be the mechanism responsible for early cell damage in acute pancreatitis.
The project will capitalise on the recent molecular identification of the MCU. In our study we will examine the potential role of MCU in acute pancreatitis, determine the specific underlying cellular mechanisms and potential of targeting these mechanisms for the development of treatment against this disease. The first objective of our study will be to determine the relevance of the MCU for acute pancreatitis. We expect that this will be revealed by characterising the development of acute pancreatitis in MCU knockout (MCU KO) animals. Our group has considerable experience in experimental acute pancreatitis, routinely assessing multiple models. In experiments with MCU KO animals we will utilise three different models of acute pancreatitis (involving repeated cerulein injections, infusion of taurolithocholic acid 3-sulphate into pancreatic duct and combined injection of alcohol and fatty acids).
Our preliminary experiments on isolated pancreatic acinar cells from MCU KO animals revealed strongly reduced Ca2+ entry into mitochondria and protection of these cells against damage induced by a supramaximal concentration of Ca2+- releasing secretagogue cholecystokinin. These experiments indicate that Ca2+ entry via MCU in pancreatic acinar cells could be important for the development of acute pancreatitis. Our next objective will be therefore to determine the specific role of MCU-dependent damage to pancreatic acinar cells in the development of acute pancreatitis. Experiments on cell specific MCU KO mice (MCU knocked out specifically in the pancreatic acinar cells) will be utilised in this part of the project.
In parallel with in vivo experiments on transgenic animals we will conduct experiments on isolated cells. The objective of these experiments will be to characterise Ca2+ entry into mitochondria of the acinar cells isolated from MCU knockout animals and treated with the inducers of acute pancreatitis. This is essential for interpreting experiments involving animal models of acute pancreatitis. In experiments on isolated cells we will also determine the role of MCU-mediated Ca2+ entry in cellular bioenergetics, apoptosis and necrosis. These experiments are required to reveal the cellular mechanisms defining the role of MCU in acute pancreatitis. The effects of pharmacological MCU inhibition on bioenergetics, apoptosis and necrosis of pancreatic acinar cells will help us to interpret the results of experiments with genetically modified cells and animals. Experiments with pharmacological inhibition of MCU should also help us to evaluate the potential of MCU inhibitors for the development of treatment against acute pancreatitis.

Acute pancreatitis is a frequent, debilitating and sometimes fatal disease. The incidence of acute pancreatitis is rising sharply but there is no specific treatment against this condition. During the last few years a number of research groups (including our own) have identified mitochondrial damage as an important contributing factor to the initiation of acute pancreatitis.
We hypothesise that the mechanism of the mitochondrial damage in conditions of acute pancreatitis involves Ca2+ overload of mitochondria in pancreatic acinar cells, which develops due to Ca2+ entry via the mitochondrial Ca2+ uniporter (MCU). In this study we will aim to identify the role of MCU in acute pancreatitis and characterise specific cellular mechanisms that determine this role. In particular, using MCU knockout animals and animal models of acute pancreatitis we will determine if MCU is important for the development of this disease. Standard procedures of histological and biochemical characterisation of acute pancreatitis will be used in these experiments. To determine the putative cellular mechanism of MCU contribution to acute pancreatitis we will characterise its role in mitochondrial Ca2+ entry, bioenergetics, vacuolisation, trypsinogen activation, apoptosis and necrosis of pancreatic acinar cells. We will combine advanced video imaging, electron microscopy and genetics approaches with molecular biology techniques to attain these objectives. We will also test if MCU inhibitors could protect pancreatic acinar cells against damage inflicted by the inducers of acute pancreatitis.
The study will involve close collaboration with NIHR Liverpool Pancreas Biomedical Research Unit (NIHR PBRU). In the framework of this collaboration the outcome of our experiments will be shared with clinicians, drug safety researchers and medicinal chemists associated with NIHR PBRU. We anticipate that the results of our investigation will contribute to the translational efforts of this unit.

The MRC-funded pancreatic research group in Liverpool primarily concentrates on studies of fundamental cellular mechanisms relevant to the pathophysiology of acute pancreatitis and pancreatic cancer. The work of the group is closely co-ordinated with the NIHR Liverpool Pancreas Biomedical Research Unit (NIHR PBRU), the only UK Research Unit focused on translational pancreatic research. The MRC funding will underpin conceptual and technological developments in the group necessary for the translational efforts of NIHR unit. The primary function of MRC-funded pancreatic research group is to discover fundamental mechanisms of pancreatic diseases and evaluate potential avenues for the development of treatment. NIHR PBRU coordinates the efforts of clinicians, medicinal chemists, structural biologists, biochemists, physiologists (our group) and pharmacologists of the University of Liverpool aiming to develop drugs against acute pancreatitis and other pancreatic diseases.
In the framework of this project we will test the hypothesis that Ca2+ entry into mitochondria of pancreatic acinar cells via mitochondrial Ca2+ uniporter is a critically important cellular mechanism underlying the development of acute pancreatitis and therefore a potential target for pharmaceutical intervention. The information obtained as a result of our experiments will be regularly transferred to laboratories collaborating within the framework of NIHR PBRU, comprehensively discussed and utilised to formulate the strategy of translational research activity of NIHR PBRU, including synthesis of new biologically-active compounds (in the case of this project synthesis of plasma membrane-permeant inhibitors of mitochondrial Ca2+ uniporter or proteins regulating the uniporter).
The MRC -funded research group serves as an important training hub for PhD students with interests in pancreatic physiology and pathophysiology (with emphases on acute pancreatitis and pancreatic cancer). During the last five years three MRC-funded PhD students and four Wellcome Trust - funded PhD students successfully completed their training in our group. Currently five PhD students are conducting research in our laboratory (all co-supervised by our colleagues from NIHR PBRU). The MRC support for the salary of an experienced postdoctoral scientist is invaluable for maintaining technical expertise which will be transferred to our PhD students. The MRC contribution to equipment maintenance allows us to retain a vital item of equipment (confocal microscope) which will be used for the project itself and for training of PhD students.
Within the framework of the current MRC-funded project we have established productive collaboration with two companies - GlaxoSmithKline and CalciMedica; these collaborations resulted in two important publications [1, 2]. We hope that the current project will allow us to enhance these industrial collaborations and advance the development of treatment against acute pancreatitis.
1 Voronina, S., Collier, D., Chvanov, M., Middlehurst, B., Beckett, A. J., Prior, I. A., Criddle, D. N., Begg, M., Mikoshiba, K., Sutton, R. and Tepikin, A. V. (2015) The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells. The Biochemical journal. 465, 405-412
2 Wen, L., Voronina, S., Javed, M. A., Awais, M., Szatmary, P., Latawiec, D., Chvanov, M., Collier, D., Barrett, J., Begg, M., Stauderman, K., Roos, J., Grigoryev, S., Ramos, S., Rogers, E., Whitten, J., Velicelebi, G., Dunn, M., Tepikin, A. V., Criddle, D. N. and Sutton, R. (2015) Inhibitors of ORAI1 Prevent Cytosolic Calcium-associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models. Gastroenterology