Cerebral ischemic reperfusion (I/R) injury is a common characteristic of ischemic stroke. It occurs when blood supply is restored after a period of ischemia and damages on neuron cells. Consequently, the recovery of the function in neurons damaged by ischemia could be limited even though reperfusion is the main treatment for acute ischemic stroke (AIS). On the contrary, the phenomenon of acquiring the ischemic tolerance called “ischemic preconditioning” is known for the remarkable neuroprotective effect against I/R injury, which is triggered by giving a mild intermittent ischemic load to brain before fatal ischemic assault [1–3]. However, in the clinical situation, it is exceedingly difficult to predict when AIS occurs and impossible to make the application of ischemic preconditioning for patient before the onset. Similarly, even after severe ischemic assault, by giving a mild intermittent ischemic load the neuroprotective effect called ischemic postconditioning (PostC) could be acquired [4], [5].
This concept of PostC can be applied as a new therapeutic approach to AIS as well as intravenous tissue-plasminogen activator and mechanical thrombectomy. Previous studies have shown that PostC is triggered through the opening of mitochondrial ATP-dependent potassium (mito-KATP) channels and suppresses synaptic glutamate over-release in I/R injury [6], [7]. And besides, one of the key processes in I/R injury is the excessive activation of N-methyl-D-aspartate receptor (NMDAR). Because this activation induces an excessive influx of Ca2+ into cytoplasm via opening of NMDAR, it leads to cytoplasmic Ca2+ overload and activation of various proteins such as caspases and endonucleases via high conductance opening of mitochondrial permeability transition pore (mPTP) [8]. In PostC, opening of the mito-KATP channel causes depolarization of mitochondrial membrane potential and induces reduction of NMDAR currents through low-conductance opening of mPTP, and in consequence that suppresses Ca2+ influx into the cytoplasm, leading to neuroprotection against I/R injury[6], [7].
Revealing the mechanism of PostC is expected to give the beneficial information of a new treatment of AIS for us and in this research, we focused on mitochondrial calcium uniporter (MCU). As mentioned above, regulatory of the cytoplasmic Ca2+ concentration is essential for I/R injury and PostC. Mitochondrial Ca2+ trafficking is also important for playing a key role in many bioenergetic processes, such as mitochondrial respiration and production of ATP. MCU is known as the highly sensitive channel for uptake of Ca2+ inwardly placed on the inner mitochondrial membrane and regulates mitochondrial Ca2+ concentration [9], [10]. Recently lots of studies have reported the structural data of MCU, which is composed of some subunits: mitochondrial calcium uptake (MICU) 1, MICU2, MICU3, MCUb, mitochondrial calcium uniporter regulator (MCUR) 1 and essential MCU regulatory element (EMRE) [10–16]. Each subunit regulates one another to promote or suppress Ca2+ transport into mitochondria [17], [18]. Furthermore, numerous pathological conditions of the cell activity such as ischemic stroke, neurodegenerative disease and cancer are caused by the dysregulation of Ca2+ uptake by MCU [19–22], which has been attracting attention as a new target of various treatments in recent years.[23–25]
To our best knowledge, however, there are few reports on involvement of MCU in PostC. We hypothesized that MCU plays a role as a driving trigger of PostC and in this study, to investigate how involving between PostC and MCU, we examined the following: (1) whether MCU is involved in the mechanism of PostC, (2) how Ca2+ kinetics with MCU is involved in the process, (3) whether downregulation of NMDAR currents is occurred in PostC without MCU, (4) how different mitochondrial membrane depolarization is between PostC and PostC without MCU. We analyzed the changes of sEPSC, NMDAR current, cytosolic Ca2+ concentration, and mitochondrial membrane potential under inhibition of MCU with Ru265 and revealed the relationship between PostC and MCU in hippocampal pyramidal neurons by using whole-cell patch clamp technique.