Mitochondrial Calcium Uniporter (MCU) Involves in Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mouse

Mitochondrial Calcium Uniporter (MCU) Involves in Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mouse | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial Calcium Uniporter (MCU) Involves in Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mouse Hiromitsu Sasaki, Ichiro Nakagawa, Takanori Furuta, Shohei Yokoyama, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3279580/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Apr, 2024 Read the published version in Cellular and Molecular Neurobiology → Version 1 posted 4 You are reading this latest preprint version Abstract Ischemic postconditioning (PostC) phenomenon is known as the neuroprotection against ischemic reperfusion (I/R) injury. One of the key processes in PostC is opening of mitochondrial ATP dependent potassium (mito-K ATP ) channel and depolarization of mitochondrial membrane potential, which triggers the release of calcium ion from mitochondria through the low conductance opening of mitochondrial permeability transition pore (mPTP). Mitochondrial calcium uniporter (MCU) is known as the highly sensitive transporter for uptake of Ca 2+ inwardly existed on the inner mitochondrial membrane. Furthermore, it has attracted attention as a new target of treatments in disease such as neurodegenerative disease, cancer and ischemic stroke. Thus, we considered that MCU may involve in PostC and trigger its mechanism. In this research, we used the whole-cell patch clamp technique to hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca 2+ concentration, mitochondrial membrane potential and N-methyl-D-aspartate receptor (NMDAR) currents under the inhibition of MCU by Ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased sEPSCs occurrence (p = 0.008), NMDAR currents (p < 0.001), intracellular Ca 2+ concentration (p < 0.001) and dead cells (p < 0.001) significantly after reperfusion, indicating the removal of the neuroprotective effects in PostC. Moreover, the mitochondrial depolarization in PostC with Ru265 was weakened, compared to it in PostC (p = 0.03). These results suggest that MCU affects the mitochondrial depolarization in the PostC mechanism to suppress NMDAR over-activation and prevent the elevation of intracellular Ca 2+ concentration against I/R injury. ischemic postconditioning (PostC) mitochondrial calcium uniporter (MCU) NMDA receptor (NMDAR) mitochondrial permeability transition pore (mPTP) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction 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 (Kitagawa et al., 1990 ; Nakagawa et al., 2002 ; Yin et al., 2005 ). However, in the clinical situation, it is very 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 (Pignataro et al., 2009 ; Xing et al., 2008 ). 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-K ATP ) channels and suppresses synaptic glutamate over-release in I/R injury (Morisaki et al., 2022 ; Yokoyama et al., 2019 ). 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 Ca 2+ into cytoplasm via opening of NMDAR, it leads to cytoplasmic Ca 2+ overload and activation of various proteins such as caspases and endonucleases via high conductance opening of mitochondrial permeability transition pore (mPTP) (Hawrysh & Buck, 2013 ). In PostC, opening of the mito-K ATP channel causes depolarization of mitochondrial membrane potential and induces reduction of NMDAR currents through low conductance opening of mPTP, and in consequence that suppresses Ca 2+ influx into the cytoplasm, leading to neuroprotection against I/R injury(Morisaki et al., 2022 ; Yokoyama et al., 2019 ). 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 Ca 2+ concentration is essential for I/R injury and PostC. Mitochondrial Ca 2+ 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 Ca 2+ inwardly placed on the inner mitochondrial membrane and regulates mitochondrial Ca 2+ concentration (Clapham, 2007 ; de Stefani et al., 2011 ). Recently a lot 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) (Baughman et al., 2011 ; de Stefani et al., 2011 ; Mallilankaraman et al., 2012 ; Perocchi et al., 2010 ; Plovanich et al., 2013 ; Raffaello et al., 2013 ; Sancak et al., 2013 ). Each subunit regulates one another to promote or suppress Ca 2+ transport into mitochondria (Lambert et al., 2019 ; Patron et al., 2014 ). Furthermore, numerous pathological conditions of the cell activity such as ischemic stroke, neurodegenerative disease and cancer are caused by the dysregulation of Ca 2+ uptake by MCU (Pchitskaya et al., 2018 ; Polster et al., 2017 ; Vultur et al., 2018 ; Woods & Wilson, 2019 ), which has been attracting attention as a new target of various treatments in recent years.(Giorgi et al., 2012 ; Liao et al., 2017 ; Modesti et al., 2021 ) 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 Ca 2+ 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 Ca 2+ 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. Materials and Methods Procedure of mouse hippocampal slices All our experiments were approved by the Animal Care and Use Committee of our University (approval no.13411) and performed all procedures in accordance with the Guidelines for the Proper Conduct of Animal Experiments. 4- to 8-week-old wild type C57BL/6J mice (58 males) weighing about 18–24 grams were used for our experiments. Mice lived in their cage with light and dark cycle in a half of day and were able to access their food and water freely for any time a day. Mice were decapitated after we checked disappearance of their postural reflex and their shallow and fast breath changing to deep and slow one under general anesthesia with isoflurane (0.05 v/v , administered by inhalation). Then the brains were removed quickly into ice-cold solution composed with sucrose 230mM, KCl 2.5 mM, NaHCO 3 25 mM, NaH 2 PO 4 1.25 mM, CaCl 2 0.5 mM, MgSO 4 10 mM, D-glucose 10 mM and bubbled with 95% O 2 / 5% CO 2 . In this solution the brains were cut into horizontal slices of the hippocampal at a thickness of 350 µm, using a linear slicer (PRO7; DOSAKA EM, Kyoto, Japan). Then slices were incubated in standard artificial cerebrospinal fluid (ACSF) composed with NaCl 125 mM, KCl 2.5 mM, NaHCO 3 25 mM, NaH 2 PO 4 1.25 mM, CaCl 2 2.0 mM, MgCl 2 1.0 mM, D-glucose 10 mM and bubbled with the same gas mixture and kept for at least 1 hour at 32℃. After this procedure slices were kept in ACSF at 27℃. We used about 5 to 7 slices per one mouse. Perfusion protocols and Patch-clamp recording Patch pipettes were made from thick-walled borosilicate glass capillaries and filled with the internal solution which was consisted of Cs-gluconate (141 mM), CsCl (4.0 mM), MgCl 2 (2.0 mM), HEPES (10.0 mM), Mg-ATP (2.0 mM), Na-GTP (0.3 mM), EGTA (0.2 mM) and adjusted to pH 7.25 with CsOH. Each slice was placed on an 800µL recording chamber mounted on a BX50WI upright microscope (Olympus, Tokyo, Japan) which was equipped with an infrared differential interference microscope and epifluorescence imaging apparatuses. And in this chamber gas-saturated ACSF was perfused at a 2.0mL/min flow and kept at 31–33℃ by a controlled heater. Hippocampal slices were put into this chamber and randomly assigned to the following three groups (Fig. 1 A): 1) control group: slices were reperfused with ACSF for 20 minutes after the anoxic period, 2) PostC group: slices were exposed after the anoxic period to intermittent the normoxic and anoxic periods based on the PostC protocol as mentioned below, 3) PostC administered with Ruthenium Red 265 (Ru265) (PostC + Ru265) group: slices in the PostC protocol were supplemented with 1µM, 10µM and 50 µM of Ru265 respectively which was the novel selective inhibitor of the mitochondrial calcium uniporter (MCU). In the control group, the baseline time schedule includes 5 minutes of normoxia, 7.5 minutes of anoxic and 20 minutes of reperfusion periods. 7.5 minutes of the severe anoxic load was simulated as exposing slices to ACSF in which glucose and oxygen were replaced to sucrose and nitrogen. In accordance with previous studies, the protocol of PostC was started from 30 seconds after the anoxic period and consisted of three times of 15 seconds of anoxia separated by 15 seconds of normoxia (Yokoyama et al., 2019 ). In the PostC + Ru265 group, ACSF containing of Ru265 was perfused from the start of the time schedule (Fig. 1 A, B). Then we confirmed visually CA1 pyramidal cells and made whole-cell voltage-clamp recordings using an EPC-9 patch clamp amplifier (Heka, Lambrecht/Pfalz, Germany). The holding potential of voltage clamp was set at -70 mV and the resistance of the pipette was kept from 2.5 to 3.5 MΩ. And to detect the glutamatergic excitatory post-synaptic currents clearly, the GABAA and GABAB antagonist picrotoxin (50 µM) was supplemented into ACSF during the recordings. We counted the number of sEPSCs in each group and calculated it as a percentage of the number of the sEPSCs occurrence during the anoxic and the reperfusion periods to that in the first normoxic period for 5 minutes before the anoxic period. To determine the optimal concentration of Ru265 in this experiment, we tried with three different types of the Ru265 concentration at 1 µM, 10 µM, and 50 µM. Ru265 and picrotoxin were purchased from Sigma-Aldrich. Recording of whole-cell currents in response to NMDA application To assess the NMDAR currents in each group, we recorded whole-cell currents to NMDA application. With another micropipette similar of one for whole-cell recordings, 5µM of NMDA was puffed to the recording cell. And the holding potential of voltage-clamp was set at -55mV during the pre- (1 second) and post-stimulation period (6 seconds) for the purpose of suppressing the Mg 2+ block to NMDAR channels. All NMDAR currents were recorded every 30 seconds during the experiments for about 30–35 minutes. Cell staining To investigate the effects of Ru265 in PostC, we counted the number of dead cells in hippocampal slices after ischemic injury. Dead cells were dyed by propidium iodide (PI) and SYTOX-blue, which are impermeable to cell membrane and used for nuclear staining. At least 45 min before the perfusion protocol started, slices were incubated for 15 min in ACSF containing 3 µM PI. Thus, PI staining was performed after the slice preparation until the perfusion protocol started, that is, dead cells, which membranes were broken during the process of making slices were stained red. Subsequently, PI was removed before the perfusion protocol started, not to dye dead cells due to I/R injury during the perfusion protocol. According to perfusion protocol, slices in the control group were loaded with the anoxic period for 7.5 min and with reperfusion period for 20 min, and slices in the PostC group and the PostC + Ru265 group were loaded with the intermittent anoxic periods after 7.5 min anoxia. After these perfusion protocols, slices were transferred to the incubation chamber and stained with 6 µM SYTOX-blue contained in ACSF for 3 h at 32℃. In this section, by staining cells with SYTOX-blue, both dead cells already stained red with PI and new dead cells after the perfusion protocol were dyed blue. Therefore, dead cells in the slice preparation procedure were dyed both red and blue, while the ones died due to I/R injury were dyed only blue. SYTOX-blue was excited at 408 nm and the blue fluorescence emission was bandpass filtered from 417 to 477 nm by using confocal microscopy (C2plus; Nikon, Japan). To detect the initial number of dead cells before ischemic stress, we also checked dead cells stained with PI, which were excited at 561 nm and observed as the bandpass of red fluorescence emission filtered from 552 to 617 nm. Thus, we compared the number of dead cells in each group showing only blue fluorescence with SYTOX-blue. Fluorometric assessment of cytosolic Ca 2+ changes To evaluate changes of the cytosolic Ca 2+ concentration, the Fura-2 (DOJINDO, Kumamoto, Japan) fluorescence signals of whole-cell voltage-clamped pyramidal cells were measured by adding 15 µmol/L of Fura-2 to the internal pipette solution. Using a fast-switching multi-wavelength illumination system (Lambda DG-4; Sutter Instruments, CA, USA), the Fura-2 fluorescence signals were excited at 340 nm and 380 nm every 10 seconds and their emission was filtered at 510 nm with the 500nm- dichroic mirror. For acquiring these images, a ×40 water immersion objective lens (LUMPlanFI/IR, Olympus, Japan) and a CCD camera (ORCA-flash 4.0 v3; Hamamatsu Photonics, Shizuoka, Japan) were used. All images were saved in MetaMorph software (Molecular Devices, CA) and for analysis we measured the region of interest (ROI) defined as a circular area of 5µm in diameter with the maximum fluorescence intensity near the center of the recording cell. The average of fluorescence intensity ratio of 340 nm excitation and 380 nm excitation in the ROI was calculated. Fluorometric assessment of mitochondrial membrane potential To evaluate changes of the mitochondrial membrane potential during the anoxic and reperfusion periods, we used a JC1 (Cayman Chemical, MI) fluorescence of which the emission wavelength changes depending on the mitochondrial membrane potential and loaded it into the cytoplasm via a patch pipette. The tip of the patch pipette was filled with an internal solution and from the back of the pipette an internal solution containing with 2.0µM of JC1 was put in immediately before use. The red fluorescence of JC1 represents the J-aggregated state excited at 548 nm with a 580-nm dichroic mirror and fluoresced at 590 nm with a long-pass filter. And the green fluorescence of JC1 represents monomeric state excited at 477 nm with a 500-nm dichroic mirror and fluoresced with a bandpass filter at 515–565 nm. Fluorescence images were acquired at every 30 seconds using the same apparatus as Fura2. For the measurement of the fluorescence the ROI was defined as a hand-drawn polygonal area covering the region of high red fluorescence because the red fluorescence was eccentrically distributed around the nucleus and was often crescent-shaped and each average of the green and red fluorescence in the ROI was calculated as the green/red ratio for analysis. The increase of this green/red ratio indicates the mitochondrial depolarization. Statistical analysis All data were calculated as mean ± standard error of the mean. For multiple testing, we used one-way analysis of variance and significant effects were further tested with a post-hoc multiple comparison test of Tukey-Kramer method and Steel-Dwass method as appropriate. Significant difference was set at the level of * p < 0.05 and ** p < 0.01. Results Changes of the sEPSCs surge The frequency of sEPSCs gradually increased during the anoxic period in all groups. After reperfusion at 7.5 min of anoxic load, in the control group and the PostC + Ru265 groups, sEPSCs occurrence was sharply increased, however in the PostC group, increase of sEPSCs was not observed (Fig. 2 A). Furthermore, to examine the optimal concentration of Ru265 in this experimental setting, we set three different concentrations of Ru265 administered to ACSF: low (1 µM; n = 5); medium (10 µM; n = 5); and high (50 µM; n = 5). The effect of Ru265 to the suppression of sEPSCs by PostC was observed at 10 µM and 50µM (Fig. 2 B). We determined Ru265 at 10µM for the optimal concentration in this experiment. The percentage of cumulative sEPSCs occurrence at 20 min in the PostC + Ru265 10µM group (n = 5) was significantly higher than that of the PostC group (n = 5) (38.78 ± 3.39 ×10 2 % vs 17.64 ± 3.57 × 10 2 %, p = 0.008) and the PostC + Ru265 1µM group (38.78 ± 3.39 ×10 2 % vs 15.68 ± 1.79 × 10 2 %, p = 0.003), similar to that of PostC + Ru265 50µM group was (43.6 ± 6.46 ×10 2 % vs 17.64 ± 3.57 × 10 2 %, p = 0.001 and 43.6 ± 6.46 ×10 2 % vs 15.68 ± 1.79 × 10 2 %, p < 0.001) (Fig. 2 C). On the contrary, the percentage of cumulative sEPSCs occurrence at 20 min in the PostC + Ru265 1µM group (n = 5) was not significantly difference from that of the PostC group (15.68 ± 1.79 × 10 2 % vs 17.64 ± 3.57 × 10 2 %, p = 0.996) (Fig. 2 C). Inhibition of MCU by Ru265 canceling the suppression of NMDAR currents by PostC We recorded NMDAR currents per 30 seconds. When NMDA was locally applied to CA1 pyramidal cells, NMDAR currents with slow decay for several seconds were observed in all three groups (Fig. 3 A). After reperfusion, NMDAR currents decreased in the PostC group (n = 12) while those increased in the PostC + Ru265 group (n = 11) and the control group (n = 9). We analyzed the change in NMDAR currents between peak and minimum amplitude from 10 min to 20 min after reperfusion in each group. In the PostC + Ru265 group, the percentage of NMDAR currents amplitude after reperfusion was significantly larger than that in the PostC group (135.64 ± 5.17% vs 92.33 ± 1.75%, p < 0.001) (Fig. 3 B). Therefore, these results indicate that inhibition of MCU by Ru265 canceled the effect of suppressing NMDAR currents by PostC. Inhibition of MCU by Ru265 in PostC increasing the number of dead CA1 neurons According to these results, it is thought that inhibition of MCU reduces the neuroprotection in PostC and dead cells may increase after reperfusion by inhibiting MCU in PostC. Thus, we counted the number of dead CA1 neurons in vivo of all groups after reperfusion. To distinguish dead cells caused by I/R injury from all dead cells, we used two kinds of membrane impermeable dyes for nuclear staining with different fluorescent wavelengths. We counted the number of CA1 neurons died during 20 min to 3 h of reperfusion period after 7.5 min anoxic insult (Fig. 4 A-C). There were significantly difference between the PostC group (87.8 ± 6.75/mm, n = 20) and the PostC + Ru265 group (194.56 ± 21.05/mm, n = 9, p < 0.001) (Fig. 4 D). Thus, CA1 dead neurons were significantly increased in the PostC + Ru265 group, similar to that in the control group (n = 11). This result indicated that in PostC, inhibition of MCU weakened its neuroprotective effect against I/R injury and leads to cell death because MCU mediated the mechanism of PostC. Ru265 inhibits the suppression of cytosolic Ca 2+ increase by PostC We examined the involvement of MCU of the change in cytosolic [Ca 2+ ] concentration by PostC. In all groups, the Fura2 ratio gradually increased during the anoxic period, indicating cytosolic [Ca 2+ ] increased. After reperfusion, the ratio started to decrease gradually in the PostC group while in the PostC + Ru265 group, it started to increase immediately until 2.5 min after reperfusion, indicating cytosolic [Ca 2+ ] increased (Fig. 5 A). We used the change in the Fura2 ratio from 0min until 5min after the anoxic period for analysis. In the PostC + Ru265 group (n = 8), the percentage of change in the Fura2 ratio was significantly higher than that in PostC group (n = 6), similar to that in the control group (n = 7) from 0min to 5min after the anoxic period (PostC vs PostC + Ru265; 5.09 ± 0.49% vs 12.49 ± 1.00%, p < 0.001, PostC vs Control; 5.09 ± 0.49% vs 12.67 ± 0.79%, p < 0.001) (Fig. 5 B). This result suggests that the suppression of cytosolic [Ca 2+ ] increase in the early stage of reperfusion by PostC is canceled due to inhibition of MCU activity by Ru265. Ru265 inhibits the interaction between the mitochondrial depolarization by PostC and MCU To examine whether MCU mediates depolarization of mitochondrial membrane potential induced by PostC or not, we estimated the mitochondrial membrane potential with JC1 fluorescence. The elevation of the green/red ratio that represents depolarization of mitochondrial membrane potential increased immediately after the anoxic period. Especially in the PostC and the PostC + Ru265 groups, the ratio sharply increased. Subsequently, this ratio was decreased in the PostC + Ru265 group, as well as it in the control group (Fig. 6 A). Change in the green/red ratio from 0min until 5min after the anoxic period were used for analysis (Fig. 6 B). The green/red ratio in the early phase from 0 min to 5min after the anoxic period was significantly different between the PostC group (n = 7) and the PostC + Ru265 group (n = 6), similar to the control group (n = 9) (PostC vs PostC + Ru265; 16.62 ± 1.55% vs 12.6 ± 1.21%, p = 0.03, PostC vs Control; 16.62 ± 1.55% vs 8.58 ± 0.69%, p < 0.001, PostC + Ru265 vs Control; 12.61 ± 1.22% vs 8.58 ± 0.69%, p = 0.13). These results suggest that in the PostC + Ru265 group, the mitochondrial depolarization by PostC is weakened. Discussion This study demonstrates the role of MCU in the Post-Conditioning (PostC) mechanism against I/R injury, using the whole-cell patch clamp technique on mouse hippocampal CA1 pyramidal cells. Blocking MCU with Ru265 weakened the effect of PostC, increasing EPSC occurrence and NMDAR currents after reperfusion. As a result, the number of dead cells in the CA1 region increased because the effect of reducing Ca2 + influx into the cell cytosol via NMDAR after reperfusion in PostC was weakened by blocking MCU. Moreover, in PostC with an inhibitor of MCU, the depolarization of the mitochondrial membrane potential was sharply elevated in the early phase after reperfusion, however, it was weakened immediately. These results suggest that interaction between the mitochondrial depolarization and the MCU activation is involved in the PostC mechanism, leading to neuroprotection through the reduction of NMDAR currents and Ca2 + influx into the cytosol. Regulation of MCU affects the PostC pathway and reduces its neuroprotective effect in PostC after reperfusion In our previous research, we have reported that PostC induces an interaction between mitochondria and NMDA receptor, and that the increase of sEPSCs occurrence was observed as a result of this interaction process indirectly(Morisaki et al., 2022 ; Yokoyama et al., 2019 ). Thus, in this study, we initially checked the sEPSCs occurrence in each group to confirm whether adjustments to this interaction process of PostC could be made by adjusting MCU with MCU blocker. The sEPSCs occurrence in the PostC + Ru265 groups was sharply increased, compared to the PostC group, as shown in the Fig. 2 . This result suggests that MCU plays a role as one of the key factors in the PostC pathway. During I/R injury, an excessive release of glutamate occurs, leading to over-activation of NMDAR (Bonova et al., 2013 ; Dávalos et al., 1997 ; Soria et al., 2014 ). This cascade leads to cell necrosis or apoptosis by excessing Ca 2+ influx into the neurons, subsequently triggering a range of downstream pro-death signaling events such as calpain activation, reactive oxygen species (ROS) generation and mitochondrial damage (Curcio et al., 2016 ; Kristián & Siesjö, 1998 ; Lau & Tymianski, 2010 ). Zhang et al. suggested that NMDAR mediates the PostC-induced neuroprotection (Yin et al., 2005 ) and Morisaki.Y et al. also reported that low conductance opening of mPTP by PostC induces extrusion of a few Ca 2+ into cell cytosol and subsequently some chemical mediators are predicted to inhibit NMDAR over-activation against excessing Ca 2+ influx into the neurons (Morisaki et al., 2022 ). Moreover, in their experiments, the increase of intracellular Ca 2+ concentration after reperfusion was not observed in either the control or the PostC group when the extracellular Ca 2+ concentration was set to 0. From this result, they concluded that in PostC the suppression of NMDAR over-activation during reperfusion inhibited Ca 2+ influx into the cytoplasm from the extracellular space, resulting in a reduction of Ca 2+ overload in the cytoplasm and cellular damage (Morisaki et al., 2022 ). Therefore, this death-signaling by NMDAR over-activation is considered as the most important key to the neuroprotective mechanism in PostC. Our present study showed that the cytosolic Ca 2+ concentration during the anoxic to reperfusion period sharply increased due to inhibition of MCU with Ru265, similar to that observed in the control group, while these changes was not observed in PostC after reperfusion in the Fig. 5 . Additionally, the reduction of NMDAR currents was also weakened by inhibiting MCU in the Fig. 3 . Thus, the results of our research also suggest that the inhibition of the MCU-mediated Ca 2+ influx into the mitochondria induces disruptions in the PostC process, resulting in the loss of PostC-induced neuroprotection against the extracellular Ca 2+ influx through NMDAR over-activation. Under physiological conditions, cytosolic Ca 2+ concentration is kept in range of 100nM by intracellular Ca 2+ stores, plasma membrane and organelle membrane Ca 2+ channels and pumps, and Ca 2+ binding proteins (Babcock et al., 1997 ; Berridge, 2016 ; Carvalho et al., 2020 ; Chen et al., 2020 ; Enomoto M et al., 2017 ). The endoplasmic reticulum (ER) is known to be the largest intracellular Ca 2+ store, but mitochondria, lysosomes and the nucleus are also involved in regulating intracellular Ca 2+ concentration (Kaufman & Malhotra, 2014 ; S. Smaili et al., 2013 ; Samanta & Parekh, 2017 ). However, the cytosolic Ca 2+ influx into the mitochondrial matrix via MCU, in particular, leads to the elevation of mitochondrial Ca 2+ concentration and influences various mitochondrial metabolic processes, including mitochondrial respiration, adenosine triphosphate (ATP) production, mitophagy / autophagy and even the death pathway of apoptosis or necrosis (Duchen, 1999 ; East & Campanella, 2013 ; Gottlieb & Bernstein, 2016 ). Furthermore, especially in the ischemic conditions, the anaerobic glycolysis in cells starts to work as the primary metabolic pathway, which leads to an increase in lactic acids and a decrease in cytosolic pH. As cytosolic pH decreases, the mitochondrial membrane potential is also diminished due to the cessation of oxygen-dependent oxidative phosphorylation. Subsequently, after reperfusion, the rapid restoration of the mitochondrial membrane potential due to the return of oxygen into cytosol provides a strong driving force for the entry of the cytosolic Ca 2+ into the mitochondria via MCU (Shintani-Ishida et al., 2012 ). This results in mitochondrial Ca 2+ overload, leading to cell damage or death associated with opening of mPTP. This process generates toxic products such as cytochrome C and swelling the matrix, eventually leading to the OMM rupture (Bonora et al., 2017 ; Hawrysh & Buck, 2013 ; Morciano et al., 2015 ; Shintani-Ishida et al., 2012 ). Thus, some research has reported that the inhibition of MCU reduces the cell damage caused by mitochondrial Ca 2+ overload and may confer neuroprotection (Novorolsky et al., 2020 ; Woods & Wilson, 2019 ; Zhao et al., 2015 ). However, our results in the Fig. 4 may initially appear to contradict these reports because they showed that an increase in dead cells in CA1 region by inhibiting MCU in PostC. Nonetheless, inhibition of MCU cannot prevent other toxicity caused by cytosolic Ca 2+ overload (Curcio et al., 2016 ; Kristián & Siesjö, 1998 ; Lau & Tymianski, 2010 ). Thus, it might be the reason why dead cells in CA1 region increased by inhibiting MCU in PostC that other death signals due to cytosolic Ca 2+ overload, not through mitochondrial Ca 2+ overload via MCU, were activated and led to cell death finally. MCU plays a role as one of the key factors in the interacting process of mitochondrial depolarization in PostC In our previous research, Morisaki.Y et al. reported that the opening of mito-K ATP channel triggered the PostC mechanism and caused the mitochondrial depolarization which was observed during the early period of reperfusion in the PostC group but, not in the control group (Morisaki et al., 2022 ). Moreover, we also have reported that one of the key factors in neuroprotection of PostC was the low conductance opening of mPTP. The catastrophic process of cell injury is induced through the high conductance mode of mPTP opening, which allows for the passage of ions, including calcium, leading to the dissipation of mitochondrial membrane potential, and eventually resulting in the cell death (Brenner & Moulin, 2012 ). Whereas under physiological conditions it is reported that the mPTP could exhibit intermittent opening in low conductance mode, contributing to intracellular Ca 2+ homeostasis and the regulation of mitochondrial function (Giorgio et al., 2013 ). Morisaki.Y et al. also reported that the low conductance mode of mPTP opening prevented the high conductance mode opening and reduced NMDAR conductance (Morisaki et al., 2022 ). Furthermore, the mitochondrial membrane potential switches these two modes with the threshold value controlled by mitochondrial Ca 2+ concentration (Bazil et al., 2010 ). The opening of mito-K ATP channel is triggered by the local decrease of ATP in cytosol due to ischemic stress and subsequently, leading to an increase in K + influx into the mitochondrial matrix (Hawrysh & Buck, 2013 ; Pamenter et al., 2008 ). In PostC opening of this channel leads to mitochondrial depolarization, reducing the driving force of the Ca 2+ influx into mitochondria matrix and would abrogate the excessive Ca 2+ accumulation in the matrix, thus avoiding the high conductance mode of mPTP opening (Hawrysh & Buck, 2013 ; Morisaki et al., 2022 ). Therefore, the mitochondrial depolarization is also one of the key factors in neuroprotection of PostC. In the present study, our data shows that the depolarization of mitochondrial membrane potential was weakened after reperfusion in the PostC with Ru265 group, compared to it in the PostC group. These data suggest that in the PostC mechanism, the mitochondrial depolarization is triggered by opening of mito-K ATP channel, however, some interacting process between mito-K ATP channel and MCU was blocked by Ru265, resulting in the loss of the neuroprotective effect through the mitochondrial membrane potential in PostC. Furthermore, it also suggests that the mild interactive regulation of MCU, provided by the driving force given from mito-K ATP channel would be necessary for maintaining of the mitochondrial depolarization in PostC. Pharmacological Approach to MCU In the present study, we used Ruthenium Red 265 to inhibit MCU, a novel selective inhibitor against MCU. The most well-known and commonly used MCU inhibitor is Ru360 named for its strong absorbance at 360nm (Emerson et al., 1993 ). However, Ru360 is poorly permeable and unstable in aqueous solution, losing its activity in days (Hajnóczky et al., 2006 ; Márta et al., 2021 ). On the other hand, Joshua J. et al. reported that in the lysate of HEK293 or Hela cells, Ru265 was taken up into these cells from two to ten times more than other structural analogs of ruthenium after these cells were incubated with each ruthenium complex at 50µM for 24h, and moreover, the rise of mitochondrial Ca 2+ concentration was observed soon after only 30minutes of incubation (Novorolsky et al., 2020 ; Woods et al., 2019 ). For our protocol of PostC in the previous research, we observed each single cell action during a very short time course, including normoxia and anoxia within about 30min to 1 hour. Therefore, it was necessary to use the quick permeable drug for this protocol, and from this point of view, we regarded Ru265 as suitable for our research because of its quick permeability (Novorolsky et al., 2020 ). Furthermore, ruthenium compounds are also known for its cytotoxicity (Alessio, 2017 ; Dutta et al., 2008 ; F. A. Peacock et al., 2006 ; Hartinger et al., 2008 ; Lameijer et al., 2017 ; Mühlgassner et al., 2012 ; Süss-Fink, 2010 ; Wachter et al., 2012 ; Wang et al., 2003 ; Wee & Dyson, 2006 ), while Ru265 is a low toxic compound without any effect on mitochondrial membrane potential and other intracellular Ca 2+ dynamics reportedly (Woods et al., 2019 ). ‘Safe pharmacological PostC’ is our central concern for clinical application, however, Diazoxide as the opener of mito-K ATP channel, is not suitable because of its toxic side effects (Kumar et al., 1976 ). In our previous study, Furuta.T et al. reported on the neuroprotective mechanism in melatonin-induced pharmacological PostC (Furuta et al., 2022 ) and melatonin could be the new candidate of alternative drugs for pharmacological PostC without any side effects. Thus, for our future direction, further studies in vitro and in vivo are needed to explore ways to deliver melatonin to the brain, such as using catheter to the MCAO models. Conclusion MCU plays an important role related to the depolarization of the mitochondrial membrane potential in PostC, leading to reduce dead cells in CA1 region through downregulation of NMDAR currents occurrence and reduction of Ca 2+ influx into cytosol. Abbreviations AIS Acute ischemic stroke I/R Ischemic reperfusion PostC Ischemic postconditioning ATP Adenosine triphosphate Mito-K ATP Mitochondrial ATP-depemdent potassium NMDA N-methyl-D-aspartate NMDAR NMDA receptor mPTP Mitochondrial permeability transition pore MCU Mitochondrial calcium uniporter Ru265 Ruthenium red 265 sEPSCs Spontaneous excitatory post-synaptic currents Declarations Author Contributions Conceptualization and design or analysis and interpretation of data, or both, T.F., I.N. and S.Y.; writing—original draft preparation or critical revision for important intellectual content, T.F., I.N., S.Y., Y.M., Y.S. and H.N.; final approval of the submitted manuscript, I.N., Y.S. and H.N. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by JSPS KAKENHI (grant number JP22K09240). Institutional Review Board Statement All experimental procedures were approved by the Animal Care and Use Committee of Nara Medical University (approval no. 13411) and were performed in accordance with the Guidelines for the Proper Conduct of Animal Experiments. Institutional Review Board Statement Not applicable. Data Availability Statement The datasets of the current study are available upon request with no restriction. Acknowledgments The authors thank Yoichi Ogawa for his technical assistance. Conflicts of Interest None. References Alessio E (2017) Thirty Years of the Drug Candidate NAMI-A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective. In European Journal of Inorganic Chemistry (Vol. 2017, Issue 12, pp. 1549–1560). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3279580","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":228029905,"identity":"1e5a0c21-a70d-48c9-9373-e300df43a7e6","order_by":0,"name":"Hiromitsu Sasaki","email":"","orcid":"","institution":"Nara Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hiromitsu","middleName":"","lastName":"Sasaki","suffix":""},{"id":228029909,"identity":"797cd727-569b-4cf7-bba9-a7e61a01388b","order_by":1,"name":"Ichiro Nakagawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYFACNgjFD+UyNqAI49Mi2UCyFoMDqFpwA3P2Y4kfv9Qclje+3fzsw88cG9kGiQTGDz8Y+PJwabHsSTssLXPssOG2O8eMZ/ZuSzMGamGW7GFgK8alxeBAeoO0BNttxm03EowZeLcdTtx/I4FBGujeRFwuNDj/vPm3xL/b9ptnpH9m/LvtfyLIlt94tdxIOyb5se124gaJHGNm3m0HQFrY8Nty41maNWPf/+QZN3KKmWW3JRs38Dxss+wxwOOX82nGN398S7Ptn5G+mfHtNjvZBvbkwzd+VBzDGWIgwMyDygdFjcGxBHxaGH9gEazBq2UUjIJRMApGFAAA0/pbgycRqvYAAAAASUVORK5CYII=","orcid":"","institution":"Nara Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ichiro","middleName":"","lastName":"Nakagawa","suffix":""},{"id":228029913,"identity":"64c67a25-b96d-4e69-aa06-5a7409fea0f4","order_by":2,"name":"Takanori Furuta","email":"","orcid":"","institution":"Nara Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takanori","middleName":"","lastName":"Furuta","suffix":""},{"id":228029915,"identity":"aba1b94d-85c6-45fd-a7eb-26b9eb055b7f","order_by":3,"name":"Shohei Yokoyama","email":"","orcid":"","institution":"Nara Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shohei","middleName":"","lastName":"Yokoyama","suffix":""},{"id":228029917,"identity":"386a2f65-4921-463a-91b0-9b9eed73c9e5","order_by":4,"name":"Yudai Morisaki","email":"","orcid":"","institution":"Nara Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yudai","middleName":"","lastName":"Morisaki","suffix":""},{"id":228029919,"identity":"fe472364-6036-477a-af74-5b2c454f4f3d","order_by":5,"name":"Yasuhiko Saito","email":"","orcid":"","institution":"Nara Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasuhiko","middleName":"","lastName":"Saito","suffix":""},{"id":228029922,"identity":"98bb1eba-d088-4eec-91d2-ed7b9cc47c02","order_by":6,"name":"Hiroyuki Nakase","email":"","orcid":"","institution":"Nara Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Nakase","suffix":""}],"badges":[],"createdAt":"2023-08-20 11:29:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3279580/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3279580/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10571-024-01464-7","type":"published","date":"2024-04-03T15:01:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":42150900,"identity":"2ec2702c-12da-4b07-af62-ab31fa8f1ee0","added_by":"auto","created_at":"2023-08-25 17:26:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":198656,"visible":true,"origin":"","legend":"\u003cp\u003ePerfusion protocols and its method \u003cstrong\u003eA\u003c/strong\u003e Diagram shows the protocol for anoxia-reperfusion, PostC and drug administration in each group. The horizontal arrow indicates time schedule, and the black band indicates the anoxic period in the control group, the PostC group and the PostC administrated with Ru265 (PostC+Ru265) group. In each group, data is recorded for 32.5 reperfusion period after anoxia in the control group: in the PostC and the PostC+Ru265 group, min: 5 min normoxia before the start of 7.5 min anoxia and 20 min PostC procedure included three cycles of 15 s of anoxia-perfusion with an intermittent 15 s of reperfusion (Yokoyama et al., 2019), is started 30 s after 7.5 min anoxia. The red band indicates Ru265 perfusion in ACSF. \u003cstrong\u003eB \u003c/strong\u003eSchema shows the way of Patch-clamp record. According to the PostC protocol in our previous study (Yokoyama et al., 2019), each patch-clamp record was performed on the chamber while switching the flow of normoxic and anoxic ACSF with administering proper drugs or not. PostC; ischemic postconditioning, Ru265; Ruthenium red 265\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/fc55e9b151a925bb919610cb.png"},{"id":42153143,"identity":"b4c65708-2c17-4eef-b2d4-c558a4d44ae7","added_by":"auto","created_at":"2023-08-25 17:42:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":215499,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of spontaneous excitatory post-synaptic currents (sEPSCs). \u003cstrong\u003eA\u003c/strong\u003e Representative case of sEPSCs for the control (upper), the PostC (middle) and the PostC+Ru265 10µM (lower) groups during the pre-anoxia, the anoxic and reperfusion periods. In each trace, sEPSCs caused by synaptic glutamate releases are observed as transient downward deflections (inward currents). For all groups, occurrence of sEPSCs began to increase approximately 7 min after anoxia-perfusion and only in the PostC group, increased occurrence of sEPSCs receded to pre-anoxia levels immediately after reperfusion. On the other hand, in traces of the control group and PostC+Ru265 10µM groups, explosive increase of sEPSCs frequency was seen 2 min after reperfusion. \u003cstrong\u003eB\u003c/strong\u003e Time course of cumulative sEPSCs occurrence in the control group, the PostC group and the PostC+Ru265 groups. In each group, the markers and error bars represent their mean value and standard error of mean (SEM). The horizontal axis and the vertical axis represent time course and cumulative sEPSCs occurrence. The cumulative number of sEPSCs occurrence is expressed as a percentage of the total number of sEPSCs during the 5 minutes before anoxia under PostC with Ru265 perfusion at 1µM, 10µM, and 50µM concentrations. In the first 5 min after reperfusion, it shows the majority of sEPSCs occurrence in the control group and the PostC+Ru265 10µM and 50µM. We used the cumulative occurrence at 12.5 min after reperfusion for statistical analysis. \u003cstrong\u003eC\u003c/strong\u003eComparison of each mean and SEM in the control, the PostC and PostC+Ru265 1µM, 10µM and 50µM groups. Each vertical rectangle and error bar shows indicate cumulative sEPSCs occurrence at 20 min after onset of anoxia (12.5 min after reperfusion) and SEM, respectively. Asterisks indicate significant difference in Tukey multiple comparisons test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). sEPSCs;spontaneous excitatory post-synaptic currents, PostC; ischemic postconditioning, Ru265; Ruthenium red 265\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/a241ffaf97d39996620d7c05.png"},{"id":42152234,"identity":"cac1c930-84e1-43c2-ae33-a5a46176fe0e","added_by":"auto","created_at":"2023-08-25 17:34:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106658,"visible":true,"origin":"","legend":"\u003cp\u003eRecording of N-methyl-D-aspartate (NMDA)-induced currents. \u003cstrong\u003eA\u003c/strong\u003e Representative traces of NMDA receptor (NMDAR) currents in the pre- and post-anoxic period in the control, the PostC and the PostC+Ru265 groups recorded from voltage clamped hippocampal CA1 pyramidal neurons. In the PostC group, decrease of NMDAR currents was seen after 5 min of reperfusion. However, in the PostC+Ru265 group, no change or rather increase of NMDAR currents was seen after 5 min of reperfusion as well as it was in the control group. \u003cstrong\u003eB\u003c/strong\u003eBar graph shows change of mean peak amplitude in NMDAR currents from 10 min to 20 min after reperfusion in the control group, the PostC group and the PostC+Ru265 group. Values are calculated as change of the percentage relative to mean peak amplitude during 5 min normoxia before the anoxic period. Asterisks indicate significant difference in Steel Dwass multiple comparisons test (**\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01). NMDA; N-methyl-D-aspartate; PostC; ischemic postconditioning, Ru265; Ruthenium red 265\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/770398f733393841efcc9837.png"},{"id":42152235,"identity":"d6320be0-3e8a-4a08-ab5e-3d6835c269fe","added_by":"auto","created_at":"2023-08-25 17:34:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":465509,"visible":true,"origin":"","legend":"\u003cp\u003eDead cell count in the CA1 region. \u003cstrong\u003eA, B, C\u003c/strong\u003eRepresentative microscopic view in the CA1 region and the nuclei of dead cells in each group. Dead cells due to preparation procedure of hippocampal slices are observed as magenta cells stained with both propidium iodide and SYTOX-blue. Blue cells stained only with SYTOX-blue are considered as dead cells due to ischemic reperfusion injury. The white scale bar in each view equals 50 µm. \u003cstrong\u003eD\u003c/strong\u003e Comparison of the number of dead neurons due to ischemic reperfusion injury in each group. Bar graph shows the number of dead neurons per 1 mm of the CA1 region in each group. The number of dead neurons is significantly higher in the PostC+Ru265 group compared to that in the PostC group. Asterisks indicate significant difference in Steel Dwass multiple comparisons test (**\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01).PostC; ischemic postconditioning, Ru265; Ruthenium red 265\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/5b0b045de8af9306a52d78f9.png"},{"id":42150904,"identity":"1a847413-dff4-40a1-b80a-fa56a76c2854","added_by":"auto","created_at":"2023-08-25 17:26:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":146488,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the Fura2 ratio and cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration. \u003cstrong\u003eA\u003c/strong\u003e Time course of the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration change in the Fura2. Change in the Fura2 ratio (340/380 ratio) was calculated relative to the mean value observed during the 5-min pre-anoxic period. The elevation of this ratio represents an increase of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration. After reperfusion, the increase of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration was suppressed in the PostC group, but Ru265 inhibited this effect. The pink band indicates the period from 0 min to 5 min after the anoxic period. Each band was used for statistical analysis. \u003cstrong\u003eB\u003c/strong\u003e Statistical analysis in the portion of the pink band. Each vertical rectangle and error bar indicate the percentage of change in the Fura2 ratio during 0–5 min after 7.5 min of anoxia and SEM, respectively. Asterisks indicate significant difference in Steel Dwass multiple comparisons test (**\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01). PostC; ischemic postconditioning, Ru265; Ruthenium red 265\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/59acedc7484cb54458d24742.png"},{"id":42152237,"identity":"181ee379-eb03-4bfc-81c4-fddf7be068a6","added_by":"auto","created_at":"2023-08-25 17:34:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":143570,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of mitochondria membrane potential in each group. \u003cstrong\u003eA\u003c/strong\u003eTime course of change in mitochondrial membrane potential estimated with JC1 fluorescence. Change in the mitochondrial membrane potential was estimated with JC1 fluorescence before the anoxic period, during the anoxic period and the reperfusion period. The increase of the JC1 green/red ratio indicates the depolarization of mitochondrial membrane potential.Percentages were calculated relative to the mean value observed during the 5 min pre-anoxic period. After the anoxicperiod at 7.5 min, the ratio was immediately and sharply increased in in the PostC and the PostC+Ru265 groups. In subsequence, it started to decrease in the PostC+Ru265 group as opposed to it in the PostC group. The pink band indicates the period from 0 min to 5 min after the anoxic period, which was used for statistical analysis. \u003cstrong\u003eB\u003c/strong\u003e Statistical analysis in the portion of the pink band. Each vertical rectangle and error bar indicate change in the percentage of JC1 fluorescence (green/red ratio) during 0–5 min after 7.5 min of anoxia and SEM. Asterisks indicate significant difference in Steel Dwass multiple comparisons test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01). PostC; ischemic postconditioning, Ru265; Ruthenium red 265\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/dae60a748e1aa149364bf3f4.png"},{"id":54304400,"identity":"08f246e8-f489-421f-adc2-76f52653614e","added_by":"auto","created_at":"2024-04-08 15:15:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1400749,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/e0a4348e-b63f-4118-9bf4-d12a27d699af.pdf"},{"id":42150906,"identity":"f1e895a3-bc2a-4b64-a041-feb26e2b732c","added_by":"auto","created_at":"2023-08-25 17:26:31","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16635288,"visible":true,"origin":"","legend":"","description":"","filename":"GA.tif","url":"https://assets-eu.researchsquare.com/files/rs-3279580/v1/b343dd0a7f4ab551b8157caa.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial Calcium Uniporter (MCU) Involves in Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mouse","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCerebral 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 \u0026ldquo;ischemic preconditioning\u0026rdquo; 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 (Kitagawa et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Nakagawa et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, in the clinical situation, it is very 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 (Pignataro et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Xing et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis 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-K\u003csub\u003eATP\u003c/sub\u003e) channels and suppresses synaptic glutamate over-release in I/R injury (Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yokoyama et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 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 Ca\u003csup\u003e2+\u003c/sup\u003e into cytoplasm via opening of NMDAR, it leads to cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e overload and activation of various proteins such as caspases and endonucleases via high conductance opening of mitochondrial permeability transition pore (mPTP) (Hawrysh \u0026amp; Buck, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In PostC, opening of the mito-K\u003csub\u003eATP\u003c/sub\u003e channel causes depolarization of mitochondrial membrane potential and induces reduction of NMDAR currents through low conductance opening of mPTP, and in consequence that suppresses Ca\u003csup\u003e2+\u003c/sup\u003e influx into the cytoplasm, leading to neuroprotection against I/R injury(Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yokoyama et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRevealing 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 Ca\u003csup\u003e2+\u003c/sup\u003e concentration is essential for I/R injury and PostC. Mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e 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 Ca\u003csup\u003e2+\u003c/sup\u003e inwardly placed on the inner mitochondrial membrane and regulates mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration (Clapham, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; de Stefani et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Recently a lot 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) (Baughman et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; de Stefani et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mallilankaraman et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Perocchi et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Plovanich et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Raffaello et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sancak et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Each subunit regulates one another to promote or suppress Ca\u003csup\u003e2+\u003c/sup\u003e transport into mitochondria (Lambert et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Patron et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, numerous pathological conditions of the cell activity such as ischemic stroke, neurodegenerative disease and cancer are caused by the dysregulation of Ca\u003csup\u003e2+\u003c/sup\u003e uptake by MCU (Pchitskaya et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Polster et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Vultur et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Woods \u0026amp; Wilson, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which has been attracting attention as a new target of various treatments in recent years.(Giorgi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Liao et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Modesti et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTo 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 Ca\u003csup\u003e2+\u003c/sup\u003e 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 Ca\u003csup\u003e2+\u003c/sup\u003e 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.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProcedure of mouse hippocampal slices\u003c/h2\u003e \u003cp\u003e All our experiments were approved by the Animal Care and Use Committee of our University (approval no.13411) and performed all procedures in accordance with the Guidelines for the Proper Conduct of Animal Experiments. 4- to 8-week-old wild type C57BL/6J mice (58 males) weighing about 18\u0026ndash;24 grams were used for our experiments. Mice lived in their cage with light and dark cycle in a half of day and were able to access their food and water freely for any time a day. Mice were decapitated after we checked disappearance of their postural reflex and their shallow and fast breath changing to deep and slow one under general anesthesia with isoflurane (0.05 \u003cem\u003ev/v\u003c/em\u003e, administered by inhalation). Then the brains were removed quickly into ice-cold solution composed with sucrose 230mM, KCl 2.5 mM, NaHCO\u003csub\u003e3\u003c/sub\u003e 25 mM, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.25 mM, CaCl\u003csub\u003e2\u003c/sub\u003e 0.5 mM, MgSO\u003csub\u003e4\u003c/sub\u003e 10 mM, D-glucose 10 mM and bubbled with 95% O\u003csub\u003e2\u003c/sub\u003e / 5% CO\u003csub\u003e2\u003c/sub\u003e. In this solution the brains were cut into horizontal slices of the hippocampal at a thickness of 350 \u0026micro;m, using a linear slicer (PRO7; DOSAKA EM, Kyoto, Japan). Then slices were incubated in standard artificial cerebrospinal fluid (ACSF) composed with NaCl 125 mM, KCl 2.5 mM, NaHCO\u003csub\u003e3\u003c/sub\u003e 25 mM, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.25 mM, CaCl\u003csub\u003e2\u003c/sub\u003e 2.0 mM, MgCl\u003csub\u003e2\u003c/sub\u003e 1.0 mM, D-glucose 10 mM and bubbled with the same gas mixture and kept for at least 1 hour at 32℃. After this procedure slices were kept in ACSF at 27℃. We used about 5 to 7 slices per one mouse.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePerfusion protocols and Patch-clamp recording\u003c/h3\u003e\n\u003cp\u003ePatch pipettes were made from thick-walled borosilicate glass capillaries and filled with the internal solution which was consisted of Cs-gluconate (141 mM), CsCl (4.0 mM), MgCl\u003csub\u003e2\u003c/sub\u003e (2.0 mM), HEPES (10.0 mM), Mg-ATP (2.0 mM), Na-GTP (0.3 mM), EGTA (0.2 mM) and adjusted to pH 7.25 with CsOH. Each slice was placed on an 800\u0026micro;L recording chamber mounted on a BX50WI upright microscope (Olympus, Tokyo, Japan) which was equipped with an infrared differential interference microscope and epifluorescence imaging apparatuses. And in this chamber gas-saturated ACSF was perfused at a 2.0mL/min flow and kept at 31\u0026ndash;33℃ by a controlled heater. Hippocampal slices were put into this chamber and randomly assigned to the following three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA): 1) control group: slices were reperfused with ACSF for 20 minutes after the anoxic period, 2) PostC group: slices were exposed after the anoxic period to intermittent the normoxic and anoxic periods based on the PostC protocol as mentioned below, 3) PostC administered with Ruthenium Red 265 (Ru265) (PostC\u0026thinsp;+\u0026thinsp;Ru265) group: slices in the PostC protocol were supplemented with 1\u0026micro;M, 10\u0026micro;M and 50 \u0026micro;M of Ru265 respectively which was the novel selective inhibitor of the mitochondrial calcium uniporter (MCU). In the control group, the baseline time schedule includes 5 minutes of normoxia, 7.5 minutes of anoxic and 20 minutes of reperfusion periods. 7.5 minutes of the severe anoxic load was simulated as exposing slices to ACSF in which glucose and oxygen were replaced to sucrose and nitrogen. In accordance with previous studies, the protocol of PostC was started from 30 seconds after the anoxic period and consisted of three times of 15 seconds of anoxia separated by 15 seconds of normoxia (Yokoyama et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the PostC\u0026thinsp;+\u0026thinsp;Ru265 group, ACSF containing of Ru265 was perfused from the start of the time schedule (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Then we confirmed visually CA1 pyramidal cells and made whole-cell voltage-clamp recordings using an EPC-9 patch clamp amplifier (Heka, Lambrecht/Pfalz, Germany). The holding potential of voltage clamp was set at -70 mV and the resistance of the pipette was kept from 2.5 to 3.5 MΩ. And to detect the glutamatergic excitatory post-synaptic currents clearly, the GABAA and GABAB antagonist picrotoxin (50 \u0026micro;M) was supplemented into ACSF during the recordings. We counted the number of sEPSCs in each group and calculated it as a percentage of the number of the sEPSCs occurrence during the anoxic and the reperfusion periods to that in the first normoxic period for 5 minutes before the anoxic period. To determine the optimal concentration of Ru265 in this experiment, we tried with three different types of the Ru265 concentration at 1 \u0026micro;M, 10 \u0026micro;M, and 50 \u0026micro;M. Ru265 and picrotoxin were purchased from Sigma-Aldrich.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRecording of whole-cell currents in response to NMDA application\u003c/h2\u003e \u003cp\u003eTo assess the NMDAR currents in each group, we recorded whole-cell currents to NMDA application. With another micropipette similar of one for whole-cell recordings, 5\u0026micro;M of NMDA was puffed to the recording cell. And the holding potential of voltage-clamp was set at -55mV during the pre- (1 second) and post-stimulation period (6 seconds) for the purpose of suppressing the Mg\u003csup\u003e2+\u003c/sup\u003e block to NMDAR channels. All NMDAR currents were recorded every 30 seconds during the experiments for about 30\u0026ndash;35 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell staining\u003c/h2\u003e \u003cp\u003eTo investigate the effects of Ru265 in PostC, we counted the number of dead cells in hippocampal slices after ischemic injury. Dead cells were dyed by propidium iodide (PI) and SYTOX-blue, which are impermeable to cell membrane and used for nuclear staining. At least 45 min before the perfusion protocol started, slices were incubated for 15 min in ACSF containing 3 \u0026micro;M PI. Thus, PI staining was performed after the slice preparation until the perfusion protocol started, that is, dead cells, which membranes were broken during the process of making slices were stained red. Subsequently, PI was removed before the perfusion protocol started, not to dye dead cells due to I/R injury during the perfusion protocol. According to perfusion protocol, slices in the control group were loaded with the anoxic period for 7.5 min and with reperfusion period for 20 min, and slices in the PostC group and the PostC\u0026thinsp;+\u0026thinsp;Ru265 group were loaded with the intermittent anoxic periods after 7.5 min anoxia. After these perfusion protocols, slices were transferred to the incubation chamber and stained with 6 \u0026micro;M SYTOX-blue contained in ACSF for 3 h at 32℃. In this section, by staining cells with SYTOX-blue, both dead cells already stained red with PI and new dead cells after the perfusion protocol were dyed blue. Therefore, dead cells in the slice preparation procedure were dyed both red and blue, while the ones died due to I/R injury were dyed only blue. SYTOX-blue was excited at 408 nm and the blue fluorescence emission was bandpass filtered from 417 to 477 nm by using confocal microscopy (C2plus; Nikon, Japan). To detect the initial number of dead cells before ischemic stress, we also checked dead cells stained with PI, which were excited at 561 nm and observed as the bandpass of red fluorescence emission filtered from 552 to 617 nm. Thus, we compared the number of dead cells in each group showing only blue fluorescence with SYTOX-blue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFluorometric assessment of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e changes\u003c/h2\u003e \u003cp\u003eTo evaluate changes of the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration, the Fura-2 (DOJINDO, Kumamoto, Japan) fluorescence signals of whole-cell voltage-clamped pyramidal cells were measured by adding 15 \u0026micro;mol/L of Fura-2 to the internal pipette solution. Using a fast-switching multi-wavelength illumination system (Lambda DG-4; Sutter Instruments, CA, USA), the Fura-2 fluorescence signals were excited at 340 nm and 380 nm every 10 seconds and their emission was filtered at 510 nm with the 500nm- dichroic mirror. For acquiring these images, a \u0026times;40 water immersion objective lens (LUMPlanFI/IR, Olympus, Japan) and a CCD camera (ORCA-flash 4.0 v3; Hamamatsu Photonics, Shizuoka, Japan) were used. All images were saved in MetaMorph software (Molecular Devices, CA) and for analysis we measured the region of interest (ROI) defined as a circular area of 5\u0026micro;m in diameter with the maximum fluorescence intensity near the center of the recording cell. The average of fluorescence intensity ratio of 340 nm excitation and 380 nm excitation in the ROI was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFluorometric assessment of mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eTo evaluate changes of the mitochondrial membrane potential during the anoxic and reperfusion periods, we used a JC1 (Cayman Chemical, MI) fluorescence of which the emission wavelength changes depending on the mitochondrial membrane potential and loaded it into the cytoplasm via a patch pipette. The tip of the patch pipette was filled with an internal solution and from the back of the pipette an internal solution containing with 2.0\u0026micro;M of JC1 was put in immediately before use. The red fluorescence of JC1 represents the J-aggregated state excited at 548 nm with a 580-nm dichroic mirror and fluoresced at 590 nm with a long-pass filter. And the green fluorescence of JC1 represents monomeric state excited at 477 nm with a 500-nm dichroic mirror and fluoresced with a bandpass filter at 515\u0026ndash;565 nm. Fluorescence images were acquired at every 30 seconds using the same apparatus as Fura2. For the measurement of the fluorescence the ROI was defined as a hand-drawn polygonal area covering the region of high red fluorescence because the red fluorescence was eccentrically distributed around the nucleus and was often crescent-shaped and each average of the green and red fluorescence in the ROI was calculated as the green/red ratio for analysis. The increase of this green/red ratio indicates the mitochondrial depolarization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were calculated as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. For multiple testing, we used one-way analysis of variance and significant effects were further tested with a post-hoc multiple comparison test of Tukey-Kramer method and Steel-Dwass method as appropriate. Significant difference was set at the level of *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChanges of the sEPSCs surge\u003c/h2\u003e \u003cp\u003eThe frequency of sEPSCs gradually increased during the anoxic period in all groups. After reperfusion at 7.5 min of anoxic load, in the control group and the PostC\u0026thinsp;+\u0026thinsp;Ru265 groups, sEPSCs occurrence was sharply increased, however in the PostC group, increase of sEPSCs was not observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, to examine the optimal concentration of Ru265 in this experimental setting, we set three different concentrations of Ru265 administered to ACSF: low (1 \u0026micro;M; n\u0026thinsp;=\u0026thinsp;5); medium (10 \u0026micro;M; n\u0026thinsp;=\u0026thinsp;5); and high (50 \u0026micro;M; n\u0026thinsp;=\u0026thinsp;5). The effect of Ru265 to the suppression of sEPSCs by PostC was observed at 10 \u0026micro;M and 50\u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We determined Ru265 at 10\u0026micro;M for the optimal concentration in this experiment. The percentage of cumulative sEPSCs occurrence at 20 min in the PostC\u0026thinsp;+\u0026thinsp;Ru265 10\u0026micro;M group (n\u0026thinsp;=\u0026thinsp;5) was significantly higher than that of the PostC group (n\u0026thinsp;=\u0026thinsp;5) (38.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.39 \u0026times;10\u003csup\u003e2\u003c/sup\u003e% vs 17.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e%, p\u0026thinsp;=\u0026thinsp;0.008) and the PostC\u0026thinsp;+\u0026thinsp;Ru265 1\u0026micro;M group (38.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.39 \u0026times;10\u003csup\u003e2\u003c/sup\u003e% vs 15.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e%, p\u0026thinsp;=\u0026thinsp;0.003), similar to that of PostC\u0026thinsp;+\u0026thinsp;Ru265 50\u0026micro;M group was (43.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.46 \u0026times;10\u003csup\u003e2\u003c/sup\u003e% vs 17.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e%, p\u0026thinsp;=\u0026thinsp;0.001 and 43.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.46 \u0026times;10\u003csup\u003e2\u003c/sup\u003e% vs 15.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). On the contrary, the percentage of cumulative sEPSCs occurrence at 20 min in the PostC\u0026thinsp;+\u0026thinsp;Ru265 1\u0026micro;M group (n\u0026thinsp;=\u0026thinsp;5) was not significantly difference from that of the PostC group (15.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e% vs 17.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e%, p\u0026thinsp;=\u0026thinsp;0.996) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of MCU by Ru265 canceling the suppression of NMDAR currents by PostC\u003c/h2\u003e \u003cp\u003eWe recorded NMDAR currents per 30 seconds. When NMDA was locally applied to CA1 pyramidal cells, NMDAR currents with slow decay for several seconds were observed in all three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). After reperfusion, NMDAR currents decreased in the PostC group (n\u0026thinsp;=\u0026thinsp;12) while those increased in the PostC\u0026thinsp;+\u0026thinsp;Ru265 group (n\u0026thinsp;=\u0026thinsp;11) and the control group (n\u0026thinsp;=\u0026thinsp;9). We analyzed the change in NMDAR currents between peak and minimum amplitude from 10 min to 20 min after reperfusion in each group. In the PostC\u0026thinsp;+\u0026thinsp;Ru265 group, the percentage of NMDAR currents amplitude after reperfusion was significantly larger than that in the PostC group (135.64\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17% vs 92.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Therefore, these results indicate that inhibition of MCU by Ru265 canceled the effect of suppressing NMDAR currents by PostC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of MCU by Ru265 in PostC increasing the number of dead CA1 neurons\u003c/h2\u003e \u003cp\u003e According to these results, it is thought that inhibition of MCU reduces the neuroprotection in PostC and dead cells may increase after reperfusion by inhibiting MCU in PostC. Thus, we counted the number of dead CA1 neurons \u003cem\u003ein vivo\u003c/em\u003e of all groups after reperfusion. To distinguish dead cells caused by I/R injury from all dead cells, we used two kinds of membrane impermeable dyes for nuclear staining with different fluorescent wavelengths. We counted the number of CA1 neurons died during 20 min to 3 h of reperfusion period after 7.5 min anoxic insult (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). There were significantly difference between the PostC group (87.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.75/mm, n\u0026thinsp;=\u0026thinsp;20) and the PostC\u0026thinsp;+\u0026thinsp;Ru265 group (194.56\u0026thinsp;\u0026plusmn;\u0026thinsp;21.05/mm, n\u0026thinsp;=\u0026thinsp;9, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Thus, CA1 dead neurons were significantly increased in the PostC\u0026thinsp;+\u0026thinsp;Ru265 group, similar to that in the control group (n\u0026thinsp;=\u0026thinsp;11). This result indicated that in PostC, inhibition of MCU weakened its neuroprotective effect against I/R injury and leads to cell death because MCU mediated the mechanism of PostC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRu265 inhibits the suppression of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e increase by PostC\u003c/h2\u003e \u003cp\u003eWe examined the involvement of MCU of the change in cytosolic [Ca\u003csup\u003e2+\u003c/sup\u003e] concentration by PostC. In all groups, the Fura2 ratio gradually increased during the anoxic period, indicating cytosolic [Ca\u003csup\u003e2+\u003c/sup\u003e] increased. After reperfusion, the ratio started to decrease gradually in the PostC group while in the PostC\u0026thinsp;+\u0026thinsp;Ru265 group, it started to increase immediately until 2.5 min after reperfusion, indicating cytosolic [Ca\u003csup\u003e2+\u003c/sup\u003e] increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We used the change in the Fura2 ratio from 0min until 5min after the anoxic period for analysis. In the PostC\u0026thinsp;+\u0026thinsp;Ru265 group (n\u0026thinsp;=\u0026thinsp;8), the percentage of change in the Fura2 ratio was significantly higher than that in PostC group (n\u0026thinsp;=\u0026thinsp;6), similar to that in the control group (n\u0026thinsp;=\u0026thinsp;7) from 0min to 5min after the anoxic period (PostC vs PostC\u0026thinsp;+\u0026thinsp;Ru265; 5.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49% vs 12.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, PostC vs Control; 5.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49% vs 12.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This result suggests that the suppression of cytosolic [Ca\u003csup\u003e2+\u003c/sup\u003e] increase in the early stage of reperfusion by PostC is canceled due to inhibition of MCU activity by Ru265.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRu265 inhibits the interaction between the mitochondrial depolarization by PostC and MCU\u003c/h2\u003e \u003cp\u003eTo examine whether MCU mediates depolarization of mitochondrial membrane potential induced by PostC or not, we estimated the mitochondrial membrane potential with JC1 fluorescence. The elevation of the green/red ratio that represents depolarization of mitochondrial membrane potential increased immediately after the anoxic period. Especially in the PostC and the PostC\u0026thinsp;+\u0026thinsp;Ru265 groups, the ratio sharply increased. Subsequently, this ratio was decreased in the PostC\u0026thinsp;+\u0026thinsp;Ru265 group, as well as it in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Change in the green/red ratio from 0min until 5min after the anoxic period were used for analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The green/red ratio in the early phase from 0 min to 5min after the anoxic period was significantly different between the PostC group (n\u0026thinsp;=\u0026thinsp;7) and the PostC\u0026thinsp;+\u0026thinsp;Ru265 group (n\u0026thinsp;=\u0026thinsp;6), similar to the control group (n\u0026thinsp;=\u0026thinsp;9) (PostC vs PostC\u0026thinsp;+\u0026thinsp;Ru265; 16.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55% vs 12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21%, p\u0026thinsp;=\u0026thinsp;0.03, PostC vs Control; 16.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55% vs 8.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, PostC\u0026thinsp;+\u0026thinsp;Ru265 vs Control; 12.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22% vs 8.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69%, p\u0026thinsp;=\u0026thinsp;0.13). These results suggest that in the PostC\u0026thinsp;+\u0026thinsp;Ru265 group, the mitochondrial depolarization by PostC is weakened.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates the role of MCU in the Post-Conditioning (PostC) mechanism against I/R injury, using the whole-cell patch clamp technique on mouse hippocampal CA1 pyramidal cells. Blocking MCU with Ru265 weakened the effect of PostC, increasing EPSC occurrence and NMDAR currents after reperfusion. As a result, the number of dead cells in the CA1 region increased because the effect of reducing Ca2\u0026thinsp;+\u0026thinsp;influx into the cell cytosol via NMDAR after reperfusion in PostC was weakened by blocking MCU. Moreover, in PostC with an inhibitor of MCU, the depolarization of the mitochondrial membrane potential was sharply elevated in the early phase after reperfusion, however, it was weakened immediately. These results suggest that interaction between the mitochondrial depolarization and the MCU activation is involved in the PostC mechanism, leading to neuroprotection through the reduction of NMDAR currents and Ca2\u0026thinsp;+\u0026thinsp;influx into the cytosol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRegulation of MCU affects the PostC pathway and reduces its neuroprotective effect in PostC after reperfusion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our previous research, we have reported that PostC induces an interaction between mitochondria and NMDA receptor, and that the increase of sEPSCs occurrence was observed as a result of this interaction process indirectly(Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yokoyama et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, in this study, we initially checked the sEPSCs occurrence in each group to confirm whether adjustments to this interaction process of PostC could be made by adjusting MCU with MCU blocker. The sEPSCs occurrence in the PostC\u0026thinsp;+\u0026thinsp;Ru265 groups was sharply increased, compared to the PostC group, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This result suggests that MCU plays a role as one of the key factors in the PostC pathway. During I/R injury, an excessive release of glutamate occurs, leading to over-activation of NMDAR (Bonova et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; D\u0026aacute;valos et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Soria et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This cascade leads to cell necrosis or apoptosis by excessing Ca\u003csup\u003e2+\u003c/sup\u003e influx into the neurons, subsequently triggering a range of downstream pro-death signaling events such as calpain activation, reactive oxygen species (ROS) generation and mitochondrial damage (Curcio et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kristi\u0026aacute;n \u0026amp; Siesj\u0026ouml;, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Lau \u0026amp; Tymianski, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Zhang et al. suggested that NMDAR mediates the PostC-induced neuroprotection (Yin et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and Morisaki.Y et al. also reported that low conductance opening of mPTP by PostC induces extrusion of a few Ca\u003csup\u003e2+\u003c/sup\u003e into cell cytosol and subsequently some chemical mediators are predicted to inhibit NMDAR over-activation against excessing Ca\u003csup\u003e2+\u003c/sup\u003e influx into the neurons (Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, in their experiments, the increase of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration after reperfusion was not observed in either the control or the PostC group when the extracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration was set to 0. From this result, they concluded that in PostC the suppression of NMDAR over-activation during reperfusion inhibited Ca\u003csup\u003e2+\u003c/sup\u003e influx into the cytoplasm from the extracellular space, resulting in a reduction of Ca\u003csup\u003e2+\u003c/sup\u003e overload in the cytoplasm and cellular damage (Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, this death-signaling by NMDAR over-activation is considered as the most important key to the neuroprotective mechanism in PostC. Our present study showed that the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration during the anoxic to reperfusion period sharply increased due to inhibition of MCU with Ru265, similar to that observed in the control group, while these changes was not observed in PostC after reperfusion in the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Additionally, the reduction of NMDAR currents was also weakened by inhibiting MCU in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Thus, the results of our research also suggest that the inhibition of the MCU-mediated Ca\u003csup\u003e2+\u003c/sup\u003e influx into the mitochondria induces disruptions in the PostC process, resulting in the loss of PostC-induced neuroprotection against the extracellular Ca\u003csup\u003e2+\u003c/sup\u003e influx through NMDAR over-activation.\u003c/p\u003e \u003cp\u003eUnder physiological conditions, cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration is kept in range of 100nM by intracellular Ca\u003csup\u003e2+\u003c/sup\u003e stores, plasma membrane and organelle membrane Ca\u003csup\u003e2+\u003c/sup\u003e channels and pumps, and Ca\u003csup\u003e2+\u003c/sup\u003e binding proteins (Babcock et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Berridge, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Carvalho et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Enomoto M et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The endoplasmic reticulum (ER) is known to be the largest intracellular Ca\u003csup\u003e2+\u003c/sup\u003e store, but mitochondria, lysosomes and the nucleus are also involved in regulating intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration (Kaufman \u0026amp; Malhotra, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; S. Smaili et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Samanta \u0026amp; Parekh, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e influx into the mitochondrial matrix via MCU, in particular, leads to the elevation of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration and influences various mitochondrial metabolic processes, including mitochondrial respiration, adenosine triphosphate (ATP) production, mitophagy / autophagy and even the death pathway of apoptosis or necrosis (Duchen, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; East \u0026amp; Campanella, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gottlieb \u0026amp; Bernstein, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, especially in the ischemic conditions, the anaerobic glycolysis in cells starts to work as the primary metabolic pathway, which leads to an increase in lactic acids and a decrease in cytosolic pH. As cytosolic pH decreases, the mitochondrial membrane potential is also diminished due to the cessation of oxygen-dependent oxidative phosphorylation. Subsequently, after reperfusion, the rapid restoration of the mitochondrial membrane potential due to the return of oxygen into cytosol provides a strong driving force for the entry of the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e into the mitochondria via MCU (Shintani-Ishida et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This results in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload, leading to cell damage or death associated with opening of mPTP. This process generates toxic products such as cytochrome C and swelling the matrix, eventually leading to the OMM rupture (Bonora et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hawrysh \u0026amp; Buck, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Morciano et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shintani-Ishida et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus, some research has reported that the inhibition of MCU reduces the cell damage caused by mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload and may confer neuroprotection (Novorolsky et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Woods \u0026amp; Wilson, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, our results in the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e may initially appear to contradict these reports because they showed that an increase in dead cells in CA1 region by inhibiting MCU in PostC. Nonetheless, inhibition of MCU cannot prevent other toxicity caused by cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e overload (Curcio et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kristi\u0026aacute;n \u0026amp; Siesj\u0026ouml;, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Lau \u0026amp; Tymianski, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thus, it might be the reason why dead cells in CA1 region increased by inhibiting MCU in PostC that other death signals due to cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e overload, not through mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload via MCU, were activated and led to cell death finally.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMCU plays a role as one of the key factors in the interacting process of mitochondrial depolarization in PostC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our previous research, Morisaki.Y et al. reported that the opening of mito-K\u003csub\u003eATP\u003c/sub\u003e channel triggered the PostC mechanism and caused the mitochondrial depolarization which was observed during the early period of reperfusion in the PostC group but, not in the control group (Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, we also have reported that one of the key factors in neuroprotection of PostC was the low conductance opening of mPTP. The catastrophic process of cell injury is induced through the high conductance mode of mPTP opening, which allows for the passage of ions, including calcium, leading to the dissipation of mitochondrial membrane potential, and eventually resulting in the cell death (Brenner \u0026amp; Moulin, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Whereas under physiological conditions it is reported that the mPTP could exhibit intermittent opening in low conductance mode, contributing to intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis and the regulation of mitochondrial function (Giorgio et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Morisaki.Y et al. also reported that the low conductance mode of mPTP opening prevented the high conductance mode opening and reduced NMDAR conductance (Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, the mitochondrial membrane potential switches these two modes with the threshold value controlled by mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration (Bazil et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The opening of mito-K\u003csub\u003eATP\u003c/sub\u003e channel is triggered by the local decrease of ATP in cytosol due to ischemic stress and subsequently, leading to an increase in K\u003csup\u003e+\u003c/sup\u003e influx into the mitochondrial matrix (Hawrysh \u0026amp; Buck, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pamenter et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In PostC opening of this channel leads to mitochondrial depolarization, reducing the driving force of the Ca\u003csup\u003e2+\u003c/sup\u003e influx into mitochondria matrix and would abrogate the excessive Ca\u003csup\u003e2+\u003c/sup\u003e accumulation in the matrix, thus avoiding the high conductance mode of mPTP opening (Hawrysh \u0026amp; Buck, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Morisaki et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the mitochondrial depolarization is also one of the key factors in neuroprotection of PostC. In the present study, our data shows that the depolarization of mitochondrial membrane potential was weakened after reperfusion in the PostC with Ru265 group, compared to it in the PostC group. These data suggest that in the PostC mechanism, the mitochondrial depolarization is triggered by opening of mito-K\u003csub\u003eATP\u003c/sub\u003e channel, however, some interacting process between mito-K\u003csub\u003eATP\u003c/sub\u003e channel and MCU was blocked by Ru265, resulting in the loss of the neuroprotective effect through the mitochondrial membrane potential in PostC. Furthermore, it also suggests that the mild interactive regulation of MCU, provided by the driving force given from mito-K\u003csub\u003eATP\u003c/sub\u003e channel would be necessary for maintaining of the mitochondrial depolarization in PostC.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePharmacological Approach to MCU\u003c/h2\u003e \u003cp\u003eIn the present study, we used Ruthenium Red 265 to inhibit MCU, a novel selective inhibitor against MCU. The most well-known and commonly used MCU inhibitor is Ru360 named for its strong absorbance at 360nm (Emerson et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). However, Ru360 is poorly permeable and unstable in aqueous solution, losing its activity in days (Hajn\u0026oacute;czky et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; M\u0026aacute;rta et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, Joshua J. et al. reported that in the lysate of HEK293 or Hela cells, Ru265 was taken up into these cells from two to ten times more than other structural analogs of ruthenium after these cells were incubated with each ruthenium complex at 50\u0026micro;M for 24h, and moreover, the rise of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration was observed soon after only 30minutes of incubation (Novorolsky et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Woods et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For our protocol of PostC in the previous research, we observed each single cell action during a very short time course, including normoxia and anoxia within about 30min to 1 hour. Therefore, it was necessary to use the quick permeable drug for this protocol, and from this point of view, we regarded Ru265 as suitable for our research because of its quick permeability (Novorolsky et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, ruthenium compounds are also known for its cytotoxicity (Alessio, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dutta et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; F. A. Peacock et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hartinger et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lameijer et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; M\u0026uuml;hlgassner et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; S\u0026uuml;ss-Fink, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wachter et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Wee \u0026amp; Dyson, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), while Ru265 is a low toxic compound without any effect on mitochondrial membrane potential and other intracellular Ca\u003csup\u003e2+\u003c/sup\u003e dynamics reportedly (Woods et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e\u0026lsquo;Safe pharmacological PostC\u0026rsquo; is our central concern for clinical application, however, Diazoxide as the opener of mito-K\u003csub\u003eATP\u003c/sub\u003e channel, is not suitable because of its toxic side effects (Kumar et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). In our previous study, Furuta.T et al. reported on the neuroprotective mechanism in melatonin-induced pharmacological PostC (Furuta et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and melatonin could be the new candidate of alternative drugs for pharmacological PostC without any side effects. Thus, for our future direction, further studies \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e are needed to explore ways to deliver melatonin to the brain, such as using catheter to the MCAO models.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMCU plays an important role related to the depolarization of the mitochondrial membrane potential in PostC, leading to reduce dead cells in CA1 region through downregulation of NMDAR currents occurrence and reduction of Ca\u003csup\u003e2+\u003c/sup\u003e influx into cytosol.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAIS\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Acute ischemic stroke\u003c/p\u003e\n\u003cp\u003eI/R\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Ischemic reperfusion\u003c/p\u003e\n\u003cp\u003ePostC\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Ischemic postconditioning\u003c/p\u003e\n\u003cp\u003eATP\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Adenosine triphosphate\u003c/p\u003e\n\u003cp\u003eMito-K\u003csub\u003eATP\u003c/sub\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mitochondrial ATP-depemdent potassium\u003c/p\u003e\n\u003cp\u003eNMDA\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; N-methyl-D-aspartate\u003c/p\u003e\n\u003cp\u003eNMDAR\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; NMDA receptor\u003c/p\u003e\n\u003cp\u003emPTP\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mitochondrial permeability transition pore\u003c/p\u003e\n\u003cp\u003eMCU\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Mitochondrial calcium uniporter\u003c/p\u003e\n\u003cp\u003eRu265 \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Ruthenium red 265\u003c/p\u003e\n\u003cp\u003esEPSCs \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Spontaneous excitatory post-synaptic currents\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization and design or analysis and interpretation of data, or both, T.F., I.N. and S.Y.; writing—original draft preparation or critical revision for important intellectual content, T.F., I.N., S.Y., Y.M., Y.S. and H.N.; final approval of the submitted manuscript, I.N., Y.S. and H.N. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI (grant number JP22K09240).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Animal Care and Use Committee of Nara Medical University (approval no. 13411) and were performed in accordance with the Guidelines for the Proper Conduct of Animal Experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets of the current study are available upon request with no restriction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Yoichi Ogawa for his technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlessio E (2017) Thirty Years of the Drug Candidate NAMI-A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective. 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Biochem Biophys Res Commun 461(3):537\u0026ndash;542. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2015.04.066\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2015.04.066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cemn","sideBox":"Learn more about [Cellular and Molecular Neurobiology](https://www.springer.com/journal/10571)","snPcode":"10571","submissionUrl":"https://submission.nature.com/new-submission/10571/3","title":"Cellular and Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ischemic postconditioning (PostC), mitochondrial calcium uniporter (MCU), NMDA receptor (NMDAR), mitochondrial permeability transition pore (mPTP)","lastPublishedDoi":"10.21203/rs.3.rs-3279580/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3279580/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemic postconditioning (PostC) phenomenon is known as the neuroprotection against ischemic reperfusion (I/R) injury. One of the key processes in PostC is opening of mitochondrial ATP dependent potassium (mito-K\u003csub\u003eATP\u003c/sub\u003e) channel and depolarization of mitochondrial membrane potential, which triggers the release of calcium ion from mitochondria through the low conductance opening of mitochondrial permeability transition pore (mPTP). Mitochondrial calcium uniporter (MCU) is known as the highly sensitive transporter for uptake of Ca\u003csup\u003e2+\u003c/sup\u003e inwardly existed on the inner mitochondrial membrane. Furthermore, it has attracted attention as a new target of treatments in disease such as neurodegenerative disease, cancer and ischemic stroke. Thus, we considered that MCU may involve in PostC and trigger its mechanism. In this research, we used the whole-cell patch clamp technique to hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration, mitochondrial membrane potential and N-methyl-D-aspartate receptor (NMDAR) currents under the inhibition of MCU by Ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased sEPSCs occurrence (p\u0026thinsp;=\u0026thinsp;0.008), NMDAR currents (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and dead cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) significantly after reperfusion, indicating the removal of the neuroprotective effects in PostC. Moreover, the mitochondrial depolarization in PostC with Ru265 was weakened, compared to it in PostC (p\u0026thinsp;=\u0026thinsp;0.03). These results suggest that MCU affects the mitochondrial depolarization in the PostC mechanism to suppress NMDAR over-activation and prevent the elevation of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration against I/R injury.\u003c/p\u003e","manuscriptTitle":"Mitochondrial Calcium Uniporter (MCU) Involves in Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mouse","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-25 17:26:26","doi":"10.21203/rs.3.rs-3279580/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-08-23T18:48:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-23T18:28:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-22T05:10:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Neurobiology","date":"2023-08-20T11:24:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cemn","sideBox":"Learn more about [Cellular and Molecular Neurobiology](https://www.springer.com/journal/10571)","snPcode":"10571","submissionUrl":"https://submission.nature.com/new-submission/10571/3","title":"Cellular and Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3676bb94-6ff7-40ed-9ff8-e5d465d413d7","owner":[],"postedDate":"August 25th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-08T15:12:19+00:00","versionOfRecord":{"articleIdentity":"rs-3279580","link":"https://doi.org/10.1007/s10571-024-01464-7","journal":{"identity":"cellular-and-molecular-neurobiology","isVorOnly":false,"title":"Cellular and Molecular Neurobiology"},"publishedOn":"2024-04-03 15:01:32","publishedOnDateReadable":"April 3rd, 2024"},"versionCreatedAt":"2023-08-25 17:26:26","video":"","vorDoi":"10.1007/s10571-024-01464-7","vorDoiUrl":"https://doi.org/10.1007/s10571-024-01464-7","workflowStages":[]},"version":"v1","identity":"rs-3279580","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3279580","identity":"rs-3279580","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}