This study demonstrates the role of MCU in the mechanisms underlying PostC protection 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. As a result, the number of dead cells in the CA1 region increased because inhibition of MCU weakened the effect of reducing Ca2+ influx into the cytosol via NMDAR by PostC. Moreover, in PostC with an inhibitor of MCU, depolarization of the mitochondrial membrane was elevated in the early phase after reperfusion, but weakened immediately. These results suggest that interactions between mitochondrial depolarization and MCU activation are involved in the PostC mechanism, leading to neuroprotection by reducing NMDAR currents and Ca2+ influx into the cytosol.

Regulation of MCU Affects the PostC Pathway and Reduces Neuroprotective Effects in PostC After Reperfusion

In our previous research, we reported that PostC induces interactions between mitochondria and NMDAR, and that the observed increase in sEPSCs was an indirect result of this interaction (Morisaki et al. 2022; Yokoyama et al. 2019). In this study, we initially checked the occurrence of sEPSCs in each group to confirm whether adjustments to this interaction process in PostC could be made by adjusting the MCU with MCU blockers. The occurrence of sEPSCs in the PostC + Ru265 group was sharply increased, compared to the PostC group, as shown in Fig. 2. This result suggests that MCU plays a key role 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 excess Ca2+ influx into the neuron, triggering a range of downstream pro-death signaling events such as calpain activation, generation of reactive oxygen species and damage to mitochondria (Curcio et al. 2016; Kristián and Siesjö 1998; Lau and Tymianski 2010). Yin et al. suggested that NMDAR mediates PostC-induced neuroprotection (Yin et al. 2005) and Morisaki et al. reported that low-conductance opening of mPTP by PostC induces extrusion of small amounts of Ca2+ into the cell cytosol, so some chemical mediators were predicted to inhibit NMDAR over-activation against excess Ca2+ influx into neurons (Morisaki et al. 2022). Moreover, in their experiments, increased intracellular Ca2+ concentrations after reperfusion were not observed in either control or PostC group when the extracellular Ca2+ concentration was set to 0. From this result, they concluded that in PostC, suppression of NMDAR over-activation during reperfusion inhibited Ca2+ influx into the cytoplasm from the extracellular space, reducing Ca2+ overload in the cytoplasm and cellular damage (Morisaki et al. 2022). Therefore, death signaling from NMDAR over-activation is considered the key to the neuroprotective mechanisms of PostC. The present study showed that cytosolic Ca2+ concentrations during the anoxic-to-reperfusion period increased sharply due to inhibition of the MCU by Ru265, similar to that observed in the control group, while no such changes were observed in PostC after reperfusion (Fig. 5). In addition, the reduction of NMDAR currents was also weakened by inhibiting the MCU (Fig. 3). Thus, our results also suggest that the inhibition of MCU-mediated Ca2+ influx into the mitochondria disrupts the PostC process, resulting in a loss of PostC-induced neuroprotection against extracellular Ca2+ influx through NMDAR over-activation.

Under physiological conditions, cytosolic Ca2+ concentrations are kept at around 100 nM by intracellular Ca2+ stores, Ca2+ channels and pumps in the plasma membrane and organelle membrane, and Ca2+-binding proteins (Babcock et al. 1997; Berridge 2016; Carvalho et al. 2020; Chen et al. 2020; Enomoto et al. 2017). The endoplasmic reticulum is known as the largest intracellular Ca2+ store, but mitochondria, lysosomes, and the nucleus are also involved in regulating intracellular Ca2+ concentrations (Kaufman and Malhotra 2014; Smaili et al. 2013; Samanta and Parekh 2017). However, the influx of cytosolic Ca2+ into the mitochondrial matrix via the MCU, in particular, leads to elevated mitochondrial Ca2+ concentrations and influences various mitochondrial metabolic processes, including mitochondrial respiration, ATP production, mitophagy/autophagy, and even the death pathways of apoptosis or necrosis (Duchen 1999; East and Campanella 2013; Gottlieb and Bernstein 2016). Furthermore, particularly under ischemic conditions, anaerobic glycolysis in cells starts to work as the primary metabolic pathway, leading to an increase in lactic acids and a decrease in cytosolic pH. While cytosolic pH decreases, during ischemia the mitochondrial membrane potential is diminished and, in case of glial cells, glutamate increases the mitochondrial respiration rate and pH-gradient of mitochondrial matrix and leads to the supportive effect to oxidative phosphorylation (Krasil’nikova et al. 2019; Surin et al. 2022). After reperfusion, the rapid restoration of the mitochondrial membrane potential with the return of oxygen into the cytosol provides a strong driving force for the entry of cytosolic Ca2+ into the mitochondrion via the MCU (Shintani-Ishida et al. 2012). This results in mitochondrial Ca2+ overload, leading to cell damage or death associated with opening of the mPTP. This process generates toxic products such as cytochrome C and swelling of the matrix, eventually causing outer mitochondrial membrane rupture (Bonora et al. 2017; Hawrysh and Buck 2013; Morciano et al. 2015; Shintani-Ishida et al. 2012). Thus, some reports have stated that inhibition of the MCU reduces the cell damage caused by mitochondrial Ca2+ overload and may confer neuroprotection (Novorolsky et al. 2020; Woods and Wilson 2019; Zhao et al. 2015). However, our results (Fig. 4) initially appear to contradict this contention, showing an increase in dead cells in the CA1 region after inhibition of the MCU in PostC. Nonetheless, inhibition of the MCU cannot prevent other toxicity caused by cytosolic Ca2+ overload (Curcio et al. 2016; Kristián and Siesjö 1998; Lau and Tymianski 2010). Thus, the reason dead cells in the CA1 region are increased with MCU inhibition in PostC may be that other death signals related to cytosolic Ca2+ overload, not simply mitochondrial Ca2+ overload via the MCU, were activated and led to cell death.

MCU Plays a Role as a Key Factor in the Interactive Processes of Mitochondrial Depolarization in PostC

In our previous research, we reported that opening of the mito-KATP channel triggered the PostC mechanism and caused the mitochondrial depolarization observed during the early period of reperfusion in the PostC group, but not in the control group (Morisaki et al. 2022). Moreover, we also reported that a key factor in the neuroprotection conferred by PostC was the low-conductance opening of the mPTP. The catastrophic process of cell injury is induced through the high-conductance mode of mPTP opening, which allows the passage of ions, including Ca2+, leading to dissipation of the mitochondrial membrane potential and eventually resulting in cell death (Brenner and Moulin 2012). Conversely, under physiological conditions, the mPTP could exhibit intermittent opening in low-conductance mode, contributing to intracellular Ca2+ homeostasis and the regulation of mitochondrial function (Giorgio et al. 2013). We also reported that the low-conductance mode of mPTP opening prevented opening in high-conductance mode and reduced NMDAR conductance (Morisaki et al. 2022). Furthermore, mitochondrial membrane potential switches between these two modes, with the threshold value controlled by mitochondrial Ca2+ concentration (Bazil et al. 2010). Opening of the mito-KATP channel is triggered by a local decrease in cytosolic ATP under ischemic stress, subsequently leading to increased K+ influx into the mitochondrial matrix (Hawrysh and Buck 2013; Pamenter et al. 2008). In PostC, opening of this channel leads to mitochondrial depolarization, reducing the driving force of Ca2+ influx into the mitochondrial matrix and abrogating excessive Ca2+ accumulation in the matrix, thus avoiding the high-conductance mode of mPTP opening (Hawrysh and Buck 2013; Morisaki et al. 2022). Mitochondrial depolarization is thus another key factor in the neuroprotection provided by PostC. In the present study, depolarization of the mitochondrial membrane was weakened after reperfusion in the PostC + Ru265 group, compared to in the PostC group. Such data suggest that in PostC, mitochondrial depolarization is triggered by opening of the mito-KATP channel, but some form of interaction between the mito-KATP channel and MCU was blocked by Ru265, resulting in a loss of neuroprotective effects through mitochondrial membrane potential in PostC. Furthermore, our findings also suggest that mild interactive regulation of the MCU provided by the driving force from the mito-KATP channel is necessary for maintaining mitochondrial depolarization in PostC. Thus, we consider that it is necessary to investigate more specifically how the mitochondrial metabolism in PostC is regulated with further research about, such as the mitochondrial Ca2+ level and the mitochondrial respiration in future.

Pharmacological Approach to the MCU

The present study used ruthenium red 265 as a novel selective inhibitor of the MCU. The most well-known and commonly used MCU inhibitor is Ru360, named for its strong absorbance at 360 nm (Emerson et al. 1993). However, Ru360 is poorly permeable and unstable in aqueous solution, losing activity within days (Hajnóczky et al. 2006; Márta et al. 2021). On the other hand, Novorolsky et al. reported that in a lysate of HEK293 or Hela cells, Ru265 was taken into these cells at 2–10 times greater rates than other structural analogs of ruthenium after these cells were incubated with each ruthenium complex at 50 µM for 24 h; moreover, the rise in mitochondrial Ca2+ concentration was observed rapidly, after only 30 min of incubation (Novorolsky et al. 2020; Woods et al. 2019). With our PostC protocol in previous research, we observed each single-cell action during a very short time course, including normoxia and anoxia within about 30 min to 1 h. Use of a rapidly permeable drug was thus necessary for this protocol, and from this point of view, we regarded Ru265 as suitable for our research (Novorolsky et al. 2020). Furthermore, ruthenium compounds are also generally known for their cytotoxicity (Alessio 2017; Dutta et al. 2008; 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 and Dyson 2006), while Ru265 is a less toxic compound without observable effects on mitochondrial membrane potential or other intracellular Ca2+ dynamics reportedly (Woods et al. 2019). Thus, in our present study, it was also observed that Ru265 itself did induce no change to all the parameters such as the EPSC occurrence, NMDAR currents, intracellular Ca2+ concentration and mitochondrial depolarization in the sham (Control + Ru265 10 µM) group, similar to the control group. These results suggest that Ru265 had less affection or toxicity to cell membranes or some other cell organelles.

Limitations and Future Direction

In our previous and present studies, we used the hippocampal pyramidal cell of male mice only. Recently in the clinical and experimental studies, it is discussed about the gender-dependent difference of the brain injury. They reported that the male neonates were more susceptible to the damages related to brain ischemia, resulting in more severe neurological outcome compared to females (Cikla et al. 2016; Hill and Fitch 2012; Uluç et al. 2013). Thus, our results might be different if we had used female mice and for the future prospection, we should consider about the ‘gender specificity in PostC.’

In our laboratory, ‘Safe pharmacological PostC’ represents our central concern for achieving clinical application. Diazoxide as the opener of the mito-KATP channel is unsuitable because of its toxic side effects (Kumar et al. 1976), while in our previous study, Furuta et al. reported on the neuroprotective mechanisms of melatonin-induced pharmacological PostC (Furuta et al. 2022) and melatonin could be a new candidate for pharmacological PostC without side effects. Thus, as future directions, in vitro and in vivo studies are needed to explore methods of delivering melatonin to the brain, such as catheterization of middle cerebral artery occlusion models. In addition, it is also necessary to find other PostC-like neuroprotective and less toxicity chemical compounds or drugs.