Mitochondrial permeability transition (mPT) is a phenomenon of a sudden increase in the permeability of the inner mitochondrial membrane (IMM). Patch-clamp experiments on isolated mitochondria identified the mPT mechanism as the Ca²⁺– and ROS-stimulated opening of a large, unspecific pore in the IMM, known as the permeability transition pore (mPTP). The opening of the mPTP disrupts normal mitochondrial function and has been implicated as the hallmark of mitochondria-driven cell death in many diseases, including ischemia-reperfusion injuries of organs.

Here, we applied advanced whole-mitoplast patch-clamp and ex vivo patch clamp approaches to investigate the still-understood mechanism of mitochondrial dysfunction associated with mPTP. The whole-mitoplast configuration allows us to observe mPTP currents at the level of the whole mitochondrion (Neginskaya & Pavlov, 2023), and ex vivo patch clamp detects the changes in IMM permeability induced by in vivo stress, contrasting with the stress applied to mitochondrial membranes after isolation (Niatsetskaya et al., 2020). Patch-clamp findings were supported by holographic imaging (HI) in cultured MEF cells. HI detects changes in the refractive indexes of mitochondria upon mPTP opening, enabling the observation of mPTP within the living cell, independently of mitochondrial depolarization (Neginskaya, Morris, & Pavlov, 2022).

Whole-mitoplast recordings from isolated cardiac mitochondria demonstrated that Ca²⁺-induced currents were partially blocked by CSA (42%, n=12) and by ADP (100%, n=2), but not sensitive to bongkrekic acid (BA), an inhibitor of the Adenine Nucleotide Translocator. In contrast, the ROS-stimulated whole-mitoplast current was transiently blocked by BA (n=1), suggesting that the mechanisms of IMM permeability might vary depending on stress conditions.

Ex vivo patch-clamping of brain mitochondria isolated from neonatal (p10) mice exposed to hypoxia-ischemia brain injury detected elevated IMM permeability after 30 min of reperfusion (420±40 pS, n=54 vs. 250±40 pS in control, n=18; p=0.05, Kruskal-Wallis). This elevated IMM permeability did not exhibit the classic CSA-sensitive mPTP current behavior. Neuroprotective hypothermia applied during reperfusion attenuated the infarct volume of brain (n=15, 36.7±6.0% to 19.2±5.0%) and normalized the associated elevation of IMM permeability (420±40 pS, n=54 to 290±50 pS, n=30, p=0.03, Kruskal-Wallis). These results suggest the existence of a non-mPTP mechanism of mitochondrial damage contributing to the injury. Interestingly, hyperoxia during reperfusion resulted in the activation of the classical CSA-sensitive mPTP (80% of CSA-sensitive channels (n=10) vs. 14% (n=14) in normoxia, p=0.01, χ2 test) and exacerbated post-ischemic injury.

Finally, HI demonstrated that mitochondrial Ca²⁺ overload induced by the ionophore ferutinin stimulates classic CSA-sensitive mPTP activation (Neginskaya et al., 2022), while ionomycin-induced Ca²⁺ dysregulation triggered mitochondrial depolarization and swelling but not the mPTP opening (n=4). CSA did not inhibit ionomycin-induced mitochondrial depolarization and swelling (n=3), or subsequent cell death (n=2), but it abolished the effect of ferutinin.

Therefore, we have demonstrated the existence of at least two mechanisms: mPTP and non-mPTP mitochondrial dysfunction that might contribute to acute ischemia-reperfusion injuries, with each pathway's impact determined by the unique stress conditions.