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A modified calcium retention capacity assay clarifies the roles of extra- and intracellular calcium pools in mitochondrial permeability transition pore opening
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Edité par CCSD ; American Society for Biochemistry and Molecular Biology -
International audience. Calcium homeostasis is essential for cell survival and is precisely controlled by several cellular actors such as the sarco/endoplasmic reticulum and mitochondria. Upon stress induction, Ca(2+) released from sarco/endoplasmic reticulum stores and from extracellular Ca(2+) pools accumulates in the cytosol and in the mitochondria. This induces Ca(2+) overload and ultimately the opening of the mitochondrial permeability transition pore (mPTP), promoting cell death. Currently, it is unclear whether intracellular Ca(2+) stores are sufficient to promote the mPTP opening. Ca(2+) retention capacity (CRC) corresponds to the maximal Ca(2+) uptake by the mitochondria before mPTP opening. In this study, using permeabilized cardiomyocytes isolated from adult mice, we modified the standard CRC assay by specifically inducing reticular Ca(2+) release to investigate the respective contributions of reticular Ca(2+) and extracellular Ca(2+) to mPTP opening in normoxic conditions or after anoxia-reoxygenation. Our experiments revealed that Ca(2+) released from the sarco/endoplasmic reticulum is not sufficient to trigger mPTP opening and corresponds to approximately 50% of the total Ca(2+) levels required to open the mPTP. We also studied mPTP opening after anoxia-reoxygenation in the presence or absence of extracellular Ca(2+) In both conditions, Ca(2+) leakage from internal stores could not trigger mPTP opening by itself but significantly decreased the CRC. Our findings highlight how a modified CRC assay enables the investigation of the role of reticular and extracellular Ca(2+) pools in the regulation of the mPTP. We propose that this method may be useful for screening molecules of interest implicated in mPTP regulation.
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