Increased mitochondrial free Ca2+ during ischemia is suppressed, but not eliminated by, germline deletion of the mitochondrial Ca2+ uniporter

Mitochondrial Ca2+ overload is proposed to regulate cell death via opening of the mitochondrial permeability transition pore. It is hypothesized that inhibition of the mitochondrial Ca2+ uniporter (MCU) will prevent Ca2+ accumulation during ischemia/reperfusion and thereby reduce cell death. To address this, we evaluate mitochondrial Ca2+ in ex-vivo-perfused hearts from germline MCU-knockout (KO) and wild-type (WT) mice using transmural spectroscopy. Matrix Ca2+ levels are measured with a genetically encoded, red fluorescent Ca2+ indicator (R-GECO1) using an adeno-associated viral vector (AAV9) for delivery. Due to the pH sensitivity of R-GECO1 and the known fall in pH during ischemia, hearts are glycogen depleted to decrease the ischemic fall in pH. At 20 min of ischemia, there is significantly less mitochondrial Ca2+ in MCU-KO hearts compared with MCU-WT controls. However, an increase in mitochondrial Ca2+ is present in MCU-KO hearts, suggesting that mitochondrial Ca2+ overload during ischemia is not solely dependent on MCU. [Display omitted] •Inhibition of MCU is proposed to reduce mitochondrial Ca2+ accumulation during I/R•Deletion of MCU significantly reduces the accumulation of mitochondrial Ca2+ during I/R•Mitochondrial Ca2+ increases in MCU-KO hearts during I/R•MCU is the main Ca2+ uptake pathway during I/R, but alternative pathways exist In brief, Petersen et al. measure mitochondrial Ca2+ with a genetically encoded fluorescent Ca2+ indicator (R-GECO1) and demonstrate that germline deletion of MCU significantly reduces, but does not eliminate, the accumulation of mitochondrial Ca2+ during I/R. Thus, MCU is the major Ca2+ uptake pathway during ischemia; however, MCU-independent mechanisms also contribute.