Data from Petersen et al The rise in mitochondrial free Ca2+ during ischemia... Cell Reports 2.zip

 Summary 

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-KO and WT mice using transmural spectroscopy. Matrix Ca2+ levels are measured with the 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 known fall in pH during ischemia, hearts are glycogen depletion to decrease the ischemic fall in pH. At 20 mins of ischemia, there is  significantly less mitochondrial Ca2+ in MCU-KO hearts compared to 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.