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

Abstract

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.

Keywords: CP: Developmental biology; MCU; calcium; cardioprotection; ischemia-reperfusion; mitochondria.