Thapsigargin induces biphasic fragmentation of mitochondria through calcium-mediated mitochondrial fission and apoptosis

Mitochondrial fission and fusion are the main components mediating the dynamic change of mitochondrial morphology observed in living cells. While many protein factors directly participating in mitochondrial dynamics have been identified, upstream signals that regulate mitochondrial morphology are not well understood. In this study, we tested the role of intracellular Ca2+ in regulating mitochondrial morphology. We found that treating cells with the ER Ca2+ -ATPase inhibitor thapsigargin (TG) induced two phases of mitochondrial fragmentation. The initial fragmentation of mitochondria occurs rapidly within minutes dependent on an increase in intracellular Ca2+ levels, and Ca2+ influx into mitochondria is necessary for inducing mitochondrial fragmentation. The initial mitochondrial fragmentation is a transient event, as tubular mitochondrial morphology was restored as the Ca2+ level decreased. We were able to block the TG-induced mitochondrial fragmentation by inhibiting mitochondrial fission proteins DLPI/Drpl or hFisI, suggesting that increased mitochondrial Ca2+ acts upstream to activate the cellular mitochondrial fission machinery. We also found that prolonged incubation with TG induced the second phase of mitochondrial fragmentation, which was non-reversible and led to cell death as reported previously. These results suggest that Ca2+ is involved in controlling mitochondrial morphology via intra-mitochondrial Ca2+ signaling as well as the apoptotic process.