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 Ca ²⁺ level decreased. We were able to block the TG-induced mitochondrial fragmentation by inhibiting mitochondrial fission proteins DLP1/Drp1 or hFis1, 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.