Mitochondrial calcium drives clock gene-dependent activation of pyruvate dehydrogenase and of oxidative phosphorylation.

Abstract Regulation of metabolism is emerging as a major output of circadian clock circuitry in mammals. Accordingly, mitochondrial oxidative metabolism undergoes both in vivo and in vitro daily oscillatory activities. In the present study we show that both glycolysis and mitochondrial oxygen consumption display a similar time-resolved rhythmic activity in synchronized HepG2 cell cultures, which translates in overall bioenergetic changes as documented by measurement of the ATP level. Treatment of synchronized cells with specific metabolic inhibitors unveiled pyruvate as a major source of reducing equivalents to the respiratory chain with its oxidation driven by the rhythmic (de)phosphorylation of pyruvate dehydrogenase. Further investigation enabled to causally link the autonomous cadenced mitochondrial respiration to a synchronous increase of the mitochondrial Ca2+. The rhythmic change of the mitochondrial respiration was dampened by inhibitors of the mitochondrial Ca2+ uniporter as well as of the ryanodine receptor Ca2+ channel or the ADPR cyclase, indicating that the mitochondrial Ca2+ influx originated from the ER store, likely at contact sites with the mitochondrial compartment. Notably, blockage of the mitochondrial Ca2+ influx resulted in deregulation of the expression of canonical clock genes such as BMALl1, CLOCK, NR1D1. All together our findings unveil a hitherto unexplored function of Ca2+-mediated signaling in time keeping the mitochondrial metabolism and in its feed-back modulation of the circadian clockwork.