The effects of mitochondrial Ca²⁺ transport on intracellular Ca²⁺ waves in cardiomyocytes

Background

Recent studies have implicated that mitochondria play important roles in intracellular Ca²⁺ homeostasis of cardiac myocytes. The major pathways for mitochondrial Ca²⁺ transport include mitochondrial Ca²⁺ uniporter and Na⁺/Ca²⁺ exchanger, as well as mitochondrial permeability transition pore (mPTP) under certain pathophysiological conditions. However, it is still unclear if mitochondrial Ca²⁺ flux can affect the generation of Ca²⁺ waves and triggered activities in cardiomyocytes.

Methods and results

Cytosolic Ca²⁺ (Ca_i²⁺) was imaged in fluo-4-AM loaded ventricular myocytes isolated from mice. Spontaneous SR Ca²⁺ release and Ca²⁺ waves (CaWs) were induced in the presence of high external Ca²⁺ (Ca_o²⁺, 4 mM). The protonophore carbonyl cyanide p - (trifluoromethoxy) phenylhydrazone (FCCP) reversibly raised basal Ca_i²⁺ levels in the presence, as well as absence of Ca_o²⁺, suggesting Ca²⁺ release from intracellular stores. Mitochondrial membrane potential (ΔΨm) was monitored by TMRM fluorescence. FCCP at 0.01- 0.1 µM, which partially depolarized ΔΨm, increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed after depolarization amplitudes and frequencies, and induction of triggered action potentials. On the contrary, FCCP at higher concentrations (>0.5 µM), which completely dissipated ΔΨm, eliminated CaWs while the basal Ca_i²⁺ remained high. The cease of CaWs was most likely due to the reduction of SR Ca²⁺ content as evaluated by rapid exposure to10 mM caffeine. Blocking sarcolemmal Na⁺-Ca²⁺ exchanger by substituting Na⁺ with Li⁺ in the perfusant further elevated basal Ca_i²⁺ and restored CaWs. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting ΔΨm) or Ru360 (a mitochondrial Ca²⁺ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca²⁺ uniporter activator kaempferol.

Conclusions

Mitochondrial Ca²⁺ release and uptake control plasma Ca²⁺ levels and plays an important role in regulation of intracellular CaWs and arrhythmogenesis.