Adrenergic Stimulation Accelerates Mitochondrial Ca2+ uptake by PYK2-Dependent Phosphorylation of Mitochondrial Ca2+ Uniporter in Cardiac H9C2 Cells

Abstract

Background: Recent break-through discovery in the molecular identity of mitochondrial Ca2+ uniporter (MCU) opens the new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported in human sample mass spectroscopy, but the post translational modifications of MCU are completely unknown.Hypothesis: Tyrosine phosphorylation of MCU can modulate the mitochondrial Ca2+-uptake rate in cardiac cells.Methods: MCU was transiently or stably overexpressed in cardiac H9C2 cells. Tyrosine phosphorylation of MCU was detected by a general anti-phospho-tyrosine antibody. Mitochondrial Ca2+ concentration ([Ca2+]m) was measured by mitochondrial matrix-targeted Ca2+-sensitive inverse pericam (Mitycam).Results: α1-adrenergic stimulation by phenylephrine enhanced the translocation of a Ca2+-dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) from cytosol to mitochondria followed by the increase in mitochondrial Pyk2 activity. Overexpressed MCU was exclusively localized at mitochondria and tyrosine residues in MCU were phosphorylated after Pyk2 activation. In addition, Pyk2 was bound to MCU at the basal condition and this interaction was enhanced by phenylephrine treatment. Moreover, Pyk2-dependent phosphorylation of MCU enhances MCU olgomerization observed by a conventional native PAGE. These effects were abolished by the co-transfection of kinase-dead Pyk2. In MCU-overexpressed cells, [Ca2+]m increased rapidly and reached to higher levels in response to cytosolic Ca2+ transients evoked by thapsigargin compared to non-transfected cells. Moreover, peak [Ca2+]m in MCU-overexpressed cells reached to much higher levels by phenylephrine pretreatment compared to non-treated cells.Conclusion: α1-adrenergic stimulation accelerates mitochondrial Ca2+ uptake through Pyk2-dependent direct phospholylation of MCU, which promotes the formation of tetrametric MCU channel pore. Our findings open up an exciting opportunity for investigating the first candidate cell signaling pathway for the MCU post translational modifications in cardiac cells.