Phosphorylation of cyclophilin D at serine 191 regulates mitochondrial permeability transition pore opening and cell death after ischemia-reperfusion

Abstract

The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivityconferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.

Mitochondrial Quality Control in Cardiac-Conditioning Strategies against Ischemia-Reperfusion Injury

“…However, this has not been the end of the story, as the post-translational regulation of mPTP by phosphorylation and S-acylation of CsA is receiving special attention. Preventing CypD phosphorylation at S191 by inhibiting the glycogen synthase kinase-3β (GSK3β) reduces the translocation and binding of CypD to OSCP, avoiding increased ROS production, mPTP opening and subsequent cell death at reperfusion [234]. Moreover, it has been demonstrated that C202 in CypD is S-acylated and that ischemia induces deacylation and Ca 2+ overload.…”

Molecular Signaling to Preserve Mitochondrial Integrity against Ischemic Stress in the Heart: Rescue or Remove Mitochondria in Danger

“…A regulatory role of Cyp-D to mPTP-opening was first suggested by the inhibitory effect of cyclosporin A (Cs-A) on Ca 2+ -induced mPTP opening and by the subsequent discovery that Cs-A binds to Cyp-D [ 32 , 33 ]. It has recently been reported that the mPTP sensitizing effect of Cyp-D is potentiated by the phosphorylation of Cyp-D at Ser191 [ 34 ]. It would be of importance to identify responsible kinases for this phosphorylation ( Figure 2 B).…”

Dynamic Regulation of Cysteine Oxidation and Phosphorylation in Myocardial Ischemia–Reperfusion Injury

“…Opening of the pore depolarizes the mitochondrial membrane and initiates cell death [ 39 ]. Kinases can mitigate pore opening and reduce the detrimental effects of oxidative stress by activating cell survival pathways [ 40 , 41 ]. These studies implicate a crucial role for kinases in I/R injury and cardioprotection.…”