Cyclophilin D phosphorylation is critical for mitochondrial calcium uniporter regulated permeability transition pore sensitivity

This editorial refers to ‘Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter’ by R.J. Parks et al., pp. 385–394. Mitochondrial oxidative metabolism provides the fuel for contractility of cardiac muscles, but mitochondria can also serve as a platform for execution of apoptosis and necrosis through mPTP opening. The mPTP plays a critical role in pathogenesis of several neurodegenerative and cardiovascular disease conditions including ischaemia/reperfusion (I/R). Early descriptions by Hunter and Haworth1 in 1979 identified the mPTP as a channel on the mitochondrial inner membrane permeable to solutes with molecular mass <1.5 kDa. Since these initial investigations, it is well appreciated that mPTP opening is regulated by elevated calcium levels within the mitochondrial matrix. In fact, the connection between high levels of mitochondrial matrix calcium and mitochondrial swelling was known more than 6 decades ago. Notably, calcium-mediated mPTP opening is potentiated by increased reactive oxygen species (ROS) production, depletion of adenosine triphosphate (ATP), and increased inorganic phosphate, many of which occur upon the reperfusion phase of I/R.2 While the exact structural identity of the mPTP remains to be fully elucidated, early models predicted the mPTP as a large multi-protein complex that spanned the outer and inner mitochondrial membranes comprised of the voltage dependent anion channel, adenine nucleotide transporter, hexokinase II, mitochondrial phosphate carrier, and cyclophilin D (CypD). While gene knockout studies in mice have refuted many of these proteins as major constituents of mPTP,3,4 these studies do not preclude the involvement of some of these proteins as mPTP modulators such as in the case for CypD.5–7 Recently, very elegant studies by Bernardi’s group provided compelling evidence for the mitochondrial F0-F1 ATPase complex V of the respiratory chain as the most likely candidate of the mPTP.7,8 However, the F0-F1 ATPase as the mPTP itself or as a major constituent of mPTP has been challenged by others and with the controversy over the mPTP not settled. Irrespective of the mPTP identity, it is commonly accepted that high levels of mitochondrial matrix calcium through protein association between CypD and F0-F1 ATPase can reportedly trigger mPTP opening and mitochondrial dysfunction in brain and heart.9 Ostensibly, loss of inner membrane integrity from mPTP channel opening would result in accumulation of solutes and ions, would impair mitochondrial respiration and ATP synthesis resulting in cell death.10,11 During I/R, excessive calcium entry into the mitochondria via mitochondrial calcium uniporter (MCU) has been suggested as a trigger point for mPTP opening and necrotic cell death.12 However, recent reports demonstrating increased sensitivity to I/R injury in the mice lacking MCU, have challenged the notion that mitochondrial calcium overload is critical for mPTP opening. If true, this would suggest that at least operationally MCU together with other mitochondrial perturbations such as ROS production may be required for mPTP opening during I/R. The study by Park et al.13 provides molecular insight into the mechanisms for mPTP opening that involve the site-specific phosphorylation of CypD. The study by Parks et al.13 demonstrates that phosphorylation of CypD at serine 42 is critical for sensitizing cardiomyocytes to I/R injury. Using a global MCU knockout mouse model and induced pluripotent stem cells-derived cardiomyocytes, the authors showed that despite low mitochondrial matrix calcium levels from lack of MCU, MCU deficient mitochondria readily underwent mPTP opening and were more susceptible to I/R injury than corresponding wild type controls. To systematically address the underlying mechanism for the increased sensitivity of MCU deficient mitochondria to I/R as well as the mode of cell death induced in the MCU-knock out (MCU-KO) mice following I/R stress, the authors first investigated whether necroptosis pathway proteins RIP1 and RIP3 were accountable for this finding. Using an ex vivo Langendorff model of I/R stress, the authors tested the effects of the RIP1 inhibitor, Necrostatin-1(Nec-1) on cardiac functional recovery as an index for cardiac dysfunction following I/R. Interestingly, in wild type mice hearts, Nec-1 resulted in improved functional recovery of left ventricular developed pressure (LVDP) and smaller infarcts sizes yet had no protective effect on these study endpoints in MCU-KO hearts. Based on these observations, the authors concluded that the increased sensitivity of MCU-KO mitochondria to I/R injury was independent of RIP1. Next, to determine the involvement of RIP3, the authors crossed RIP3-KO mice with MCU-KO mice, but again no improvement in functional recovery was observed in MCU-KO/RIP3-KO double knockout mice—excluding RIP3 and necroptosis pathway for the increased sensitivity of MCU-KO hearts to I/R injury. Next the authors explored the involvement of mPTP opening as a potential mechanism to explain the sensitivity of MCU-KO mice to I/R injury. For these studies, the authors assessed mitochondrial calcium in mitochondria derived from wild type and MCU-KO mice hearts, by using calcium ionophore ETH-129. Interestingly, while the MCU-KO derived mitochondria were responsive to calcium uptake following repeated boluses of calcium, the calcium retention capacity (CRC) in these mitochondria was significantly reduced, and much lower concentrations of calcium were required to trigger mPTP opening compared to wild type controls. Mitochondrial phospho-proteome screening conducted on wild type and MCU-KO hearts revealed altered phosphorylation of several mitochondrial proteins in MCU-KO hearts including CypD. Notably, a marked increased interaction between phosphorylated CypD and F0/F1ATPase was observed in MCU-KO hearts. Phosphorylation levels of CypD in MCU-KO hearts were confirmed by mass spectroscopy and immunoprecipitation. Based on series of carefully designed experiments and the information derived from Scaffold PTM software, the authors identified CypD to be uniquely phosphorylated on serine 42. Next, the authors generated a series of charge to neutral mutations of CypD where serine 42 was exchanged either with phospho-resistant alanine (S42A) or with phospho-mimetic glutamine (S42D). CypD mutants were then tested on mitochondrial CRC in reconstituted CypD-KO MEFS. MEFs transfected with phospho-mimetic mutant CypD S42D demonstrated marked reduction in CRC compared to transfected controls or MEFs transfected with phospho-resistant mutant of CypD S42A. Moreover, an increase in CypD-F0/F1ATPase interaction, mPTP opening, and cell death was observed following H2O2 treatment in the presence of phospho-mimetic mutant CypD S42—consistent with author’s notion that phosphorylation of CypD at S42 is a requisite event for interaction of CypD with F0/F1ATPase for mPTP opening and cell death (Figure 1). Phosphorylation of cyclophilin D at serine 42 triggers permeability transition pore opening. In the absence of MCU, CypD is phosphorylated at serine 42 and provokes mitochondrial perturbations and cell death. Left panel: Mitochondria with intact MCU, demonstrate regulated calcium (Ca2+) entry into mitochondrial matrix. Absence of CypD phosphorylation coincides with high CRC and mPTP closure. Right panel: In MCU-KO hearts, CypD is phosphorylated at serine 42, and this modification results in association between CypD and F0/F1ATPase. CypD S42 phosphorylation leads to reduced CRC of mitochondria, mPTP opening, and cell death. The findings that phosphorylation of S42 of CypD is critical step for mPTP opening and cell death is novel, but raises the question, how does CypD get phosphorylated in the MCU-KO mitochondria in the first place? This point requires careful consideration. First, authors used an MCU germ line deficient mice model for most of the experiments, at this point we do not know whether the findings derived from this study will hold true under physiological relevant or disease conditions where MCU is rendered inactive or down-regulated. Further, it remains unclear whether CypD S42 phosphorylation is restricted to conditions where MCU is deregulated or whether it is a universal phenomenon in response to other forms of cell stress that involves mPTP opening. Second, exact mechanism of how cells die in response to I/R in mice lacking MCU is not clear; while necrosis is considered the primary mode of cell death during I/R, Nec-1 conferred cardioprotection only in wild type mice but not in MCU-KO. Based on this observation, the authors concluded that increased sensitivity observed in MCU-KO during I/R injury is not due to RIP1 signalling. However, lack of Nec-1 mediated protection in absence of MCU, could also occur if the uniporter itself or a regulator of MCU was a target of Nec-1. Moreover, there exist a disparity in literature in terms of cardio-protection against I/R in response to MCU loss, and requires careful investigation. Cardiac-specific ablation of MCU in adult mice was better protected against I/R.14 However a dominant negative DN-MCU model, by a different group and germ line MCU-KO mouse model shown in present study demonstrated increased cardiotoxicity against I/R.13,15 Therefore, the role of MCU seems to be more complex and variable in settings of acute and chronic stress. Timing of activation of MCU (perinatal vs. post-natal) may also be important, one possibility exists that different outcomes in cardio-protection in response to loss of MCU could be due to differential role of MCU in cardiomyocytes vs. other cell types such as fibroblasts, smooth muscles, and blood cells. This disparity in the literature needs to be resolved for better understanding of the role of MCU in I/R stress. Also, it would be important to know whether there is a link between MCU and calcium in regulation of mitochondrial dynamics or mitochondrial respiration during I/R and how these processes connect, with MCU and mPTP opening. Despite these issues, the study by Parks et al. highlights novel phosphorylation of CypD at serine 42 to be critical for CRC and mPTP opening and provides new insight into the regulation of the mPTP by CyPD and MCU. Therefore, selective modulation of CypD or approaches that target phosphorylation of CypD at critical S42 may prove beneficial in overcoming mPTP-induced necrotic cell death during I/R. We are grateful to Dr Inna Rabinovich-Nikitin for critical comments on the manuscript. Conflict of interest: none declared. This work was supported by grants to L.A.K. from the CIHR, and St. Boniface Hospital Research Foundation, L.A.K. holds a Canada Research Chair in Molecular Cardiology.

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