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. 2019 Feb 1;115(2):385-394.
doi: 10.1093/cvr/cvy218.

Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter

Randi J Parks  1 , Sara Menazza  1 , Kira M Holmström  2 , Georgios Amanakis  1 , Maria Fergusson  1 , Hanley Ma  1 , Angel M Aponte  2 , Paolo Bernardi  3 , Toren Finkel  2 , Elizabeth Murphy  1
Affiliations

Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter

Randi J Parks et al. Cardiovasc Res. .

Abstract

Aims: Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice abrogates mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice are not protected from ischaemic injury. We aimed to investigate whether adaptive alterations occur in cell death signalling pathways in the hearts of global MCU-KO mice.

Methods and results: First, we examined whether cell death may occur via an upregulation in necroptosis in MCU-KO mice. However, our results show that neither RIP1 inhibition nor RIP3 knockout afford protection against ischaemia-reperfusion injury in MCU-KO as in wildtype (WT) hearts, indicating that the lack of protection cannot be explained by upregulation of necroptosis. Instead, we have identified alterations in cyclophilin D (CypD) signalling in MCU-KO hearts. In the presence of a calcium ionophore, MCU-KO mitochondria take up calcium and do undergo PTP opening. Furthermore, PTP opening in MCU-KO mitochondria has a lower calcium retention capacity (CRC), suggesting that the calcium sensitivity of PTP is higher. Phosphoproteomics identified an increase in phosphorylation of CypD-S42 in MCU-KO. We investigated the interaction of CypD with the putative PTP component ATP synthase and identified an approximately 50% increase in this interaction in MCU-KO cardiac mitochondria. Mutation of the novel CypD phosphorylation site S42 to a phosphomimic reduced CRC, increased CypD-ATP synthase interaction by approximately 50%, and increased cell death in comparison to a phospho-resistant mutant.

Conclusion: Taken together these data suggest that MCU-KO mitochondria exhibit an increase in phosphorylation of CypD-S42 which decreases PTP calcium sensitivity thus allowing activation of PTP in the absence of an MCU-mediated increase in matrix calcium.

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Figures

Figure 1
Figure 1
Necroptosis is not up-regulated in MCU-KO hearts. (A) The top panel shows the ischaemia-reperfusion protocol, including treatment with 30 µM necrostatin-1 for 5 min prior to ischaemia. RIP1 inhibition with necrostatin-1 increased LVDP in WT, but not in hearts from MCU-KO mice. (B) RPP was increased in WT with necrostatin-1 treatment, but had no effect in MCU-KO. (C) Infarct size was decreased by necrostatin-1 in WT hearts, but remained unchanged in MCU-KO. Data are represented as mean ± SEM. *P < 0.01 (two-way ANOVA), n = 4–5 mice per group for necrostatin-1 experiments. (D) The top panel depicts the ischaemia-reperfusion protocol followed in these experiments. KO of RIP3 increased LVDP at the end of reperfusion, but double KO of RIP3 and MCU had no effect in comparison to WT. (E) RPP followed the same trend, in that RIP3-KO increased functional recovery in WT hearts, but had no effect in RIP3 and MCU double KO hearts. (F) Infarct size was reduced in RIP3-KO hearts, but this protection was not present in hearts from double KO mice. Data are represented as mean ± SEM. *P < 0.01 (two-way ANOVA), n = 5–7 mice per group for RIP3-KO experiments.
Figure 2
Figure 2
iPSC-derived cardiomyocytes lacking MCU are protected against RIP3-induced necroptosis. (A) KO of MCU in iPSC clones was confirmed with Western blot, where no MCU protein is present in comparison to WT control. β-actin was used as a loading control. (B) Representative western blot analysis of RIP3 protein levels in iPS-cardiomyocytes infected with Ad-GFP (used as control) or Ad-RIP3. Similar expression of RIP3 was seen using an MOI of 20 or 100. (C) Cell viability as assessed by LDH activity after infection with Ad-GFP or Ad-RIP3; data obtained with an MOI of 20 or 100 were pooled since RIP3 expression was similar. Overexpression of RIP3 increased cell death in WT but not in MCU-KO cells. Data are represented as mean ± SEM. *P < 0.0001 (two-way ANOVA), n = 10 samples.
Figure 3
Figure 3
PTP opening can occur in the absence of MCU. (A) The calcium ionophore ETH-129 permitted calcium entry into mitochondria from MCU-KO. (B) MCU-KO underwent mitochondrial swelling in the presence of ETH. (C) Following depletion of endogenous calcium, mitochondria from MCU-KO underwent pore opening at a lower concentration of calcium than WT. The inset shows that mean calcium retention capacity in MCU-KO is lower than WT. Data are represented as mean ± SEM. *P = 0.04 (Student’s t-test), n = 5 WT and 5 MCU-KO mice.
Figure 4
Figure 4
Phosphoprotein immunoprecipitation showed an increase in CypD phosphorylation in MCU-KO vs. WT. (A) CypD protein levels were similar in mitochondria from WT and MCU-KO hearts (blot representative of n = 4). (B) Mitochondria immunoprecipitated for phospho-S/T/Y were probed for CypD, with IgG used as IP antibody control. (C) Mean data demonstrate that there was an increase in the amount of CypD pulled-down by phospho antibody in MCU-KO compared with WT. Data are represented as mean ± SEM. *P = 0.005 (Student’s t-test), n = 8 WT and 10 MCU-KO mice.
Figure 5
Figure 5
Pull-down of ATP synthase revealed an increase in association with CypD in MCU-KO mitochondria. (A) Representative blots of samples that were immunoprecipitated with an F1/F0-ATP synthase antibody, and probed for CypD and ATP synthase subunit α as a pull-down control. (B) There was a significant increase in the amount of CypD associated with ATP synthase in MCU-KO vs. WT. Data are represented as mean ± SEM. *P = 0.02 (Student’s t-test), n = 6 WT and 6 MCU-KO mice.
Figure 6
Figure 6
Phosphorylation of S42-CypD decreases CRC and increases CypD association with ATP synthase and cell death. (A) CypD levels were measured between different stable cell lines containing S42A or S42D mutations. ATP synthase subunit α was used as a loading control. (B) Representative calcium uptake assay in CypD-KO MEFs infected with lentivirus containing mutated CypD, either phospho-resistant S42A or phosphomimic S42D. (C) Mean CRC values, measured as nmol of calcium per mg of protein, indicated that phosphorylation of CypD at S42 decreased CRC. Data are represented as mean ± SEM. *P = 0.005 (two-way ANOVA), n = 5 independent experiments. (D) Immunoprecipitation of ATP synthase pulled down more CypD in the S42D mutant than S42A. Data are represented as mean ± SEM. *P = 0.02 (Student’s t-test), n = 5 independent experiments. (E) Oxidative stress induced with 1 mM H2O2 caused more cell death in the phosphomimic S42D mutant than in the phospho-resistant S42A mutant. P < 0.001 (Two-way ANOVA), *indicates within time point, and †indicates overall effect of cell line; n = 6–7 independent experiments.
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