Properties of the Permeability Transition Pore in Mitochondria Devoid of Cyclophilin D

We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif–/– mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif–/– mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo. We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif–/– mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif–/– mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo. The “permeability transition” is a sudden increase of the inner mitochondrial membrane permeability to ions and solutes, which causes dissipation of Δψm, 1The abbreviations used are: Δψm, mitochondrial membrane potential; CsA, cyclosporin A; CRC, calcium retention capacity; CyP-D, cyclophilin D; Δp, proton electrochemical gradient; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; MOPS, 4-morpholinepropanesulfonic acid; PTP, permeability transition pore; Ub0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone (ubiquinone 0). loss of mitochondrial ion homeostasis, impairment of ATP synthesis, and diffusion of solutes down their concentration gradient (1Hunter D.R. Haworth R.A. Arch. Biochem. Biophys. 1979; 195: 453-459Crossref PubMed Scopus (595) Google Scholar). In vitro, at least, this is followed by an osmotically obligatory water flux across the inner membrane with passive swelling, outer membrane rupture, and cytochrome c release (2Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1343) Google Scholar). This complex phenomenon is due to opening of a regulated, high conductance channel of unknown molecular structure, the PTP. PTP opening requires matrix Ca2+, and its open-closed transitions are affected by a striking number of agents that may converge on a set of control elements such as the Δψm (3Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar), matrix pH (4Nicolli A. Petronilli V. Bernardi P. Biochemistry. 1993; 32: 4461-4465Crossref PubMed Scopus (151) Google Scholar), adenine nucleotides (1Hunter D.R. Haworth R.A. Arch. Biochem. Biophys. 1979; 195: 453-459Crossref PubMed Scopus (595) Google Scholar), and the redox potential (5Petronilli V. Costantini P. Scorrano L. Colonna R. Passamonti S. Bernardi P. J. Biol. Chem. 1994; 269: 16638-16642Abstract Full Text PDF PubMed Google Scholar). Interest in the permeability transition as an executioner mechanism of cell death through Ca2+ deregulation and ATP depletion dates to the early 1990s (6Broekemeier K.M. Carpenter Deyo L. Reed D.J. Pfeiffer D.R. FEBS Lett. 1992; 304: 192-194Crossref PubMed Scopus (83) Google Scholar, 7Duchen M.R. McGuinness O. Brown L.A. Crompton M. Cardiovasc. Res. 1993; 27: 1790-1794Crossref PubMed Scopus (265) Google Scholar, 8Pastorino J.G. Snyder J.W. Serroni A. Hoek J.B. Farber J.L. J. Biol. Chem. 1993; 268: 13791-13798Abstract Full Text PDF PubMed Google Scholar, 9Zoeteweij J.P. van de Water B. de Bont H.J. Mulder G.J. Nagelkerke J.F. J. Biol. Chem. 1993; 268: 3384-3388Abstract Full Text PDF PubMed Google Scholar, 10Snyder J.W. Pastorino J.G. Attie A.M. Farber J.L. Biochem. Pharmacol. 1992; 44: 833-835Crossref PubMed Scopus (41) Google Scholar, 11Imberti R. Nieminen A.L. Herman B. Lemasters J.J. J. Pharmacol. Exp. 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The putative receptor for CsA is CyP-D, a matrix peptidyl-prolyl cis-trans isomerase that is inhibited by CsA in the same range of concentrations that inhibits the pore (16Davidson A.M. Halestrap A.P. Biochem. J. 1990; 268: 147-152Crossref PubMed Scopus (79) Google Scholar) through an effect that does not require calcineurin inhibition (17Nicolli A. Basso E. Petronilli V. Wenger R.M. Bernardi P. J. Biol. Chem. 1996; 271: 2185-2192Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Largely through the use of CsA key advances have been made in understanding the role of the PTP in several ex vivo and in vivo models of disease such as ischemia-reperfusion injury of the heart (18Griffiths E.J. Halestrap A.P. J. Mol. Cell Cardiol. 1993; 25: 1461-1469Abstract Full Text PDF PubMed Scopus (506) Google Scholar, 19Di Lisa F. Menabò R. Canton M. Barile M. Bernardi P. J. Biol. 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Sci. U. S. A. 2003; 100: 10014-10019Crossref PubMed Scopus (51) Google Scholar), and fulminant hepatitis mediated by TNFα or Fas (32Crouser E.D. Julian M.W. Blaho D.V. Pfeiffer D.R. Crit. Care Med. 2002; 30: 276-284Crossref PubMed Scopus (224) Google Scholar, 33Soriano M.E. Nicolosi L. Bernardi P. J. Biol. Chem. 2004; 279: 36803-36808Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 34Feldmann G. Haouzi D. Moreau A. Durand S.A. Bringuier A. Berson A. Mansouri A. Fau D. Pessayre D. Hepatology. 2000; 31: 674-683Crossref PubMed Scopus (123) Google Scholar). Evidence that CyP-D is a modulator of the PTP remains indirect, however, and CyP-D overexpression did not cause the expected sensitization to cell death but rather protected from cell death induced by oxidative stress and mediated by mitochondria (35Lin D.T. Lechleiter J.D. J. Biol. Chem. 2002; 277: 31134-31141Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). To unambiguously resolve basic questions related to the role of CyP-D in PTP regulation, and the function of the permeability transition in normal biological processes like programmed and accidental cell death, we have generated a mouse line in which the expression of CyP-D has been eliminated by “knock-out” of the Ppif gene. We report here for the first time the properties of the permeability transition in mitochondria from Ppif–/– animals, which are devoid of CyP-D. Part of these results have already been presented in abstract form (36Basso E. Bernardi P. Forte M. Biophys. J. 2004; 86: 357AGoogle Scholar). Generation and Characterization of Ppif–/–Mice—To identify mouse homologs of human CyP-D gene, degenerate oligonucleotide primers representing sequences encoding the unique N terminus of this molecule (amino acid sequence (residues 30–47) obtained from the purified human protein) and the common C terminus (amino acids 200–208) were used to PCR-amplify sequences encoding this molecule from cDNA generated from mouse liver RNA. To identify and characterize the region of the mouse genome encoding CyP-D, a unique set of PCR probes was generated from full-length mouse cDNA and used to screen a mouse bacterial artificial chromosome genomic library prepared from 129Sv ES cells. Several overlapping bacterial artificial chromosome clones were identified and genomic regions encoding Ppif identified by restriction enzyme digestion, PCR analysis, and Southern blotting. Eventually, a 23.5-kb fragment of genomic sequence containing the Ppif gene was characterized. To generate mice in which the expression of Ppif has been eliminated, ES cells were cultured using standard conditions, transfected by electroporation with the targeting construct, transfectants selected with appropriate antibiotics (G418 and ganciclovir), and candidate ES cells screened by PCR analysis and Southern blotting for replacement of the endogenous Ppif gene with the targeting construct. Male chimeras were subsequently mated with black, non-agouti C57BL/6 female mice and offspring evaluated for germ line transmission. Several of the chimeric males were able to pass the Ppif knock-out gene to progeny as assessed by both PCR analysis and Southern blotting. F1 heterozygotes were then back-crossed for eight generations into a C57BL/6 genetic background and isogenic heterozygotes intercrossed to generate homozygous wild-type and Ppif–/– animals. To assess the expression of CyP-D protein in mice of the indicated genotypes, mitochondria were prepared from liver, heart, and kidney by homogenization and differential centrifugation following published protocols. Mitochondrial proteins were then separated on 15% SDS-polyacrylamide gels, proteins transferred to nitrocellulose, and blots probed for CyP-D using an antibody generated to unique N-terminal epitope from Affinity Bioreagents (Golden, CO) or the antibody described by Lin and Lechleiter (35Lin D.T. Lechleiter J.D. J. Biol. Chem. 2002; 277: 31134-31141Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). An anti-voltage-dependent anion channel antibody kindly provided by Dr. William Craigen (Baylor College of Medicine) was used as a control for loading. Assays on Isolated Mouse Liver Mitochondria—Mitochondria were isolated from the livers of wild-type and Ppif–/– C57BL/6 mice exactly as described previously for rat liver mitochondria (37Costantini P. Petronilli V. Colonna R. Bernardi P. Toxicology. 1995; 99: 77-88Crossref PubMed Scopus (129) Google Scholar) except that 10 ml of isolation medium per liver were used. The CRC of mitochondrial preparations was assessed fluorimetrically in the presence of the Ca2+ indicator Calcium Green-5N (Molecular Probes) with a PerkinElmer Life Sciences LS50B spectrofluorimeter exactly as described previously (38Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Oxygen consumption was measured polarographically with a Clark oxygen electrode in a closed 2-ml vessel. Mitochondrial swelling was measured as the decrease of 90° light scattering at 540 nm in a PerkinElmer Life Sciences 650-40 spectrofluorimeter. All instruments were equipped with magnetic stirring and thermostatic control. The incubation conditions are specified in the figure legends. CyP-D (also known as CyP-F in mice) is a member of the larger cyclophilin family and is equivalent to the previously cloned cDNA encoding human CyP-3 (39Bergsma D.J. Eder C. Gross M. Kersten H. Sylvester D. Appelbaum E. Cusimano D. Livi G.P. McLaughlin M.M. Kasyan K. J. Biol. Chem. 1991; 266: 23204-23214Abstract Full Text PDF PubMed Google Scholar). CyP-D is nuclearly encoded and contains a mitochondrial targeting presequence that is cleaved after translocation of the protein into the matrix (39Bergsma D.J. Eder C. Gross M. Kersten H. Sylvester D. Appelbaum E. Cusimano D. Livi G.P. McLaughlin M.M. Kasyan K. J. Biol. Chem. 1991; 266: 23204-23214Abstract Full Text PDF PubMed Google Scholar). In addition, mature forms of the protein also contain a unique N terminus, which serves to identify CyP-D from other CyP isoforms. Probes generated from sequences encoding human CyP-D were used to identify cDNAs encoding murine CyP-D. Subsequently, sequences representing the Ppif gene encoding CyP-D were characterized (see “Experimental Procedures”) and included 11 and of the and The Ppif gene of genomic and of separated by of human and mouse genome that a gene CyP-D in and that the murine Ppif gene is on To generate of mice in CyP-D, a targeting was generated in which the gene was so that the first of the Ppif gene, of the were eliminated The targeting of Ppif of the and of genomic sequence the Ppif gene the gene. The gene was so that its from the to that used to for ES cell in which the Ppif gene had been were identified by PCR analysis of genomic and Southern blotting not ES cells were then into and chimeric offspring evaluated for the to generate F1 heterozygotes the were back-crossed eight to C57BL/6 and heterozygotes then intercrossed to generate isogenic wild-type and Ppif–/– mice. PCR analysis of genomic from and Ppif–/– mice that the Ppif gene had been of CyP-D at the protein that expression was to in liver mitochondria from and that no CyP-D protein was in mitochondria prepared from liver or heart not of Ppif–/– mice. the absence of CyP-D, mitochondria from Ppif–/– mice displayed and of that were from of mitochondria prepared from wild-type mice that CyP-D does not and ATP We then the properties of the permeability transition in liver mitochondria from wild-type and Ppif–/– mice with the CRC which the Ca2+ required to open the PTP in a of mitochondria in The experiments of that mitochondria from Ppif–/– mice required about twice the of Ca2+ necessary to open the PTP in wild-type mitochondria a and of CsA caused the expected increase of CRC in wild-type but not in Ppif–/– mitochondria both were to c and (see These experiments that CyP-D modulates the affinity of the PTP for Ca2+ and the first that CyP-D is the target for the of CsA on the PTP. The PTP is by the Δp, in the that the pore open as the membrane potential and at matrix pH (3Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar). To assess the of CyP-D affected the PTP we the effect of the of to mitochondria with a of Ca2+ that is not to open the PTP per but is for the opening by mitochondria from wild-type animals, mitochondria from Ppif–/– mice the PTP the of a and from the of wild-type the swelling response of Ppif–/– mitochondria was insensitive to CsA and was as to c and that no effect was in Ppif–/– mitochondria the concentration of CsA was to not The PTP open also a on the pH of the incubation medium (1Hunter D.R. Haworth R.A. Arch. Biochem. Biophys. 1979; 195: 453-459Crossref PubMed Scopus (595) Google Scholar, A.P. Biochem. J. 1991; PubMed Scopus Google Scholar). PTP inhibition as pH is from to and we have that the effect is from the matrix of the inner membrane (3Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar) through of (4Nicolli A. Petronilli V. Bernardi P. Biochemistry. 1993; 32: 4461-4465Crossref PubMed Scopus (151) Google Scholar). The PTP is also inhibited as the pH is increased through an mechanism (4Nicolli A. Petronilli V. Bernardi P. Biochemistry. 1993; 32: 4461-4465Crossref PubMed Scopus (151) Google Scholar). To assess the of PTP opening on matrix pH we used mitochondria in a medium (4Nicolli A. Petronilli V. Bernardi P. Biochemistry. 1993; 32: 4461-4465Crossref PubMed Scopus (151) Google Scholar). In this Ca2+ is by the diffusion potential A.P. FEBS Lett. 10: PubMed Scopus Google Scholar), and Ca2+ of matrix pH, which these conditions pH (4Nicolli A. Petronilli V. Bernardi P. Biochemistry. 1993; 32: 4461-4465Crossref PubMed Scopus (151) Google Scholar). In these the PTP response was in Ppif–/– and wild-type mitochondria in the pH range the Ppif–/– mitochondria were to inhibition by pH These results that CyP-D does not the of matrix pH on the PTP and that the that can by (4Nicolli A. Petronilli V. Bernardi P. Biochemistry. 1993; 32: 4461-4465Crossref PubMed Scopus (151) Google Scholar) are not on CyP-D. The PTP is to oxidative and a at is of in the PTP response to The PTP is as the is to a (5Petronilli V. Costantini P. Scorrano L. Colonna R. Passamonti S. Bernardi P. J. Biol. Chem. 1994; 269: 16638-16642Abstract Full Text PDF PubMed Google Scholar). Mitochondria from Ppif–/– mice were as as from wild-type to the of which through the (5Petronilli V. Costantini P. Scorrano L. Colonna R. Passamonti S. Bernardi P. J. Biol. Chem. 1994; 269: 16638-16642Abstract Full Text PDF PubMed Google Scholar). Ppif–/– mitochondria to require concentrations of the effect was to that in wild-type mitochondria (see also of the on the CRC already described for CsA, Ub0, and is presented in which also the of which inhibits the PTP (1Hunter D.R. Haworth R.A. Arch. Biochem. Biophys. 1979; 195: 453-459Crossref PubMed Scopus (595) Google Scholar), and of the a PTP that is in the absence of Ca2+ (3Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar, E. Bernardi P. J. Bioenerg. Biomembr. 1991; PubMed Scopus Google Scholar). can that Ppif–/– mitochondria were to Ub0, which inhibits the pore through a E. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (224) Google Scholar), and to which may through a effect on the adenine by in the (2Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1343) Google Scholar). The that and are of the PTP in Ppif–/– in wild-type mitochondria on their the CRC are from obtained in the presence of or of the of these are not in the of animals. that both and affected the PTP in wild-type and Ppif–/– mitochondria. In our experiments demonstrate that the PTP can form and open in the absence of CyP-D. The of of CsA on the PTP in Ppif–/– mitochondria that CyP-D represents the unique target for PTP inhibition by A from our is that CyP-D modulates the PTP sensitivity to The Ca2+ of the Ppif–/– mitochondria is similar to that in mitochondria from mice with genetic of the adenine J. 2004; PubMed Scopus Google Scholar), that the PTP sensitivity to Ca2+ may on is to stress that the PTP of Ppif–/– mitochondria its basic by the and its sensitivity to other Ca2+ and to other Ppif–/– were at the expected and were from C57BL/6 that CyP-D is for and of mice. is that this of an may due to the sensitivity of the PTP to Ca2+ is by that are not in isolated mitochondria. An is by the that CyP-D overexpression cells from that CyP-D may also a role as a cell molecule on or other the PTP (35Lin D.T. Lechleiter J.D. J. Biol. Chem. 2002; 277: 31134-31141Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). This function of CyP-D that the and may in the Ppif–/– and that the of in Ppif–/– may require the of in vivo disease