Circulation Journal REVIEW Official Journal of the Japanese Circulation Society http://www.j-circ.or.jp Physiologic Functions of Cyclophilin D and the Mitochondrial Permeability Transition Pore John W. Elrod, PhD; Jeffery D. Molkentin, PhD This review focuses on the role of cyclophilin D (CypD) as a prominent mediator of the mitochondrial permeability transition pore (MPTP) and subsequent effects on cardiovascular physiology and pathology. Although a great num- ber of reviews have been written on the MPTP and its effects on cell death, we focus on the biology surrounding CypD itself and the non-cell death physiologic functions of the MPTP. A greater understanding of the physiologic functions of the MPTP and its regulation by CypD will likely suggest novel therapeutic approaches for cardiovascu- lar disease, both dependent and independent of programmed necrotic cell death mechanisms. (Circ J 2013; 77: 1111 – 1122) Key Words: Calcium; Cyclophilin D; Ischemic injury; Metabolism; Mitochondria CypD, including the first 29 AA that encode the N-terminal PPIF Gene: Domains and Structure mitochondrial targeting sequence and other extra-PPIase re- Cyclophilin D (CypD) is a mitochondrial matrix protein en- gions that may impart isomerase target specificity or binding coded by the peptidyl-prolyl cis-trans isomerase F gene (PPIF), regions for modulatory partners.1 The crystal structure of human a member of the greater cyclophilin gene family. Cyclophilins CypD has been elucidated in complex with CsA.6 Kajitani et are conserved through all eukaryotic and even bacterial king- al reported that CypD contained 8 β-strands, 2 α-helices, and doms and all exhibit peptidyl-prolyl cis-trans isomerase (PPIase) 1 310 helix, representing a structure similar to that of the other activity, with most members directly binding the immunosup- known cyclophilin family members.6 The authors also con- pressive drug cyclosporin A (CsA). There are 17 cyclophilins firmed that CsA binding sites in CypD were analogous among in the human genome and all are primarily implicated in pro- other cyclophilins and that binding did not require a confor- tein folding and chaperone function.1 PPIF, located on human mational change. chromosome 10, consists of 6 exons (all coding) and is highly conserved to the yeast homolog.2,3 As an aside, the HUGO gene nomenclature committee incorrectly annotated the CypD Phylogenetic Conservation protein as PPIF (peptidyl-prolyl isomerase F), whereas it As previously stated, PPIF is well conserved across many should have been named PPID. The full-length CypD protein organisms with predicted homology noted even in plants and consists of 207 AA (22 kDa) with the most prominent feature fungi (Table 1).2,7,8 In vertebrates, the gene is extremely well being a 109 AA cyclophilin domain (cl00197, IPR002130) conserved, with the zebrafish (Danio rerio) homologous gene that imparts prolyl-isomerization activity that is conserved (ppifb) having a 78% protein identity match with Homo sapi- among most cyclophilins.4 To illustrate the importance of the ens and functionally being linked to mitochondrial permeabil- isomerase domain, Baines et al showed that an isomerase- ity transition.9 The interest in mitochondrial cyclophilin in other deficient mutant of cyclophilin D (R96G) was unable to rescue species began with the observation that permeability transition mitochondrial swelling or ROS-induced cell death in Ppif–/– may be a highly conserved phenomenon. Indeed, permeability mouse embryonic fibroblasts (MEFs), yet the wildtype (WT) transition has been noted in Drosophila, yeast, plants, fish and version fully rescued.5 These results suggest that the isomerase amphibians. (For an excellent review on this topic see Azzolin domain of CypD is necessary for modulation of the mitochon- et al.10) In addition to academically deciphering the relevance drial permeability transition pore (MPTP) (detailed more ex- in situ, studying pore function in lower organisms allows high- tensively below). throughput screening efforts in the search for novel permeabil- The remaining coding regions impart properties unique to ity transition inhibitors (drug discovery) and the pursuit of the Received March 4, 2013; accepted March 5, 2013; released online March 29, 2013 Center for Translational Medicine, Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA (J.W.E.); De- partment of Pediatrics, University of Cincinnati, Cincinnati Children’s Hospital Medical Center, Howard Hughes Medical Institute, Cincinnati, OH (J.D.M.), USA Mailing address: John W. Elrod, PhD, Center for Translational Medicine, Temple University School of Medicine, 3500 N Broad St, MERB 949, Philadelphia, PA 19140, USA. E-mail: [email protected] and Jeffery D. Molkentin, PhD, Cincinnati Children’s Hospital Medical Center, Howard Hughes Medical Institute, Molecular Cardiovascular Biology, 240 Albert Sabin Way, MLC 7020, Cincinnati, OH 45229, USA. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-13-0321 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Circulation Journal Vol.77, May 2013 1112 ELROD JW et al. Table 1. PPIF Homology Across Species HomoloGene pairwise alignment scores Identity % Species Symbol Protein DNA Homo sapiens PPIF vs. Pan troglodytes PPIF 99.5 99.5 vs. Macaca mulatta PPIF 97.1 96.9 vs. Bos taurus PPIF 91.4 88.3 vs. Mus musculus Ppif 90.3 89 vs. Rattus norvegicus Ppif 89.8 88.7 vs. Gallus gallus PPIF 83.1 72.6 vs. Danio rerio ppifb 78.1 69.7 vs. Drosophila melanogaster Cyp1 72.4 68.1 vs. Anopheles gambiae AgaP_AGAP000462 71.9 68.9 vs. Saccharomyces cerevisiae CPR1 70.4 64.6 vs. Kluyveromyces lactis KLLA0D16676 g 69.8 65.2 vs. Eremothecium gossypii AGOS_AGL177C 70.4 67.5 vs. Schizosaccharomyces pombe ppi1 75.6 66.2 vs. Magnaporthe oryzae MGG_10447 63.9 64.3 vs. Neurospora crassa NCU00726 61.1 61.4 vs. Arabidopsis thaliana CYP20–2 72.3 61.6 vs. Oryza sativa Os05 g0103200 66.1 62.6 identity of the elusive ‘pore-forming’ components of the MPTP red-sensitive mechanism (now known to be the mitochondrial with genetic screening approaches. calcium uniporter (MCU), the genetic identity of which was recently discovered14–16) and that permeability transition re- sulted in the uncoupling of oxidative phosphorylation. The Mitochondrial Targeting and Import same group followed this discovery with numerous reports The mitochondrial targeting sequence for human CypD is detailing the nature of permeability transition and its role in encoded by the following N-terminal leader sequence: (ML- physiology that have largely held to date. The authors concluded ALRCGSRWLGLLSVPRSVPLRLPAARA). The sequence in subsequent studies that the channel is gated in a Ca2+-spe- computationally predicts to be a mitochondrial transit peptide cific manner and that its opening imparts permeability to sol- recognized at the outer mitochondrial membrane (OMM) where utes up to 1.5 kDa in size.17 Hunter et al18–20 also noted that processing allows import into the matrix. This post-transla- MPTP open probability is reduced by ADP, adenine nucleo- tional processing results in the mitochondrial mature form tides, and other divalent cations such as Mg2+ (Sr2+, Ba2+ and (~18 kDa) that accounts for all CypD function. It had been Mn2+ have also been shown to inhibit permeability transition). previously hypothesized that multiple mitochondrial isoforms Also see an excellent accounting of these seminal findings, of cyclophilin exist, but expression of [35S]-labeled CypD by including work predating Hunter and Haworth, in the expert Johnson et al experimentally confirmed cleavage of the pre- reviews of Dr. Paolo Bernardi and colleagues.21,22 dicted mitochondrial localization sequence and mitochondrial 11 import of a single isoform. With careful western blotting tech- Historical Discovery of CypD nique we can visualize both the full-length, cytosolic CypD (~22 kDa) and also the truncated, mitochondrial-localized iso- as a Component of the MPTP form (~18 kDa) in cardiac protein extracts. Although there is Mitochondrial permeability transition is best defined as the little evidence for transcriptional regulation of CypD, Matas collapse of the chemiosmotic gradient across the inner mito- et al reported alterations in the subcellular distribution of CypD chondrial membrane (IMM) mediated by opening of a large- (mitochondrial vs. cytosolic) in rat hearts following volume- conductance pore that we call the MPTP. This is not to be overload induced hypertrophy, suggesting another mode of confused with OMM rupture, which results in the release of potential regulation.12 cytochrome c and apoptogens from the inner membrane space (IMS), potentiating apoptotic cell death signaling (see the in- depth review on OMM permeability by Kroemer et al23). The Historical Discovery of the MPTP MPTP has long been postulated to be a channel with finite The first description of permeability transition can be found in properties that spans the IMM and OMM simultaneously, al- a 1975 manuscript published in The Journal of Biological though the molecular components that actually constitute this Chemistry by Douglas Hunter and Robert Haworth where they presumed structure remain undefined. Perhaps the most ex- concluded that Ca2+ addition to isolated cow heart mitochon- perimentally manipulated feature of the MPTP is that open dria induced a “…nonspecific increase in the permeability of probability is reduced by CsA, which is a potent immunosup- the inner membrane,