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Supplemental Figure Legends (Baines et al) Supplemental Figure 1. Excessive Ca2+ induces mitochondrial permeability transition independent of cyclophilin D. a, Low Ca2+-induced, and b, high Ca2+-induced swelling in cardiac mitochondria from Ppif+/+ and Ppif-/- mice. Data were compiled from 3-5 mitochondrial preparations per group. Error bars indicate ±sem. The low concentration of Ca2+ (250 M) induced a permeability transition in Ppif+/+ mitochondria that was completely reversed by 1 M cyclosporine-A (CsA). However, high Ca2+ (2 mM) could completely overcome the inhibitory effects of CsA. Ppif-/- mitochondria did not exhibit permeability transition in response to low Ca2+. However, similar to Ppif+/+ mitochondria, Ppif-/- mitochondria were able to undergo a CsAinsensitive permeability transition in response to high Ca2+. He and Lemasters (FEBS Letters. 2002, 512:1-7) proposed an interesting model whereby the mitochondrial pore can exist in two conductance modes: a regulated CsA-sensitive mode that requires cyclophilin D binding to the inner membrane, and an unregulated CsA-insensitive mode that is independent of cyclophilin D binding sue to accumulated unfolded proteins. Our findings support such a model and implicate cyclophilin D as an essential mediator of regulated, but not unregulated pore opening. Supplemental Figure 2. Ca2+ uptake in a, Ppif+/+ and b, Ppif-/- liver mitochondria. Isolated liver mitochondria (0.5 mg/ml) were incubated at 25 °C in buffer containing 120 mM KCl, 10 mM Tris pH 7.6, 5 mM succinate, 2 mM KH2PO4, and 1 M Ca2+ green-5N. Pulses of 4 M Ca2+ were added every 2 min (arrows) and fluorescence measured using a spectrofluorometer (excitation 506 nm, emission 532 nm). The Ca2+ green-5N dye cannot penetrate the mitochondria and therefore measures extramitochondrial Ca2+. Each pulse of Ca2+ induces a rapid increase in fluorescence. The signal then decays as the mitochondria sequester the Ca2+ away from the dye. Eventually the mitochondria reach a critical Ca2+ concentration that induces permeability 1 transition, thus releasing Ca2+ back into the medium and causing a large increase in fluorescence. Mitochondria from both Ppif+/+ and Ppif-/- mice were able to take up a given pulse of Ca2+ at a similar rate. However, the Ppif-/- mitochondria were able to take up a greater number of Ca2+ pulses than Ppif+/+ mitochondria before permeability transition was achieved, consistent with the data shown in Figure 1. These data demonstrate that the resistance of Ppif-/- mitochondria to Ca2+-induced permeability transition is not due to alterations in the rate of Ca2+ uptake, and that Ppif-/- mitochondria may have a greater overall capacity for Ca2+. Supplemental Figure 3. Histological and functional assessment of Ppif-/- mouse hearts. a, Histology of Ppif+/+, Ppif+/-, and Ppif-/- mouse hearts. Hearts from 2-month old mice of each genotype were fixed in buffered formalin, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin. There were no overt morphological differences between the three genotypes. b, Quantification of heart-weight to body-weight ratios in Ppif+/+, Ppif+/-, and Ppif-/mice at 2-months of age (N=6 mice per group). Error bars indicate ±sem. c, Echocardiographic analyses of left-ventricular end-diastolic dimension (LVED), d, Left-ventricular end-systolic dimension (LVES), e, Heart rate, and f, Shortening fraction in 2-month old Ppif+/+, Ppif+/-, and Ppif-/- mice (c-f were performed on 4 separate mice per group, each in triplicate). Error bars indicate ±sem. Mice were anesthetized with isofluorane and M-mode echocardiograms were obtained using a Hewlett-Packard Sonos 5500 instrument with a 15 MHz transducer. These functional analyses revealed no difference in cardiac chamber size, heart rate, and contractility between the three genotypes. Supplemental Figure 4 H2O2-induced mitochondrial dysfunction and death in Ppif-/- cells. a, Mitochondrial in Ppif+/+ and Ppif-/- MEFs. MEFs of both genotypes were treated with 250 2 M H2O2 for 6 hrs, stained with the mitochondrial potentiometric dye TMRE, and imaged by confocal microscopy in live cells. Opening of the mitochondrial pore uncouples mitochondria, which in turn dissipates the m signal. H2O2 treatment almost completely abolished mitochondrial in Ppif+/+ MEFs, as exhibited by a decrease in TMRE fluorescence, whereas m was preserved in Ppif-/- MEFs. As a positive control, addition of 50 M carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a mitochondrial uncoupler, was able to dissipate mitochondrial in both Ppif+/+ and Ppif-/- MEFs (data were collected from 2 independent experiments each performed in duplicate). b, Mitochondrial pore opening in Ppif+/+ and Ppif-/MEFs treated as in a. Following H2O2 exposure, MEFs were stained with calcein-AM in the presence of CoCl2, which results in selective staining of mitochondria that is imaged by confocal microscopy. Pore opening results in a movement of calcein out of the mitochondria, where it is quenched by CoCl2. Treatment of Ppif+/+ MEFs with H2O2 caused a reduction in calcein fluorescence that was associated with a more diffuse cytosolic staining, indicative of pore opening. In contrast, calcein fluorescence was retained within the mitochondria of Ppif-/- MEFs, indicative of pore inhibition (data were collected from two independent experiments each performed in duplicate). c, Propidium iodide (PI) staining in H2O2 treated Ppif+/+ and Ppif-/MEFs. MEFs were treated with increasing concentrations of H2O2 for 6 hrs and then incubated with PI for 10 min at room temperature. Under these conditions, PI can only stain the nuclei of cells that have undergone membrane failure, and therefore is mostly an indicator of necrotic cell death. The cells were then fixed in paraformaldehyde, all nuclei counterstained with bisbenzamide, and the number of PI-positive cells determined by an operator blinded to both the genotype and experimental intervention. H2O2 concentration-dependently induced cell death in both Ppif+/+ and Ppif-/- MEFs. However, the number of dead cells was significantly less in Ppif-/MEFs at all concentrations tested (N=3). d, TUNEL staining in H2O2 treated Ppif+/+ and Ppif-/3 MEFs. MEFs were treated as in c, and were then fixed in paraformaldehyde, permeabilized, and then stained using a commercially available TUNEL detection kit (Roche). Similar to the PI staining, H2O2 concentration-dependently induced cell death in both genotypes, but with significantly less cell mortality in Ppif-/- MEFs (N=3). e, Annexin V-FITC staining in H2O2 treated Ppif+/+ and Ppif-/- MEFs. MEFs were treated with 500 M H2O2 for 6 hrs. The supernatants were collected, the remainder of the cells were released from the culture plates, and the cell pellets were combined and incubated with Annexin V-FITC for 30 min at room temperature in Annexin V binding buffer (Molecular Probes). Labeled cells were then subjected to FACS analysis for quantitation of 10,000 cells in each group (N=2). Error bars indicate ±sem. Supplemental Figure 5 Histological and functional assessment of cyclophilin D transgenic mouse hearts. a, Histology of adult hearts from non-transgenic (NTG) and cyclophilin D transgenic lines 26.2 and 25.4. Hearts from 2-month old mice of each genotype were fixed in buffered formalin, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin. The 26.2 line showed no morphological differences to NTG hearts at this young age. However, the 25.4 line exhibited grossly enlarged hearts, associated with enlargement of the ventricular chambers. b, Quantification of heart weight to body weight ratios in NTG, line 26.2 and line 25.4 mice confirmed the enlarged heart phenotype in the 25.4 transgenic animals (N=6). *P<0.05 vs. NTG. Error bars indicate ±sem. c, Echocardiographic analyses of left-ventricular end-diastolic dimension (LVED), d, Left-ventricular end-systolic dimension (LVES), e, Heart rate, and f, Shortening fraction in 6-month old NTG, 26.2 and 25.4 mice. The 26.2 transgenic line exhibited reduced systolic function as evidenced by an increased LVES and reduced shortening fraction compared to NTG controls. The 25.4 line was associated with overt chamber dilation at both diastole and systole, reduced contractility, and reduced heart rate. *P<0.05 vs. NTG. Error bars 4 indicate ±sem. The data shown in c-f were generated from 4 individual mice, each analyzed in triplicate. g, Western blots of cytosolic lysates for cytochrome c and cleaved caspase-9 in NTG and 25.4 transgenic mouse hearts. Release of cytochrome c and processing of caspase-9 was significantly increased in the 25.4 transgenic hearts, indicative of activation of the mitochondrial death pathway. 5