www.sciencemag.org/content/350/6265/aad2459 /suppl/DC1

Supplementary Materials for

Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice

Guohua Gong, Moshi Song, Gyorgy Csordas, Daniel P. Kelly, Scot J. Matkovich, Gerald W. Dorn II*

*Corresponding author. E-mail: gdorn@dom.wustl.edu

Published 4 December 2015, Science 350, aad2459 (2015) DOI: 10.1126/science.aad2459

This PDF file includes:

Materials and Methods Figs. S1 to S19 References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/350/6265/aad2459/DC1)

Data set S1: Gene lists by functional category Data set S2: Regulated gene details

Materials and Methods Mfn2 KO MEFs were purchased from ATCC (CRL-2993). All other MEFs were

derived from E13.5 embryos of wild-type C57BL/6J, PINK1, or Parkin knockout mice (38, 39). Briefly, after removing the head, visceral tissues and gonads, the bodies were rinsed with PBS twice and digested with 10 mL 0.05% trypsin-EDTA three times for 10 min at 37 °C. Dissociated cells were pelleted by centrifugation at 200g and plated on 150 mm plastic culture dishes prior to incubation at 37 °C with 5% CO2. Here, primary MEFs were used within 5 passages to avoid replicative senescence. All cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1x nonessential amino acids, 1 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen).

Adenoviruses expressing wild-type human Mfn2 (Mfn2-WT), Mfn2-AA, Mfn2-EE mutants, or Parkin were generated using the pENTR Directional TOPO cloning kit (Invitrogen, K2400-20) followed by recombination into the pAd/CMV/V5-DEST Gateway vector (Invitrogen, V493-20). Empty adenovirus was purchased from Vector Biolabs. Adeno-mCherry-Parkin was a gift from Dr. Asa Gustafsson, University of California San Diego. MEFs (0.1 million) were plated on 25 mm round coverslips in 6-well plates. After cell attachment, adenoviruses were added at a multiplicity of infection (MOI) of 20-100. MEFs were maintained in culture for 48 hours or as indicated prior to imaging. Cardiomyocyte-specific human Mfn2-WT transgenic mice were generated with the Myh6 promoter system. Cardiomyocyte-specific human Mfn2-AA and Mfn2-EE transgenic mice were generated with the Myh6 promoter-driven doxycycline suppressible (“tet-off”) bi-transgenic system (20). Doxycycline food was withdrawn at 3 weeks, 8 weeks or never administered to dams and pups for induction of transgene expression. M-mode echocardiography, histopathological studies, and all other analyses were performed on age-matched non-transgenic or tet-off littermates. ParkinloxP/loxP mice (11) were obtained from Lexicon Pharmaceuticals and bred with mice carrying a Myh6 promoter-driven nuclear-directed modified estrogen receptor (MER)-Cre transgene (40) to achieve tamoxifen-inducible gene deletion. A single 1 mg dose of tamoxifen (Sigma, T5648), dissolved in 90% sesame oil (Sigma, S3547) and 10% ethanol, was administered subcutaneously to one-day neonatal mice (41). The mouse hearts were harvested at 21 days. To detect the conditional cardiac-specific Parkin ablation, in which exon 7 was flanked by loxP sites, PCR was performed using heart DNA extracted using the DNeasy blood & tissue kit (Qiagen, 69504). The amplicon for wild-type Parkin is 365 bp, Parkin floxed allele 433 bp, and Parkin knockout 262 bp. Histopathological and electron microscopic studies were performed on age-matched ParkinloxP/loxP littermates. All experimental procedures were approved by the Animal Studies Committee at Washington University School of Medicine.

Immunoblot analyses were performed using cultured cells or mouse hearts. Adenovirally-infected cultured MEFs were harvested in lysis buffer (Cell Signaling, #9803S) 48 hours after gene transfer. Cardiac tissues were collected, snap-frozen in liquid nitrogen, homogenized in either tissue extraction reagent (Invitrogen, FNN0071) or a homogenizing buffer (10 mM HEPES, 320 mM sucrose, 3 mM MgCl2 and 1 mM DTT), supplemented with protease inhibitors (Roche, 05892970001) and phosphatase

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inhibitors (Roche, 04906837001). Mitochondrial fractions and post-mitochondrial fractions were separated and immunoblotting performed as previously described (18, 21). Mouse monoclonal anti-Flag (F3165, 1:1000) was from Sigma Aldrich. Rabbit polyclonal anti-Mfn1 (sc-50330, 1:200), rabbit polyclonal anti-ANT (sc-11433, 1:300), mouse monoclonal anti-ubiquitin (sc-8017, 1:200), rabbit polyclonal anti-Miro2 (sc-135387, 1:100) and rabbit polyclonal anti-AFG3L2 (sc-84687, 1:500) were from Santa Cruz Biotechnology. Mouse monoclonal anti-Mfn2 (ab56889, 1:500), mouse monoclonal anti-Drp1 (ab56788, 1:1000), mouse monoclonal anti-TOMM20 (ab56783, 1:1000), mouse monoclonal anti-p62 (ab56416, 1:1000), mouse monoclonal anti-COX IV (ab14744, 1:1000), rabbit polyclonal anti-VDAC1 (ab15895, 1:1000), rabbit monoclonal anti-MFF (ab109275, 1:500), mouse monoclonal total OXPHOS rodent WB antibody cocktail (ab110413, 1:1000), rabbit monoclonal anti-FGF21 (ab171941, 1:1000), rabbit polyclonal anti-LONP1 (ab103809, 1:250), rabbit polyclonal anti-Hsp60 (ab46798, 1:2000), mouse monoclonal anti-Miro1 (ab55035, 1:250), and mouse monoclonal anti-GAPDH (ab8245, 1:3,000) were from Abcam. Rabbit polyclonal anti-Parkin (CS2132, 1:1000), rabbit polyclonal anti-Caspase 3 (#9665, 1:1000) and mouse monoclonal anti-Myc-Tag (CS2276, 1:1000) were from Cell Signaling. Secondary antibodies were horseradish peroxidase-conjugated anti-mouse secondary antibody (Cell Signaling, 7076S, 1:4000) and goat anti-rabbit secondary antibody (Thermo Scientific, 31460, 1:4000). Membranes were visualized using ECL enhanced chemiluminescence substrate (PerkinElmer, NEL105001EA) on an Odyssey system (Li-COR). Signal intensity was quantified using Image Studio v3.1 software (Li-COR) with background subtraction.

The substrate for in vitro PINK1 phosphorylation assays was recombinant Mfn2 obtained by FLAG immunoprecipitation. The plasmid containing Flag-tagged human wild-type Mfn2 (Flag-Mfn2) was previously described (12). HEK293 cells were transfected using FuGENE HD transfection reagent (Promega, E2311) with the plasmid for 48 hours. The cells were harvested and lysed in cell extraction buffer (Invitrogen, FNN0011) supplemented with 1 mM PMSF and complete mini protease inhibitor cocktail tablet (Roche, 05892970001). Flag-Mfn2 was immunoprecipitated from ~2 mg of cell lysate with 5 µg of mouse monoclonal anti-Flag antibody (Sigma, F3165) using Dynabeads protein G immunoprecipitation kit according to the manufacturer’s protocol (Invitrogen, 10007D). ~0.75 µg of Flag-Mfn2 was incubated with 14 µg of recombinant WT MBP-TcPINK1 (MRC-PPU Reagents, DU34701) or KD MBP-TcPINK1 D359/A (MRC-PPU Reagents, DU34832) (resulting in a 25:1 molar ratio of TcPINK1/Mfn2) in kinase buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM DTT, 0.1 mM EGTA, 10 mM MgOAc, and 100 µM ATP for 45 min at 30°C. Another ~0.75 µg of Flag-Mfn2 alone was incubated with 50 units of calf intestinal phosphatase (CIP) for 45 min at 30°C. After the kinase assay, samples were immediately mixed with 2x Laemmli buffer and loaded onto 7.5% polyacrylamide gel. Mobility shift detection of phosphorylated Mfn2 using Phos-tag SDS-PAGE was performed as previously described (12). Briefly, samples were loaded onto 7.5% polyacrylamide gels containing 25 µM phos-tag acrylamide (Wako Chemicals USA, Richmond, VA) and 50 µM MnCl2. Gels were washed twice with 1x transfer buffer containing 1mM EDTA and once with 1x transfer buffer without EDTA for 10 min at room temperature. Proteins were transferred to PVDF membranes and immunoblotting for Mfn2 was performed as described above.

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Mfn2-Miro1/Miro2/Parkin coimmunoprecipitation used HEK293 cells transfected using using FuGENE HD transfection reagent (Promega, E2311) with constructs containing Flag-Mfn2-WT, Flag-Mfn2-AA, Flag-Mfn2-EE, myc-Miro1, myc-Miro2, HA-Parkin, or pcDNA3.1(+) plasmid as a vector control for 48 hours. Cells were harvested and lysed, and Flag-Mfn2 was immunoprecipitated from ~0.5 mg of cell lysate with 5 µg of mouse monoclonal anti-Flag antibody (Sigma, F3165) as described above. Beads were boiled in 2x Laemmli buffer before separation by SDS-PAGE.

Live cell imaging used a Nikon Ti confocal microscope equipped with a 60x 1.3NA oil immersion objective as previously described (21). For visualization of mitochondria, MEFs were stained with 200 nM MitoTracker Green (Invitrogen, M-7514) and 10 μg/mL Hoechst dye (Invitrogen, 33342). To assay Parkin translocation, MEFs were infected with adeno-mCherry Parkin for 48 hours. For assessment of lysosomal engulfment of mitochondria, MEFs were stained with 50 nM LysoTracker Red (Invitrogen, L-7528) and 200 nM MitoTracker Green. When indicated, 10 µM FCCP or Antimycin A were applied for 1 hour to promote mitochondrial depolarization.

Histological studies were performed on mouse hearts fixed with 4% formaldehyde solution in PBS. Tissues were paraffin-embedded and sectioned at a thickness of 5 μm on a Leica RM2255 rotary microtome. The sections were de-paraffinized in xylene and rehydrated with a gradient (100-50%) of ethanol, followed by a wash in distilled water. Masson’s trichrome staining (Sigma, HT15, HT10132 & 34256) and TUNEL assay (Promega, G3250) were performed according to the manufacturers’ protocols. Wheat germ agglutinin staining (WGA) was performed with 10 μg/mL FITC conjugated-wheat germ agglutinin (Invitrogen, W834) for 30 min at 37°C. For Evans blue studies (EBD), mice were intraperitoneally injected with 1% Evans blue dye PBS (Sigma, E2129) solution at 1% volume relative (mL) to body mass (g) ~24 h prior to tissue collection. Evans blue positive cardiomyocytes fluoresce with red color. Hematoxylin and eosin (HE) (Biocare Medical, 080514, 103114A) staining was performed using standard methods .

Ultrastructural examination of osmium tetroxide/uranyl acetate stained mouse heart thin sections (90 nm) used a Jeol electron microscope (JEM-1400) at 2,000 - 5, 000x direct magnification (JEOL, Tokyo, Japan). Mitochondrial aspect ratio (the ratio of length/width), content (% of mitochondrial area compared to whole-cell area) and individual mitochondrial size were quantified using the open-source image analysis program ImageJ (NIH). In live cell studies, Parkin aggregation was calculated as % of cells with clumped mCherry-Parkin compared to all cells present. Lysosomal engulfment of mitochondria was calculated by counting the number of co-localized lysosomes and mitochondria per cell detected by confocal co-localization of LysoTracker Red and MitoTracker Green (21).

Mitochondria were isolated from minced mouse hearts, livers, or gastrocnemius digested with trypsin, homogenized with a glass/Teflon Potter Elvejhem homogenizer, and then centrifuged at 800g for 10 min at 4 °C. The supernatants were centrifuged at 8,000g for 10 min at 4 °C and the resulting supernatants were discarded. The pellet containing the mitochondria was washed and centrifuged at 8,000g for 10 min at 4 °C before resuspension. Mitochondrial protein concentration was colorimetrically determined using Bio-Rad protein assay dye reagent (Bio-Rad, 500-0006). Mitochondrial respiration (oxygen consumption) was measured in freshly isolated cardiac

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mitochondria using a Strathkelvin 782 apparatus (Strathkelvin Instruments Limited, North Lanarkshire, Scotland). Briefly, 100 μL of IBm3 buffer (0.25 M sucrose, 0.01 M Tris-HCl, 5 mM MgCl2, 20 μM EGTA and 2 mM KH2PO4, pH adjusted to 7.4) with substrate glutamate/malate (5 mM/2.5 mM, Sigma, 27647/M1000) was added into the MT200/MT200A respirometer chamber. Upon stabilization, 20 μg of mitochondria were added, basal oxygen consumption rate (state 2) and ADP (400 μM, Sigma, A5285) stimulated oxygen consumption rate (state 3) were measured. Oligomycin (0.6 μg/mL, Sigma, 75351) was added to inhibit ATP-synthase. The maximum respiration was stimulated with 50 nM FCCP. Mitochondrial H2O2 production was assessed with the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen, A22188) according to the manufacturer’s instructions. Calcium-induced mitochondrial permeability transition pore opening was determined in freshly isolated cardiac mitochondria. Mitochondria were suspended in 200 μL reaction buffer (120 mM KCl, 10 mM Tris, pH 7.6 and 5 mM KH2PO4) at a concentration of 250 μg/mL and stimulated by the addition of 250 μM CaCl2. The absorbance was measured at intervals of 30 sec for 20 min at room temperature using a Spectra MAX M5 (Molecular Devices, Sunnyvale, CA) 96 well plate reader at 540 nm. For palmitoylcarnitine- and pyruvate-stimulated respiration measurement, mouse cardiac myocytes were isolated from 5 week-old mice following a previously reported protocol (42). Cells were suspended in respiration buffer (125 mM KCl, 20 mM HEPES, 3 mM Mg-acetate, 5 mM KH2PO4, 0.4 mM EGTA, 0.3 mM DTT and 2 mg/ml BSA, pH 7.1). For palmitoyl-L-carnitine (PC) respiration, 20 μM PC and 5 mM malate were added and followed with 1 mM ADP and oligomycin. For pyruvate respiration, 10 mM pyruvate and 5 mM malate were added and followed with ADP and 0.6 µg/mL oligomycin.

Flow cytometric analysis of cardiac mitochondria was performed after labeling with 200 nM MitoTracker Green (Invitrogen, M-7514) or 2.5 μM MitoSOX red (Invitrogen, M36008) for 20 min at room temperature. Mitochondrial size (forward scatter, FSC) or mitochondrial superoxide level (MitoSOX red fluorescence intensity detected by PE channel) were assessed using BD LSR II Flow Cytometer (Becton Dickinson, San Jose, CA). Data are shown as histograms for, and as bar graph of average signal intensity of, ~50,000 ungated events.

Transcriptomics analyses were performed using RNA-sequencing. Total RNA was isolated from flash-frozen hearts using Trizol (Invitrogen). Polyadenylated RNA selection on oligo-dT Dynabeads, fragmentation, first and second strand cDNA synthesis, and ligation of indexed adapters for Illumina sequencing were carried out as previously described (43, 44). Single-end, 50 nt reads were obtained on an Illumina HiSeq 2500. A large number of RNA-sequencing libraries (total 56) were prepared to analyze 4 different genotypes (nontransgenic, Mfn2wt-TG, tet-off, and Mfn2AA-TG) at 3 different time points (P1, P21 and 5 wk), which necessitated preparation of libraries via separated bench procedures and analysis of multiplexed libraries in separate sequencing lanes. All libraries for a particular time point were prepared and analyzed together. To minimize ‘batch effects’ when analyzing progressive alteration in gene expression in nontransgenic animals over time, new sequencing libraries were prepared from nontransgenic total RNA aliquots from each time point, and multiplexed and analyzed together on the same sequencing lane. Total reads per individual heart sample averaged 14.3 million, with

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66% alignment to the mouse transcriptome defined by the Illumina iGenomes annotation for UCSC mm10 (http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn). Mapping of sequencing reads to the mouse transcriptome was performed using Tophat 2.0.10 and a command of the following form, which instructs Tophat to consider only exon-exon junctions defined in the supplied annotation gtf and to allow a maximum of 1 mismatched nucleotide:tophat --segment-length 25 --segment-mismatches 0 --no-novel-indels -o output_directory -T -n 1 –-transcriptome-index=transcriptome-data/known-mouse-genes bowtie-index/mmus-genome heart-sample.fastq. The number of reads pertaining to each known transcriptome feature (“gene”) was obtained from alignment (.bam/.sam) files produced by Tophat, using htseq-count (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.htmL). Genes with fewer than an average of 1/100,000th of the aligned reads in at least one genotype or treatment group were eliminated, leaving ~9,000 genes (depending on the group of samples undergoing simultaneous comparison) for downstream analyses.

After removal of a total of 6 libraries that exhibited outlier properties in principal components analysis and/or alignment to the defined mouse transcriptome <40%, biological replicate mRNA-sequencing libraries at each time point were as follows:

P1: 6 nontransgenic, 4 Mfn2 WT, 5 tet-off, 3 Mfn2AA P21: 6 nontransgenic, 6 Mfn2 WT, 5 tet-off, 6 Mfn2AA 5 wk: 4 nontransgenic, 4 Mfn2 WT, 3 tet-off, 4 Mfn2AA Statistical analyses of differentially expressed mRNAs used the R/Bioconductor

package DESeq 1.9 (45) (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html). Read counts were normalized for library depth, and pairwise comparisons between groups, measuring fold-differences, uncorrected p-values from the negative binomial distribution, and adjusted p-values (false discovery rate; FDR) were obtained. Partek Genomics Suite v6.6 (Partek, St. Louis, MO) was used for quality control by principal components analysis and for heat map construction. To visualize differentially expressed mRNAs, normalized read counts from DESeq were subjected to unsupervised hierarchical clustering with Euclidean distance and average linkage using Partek Genomics Suite v6.6. Unless otherwise specified, standardized heatmaps are shown in which map colors represent the extent of deviation from the row average. Significant comparisons were defined as those with a fold-difference of at least ±25% and FDR < 0.02, unless otherwise specified. For clarity of presentation and to enable comparison with other studies, individual gene expression levels are rendered as FPKM (Fragments Per Kilobase of exon per Million reads mapped to the transcriptome) calculated using Cufflinks 2.1.1 (46). However, all fold-differences and statistics were calculated using DESeq.

To focus on mitochondrially-localized proteins and their coding genes, we obtained a list of 1098 genes from mouse MitoCarta (http://www.broadinstitute.org/pubs/MitoCarta/) (47). 873 of these genes were expressed at FPKM≥3 in a compendium of nontransgenic, adult, FVB/N mouse hearts (48) (NCBI GEO GSE55792) or in E13.5 hearts (NCBI GEO GSE58455) (49) in the current data set, and were used for heatmap construction. To compare the expression profiles of

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metabolic genes, mRNAs were classified into the following groups based on existing KEGG pathways (50): glycolysis #00010, fatty acid entry into the citric acid cycle #00071, branched-chain amino acid entry into the citric acid cycle #00280, the citric acid cycle itself #00020, and oxidative phosphorylation #00190. A full list of the genes assigned to each category is provided in Supplemental dataset 1. Pathway-specific gene regulation in figure 4C between 5 wk and P1 hearts, for WT Mfn2 and Mfn2 AA groups, was compared via 2-way ANOVA on log2-transformed 5 wk / P1 ratios. Significance between WT Mfn2 and Mfn2 AA gene regulation was taken at p<0.05. For biological and biochemical studies the data are reported as mean ± SEM. Statistical comparisons used unpaired Student t-test or one-way ANOVA with Bonferroni corrections, as appropriate. P<0.05 was defined as significant.

Metabolomic analyses were performed on acylcarnitines and organic acids from nontransgenic P1, and 3 week-old mouse hearts. Flash-frozen biventricular tissue was lyophilized to dryness and powdered using a Precellys (bead-based) system, then subsequently homogenized in 1:1 acetonitrile/0.3% formic acid. Both acylcarnitine and organic acid abundances were normalized to mg of dry heart powder, and 1-way ANOVA was used to test for differences between genotypes (at α=0.05). For acylcarnitines, a 100 μL aliquot of mouse heart homogenate was spiked with a 10 µL mixture of heavy isotope-labeled acylcarnitine internal standards (C2, C3, C4 butyryl, C5 isovaleryl, C6, C8, C10, C12, C14, C16, and C18 acylcarnitines; Sigma-Aldrich, St. Louis, MO; Toronto Research Chemicals, Toronto, Canada; Larodan, Solna, Sweden). Samples were treated with 800 µL of ice-cold methanol and centrifuged to pellet precipitated protein. The methanolic extract was then dried under nitrogen. Acylcarnitines were derivatized by reconstituting the dried methanolic extract in 100 μL of 0.2 M O-benzylhydroxylamine (Sigma-Aldrich, St. Louis, MO) and 10 μL of 2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich, St. Louis, MO). Reconstituted samples were vortexed at 30 sec and allowed to incubate at room temperature for 10 min prior to analysis by LC/MS/MS. Derivatized acylcarnitines were separated on a 2.1 x 100 mm, 1.7 μm Waters Acquity UPLC BEH C18 column maintained at 50°C using a 12.5-min linear gradient with 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.4 mL/min. Quantitation of derivatized acylcarnitines was achieved using multiple reaction monitoring on an Agilent 1290 Infinity HPLC/6490 triple quadrupole mass spectrometer (Agilent, Wilmington, DE). A standard calibration curve (0.025-50 µM for C2; 0.0025-5 μM for all other acylcarnitines) for derivatized acylcarnitines was prepared by spiking 10 µL aliquots of C2, C3, C4 butyryl, C5 isovaleryl, C6, C8, C10, C12, C14, C16, and C18 acylcarnitines (Sigma-Aldrich, St. Louis, MO; Toronto Research Chemicals, Toronto, Canada; Larodan, Solna, Sweden; R&D Systems, Minneapolis, MN) and their corresponding heavy isotope-labeled internal standards (Sigma-Aldrich, St. Louis, MO; Toronto Research Chemicals, Toronto, Canada; Larodan, Solna, Sweden) into 100-µL aliquots of a 1:1 acetonitrile/0.3% formic acid solution. Samples were derivatized and extracted similarly to acylcarnitines in mouse heart homogenate.

For organic acids, a 50 μL aliquot of mouse heart homogenate was spiked with a 10 μL mixture of heavy isotope-labeled organic acid internal standards (lactate, pyruvate, 3-hydroxybutyrate, succinate, fumarate, malate, α-ketoglutarate, and citrate; Sigma-Aldrich, St Louis, MO; Cambridge Isotopes, Cambridge, MA; CDN Isotopes, Quebec,

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Canada). This was followed by the addition of 50 μL of 0.4 M O-benzylhydroxylamine and 10 μL of 2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Samples were vortexed thoroughly and derivatized at room temperature for 10 min. The derivatized organic acids were then extracted from the homogenate by liquid-liquid extraction using 100 μL of water and 600 μL of ethyl acetate. Samples were vortexed for 5 sec and then centrifuged at 18,000 x g for 5 min at 10°C. A 100 μL aliquot of the ethyl acetate layer was dried under nitrogen and reconstituted in 1 mL of 1:1 methanol/water prior to LC/MS/MS analysis. Derivatized organic acids were separated on a 2.1 x 100 mm, 1.7 μm Waters Acquity UPLC BEH C18 column (T = 45°C) using a 7.5-min linear gradient with 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min. Quantitation of derivatized organic acids was achieved using multiple reaction monitoring on an Dionex UltiMate 3000 HPLC/Thermo Scientific Quantiva triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA). A standard calibration curve (1-5000 μM for lactate; 0.2-1000 μM for 3-hydroxybutyrate; 0.05-250 μM for pyruvate, succinate, fumarate, malate, and citrate; 0.02-100 μM for α-ketoglutarate) for derivatized organic acids was prepared by spiking 10-µL aliquots of organic acids (Sigma-Aldrich, St. Louis, MO) and internal standards (Sigma-Aldrich, St Louis, MO; Cambridge Isotopes, Cambridge, MA; CDN Isotopes, Quebec, Canada) into 50-µL aliquots of a 1:1 acetonitrile/0.3% formic acid solution. Calibration samples were derivatized and extracted similarly to organic acids in mouse heart homogenate.

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Fig. S1. Manipulation of Parkin and mitophagy in Mfn2 null MEFs by phospho-Mfn2 mutants. (A) Confocal studies showing mCherry-Parkin translocation to FCCP (10 µM, 60 min) treated mitochondria. (B) Spontaneous Parkin translocation (dark aggregates) provoked by adeno-Mfn2 EE, and FCCP-mediated Parkin translocation suppressed by adeno-Mfn2 AA. To the right is immunoblot analysis of Mfn2 expression. (C) Lysosomal-mitochondrial interactions (white squares) provoked by adeno-Mfn2 EE and suppressed by adeno-Mfn2 AA. (D) Mitochondrial elongation (aspect ratio) inhibited by adeno-Mfn2 EE and stimulated by adeno-Mfn2 AA. WT is wild type adeno-Mfn2. All studies n=3 independent experiments with 12-15 biological replicates. * is p<0.05 vs empty virus control (Ctrl); # is p<0.05 vs same condition WT adeno-Mfn2.

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Fig. S2. Fusion modulation without mitophagy regulation by phospho-Mfn2 mutants in Parkin null MEFs. (A) Mitochondrial elongation is inhibited by adeno-Mfn2 EE and stimulated by adeno-Mfn2 AA. + Parkin is 48 hours after adeno-Parkin. (B) Lysosomal-mitochondrial interactions. All studies n=3 independent experiments with 12-15 biological replicates. * is p<0.05 vs empty virus control (Ctrl).

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Fig. S3. Regulation of Parkin recruitment by phospho Mfn2 mutants in PINK1 null MEFs. (A) Mitochondrial elongation is stimulated by adeno-Mfn2 AA. (B) PINK1 null MEFs have diminished FCCP-stimulated mitophagy response (compare to Fig 1 and fig. S2), but Parkin translocation is increased by adeno-Mfn2 EE and FCCP-mediated Parkin translocation is suppressed by adeno-Mfn2 AA. (C) Absence of lysosomal-mitochondrial interactions in PINK1 deficient MEFs. All studies n=3 independent experiments with 12-15 biological replicates. * is p<0.05 vs empty virus control (Ctrl).

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Fig. S4. Fusion-defective Mfn2 mutant R400Q does not stimulate Parkin recruitment or mitophagy. (A) Confocal studies of mitochondrial morphometry (top), mcParkin mitochondrial translocation, and lysosomal-mitochondria overlay in empty adenovirus (left), adeno WT-Mfn2 (middle), and adeno Mfn2 R400Q infected MEFs. (B) Quantitative data for studies in (A). All studies n=3 independent experiments with 12-15 biological replicates. * is p<0.05 vs empty virus control (Ctrl).

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Fig. S5. Mutation of PINK1 phosphorylation sites does not prevent Mfn2 binding to Miro proteins. Immunoprecipitation of Flag-WT Mfn2, non-phosphorylatable Flag-Mfn2 AA, and phosphomimic Flag-Mfn2 EE with myc-Miro 1 or myc-Miro 2.

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Fig. S6. Cardiac effects of cardiomyocyte-directed perinatal expression of Parkin-binding Mfn2 EE. (A) Representative four chamber views of 6 week old mice. (B) Serial echocardiographic data of 4-6 week old mice. (C) Representative transmission electron micrographs of 6 week old mice with quantitative results. (D) Representative histological studies with quantitative results. There were no significant differences.

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Fig. S7. Cardiomyocyte death studies in perinatal Mfn2 AA expressing hearts. (A) Myocardial TUNEL labeling in 6 week old mice. (B) In vivo Evans blue staining of mouse myocardium. (C) Intact and processed myocardial caspase 3 levels in nontransgenic control (NTG), WT-Mfn2, and both lines of cardiac Mfn2 AA expressing mice. GAPDH is a loading control. (D) Mitochondrial perimeter, transversal side length (T side) and transversal side segment where the outer mitochondrial membrane was at <50 nm radial distance from the interfacing jSR (interface length) determined via TEM (15,000x; n=4 WT and 3 AA mice with 10 mitochondria-SR junctions analyzed in each of 6-7 cardiomyocytes). * is p<0.05 vs Ctrl and WT.

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Fig. S8. Cardiac effects of Mfn2 AA expressed at weaning or in young adults. (A) Representative hearts of mice expressing cardiomyocyte-directed Mfn2 AA from 3 weeks age at sacrifice (14 weeks) on left; serial echocardiography studies in middle; gravimetric data on right. (B) As in (A) except Mfn2 AA expression induced at 8 weeks and mice sacrificed at 21 weeks. (C) Inset showing immunoblot analysis of cardiac Mfn2 AA expression when induced at 3 weeks or 8 weeks. Ctrl is non-transgenic littermates. There were no significant differences.

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Fig. S9. DNA sequencing of human Mfn2 AA transgene from a Mfn2 AA line 1 mouse. On the left are Sanger sequence pherograms for WT Mfn2 and Mfn2 AA regions of interest of the respective mouse lines. To the right are the complete nucleotide and in vitro translated amino acid sequences of the Mfn2 AA mouse, aligned to reference human Mfn2 cDNA sequence. No unintended mutations are identified.

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Fig. S10. Mitochondrial stress is increased in young adult Mfn2 AA mice. On the left are representative immunoblots; quantitative group data are to the right. Levels of the mitocytokine fibroblast growth factor 21 (FGF21), mitochondrial heat shock protein 60 (Hsp60), and mediators of the mitochondrial uncoupled protein response LON peptidase 1 (LONP1) and AFG-like 2 (AFGL2) are increased in both independent lines of Mfn2 AA mice at young adulthood. * is p<0.05 vs nontransgenic Ctrl; # is p<0.05 vs WT Mfn2.

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Fig. S11. Mitochondrial studies in skeletal muscle and liver from young adult cardiac Mfn2 AA mice and controls. (A) PCR analysis of Myh6-Mfn2 AA gene expression in mouse heart, gastrocnemius skeletal muscle, and liver. RT is reverse transcription mix. Mitochondrial studies: Gastrocnemius muscle is B-F; liver is G-K. (B, G) Glutamate/malate-stimulated respiration. (C, H) Flow cytometric forward scatter. (D, I) Flow cytometric MitoSOX fluorescence (O2

-). (E, J) Calcium-stimulated mitochondrial swelling. (F, K) Representative TEM studies. N=3 mice per group; there were no significant between-group differences.

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Fig. S12. Immunoblot analysis of mitochondrial proteins in WT Mfn2 and Mfn2 AA expressing mouse hearts. To the left are representative immunoblots. To the right are quantitative group data. Fusion protein Mfn1 appears counter-regulated to overexpressed Mfn2. Mitochondrial outer membrane transport protein Tomm20, adenine nucleotide translocase 1 (ANT), voltage dependent anion channel (VDAC), and mitochondrial fission factor (Mff) are decreased, reflecting loss of mitochondria; cytosolic fission protein Drp1 is unchanged by Mfn2 AA. Same protein samples and GAPDH loading control used for fig. S6C. * is p<0.05 vs nontransgenic Ctrl; # is p<0.05 vs WT Mfn2.

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Fig. S13. Ultrastructural studies of cardiomyocyte mitochondria in 6 week old mice. Each electron micrograph is from a different nontransgenic control (Mfn2 AA littermate), WT Mfn2, or Mfn2 AA mouse (n=3 each). Quantitative data on mitochondrial content (% area occupied by mitochondria), mitochondrial area (mean area of individual organelles), and aspect ratio (long axis/short axis) are reported in Fig. 4F.

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Fig. S14. Comparative cardiac mitochondrial function in cardiac Mfn2 AA mice expressed at weaning (3 weeks; A-E) or at birth (F-J). (A, F) Glutamate/malate-stimulated respiration. (B, G) Flow cytometric forward scatter. (C, H) Flow cytometric MitoSOX fluorescence (O2

-). (D, I) Amplex Red fluorescence (H2O2). (E, J) Calcium-stimulated mitochondrial swelling. Except (J), perinatal Mfn2 AA data are reproduced from Fig. 4 for comparison.

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Fig. S15. Postnatal dilated cardiomyopathy development in Mfn2 AA mice. Above are M-mode echocardiographic tracings from n=5 individual mice per group with littermate controls. Below are quantitative group data. * = p<0.05 vs respective littermate controls; # = p<0.05 vs WT Mfn2.

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Fig. S16. Developmental evolution of cardiac mitochondrial gene expression from embryonic to adult hearts. Heat map display for unsupervised hierarchical clustering of RNA sequence data for 873 mitochondrial genes (from Mouse MitoCarta inventory of 1098 mitochondrial genes [41]) expressed at FPKM of 3 or greater in mouse hearts at any of the developmental stages studied.

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Fig. S17. Expression profile 449 mitochondrial genes regulated during the perinatal-adult transition in 5 week old tet-off Ctrl and Mfn2 AA transgenic (TG) mice, compared to sham-operated and 4 week post transverse aortic constriction (TAC). A. Heat map of mRNA levels. Mfn2 AA and TO control RNA sequence data are from Fig 4B, but here are clustered by genotype. Sham and TAC data are from (45). B. Comparative regulation of cardiac hypertrophy and failure-associated mRNAs. Mfn2 AA TG mice exhibit a perinatal transcriptional signature that is not recapitulated in pressure overloaded adult hearts, despite similar expression of genetic markers for cardiac pathology. * = FDR<0.02 vs Ctrl or Sham.

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Fig. S18. Postnatal reprogramming of mitochondrial genes by function. Genes that promote branched chain amino acid entry into the TCA cycle. Bars are mean values from RNA-sequencing results depicted in Fig 6B. Scale is log(2) of gene expression in 5 week old/P1 hearts for Ctrl (top), WT Mfn2 (middle) and Mfn2 AA (lower) hearts. Blue and red bars are significantly down and upregulated, respectively (1.25 fold, FDR <0.02); black bars are not significantly regulated. # = p<0.0001 vs WT Mfn2 (2-way ANOVA).

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Fig. S19. Schematic depiction of mechanisms underlying cardiac mitochondrial dysfunction in young adult Mfn2 AA mice as determined by integration of results from RNA-sequencing and metabolic profiling studies. Illustration depicts mRNAs (free red text) and metabolites (text in ovals) differing between 5 week Mfn2 AA and 5 week WT Mfn2 hearts, and similar between 5 week Mfn2 AA hearts and normal P1 controls. Signaling pathways pertaining to fatty acid beta-oxidation (KEGG #mmu00071), glycolysis (KEGG #mmu00010), TCA cycle (KEGG #mmu00020) and branched-chain amino acid (BCAA) catabolism (KEGG #mmu00280) are heat mapped according to degree of transcriptional perturbation (red = maximum, orange = moderate, yellow = mild, and white = no change). Dysregulation of genes within the pyruvate dehydrogenase complex (PDH) and complexes I-V of the oxidative phosphorylation (OXPHOS) process is similarly heat mapped. Key transcriptional regulators are indicated with bold text. Acadm, acyl-Coenzyme A dehydrogenase, medium chain; Acads, acyl-Coenzyme A dehydrogenase, short chain; Aco2, aconitase 2, mitochondrial; Acsf2, acyl-CoA synthetase family member 2; Aldh1a2, aldehyde dehydrogenase family 1, subfamily A2; Aldh6a1, Aldh1a2, aldehyde dehydrogenase family 6, subfamily A1; Bcat2, branched chain aminotransferase 2, mitochondrial; Bckdhb, branched chain ketoacid

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dehydrogenase E1, beta polypeptide; Cox10, COX10 homolog, cytochrome c oxidase assembly protein, heme A; Cox11, COX11 homolog, cytochrome c oxidase assembly protein (yeast); Cox15, COX15 homolog, cytochrome c oxidase assembly protein (yeast); Cox5a, cytochrome c oxidase, subunit Va; Cox6a2, cytochrome c oxidase, subunit VI a, polypeptide 2; Cox7a1, cytochrome c oxidase, subunit VIIa 1; Cox8b, cytochrome c oxidase, subunit VIIIb; Cyb5r2, cytochrome b5 reductase 2; Dlat, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase); Dlst, dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex); Echs1, enoyl Coenzyme A hydratase, short chain, 1, mitochondrial; Esrra, estrogen related receptor, alpha; Esrrg, estrogen-related receptor gamma; Fads1, fatty acid desaturase 1; Hibadh, 3-hydroxyisobutyrate dehydrogenase; Hsd17b10, hydroxysteroid (17-beta) dehydrogenase 10; Idh3g, isocitrate dehydrogenase 3 (NAD+), gamma; Ivd, isovaleryl coenzyme A dehydrogenase; Ldhb, lactate dehydrogenase B; Mccc1, methylcrotonoyl-Coenzyme A carboxylase 1 (alpha); Mccc2, methylcrotonoyl-Coenzyme A carboxylase 2 (beta); Mdh2, malate dehydrogenase 2, NAD (mitochondrial); mt-Co1, cytochrome c oxidase I, mitochondrial; Mut, methylmalonyl-Coenzyme A mutase; Ndufan, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1/3/6/8/10; Ndufbn, NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7/8/10; Ndufsn NADH dehydrogenase (ubiquinone) Fe-S protein 2/3/6/7/8; Ndufv1, NADH dehydrogenase (ubiquinone) flavoprotein 1; Pcca, propionyl-Coenzyme A carboxylase, alpha polypeptide; Pdha1, pyruvate dehydrogenase E1 alpha 1; Pdhx, pyruvate dehydrogenase complex, component X; Pdk2, pyruvate dehydrogenase kinase, isoenzyme 2; Pdk4, pyruvate dehydrogenase kinase, isoenzyme 4; Pdp2, pyruvate dehyrogenase phosphatase catalytic subunit 2; Pdpr, pyruvate dehydrogenase phosphatase regulatory subunit; Ppara, peroxisome proliferator activated receptor alpha; Sdhb, succinate dehydrogenase complex, subunit B, iron sulfur (Ip); Sdhc, succinate dehydrogenase complex, subunit C, integral membrane protein; Slc27a1, solute carrier family 27 (fatty acid transporter), member 1.

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