RESEARCH ARTICLE SUMMARY◥

HEART MITOCHONDRIA

Parkin-mediated mitophagy directsperinatal cardiac metabolicmaturation in miceGuohua Gong, Moshi Song, Gyorgy Csordas, Daniel P. Kelly,Scot J. Matkovich, Gerald W. Dorn II*

INTRODUCTION: During heart development,increased oxygenationduring the early perinatalperiod and a change in nutrient availabilityevokes a switch inmitochondrial substrate pref-erence from carbohydrates to fatty acids. Thismetabolic switching is reversed in adult heartdisease. Genetic “reprogramming” ofmitochon-dria plays a role in developmental and disease-related metabolic transitioning, buthow mitochondrial plasticity is gov-erned is unclear. Here, we foundthat mitophagy induced by PINK1-mitofusin 2 (Mfn2) –Parkin signalingwas central to perinatal switchingfrom glycolytic to fatty acid metabolism inmouse hearts. The Mfn2-Parkin interaction pro-voked generalized mitophagic removal of fetalcardiomyocyte mitochondria during the first3 weeks of life and was a prerequisite for in-

troduction of mature cardiac mitochondriaoptimized for fatty acid metabolism.

RATIONALE: We considered that the highlyordered paracrystallar structure of ATP bio-synthetic pathwaysmakes it unlikely thatmito-chondria can behave as flexible fuel organelles,readily adjusting their metabolism to differ-

ing substrate availability. Rather, weposited that mitochondria optimizedfor a given metabolic milieu must bereplaced when conditions change, asduring the perinatal period. In sup-port of this notion, late fetal and adult

cardiomyocyte mitochondria have distinct mor-phologies as well as metabolic preferences.Because targeted autophagic elimination of in-dividual damaged mitochondria (mitophagy) ismediated by the Parkinson’s disease factors

PINK1andParkin,weexaminedtheconsequencesof cardiac-specific Parkin loss-of-function onperinatal mitochondrial maturation and meta-bolic transitioning in mouse hearts. WhereasParkin deletion from adult hearts had no dis-cernible adverse effects, cardiomyocyte-specificParkin ablation from the first day of life waslethal in most mice before 3 weeks of age; insurvivingmice, mitochondrial maturationwasarrested at the fetal stage.

RESULTS: To interrupt Parkin-mediatedmito-phagy with more precision than gene ablation,we expressed PINK1 T111 and S442 phosphoryl-ation site Mfn2mutants. In cultured fibroblasts,the glutamic acid (E) substituted phosphomimicmutantMfn2EE spontaneously recruitedParkinto mitochondria and promoted mitophagy,whereas alanine (A) substituted nonphosphoryl-atable Mfn2 AA prevented Parkin translocationand interruptedmitophagy stimulated bymito-chondrial depolarization.We expressedwild-typeMfn2,Mfn2 EE, and

Mfn2 AA in mouse hearts. Mfn2 AA, when ex-pressed perinatally but not at or after wean-ing, provoked cardiomyopathy that was lethalby 7 to 8 weeks. Cardiomyocyte mitochondriaof surviving young adult Mfn2 AA mice hadan eccentric morphology and impaired pal-mitoylcarnitine use, which are typical featuresof fetal heart mitochondria. The transcriptionalsignature of juvenile Mfn2 AA hearts was dis-tinguished from age-matched controls by de-pressed abundance of fatty acid and branchedchain amino acidmetabolismmessenger RNAs,again resembling fetal hearts. Mitochondrialbiogenesis was impaired, and metabolite pro-filing of young adult Mfn2 AA hearts revealeddevelopmental metabolic arrest at the peri-natal stage—that is, impaired fatty acid useand preserved glycolytic function. Thus, inter-ruptingParkin-mediatedmitophagy inperinatalmouse hearts prevented normal maturationalmetabolic transitioning to fatty acids throughretention of fetal cardiomyocyte mitochondria.Mitophagy was a prerequisite for mitochon-drial biogenesis in this context.

CONCLUSION: Fetal cardiomyocytemitochon-dria undergo perinatal PINK1-Mfn2-Parkin–mediated mitophagy and replacement bymature adult mitochondria, rather than tran-scriptional reprogramming. Mitophagic mito-chondrial removal underlies developmentalcardiomyocyte mitochondrial plasticity andmetabolic transitioning. Facilitating develop-mentally programmed mitochondrial turnoveris functionally distinct from canonical selectivetargeting and removal of damaged mitochon-dria by Parkin in other contexts.▪

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Mitochondrial maturation fails whenmitophagy is interrupted.Normal perinatal mitochondrialmaturation is shown on the left: Heart sections from neonatal and 5-week-old hearts are super-imposed on their electron micrographs.To the right are similar images from hearts expressing thedominant negative mitochondrial Parkin receptor, Mfn2 AA. Retention of fetal cardiomyocytemitochondria in mitophagically impaired hearts was lethal.

The list of author affiliations is available in the full article online.*Corresponding author. E-mail: gdorn@dom.wustl.eduCite this article as G. Gong et al., Science 350, aad2459(2015). DOI: 10.1126/science.aad2459

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RESEARCH ARTICLE◥

HEART MITOCHONDRIA

Parkin-mediated mitophagy directsperinatal cardiac metabolicmaturation in miceGuohua Gong,1* Moshi Song,1* Gyorgy Csordas,2 Daniel P. Kelly,3

Scot J. Matkovich,1 Gerald W. Dorn II1†

In developing hearts, changes in the cardiac metabolic milieu during the perinatalperiod redirect mitochondrial substrate preference from carbohydrates to fatty acids.Mechanisms responsible for this mitochondrial plasticity are unknown. Here, we found thatPINK1-Mfn2-Parkin–mediated mitophagy directs this metabolic transformation in mousehearts. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary forParkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressingmitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expressionof Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrialmaturation essential to survival. Five-week-old Mfn2 AA hearts retained a fetalmitochondrial transcriptional signature without normal increases in fatty acid metabolismand mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels andcardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed.Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondriaundergo perinatal Parkin-mediated mitophagy and replacement by mature adultmitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocytemitochondrial plasticity and metabolic transitioning of perinatal hearts.

Mammalian hearts depend on mitochon-drial oxidative phosphorylation to fuelmyocardial contraction and pump func-tion; catabolism of carbohydrates or fatsgenerates adenosine triphosphate (ATP)

that powers excitation-contraction coupling. Un-der conditions of optimal intrinsic mitochon-drial functioning—i.e., when mitochondria are“fit”—energy demands and access to metabolicsubstrates and oxygen are central determinantsof mitochondrial respiration. During organismdevelopment, both substrate availability and tis-sue oxygen content change. Accordingly, the in-crease in transplacental oxygen exchange in earlyembryos provokes a shift from anaerobic gly-colysis to aerobic mitochondrial respiration (1).After birth, loss of transplacental carbohydratesubstrates promotes a further transition to fattyacidmetabolism (2) as small, fetal cardiomyocytemitochondria are supplanted by adult organelles(3). The normal perinatal developmental conver-sion from glucose to fatty acid cardiac metabo-lism, and its maladaptive reversal back towardglucose in diseased adult hearts, have been linked

to changes inmetabolic gene expression, so-called“metabolic reprogramming” (4). Cellular mecha-nisms underlying these cardiac metabolic tran-sitions are poorly described, and conventionalwisdom has been that mitochondria are “flexiblefuel” organelles capable of switching back andforth between carbohydrate and fatty acid me-tabolism (5).Stochastic damage to cardiomyocytemitochon-

dria places hearts at risk from bioenergetic insuffi-ciency or reactive oxygen species (ROS)–mediatedcytotoxicity (6). It is believed that maintainingmitochondrial functional integrity requires con-tinuous surveillance and culling of dysfunctionalorganelles. In cultured fibroblasts, depolarizedmitochondria are identified, sequestered, andeliminated through directed autophagy followedby lysosomal destruction (mitophagy). This formof mitochondrial quality control is mediated inpart by two Parkinson’s disease factors, the E3ubiquitin ligase Parkin and its upstream activatingkinase, phosphatase and tensin homolog (PTEN)–induced putative kinase 1 (PINK1) (7, 8). By remov-ing impaired organelles, the overall fitness of themitochondrial collective is preserved.The observation that glycolytic metabolism is

preferred in fetal hearts, but is maladaptive indiseased adult hearts (4), indicates that mito-chondrial fitness is neither a specific nor a uniquecondition. Rather, mitochondria are “fit” whentheydemonstrate optimal functional compatibilityfor a given developmental or pathophysiologicalmilieu. In this context, the idea that mitochon-

drial quality must be actively controlled appliesnot just to selective culling of individual damagedmitochondria but also to generalized cell- andorgan-wide promotion ofmitochondrial turnoverduring developmental or disease-related tran-sitions of cellular fuel and energymetabolism. Apossible role for mitophagic mitochondrial re-placement during metabolic transitions has notbeen addressed, in part because developmentalphenotypes are not observed in otherwise nor-malmice systemically lacking Parkin (9, 10). How-ever, absence of phenotypes in Parkin-deficientmice may simply reflect opportunistic compen-sation by Parkin-independent mitophagy path-ways (11).Here, we set out to clarify the role of Parkin-

mediated mitochondrial turnover in the normaldevelopmental switch from carbohydrate to fattyacid–basedmetabolism inperinatalmousehearts.To avoid confounding effects of germline andcardiac-specific Parkin ablation, we developedand deployed in vitro and in vivo systems inwhichexpression of amitofusin 2 (Mfn2)mutant lackingPINK1 phosphorylation sites essential for its bind-ing of Parkin suppressed Parkin translocation tomitochondria, thus specifically interruptingParkin-dependent mitophagy.

Parkin is essential for perinatalmitochondrial maturationin cardiomyocytes

Cardiomyocyte mitochondria of 1-day-old miceexhibit the typical elongated, curvilinear mor-phology of human fetal heart mitochondria (3)but mature over 3 weeks into the larger ovoidmitochondrial structure and denser collectivethat is characteristic of adult mammalian hearts(Fig. 1A). Cardiomyocyte-specific ablation of Park2,encoding Parkin, on perinatal day 1 (P1) was rap-idly lethal inmostmice. However, a small numberof “escapers” survived until P21. Polymerase chainreaction (PCR) analysis revealed incomplete Cre-mediated Park2 gene recombination in the heartsof these mice (Fig. 1B), suggesting that partialParkin insufficiency induced a forme fruste ofthe early lethal cardiac Parkin deletion pheno-type. Compared with normal littermates, P21Parkin-insufficient hearts appeared slightly smaller(Fig. 1, C and D). Myocardial histology of Parkin-deficient hearts was unremarkable (Fig. 1, C andD), but ultrastructural imaging of myocardiumfrom surviving 3-week-old perinatal cardiacParkin-insufficient mice revealed mitochondriahaving a morphology typical of fetal hearts (Fig.1C), whereas control (Park2 fl/fl) littermatemitochondria had the morphology of normaladult hearts (Fig. 1D). Furthermore, perinatalParkin-deficient hearts had abundant homoge-nous inclusions with smooth borders, charac-teristic of lipid droplets (Fig. 1C). Lethality ofperinatal cardiac-specific Park2 ablation, to-gether with findings of surviving escaper mice,suggested that Parkin may be essential formatu-rational development of cardiomyocyte mito-chondria. However, the aggressive phenotypeconfounded attempts to determine underlyingmechanisms. Accordingly, we built upon previous

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1Center for Pharmacogenomics, Department of InternalMedicine, Washington University School of Medicine, St.Louis, MO, USA. 2Department of Pathology, Anatomy, andCell Biology, Thomas Jefferson University, Philadelphia, PA,USA. 3Center for Metabolic Origins of Disease, CardiovascularMetabolism Program, Sanford Burnham Prebys MedicalDiscovery Institute, Orlando, FL, USA.*These authors contributed equally to this work. †Correspondingauthor. E-mail: gdorn@dom.wustl.edu

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observations that Parkin fails to translocate todepolarized mitochondria of Mfn2-deficient mice(12, 13), and the discovery that PINK1 phospho-rylation of Mfn2 promotes Mfn2-Parkin binding(12), to develop a system inwhich Parkin-mediatedmitophagy could be conditionally interrupted invitro or in vivo without primarily targeting eitherParkin or PINK1.

Mutational interdiction of Mfn2phosphorylation by PINK1 inhibitsParkin-mediated mitophagy

PINK1 is stabilized and accumulates in depolar-izedmitochondria (14) and phosphorylatesMfn2(Fig. 2A), enabling its binding to Parkin (12).Mimicking Mfn2 phosphorylation by substitutingglutamic acid (E) for the critical threonine andserine (Mfn2 T111E/S442E; Mfn2 EE) inducesPINK1-independent Mfn2-Parkin association(12) and promotes spontaneous Parkin recruit-ment to mitochondria of cultured fibroblasts(Fig. 2B and fig. S1). Nonphosphorylatable ala-nine (A) substitution at the same sites (Mfn2AA) had reciprocal effects, preventing PINK1-mediated Mfn2-Parkin binding (12) and sup-pressing Parkin translocation provoked by the

mitochondrial uncoupling agent carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) (Fig.2B and fig. S1). Effects of Mfn2 EE and Mfn2 AAon Parkin translocation were concordant withstimulation or inhibition, respectively, of mito-chondrial-lysosomal interactions (Fig. 2C andfig. S1). In agreement with Mfn2 EE functioningas a constitutive mitochondrial Parkin receptor,it failed to evoke mitophagy in Parkin-deficientcells (fig. S2). Although the mitophagy responseto FCCP was reduced in PINK1-deficient cells,Mfn2 EE nevertheless promoted spontaneousParkin translocation (fig. S3). Because nonphos-phorylatable Mfn2 AA inhibited Parkin local-ization tomitochondriawithoutprimarily affectingPINK1 or Parkin, we used it to interrupt PINK1-Parkin–mediated mitophagy without adversely af-fecting nonmitophagic Parkin functionality (15).We asked whether Mfn2 phosphorylation of

T111 and S442 modified other cellular actionsof Mfn2. Mfn2 promotes outer mitochondrialmembrane tethering and fusion (16). Nonphos-phorylatable Mfn2 AA was as effective as wild-type (WT)Mfn2 for inducingmitochondrial fusionin eitherWT orMfn2-deficient fibroblasts; pseudo-phosphorylated Mfn2 EE did not promote fusion,

instead evoking mitochondrial shortening (Fig.2D and figs. S1 to S3). Thus, Parkin binding andmitochondrial outermembrane fusion aremutu-ally exclusive functions of Mfn2 regulated byPINK1-mediated phosphorylation.Mitochondrial fragmentation is functionally

linked to mitophagy (17). We asked whether theabsence of fusion-promoting activity for the phos-phomimic Mfn2 EE was sufficient to provokemitophagy. FCCP-stimulated Parkin translocationand lysosomal-mitochondrial colocalization wereexamined in cells expressing a naturally occur-ring fusion-defective humanMfn2mutant, R400Q,in which PINK1 phosphorylation sites are intact(18). Mfn2 R400Q induced mitochondrial short-ening similar to Mfn2 EE but did not promoteParkin translocation or mitophagy (fig. S4). Thus,inhibition of mitochondrial fusion andmitochon-drial recruitment of Parkin by Mfn2 are not func-tionally coupled, except through PINK1-mediatedMfn2 phosphorylation. Finally, we determinedwhether phosphorylation of Mfn2 on T111 andS442 affected its binding of Miro mitochondrialtransport proteins (19). WT Mfn2, Mfn2 AA, andMfn2 EE each bound Miro1 and Miro2, exhibit-ing the previously reported preference for Miro2(fig. S5). Mitochondrial fusion and Parkin trans-location are therefore reciprocally and uniquelyregulated by PINK1-mediated phosphorylation ofMfn2 on T111 and S442.

Perinatal inhibition ofParkin-mediated mitophagy induceslethal cardiomyopathy

BecauseMfn2 AA promotedmitochondrial fusionsimilar to WT Mfn2, but inhibited mitochondrialParkin localization, we used it to dissect the role ofParkin-mediatedmitophagy inmousehearts. Bothseparation-of-functionMfn2mutants (EE andAA)were expressed using a bitransgenic doxycycline-suppressible cardiomyocyte-specificMyh6 promot-er system (Fig. 3A); cardiac transgene expressioncan be suppressed with doxycycline, but in its ab-sence, such expression begins shortly after birth(20). WT Mfn2 was expressed separately as acontrol for increased myocardial Mfn2 content(Fig. 3A, top). In vivo regulation of cardiac mi-tophagy by Mfn2 EE and Mfn2 AA was assessedby crossingMfn2 EE andMfn2 AAmice to miceconditionally expressing Parkin using the samedoxycycline-suppressible Myh6-driven expres-sion system (21) and concomitantly inducingthe Mfn2 and Parkin transgenes in adult mice.Mitochondrial localization of Parkin, increasedubiquitination of mitochondrial proteins, andmitochondrial localization of the autophagosomaldocking protein, p62/Sequestosome 1 (SQSTM1),were enhanced in Mfn2 EE/Parkin hearts but re-duced in Mfn2 AA/Parkin hearts, compared withParkin-overexpressing controls (Fig. 3A, bottom).These in vivo results recapitulate the in vitroeffects of the Mfn2 phosphorylation mutants onmitophagy.Mice expressing WT Mfn2 or Mfn2 EE from

birth (i.e., those that never received doxycycline)appeared normal (Fig. 3, B to E, and fig. S6). Incontrast, cardiac expression of Mfn2 AA at similar

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Fig. 1. Early lethality of perinatal cardiomyocyte-specific Parkin-deficient mice. (A) Transmissionelectron microscopy (TEM) showing normal cardiomyocyte mitochondria on the first (P1) and 21st (P21)day of life. Enlargement shows structural details at P1. (B) PCR genotyping of floxed Park2 gene (top) andtamoxifen-inducible cardiac Cre (bottom) of surviving mice from a representative litter at P21; three micedied beforeweaning. KO indicates Cre recombined Park2 fl/fl allele.T is tail DNA; H is heart DNA. (C andD)Representative (of three) hearts, histological sections, and TEMs from P21 cardiac Parkin–deficient (top)and control (bottom) mice. Scale bars for hearts are 2 mm.

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levels from birth (Fig. 3A) was uniformly lethalby 7 to 8 weeks of age in two independent trans-genic lines (Fig. 3, B and C). Cardiac dilatation(Fig. 3, B and D), worsening pump function (Fig.3D), and pulmonary congestion (Fig. 3E) iden-tified progressive cardiomyopathy as the underlyingprocess and heart failure as the terminal event.Myocardium from surviving 6-week-old Mfn2 AAmice exhibited cardiomyocyte enlargement andreplacement fibrosis (Fig. 3F). TUNEL (terminaldeoxynucleotidyl transferase–mediated deoxyuri-dine triphosphate nick end labeling) staining was

increased proportionally to in vivo Evans blue la-beling without caspase 3 processing, revealingnecrosis to be the likely mechanism for Mfn2AA–induced cardiomyocyte dropout (fig. S7, A toC). However, there were no differences in the sizeof Mfn2-dependent mitochondrial-sarcoplasmicreticulum interfaces (22) inWT-Mfn2 andMfn2AA–expressing mice (fig. S7D), showing thatinterorganelle tethering by Mfn2 (23) was notperturbed by preventing T111 and S442 phospho-rylation. Remarkably, cardiac Mfn2 AA induc-tion at the time of weaning (~3 weeks) or in

young adult mice (8 weeks) evoked no cardiacpathology (fig. S8). Thus, Parkin localization toMfn2 on cardiomyocyte mitochondria is essen-tial only between birth and weaning.

Parkin-mediated mitophagy is essentialfor perinatal cardiomyocytemitochondrial maturation

The only molecular differences between cardiacWTMfn2 andMfn2 AAmice were two nonphos-phorylatable amino acids (fig. S9). Yet, the cardiacphenotype of the former was benign, whereas thelatter developed lethal juvenile cardiomyopathy.BecauseMfn2AApromotedmitochondrial fusionaswell asWTMfn2 (Fig. 2D and fig. S1D), and theMfn2 AA mutation did not affect other Mfn2functions (figs. S5 and S7), we reasoned that theMfn2 AA cardiomyopathy was caused by sup-pressing Parkin-mediated mitophagy. In agree-mentwith this idea,mitochondria-associatedParkinand p62/SQSTM1were both reduced in the heartsof 2- to 3-week-old Mfn2 AA mice, comparedwith age-matched WT Mfn2 mice (Fig. 4A, top).Mitochondrial Parkin association was likewisedepressedbyMfn2AA inhearts of 2- to 3-week-oldfood-deprived mice, but mitochondrial-associatedp62/SQSTMI was increased (Fig. 4A, bottom).Thus, Mfn2 AA inhibits developmentally pro-grammed Parkin-mediatedmitophagy, but notParkin-independent mitochondrial autophagyprovoked by starvation.Multiple lines of evidence revealed a deterio-

ration in mitochondrial quality specific to Mfn2AAhearts, including impairedmaximalmitochon-drial respiration (Fig. 4B), increased mitochon-drial production of O2

– and H2O2 (Fig. 4C), andmodest increases in levels of the mitochondrialstress proteins fibroblast growth factor 21 (FGF21),heat shock protein 60 (Hsp60), Lon peptidase1 (LONP1), and adenosine triphosphatase familygene 3–like 2 (AFG3L2) (fig. S10). By compari-son, liver and skeletalmusclemitochondria fromcardiac-expressing Mfn2 AA mice were normal(fig. S11).Reducedmyocardial content of mitochondrial

proteins (Fig. 4D and fig. S12) and decreased flowcytometric forward scatter (Fig. 4E) pointed toabnormalities in the abundance and morphol-ogy of cardiac mitochondria of 6-week-old Mfn2AA mice. Ultrastructural examination revealedsmall, unusually shaped cardiomyocytemitochon-dria (Fig. 4F and fig. S13). Decreased respiratorycomplex protein abundance reflected lowermitochondrial content in Mfn2 AA hearts (Fig.4G). In agreementwith absence of cardiac pheno-types when Mfn2 AA expression was induced ator after weaning (fig. S8A), mitochondrial respi-ration, morphometry, and ROS production werenormal in those mice (fig. S14).Mitochondrial morphology of young adult

Mfn2 AA mouse hearts resembled that of bothnormal postnatal (P1) mouse hearts and P21 car-diac Parkin-deficientmouse hearts (compare Fig.4F to Fig. 1, A and D). The transition of mousecardiac mitochondria from fetal to adult mor-phology normally occurs within the first 3 weeksof life (Fig. 1), concurrent with a functional

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Fig. 2. Mitochondrial Parkinmobilization directed by pseudo-PINK1 phosphorylatedMfn2. (A) Phos-phorylation of Mfn2 by recombinant PINK1 in a cell-free system. First three panels show enrichment ofFLAG-Mfn2 by immunoprecipitation (IP) with antibody to FLAG (anti-FLAG); left is Coomassie blue stainedgel, middle is anti-Mfn2 immunoblot, right is anti-FLAG immunoblot. Fourth panel shows anti-Mfn2 Phos-tag immunoblot of in vitro PINK1 phosphorylation reactants; KD is kinase dead PINK1; CIP is calf intestinalphosphatase. Arrowheads show FLAG-Mfn2; bold arrow indicates phospho-Mfn2. (B) Spontaneousmaraschino cherry (mc) Parkin translocation in MEFs provoked by adeno-Mfn2 EE, and FCCP-mediatedmcParkin translocation suppressed by adeno-Mfn2AA.To the left is an immunoblot ofMfn2. (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. In (B) and (C), gray bars are basal; the black bars are 60 min after FCCP orantimycinA. In (D), gray bars are 24hours andblack bars are48hours after adeno-Mfn2 virus infection. *,P<0.05 versus adeno b-gal control (Ctrl); #, P < 0.05 versus same condition WTadeno-Mfn2.

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shift favoring fatty acid metabolism (5, 24, 25).We posited that suppression of Parkin-mediatedmitophagy by Mfn2 AA caused fetal mitochon-dria to be retained. Consistent with this idea,Mfn2 AA cardiac mitochondria did not undergothe time-dependent transformation in size andshape observed by P21 in WT Mfn2 and controlhearts (Fig. 5A). Interruption of normal mito-chondrial morphological maturation that is nor-mally complete by P21was associatedwith dilatedcardiomyopathy that developed within 2 weeksthereafter (Figs. 3D and 5, B and C and fig. S15).

Mitophagy is essential for the perinataltransformation of cardiac metabolism

Cardiac metabolic transitioning after birth islinked to increased expression of mitochondrialreplication and transcription factors—i.e., mito-chondrial biogenesis (26).Wemeasured the changesin transcript levels for more than 400 cardiac-

expressed mitochondrial proteins from late em-bryo through adulthood, thereby defining nor-mal transcriptional reprogramming of cardiacmetabolism (fig. S16). RNA sequencing of peri-natal day 1 (P1), day 21 (P21), and 5-week-oldhearts showed that WT Mfn2 did not perturbnormal mitochondrial gene reprogramming. Bycontrast, the mitochondrial transcript profile of5-week-oldMfn2 AA hearts cosegregated with P1hearts (Fig. 6A), driven largely by failure of elec-tron transport, fatty acid catabolism, and ketonebody metabolism gene abundance to increaseduring the perinatal-to-adult transition (Fig. 6Aand supplementary data sets 1 and 2). Hemo-dynamic stress in adult hearts did not fully re-capitulate fetal metabolic gene expression (fig.S17), demonstrating that Mfn2 AA caused trueretention of the embryonic metabolic transcrip-tome and not cardiomyopathy-related reexpres-sion of embryonic genes (25). Suppression of

metabolic gene reprogramming in 5-week-oldMfn2 AA hearts affected genes encoding fattyacid (Fig. 6B) and branched chain amino acid(fig. S18) tricarboxylic acid (TCA) cycle entry fac-tors and some TCA cycle enzymes themselves(Fig. 6B), while largely sparing glycolysis genes(Fig. 6B); effects on oxidative phosphorylation(OXPHOS) genes were variable (Fig. 6B). Char-acteristic perinatal increases in the abundance oftranscriptional activators ofmetabolism andmito-chondrial replication factors—i.e., mitochondrialbiogenesis genes—were also suppressed in Mfn2AA hearts (Fig. 6C).To define the metabolic consequences of in-

terrupting Parkin-mediated mitophagy in Mfn2AA mouse hearts, we compared mitochondrialrespiration stimulated by the fetal-preferred gly-colytic substrate pyruvate to respiration stimu-lated by the adult-preferred fatty acid substratepalmitoylcarnitine (2, 4, 24). Oxygen consumption

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Fig. 3. Perinatal cardiomyopathy evoked by nonphosphorylated Mfn2 AA.(A) Immunoblot analysis of Mfn2 expression (top) and mitochondrial Parkinlocalization (bottom) in transgenic mouse hearts. (Top) Top pair is cardiachomogenate; bottom pair is mitochondrial-enriched 10,000 g pellet (cyto-chrome oxidase IV; COX IV) and postmitochondrial supernatant [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. (Bottom) Immunoblot analysis ofmitochondrial-associated Parkin and downstream mitophagy events and their

modulation by cardiac-expressed Mfn2 EE and Mfn2 AA. (B) Representativehearts of 6-week-old mice. (C) Survival. (D) Serial echocardiographic data of4- to 6-week-oldmice; white bars are Ctrl, gray isWTMfn2, and black isMfn2AA. (E) Heart (top) and lung (bottom) weights of 6-week-old mice indexed tobody weight (BW). (F) Histological studies of cardiomyocyte cross-sectionalarea (top) and myocardial fibrosis (bottom); quantitative data are on the right.*, P < 0.05 versus WT Mfn2 and nontransgenic (NTG) control.

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by permeabilized cardiomyocytes was similarin control, WT-Mfn2, and Mfn2 AA cells givenpyruvate. However, Mfn2 AA cardiomyocytesexhibited impaired respiration when providedwith palmitoylcarnitine (Fig. 7A). Moreover, myo-cardial metabolite profiling revealed abnormallylow levels of multiple fatty acid acylcarnitines,which are products of mitochondrial fatty acidoxidative flux, in comparison with age-matchedcontrol and WT-Mfn2 hearts. Specifically, acylcar-nitine levels in 5-week-old Mfn2 AA hearts werecomparable to those of normal P1 hearts (Fig. 7, Bto D). By contrast, myocardial abundance of or-ganic acids in young adult Mfn2 AA and controlhearts was similar. The concordant abnormal-ities of mitochondrial gene expression, substratepreference formitochondrial respiration, andmeta-bolic profile exhibited by cardiac Mfn2 AA mice(fig. S19) illustrate the global effect of Parkin-mediatedmitochondrial removal onnormal devel-opmental metabolic transitioning of the perinatalheart. Taken together, the data point to a mis-match between mitochondrial programmingand metabolic substrate availability as the under-lying cause of progressive cardiomyopathy in ju-venileMfn2AAmicewith defective cardiomyocytemitophagy.

Discussion

Wehave shown that Parkin-mediatedmitophagyis essential for normal perinatal cardiacmitochon-

drial and metabolic maturation. By expressingfrom birth an engineeredMfn2 AAmutant thatcannot be PINK1-phosphorylated on T111 andS442 as required forMfn2-Parkin binding (12), thenormal developmental perinatal transformationof cardiacmetabolismwas disrupted. Persistenceof fetal carbohydrate-metabolizingmitochondriain adultMfn2AAhearts revealed the requirementfor organelle removal through the PINK1-Mfn2-Parkin mitophagy mechanism before mitochon-drial transitioning to normal adult fatty acidmetabolism. Even the genetic program encodingcritical fatty acid metabolism pathways was re-pressed when fetal mitochondria were retained.Parkin thus promotesmitochondrial removal andsuppresses biogenesis, consistent with previous-ly described Parkin-dependent regulation of themitochondrial biogenesis factors PARIS and PGC-1a (11). Organelle replacement, rather than simplereprogramming,may be necessary becausemito-chondrial respiratory supercomplexes are orga-nized as paracrystalline arrays (27, 28) whosedisassembly, reorganization, and reassembly inpreexisting embryonic cardiacmitochondria couldbe disruptive (7). An alternatemeans of mitochon-drial transitioning might be through cycles offission and fusion. However, the normal half-time for turning over adult cardiomyocyte mito-chondria through mitochondrial dynamism is~3 weeks (29), within which time our studies re-veal the perinatal transformation from fetal to

adult mitochondria to be complete. Thus, we pro-pose that the Parkin-Mfn2 interaction drives gen-eral mitophagic turnover of fetal mitochondria inthe perinatal heart, enabling their replacementwith mitochondria incorporating biogenicallyderived metabolic systems optimized for the highenergetic demands of contracting adult hearts.Themetabolic preference for glycolysis in grow-

ing fetal hearts is reminiscent of the Warburgeffect observed in cancer, wherein the transfor-mation from restrained to malignant growth isassociated with a transition to increased glycoly-sis and less dependence on aerobic mitochon-drial ATP production (30). Although the specificmolecular determinants of glycolyticmetabolismin fetal hearts undoubtedly differ from those oftumors, in both instances, increased glycolysis isadaptive for a hypoxic environment and is opti-mized for increasing biomass—i.e., for promot-ing cell growth (31). Thus, the growing fetal heartderives twobenefits fromapredominantly glycoly-tic metabolism: (i) the ability to generate ATPsufficient for the comparatively modest needs offetal heart contraction despite a relatively hypoxicenvironment; and (ii) a metabolism that facili-tates uptake and incorporation of amino acids andfatty acids into new cellular structures for cell andorgan growth, rather thanmetabolizing them forenergy production. On the other hand, fatty acidmetabolism in fully grown adult hearts providesmore efficient ATP production to fuel increased

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Fig. 4. Abnormalities in mitophagy and mitochondria induced by peri-natal cardiac Mfn2 AA. (A) Immunoreactive Parkin and p62/SQSTM1 in 2- to3-week-old mitochondrial-enriched mouse heart fractions (mito-) of fed mice(top) and food-deprived mice (bottom). Cyto-p62 is p62/SQSTM1 in the cy-tosolic fraction. (B) Substrate-stimulated (left) and maximum uncoupled(right) respiration of isolated cardiac mitochondria. (C) Isolated mitochon-drial O2

– (MitoSOX; left) and H2O2 (Amplex red; right) production studies.

(D) Cardiac mitochondrial protein content. (E) Flow cytometric mitochondrialforward scatter. (F) Ultrastructural studies of cardiomyocyte mitochondria.Mitochondrial content is the percentage of cell area occupied bymitochondria;mitochondrial area is the mean area of individual organelles; mitochondrialaspect ratio is long axis/short axis. (G) Immunoblot analysis of respiratorycomplex proteins.Quantitative data to right are n = 4 individual mouse hearts.*, P < 0.05 versus Ctrl; #, P < 0.05 versus WT Mfn2.

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contractile demand. Our findings reveal this car-diacmetabolic transition to be an essential adap-tation for extra-uterine life.The developmental role for Parkin-dependent

mitophagy described herein was unsuspectedbecause germline Parkin gene ablation in micehas not produced robust phenotypes (9, 10). Asdeployed here, Mfn2 AA perturbed endogenousParkin strictly by interdicting its localization tomitochondria—i.e., by acting as a nonfunctioningcardiomyocyte Parkin receptor. The lethal cardio-myopathy evoked byMfn2 AA, but notWTMfn2,exposed novel Parkin functionality specific tothe postnatal period, during which myocardialcarbohydrate-dependent metabolism transitionsto fatty acid dependence. Early lethality after peri-natal cardiomyocyte-specific Parkin gene deletionlikewise demonstrated an essential role for Parkinin postnatal hearts. However, perinatal cardio-myocyte Parkin deficiency was generally fatalwithin the first 3 weeks of life, whereas perinatalcardiac Mfn2 AA expression provoked a slower-

progressing (but ultimately equally deadly) car-diomyopathy. The aggressive phenotype inducedby cardiomyocyte-directed Parkin deletion likelyresults from complete disruption of Parkin activ-ity that, in addition to promoting mitophagy, in-cludes regulation of mitochondrial dynamism,modulation of subcellular mitochondrial motil-ity, mediation of mitochondrial protein degrada-tion, and stimulation of mitochondrial biogenesis(15). By comparison, expressing Mfn2 AA is a moreprecise intervention that only inhibits Parkinactivity dependent upon its binding to PINK1-phosphorylatedMfn2—i.e.,mitophagy. Indeed, be-cause endogenous Mfn2, PINK1, and Parkin arestill present,mitophagywasnot entirely abrogatedby Mfn2 AA. Accordingly, cardiomyocyte-specificperinatal Parkin gene deletion and Mfn2 AA ex-pression had similar effects on mitochondrialmaturation in the heart, but the Mfn2 AA phe-notype was an attenuated form of the full-blownphenotype that can be evoked by perinatal mito-phagic dysfunction.

Our findings add to the evidence that in vivoParkin functionality is underestimated fromconventional systemic Parkin deletion. Whereasgermline Parkin knockout mice exhibit fewmani-festations of Parkinson’s disease or cardiac in-volvement at baseline (9, 10, 32), conditionalParkin ablation in the substantia nigra recapit-ulates a central pathological feature of Parkinson’sdisease, degeneration of dopaminergic neurons(11). Likewise, short hairpin RNA–mediated Parkinsuppression reduces mitophagy in mouse livers,whereas mitophagy is intact in germline Parkinknockoutmouse livers (33). Finally, cardiomyocyte-specific Parkin ablation alleviates the cardio-myopathy induced by dynamin-related protein1 (Drp1) deficiency, whereas global Parkin defi-ciency fails to rescue a similar cardiac Drp1-deficient model (21, 34).These results also clarify the specific function

of PINK1-Mfn2-Parkin signaling in mitophagy.The need for PINK1-phosphorylated Mfn2 tofunctionas aParkinbindingprotein inmitophagicmitochondrial clearance is not absolute, becauseParkin will translocate to mitochondria of FCCP-treated cells lacking Mfn2 (fig. S1). Thus, just asthere are Parkin-independent pathways formito-chondrial elimination (35), we would infer oneor more Mfn2-independent means of recruitingParkin to depolarized mitochondria. Nonetheless,innormal andMfn2-deficient fibroblasts, nonphos-phorylatable Mfn2 AA abolished Parkin trans-location to, and lysosomal engulfmentof, depolarizedmitochondria without affecting other Mfn2 activ-ities such asmitochondrial fusion,mitochondrial-sarcoplasmic reticulum tethering, and Miro binding.These results define a central role for the PINK1-Mfn2 phosphorylation interaction in conven-tional mitochondrial quality control and suggesta mechanism whereby PINK1-phosphorylatedMfn2 induces primary mitochondrial Parkinbinding, with PINK1-phosphorylated ubiquitinas the preferred substrate for mitochondrial lo-calized Parkin (36).The current findings suggest why Mfn2 and

Parkin are present in hearts that seemingly havelittle use for the canonical functions assignedto both proteins. In adult hearts, cardiomyocytemitochondria are static, distinct, rounded organ-elles. Lacking extensive interconnectedmitochon-drial networks, adult cardiomyocytes have littlerequirement for the mitochondrial remodelingthat would be promoted by a redundant mito-chondrial fusion protein. Indeed, in our hands,Mfn1 (but notMfn2) is completely dispensable toadult mouse hearts (12). Likewise, the PINK1-Parkin pathway clearlymediates autophagic elim-ination of damaged mitochondria in culturedmammalian cells (14), but evidence fromgermlinePark2 knockout mice (9), adult cardiomyocyte-specific Parkin-deficient mice (21), and from in-terrupting Parkin signaling in adult mice withMfn2 AA (current study) does not support animportant role for Parkin-mediated mitophagyas a homeostatic mitochondrial quality-controlmechanism in hearts; normal surveillance andculling of individual damagedmitochondria seemsto be accomplished through Parkin-independent

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Fig. 5. Fetal mitochondria persist in young adult Mfn2 AA mouse hearts. (A) Representative four-chamber heart sections and transmission electron micrographs of cardiomyocyte mitochondria from P1,P21, and 5-week-old mouse hearts. NTG controls are top row,WT Mfn2 middle row, Mfn2 AA bottom row.Quantitative data for heart weights are in (B) and formitochondrial ultrastructure in (C). *, P < 0.05 versusP1; #, P < 0.05 versus WT Mfn2 at the same stage.

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mechanisms (35).Rather, Parkin-directedmitophagyis invoked as needed to accelerate generalizedmitochondrial turnover during developmental tran-sitions of myocardial metabolism (current study),after myocardial injury (9), and when mitochon-drial homeostasis is disrupted (21).Our findings demonstrate that mitochondria

are not simply flexible fuel organelles readilyswitching between carbohydrate and fatty acidsubstrates. Rather, they have intrinsic metabolicand respiratory systems optimized for differentsubstrate-specificmetabolic pathways, andmeta-bolic transitioning requires existing organelles tobemitophagically removed, enabling their replace-ment with mitochondria appropriate for a givenbiological context. Parkin and PINK1 gene muta-tions cause hereditary Parkinson’s disease, andMfn2mutations cause Charcot-Marie-Tooth syn-drome type IIa. These are chronic neuromusculardisorders inwhich cardiac involvement is uncom-mon (37). It remains to be determined whetherPINK1-Parkin-Mfn2–mediated mitophagy regu-lates metabolic function in neurons or skeletalmuscle as it does in perinatal hearts. The discov-ery that PINK1-Mfn2-Parkin directed–mitophagyis essential to cardiomyocytemetabolic remodeling

only during the brief period following birth sup-ports intensive genetic evaluation of the Parkinsignaling pathway in neonatal, in addition toadult, cardiomyopathy.

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Fig. 6. Failure of metabolic gene reprogramming after perinatal Mfn2AA expression. (A) Heat map of mitochondrial gene expression in P1, P21,and 5-week-old mouse hearts; functional annotation of Mfn2 AA gene clustersis to the right. (B) Postnatal reprogramming of mitochondrial genes by meta-bolic function. Bars are mean values from results in Fig. 4B; log(2) gene ex-pression at 5 weeks versus P1 for Ctrl (top),WT Mfn2 (middle), and Mfn2 AA

(bottom) hearts. Blue and red bars are significantly down- and up-regulated,respectively [1.25-fold; false discovery (FDR) < 0.02]; black bars are not sig-nificantly regulated. (C) Regulated expression of mitochondrial biogenesis andreplication genes during the perinatal-adult transition. *, FDR < 0.02 versuslittermate control mice (TO); #, P < 0.0001 versusWTMfn2 [two-way analysisof variance (ANOVA)].

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Fig. 7. Adult Mfn2 AA hearts retain a fetal-like glycolytic metabolism.(A) Palmitoylcarnitine-stimulated (top) and pyruvate-stimulated (bottom)respiration of isolated permeabilized cardiomyocytes. (B and C) Standardizedheat map showing unsupervised clustering of myocardial acylcarnitine (B) andorganic acid (C) metabolite content in P1 NTG and 5-week-old NTG,WTMfn2,and Mfn2 AA mouse hearts. Vertical lines to the right of (B) indicate sig-

nificantly dysregulated metabolites in 5-week-old Mfn2 AA hearts versuscontemporaneous controls. (D) Quantitative data for absolute myocardialcontent of dysregulated metabolites in (B). -DC and -OH designate mono-hydroxylated and dicarboxylic acid acylcarnitine species, respectively. Com-mon names for parent species are in parentheses. *, P < 0.05 versus WTMfn2(ANOVA).

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ACKNOWLEDGMENTS

We gratefully acknowledge the invaluable contributions of S. J. Gardellof the Sanford Burnham Prebys Medical Discovery Institute for liquid

chromatography tandem mass spectrometry studies; and M. Levy ofthe Laboratory of Electron Microscopy Sciences, Department of CellBiology, Washington University School of Medicine, S. de la Fuenta,J. Mishra, and D. Weaver of the Thomas Jefferson University Mito CareCenter for TEM analyses. Park2 floxed allele mice were obtained fromand used under a Materials Transfer Agreement with LexiconPharmaceuticals. The authors declare no conflicts. The data presentedin this manuscript are tabulated in the supplementary materials. Bothunprocessed and transcriptome-aligned RNA-sequencing reads havebeen deposited in the National Center for Biotechnology InformationGene Expression Omnibus with accession number GSE68921. Thiswork was supported by NIH HL59888, HL108943, HL128071 (G.W.D.),

HL122124 (G.C.), HL058493 (D.P.K.), and American Heart Associationgrants 14PRE18970093 (M.S.) and 14GRNT20410000 (S.J.M.).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/350/6265/aad2459/suppl/DC1Materials and MethodsFigs. S1 to S19Data sets S1 and S2References (38–50)

13 August 2015; accepted 16 October 201510.1126/science.aad2459

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Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in miceGuohua Gong, Moshi Song, Gyorgy Csordas, Daniel P. Kelly, Scot J. Matkovich and Gerald W. Dorn II

DOI: 10.1126/science.aad2459 (6265), aad2459.350Science 

, this issue p. 10.1126/science.aad2459, p. 10.1126/science.aad0116; see also p. 1162Sciencediet, which altered cardiac metabolism.a form of dilated cardiomyopathy. Mice destined to develop cardiomyopathy were protected by feeding with a high-fat

frommitochondrial fission and fusion in mouse cardiomyocytes. Disruption of these processes led to ''middle-aged'' death examined the role ofet al.quality-control pathway to facilitate a major developmental transition after birth. Wai

cardiomyocytes prevented the normal metabolic transition and caused heart failure. Thus, the heart has coopted a the first 3 weeks after birth. Preventing this turnover by interfering with parkin-mediated mitophagy specifically inmouse heart mitochondria soon after birth. Mitochondria were replaced wholesale via mitophagy in cardiomyocytes over

studied the developmental transitions inet al.organs and tissues changes. This invokes changes in metabolism. Gong heart muscle cells (see the Perspective by Gottlieb and Bernstein). After birth, the availability of oxygen and nutrients to

Mitochondria provide an essential source of energy to drive cellular processes and are particularly important inA change of heart (mitochondria)

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