Bax regulates primary necrosis throughmitochondrial dynamicsRussell S. Whelana,b,c,1, Klitos Konstantinidisa,b,c,1, An-Chi Weid, Yun Chene, Denis E. Reynac,f, Saurabh Jhaa,c,Ying Yanga,b,c, John W. Calvertg, Tullia Lindstenh, Craig B. Thompsonh, Michael T. Crowi, Evripidis Gavathiotisc,f,j,Gerald W. Dorn IIe, Brian O’Rourked, and Richard N. Kitsisa,b,c,j,k,2

Departments of aMedicine, bCell Biology, and fBiochemistry, cWilf Family Cardiovascular Research Institute, jAlbert Einstein Cancer Center, and kDiabetesResearch Center, Albert Einstein College of Medicine, Bronx, NY 10461; Division of dCardiology Johns Hopkins University School of Medicine, Baltimore,MD 21205; iPulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224; eCenter for Pharmacogenomics,Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110; gDivision of Cardiothoracic Surgery, Department ofSurgery, Carlyle Fraser Heart Center, Emory University School of Medicine, Atlanta, GA 30308; and hCancer Biology and Genetics and Immunology Programs,Memorial Sloan-Kettering Cancer Center, New York, NY 10065

Edited* by Andrew R. Marks, Columbia University College of Physicians and Surgeons, New York, NY, and approved March 9, 2012 (received for reviewFebruary 1, 2012)

The defining event in apoptosis is mitochondrial outer membranepermeabilization (MOMP), allowing apoptogen release. In contrast,the triggering event in primary necrosis is early opening of the innermembrane mitochondrial permeability transition pore (mPTP), pre-cipitatingmitochondrial dysfunction and cessationofATP synthesis.Bcl-2 proteins Bax and Bak are the principal activators ofMOMP andapoptosis. Unexpectedly, we find that deletion of Bax and Bak dra-matically reduces necrotic injury during myocardial infarction invivo. Triple knockout mice lacking Bax/Bak and cyclophilin D, akey regulator of necrosis, fail to show further reduction in infarctsize over those deficient in Bax/Bak. Absence of Bax/Bak renderscells resistant to mPTP opening and necrosis, effects confirmed inisolatedmitochondria. Reconstitution of these cells or mitochondriawith wild-type Bax, or an oligomerization-deficient mutant thatcannot support MOMP and apoptosis, restores mPTP opening andnecrosis, implicating distinctmechanisms for Bax-regulated necrosisand apoptosis. Both forms of Bax restore mitochondrial fusion inBax/Bak-null cells, which otherwise exhibit fragmented mitochon-dria. Cells lacking mitofusin 2 (Mfn2), which exhibit similar fusiondefects, are protected to the same extent as Bax/Bak-null cells. Con-versely, restoration of fused mitochondria through inhibition offission potentiates mPTP opening in the absence of Bax/Bak orMfn2, indicating that the fused state itself is critical. These datademonstrate that Bax-driven fusion lowers the threshold for mPTPopening and necrosis. Thus, Bax and Bak play wider roles in celldeath than previously appreciated and may be optimal therapeutictargets for diseases that involve both forms of cell death.

Cells die primarily by apoptosis or necrosis, and mitochondriaplay major roles in both processes (1, 2). Apoptosis is char-

acterized by cell shrinkage, fragmentation, and phagocytosis,maintenance of plasma membrane integrity and ATP levels, andabsence of an inflammatory response. In contrast, central fea-tures of necrosis include cellular and organelle swelling, markeddepletion of ATP, disruption of membranes, and inflammation.Apoptosis has long been recognized as a highly regulated, gene-directed process, whereas, until recently, necrosis was consideredan unregulated form of cell death. Studies over the past decadehave challenged this view and demonstrated that a significantportion of necrotic deaths also occur through highly regulatedmechanisms (3, 4).Apoptosis and necrosis aremediated by distinct but overlapping

pathways involving cell surface death receptors and mitochondria/endoplasmic reticulum (1, 3). The critical mitochondrial event inapoptosis is mitochondrial outer membrane permeabilization(MOMP), which permits release of cytochrome c and otherapoptogens leading to caspase activation. In contrast, the keymitochondrial event in primary necrosis is early opening of themitochondrial permeability transition pore (mPTP) in the innermembrane, which occurs in the absence of cytochrome c release.

Opening of the mPTP causes immediate dissipation of the elec-trical potential difference across the inner membrane (Δψm)leading to cessation of ATP synthesis and massive inflow of waterinto the solute-rich matrix causing severe mitochondrial swelling.In contrast to primary necrosis, secondary necrosis follows apo-ptosis if the removal of apoptotic bodies is delayed or nonexistentas in cell culture (5). In this case, necrotic events, such as loss ofΔψm, occur coincident or after cytochrome c release (6).Mitochondrial morphology is determined by a dynamic equilib-

rium between fission and fusion, repeated cycles of which re-distribute mitochondrial constituents, including DNA, to maintainmitochondrial structure and function (7). Fission is mediated bydynamin-related protein 1 (Drp1), a GTPase that transits from cy-tosol to mitochondria, and Fis1, an outer mitochondrial membraneprotein. Fusion is controlled by three dynamin-related GTPases:Mfn1 andMfn2 in the outer mitochondrial membrane andOpa1 inthe inner mitochondrial membrane. The relationship betweenmitochondrial dynamics and cell death is poorly understood.The Bcl-2 family consists of pro- and antiapoptotic members

that engage in a complex set of interactions to regulate apoptosis(1). Apoptotic signals ultimately converge on Bax and Bak, mul-tidomain proapoptotic proteins to promote MOMP, subsequentcaspase activation, and apoptotic cell death. An additional func-tion of Bax and Bak is to promote fusion in healthy cells, and cellsdeficient in these proteins contain fragmented mitochondria (8, 9).Prior studies have provided hints that Bcl-2 proteinsmay regulate

cell death in situations where necrosis was thought to be involved(10–12), but molecular events and mechanisms have not yet beenelucidated. Here we demonstrate that Bax regulates the sensitivityof cells to undergo primary necrosis. This effect of Bax occursthrough a mechanism that is distinct from the role of Bax in apo-ptosis. Unexpectedly, Bax-driven fusion is critical for these effects.

ResultsAbsence of Bax and Bak Decreases Necrosis in Vivo. To assess therole of Bax and Bak in regulating necrosis in vivo, we used amousemodel of myocardial infarction, a disease process characterized bya mixture of necrotic and apoptotic cardiac myocyte death (13).

Author contributions: R.S.W., K.K., J.W.C., T.L., C.B.T., M.T.C., E.G., G.W.D., B.O., and R.N.K.designed research; R.S.W., K.K., A.-C.W., Y.C., D.E.R., S.J., Y.Y., J.W.C., and E.G. performedresearch; T.L., C.B.T., M.T.C., and E.G. contributed new reagents/analytic tools; R.S.W., K.K.,A.-C.W., Y.C., Y.Y., J.W.C., G.W.D., B.O., and R.N.K. analyzed data; and R.S.W., K.K., andR.N.K. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1R.S.W. and K.K. contributed equally to this work.2To whom correspondence should be addressed. E-mail: richard.kitsis@einstein.yu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1201608109/-/DCSupplemental.

6566–6571 | PNAS | April 24, 2012 | vol. 109 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1201608109

Combined deletion of Bax and Bak significantly reduced infarctsize, a measure of total cell death (Fig. 1A and Fig. S1). Consistentwith the known roles of Bax and Bak in apoptosis (14), this wasaccompanied by a decrease in the percentage of TUNEL-positivecardiacmyocytes (Fig. 1B). Unexpectedly, absence of Bax andBakmarkedly diminished characteristic features of necrosis, includingamorphous mitochondrial densities, poorly defined cristae, swol-len and ruptured mitochondria, and sarcomeric disorganization(Fig. 1C). Strikingly, triple knockout mice lacking Bax/Bak andcyclophilin D, a key regulator of mPTP opening and necrosis (15–17), failed to show any further reduction in infarct size over thosedeficient in Bax/Bak (Fig. 1A). These data indicate that Bax andBak regulate necrosis in vivo and suggest a connection betweenmitochondrial events that mediate apoptosis and necrosis.

Bax/Bak-Null Cells Are Protected from Necrosis, and Susceptibility IsRestored by Reconstitution with Bax. To determine whether thedefining events of necrosis are abrogated by the absence of Baxand Bak, we modeled necrosis and apoptosis in mouse embryofibroblasts (MEFs). As Ca2+ is a primary regulator of mPTPopening, the Ca2+ ionophore ionomycin, was used to inducenecrosis. Treatment with ionomycin triggered mPTP opening asearly as 1 h, as assessed by loss of Δψm. Moreover, this occurredin the absence of cytochrome c release, indicative of primarynecrosis (Fig. 2 A and B). In contrast, the initial event following

treatment with the apoptosis inducer staurosporine was cyto-chrome c release, minimally detectable at 4 h and becomingmaximal at 8 h (Fig. 2B), and only then was loss of Δψm ob-served, consistent with apoptosis (6) (Fig. 2A). Ionomycin, butnot staurosporine, resulted in loss of plasma membrane integrity,a hallmark of necrosis (Fig. 2C). Thus, early mPTP openingwithout cytochrome c release indicates primary necrosis, whereaslater release of cytochrome c coincident with mPTP openingdemarcates apoptosis.Consistent with previous studies (14), cells lacking Bax and

Bak did not undergo apoptosis, as assessed by cytochrome crelease in response to staurosporine (Fig. 2B). Importantly, ab-sence of Bax and Bak abrogated ionomycin-induced necrosis, asassessed by early mPTP opening and loss of plasma membraneintegrity (Fig. 2 A and C). Similarly, isolated mitochondria fromBax/Bak-null hearts required a greater Ca2+ load to inducemPTP opening and swelling compared with wild-type mito-chondria (Fig. 2D).We focused on the role that Bax may play in this process.

Reconstitution of Bax/Bak-null MEFs with Bax restored ion-omycin-induced mPTP opening (Fig. 3B). Similarly, recombinantBax restored Ca2+-induced mPTP opening in Bax/Bak-null iso-lated cardiac mitochondria (Fig. 3C). These results demonstratethat absence of Bax and Bak abrogates necrosis, and sensitivity isrestored by reconstitution with Bax.

A B

CRemote Infarct Infarct

WT-I/R DKO-I/R

Fig. 1. Deletion of Bax and Bak markedly reduces necrotic injury during myocardial infarction in vivo. (A) Infarct size following 45 min of left coronary arteryocclusion followed by 24 h of reperfusion (I/R). AAR/LV, area at risk/left ventricle; INF/AAR, infarct size normalized to AAR; (Left graph) WT, wild-type mice;DKO, double knockout mice lacking Bax and Bak; TKO, triple knockout mice lacking Bax, Bak, and cyclophilin D. (Right graph) WT, wild-type mice; Ppif KO,mice lacking cyclophilin D. Numbers of animals indicated in circles. Confirmation of knockouts in Fig. S1. (B) Apoptosis assessed within the AAR of heartsections from mice subjected to sham operation or 45 min ischemia/10 h reperfusion using TUNEL and costaining with troponin I to identify cardiac myocytesand DAPI. (C) Transmission electron microscopy of infarct zone and remote myocardium following 45 min ischemia/24 h reperfusion. Key features ofmyocardial necrosis including amorphous mitochondrial densities (red arrow), poorly defined cristae (black arrow), mitochondria swelling, and rupture,sarcomeric disorganization (on lower power images). Representative of at least 10 randomly selected fields for each genotype. Data mean ± SEM. ***P <0.001, *P < 0.05, compared with WT. †No significant difference compared with DKO.

Whelan et al. PNAS | April 24, 2012 | vol. 109 | no. 17 | 6567

CELL

BIOLO

GY

Oligomerization-Deficient Bax Restores mPTP Opening in Bax/Bak-Null Cells and Mitochondria. The precise roles of Bax and Bak inMOMP are incompletely understood, but homo- and/or hetero-oligomerization of these proteins is involved (18–20), and olig-omerization-deficient Bax mutants cannot support MOMP andapoptosis (21, 22). Consistent with these observations, treatmentof cells with staurosporine shifted Bax into high molecularweight complexes (Fig. 3A). In contrast, treatment of cells withionomycin did not induce Bax oligomerization (Fig. 3A). Ac-cordingly, we tested whether oligomerized Bax is needed formPTP opening in response to ionomycin. Bax(63-65)A, harbor-ing L63A, R64A, and R65A mutations in the BH3 domain, isunable to oligomerize (21). Equivalent reconstitution of Bax/Bak-null cells or isolated cardiac mitochondria with this mutantor wild-type Bax restored Ca2+-induced mPTP opening to thesame extent (Fig. 3 B and C). These experiments demonstratethat nonoligomerized Bax, although unable to mediate apopto-sis, is sufficient to mediate necrosis.

Mitochondrial Shape Regulates Sensitivity of mPTP Opening. Bothwild-type and oligomerization-deficient Bax restore mitochon-drial fusion in Bax/Bak-null cells (9), which contain fragmentedmitochondria (Fig. 4A). Similarly, cells lacking Mfn2 also exhibit

fragmented mitochondria (8, 9) (Fig. 4A), and these cells wereprotected from Ca2+-induced mPTP opening to a similar extentas Bax/Bak-null cells (Fig. 4B). Given that Bax/Bak-null andMfn2-null cells exhibit a common fragmented mitochondrialmorphology, we tested whether inhibition of mPTP opening ismediated by deficiencies in these specific proteins or, more gen-erally, by changes in mitochondrial morphology. Attenuation ofmitochondrial fission with the small molecule Mdivi-1 (23), aninhibitor of the fission protein Drp1, restored both the fused stateand sensitivity to ionomycin-induced mPTP opening in Bax/Bak-null and Mfn2-null cells (Fig. 4 A and B). These results indicatethat the shift to the fused state per se potentiates Ca2+-inducedmPTP opening.

DiscussionUsing an in vivo model of myocardial infarction, MEFs, andisolated cardiac mitochondria, these data demonstrate that theabsence of Bax and Bak confers resistance to necrotic cell death,and sensitivity can be restored by reconstitution with Bax. Celldeath in these studies occurred by primary necrosis—not ne-crosis secondary to apoptosis—because the events that definenecrosis (mPTP opening, loss of plasma membrane integrity, andcell death) take place within a few hours, without Bax

Fig. 2. Absence of Bax and Bak inhibits mPTP opening and necrosis. (A) mPTP opening in WT and Bax/Bak DKO MEFs treated with ionomycin (Iono) (10 μM) orstaurosporine (STS) (2μM)asassessedby loss ofΔψmusingflowcytometryof live cells stainedwith tetramethyl rhodamineethyl ester (TMRE). ***P<0.001 comparedwith zero timepoint. (B) Cytochrome c (cyt c) assessedby immunoblot of the cytosolic fractionofWTandBax/BakDKOMEFs following stimulationwith STS (2 μM)orIono (10 μM).GAPDH (cytosolic) and complexVα (CVα) (innermitochondrialmembrane)markers. (C) LDH release by enzymatic assay of themedia ofWTandBax/BakDKOMEFs following stimulation with STS (2 μM) or Iono (10 μM). **P < 0.01 comparedwith zero time point. (D) Isolated cardiac mitochondria loaded by repetitiveadditions of CaCl2 (35 μMfirst addition, 25 μMsubsequent additions) to the cuvettete as shownbygreen spikes. Dotted lines,WT; solid lines, Bax/BakDKO;green, CaGreen; red, Δψm; gray, mitochondrial swelling. Δψm lost in WT during Ca2+ load 5 and in DKO after load 9. n ≥ 3 independent experiments for each panel.

6568 | www.pnas.org/cgi/doi/10.1073/pnas.1201608109 Whelan et al.

oligomerization, in the absence of cytochrome c release, and canbe reconstituted with oligomerization-deficient Bax(63-65)A,which cannot support MOMP and apoptosis (21). Moreover, theability of this mutant to restore sensitization to necrosis, but notapoptosis, indicates that Bax modulates necrosis through mech-anisms distinct from apoptosis.In addition to their abilities to rescue sensitivity to necrosis in

Bax/Bak-deficient cells, both wild-type Bax and Bax(63-65)A re-store mitochondrial fusion in these cells, which exhibit baselinemitochondrial fragmentation (8, 9). These observations suggest thehypothesis that Bax-regulated fusion mediates sensitivity to ne-crotic cell death. Consistent with this possibility, cells lacking thefusion proteinMfn2, which also show fragmentedmitochondria (8,9), phenocopy the resistance to necrosis observed in Bax/Bak-nullcells (Fig. 4B). To test the causality of these observations, andwhether the fusion process itself or the fused mitochondrial mor-phology is critical, we restored the fused morphology in Bax/Bak-deficient cells through an independent means, by opposing Drp1-

mediated fission with Mdivi-1. Restoration of the fused morphol-ogy reinstituted susceptibility to necrosis. Moreover, the sameresults were observed with inhibition of fission in Mfn2-null cells.These data strongly suggest that the fused morphology, regardlessof how it is achieved, mediates sensitivity to necrosis.The mechanistic connections between mitochondrial mor-

phology and cell death remain unclear. Previous work haslinked mitochondrial fission with apoptosis (24). Inhibition offission genetically or pharmacologically with Mdivi-1 decreasesor delays apoptosis. Moreover, during apoptosis, Bax oligomersin the outer mitochondrial membrane are associated with fis-sion, although a causal connection has not been demonstrated.In contrast, nonoligomerized Bax is known to drive fusion incells that are considered healthy—defined as not having beensubjected to an apoptotic stimulus (9). The current studyintroduces the concept that the fused mitochondrial state ren-ders these cells poised to undergo necrosis, if presented with anappropriate stimulus.

Fig. 3. Reconstitution of Bax/Bak-null MEFs or mitochondria with wild type or oligomerization-deficient Bax restores mPTP opening. (A) Size fractionation ofcellular lysates from wild-type MEFs not treated or treated for 8 h with staurosporine (1 μM) or ionomycin (10 μM) [all conditions in the presence of z-VADfmk(50 μM)] followed by immunoblotting of fractions for endogenous Bax. Representative of two independent experiments. (B, Left) Reconstitution of Bax/Bak-null MEFs by transfection with GFP, GFP-Bax (WT), or GFP-Bax(63-65)A in the presence of z-VADfmk (40 μM). Flow cytometric analysis of Δψm loss intransfected cells gated for GFP. (Right) Flow cytometric analysis of GFP intensities indicate similar levels of expression of GFP-Bax (WT) and GFP-Bax(63-65)A.***P < 0.001, compared with no ionomycin. (C) Measurement of Ca2+ load required for Δψm loss and swelling following reconstitution of Bax/Bak DKOisolated cardiac mitochondria with 100 nM recombinant WT Bax or Bax(63-65)A. *P < 0.05, compared with no treatment. n ≥ 3 independent experiments.

Whelan et al. PNAS | April 24, 2012 | vol. 109 | no. 17 | 6569

CELL

BIOLO

GY

This model is consonant with previous observations showingthat diffusion of Ca2+ waves is more efficient in fused versusfragmented mitochondria (25). In addition, it is consistent withobservations that absence of Mfn2 inhibits cardiac myocyte death(26). Our findings conflict, however, with those of Ong et al. (27)who reported that inhibition of fission with Mdivi-1 delays mPTPopening and protects cardiac myocytes against ischemia/reperfu-sion injury. Although the reasons for this discrepancy are not clear,cell death in this other study was scored using plasma membraneintegrity at late time points, raising the possibility of necrosissecondary to apoptosis. In addition, Mdivi-1 was used to treatwild-type cells, in which a substantial proportion of mitochondriaare already fused. In contrast, the present study uses geneticmodels of defective mitochondrial fusion to delineate the role ofthe fused morphology in setting the baseline susceptibility of wild-type cells to undergo necrosis. Elucidation of the molecular andbiophysical mechanisms by which the fused state sensitizes cells tomPTP opening and necrosis will likely require identification of the

components of this pore, none of which have yet been determinedwith certainty (28).Cell death in ischemic syndromes, such as myocardial in-

farction and stroke, is characterized by a spatially and temporallycomplex pattern of necrosis and apoptosis (4, 29). Whereas in-hibition of either form of cell death reduces infarct size, optimalamelioration of both the acute injury in the central infarct zoneand the subsequent cell death in immediately surrounding areasrequires inhibition of both necrosis and apoptosis. Bax and Bakmay provide especially potent therapeutic targets to achieve thisgoal in these common and lethal syndromes.

Materials and MethodsMyocardial Infarction Model and Analysis. Bax(flox/flox); Bak−/− mice (30) werecrossed with α-myosin heavy chain-Cre transgenic mice (31) to generate micewith cardiac myocyte-specific deletion of Bax and generalized deletion ofBak. These mice were crossed with Ppif−/− mice (15) to generate mice withcardiac myocyte-specific deletion of Bax and generalized deletion of Bak andPpif. Ischemia/reperfusion was induced in 8- to 12-wk-old male mice by

No tx

Mdivi-1

A

FSC-AFSC-AFSC-A

Nu

mb

er

of

even

ts

Nu

mb

er

of

even

ts

Nu

mb

er

of

even

ts

B

FSC AFSC A

Fig. 4. Cells with fragmented mitochondria are resistant to mPTP opening, which can be reversed by restoration of fused morphology. (A) Analysis ofmitochondrial morphology by confocal microscopy of WT, Bax/Bak DKO, and Mfn2 KO MEFs not treated or treated for 6 h with Mdivi-1 (50 μM). Cells stainedwith MitoTracker Red and DAPI. Quantification of fused mitochondrial morphology by flow cytometry of mitochondria. FSC-A, forward scatter area. (B)Ionomycin-induced mPTP opening in the same groups except ionomycin (10 μM) added 2 h before analysis. n = 3 independent experiments. ***P < 0.001,compared with wild-type cells that were treated with ionomycin but not Mdivi-1. §§§P < 0.001 compared with cells of the same genotype that were treatedwith ionomycin but not Mdivi-1.

6570 | www.pnas.org/cgi/doi/10.1073/pnas.1201608109 Whelan et al.

ligating the left coronary artery for 45 min followed by 24-h reperfusion. Thearea at risk (AAR) was assessed by Evan’s blue dye, and the area of infarct(INF) was determined by staining with 2,3,5-triphenyltetrazolium chloride asdescribed (32). TUNEL was performed as described (33) and sections werecounterstained for troponin I (Santa Cruz) and DAPI (Vector Laboratories).Transmission electron microscopy was performed on samples fixed with 2%(vol/vol) paraformaldehyde, 2.5% (vol/vol) glutaraldehyde in 0.1 M sodiumcacodylate, postfixed with 1% (wt/vol) osmium tetroxide, followed by 1%(wt/vol) uranyl acetate, dehydrated, and embedded in LX112 resin. Ultrathinsections stained with uranyl acetate followed by lead citrate were viewed ona JEOL 1200EX transmission electron microscope at 80 kV.

Immunoblotting. Bax and Bak antisera were from Cell Signaling. Cyclophilin D,cytochrome c, complex Vα, and GAPDH antibodies were from Mitosciences/Abcam.

Analysis of Δψm.MEFs were seeded at a density of 310 cells per mm2, and thenext day treated with stimuli as indicated, incubated with tetramethylrhodamine ethyl ester (TMRE) (20 nM) for 30 min, trypsinized, collected, andanalyzed by flow cytometry using a FACS Calibur DXP10.

Subcellular Fractionation. Cells were harvested and resuspended in 10mMKCl,5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 20 mM HepespH 7.2, 0.025% (wt/vol) digitonin, and protease inhibitors. Following 5 minincubation on ice, the lysate was spun down at 15,000 × g for 10 min at 4 °C,and the supernatant containing the cytosol was stored. The pellet was lysedwith 1% (vol/vol) Triton X-100 in PBS for 1 h at 4 °C. Fractional purity wasdetermined by blotting with GAPDH (cytosolic) and complex Vα (inner mi-tochondrial membrane) markers.

LDH Release. This was quantified using CytoTOX-One homogeneous mem-brane integrity assay (Promega).

Ca2+ Loading Assay. Cardiac mitochondria were isolated from adult mice andincubated with Ca-Green and TMRE (Invitrogen) to determine the Ca2+ loadthat triggers Δψm loss and swelling (34). For reconstitution experiments,recombinant WT Bax or Bax(63-65)A were added to the mitochondria ata final concentration of 100 nM and measurements made 10 min later.

FPLC. Total cellular proteinwas size fractionatedonaSuperose 6 sizing columnusing the AKTA FPLC system (35), and fractions immunoblotted for Bax.

Transfection. Cells were transfected using TransIT-LT1 (Mirus Bio).

Recombinant Bax Production. WT Bax and Bax(63-65)A Bax were generatedusing the CBP-intein system (36). Following purification, monomeric Bax indetergent-free buffer was isolated by size-exclusion chromatography.

Mitochondrial FACS. This procedure was perfomed as described (37).

ACKNOWLEDGMENTS. We thank Drs. Emily H.-Y. Cheng and Xu Luo forconstructs and cell lines, Drs. Nina Kaludercic and Fabio DiLisa for adviceregarding mitochondrial assays, Drs. Charles J. Steenbergen and Stephen M.Factor for advice regarding cardiac pathology, and Chad K. Nicholson fortechnical assistance.We also thank theWilf family for their ongoing generosityand support. This work was supported by Grants 5R01HL60665-13 (to R.N.K.),5P30CA013330-39 (to R.N.K. and E.G.), 5P60DK020541-34 (to R.N.K.),5R37HL054598-16 (to B.O.), 5R01HL059888-12 (to G.W.D.), 4R00HL095929-02(to E.G.), American Heart Association Grant GRNT2290168 (to M.T.C.),5T32AG023475-08 (to R.S.W.), and the A. G. Leventis Foundation (K.K.). R.N.K.is supported by The Dr. Gerald andMyra Dorros Chair in Cardiovascular Disease.

1. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR (2010) The BCL-2 family

reunion. Mol Cell 37:299–310.2. Zong WX, Thompson CB (2006) Necrotic death as a cell fate. Genes Dev 20:1–15.3. Golstein P, Kroemer G (2007) Cell death by necrosis: Towards a molecular definition.

Trends Biochem Sci 32:37–43.4. Kung G, Konstantinidis K, Kitsis RN (2011) Programmed necrosis, not apoptosis, in the

heart. Circ Res 108:1017–1036.5. Zitvogel L, Kepp O, Kroemer G (2010) Decoding cell death signals in inflammation and

immunity. Cell 140:798–804.6. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR (2000) The coordinate release

of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell

Biol 2:156–162.7. Detmer SA, Chan DC (2007) Functions and dysfunctions of mitochondrial dynamics.

Nat Rev Mol Cell Biol 8:870–879.8. Karbowski M, Norris KL, Cleland MM, Jeong SY, Youle RJ (2006) Role of Bax and Bak

in mitochondrial morphogenesis. Nature 443:658–662.9. Hoppins S, et al. (2011) The soluble form of Bax regulates mitochondrial fusion via

MFN2 homotypic complexes. Mol Cell 41:150–160.10. Martinou JC, et al. (1994) Overexpression of BCL-2 in transgenic mice protects neurons

from naturally occurring cell death and experimental ischemia. Neuron 13:1017–1030.11. Hochhauser E, et al. (2003) Bax ablation protects against myocardial ischemia-re-

perfusion injury in transgenic mice. Am J Physiol Heart Circ Physiol 284:H2351–H2359.12. Pastorino JG, et al. (1999) Functional consequences of the sustained or transient ac-

tivation by Bax of the mitochondrial permeability transition pore. J Biol Chem 274:

31734–31739.13. Whelan RS, Kaplinskiy V, Kitsis RN (2010) Cell death in the pathogenesis of heart

disease: Mechanisms and significance. Annu Rev Physiol 72:19–44.14. Wei MC, et al. (2001) Proapoptotic BAX and BAK: A requisite gateway to mito-

chondrial dysfunction and death. Science 292:727–730.15. Baines CP, et al. (2005) Loss of cyclophilin D reveals a critical role for mitochondrial

permeability transition in cell death. Nature 434:658–662.16. Nakagawa T, et al. (2005) Cyclophilin D-dependent mitochondrial permeability

transition regulates some necrotic but not apoptotic cell death. Nature 434:652–658.17. Schinzel AC, et al. (2005) Cyclophilin D is a component of mitochondrial permeability

transition and mediates neuronal cell death after focal cerebral ischemia. Proc Natl

Acad Sci USA 102:12005–12010.18. Gross A, Jockel J, Wei MC, Korsmeyer SJ (1998) Enforced dimerization of BAX results

in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 17:3878–3885.19. Antonsson B, Montessuit S, Lauper S, Eskes R, Martinou JC (2000) Bax oligomerization

is required for channel-forming activity in liposomes and to trigger cytochrome c

release from mitochondria. Biochem J 345:271–278.

20. Mikhailov V, et al. (2003) Association of Bax and Bak homo-oligomers in mitochon-dria. Bax requirement for Bak reorganization and cytochrome c release. J Biol Chem278:5367–5376.

21. George NM, Evans JJ, Luo X (2007) A three-helix homo-oligomerization domaincontaining BH3 and BH1 is responsible for the apoptotic activity of Bax. Genes Dev 21:1937–1948.

22. Kim H, et al. (2009) Stepwise activation of BAX and BAK by tBID, BIM, and PUMAinitiates mitochondrial apoptosis. Mol Cell 36:487–499.

23. Cassidy-Stone A, et al. (2008) Chemical inhibition of the mitochondrial divisiondynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane per-meabilization. Dev Cell 14:193–204.

24. Martinou JC, Youle RJ (2011) Mitochondria in apoptosis: Bcl-2 family members andmitochondrial dynamics. Dev Cell 21:92–101.

25. Szabadkai G, et al. (2004) Drp-1-dependent division of the mitochondrial networkblocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis.Mol Cell 16:59–68.

26. Papanicolaou KN, et al. (2011) Mitofusin-2 maintains mitochondrial structure andcontributes to stress-induced permeability transition in cardiac myocytes.Mol Cell Biol31:1309–1328.

27. Ong SB, et al. (2010) Inhibiting mitochondrial fission protects the heart against is-chemia/reperfusion injury. Circulation 121:2012–2022.

28. Halestrap AP (2010) A pore way to die: The role of mitochondria in reperfusion injuryand cardioprotection. Biochem Soc Trans 38:841–860.

29. Moskowitz MA, Lo EH, Iadecola C (2010) The science of stroke: Mechanisms in searchof treatments. Neuron 67:181–198.

30. Takeuchi O, et al. (2005) Essential role of BAX,BAK in B cell homeostasis and pre-vention of autoimmune disease. Proc Natl Acad Sci USA 102:11272–11277.

31. Oka T, et al. (2006) Cardiac-specific deletion of Gata4 reveals its requirement forhypertrophy, compensation, and myocyte viability. Circ Res 98:837–845.

32. Calvert JW, et al. (2011) Exercise protects against myocardial ischemia-reperfusioninjury via stimulation of β(3)-adrenergic receptors and increased nitric oxide signaling:Role of nitrite and nitrosothiols. Circ Res 108:1448–1458.

33. Lee P, et al. (2003) Fas pathway is a critical mediator of cardiac myocyte death and MIduring ischemia-reperfusion in vivo. Am J Physiol Heart Circ Physiol 284:H456–H463.

34. Wei AC, Liu T, Cortassa S, Winslow RL, O’Rourke B (2011) Mitochondrial Ca2+ influxand efflux rates in guinea pig cardiac mitochondria: Low and high affinity effects ofcyclosporine A. Biochim Biophys Acta 1813:1373–1381.

35. Zheng R, et al. (2010) Analysis of the interactions between the C-terminal cytoplasmicdomains of KCNQ1 and KCNE1 channel subunits. Biochem J 428:75–84.

36. Gavathiotis E, et al. (2008) BAX activation is initiated at a novel interaction site.Nature 455:1076–1081.

37. Chen Y, Liu Y, Dorn GW, 2nd (2011) Mitochondrial fusion is essential for organellefunction and cardiac homeostasis. Circ Res 109:1327–1331.

Whelan et al. PNAS | April 24, 2012 | vol. 109 | no. 17 | 6571

CELL

BIOLO

GY