mitofusin-2 maintains mitochondrial structure and contributes to

MOLECULAR AND CELLULAR BIOLOGY, Mar. 2011, p. 1309–1328 Vol. 31, No. 60270-7306/11/$12.00 doi:10.1128/MCB.00911-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Mitofusin-2 Maintains Mitochondrial Structure and Contributes toStress-Induced Permeability Transition in Cardiac Myocytes�†

Kyriakos N. Papanicolaou,1 Ramzi J. Khairallah,2 Gladys A. Ngoh,1 Aristide Chikando,4 Ivan Luptak,1,3

Karen M. O’Shea,2 Dushon D. Riley,4 Jesse J. Lugus,1 Wilson S. Colucci,1,3

W. Jonathan Lederer,4 William C. Stanley,2 and Kenneth Walsh1*Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany Street, W611, Boston, Massachusetts 021181;

Division of Cardiology and Department of Medicine, University of Maryland, 20 Penn Street, HSF2, Room S022, Baltimore,Maryland 212012; Cardiovascular Medicine Section and Myocardial Biology Unit, Boston University Medical Center,

715 Albany Street, X704, Boston, Massachusetts 021183; and Center for Biomedical Engineering and Technology,University of Maryland Baltimore, 725 W. Lombard Street, Baltimore, Maryland 212014

Received 5 August 2010/Returned for modification 10 September 2010/Accepted 17 December 2010

Mitofusin-2 (Mfn-2) is a dynamin-like protein that is involved in the rearrangement of the outermitochondrial membrane. Research using various experimental systems has shown that Mfn-2 is amediator of mitochondrial fusion, an evolutionarily conserved process responsible for the surveillance ofmitochondrial homeostasis. Here, we find that cardiac myocyte mitochondria lacking Mfn-2 are pleiomor-phic and have the propensity to become enlarged. Consistent with an underlying mild mitochondrialdysfunction, Mfn-2-deficient mice display modest cardiac hypertrophy accompanied by slight functionaldeterioration. The absence of Mfn-2 is associated with a marked delay in mitochondrial permeabilitytransition downstream of Ca2� stimulation or due to local generation of reactive oxygen species (ROS).Consequently, Mfn-2-deficient adult cardiomyocytes are protected from a number of cell death-inducingstimuli and Mfn-2 knockout hearts display better recovery following reperfusion injury. We conclude thatin cardiac myocytes, Mfn-2 controls mitochondrial morphogenesis and serves to predispose cells tomitochondrial permeability transition and to trigger cell death.

Mitochondria from a variety of organisms and tissues havebeen described as dynamic organelles that change their shapeand size and remodel their internal membranes or move todistinct cellular locations (11, 36, 54). These morphologicaltransitions are greatly influenced by fusion and fission of themitochondrial membranes and have been referred to as mito-chondrial dynamics (29, 46). In adult cardiac myocytes, mito-chondria do not display significant motility and they are inclose contact with each other (10, 81). Their morphologicalvariability is confined and depends upon the myocyte compart-ment that they occupy (e.g., interfibrillar versus subsarcolem-mal mitochondria [IFM and SSM, respectively]) (2, 53, 76).Furthermore, it has been recognized that cardiac mitochondriaare arranged in a highly organized pattern and under localizedstress conditions can coordinate their membrane potential andpropagate depolarizing events throughout the cell, suggestingthe existence of interorganellar communication mechanisms(4, 14, 15, 85). Therefore, questions remain as to what are theunique features of mitochondrial dynamics in fully differenti-ated cardiac myocytes and what is their impact on mitochon-drial structure and energetics.

Mitochondrial fusion requires membrane potential, GTP hy-

drolysis, and the assembling activities of mitofusins 1 and 2(Mfn-1 and Mfn-2, respectively) and optic atrophy protein 1(Opa-1) (17, 18, 21, 43, 55, 74). Mfn-1 and Mfn-2 are integralto the outer mitochondrial membrane (OMM), whereas Opa-1can be integral or associated with the inner mitochondrialmembrane (IMM) (50, 62). Mitochondrial fission requires dy-namin-related protein 1 (Drp-1), which is detected primarily inthe cytosol but translocates to the OMM after interacting withfission protein 1 (Fis-1) (78, 84). All of these mitochondrion-shaping proteins are expressed in the mammalian heart (28, 32,42, 73), but their roles in regulating organelle structure andfunction in this tissue remain to be elucidated.

Mfn-2 is a large GTPase that is essential for mitochondrialfusion during embryonic development and neuronal differen-tiation (16, 18, 19). In the human population, mutations in theMFN-2 locus are linked to Charcot-Marie-Tooth type 2a(CMT2a) neuropathy (86). Mfn-2 is robustly expressed in theheart (5), and Mfn-2 insufficiency and associated fragmenta-tion of the mitochondrial network in cultured neonatal cardiacmyocytes have been reported to promote early apoptoticevents (66). In a different experimental setting, however,Mfn-2 is reported to induce death in neonatal cardiomyocytesand in H9C2 cells through the intrinsic mitochondrion-depen-dent pathway (75). This apparent controversy may be due tocell type-specific effects or may be reflective of the multipleroles ascribed to Mfn-2 (27). More recently, Mfn-2, in additionto its targeting on mitochondria, was shown to reside on en-doplasmic reticulum (ER) membranes, and this dual localiza-tion is thought to facilitate transfer of Ca2� from the ER intothe adjacent mitochondria (26). This could potentially expose

* Corresponding author. Mailing address: Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Med-icine, 715 Albany Street, W611, Boston, MA 02118. Phone: (617)414-2390. Fax: (617) 414-2391. E-mail: [email protected].

† Supplemental material for this article may be found at http://mcb.asm.org/.

� Published ahead of print on 18 January 2011.

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mitochondria to very high local Ca2� concentrations, as sug-gested by the Ca2�-microdomain hypothesis (70, 71).

In parallel with their crucial role in fuel oxidation and energyconversion, cardiac mitochondria are also centrally involved incell death cascades (37). The mitochondrial permeability tran-sition pore (MPTP), classically activated by Ca2� and reactiveoxygen species (ROS), is an important determinant of myocyteloss, especially in the context of ischemia and reperfusion in-jury (7, 30, 38), but its molecular composition and regulationremain controversial (40). The initial working model suggestedthat the pore is made of the outer mitochondrial membranevoltage-dependent anion channel (VDAC), the inner mito-chondrial membrane adenine nucleotide translocase (ANT),and the matrix protein cyclophilin D (Cyp-D) (23). However,genetic studies have challenged this model and showed thatonly Cyp-D is a critical member of the pore (8, 60), whereasANT appears to perform a regulatory rather than a structuralrole (49). Finally, the outer mitochondrial membrane compo-nent of the pore remains elusive, as all isoforms of VDAC wereshown to be dispensable for MPTP function (9).

In the present study, we find that the conditional deletion ofMfn-2 increases the proportion of enlarged mitochondria incardiac myocytes but does not lead to a major impairment ofcardiac function. In addition, Mfn-2-depleted mitochondriawere found to be more tolerant to Ca2�-induced MPTP open-ing, and isolated Mfn-2-knockout myocytes were protectedfrom local generation of ROS and subsequent MPTP activa-tion. Finally, Mfn-2 knockout hearts were able to develophigher pressures during postischemic reperfusion and exhib-ited diminished cell death following in vivo regional ischemiaand reperfusion injury. These data illustrate that Mfn-2 notonly serves to maintain mitochondrial morphology in cardiacmyocytes but also promotes MPTP opening in the heart underconditions of stress.

MATERIALS AND METHODS

Mice. All procedures that involved animal handling were approved by theInstitutional Animal Care and Use Committee at the Boston University Schoolof Medicine or the University of Maryland School of Medicine. The mice werehoused in a 12-hour light/dark cycle and temperature-controlled room withaccess to water and food ad libitum. Genotyping was performed to differentiatebetween the wild-type Mfn-2 allele (Mfn-2�) and the conditional Mfn-2flox (Mfn-2F) allele as previously described (19). Similarly, to differentiate between micewith the alpha myosin heavy chain (�-MHC) transgene (cre�) present and absent(cre0), genotyping was performed as previously described (1). Mice with thegenotype Mfn-2flox/flox; cre� are termed F/F;cre, whereas their littermates with thegenotype Mfn-2F/F; cre0 are termed F/F;�. In addition, mice with the genotypeMfn-2�/�; cre� were derived from different crossings within the same lineage andare termed �/�;cre. The mice examined in this study are on a mixed geneticbackground (129S/C57BL6/Black Swiss). The macrophage-specific Mfn-2-defi-cient strain was generated by crossing the Mfn-2F/F line to a strain expressing creunder the control of the endogenous promoter of lysozyme M (LyzM-cre strain;Jackson Laboratory). These mice are referred as F/F;creLysM.

Echocardiography and invasive hemodynamic analysis of the LV. Mice wereanesthetized with 1% isoflurane and fixed in the supine position on a heating padequipped with electrocardiogram (ECG) surface lead II, and preheated sono-graphic gel (Aquasonic) was applied to their shaved chests. The Vevo 770 systemwith probe 707 was used to obtain long-axis parasternal views of the left ventricle(LV). The endocardium and epicardium were traced in long-axis frames tocalculate the LV mass according to a built-in formula that utilizes the average LVwall thickness, the epicardial and endocardial dimensions in the diastole, and thespecific gravity constant of myocardium (1.05 g/ml). Using the ECG trace, weidentified the LV frames that corresponded to the end diastole (LVd) and endsystole (LVs). These measurements from the long axis were combined with

measurements from four different levels from the short axis in order to calculatethe end-systolic and end-diastolic volumes (ESV and EDV, respectively) accord-ing to Simpson’s equation. Subsequently, the ejection fraction (EF) was calcu-lated as the stroke volume (SV � EDV � ESV) over the EDV. Peak flowvelocities at the pulmonic and aortic valves (PPF and PAF, respectively) werealso recorded from the long-axis position using the pulsed-wave (PW) Dopplertransducer. The short-axis M-mode view at the level of the papillary muscles wasused to evaluate cardiac contractility in terms of fractional shortening (FS) basedon the dimensions of the LV in diastole (LVIDd) and systole (LVIDs). At thefour-chamber apical view, we recorded mitral flow velocities, and relevant wave-forms were used to measure early (E) and late (A) filling velocities and ejectiontime (ET). From the same anatomic position, we also recorded the motion of themitral annulus using tissue Doppler (TD), and E� and A� values were measured.

Hemodynamic analysis of the heart in situ was performed on isoflurane-anesthetized and constantly ventilated mice as previously described (65).Briefly, the 1.4F SPR-839 catheter (Millar), connected to a PowerLab/8SP(ADInstruments), was inserted in the LV via the right carotid artery. Pres-sure-volume (PV) loops at baseline and after vena cava occlusion wererecorded and subsequently analyzed offline using PVAN 3.2 software. Thevolume signal was corrected for parallel conductance using the saline bolusinjection technique and was converted to �l according to a standard curvegenerated with a calibration cuvette. In a separate set of experiments, thejugular vein was cannulated for isoproterenol infusions and the catheter wasinserted in the LV of anesthetized mechanically ventilated mice via an apicalstub. Following stabilization, baseline recordings were taken and isoproter-enol (Calbiochem) dissolved in normal saline (0.2 �g/ml) was continuouslyinfused for 5 min with a syringe pump (Harvard Apparatus) at a rate of 5ng/kg/min. At the end of the infusion, PV loops were recorded to calculatethe various hemodynamic parameters offline.

Isolated heart ischemia and reperfusion model. Mice were heparinized andanesthetized with sodium pentobarbital (150 mg/kg of body weight intraperito-neally [i.p.]), and their hearts were quickly removed. Hearts were perfused in theLangendorff mode with phosphate-free Krebs-Henseleit buffer containing 118mM NaCl, 25 mM NaHCO3, 5.3 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 0.5mM EDTA, 5 mM glucose, and 0.5 mM pyruvate at 37.5°C as previously de-scribed (57). The perfusate was equilibrated with 95% O2 and 5% CO2 (pH 7.4).All hearts were stabilized for 25 min at a constant perfusion pressure of 80 mmHg. A water-filled balloon was inserted into the LV to record ventricular pres-sure and heart rate. After stabilization, balloon volume was adjusted to achievethe end diastolic pressure (8 to 10 mm Hg). Baseline tracings were acquired for10 min, and hearts were subjected to no-flow global ischemia for 10 min andreperfused for 20 min (57).

In vivo myocardial reperfusion injury and determination of cell death. Themice were anesthetized with intraperitoneal injection of 50-mg/kg sodium pen-tobarbital (Nembutal), mechanically ventilated at 130 breaths/min, and con-nected to a PowerLab/8SP for constant monitoring of heart rate and ECGpatterns as previously described (65). Core body temperature was monitoredwith a rectal probe connected to a temperature controller (Harvard Apparatus)and maintained between 37.0 and 37.2°C. The chest was opened, and the leftanterior descending (LAD) coronary artery was tied in line with a snare occluderusing a monofilament suture (8-0; S&T) to produce regional ischemia. Reper-fusion injury was induced by removing the occluder 30 min later. During ma-nipulations of the LAD coronary artery, the ECG pattern was used to confirmischemia and reperfusion. The animals were allowed to recover, and 2 h afterreperfusion, the LAD coronary artery was reoccluded and 0.15 ml Evans blue(5%, wt/vol) was injected into the jugular vein to delineate the ischemic from thenonischemic area. The hearts were then collected and cut into 5 slices, whichwere weighed and subsequently incubated in triphenyl-tetrazolium-chloride(TTC; 1%, wt/vol) at 37°C for 5 min to highlight the infarct areas (IA). Imagingand calculations of the different portions of the heart (i.e., area at risk [AAR] andIA) were carried out as previously described (65). In addition, some slices werefixed in formalin, and 5-�m-thick sections were collected from paraffin-embed-ded heart samples and were stained to detect double-stranded DNA breaks usingthe terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label-ing (TUNEL) assay according to manufacturer’s specifications (Roche).

Tissue processing for electron microscopy and micrograph analysis. For elec-tron microscopy, the hearts were perfused through the apex with Sorensen’sphosphate buffer (0.2 M, pH 7.2) and excised and the LV free wall was dissectedand cut into 4 longitudinal rod-shaped pieces (�1 mm in diameter). The pieceswere placed in Karnovsky fixative (2% paraformaldehyde and 2.5% glutaralde-hyde in 0.1 M phosphate buffer; Electron Microscopy Sciences) for 2 h. Follow-ing postfixation in 2% OsO4, the pieces were dehydrated through ethanol andembedded in Epon plastic. Toluidine blue staining of semithin sections was used

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to evaluate the orientations of the sections and to select the areas containingpredominantly longitudinal myofibers. Preparation of ultrathin sections (�70nm) perpendicular to the long axis of the tissue was performed with a diamondknife microtome (Sorvall). Sections were stained with lead citrate and uranylacetate and visualized using a Philips CM12 transmission electron microscope.Ten to 18 fields containing longitudinally arrayed myofibrils, excluding nuclei,were photographed from each section at the �6,300 or �35,000 magnification.Mitochondrial and myofibrillar volume density was assessed directly from themicrographs using the grid method (59). Using ImageJ (Wayne Rasband, NIH),a mask was generated for each field photographed and cross-sectional areas andmaximum/minimum Feret’s diameters of individual mitochondria were mea-sured. Kolmogorov-Smirnov analysis was used to evaluate differences in thediameter distributions between the two genotypic groups.

Live-cell imaging with confocal microscopy and morphological analysis. Insome experiments, cardiac myocytes were isolated from adult mice and seededon glass bottom dishes (MatTek) that had been coated with laminin (BD Bio-sciences; 240 �g/dish) in minimum essential medium (MEM; Invitrogen) sup-plemented with 20 mM butanedione monoxime (BDM; Sigma) and 5% (vol/vol)calf serum (Invitrogen) as previously described (65). After an initial plating stepfor at least 30 min, cells were exposed to fresh medium containing 2 nM tetra-methylrhodamine ethyl ester (TMRE; Molecular Probes) for 15 min at 37°C. Themedium was replaced with dye-free medium, and cells were visualized with aZeiss LSM 710 confocal microscope. To analyze mitochondrial morphology, weused the 100� oil immersion lens (Plan-Apochromat; numerical aperture [NA],1.4), TMRE was excited with the 543-nm laser set at 0.2% power, and myocyteswere scanned in their z axis in 15 to 20 slices spaced by 0.38 �m. Volumerenderings were performed with the three-dimensional (3D) viewer plug-in ofImageJ (M. Abramoff). In other experiments, 100 nM tetramethylrhodaminemethyl ester (TMRM) was loaded onto myocytes for 15 min at 37°C, and afterthe monocytes were switched to dye-free medium, they were exposed to 200 �MH2O2 at time zero (t0) and imaged at 1-min intervals with the 63� objective (1.40oil differential inference contrast [DIC]) and with the 543-nm excitation laser setat 1.8% power, for a total duration of 25 min. The images were analyzed offlinefor changes in fluorescence intensity over time from multiple regions of interest(ROI) (8 by 8 �m) per myocyte using ImageJ. To assess mitochondrial mem-brane potential (m), freshly prepared myocytes were incubated for 30 min at37°C in medium containing 5 �M JC-1, which was subsequently replaced bydye-free medium. Stained myocytes were individually imaged using the 100�objective; the monomeric form of JC-1 was excited using the 488-nm laser, andthe aggregate form was excited using the 543-nm laser. Acquired images wereanalyzed offline using ImageJ, and the fluorescence intensity for each JC-1 formwas determined for multiple regions of interest per myocyte. In other experi-ments, fresh myocytes were exposed to 500 nM MitoTracker Red (CMXRos;Molecular Probes) or 100 nM TMRM for 15 min at 37°C, after which they wereswitched to dye-free medium. The m-dependent accumulation of fluorophoremolecules in mitochondria was determined by exciting with the 561-nm (Mito-Tracker Red) or 543-nm (TMRM) laser, and the fluorescence intensity wasdetermined from multiple ROI per myocyte and analyzed offline with ImageJsoftware.

In other experiments, adult mouse cardiomyocytes were isolated using previ-ously described methods (77). In brief, mice were heparinized and then anes-thetized using a lethal dose of i.p. pentobarbital (100 mg/kg). The heart wasquickly excised and placed in an ice-cold, Ca2�-free physiologic buffer solution.Following aortic cannulation, the heart was perfused with Ca2�-free physiolog-ical saline and then with a solution containing 1 mg/ml collagenase and 0.05 �MCa2�. The ventricles were cut, minced, and treated with a second enzymaticsolution containing 0.67 mg/ml collagenase, 0.13 mg/ml protease, 0.05 �M Ca2�,and 16.67 mg/ml bovine serum albumin (BSA). Ventricular tissue was lightlytriturated with a coated glass pipette. Cells were washed through a large meshfilter (300 �m) and lightly centrifuged at 200 rpm for 2 min. The cells weregradually reintroduced to physiologic Ca2� concentration and stored in physio-logical saline at room temperature.

Freshly isolated cardiomyocytes were bathed at room temperature with aphysiological saline solution that contained 100 nM TMRM for 20 min. Follow-ing the loading period, cells were transferred to a TMRM-free physiologicalsolution and stored until use. Imaging was carried out in living TMRM-loadedcardiomyocytes placed on laminin-coated glass coverslips in a custom-designedperfusion chamber. A laser scanning confocal microscope (Zeiss LSM 510, 100�oil immersion lens; NA, 1.3) was used to image the cells with 543-nm excitation,and cells were viewed through a 560LP emission filter. Repetitive imaging wascarried out at 0.7 Hz with constant illumination intensity and a constant size (30by 35 �m) of the region of interest.

To measure cell contractility, isolated myocytes were loaded with the Ca2�-

sensitive indicator fluo-4 AM (10 �M) for 15 min before they were washed andstored in physiological saline. Cells were imaged using a 488-nm excitation laserand imaged with a model 505LP optical filter. Longitudinal line scan imagesalong the full length of each cardiomyocyte were acquired at a rate of 520 scansper second. While being imaged, cardiomyocytes were stimulated by field shocks(MyoPacer [IonOptix], 20 to 40 V for 1 to 2 ms). Cells were stimulated at a 1-Hzfrequency for 10 s to ensure proper sarcoplasmic reticulum (SR) loading beforeimages were acquired for 10 s, during which time cells were stimulated at 1 Hzfor 5 s, followed by 2.5 s of rest.

Analysis of mitochondrial morphology in cardiac myocytes from neonate ratsor peritoneal macrophages and time-lapse fluorescence microscopy. Neonatalrat cardiac myocytes (NRCMs) were isolated as previously described (68) andtreated with 90 nM Mfn-2-specific or unrelated small interfering RNA (siRNA)(Dharmacon) using the Lipofectamine reagent (Invitrogen) under serum-freeconditions for 48 h. For mitochondrial morphology, cells were loaded with 50 nMMitoTracker Red and incubated at 37°C for 30 min, after which cells werewashed and fixed. Imaging was performed using the 63� lens of a Nikon invertedmicroscope. Additionally, NRCMs were loaded with 1 nM TMRM for 30 minand then switched to imaging medium (Dulbecco’s modified Eagle’s medium[DMEM] with 25 mM HEPES and without phenol red and pyruvate). Oxidativestress was induced with 200 �M H2O2, and imaging was initiated immediately.Images were captured every 90 s for 60 min using a Nikon deconvolutionwide-field epifluorescence microscope system controlled with Nikon software.Similarly, peritoneal macrophages isolated from F/F;creLyzM and F/F;� micewere loaded with 1 nM TMRM for 30 min, and the medium was replaced withdye-free medium. Oxidative stress was induced with 200 �M H2O2, and imagingwas initiated immediately. Cells were visualized with a Zeiss LSM 226 710confocal microscope. The TMRM dye was excited using the 543-nm laser set at1.8% power. Images were captured at 1-min intervals.

Isolation of cardiac mitochondria. Subsarcolemmal mitochondria (SSM) andinterfibrillar mitochondria (IFM) were isolated from adult mouse hearts aspreviously described (48). Briefly, hearts were minced in ice-cold isolation buffer(IB; 100 mM KCl, 50 mM MOPS [morpholinepropanesulfonic acid], 5 mMMgSO4 � 7H2O, 1 mM EGTA, 1 mM ATP, pH 7.4), and the suspension wasdisrupted by Polytron treatment and homogenized with a Potter-Elvehjem tissuegrinder. The homogenate was centrifuged at 600 � g, and then the SSM werecollected from the supernatant at 3,000 � g and washed/resuspended in KMEbuffer (100 mM KCl, 50 mM MOPS, 0.5 mM EGTA) at a final concentration of25 mg mitochondrial protein/ml. To release IFM, the pellet was disrupted byPolytron treatment in the presence of trypsin (5 mg/ml) and centrifuged at600 � g. The IFM were collected from the supernatant at 3,000 � g and werewashed/resuspended in KME buffer at a final concentration of 25 mg mito-chondrial protein/ml.

In a separate set of experiments, total cardiac mitochondrial populations wereisolated using methods previously described (39, 61). Briefly, hearts were har-vested after CO2 asphyxiation and homogenized in 4 ml buffer A (67 mMsucrose, 50 mM Tris-HCl, 2 mM EGTA, 50 mM KCl, and 0.2% fatty acid-freeBSA, pH 7.4) using an ice-cold glass homogenizer (Kontes). The homogenatewas centrifuged at 2,000 � g, and the mitochondrion-containing supernatant wascentrifuged at 10,000 � g. This pellet was washed twice with 1 ml of buffer B(buffer A without BSA), resuspended in 1 ml of buffer B, loaded onto a 19%(vol/vol) Percoll solution, and centrifuged at 14,000 � g in 4°C. The mitochon-drial fraction was resuspended in 0.2 ml of mitochondrial swelling buffer (67 mMsucrose, 50 mM Tris-HCl, and 50 mM KCl, pH 7.4). Mitochondrial proteinconcentration was determined using the Bradford method.

To determine mitochondrial diameter, a flowmetric assay was used as previ-ously reported (24). Briefly, isolated SSM and IFM were stained with Mito-Tracker Deep Red 633 (Molecular Probes) and assessed using a BD LSR Iflowmeter (BD Biosciences). The mean output from the forward scatter detectorwas used as an index of mitochondrial size. The adjustment to actual �m wasperformed using calibration microspheres (0.5 to 6.0 �m; Invitrogen), and mi-tochondrial volume was calculated based on the diameter values.

Mitochondrial respiration measurements. SSM and IFM were kept in respi-ration buffer containing 100 mM KCl, 50 mM MOPS, 5 mM KH2PO4, 1 mMEGTA, and 0.1% fatty acid-free BSA, pH 7.0. Oxygen consumption in mito-chondrial subpopulations was assessed using a Clark-type electrode and thesubstrate combinations (i) 10 mM pyruvate plus 5 mM malate, (ii) 40 �Mpalmitoylcarnitine, and (iii) 20 mM succinate plus 3.75 �M rotenone as previ-ously described (48, 64, 72). State III respiration was measured in the presenceof 200 �M ADP, and state IV was measured after ADP consumption began.Activities of citrate synthase (CS), isocitrate dehydrogenase (IDH), and medium-chain acyl coenzyme A (acyl-CoA) dehydrogenase (MCAD) were measuredfrom heart homogenates as previously described (64).

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Ca2�-induced mitochondrial permeability transition pore (MPTP) openingassays. MPTP opening in SSM and IFM was performed as previously described(64). In short, mitochondria were resuspended in 2.0 ml assay medium contain-ing 100 mM KCl, 50 mM MOPS, 5 mM KH2PO4, 5 �M EGTA, 1 mM MgCl2,5 mM glutamate, and 5 mM malate and assayed for Ca2� uptake in a fluores-cence spectrophotometer at 37°C. CaCl2 (5 mM) was infused at a rate of 2�l/min, and the concentration of free Ca2� in the medium was calculated bymonitoring the fluorescence of the Ca2�-bound and Ca2�-free Fura-6-F (0.1 M;Molecular Probes) (the excitation wavelengths for the Ca2�-bound and Ca2�-free fluorophores were 340 and 380 nm, respectively, and the emission wave-length was 550 nm). Calibrations of Fura-6-F fluorescence were performed at theend of the experiment using 0.1 � EGTA and 0.1 M CaCl2 to establish a zerolevel and a Ca2�-saturated level of the fluorophore, respectively (64). MPTPopening was inferred from the sudden and large increase of fluorescence. Mi-tochondrial Ca2� tolerance was defined as the cumulative Ca2� load that wasrequired to induce the abrupt increase in extramitochondrial Ca2� from a semi-log plot (47).

In a separate set of experiments, Ca2� tolerance was evaluated according toprevious reports (22, 61). Briefly, mitochondria resuspended in swelling buffer (2mg/ml protein) were warmed to room temperature in a 96-well plate and CaCl2(200 �M) was added. The time-dependent decrease in absorbance at 520 nm isindicative of the tendency for MPTP opening and mitochondrial swelling. Forthe mitochondrial shrinkage assay, mitochondria were preswollen by incubatingthem at 2 �g protein/ml at room temperature for 10 min in the mitochondrialswelling buffer containing 500 �M CaCl2. Pores were closed by addition of 1 mMEGTA to chelate the added Ca2�. Five percent polyethylene glycol (PEG) wasadded to swollen mitochondria, and the absorbance at 520 nm was monitored for20 min using a spectrophotometer.

H/R and H2O2-induced myocyte death. Cardiac myocytes were isolated fromadult mice using aortic cannulation and Liberase dissociation as described above.Myocytes were seeded on laminin-coated dishes in the presence of a normoxiamedium that consists of MEM supplemented with 1.2 mM CaCl2, 12 mMNaHCO3, 2.5% (vol/vol) fetal bovine serum (FBS), 1% (vol/vol) penicillin-streptomycin, and 25 �M blebbistatin (44). Hypoxia was induced by replacing thenormoxia medium with a hypoxia medium that consisted of 118 mM NaCl, 16mM KCl, 24 mM NaHCO3, 1 mM NaHPO4, 2.5 mM CaCl2 � 2H2O, 1.2 mMMgCl2, 20 mM sodium lactate, 10 mM deoxyglucose, and 10 mM HEPES, pH 6.2(25) and placing the trays in a Billups-Rothenberg modular incubator chambersaturated with 95% N2, 5% CO2, and 1% O2. One hour later, the normoxiamedium was used to replace the hypoxia medium, and myocytes were incubatedfor two more hours in normoxic conditions. In parallel experiments, myocyteswere exposed to 20 �M H2O2 or were left untreated for 2 h. To determinemyocyte death either after hypoxia/reoxygenation (H/R) or H2O2 treatment,cells were exposed to 0.04% trypan blue-containing medium for 10 min, andphotographs of myocytes were obtained in a systematic fashion using the �10magnification of a light microscope. Cell counting was performed with ImageJsoftware. Following counting, the cells were collected in lysis buffer and totalmyocyte protein was isolated for Western blotting.

Western blotting. Cardiac samples weighing 30 mg were flash frozen in liquidnitrogen, and protein was extracted in tissue lysis buffer (tissue protein extractionreagent [T-PER buffer; Pierce] containing EDTA-free protease inhibitor[Roche]). Protein concentration in the lysates was quantified using the bicincho-ninic acid (BCA) assay (Thermo Scientific) according to the manufacturer’sspecifications. Twenty micrograms of protein from each sample was resolved on10% SDS-PAGE gels (Lonza) and transferred to polyvinylidene difluoride(PVDF) membranes (Amersham). After semidry transfer of proteins at 400 mAfor 60 min at 4°C, the membranes were blocked in 3% nonfat milk in phosphate-buffered saline (PBS) containing 0.5% Tween 20 (PBS-T) for 1 h. Primaryantibodies for mitofusin-1 (molecular mass, 80/85 kDa; Abcam), mitofusin-2 (80kDa; Sigma), VDAC-porin (30 kDa; Abcam), C-recombinase (40 kDa; NovusBiologicals), cyclophilin D (17 kDa; Thermo Scientific), ANT1/2 (35 kDa; SantaCruz), �-tubulin (55 kDa; Calbiochem), cytochrome c oxidase subunit IV (COX-IV; 18 kDa; Abcam), Bcl-2 (25 kDa; BD Transduction Laboratories), lactatedehydrogenase (LDH; 35 kDa; Cell Signaling), complex V subunit � (50 kDa;Molecular Probes), Bax (20 kDa; Cell Signaling), caspase-9 (49/39 kDa; CellSignaling), poly(ADP-ribose) polymerase (PARP-1) (fragments of 116, 89, and24 kDa; Cell Signaling), Drp-1 (75 to 80 kDa; BD Transduction Laboratories),Opa-1 (80 to 90 kDa; Abcam), and GAPDH (glyceraldehyde-3-phosphate de-hydrogenase; 37 kDa; Cell Signaling) were diluted to a 1:1,000 ratio in 3%blocking solution and incubated with the membrane overnight at 4°C. Detectionof immunoreactive bands was performed with the appropriate secondary anti-bodies conjugated with horseradish peroxidase activity (HRP) using the ECLreagent (Amersham).

Histology. Hearts were perfused through the apex with normal saline, har-vested, and cut through the long axis. Sagittal slices were fixed in 10% bufferedformalin, passed through graduated concentrations of ethanol, and embedded inparaffin according to standard protocols. Four-micrometer-thick sections wereprocessed with Harris hematoxylin and eosin (H&E) or Masson’s trichromereagents (Sigma). Sections were photographed under a light microscopeequipped with a digital camera, and images were quantified for myocyte cross-sectional area or collagen content as previously described (65).

cDNA synthesis and quantitative real-time PCR. Total cardiac mRNA wasextracted from frozen samples using the Qiagen fibrous tissue minikit accordingto the manufacturer’s specifications. Eight hundred fifty nanograms of RNA wasreverse transcribed into cDNA using the Thermoscript reverse transcription-PCR (RT-PCR) system (Invitrogen). The amounts of different cDNAs werequantified using the SYBR green reagent and the StepOne real-time system(Applied Biosystems). These quantities were expressed relatively to that of theGAPDH gene, which was used as the housekeeping gene. All primer sequencesfor genes shown in Table 3 are available upon request.

Statistical analysis. All values shown are means � standard errors of themeans (SEM) unless otherwise specified. When two groups were compared, theStudent two-tailed t test was applied (unpaired). For comparisons between 3 ormore groups, we used one-way analysis of variance (1-way ANOVA), and ifstatistically significant differences were detected, we utilized Bonferroni’s posthoc test to further identify groups with different means. Differences were con-sidered significant for P values of less than 0.05.

RESULTS

Because the heart is a mitochondrion-rich tissue, we usedthe �-MHC-Cre transgenic mouse line (1, 34) to disrupt theMfn-2 locus selectively in cardiac myocytes of Mfn-2loxP mice(19). Cre-mediated excision of Mfn-2 exon 6 is associated witha predictable loss of Mfn-2 protein (Fig. 1A), which in the caseof �-MHC-Cre � Mfn-2F/F mice reaches �90% efficiency (Fig.1A, right panel). We refer to these mice as F/F;cre and utilizetheir cre-negative F/F;� littermates or cre-only (�/�;cre)mice as age-matched controls. The protein levels of Mfn-1 didnot appear to change significantly in these heart samples. Fur-thermore, the genetic recombination is found to be specific tothe heart and is not detectable in any other tissue of F/F;cremice, nor does it appear to occur in the absence of the cretransgene (results not shown).

Histological examination of F/F;cre hearts detected cardiacenlargement that was not accompanied by overt ventriculardilatation (Fig. 1B). Microscopic analysis revealed the pres-ence of myocyte hypertrophy in F/F;cre hearts without signif-icant increases in the collagen content (Fig. 1C). We analyzedthe cardiac function of adult F/F;cre and F/F;� or �/�;cremice using noninvasive echocardiography or cardiac catheter-ization (Tables 1 and 2). There were no significant differencesin chamber dimensions, systolic function, or hemodynamic per-formance between the two groups, except for the detection ofincreased left ventricle (LV) mass in the F/F;cre group (Table1). To examine the heart function under conditions of -ad-renergic stress, we acutely infused isoproterenol (5 ng/kg/min)and monitored the hemodynamic response using LV catheter-ization. As shown in Fig. 2A and B and Table 3, this approachrevealed small but statistically significant differences betweenF/F;cre and F/F;� or �/�;cre hearts in terms of systolic func-tion (i.e., reductions in the end-systolic pressure and the max-imum rate of LV pressure rise [dP/dtmax, where P is pressureand t is time] in the F/F;cre group [Table 3]). Examination ofcontractility in isolated cardiac myocytes identified a smallreduction in fractional shortening in the F/F;cre myocytes,while the intracellular Ca2� ([Ca2�]i) transients appear to be



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similar between F/F;cre and F/F;� myocytes (Fig. 2C to F).Furthermore, the mRNA levels of various genes associatedwith stress, metabolism, and mitochondrial biogenesis or func-tion were normal or showed small changes in expression inF/F;cre hearts, with the exception of the atrial natriuretic pep-tide (ANP) mRNA, which was upregulated by 2.9-fold (Table4). Taken together, these data suggest that the lack of Mfn-2from the heart is associated with modest myocyte hypertrophyaccompanied by mild deterioration of left ventricular function.

Morphological analysis using electron microscopy of the LVwall revealed mostly round or rectangular mitochondria withdiameters ranging from 0.5 to 2 �m in F/F;� hearts (Fig. 3A).In F/F;cre samples, however, some regions had mitochondria

with normal morphology (Fig. 3B) while other areas containedenlarged mitochondria with diameters sometimes up to 3 or 4�m and, more rarely, up to 5 or 6 �m that tended to form intoclusters (Fig. 3C and D). In some cases, the enlarged mito-chondria in the F/F;cre tissue displayed further abnormalitiesin their internal structure, such as loss of cristae and formationof inner membrane vesicles (Fig. 3E). However, as shown inFig. 3F, the gross mitochondrial cross-sectional area remainsunchanged in the F/F;cre hearts, suggesting the maintenanceof overall mitochondrial mass. The copy numbers of the mito-chondrial gene for NADH dehydrogenase subunit 1 were notfound to differ significantly between the two groups (results notshown), further suggesting normal mitochondrial biogenesis in

FIG. 1. Cardiac myocyte-specific deletion of Mfn-2. (A) Hearts from mice with the indicated Mfn-2 genotypes (F/F, homozygous for Mfn-2loxP;�/F, heterozygous for Mfn-2loxP) with or without the �-MHC transgene (� or �) were analyzed by Western blotting. The panel on the right showsthe quantification of Mfn-2 band intensity relative to that for tubulin (*, P � 0.05; four samples per group; F/F;� or F/F;cre). (B, left) Grossmorphological analysis of F/F;� and F/F;cre hearts to assess cardiac growth and chamber dilation (the scale bars are 1 mm). (Right) Normalizationof total heart weight (HW) to body weight (BW) indicates that F/F;cre hearts are moderately but significantly larger than F/F;� or �/�;cre hearts.The numbers of animals are indicated in circles in their respective bars. (C) Microscopic examination of histological sections stained withhematoxylin and eosin to assess myocyte hypertrophy (the scale bars are 50 �m). The lower left panel shows average values in the myocytecross-sectional area, while the lower right panel shows average values in collagen deposition (*, P was �0.05 for F/F;� versus F/F;cre mice; #, Pwas �0.05 for �/�;cre versus F/F;cre mice by one-way ANOVA).



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F/F;cre myocytes. The myofibrillar compartment appearslargely intact in F/F;cre sections (Fig. 3F, myofibrils). Never-theless, the cross-sectional area per individual mitochondrionis found to be, on average, significantly increased in the knock-out group (Fig. 3G), in agreement with the presence of en-

larged mitochondria. Furthermore, the distribution of the max-imum and minimum mitochondrial diameters (Fig. 3H and I,respectively) was found to be significantly altered in the Mfn-2knockout group as it shifted to the right, indicative of mito-chondria with increased diameters (4 to 5 �m in the major axisand 2 to 3 �m in the minor axis). This analysis also revealedthat the number of detectable mitochondria per equal areaanalyzed was reduced in the knockout group (2,042 versus1,186). Mfn-2 has been recently implicated in the bridging ofthe outer mitochondrial membrane with the endoplasmic re-ticulum (26). Using electron microscopy on heart samples withor without Mfn-2, we examined the organization of the “Ca2�

release domains” that include the T-tubule, the junctional sar-coplasmic reticulum (jSR) and the outer mitochondrial mem-brane (13). As shown in Fig. 3J, the distance between thecenter of the T-tubule and the outer mitochondrial membranedoes not appear to change significantly in the F/F;cre group,indicating that the gross distance between the jSR and theouter mitochondrial membrane is not likely to be affected bythe absence of Mfn-2. Collectively, the electron microscopicanalysis identified the propensity in Mfn-2-deficient mitochon-dria to become fewer and enlarged without changing their

TABLE 1. Echocardiographic analysisa

ParameterMean value � SD for:

F/F;� (n � 20) F/F;cre (n � 18) �/�;cre (n � 9)

Age (days) 98.45 � 29.36 98.67 � 31.51 108.67 � 31.90BW (g) 29.24 � 5.61 29.30 � 4.24 32.80 � 4.58Calculated LV

mass (mg)b137.01 � 14.98 162.42 � 29.95 147.59 � 24.15

LV/BW (mg/g)b,c 4.77 � 0.64 5.56 � 0.83 4.53 � 0.53EDV (�l) 68.60 � 10.56 78.03 � 18.03 81.87 � 16.40ESV (�l) 35.16 � 8.16 39.05 � 15.17 45.53 � 15.27EF (%) 47.96 � 7.85 51.41 � 12.18 47.44 � 10.71CO (ml/min) 18.98 � 3.61 21.04 � 7.18 20.68 � 6.44FS (%) 39.31 � 10.08 39.08 � 8.68 34.98 � 7.84PPF (mm/s) 664.39 � 135.28 692.86 � 115.53 664.45 � 339.63PAF (mm/s) 855.07 � 208.29 856.70 � 163.82 978.49 � 208.32E (mm/s) 527.09 � 114.90 490.94 � 145.44 474.13 � 157.13A (mm/s) 342.55 � 98.66 315.49 � 104.36 324.17 � 62.26ET (ms)b 45.97 � 5.97 51.72 � 7.24 49.14 � 5.00E� (mm/s) 11.72 � 3.06 11.81 � 2.85 10.74 � 2.32A� (mm/s) 10.16 � 2.77 10.18 � 2.91 9.18 � 1.63

a BW, body weight; LV, left ventricle; EDV, end-diastolic volume; ESP, end-systolic volume; EF, ejection fraction; CO, cardiac output; FS, fractional short-ening; PPF, peak flow velocity at the pulmonic valve; PAF, peak flow velocity atthe aortic valve; E, peak early filling flow at the mitral valve; A, peak late fillingflow at the mitral valve; ET, ejection time; E�, early relaxation velocity at themitral annulus; A�, late relaxation velocity at the mitral annulus.

b There was significant difference between the means for F/F;� and F/F;cregroups at the 0.05 probability level according to ANOVA followed by the Bon-ferroni post hoc test.

c There was significant difference between the means for �/�;cre and F/F;cregroups at the 0.05 probability level according to ANOVA followed by the Bon-ferroni post hoc test.

TABLE 2. Hemodynamic analysisa

Parameter

Mean value � SD for:

F/F;� (n � 11) F/F;cre(n � 14)

�/�;cre(n � 6)

Age (days) 66.00 � 9.75 65.29 � 8.54 72.00 � 12.00BW (g) 28.64 � 3.91 28.93 � 2.53 26.83 � 3.87HW (mg)b,c 127.00 � 15.84 156.43 � 16.02 129.17 � 14.82HW/TL (mg/mm)b,c 6.51 � 0.74 8.15 � 0.81 6.69 � 0.78HR (bpm) 522.71 � 16.12 532.68 � 19.68 505.10 � 34.18ESP (mm Hg) 104.32 � 10.30 105.20 � 11.55 97.19 � 6.56EDP (mm Hg) 1.83 � 1.16 3.22 � 4.02 2.27 � 0.58CO (ml/min) 10.76 � 1.93 11.36 � 2.25 8.70 � 3.25dP/dtmax (mm Hg/s) 10,573 � 1,871 9,507 � 2,110 9,000 � 2,234dP/dtmin (mm Hg/s) �9,758 � 1,490 �8,915 � 2,044 �8,212 � 1,195PRSW (mm Hg) 60.72 � 10.29 66.23 � 16.48 66.06 � 6.90dP/dtEDV (mmHg/s � �l) 263.65 � 114.15 254.96 � 65.29 359.78 � 72.13�G (ms) 9.73 � 1.45 12.02 � 6.02 12.18 � 3.81

a BW, body weight; HW, heart weight; TL, tibia length; HR, heart rate in beatsper minute (bpm); ESP, end-systolic pressure; EDP, end-diastolic pressure; CO,cardiac output; dP/dt, peak rate of pressure increment (maximum or minimum);PRSW, preload recruitable stroke work; dP/dtEDV, dP/dt-EDV relationship; �G,relaxation time constant (tau) calculated by the Glantz equation.

b There was significant difference between the means for F/F;� and F/F;cregroups at the 0.05 level according to ANOVA followed by the Bonferroni posthoc test.

c There was significant difference between the means for �/�;cre and F/F;cregroups at the 0.05 level according to ANOVA followed by the Bonferroni posthoc test.

FIG. 2. Analysis of cardiac contractility in the presence or absenceof Mfn-2. Pressure-volume loop recordings taken before (black line) orafter (blue line) acute isoproterenol infusion in hearts with normallevels of Mfn-2 (F/F;�) (A) or in hearts where Mfn-2 had been ablated(F/F;cre) (B). For further details and a quantitative analysis, see alsoTable 3. (C) Sample [Ca2�]i and shortening records for isolated car-diac myocytes from F/F;� and F/F;cre mice. (D) Representative trac-ings of the change in [Ca2�]i for F/F;� and F/F;cre myocytes. (E) Av-erage shortening in F/F;� (0.169 � 0.053) and F/F;cre (0.126 � 0.035)myocytes (*, P � 0.05, F/F;� versus F/F;cre). (F) Average peak[Ca2�]i in F/F;� (2.85 � 0.37) and F/F;cre (3.76 � 0.75) myocytes.



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overall mass, indicating a defect in mitochondrial patterningand distribution rather than in biogenesis.

To further examine mitochondrial morphology, we per-formed confocal microscopy on intact myocytes isolated fromadult hearts. Mitochondria visualized with the membrane po-tential-sensitive dye TMRE have a rectangular shape and dis-play the typical striated appearance in myocytes expressingnormal levels of Mfn-2 (Fig. 4A). In contrast, the mitochondriafrom isolated F/F;cre myocytes are more heterogeneous inshape, often spherical, enlarged, and less precisely organizedwithin the myocyte (Fig. 4B). Three-dimensional representa-tion of mitochondria in the two groups further illustrates thepresence of enlarged mitochondria within a given area of themyocyte (Fig. 4C and D). We examined, in addition to thesemorphological defects, the levels of the mitochondrial mem-brane potential (m) in the absence of Mfn-2 using threedifferent dyes that sequester in polarized mitochondria (Fig.4E). As shown in the left panel of Fig. 4E, the ratio of the JC-1aggregate fluorescence to the JC-1 monomer fluorescence inF/F;cre myocytes is decreased, indicating a lower m in mi-tochondria lacking Mfn-2. Consistently, fluorescence intensitydue to accumulation of MitoTracker red or TMRM into mi-tochondria was found to be lower in myocytes without Mfn-2,again suggesting a partial decrease in m (Fig. 4E, middleand right panels). Taken together, results of the confocal mi-croscopy analysis of isolated myocytes indicate that in the ab-sence of Mfn-2, mitochondria lose their strict structural orga-nization within the myocyte and display an increase in sizewhich coincides with a partial reduction in membrane poten-tial.

The effect of Mfn-2 on mitochondrial morphology was alsoexamined in cultured neonatal rat cardiac myocytes treatedwith Mfn-2-specific siRNAs. This approach led to significantreductions in Mfn-2 mRNA and protein levels (results notshown). Mfn-2 downregulation was associated with fragmen-tation of the elongated and interconnected mitochondria intonumerous smaller spherical mitochondria (Fig. 4F, upper pan-

els). However, this was not the case in adult myocytes, whereMfn-2 ablation led to increased mitochondrial size (Fig. 4F,lower panels). These data suggest that the effects of Mfn-2 onmitochondrial morphology can be greatly affected by the cel-lular context.

To assess the effects of Mfn-2 ablation on mitochondrialfunction, we examined the activities of mitochondrial enzymesin whole-heart preparations or the respiratory activity of iso-lated interfibrillar mitochondria (IFM) and subsarcolemmalmitochondria (SSM) (Fig. 5). Deletion of Mfn-2 did not affectthe activities of citrate synthase (CS), isocitrate dehydrogenase(IDH), or medium-chain acyl-CoA dehydrogenase (MCAD) inwhole tissue (Fig. 5A) and in IFM and SSM (results notshown), consistent with normal biogenesis of mitochondrialmass in the absence of Mfn-2. The sizes of isolated mitochon-dria were assessed using a flowmetric approach. As shown inFig. 5B, the mitochondrial volume does not change signifi-cantly upon deletion of Mfn-2 when comparisons are madebetween IFM. Nevertheless, the absence of Mfn-2 is associatedwith a significant increase in volume in SSM (Fig. 5C), inagreement with the notion that loss of Mfn-2 results in theformation of a subset of enlarged mitochondria that coexistwith structurally normal mitochondria. Using a number of dif-ferent substrates, the rates of ADP-driven (state III) oxygenconsumption in isolated IFM and SSM were found to be sim-ilar, regardless of the presence/absence of Mfn-2 (Fig. 5D andE). Furthermore, levels of state IV respiration were found tobe similar between the two groups, and the respiratory controlratio was unaffected (results not shown), indicating that Mfn-2is dispensable for normal coupling of the respiratory chain.

Despite the relatively normal metabolic function of Mfn-2-deficient mitochondria, there was a pronounced resistance toCa2�-induced mitochondrial permeability transition (MPT).As shown in Fig. 6A, the incremental infusion of Ca2� inisolated SSM and IFM can lead to a marked increase in ex-tramitochondrial Ca2�. This is attributed to the formation ofthe high-conductance MPT pore (MPTP), which mediates

TABLE 3. Hemodynamic analysis before and after isoproterenol infusiona

Parameter

Baseline value for:

P(*)

Value afterisoproterenol

infusion (5 ng/kg/min) in controlgroup (n � 7)

P(#)

Value afterisoproterenol

infusion (5 ng/kg/min) in F/F;cregroup (n � 7)

P(#) P(*)Control mice

(n � 7)F/F;cre mice

(n � 7)

Age (days) 74.57 � 7.43 75.71 � 0.48HW/BW (mg/g) 4.58 � 0.27 5.10 � 0.36 0.011HR (bpm) 430 � 41.83 446 � 47.69 0.521 484.33 � 37.32 0.000 481.64 � 47.91 0.008 0.916ESP (mm Hg) 71.88 � 11.03 61.21 � 8.11 0.061 88.93 � 6.83 0.004 79.22 � 8.51 0.001 0.037dP/dtmax (mm Hg/s) 5,299 � 1,892 3,698 � 981 0.070 8,342 � 1,971 0.000 6,393 � 1,227 0.000 0.046dP/dtmax/IP (s�1) 117.27 � 26.06 103.96 � 16.99 0.280 146.49 � 31.86 0.003 127.00 � 20.76 0.003 0.200EDP (mmHg) 3.26 � 0.88 3.52 � 1.43 0.691 3.07 � 0.86 0.408 3.39 � 1.36 0.643 0.605dP/dtmin (mm Hg/s) �4,925 � 1,870 �3,687 � 1,323 0.178 �6,974 � 1,886 0.015 �5,966 � 1,685 0.004 0.403Adj. PMX (mW/�l2) 43.91 � 26.34 32.92 � 15.74 0.362 65.79 � 29.79 0.001 55.02 � 12.10 0.002 0.393EF (%) 43.61 � 15.31 41.40 � 10.76 0.760 50.56 � 15.77 0.002 53.21 � 5.71 0.011 0.682Cardiac output (�l/min) 8,458 � 3,149 8,397 � 2,495 0.969 10,862 � 3,730 0.001 11,561 � 2,639 0.007 0.693�G (ms) 16.18 � 6.33 15.10 � 5.10 0.730 13.84 � 4.89 0.089 12.49 � 3.77 0.171 0.573

a The control group consists of 4 F/F;� mice and 3 �/�;cre mice. Values are means � standard deviations. P(�), comparisons for significance between control andF/F;cre groups were made by two-tailed unpaired t test. P(#), comparisons for significance within a given baseline and isoproterenol-treated group were made bytwo-tailed paired t test. The P values that are below the 0.05 level of significance are shown in bold. HW/BW, heart weight to body weight ratio; HR, heart rate; ESP,end-systolic pressure; dP/dtmax, peak rate of pressure increment; dP/dtmax/IP, peak rate of pressure increment normalized by instantaneous developed pressure (IP);EDP, end-diastolic pressure; dP/dtmin, peak rate of pressure decrement; Adj. PMX, maximum power adjusted by preload; EF, ejection fraction; �G, relaxation timeconstant calculated by the Glantz equation.



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an exponential release of the previously accumulated Ca2�

(41, 51). This assay shows that the loss of Mfn-2 can signif-icantly delay the MPTP, as judged by the comparison of theCa2� release curves in control and Mfn-2-deficient mito-chondria, an effect that can be observed in both SSM andIFM (Fig. 6A). The cumulative Ca2� load required forMPTP opening (mitochondrial Ca2� tolerance) was alsoincreased in Mfn-2 SSM and IFM (Fig. 6B). This compari-son shows that Mfn-2-deficient mitochondria required ap-proximately twice the Ca2� load applied to wild-type mito-chondria to induce MPTP opening.

MPTP opening was also assessed by measuring the gradualswelling of isolated mitochondria in the presence of Ca2�.Mitochondria with or without Mfn-2 were exposed to 200 �MCa2�, and swelling was monitored as a decrease in absorbanceover time. As shown in Fig. 7A and B, the change in absor-

bance (relative to the baseline absorbance) is more pro-nounced in wild-type mitochondria than in mutant mitochon-dria, indicating that the absence of Mfn-2 is associated with anattenuated MPT response. In the presence of cyclosporine(CsA), mitochondria from both groups maintained their opti-cal density throughout the assay (results not shown), suggest-ing that the decrease in absorbance seen here is attributable toCa2�-induced MPTP opening. As an alternative way to assessMPT, we also examined the ability of mitochondria, previouslyswollen by Ca2�, to undergo shrinkage after being exposed topolyethylene glycol (PEG). As shown in Fig. 7C, the additionof PEG in F/F;� mitochondria is associated with a gradualincrease in absorbance that signifies mitochondrial shrinkagefacilitated by MPTP opening. On the other hand, the additionof PEG to preswollen Mfn-2-depleted mitochondria resultedin a lower rate of mitochondrial shrinkage, indicating a less-than-optimal MPTP opening.

To directly assess the effect of Mfn-2 deletion on the expres-sion of proteins previously associated with the function of theMPTP, we analyzed whole-heart extracts by Western blotting.As shown in Fig. 7D, the levels of the MPTP-regulatory com-ponent Cyp-D were not different between F/F;cre and F/F;�extracts. This was also the case for the other purported MPTPcomponents, such as the voltage-dependent anion channel(VDAC)-porin, and the two isoforms of the adenine nucleo-tide translocase (ANT1/2). Identical results were obtained withextracts from isolated mitochondria (results not shown). Thesedata indicate that the loss of Mfn-2 is sufficient to affect MPTin the absence of significant changes in the levels of candidateMPTP components.

To assess the consequences of Mfn-2 ablation on stress-induced MPTP opening in intact cells, myocytes were isolatedand examined for permanent mitochondrial depolarization un-der conditions of ROS generation, which is known to promoteloss of membrane potential via MPTP activation (85). Asshown in Fig. 8A, polarized mitochondria are detected asbright rectangles due to the accumulation of TMRM. Laserillumination of TMRM-loaded mitochondria, leading to atightly controlled local generation of ROS (4), allows the as-sessment of MPTP activation downstream of ROS. Represen-tative time points (1, 5, and 9 min [t1, t5, and t9]) are shown inthe middle panels of Fig. 8A, where the gradual depolarizationof the mitochondrial population is depicted. As shown in Fig.8B (also see the movie in the supplemental material), the timecourse of mitochondrial depolarization is significantly delayedin myocytes lacking Mfn-2 (blue tracing), compared to thedepolarization in myocytes with normal levels of Mfn-2 (purpletracing). To further determine the critical involvement ofMPTP in the outcome of this assay, we analyzed the effects ofCsA pretreatment (a potent inhibitor of MPTP) on mitochon-drial depolarization. As shown in Fig. 8B, the addition of CsAsignificantly delays the rate of depolarization in wild-type mi-tochondria (orange tracing), while it appears to act additivelywith the Mfn-2 deficiency to induce further delays in loss ofmembrane potential (green tracing). These actions are alsoobserved when calculating the time to half depolarization (T50)in the different groups (Fig. 8C). We further examined theresponse of adult myocytes to exogenous H2O2 as an alterna-tive source of ROS. As shown in Fig. 8D, H2O2 exposure isable to induce time-dependent mitochondrial depolarization

TABLE 4. Transcriptional regulation of genes of interest

F/F;cre vs F/F;�transcript

Foldinduction

Pvalue Functional class

Mfn-2 0.058 0.000 Mitochondrial fusion/fissionMfn-1 0.760 0.002Opa-1 0.767 0.001Drp-1 0.890 0.164Fis-1 0.830 0.053Cycs 0.869 0.303 Respiratory chain subunitsCox5b 1.323 0.009 (nuclear)Atp5o 0.898 0.132Ndufb5 0.936 0.300Cytb 0.815 0.008 Respiratory chain subunitsNd5 0.771 0.021 (mitochondrial)Cd36 0.775 0.010 Fatty acid metabolismMCAD 0.684 0.002Cpt2 0.770 0.012Pfk 0.735 0.001 GlycolysisLDH 0.689 0.002Hk2 0.746 0.001Pkm 0.795 0.001Nrf-1 0.817 0.033 Transcription factorsTfam 0.724 0.018Err� 0.931 0.304Pgc-1� 0.744 0.042ANP 2.902 0.000 Stress indicatorsBNP 1.221 0.023�-MHC 0.743 0.001�-MHC 0.727 0.001�-Sk actin 0.872 0.362Phospholamban 0.688 0.007 Ca2� handlingSerca2a 0.671 0.000Sod2 0.820 0.005 ROS detoxificationCatalase 1.428 0.000

Values represent fold reduction/induction in the Mfn-2 F/F;cre group relativeto the F/F;� group. Comparisons for statistical significance were performed withthe two-tailed Student t test. Mfn-2, mitofusin-2; Mfn-1, mitofusin-1; Opa-1,optic atrophy 1 long variant; Drp-1, dynamin-related protein 1; Fis-1, fissionprotein 1; Cycs, cytochrome c somatic; Cox5b, cytochrome c oxidase subunit 5b;Atp5o, F1Fo ATPase complex subunit 5o; Ndufb5, NADH-dehydrogenase(ubiquinone) 1 subcomplex, polypeptide 5; Cytb, cytochrome b; Nd5, NADH-dehydrogenase subunit 5; Cd36, scavenger receptor CD36; MCAD, medium-chain acyl-CoA dehydrogenase; Cpt2, carnitine palmitoyl transferase 2; Pfk,phosphofructokinase; LDH, lactate dehydrogenase; Hk2, hexokinase 2; Pkm,pyruvate kinase muscle isoform; Nrf-1, nuclear respiratory factor 1; Tfam, tran-scriptional factor A mitochondrial; Erra, estrogen receptor-related factor a;Pgc-1a, PPAR gamma coactivator 1a; ANP, atrial natriuretic peptide; BNP,brain natriuretic peptide; �-MHC, alpha myosin heavy chain; -MHC, betamyosin heavy chain; �-Sk actin, alpha skeletal actin; Serca2a, sarcoendoplasmicreticulum Ca2� ATPase type 2a; sod2, superoxide dismutase, mitochondrial.



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FIG. 3. Electron microscopy analysis of F/F;� and F/F;cre hearts. (A) The typical organization of mitochondria along the myofibrils is detectedin F/F;� hearts. (B) Region of an F/F;cre heart containing mostly normal mitochondria. (C) Different region where the mitochondria becomeabnormally enlarged. (D) Greater detail of a region of an F/F;cre heart containing enlarged spherical mitochondria. (E) In some rare cases,mitochondria are found to display further abnormalities, such as the formation of internal vesicles and crista decondensation. The scale bars are2 �m. (F) Assessment of mitochondrial (mitoch.) and myofibrillar (myof.) volume density in F/F;� and F/F;cre heart sections using the gridmethod (44 fields; size, 40 by 13 �m; three hearts per group were analyzed). (G) The area enclosed by the mitochondrial border was quantifiedin the same samples and averaged for F/F;� and F/F;cre samples. The results indicate a statistically significant increase in mitochondrial area inthe F/F;cre group (30 fields from three samples per genotypic group). (H to I) Distribution analysis of major and minor mitochondrial diametersto examine mitochondrial dimensions. The distribution analysis was performed by binning at 0.1-�m increments. The differences in diametersbetween F/F;� and F/F;cre mitochondria are significant according to the Kolmogorov-Smirnov (K-S) test (P � 0.001, F/F;� versus F/F;cre; Drepresents the value for the largest overall deviation). (J) The organization of the calcium release domains in F/F;� and F/F;cre samples isexamined in further detail. The T-tubule is indicated by a black arrow and the mitochondrial surface by a white arrow. The arrowheads indicatesacks of junctional sarcoplasmic reticulum (jSR). The distance (d, shown as a dotted line) between the center of the T-tubule (shown with a circle)and the mitochondrial surface was measured, and values were averaged for the two groups (lower right).



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FIG. 4. Confocal analysis of adult cardiac myocytes indicates the presence of enlarged/spherical mitochondria. (A) F/F;� myocytes containmitochondria with a rectangular shape that are highly ordered. (B) F/F;cre myocytes contain mitochondria with a heterogeneous morphology thatcan be spherical or enlarged. The scale bar is 10 �m. TMRE, tetramethylrhodamine ethyl ester. (C) Volume rendering of a z-stack collected froma region (13 by 31 �m) of an F/F;� myocyte. (D) Volume rendering of a z-stack selected from a region (13 by 31 �m) of an F/F;cre myocyte. (E,left) The mitochondrial membrane potential was estimated using dual mitochondrial labeling with JC-1. The fluorescence intensity of the aggregateform is divided by that of the monomeric form to measure the potential across the inner mitochondrial membrane (�, P was �0.05 for F/F;� versusF/F;cre mice by Student’s t test [33 and 43 F/F;� or F/F;cre myocytes, respectively, from two animals per group]). (Middle) F/F;� or F/F;cremyocytes were loaded with 500 nM Mitotracker Red, and absolute units of fluorescence intensity (FI) were determined as a measure of m.Multiple ROI were quantified from three experiments per genotype (totals of 61 and 70 ROI from 5 F/F;� and 6 F/F;cre myocytes, respectively).The laser power (561 nm) in these experiments was at 0.3%, 0.4%, and 0.4% for F/F;� myocytes and 0.5%, 0.7%, and 0.5% for F/F;cre myocytes.�, P was �0.05 for F/F;� versus F/F;cre myocytes by Student’s t test. (Right) Cells loaded with 100 nM TMRM, where units of absolute (abs.) FIwere determined as a measure of m. Multiple ROI were quantified from three different experiments per genotype (total of 91 and 90 ROI from8 F/F;� and 8 F/F;cre myocytes, respectively). The laser power (543 nm) in the respective experiments was at 1.8%, 1.8%, and 1.5% for F/F;�myocytes and at 1.5%, 1.8%, and 2.0% for F/F;cre myocytes. �, P was �0.05 for F/F;� versus F/F;cre myocytes by Student’s t test. (F) The structureof the mitochondrial network is analyzed in neonatal cardiac myocytes treated with unrelated or Mfn-2-specific siRNAs (upper row) and comparedwith the structure of mitochondria in adult cardiac myocytes with or without Mfn-2 (lower row). The staining of mitochondria was performed withMitotracker Red. The scale bars are 10 �m.



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and, eventually, hypercontracture in isolated myocytes. Inagreement with the observations made above, the loss of Mfn-2is associated with a reduced rate of mitochondrial depolariza-tion (Fig. 8E and F), providing additional evidence for theinvolvement of Mfn-2 in MPT.

We also examined ROS-induced MPTP in neonatal rat car-diac myocytes that were depleted of Mfn-2 via siRNA knock-down. As shown in Fig. 9A, mitochondrial depolarization andTMRM fluorescence decay in response to H2O2 exposure areaccelerated upon Mfn-2 knockdown, which is in contrast tofindings for adult myocytes. The enhanced depolarizing effectin the absence of Mfn-2 in NRCMs was also associated with anincreased release of lactate dehydrogenase (LDH), a markerof cell death (Fig. 9B). To examine the role of Mfn-2 deficiencyin an independent system, mice lacking Mfn-2 in macrophageswere constructed by breeding the Mfn-2loxP mice with theLyzM-cre transgenic mouse line. In macrophages recruited tothe peritoneum by thioglycolate treatment, Mfn-2 deficiency isprotective against mitochondrial depolarization (Fig. 9C), inagreement with the observations made with adult cardiac myo-cytes. The delay in mitochondrial depolarization in Mfn-2 mac-rophages was also associated with a reduced release of LDHinto the culture medium (Fig. 9D). Taken together, these data

show that the effect of Mfn-2 insufficiency on MPTP activationis cell type dependent.

Based on the observation that F/F;cre mice have normalcardiac function at baseline but contain mitochondria that ex-hibit resistance to MPTP opening, we examined their responseto reperfusion injury using the isolated heart configuration. Asshown in Fig. 10, F/F;� and F/F;cre hearts had similar systolicand developed pressures at baseline (Fig. 10A and B) andglobal ischemia for 10 min led to the expected reduction incardiac function in both groups. However, upon reperfusion,Mfn-2-ablated hearts were able to produce higher systolic pres-sures than control hearts, indicating a protection from theinjurious effects of the reflow (Fig. 10A). Similar observationswere also made when developed pressures were measured inthe two groups (Fig. 10B). Therefore, these experiments dem-onstrate that loss of Mfn-2 can alleviate some of the detrimen-tal effects of postischemic reperfusion, the period that is knownto coincide with the induction of MPT (35). To obtain furthermolecular details on the Mfn-2-associated cardioprotectionduring ex vivo ischemia/reperfusion (I/R), Western blot analy-sis of purified mitochondrial protein was performed. As shownin Fig. 10C, the antiapoptotic protein Bcl-2 is found to be moreabundant on Mfn-2-depleted mitochondria.

FIG. 5. Functional evaluation of mitochondria in F/F;� and F/F;cre hearts. (A) Enzymatic activities in total myocardial extracts. CS, citratesynthase; IDH, isocitrate dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase (6 and 7 preparations per group were used from 17 and21 F/F;� and F/F;cre mice, respectively). (B and C) Organelle volume assessed in cardiac mitochondria isolated from the interfibrillar andsubsarcolemmal compartments (IFM and SSM, respectively) (*, P was �0.05 for F/F;� versus F/F;cre myocytes by Student’s t test; 6 and 7preparations were used from F/F;� and F/F;cre mice, respectively). (D and E) The activity of the respiratory chain in isolated IFM and SSMmitochondria is evaluated in the presence of different substrates. These assays did not reveal statistically significant differences in state III(ADP-driven) oxygen consumption between the two genotypic groups (Student’s t test was used for each substrate, with 6 or 7 preparations pergenotype). P, pyruvate; M, malate; PCar, palmitoyl carnitine; S, succinate; Rot, rotenone; prot., protein; nA O, nanoatoms of oxygen.



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To examine the impact of Mfn-2 loss on cell death after I/Rin greater detail, an in vitro hypoxia/reoxygenation assay wasemployed. In this experiment, cardiac myocytes purified fromwild-type and Mfn-2-deficient hearts were exposed to nor-moxia only or were exposed to hypoxic conditions (1% O2, 5%CO2) and then returned to normoxic conditions (reoxygen-ation). As shown in Fig. 11A and B, the percentage of myocytesundergoing necrotic cell death (indicated by the number oftrypan blue-stained cells) decreases in the absence of Mfn-2. Infact, Mfn-2-deficient cardiac myocytes were able to better tol-erate the stress induced by the isolation process, as there werefewer cells from this genotype staining positive for trypan blueunder normoxic conditions. Consistently, the Mfn-2-deficientmyocytes retained their resistance to necrosis under the hyp-oxia/reoxygenation conditions (Fig. 11B). It has been previ-ously reported that Mfn-2 can also promote cardiomyocyteapoptosis by activating the intrinsic/mitochondrial pathway(75). We therefore examined the activation of apoptosis inF/F;� and F/F;cre myocytes from the above-described assay byWestern blotting. As shown in Fig. 11 C, the abundance ofcleaved caspase-9 and cleaved PARP-1 is decreased in F/F;cresamples compared to F/F;� samples, both at normoxia andupon hypoxia/reoxygenation, suggesting inhibition of the ap-optotic pathway in the absence of Mfn-2. The tolerance ofMfn-2-deficient myocytes to cell death was further demon-strated using H2O2 treatment. As shown in Fig. 11D, the pro-portion of dead cells in myocyte preparations left untreated orexposed to 20 �M H2O2 is consistently lower in the absence of

Mfn-2. Taken together, these observations show that loss ofMfn-2 is associated with improved cell survival in the face ofdeath-inducing stimulation.

To further assess the role of Mfn-2 in cardiac myocyteapoptosis, we examined the expression of proapoptotic andantiapoptotic proteins that are known to be associated withmitochondrial morphogenesis (45). As shown in Fig. 12A, theproapoptotic protein Bax was found to be increased in nonis-chemic heart extracts lacking Mfn-2. The antiapoptotic Bcl-2appeared to be upregulated in the same experimental group, inagreement with our previous observations (Fig. 10C). We alsoexamined the expression of the mitochondrial fission factorDrp-1, which has also been implicated in ischemia/reperfusioninjury and cell death (63). As shown in Fig. 12A, the levels ofDrp-1 protein were decreased in hearts lacking Mfn-2. Finally,the levels of the protein Opa-1, which is implicated in innermitochondrial membrane fusion and also in the regulation ofcytochrome c release, did not appear to change significantlyupon genetic deletion of Mfn-2.

Finally, we subjected Mfn-2 F/F;� and F/F;cre mice to invivo ischemia/reperfusion injury by surgically closing and re-opening the LAD coronary artery for 30 min and 2 h, respec-tively. As shown in representative images (Fig. 12B) and theaccompanying histogram (Fig. 12C), the area at risk (AAR) tothe left ventricle (LV) area is the same between the twogroups, indicating similar magnitudes of ischemic stress. How-ever, the ratio of the infarct area (IA) (shown as the white bandin Fig. 12B) to the AAR (IA/AAR) was found to be lower in

FIG. 6. Ca2� retention capacity is increased in Mfn-2-depleted mitochondria. (A) Subsarcolemmal mitochondria (SSM) (upper panel) andinterfibrillar mitochondria (IFM) (lower panel) were isolated from F/F;� and F/F;cre hearts and assessed for their ability to buffer extramito-chondrial Ca2�. Low Ca2� loads are sustainable by mitochondria; however, when a threshold Ca2� load is attained, a large and abrupt increasein extramitochondrial Ca2� that signifies mitochondrial permeability transition occurs (*, P was �0.05 by repeated-measures ANOVA, with 7preparations per group). (B) Mitochondrial Ca2� tolerance, defined as the Ca2� load that is recorded during the exponential rise of extramito-chondrial Ca2�, is significantly increased after genetic deletion of Mfn-2 from SSM and IFM (*, P was �0.05 by Student’s t test). AU, arbitraryunits.



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F/F;cre hearts, indicating a diminished cell death response. Insupport of the above conclusions, the percentage of TUNEL-positive nuclei was found to be significantly lower in F/F;crehearts than in F/F;� hearts (Fig. 12D and E), indicating thatthe apoptotic response under these conditions is mitigated inthe absence of Mfn-2.

DISCUSSION

In adult cardiac myocytes, the compact placement of mito-chondria between the myofibrils sets significant constraints interms of their positioning and structure. Thus, mitochondria inthis cell type have been described as independent units, withvery little spatial freedom and precise arrangement into a“crystal-like” pattern (10, 81). Despite these limitations, thepossibility that a functional cross talk takes place among mi-tochondria in cardiac myocytes has been documented (4, 14,15, 85), although the molecular mediators of this process re-main largely unknown. The pore that mediates mitochondrialpermeability transition (MPTP) is generally linked to celldeath events, but recent work has suggested that it may also beinvolved in physiologically relevant processes, such as mito-chondrial Ca2� efflux (31) and ROS signaling between mito-

chondria (82), thereby making it an attractive candidate me-diator of intermitochondrial cross talk.

Mfn-2 resides on the outer mitochondrial membrane andhas been previously shown to regulate mitochondrial fusion, aprocess that involves the exchange of content among adjacentmitochondria in a variety of cell types (29). Mfn-2 is highlyexpressed in the adult mammalian heart, yet its roles in thistissue remain to be determined. In the present study, we haveablated the expression of the Mfn-2 gene specifically in murinecardiac myocytes and analyzed the impact of this perturbationon mitochondrial and cardiac functions under baseline andstress conditions.

The deletion of Mfn-2 led to detectable aberrations in themitochondrial compartment, the most prominent of them be-ing an increase in mitochondrial size that was reflected bydifferences in mitochondrial cross-sectional area and volume.However, not all of the mitochondria were enlarged in a givenmyocyte or field, and it was found that the enlarged mitochon-dria occurred in the subsarcolemmal rather than the inter-fibrillar compartment. These findings for cardiac myocytes aresimilar to observations with other mouse models of Mfn-2ablation. The deletion of Mfn-2 in Purkinje cells producesmitochondria that are enlarged and spherical and appear in

FIG. 7. Calcium-induced mitochondrial swelling is delayed in the absence of Mfn-2. (A) Total cardiac mitochondria were isolated from F/F;�and F/F;cre hearts and exposed to 200 �M Ca2� to induce swelling or left untreated. The change in absorbance that occurs as a result ofmitochondrial swelling is monitored over time at 520 nm and is expressed relative to the absorbance at the beginning of the assay (6 preparationsper group; error bars indicate standard errors of the means [SEM]). (B) The maximum change in absorbance throughout the assay indicates thatF/F;cre mitochondria are less prone to Ca2� swelling than F/F;� mitochondria (�, P was �0.05 for untreated versus Ca2�-treated mitochondria;#, P was �0.05 for F/F;� versus F/F;cre mitochondria [6 preparations per group]). (C) The rate of mitochondrial shrinkage in response to PEGtreatment is delayed in the absence of Mfn-2. Isolated mitochondria from hearts with or without Mfn-2 were preloaded with Ca2� to induceswelling and were subsequently exposed to PEG to induce shrinkage (3 preparations per group; error bars indicate SEM). The rates ofmitochondrial shrinkage in response to PEG in the absence of Ca2� swelling were not different between the two groups (results not shown).(D) The expression levels of the different proteins responsible for MPT in hearts with or without Mfn-2. Cyp-D, cyclophilin-D; VDAC,voltage-dependent anion channel; ANT, adenine nucleotide translocase; COX-IV, cytochrome c oxidase subunit 4.



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clusters (19). Mfn-2-deficient mouse embryo fibroblasts con-tain enlarged mitochondria that are spherical (18). Likewise,the deletion of the two Mfn-2 alleles and one Mfn-1 allele fromskeletal muscle is reported to lead to the formation of a fewunusually large mitochondria (20). These features resemblethe mitochondrial phenotype identified in the present study,although their extent appears to be less severe here. Despitethese structural abnormalities, the total mitochondrial mass incardiac myocytes was not affected by the deletion of Mfn-2.Furthermore, the respiratory activity of isolated mitochondriawas found to be normal, and the levels of expression of genesassociated with mitochondrial biogenesis and functions weresimilar or displayed small differences between wild-type andMfn-2-depleted hearts.

Mfn-2-deficient cardiac myocytes underwent modest hyper-trophy, leading to an increase in the heart weight/body weight

ratio that was associated with elevated transcript levels ofANP. However, the function of the intact Mfn-2 null heartappeared normal in most physiological analyses, although iso-proterenol stimulation was able to unmask a mild systolic dys-function. Individual Mfn-2 null myocytes also exhibited a smalldecrease in contractility when paced ex vivo. In agreement withreports of other systems (5, 18, 67), a reduction in baselinemitochondrial membrane potential could be detected in Mfn-2-deficient cardiac myocytes. Collectively, these analyses iden-tify a modest reduction in function, likely to be attributed to anunderlying mitochondrial defect. This partial phenotype mayindicate that compensatory mechanisms operate, perhapsthrough the ability of Mfn-1, to functionally counterbalancethe loss of Mfn-2 (20).

In addition to the above-described features at baseline, mul-tiple lines of evidence in the present study suggest that Mfn-2

FIG. 8. Deletion of Mfn-2 in cardiac myocytes diminishes the rate of mitochondrial depolarization in response to generation of ROS.(A) Representative image of a TMRM-loaded myocyte and time course of photostress-dependent mitochondrial depolarization. (Top) TMRMfluorescence indicates the pattern of polarized mitochondrial in myocytes. The two nuclei are seen as dark ovals. A depolarized mitochondrion(arrow) is dark. The box indicates a preselected area (30 by 35 �m) to be repetitively imaged during the photon stress experiment. (Middle) Sampleimages of the illuminated region at 1, 5, and 9 min. (Bottom) Image of the cell showing the permanent depolarization of mitochondria in thepreselected area, while other areas of the cell contain polarized mitochondria. (B) The loss of TMRM fluorescence intensity (FI) is delayed in theabsence of Mfn-2 or by pretreatment with cyclosporine (CsA). Comparison of F/F;� (purple) and F/F;cre (blue) myocytes loaded with TMRMand subjected to photon stress shows that the loss of Mfn-2 is associated with a delayed mitochondrial depolarization. Furthermore, this effect canbe attributed to MPTP activation, as it is shown that CsA is able to delay the depolarization in F/F;� myocytes (orange) and can be additive tothe effects associated with Mfn-2 deletion (green, dotted lines represent SEM for each group). Rel, relative. (C) Times to half-depolarization (T50)in mitochondria with or without Mfn-2 in the presence of CsA (**, P was �0.05 for untreated versus CsA-treated F/F;� myocytes; ***, P was�0.05 for untreated versus CsA-treated F/F;cre myocytes). (D) Time-dependent mitochondrial depolarization in adult myocytes in response toH2O2 exposure. Myocytes were exposed to 200 �M H2O2, and images were collected at 1-min intervals (the images shown here are pseudocoloredto illustrate mitochondria). (E) Multiple areas (8 by 8 �m) were selected from 9 to 12 myocytes per group and analyzed with ImageJ for the lossof fluorescence intensity (FI) as a function of time after H2O2 treatment, and the change in fluorescence was expressed relative to the values atthe beginning of the experiment. Error bars indicate standard errors of the means (21 to 27 ROI per group). (F) The FI at 1,500 s of H2O2 exposurewas subtracted from the FI at 60 s to calculate the cumulative change that was expressed as a percentage of the baseline (t0) (*, P was �0.05, 21to 27 ROI per group).



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deficiency can delay MPTP opening in stressed adult cardiacmyocytes. First, isolated mitochondria that lack Mfn-2 are ca-pable of higher loads of Ca2� uptake. Second, the time courseof mitochondrial swelling in response to Ca2� exposure inMfn-2-depleted mitochondria is distinct from that in wild-typemitochondria. Third, water extrusion through the MPTP dueto PEG treatment is diminished in purified mitochondria lack-ing Mfn-2. Fourth, the mitochondrial depolarization in cardiacmyocytes lacking Mfn-2 is resistant to photon stress or freeradical-induced MPTP activation. Furthermore, Mfn-2-defi-cient myocytes are protected from death in hypoxia/reoxygen-ation and H2O2 exposure assays, an effect likely to be at leastin part attributable to MPTP inhibition. In addition, heartsisolated from knockout mice display improved function follow-ing global ischemia/reperfusion (I/R) injury ex vivo, and thecardiac cell death response to in vivo regional ischemia andreperfusion injury is attenuated in Mfn-2 knockout mice.

Despite this evidence that Mfn-2 deletion delays MPTPopening in adult cardiac myocytes, the opposite outcome wasobserved in cultured neonatal myocytes treated with siRNA toablate Mfn-2. In the neonatal system, Mfn-2 knockdown facil-itated the loss of mitochondrial membrane potential in re-

sponse to ROS stress. Mfn-2-deficient neonatal myocytes alsodisplayed changes in mitochondrial morphology that werestrikingly different from those seen after Mfn-2 ablation inadult myocytes (compare panels in Fig. 4F), suggesting that thebehavior of mitochondria in the neonatal myocyte system maynot be predictive of that of adult myocytes or the intact heart.To further test the effects of Mfn-2 ablation in an independentsystem, mice lacking Mfn-2 in peritoneal macrophages wereconstructed and analyzed. The loss of Mfn-2 in this cell typeresulted in a delay in mitochondrial membrane depolarizationdue to ROS stress, in agreement with observations with adultcardiac myocytes. Collectively, these experiments indicate thatthe actions of Mfn-2 on MPTP are likely to be cell type specific,perhaps depending on the extent of cellular differentiation.

The mechanisms by which Mfn-2 promotes permeabilitytransition in adult cardiac myocytes are likely to be pleiotropic.Opening of the MPTP has been linked to cataclysmic cellevents, such as apoptosis and necrotic cell death (6, 52), yet thestructure and regulation of the MPTP have not been fullyunraveled (40). The initial working model suggested that theMPTP is made of VDAC, ANT, and Cyp-D (23). However,genetic studies have challenged this model, and all isoforms of

FIG. 9. The effects of Mfn-2 on mitochondrial depolarization are context dependent. (A) Neonatal cardiac myocytes (NRCMs) were treatedwith unrelated (control) siRNA or with an Mfn-2-targeting siRNA and analyzed for mitochondrial depolarization in response to H2O2 exposure.(B) The loss of membrane potential is associated with increased cell death as assessed by the release of lactate dehydrogenase (LDH) into theculture medium (3 independent experiments per group; P was �0.05 for control versus Mfn-2 siRNA-treated cells after exposure to H2O2).(C) Peritoneal macrophages (M�) lacking Mfn-2 (F/F;creLyzM) and control macrophages (F/F;�) were loaded with TMRM and exposed to H2O2.(D) The reduction in mitochondrial depolarization as a result of Mfn-2 ablation was associated with less macrophage death and subsequent releaseof LDH in the medium (4 independent experiments per group; P was �0.05 for F/F;� versus F/F;creLyzM macrophages).



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VDAC, which is the candidate component on the outer mito-chondrial membrane, were found to be dispensable for MPTPopening (9). On the other hand, only Cyp-D appears to func-tion as a central regulator of the pore (8, 60), while ANT islikely to function as a peripheral regulator (49). In contrast toMfn-2, an integral protein of the outer membrane, Cyp-D, islocated in the matrix, where it participates in protein foldingand is inhibited by CsA. Cyp-D is thought to facilitate MPTPopening by functioning as a Ca2�-sensing element that trans-lates the Ca2� stimulus into conformational changes of anMPTP component(s) on mitochondrial membranes (56). LikeCyp-D-deficient cardiac mitochondria, Mfn-2-deficient cardiacmitochondria display enhanced capacity for Ca2� uptake.However, Cyp-D-null mitochondria are completely resistant toswelling induced by low concentrations of Ca2�, whereas Mfn-

2-depleted mitochondria undergo swelling in response to thistreatment but at substantially lower rates than wild-type mito-chondria. Furthermore, Cyp-D-null fibroblasts displayed a ro-bust resistance to H2O2-induced mitochondrial depolarization(8), while Mfn-2-null cardiac myocytes displayed a markeddelay but still underwent depolarization upon H2O2 exposure.Finally, Mfn-2-null hearts display significant protection fromischemia/reperfusion injury an effect also observed in Cyp-D-null hearts (8, 60). Thus, whereas Mfn-2 exhibits a number ofparallels with Cyp-D and could be a novel regulator of MPTP,it should be noted that the pore can still function in the ab-sence of Mfn-2, ruling out the possibility that it alone is anessential structural component of this poorly defined complex.In this regard, it would be interesting to test whether a func-tional redundancy exists between Mfn-1 and Mfn-2 as potentialregulators of MPTP.

An alternative hypothesis is that MPTP is influenced byMfn-2 ablation due to diminished coupling between the mito-chondria and the SR. Mfn-2 has been shown to regulate theefficiency of mitochondrial Ca2� uptake from intracellularstores by controlling the distance between the ER and mito-chondria (26). Therefore, it can be suggested that Mfn-2 pro-motes MPTP opening by enforcing mitochondrial Ca2� uptakefrom the SR. However, this hypothesis is difficult to reconcilewith the following observations: (i) the defect in MPTP acti-vation could be detected on isolated mitochondria that werefree from cytosolic contaminants and (ii) the gross structure ofthe “calcium release domain” was not significantly impaired inMfn-2-deficient myocytes. Although it is possible that Mfn-2can influence MPTP activation through subtle effects on SR/mitochondrial connections, this can only partly explain thephenotypes presented here. In fact, the findings from isolatedmitochondria suggest that the ability of Mfn-2 to influence theMPTP is likely owing to its localization on mitochondria per se.

Another mechanism could be that Mfn-2 facilitates the per-meability transition through its participation in OMM remod-eling and fusion. Dynamin-like proteins can introduce desta-bilization of the lipid bilayer, allowing adjacent mitochondriato merge (58, 69). It is therefore possible that, under stressconditions, Mfn-2-dependent local OMM destabilization canlead to the formation of MPTP. The potential involvement ofMfn-2 in OMM disruption can be in agreement with the find-ing that Mfn-2 functionally interacts with Bax and Bak inhealthy cells (45). Bax and Bak are thought to induce theformation of large pores on the OMM through a process termedmitochondrial outer membrane permeabilization (MOMP), akey step in mitochondrion-mediated apoptosis (3, 80). Al-though the interaction of Bax/Bak with Mfn-2 was linked tomitochondrial remodeling rather than membrane permeabili-zation (45), the possibility that Mfn-2 association with Bax/Bakpromotes MOMP in cardiac myocytes remains an open possi-bility. In this regard, we find that genetic deletion of Mfn-2 inheart attenuates the apoptotic response downstream of differ-ent ischemic insults. Interestingly, the loss of Mfn-2 was asso-ciated with increased levels of Bax but also with a concomitantincrease in Bcl-2. Our findings that loss of Mfn-2 leads toinhibition of apoptosis are in agreement with the study fromShen et al. which demonstrates that overexpression of Mfn-2 incardiac myocytes activates the mitochondrion-dependent ap-optotic pathway (75).

FIG. 10. Mfn-2 ablation results in improved cardiac performancefollowing ex vivo ischemia/reperfusion (I/R) injury. F/F;� and F/F;crehearts (5 and 4 hearts, respectively) from 10-week-old mice wereperfused in the Langendorff mode, subjected to 10 min of globalischemia, and followed up with reperfusion for 20 min. The systolic anddeveloped pressures (A and B, respectively) were recorded beforeischemia (baseline), during ischemia (10 to 20 min), and upon termi-nation of ischemia (reperfusion, 20 to 40 min) (*, P was �0.05 forF/F;� versus F/F;cre hearts; **, P was �0.01 for F/F;� versus F/F;crehearts, by repeated-measures ANOVA). This experiment was inde-pendently repeated with similar results. (C) Mitochondria from F/F;cre hearts contain higher levels of the antiapoptotic protein Bcl-2 thanF/F;� hearts. Following I/R injury, mitochondria were isolated fromF/F;� and F/F;cre hearts and protein extracts were analyzed. LDH, lac-tate dehydrogenase; V�, complex V (FoF1 ATPase), subunit �.



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Finally, the alterations in the structure of mitochondria dueto Mfn-2 ablation could potentially impact MPTP functionindirectly, perhaps due to changes in the structure of the mi-tochondria. However, it should be noted that only a portion ofmitochondria in Mfn-2 F/F;cre hearts displayed structural ab-normalities, while their delayed response to MPTP activationappeared to be a general property exhibited by mitochondriawith either normal or abnormal morphology. This can be seenin Fig. 5B and C, which show that Mfn-2 knockout IFM havethe same volume as wild-type IFM but that the SSM becomeenlarged. Despite these differences in morphology, mitochon-dria from both compartments display a significant delay inMPTP activation (Fig. 6A and B). It could also be argued thata lower m detected in Mfn-2 knockout mitochondria mayhave secondary effects on MPTP activation. However, it shouldbe noted that a lower m has been found to be a strong

inducer of MPTP (12); thus, a decrease in m in Mfn-2-deficient mitochondria should, if anything, decrease, not in-crease, their tendency for MPTP opening. Therefore, we con-clude that the ability of Mfn-2-deficient mitochondria toundergo delayed MPTP activation is distinct from their base-line structural characteristics.

A close relation between mitochondrion-shaping proteinsand apoptotic cell death has previously been recognized (79,83). For example, Opa-1, which is required for inner mitochon-drial membrane fusion, can independently regulate the releaseof cytochrome c from cristae and control apoptosis (33). Basedon the present study, it would be of interest to test whether theablation of other regulators of mitochondrial morphology/dy-namics also affects MPTP and to what extent this may lead toapoptotic or necrotic cell death. In this regard, pharmacologicinhibition of Drp-1 in adult myocytes is reported to attenuate

FIG. 11. Mfn-2-deficient cardiac myocytes are protected from cell death induced by hypoxia/reoxygenation and H2O2 exposure. (A) Adultcardiac myocytes were isolated from F/F;� and F/F;cre hearts and were exposed to either normoxic conditions or subjected to 1 h of hypoxiafollowed by 2 h of reoxygenation. At the end of the reoxygenation (or the normoxia treatment used as a control), the myocytes were stained withtrypan blue to identify myocytes that had lost their membrane integrity (blue). (B) The trypan blue-positive myocytes were counted, and theirnumber was expressed as a percentage of the total number of myocytes per field (stained and unstained). The assay indicates that loss of Mfn-2is associated with a decreased percentage of trypan blue-positive myocytes at normoxia (�, P was �0.05 for F/F;� versus F/F;cre myocytes byone-way ANOVA) and after hypoxia/reoxygenation treatment (#, P was �0.01 for F/F;� versus F/F;cre myocytes by one-way ANOVA). Thenumbers of quantified fields per group are shown in circles in their respective bars, and myocytes were isolated from 6 and 5 F/F;� and F/F;crehearts, respectively. (C) The activation of apoptosis in purified myocytes exposed to normoxia or hypoxia/reoxygenation was examined by Westernblotting. The bands of cleaved (cl) caspase-9 (detected as a 39-kDa band) and the short fragment of cleaved PARP-1 (cl-PARP, detected as an�30-kDa band) are shown. GAPDH was used as a loading control, and the blots shown here are representative of the results of experimentsperformed with myocytes isolated from three hearts per group. (D) The death of purified myocytes as a result of H2O2 exposure was also examined.In this assay, myocytes under normoxic conditions were exposed to 20 �� 2�2 for 2 h and then analyzed for trypan blue uptake. The data showthat cell death is higher in myocytes expressing normal levels of Mfn-2 than in Mfn-2-depleted myocytes (#, P was �0.01 for F/F;� versus F/F;cremyocytes by one-way ANOVA). The number of quantified fields per group is shown in circles in the respective bars.



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mitochondrial depolarization in response to hypoxia/reoxygen-ation and to protect hearts from I/R injury (63), and Mfn-2knockout hearts exhibiting resistance to I/R had consistentlylower levels of Drp-1.

In conclusion, Mfn-2 deficiency in cardiac myocytes leads toperturbations in mitochondrial morphology and to disruptionof their normal spatial orientation. Despite these changes,mitochondrial and cardiac function is normal or minimallyimpaired. Surprisingly, Mfn-2-depleted mitochondria are moretolerant of Ca2� overload, and Mfn-2 deficiency in myocytesprotects isolated cells from ROS stress and hearts from isch-emia-reperfusion injury, suggesting that Mfn-2 can function tocontrol MPTP opening. Although MPTP opening is linked tocell death events, emerging evidence suggests that it is alsoinvolved in physiologically relevant processes, such as ROS

signaling (82) and mitochondrial Ca2� unloading (31). Be-cause mitochondria within adult myocytes display very limitedmotility, Mfn-2 may primarily function as an MPTP mediatorin this context to promote intermitochondrial communication,allowing these organelles to coordinate membrane potentialunder conditions of Ca2� and ROS stress.

ACKNOWLEDGMENTS

We thank David C. Chan for the provision of the Mfn-2flox mouseline, Michael D. Schneider for the �-MHC-Cre mouse line, Donald L.Gantz for assistance with electron microscopy, and Michael T. Kirberfor help with confocal microscopy. We are also thankful to David R.Pimentel for providing neonatal rat cardiac myocytes and to AkikoHiguchi and Taina Rokotuiveikau for assistance with the animalcolony.

FIG. 12. Mfn-2 deficiency in the heart results in an altered expression of outer mitochondrial membrane-associated factors and confersprotection from apoptosis. (A) Western blot analysis of cardiac extracts from hearts with (F/F;�) or without (F/F;cre) Mfn-2. The expression ofthe proapoptotic protein Bax and the antiapoptotic protein Bcl-2 was examined. The levels of Drp-1 and Opa-1 were also assessed. (B) Heart slicestaken from mice with (F/F;�) or without (F/F;cre) cardiomyocyte Mfn-2 that were subjected to 30 min of regional ischemia and 2 h of reperfusionto induce myocardial injury and cell death. The infarct areas (IA) stain negative for TTC and appear as white areas, whereas the ischemic-but-viable myocardium stains red. The nonischemic portion of the LV is stained blue. (C) Quantification of the different regions produced by LADcoronary artery occlusion was performed by planimetry and corrected for slice weight. The IA/AAR ratio provides estimation for the extent of celldeath. AAR, area at risk; LV, left ventricle; IA, infarct area (�, P was �0.05 for F/F;� versus F/F;cre hearts by Student’s t test [7 and 8 mice,respectively]). (D) The TUNEL assay was performed to evaluate the degree of apoptosis in response to I/R in hearts with or without Mfn-2.TUNEL-positive nuclei correspond to apoptotic cells and are stained green. The total number of nuclei was determined by DAPI (4�,6-diamidino-2-phenylindole) counterstaining (blue) and was used to calculate the ratio of apoptotic nuclei in a given field. The scale bar is 100 �m.(E) Quantification of the apoptotic nuclei after I/R in the LV of mice with or without Mfn-2. Multiple fields from two or three sections per heartwere quantified for the number of green and blue nuclei, and the percentages were averaged per animal (*, P was �0.05 for F/F;� versus F/F;crehearts by Student’s t test [7 and 8 mice, respectively]).



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This study was funded by National Institutes of Health grantsHL061639, HL064750, and NO1-HV-28178 to W. S. Colucci,HL074237 to W. C. Stanley, and HL102874, AG34972, AG15052, andHL68758 to K. Walsh.

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