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Mitochondria-localizedcaveolininadaptationtocellularstressandinjury

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ImpactFactor:5.04·DOI:10.1096/fj.12-215798·Source:PubMed

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U.S.DepartmentofVeteransAffairs

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UniversityofCalifornia,SanDiego

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The FASEB Journal • Research Communication

Mitochondria-localized caveolin in adaptation to cellularstress and injury

Heidi N. Fridolfsson,*,1 Yoshitaka Kawaraguchi,*,1 Sameh S. Ali,*,§,1

Mathivadhani Panneerselvam,* Ingrid R. Niesman,* J. Cameron Finley,*Sarah E. Kellerhals,* Michael Y. Migita,*,� Hideshi Okada,†,� Ana L. Moreno,*Michelle Jennings,* Michael W. Kidd,* Jacqueline A. Bonds,* Ravi C. Balijepalli,¶

Robert S. Ross,†,� Piyush M. Patel,*,� Atsushi Miyanohara,† Qun Chen,#,**Edward J. Lesnefsky,#,** Brian P. Head,*,� David M. Roth,*,� Paul A. Insel,†,‡,2

and Hemal H. Patel*,�,2,3

*Department of Anesthesiology, †Department of Medicine, and ‡Department of Pharmacology,University of California–San Diego, La Jolla, California, USA; §Center for Aging and AssociatedDiseases, Helmy Institute for Medical Sciences, Zewail City of Science and Technology, Egypt;�Veterans Affairs San Diego Healthcare System, San Diego, California, USA; ¶Department ofMedicine, University of Wisconsin School of Medicine, Madison, Wisconsin, USA; #McGuire VeteransAffairs Medical Center, Richmond, Virginia, USA; and **Virginia Commonwealth University,Richmond, Virginia, USA

ABSTRACT We show here that the apposition ofplasma membrane caveolae and mitochondria (first notedin electron micrographs >50 yr ago) and caveolae-mito-chondria interaction regulates adaptation to cellular stressby modulating the structure and function of mitochon-dria. In C57Bl/6 mice engineered to overexpress caveolinspecifically in cardiac myocytes (Cav-3 OE), localization ofcaveolin to mitochondria increases membrane rigidity(4.2%; P<0.05), tolerance to calcium, and respiratoryfunction (72% increase in state 3 and 23% increase incomplex IV activity; P<0.05), while reducing stress-in-duced generation of reactive oxygen species (by 20% incellular superoxide and 41 and 28% in mitochondrialsuperoxide under states 4 and 3, respectively; P<0.05) inCav-3 OE vs. TGneg. By contrast, mitochondrial functionis abnormal in caveolin-knockout mice and Caenorhabditiselegans with null mutations in caveolin (60% increase freeradical in Cav-2 C. elegans mutants; P<0.05). In humancolon cancer cells, mitochondria with increased caveolinhave a 30% decrease in apoptotic stress (P<0.05), butcells with disrupted mitochondria-caveolin interaction

have a 30% increase in stress response (P<0.05). Tar-geted gene transfer of caveolin to mitochondria inC57Bl/6 mice increases cardiac mitochondria tolerance tocalcium, enhances respiratory function (increases of 90%state 4, 220% state 3, 88% complex IV activity; P<0.05), anddecreases (by 33%) cardiac damage (P<0.05). Physical asso-ciation and apparently the transfer of caveolin betweencaveolae and mitochondria is thus a conserved cellularresponse that confers protection from cellular damage in avariety of tissues and settings.—Fridolfsson, H. N., Kawara-guchi, Y., Ali, S. S., Panneerselvam, M., Niesman, I. R.,Finley, J. C., Kellerhals, S. E., Migita, M. Y., Okada, H.,Moreno, A. L., Jennings, M., Kidd, M. W., Bonds, J. A.,Balijepalli, R. C., Ross, R. S., Patel, P. M., Miyanohara,A., Chen, Q., Lesnefsky, E. J., Head, B. P., Roth, D. M.,Insel, P. A., Patel, H. H. Mitochondria-localized caveo-lin in adaptation to cellular stress and injury. FASEB J.26, 4637–4649 (2012). www.fasebj.org

Key Words: cardiac protection � cancer biology � gene transfer� reactive oxygen species � respiratory function

In addition to sensing extracellular signals that mod-ulate cellular function, the plasma membrane helpsprotect intracellular structures and activities from in-sults by the external environment. Caveolae (“littlecaves”), cholesterol- and sphingolipid-enriched invagi-nations of the plasma membrane (1), are a subset oflipid (membrane) rafts (2) that facilitate such sensing

1 These authors contributed equally to this work.2 These authors contributed equally to this work.3 Correspondence: University of California–San Diego, Depart-

ment of Anesthesiology, VASDHS (9125), 3350 La Jolla Village Dr.,San Diego, CA 92161, USA. E-mail: hepatel@ucsd.edu

doi: 10.1096/fj.12-215798

Abbreviations: 5-DSA, 5-nitroxyl stearate; AAV9, adenoassoci-ated virus 9; ANT, adenine nucleotide translocase; Cav-1, caveo-lin 1; Cav-2, caveolin 2; Cav-3, caveolin 3; CM, cardiac myocyte;DEPMPO, 5-(diisopropoxyphosphoryl)-5-ethyl-1-pyrroline-N-ox-ide; DHE, dihydroethidium; EM, electron microscopy; EPR,electron paramagnetic resonance; IFM, interfibrillary mitochon-dria; IMM, inner mitochondrial membrane; IPC, ischemic pre-conditioning; I/R, ischemia-reperfusion; KO, knockout; MIM,mitochondrial isolation medium; mito-CSD, mitochondrial-tar-geted caveolin scaffolding domain; mPTP, mitochondrial per-meability transition pore; OE, overexpressing; RFP, red fluores-cent protein; ROS, reactive oxygen species; SSM, subsarcolemmalmitochondria; TGneg, transgene negative; TMPD, 2,2,4-trimethyl-1,3-pentanediol; TRAIL, TNF-related apoptosis-inducing ligand;VDAC, voltage-dependent anion channel; WT, wild type

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and contribute to cellular responses to environmentalperturbations. Caveolins, scaffolding proteins of cave-olae, contribute to the regulation of numerous cellularfunctions, including endocytosis, calcium homeostasis,and compartmentalization of signaling components(receptors, effectors) (3). Three isoforms, caveolins1–3 (Cav-1, Cav-2, and Cav-3), function both inside andoutside of caveolae (4).

In highly metabolic organs (e.g., heart, brain, andliver), mitochondria play a key role in the adaptation tocellular stress. Lack of oxygen inhibits mitochondrialfunction, including blockade of ATP synthesis and lossof membrane potential (5). Disruption of mitochon-dria also occurs during injury as a consequence ofincreased calcium and reactive oxygen species (ROS;ref. 6), which both contribute to opening of the mito-chondrial permeability transition pore (mPTP) andapoptosis (7, 8). Mitochondrial dysfunction is a hall-mark of the pathophysiology of highly metabolic or-gans and occurs in ischemic injury and other settingsthat include heart failure, diabetes mellitus, Alzhei-mer’s disease, Parkinson’s disease, cancer, and aging.

Caveolin-deficient stromal cells have compromisedmitochondrial function (9) and mitochondria fromCav-1-knockout (KO) fibroblasts accumulate choles-terol and have severe dysfunction; such cells adaptpoorly to nutrient starvation and are predisposed toapoptosis (10). Loss of caveolin alters mitochondrialfunction in adipose tissue, suggesting a link betweencaveolins and metabolism (11). Knockdown of caveolinaccelerates neuronal aging and decreases adaptation tocerebral and cardiac injury (12–14). By contrast, cardiac-specific overexpression of Cav-3 protects the heart frominjury and pressure overload-promoted failure (15, 16).Such findings suggest a link between caveolin and mito-chondrial function but whether the two are directlyrelated has not been defined.

Tissue protection from injury may be associated withthe formation of plasma membrane signaling microdo-mains (signalosomes) that can interact with mitochondria(17, 18). For example, disruption of caveolae inhibitss-nitrosylation of mitochondrial proteins, a mechanismthat contributes to the protective phenotype (17, 18). Inaddition, caveolae can form contacts that can communi-cate membrane-derived signals to other cellular compo-nents, e.g., “nanocontacts” between caveolae and theendoplasmic reticulum in smooth muscle cells (19).

Here, we tested the hypothesis that plasma mem-brane caveolae interact with mitochondria and that thisinteraction has important functional consequences. Weused a variety of experimental approaches and cellularsystems, including cardiac and cancer cells to definegeneral features of this interaction. Use of Caenorhabdi-tis elegans as a model organism provided further supportfor this hypothesis. The results thus identify caveolae-mitochondria interaction and mitochondrial caveolinsas previously unrecognized, critical regulators in theadaptation of eukaryotic cells to stress.

MATERIALS AND METHODS

Ethics statement

Animals were treated in compliance with the U.S. NationalAcademy of Science Guide for the Care and Use of Labora-tory Animals, and protocols were approved by the VeteransAffairs San Diego Healthcare System Animal Care and UseCommittee.

Materials

Antibodies for Cav-3 (monoclonal) were from BD Biosciences(San Jose, CA, USA); antibodies for Cav-3, Cav-1 (polyclonal),and voltage-dependent anion channel (VDAC) were fromAbcam (Cambridge, MA, USA); antibodies for prohibitin,adenine nucleotide translocase (ANT), and actin were fromSanta Cruz Biotechnology (Santa Cruz, CA, USA); and anti-bodies for cytochrome c were from Imgenex (San Diego, CA,USA). FITC and Alexa-conjugated secondary antibodies werefrom Invitrogen (Carlsbad, CA, USA). Other secondary anti-bodies were obtained from Santa Cruz Biotechnology. Otherchemicals and reagents were obtained from Sigma (St. Louis,MO, USA) unless otherwise stated.

Animals

Animals were kept on a 12-h light-dark cycle in a temperature-controlled room with ad libitum access to food and water.Cav-3-overexpressing (OE) mice were produced in aC57BL/6 background as described previously (16). Cav-3-KOmice were created as reported previously and backcrossed 10generation in the C57BL/6 background (20). Transgene-negative (TGneg) siblings in the C57BL/6 backgroundserved as controls for Cav-3-OE and Cav-3-KO mice. Sprague-Dawley rats (250–300 g, male) were used for some studies.

CM preparation

Adult male Sprague-Dawley rats were anesthetized with ket-amine (100 mg/kg) and xylazine (10 mg/kg), hearts wereexcised and retrograde-perfused with medium containingcollagenase II, as described previously (21) to isolate ventric-ular CMs. A similar procedure was used for adult mouseventricular myocyte isolation with slight variations (14).

Immunofluorescence and deconvolution microscopy of CMs

Samples were prepared for immunofluorescence microscopy,and images were deconvolved as described previously (21).

Mitochondrial isolation

Mice were sacrificed, and hearts were removed. Ventricleswere placed in ice-cold mitochondrial isolation medium(MIM: 0.3 M sucrose, 10 mM HEPES, 250 uM EDTA),minced, and homogenized with a Tissuemiser (Fisher Scien-tific, Waltham, MA, USA). Homogenates were rinsed in MIM.Samples were centrifuged at 600 g to clear nuclear/mem-brane debris. The resulting supernatant was spun at 8000 gfor 15 min. The resulting pellet was resuspended in MIM inthe presence of 1 mM BSA, followed by another 8000-g spinfor 15 min. The resulting pellet was resuspended in isolationbuffer with BSA and spun again at 8000 g. Metabolically active

4638 Vol. 26 November 2012 FRIDOLFSSON ET AL.The FASEB Journal � www.fasebj.org

mitochondria were then suspended in 150 �l MIM forfunctional studies.

To isolate pure mitochondria for biochemical and electronmicroscopy (EM) analysis, the washing steps were repeatedwith MIM in a final 2-ml resuspension of the pellet inmitochondrial resuspension buffer (MRB; 500 �M EDTA, 250mM mannitol, and 5 mM HEPES). The mitochondria werelayered on top of a 30% Percoll/70% MRB solution. ThePercoll gradient was spun at 95,000 g for 30 min. Themitochondrial band was removed from the gradient, andvolume was increased 10-fold with MRB to remove the Percollby centrifugation at 8000 g for 15 min. The mitochondrialpellet was resuspended in 50–150 �l of MRB and subjected tofurther analysis.

Purification of subsarcolemmal mitochondria (SSM) andinterfibrillary mitochondria (IFM)

Cardiac SSM and IFM populations were isolated using themethod of Palmer et al. (22), modified as described previouslyby the use of Chappell–Perry buffer (100 mM KCl, 50 mMMops, 1 mM EGTA, 5 mM MgSO4, and 1 mM ATP, pH 7.4)for mitochondrial isolation (23).

Membrane fractionation of mitochondria

Purified mitochondria were lysed in buffer containing 150mM Na2CO3 (pH 11.0) and 1 mM EDTA, and then sonicatedon ice with 3 cycles of 20-s bursts. Approximately 1 ml ofhomogenate was mixed with 1 ml of 80% sucrose in 25 mM2-(N-mopholino)ethanesulfonic acid (MES) and 150 mMNaCl (MBS; pH 6.5) to form 40% sucrose and loaded at thebottom of an ultracentrifuge tube. A discontinuous sucrosegradient was generated by layering 6 ml of 35% sucroseprepared in MBS followed by 4 ml of 5% sucrose (also inMBS). Gradients were centrifuged at 175,000 g using aSW41Ti rotor (Beckman Coulter, Fullerton, CA, USA) for 3 hat 4°C. Samples were removed in 1-ml aliquots to form 12fractions and fractions probed for specific proteins.

EM

Cells or tissues were fixed with 2.5% glutaraldehyde in 0.1 Mcacodylate buffer for 2 h and postfixed in 1% OsO4 in 0.1 Mcacodylate buffer (1 h) at room temperature, and thenembedded as monolayers in LX-112 (Ladd Research, Willi-ston, VT, USA), as described previously (24). Sections werestained in uranyl acetate and lead citrate prior to EM (Jeol1200 EX-II; Jeol Ltd., Akishima, Japan; or Philips CM-10;Philips, Amsterdam, The Netherlands). Caveolae were quan-tified on random images per length of membrane. Forimmunogold labeling, purified mitochondria were fixed in4% paraformaldehyde in 10 mM phosphate buffer (pH 7.4),cryoprotected with 2.3 M sucrose, and frozen in liquidnitrogen. Ultrathin cryosections were cut at �100°C using aLeica Ultracut UCT Microtome with an EMFCS cryoattach-ment (Leica Microsystems, Wetzlar, Germany), placed onglow-discharged nickel grids, stored on 2% gelatin and PBS at4°C, and incubated with primary antibodies, followed by 5 or10 nm gold, and then goat anti-rabbit or anti-mouse IgG inPBS with 10% fetal calf serum. Mitochondrial cross-reactionswith either mouse or rabbit antibodies are commonly ob-served. Therefore, antibodies were also tested on mitochon-dria isolated from Cav-3-KO mice to test specificity. Onlyantibodies with limited background label were used in theanalysis. Grids were absorption stained with 0.2% neutraluranyl acetate and 0.2% methyl cellulose and viewed directlyon the microscope.

Calcium pulse

Opening of the MPT pore after in vitro Ca2� overload wasassessed by following changes in the membrane potential(��m) by using the fluorescent dye rhodamine 123 (50 nM;Invitrogen, Carlsbad, CA, USA) in the presence of pyruvateand malate (5 mM) (25). Fluorescence was monitored withan Infinite M200 plate reader (Tecan Group Ltd., Männe-dorf, Switzerland). Excitation and emission wavelengths wereset to 503 and 527 nm, respectively. Isolated mitochondria(0.5 mg/ml) were suspended in 1.0 ml recording buffercontaining 220 mM sucrose, 10 mM 4-[2-hydroxyethyl] piper-azine-1-ethanesulfonic acid, and 10 mM KH2 PO4 (pH 7.3).At the end of the preincubation period, pulses of 10 �MCaCl2 were administered at 60-s intervals. After sufficientCa2� loading, MPT pore opening results in a sudden collapseof ��m. To achieve complete mitochondrial depolarization, 1�M carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone(FCCP) was added to the buffer at the end of the experiment.The amount of Ca2� necessary to trigger this sudden collapseof ��m was used as an indicator of the susceptibility of theMPT pore to Ca2� overload.

Calcium swelling

Calcium swelling was measured on an Infinite M200 platereader at 540 nm over a span of 20 min. Crude mitochondria(0.5 �g/�l) in the absence of calcium were loaded onto aclear flat bottom 96-well plate and challenged with 250 �Mcalcium, with absorbance measured every 10 s. Change at 540nm was compared between samples.

Mitochondrial respiration

Mitochondrial respiratory function was studied according topublished protocols (26). Oxygen consumption was mea-sured using a Clark-type oxygen electrode (Oxygraph; Han-satech, Norfolk, UK) during the sequential additions ofsubstrates and inhibitors to purified mitochondria. Purifiedmitochondria (�100–200 �g protein) were added to theoxymetry chamber in a 300 ml solution containing 100 mMKCl, 75 mM mannitol, 25 mM sucrose, 5 mM H3PO4, 0.05mM EDTA, and 10 mM Tris-HCl, pH � 7.2 at 37°C. After 2min equilibration, 5 mM pyruvate and 5 mM malate wereadded and oxygen consumption followed for �1–2 min (state4). ADP (250 �M) was added to measure state 3 (phosphor-ylating) respiration. To switch from NAD�- to FAD�- linkedrespiration, we first eliminated complex I through the inhi-bition of the back electron transfer using 0.5 mM rotenoneand triggered complex II activity by the addition of 10 mMsuccinate. Next, we inhibited complex III by the addition of 5mM anti-mycin A. Complex IV activity was measured in thepresence of 0.5 mM 2,2,4-trimethyl-1,3-pentanediol (TMPD)and 2 mM ascorbate. Finally, to test the integrity of themitochondrial outer membrane, we followed the oxygenconsumption rate on the addition of 10 mM cytochrome c,which is expected to enhance oxygen consumption if theouter membrane was compromised during the isolation.Oxygen utilization traces and rate determinations were ob-tained using Oxygraph software and normalized to protein.

Reactive oxygen species (ROS) assessment usingdihydroethidium (DHE) staining and electron paramagneticresonance (EPR)

For DHE assay, isolated adult mouse CMs were incubated withDHE (5 �M) for 30 min at 37°C in a humidified incubator. Afterincubation, cells were washed with PBS, hypoxia medium lack-

4639CAVEOLIN AND MITOCHONDRIA IN STRESS

ing glucose was added, and cells were placed in a hypoxiachamber for 60 min. Following hypoxia, fresh medium wasadded to cells. After 20 min in fresh medium, the cells wereimaged for DHE fluorescence. As DHE is oxidized by ROS, itbecomes ethidium and accumulates in the nucleus, so wequantified nuclear specific fluorescence between the groups.For EPR studies, immediately after mixing mitochondria (0.1–0.2 mg of protein) with 70 mM 5-(diisopropoxyphosphoryl)-5-ethyl-1-pyrroline-N-oxide (DEPMPO) and appropriate combina-tions of the substrates, the mixture was loaded into 500-�l glasscapillary tubes and introduced into the EPR cavity of a MiniScopeMS300 benchtop spectrometer (Magnettech GmbH, Berlin, Ger-many). We confirmed that the detected EPR signals are sub-strate specific, and not due to redox cycling in the studiedmixtures, by lack of signals when DEPMPO was mixed withcombinations of substrates and inhibitors in the absence ofmitochondria. Assignment of the observed signals from mito-chondria was confirmed through computer-assisted spectralsimulation using the WinSim software (http://epr.niehs.nih.gov/pest.html). In most cases, a mixture of signals due toDEPMPO-OOH and DEPMPO-OH adducts, with occasionalcontribution from a carbon-centered radical, was detected, butthe complete removal of these signals on the inclusion of SODconfirmed that superoxide radical was the exclusive source ofthe observed EPR-active species. Signals were quantified bymeasuring the peak amplitudes of the observed spectra andnormalized by mitochondrial protein concentrations.

EPR for membrane fluidity

Hydrocarbon chain mobility was measured using fatty acid spinlabeling EPR analysis using 5-nitroxyl stearate (5-DSA) as a spinprobe (27, 28). The number designation indicates the relativeposition of the nitroxide on the stearic acid relative to the polarcarboxylic group. In the case of 5-DSA, the spin probe is firmlyheld in place by the head groups of the lipids, which is reflectedin broad EPR lines. Plasma membrane or purified mitochondriawas incubated for 15 min with 5-DSA (1 mM final concentra-tion) at 25°C. The mixture was then loaded into a 50-�l glasscapillary tube and inserted into the EPR cavity of a MiniScopeMS200 benchtop spectrometer (Magnettech), maintained at37°C, where the EPR spectra registered. EPR conditions werethe following: microwave power, 5 mW; modulation amplitude,2 G; modulation frequency, 100 kHz; sweep width, 150 Gcentered at 3349.0 G; scan rate, 7.5 G/s, with each spectrumrepresenting the average of 5 scans. The fluidity parameters T�and T� were used to calculate the order parameter, as describedpreviously (27, 28).

C. elegans strains

C. elegans were cultured using standard conditions, and N2was used as wild type (WT; ref. 29). The cav-1(ok2089) andcav-2(hc191) strains used in this work were provided by theCaenorhabditis Genetics Center, which is funded by the U.S.National Institutes of Health National Center for ResearchResources (NCRR).

ATP assay

Mitochondria ATP production was measured by the ATPDetermination Kit (Invitrogen, Carlsbad, CA, USA) as de-scribed by the manufacturer. Functional mitochondria werenormalized to 5 �g/�l, and 10 �l was added to 90 �l of thereaction mix. Samples were loaded into a Tecan InfiniteM200 plate reader, and luminescence was quantified over 10min.

Generation of stable cell lines

We purchased human colon cancer cell lines, HCT116 andHT29, from American Type Culture Collection (ATCC; Rock-ville, MD, USA). Both cell lines were cultured in McCoy’s 5amedium containing 10% fetal bovine serum and 1% penicil-lin/streptomycin in a 95% air, 5% CO2 humidified atmo-sphere at 37°C. Stably transfected HT29 cells were supple-mented with 800 �g/ml G418. For generation of stable celllines, mouse Cav-1 (456 bp) sequence was cloned into thepTurboRFP-mito vector (Evrogen, Moscow, Russia). The orig-inal vector containing red fluorescent protein (RFP) was usedas a control. Cells (5�105/well) were seeded on 24-well plateswithout antibiotics and cultured overnight. On the next day,0.8 �g of pTurbo-mito RFP or pTurbo-mito Cav-1 plasmid wastransfected into HT29 cells using 2 �l of Lipofectamine 2000in Opti-MEM Reduced Serum Medium following the manu-facturer’s instructions. Transfected cells were selected withG418 at 800 �g/ml for 4 wk, and the expression of Cav-1protein in selected cell clones was determined by Westernblotting. The mitochondrial-targeted caveolin scaffolding do-main (mito-CSD) was generated by direct cloning of synthe-sized DNA with selective restriction enzyme ends into thepTurboRFO vector with RFP deletion. Transient transfectionswere performed in HCT116 cells using Lipofectamine 2000.

Apoptosis assay

After 24 h treatment with TNF-related apoptosis-inducingligand (TRAIL), apoptosis was quantified by nucleosomalfragmentation (Cell Death Detection ELISA plus; RocheApplied Science, Indianapolis, IN, USA). The absorbancevalues were normalized to those from control-treated cells toderive a nucleosomal enrichment factor at all concentrationsas per the manufacturer’s protocol.

Mitochondrial membrane potential

JC-1, a lipophilic cationic dye that enters mitochondria and shiftsits fluorescence from green to red when ��m increases, was usedto assess ��m in the setting of TRAIL stress. Following treatment,5 � 105 cells were washed with PBS and incubated with 2 �MJC-1 fluorescent probe in dark for 15 min at 37°C. Cells werewashed again with PBS and analyzed immediately by flowcytometry. Detection setting was JC-1 green fluorescence at 530nm and JC-1 red fluorescence at 590 nm. The ratios of green tored fluorescence intensity values were calculated, and the per-centage increase in these ratios were expressed as measures ofpercentage increase in �m.

Generation of adenoassociated virus 9 (AAV9) vector andTaqMan assay

The construct used to express inner mitochondrial mem-brane (IMM)-targeted Cav-3 was generated by amplifyingmouse Cav-3 cDNA by PCR and inserting it into the BamHIand XbaI sites of the pTurboRFP-mito vector (Evrogen). Thisremoved the RFP sequence and replaced it with Cav-3.AAV9.IMM.Cav-3 was generated by integration of this con-struct into AAV9. To determine viral copy number, TaqManprobes were designed to bind the viral backbone of AAV9,and a TaqMan assay was performed on various tissues.

In vivo ischemia-reperfusion (I/R) injury

Pentobarbital (80 mg/kg)-anesthetized mice were mechanicallyventilated, and ischemia was produced by occluding the left

4640 Vol. 26 November 2012 FRIDOLFSSON ET AL.The FASEB Journal � www.fasebj.org

coronary artery with a 7-0 silk suture on a tapered BV-1 needle(Ethicon, Somerville, NJ, USA) for 30 min (30). After 30 minocclusion, the ligature was released and the heart was reperfusedfor 2 h. Ischemic preconditioning (IPC) was induced by occlu-sion of the left coronary artery for 5 min followed by 15 minreperfusion just prior to ischemia. The area at risk (AAR) wasdetermined by staining with 1% Evans blue (1.0 ml, Sigma) (30).The heart was immediately excised and cut into 1 mm slices(McIlwain tissue chopper; Brinkmann Instruments, Westbury,NY, USA). Left ventricle was counterstained with 1% 2,3,5,-triphenyltetrazolium chloride (Sigma). Images were analyzed byImage-Pro Plus (Media Cybernetics, Inc., Bethesda, MD, USA),and infarct size was determined by planimetry.

Statistics

All data are presented as means sem. GraphPad Prism 4software (GraphPad Software, Inc., San Diego, CA, USA) wasused for all statistical analysis. Statistical analyses were per-formed by unpaired Student’s t test (2-tailed testing) or 1-wayANOVA followed by a Bonferroni’s post hoc test.

RESULTS

Caveolae are closely apposed to mitochondria

Immunohistochemical staining revealed that Cav-3 isexpressed in a punctate pattern along the sarcolemmalmembrane and in transverse striations within the inte-rior of adult CMs. Cav-3 colocalizes with cytochrome c,

a mitochondrial marker, along the sarcolemmal mem-brane (Fig. 1A, �70% colocalization within 2 �m of thesurface). By contrast, Cav-3 and cytochrome c stain inparallel, transverse patterns with minimal colocaliza-tion in the cellular interior. EM reveals that CMs haveabundant caveolae closely apposed to SSM (Fig. 1B),which in turn are in proximity to regions of the plasmamembrane that contain caveolae (Fig. 1C): �50% oftotal caveolae per micrometer of sarcolemma associatewith SSM (Fig. 1D). A subset of caveolae on thesarcolemmal membrane is thus closely juxtaposed tomitochondria, suggesting functional interaction be-tween caveolae and mitochondria.

IPC promotes connections between caveolae andmitochondria and the transfer of Cav-3 protein tomitochondria

Caveolae contribute to signal transduction, including theprotection of CMs from I/R injury (3). Cellular signalingevents that protect from I/R injury converge on the mito-chondria (31). Given the close apposition of caveolae andmitochondria in CMs and their importance in protectionfrom I/R injury, we investigated whether caveolae andmitochondria have direct connections during ischemicstress. We thus subjected mice to 5 min of ischemia byoccluding the coronary circulation (i.e., a protective precon-ditioning stimulus involving sublethal stress; ref. 32) and

Figure 1. Caveolae are closely apposed to mitochondria, and IPC is associated with the formation of tethers between caveolaeand mitochondria. A) Caveolae (Cav-3) and mitochondria (Cyto C) show close association at the sarcolemmal membrane butnot internal regions of adult rat CMs. B) EM image shows close apposition of caveolae and mitochondria. C, D) Nearly all SSMare associated with caveolae (C), but only about half of the membrane caveolae are associated with mitochondria (D), suggestingthe existence of mitochondrial-associated and -unassociated pools of caveolae. E) IPC shows evidence of increased associationof caveolae-mitochondria. Arrows denote caveolae; triangles represent unidentified structures that link mitochondria tocaveolae. F) Higher magnification of the micrograph shown in E. G) No such structures are evident in controls hearts. Scale bars �50 nm. H) EM quantification after IPC showed increased association of caveolae and mitochondria.

4641CAVEOLIN AND MITOCHONDRIA IN STRESS

then allowed recovery for 15 min. We identified structuresbetween caveolae and mitochondria in the precondi-tioned hearts (Fig. 1E, F); i.e., in response to the IPCstimulus, but these structures are not found in un-treated hearts (Fig. 1G). Preconditioning increased theassociation of caveolae with mitochondria (Fig. 1H).IPC thus induces the formation of caveolae and in-creases their association with mitochondria.

Exposure of adult CMs to sublethal stress (10 minoxygen/glucose deprivation) also generates a connec-tion between caveolae and mitochondria; this occurs 15min after the stress but is lost by 60 min, at which timevesicles are observed in SSM (Fig. 2A). These resultssuggest that a transient connection forms betweencaveolae and mitochondria but progresses to a fusionwith mitochondrial membranes and an interminglingof constituents in caveolar and mitochondrial mem-branes.

Immunogold labeling revealed that Cav-3 localizes inthe IMM, matrix, and perhaps also on the outer mito-chondrial membrane (Fig. 2B). Probing for Cav-3 pro-tein in SSM and IFM revealed that most mitochondrialCav-3 is in SSM (Fig. 2C), implying its preferentialtransfer from plasma membrane caveolae to SSM butnot to all mitochondria.

This transfer of caveolin protein occurs in responseto cellular stress. Mice subjected to a preconditioningstimulus and allowed to recover for 60 min haveincreased mitochondrial localization of Cav-3 (Fig. 2D;protein levels normalized to ANT, an IMM protein). Inview of the important role of mitochondria in limitingI/R injury (33, 34), the association of caveolae andmitochondria following sublethal ischemia and thetransfer of caveolin to mitochondria is a previouslyunappreciated cellular response and suggests thatplasma membrane caveolae “sense” stress, perhapshelping mitochondria in proximity to the plasma mem-brane resist damage and maintain cellular function.

Mitochondria from Cav-3-OE mice have increasedCav-3 protein, improved Ca2� tolerance andrespiration, and altered mitochondrial membranestructure

Transgenic mice with CM-specific Cav-3 overexpressionhave increased sarcolemmal caveolae and improvedfunctional recovery following I/R injury; e.g., decreasedinfarct size and apoptosis than do TGneg controls (16).CM-specific Cav-3-OE mice thus have cardioprotectionakin to that of WT mice undergoing IPC (16); mitochon-dria of Cav-3-OE mice have greater Cav-3 protein levelsthan do TGneg mitochondria (Fig. 3A).

To test whether the increased Cav-3 in mitochondriaof Cav-3-OE mice contributes to enhanced cardiacprotection and improves mitochondrial function, wechallenged purified mitochondria with calcium over-load. Calcium overload in the mitochondrial matrixcan occur during I/R injury and open the mPTP,depolarize mitochondrial membrane potential, releasecytochrome c, and produce apoptosis (7, 8). We mon-itored mitochondrial membrane potential in TGnegand Cav-3-OE mice in the presence of increasing cal-cium concentration and found that mitochondria fromCav-3-OE mice have greater recovery and maintainmembrane potential at higher calcium concentrationsthan do mitochondria from TGneg mice (Fig. 3B).Cav-3-OE mitochondria are also more resistant to cal-cium swelling than are TGneg mitochondria (Fig. 3C).Thus, mitochondrial Cav-3 may help maintain calciumhomeostasis in mitochondria and prevent mitochon-dria-mediated apoptosis during I/R.

Mitochondria from the hearts of Cav-3-OE mice(compared to those from TGneg mice) have greaterrespiratory rates during state 3 in response to additionof malate, pyruvate, and ADP (Fig. 3D), indicatingmore efficient function of complex I in the electrontransport chain. Such mitochondria also have increasedcomplex IV oxygen consumption in response to TMPD

Figure 2. IPC modifies the association of cave-olae and mitochondria and Cav-3 localizationto mitochondria. A) Adult CMs under basalconditions show close apposition, which transi-tions to a physical association with sublethalstress and finally to mitochondrial internalizedstructures at 60 min poststress (denoted byarrowheads). B) Cav-3 localizes to the IMM,mitochondrial matrix, and potentially the outermitochondrial membrane. C) The majority ofCav-3 in mitochondria is present in SSM butnot IFM. D) IPC increased the mitochondriallocalization of Cav-3 (normalized to ANT); n � 4.

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and ascorbate (Fig. 3D) but no changes with succinate,suggesting limited effects on complex II. The heart isalmost entirely dependent on mitochondria-generatedATP for contractile energy; thus, improvement in mi-tochondrial oxidative phosphorylation implies bettercontractile function during reperfusion. Increased re-spiratory rates in mitochondria from Cav-3-OE mice ispredicted to increase ATP production and may accountfor the enhanced functional recovery of those miceduring I/R injury.

We used EPR and phospholipid spin probes toassess membrane fluidity and determine whethermitochondria-localized caveolin alters mitochondrialstructure (Fig. 3E). We found that membrane orderparameter, indicative of the ratio of membrane rigid-ity to fluidity, increases in the cardiac sarcolemmaand mitochondria of Cav-3-OE mice (Fig. 3F), sug-gesting that an increase in expression of caveolinalters membrane structure and may modulate mito-chondrial function.

Cav-3 overexpression reduces ROS generation in CMsand mitochondria, whereas loss of caveolin enhancesmitochondrial dysfunction

Reperfusion leads to increased utilization of cellularoxygen for generation of ROS, rather than energyproduction. ROS generation along with mitochon-

drial dysfunction contributes to myocardial I/R in-jury (35). ROS have destructive potential because oftheir high reactivity with molecules, including lipids,proteins, and DNA. CMs from Cav-3-OE mice havedecreased generation of superoxide in response tooxygen/glucose deprivation for 1 h followed by 20min recovery (Fig. 4A, B); by contrast, CMs fromCav-3-KO mouse hearts have increased superoxideproduction, which may contribute to cardiac pathol-ogy in these animals (13, 36). Consistent with thisidea, mitochondria from TGneg mouse hearts pro-duced greater superoxide signals with the addition ofmalate and pyruvate in state 3 (with ADP) and 4 (noADP) compared to mitochondria from Cav-3-OEmice (Fig. 4C, D). This result implies that complex Iis more tightly coupled in Cav-3-OE mitochondriaand is likely a source of ROS leakage in mitochondriafrom TGneg mice. No changes were observed withaddition of succinate, suggesting that complex II isnot involved in the response. These results alsoconfirm that the increased oxygen consumption ofmitochondria from Cav3-OE mice produces energyand not increased amounts of ROS.

Mitochondria in cardiac muscle are arranged infilamentous networks; changes in mitochondrial mor-phology have been noted in apoptosis (37). Fragmen-tation of mitochondria is greater in Cav-3-KO hearts,but mitochondrial structure is maintained in TGneg

Figure 3. Cav-3-OE mice show cardiac protection; their mitochondria have increased Cav-3 and exhibit Ca2� tolerance. A)Mitochondria from Cav-3-OE mice have increased Cav-3 expression (normalized to prohibitin and VDAC loading). B)Mitochondria from Cav-3-OE mice have increased tolerance to calcium pulsing. n � 4–6. C) Mitochondria isolated fromCav-3-OE mouse hearts are more resistant to calcium swelling. n � 4–6. D) Mitochondria from Cav-3-OE mouse hearts showincreased respiratory rates during state 3, as triggered by malate � pyruvate � ADP, and when complex IV is artificiallystimulated by TMPD � ascorbate (n�5–6). E) 5-DSA probes membrane rigidity close to the hydrophilic surface of themembrane, while 16-DSA probes the fluidity in the core of the phospholipid bilayer. F) Cav-3-OE mouse hearts have increasedorder parameter at the membrane interface in both the plasma and mitochondrial membranes, as revealed by the analysis of5-DSA spectra. n � 5. *P � 0.05.

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and Cav-3-OE hearts (Fig. 4E). Fragmentation of mito-chondria may contribute to increased ROS productionand cardiac pathology in Cav-3-KO mice.

We assessed C. elegans strains carrying null mutationsin caveolin genes as a further means to determinewhether caveolin deficiency can result in mitochondrialdysfunction. C. elegans express two isoforms of caveolin(38), but their role in mitochondrial function is notknown. We found that under state 3, but not state 4(data not shown) conditions, C. elegans mutants ofCav-1 and Cav-2 have compromised ATP synthesis (Fig.4F). The Cav-2 mutant has enhanced generation ofcarbon-centered free radicals, as assessed by EPR (Fig.4G, H). These data confirm a role for caveolin expres-sion in mitochondrial function and protection andextend this conclusion to an organism evolutionarilydistant from mammals.

Mitochondria-localized caveolin enhances, whereastargeted disruption of mito-CSD reduces, coloncancer cell survival in response to cellular stress

Caveolin has been considered both a tumor promoterand suppressor (39), and thus its role in cancer cellsurvival and cell death is controversial (40). We identifiedtwo colon cancer cell lines, HCT116 and HT29, whichexpress high and low/no levels of caveolin, respectively(Fig. 5A), and used these cells as another system to testwhether mitochondria-localized caveolin regulates adap-tation to cellular stress. Mitochondria isolated fromHCT116 cells are enriched in caveolin (Fig. 5A), a resultconfirmed by immunohistochemistry (Fig. 5B). We gen-erated stable HT29 cell lines that have targeted expres-sion of RFP or caveolin to mitochondria (mRFP ormCav-1; Fig. 5C–E). Treatment of these stable HT29 cellswith TRAIL revealed that cells with mitochondria-targeted

Figure 4. Cav-3 overexpression reduces the generation of ROS in CMs and mitochondria. A) DHE was used to probe superoxidewith nuclear-localized ethidium. Hypo, hypoxia/reoxygenation. B) Increased expression of Cav-3 suppresses superoxideformation, while knockdown of Cav-3 increases superoxide formation; n � 6. C, D) EPR with DEPMPO spin probe onmitochondria from Cav-3-OE hearts have reduced ROS production during NAD-linked, but not FAD-linked, respiration; n �5–6. E) EM reveals that mitochondria from Cav-3-KO, but not Cav-3-OE, mice have morphological abnormalities. F) ATPsynthesis in C. elegans caveolin mutants under state 3 conditions was suppressed; n � 5. G, H) EPR traces (G) and normalizedresults (H) show increased ROS generation in C. elegans caveolin mutants; n � 6. *P � 0.05.

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caveolin have greater resistance to TRAIL (Fig. 5F), morestable mitochondrial membrane potential (Fig. 5G), andincreased biogenesis of mitochondria (Fig. 5H, I). Inaddition, transient transfection of high-expressing, mito-chondria-localized caveolin HCT116 cells with a mito-CSD peptide vector (to compete for caveolin bindingpartners by disrupting mitochondria-specific caveolin sig-naling; Fig. 5J) enhances the sensitivity to TRAIL-inducedapoptosis (Fig. 5K). Such data suggest that the level ofmitochondrial expression of caveolin helps control cellu-lar adaptation to stress.

AAV9.IMM.Cav-3 increases cardiac mitochondriallocalization of Cav-3 and Ca2� tolerance ofmitochondria, improves mitochondrial respiration,and reduces infarct size

The results thus far show that caveolin is found inmitochondria, predominantly localizing in the IMM andimplicating a role for caveolin in stress adaptation bymaintenance of mitochondrial function. To test the hy-pothesis that a cellular stress-induced transfer of caveolinprotein from caveolae to mitochondria improves calcium

Figure 5. Mitochondria-localized and functional caveolin contribute to survival of colon cancer cells. A) Colon cancer cell lines canhave high (HCT116) or no (HT29) caveolin expression; purified mitochondria show caveolin enrichment in HCT116 cells. B)Caveolin localize to mitochondria (as observed by costaining with prohibitin) in HCT116 but not HT29 cells. C) Schematic ofmitochondria-targeted caveolin in HT29 cells (mCav-1 HT29, RFP served as control). D, E) Mitochondria from mCav-1 HT29 showenrichment of caveolin by immunoblot (D) and colocalization between caveolin and prohibitin (E). F, G) Mitochondria stable HT29cells were stressed with TRAIL and show reduced apoptosis (F) and more stable mitochondria membrane potential (G); n � 4. H,I) EM reveals increased mitochondrial density in representative (H) and quantified (I) samples of mCav-1 HT29 cells; n � 4. J) Amito-CSD peptide was designed to displace putative mitochondria-localized caveolin binding partners that confer cytoprotection. K)Transient transfection of HCT116 cells with mito-CSD enhances apoptosis in response to TRAIL treatment; n � 4. *P � 0.05.

4645CAVEOLIN AND MITOCHONDRIA IN STRESS

tolerance and respiration and reduces production of ROS,ultimately leading to cell protection, we engineered AAV9 toselectively express Cav-3 in the IMM (AAV9.IMM.Cav-3) byutilizing the mitochondrial targeting sequence for sub-unit VIII of cytochrome c oxidase. We tested this vector inan in vivo model of I/R injury. AAV9 provides efficientcardiac gene transfer compared to other serotypes (41);indeed, AAV9.IMM.Cav-3 showed cardiac-selective genetransfer (relative to most organs) 14 d after jugular veininjection (Fig. 6A). Cardiac mitochondria isolated fromAAV9.IMM.Cav-3-treated mice have increased mitochon-drial Cav-3 protein levels, indicating proper expressionand targeting (Fig. 6B). Mitochondria from mice treatedwith AAV9.IMM.Cav-3 withstand higher Ca2� concentra-tions and show delayed depolarization (Fig. 6C), resultsconsistent with delayed opening of the mPTP and stron-ger resistance to apoptosis. Mitochondria from those micehad increased oxygen consumption during all respiratory

states (Fig. 6D), indicating more efficient function ofcomplexes I, II, and IV.

Targeting Cav-3 to mitochondria thus improves mi-tochondrial function. To test whether such mitochon-dria have improved cardiac protection, we subjectedmice to 30 min ischemia and 2 h reperfusion in theabsence or presence of AAV9.IMM.Cav-3 gene transfer.We found that mitochondria-targeted Cav-3 reducesinfarct size (Fig. 6E). Direct targeting of Cav-3 tomitochondria thus mimics a preconditioning stimulusand induces stress adaptation in the heart.

DISCUSSION

These studies identify physical and functional associa-tions between caveolae/caveolin and mitochondriathat help cells adapt to stress via an evolutionarily

Figure 6. Treatment of mice with AAV9.IMM.Cav-3 increases cardiac mitochondrial Cav-3 expression and Ca2� tolerance, improvesmitochondrial respiration, and reduces infarct size 14 d after gene transfer. A) AAV9 engineered to express mitochondria-targetedCav-3 to the IMM (AAV9.IMM.Cav-3) was injected via the jugular vein, and organs were harvested 14 d later to assess viral copynumber (via TaqMan assay). The heart showed higher specific gene transfer with AAV9 relative to most other organs, although theliver showed the highest copy number. B) Isolated mitochondria from AAV9.IMM.Cav-3-treated mice have increased Cav-3 expression.C) Mitochondria from AAV9.IMM.Cav-3-treated mice have increased tolerance to calcium pulsing. D) Mitochondrial respiration,assessed using an Oxygraph, was improved in mitochondria from AAV9.IMM.Cav-3-treated mice relative to controls. E) Mice weresubjected to 30 min occlusion and 2 h reperfusion with and without gene transfer of AAV9.IMM.Cav-3. Infarct size was reduced bymitochondria-targeted Cav-3; n � 5. *P � 0.05. F) Model showing transfer of caveolin to mitochondria from caveolae and the resultantfacilitation of stress adaptation by preservation of mitochondrial function.

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conserved mechanism (Fig. 6F). Is there a precedentfor this observation? Shortly after Palade identifiedcaveolae (1), he published an image of a membranecaveola in close proximity to a mitochondrion (plate 14in ref. 42). A similar apposition of caveolae and mito-chondria is found in studies of multiple cell types, butsuch studies do not note this anatomic relationship(e.g., Fig. 5 in ref 43; Figs. 8, 18, and 32 in ref. 44; Figs.5 and 6 in ref. 45; Fig. 12 in ref. 46; Fig. 5 in ref. 47; andFigs. 1 and 2 in ref. 48). The current study thus links toobservations in the literature for over half a century that findcaveolae and mitochondria can be closely apposed.

Caveolins can localize in mitochondria (46), but thefunctional role of this localization has not been de-fined. The current studies identify such a functionalrole—modulation of mitochondrial function in theadaptation to cellular stress—and lead us to postulatethat the plasma membrane senses cellular stress and, inresponse, interacts with nearby mitochondria to facilitatecellular adaptation to maintain homeostasis. Optimalmitochondrial function may thus depend on interactionwith the plasma membrane. Caveolae-mitochondria com-munication may also provide a unifying molecular expla-nation for preconditioning, a widely studied phenome-non but whose mechanism is ill-defined; in addition,this communication may be relevant to the function ofcancer cells. Overall, our results underscore the impor-tance of mitochondria-localized caveolin as a conservedmechanism for adaptation to cellular stress.

Highly metabolic organs require an ongoing supplyof energy and oxygen. The high content of mitochon-dria in cardiac muscle provides this energy throughoxidative phosphorylation and aerobic respiration. Car-diac I/R damages mitochondria, reducing oxidativephosphorylation while promoting release of cyto-chrome c, production of ROS, and mitochondrial cal-cium overload (49). The latter two effects contribute tomPTP opening, which induces myocyte death. By pre-serving the function of mitochondria, one can decreasemyocardial injury during I/R (33, 34). Similar effectsoccur in other cell types, organ systems, and disease/injury states (50–53), but the mechanisms for mito-chondrial dysfunction or that protect them from injuryare poorly understood. The proximity of caveolae tosubsarcolemmal mitochondria may facilitate a linkagebetween stress sensors in the plasma membrane andprotective responses.

Loss of caveolins results in severe defects in varioustissues (12, 54, 55), including decreased response tostimuli that protect tissues from cellular stress states(12, 14, 21). By contrast, protective stimuli enhancecaveolae formation, and Cav-3-OE mice have a “pro-tected” phenotype (16). Conditions such as ischemia,in which a mismatch occurs between energy productionand utilization, are associated with decreased expres-sion of caveolae and/or redistribution of caveolin (56,57). However, no mechanism has linked caveolin/caveolae expression, oxidative damage, and mitochon-dria (58, 59). The current results identify the mainte-nance of mitochondrial function as a means whereby

caveolin promotes protection from I/R injury and showthat caveolin-mitochondria interaction also influencesapoptosis of cancer cells.

Our findings lead to two major questions that requirefurther study: the mechanism for translocation ofcaveolin to mitochondria; and how caveolin in mito-chondria enhance adaptation to cellular stress. Cav-3-OE mice have basal adaptation to stress and alsoenhanced inhibitory heterotrimeric G protein (Gi)signaling; in addition, interventions that activate Gisignaling (i.e., IPC) yield an increase in caveolae expres-sion (16). Moreover, pertussis toxin, which inhibits Gi,blocks adaptation to stress and decreases the localiza-tion of Cav-3 in mitochondria (unpublished results).Perhaps Gi signaling helps facilitate movement of Cav-3to mitochondria. Once present in mitochondria, caveo-lin appears to influence components of the electrontransport chain (changes oxygen consumption andROS generation; Figs. 3D, 4, and 6D) and of mitochon-drial membranes [alters mitochondrial membrane flu-idity (Fig. 3F) and loss of membrane potential inresponse to stress (Fig. 5G) and increases Ca2� toler-ance (Fig. 3B, C)]. Future studies will need to definehow caveolin influences mitochondrial components.

The numerous functions of caveolins have generallybeen thought to derive from their role in forming cave-olae, but evidence for the presence of caveolins in cellsthat lack caveolae contradict this notion (4). The translo-cation of caveolins from the plasma membrane to mito-chondria provides new evidence for localization andfunction of caveolins outside of the plasma membrane.Perhaps the caveolin content of mitochondria increaseswith cellular stress when mitochondria are vulnerable todamage. By loading mitochondria with caveolin, one canprotect cells (and mitochondria) from injury. Therapiesto alter mitochondria-localized caveolin may thus providenovel ways to influence cell death and survival.

This work was supported by grants from the AP GianniniFoundation (H.N.F.); U. S. National Institutes of Heathgrants HL091071 (H.H.P.), HL107200 (H.H.P. and D.M.R.),ARRA Supplement 3R01HL91071 (H.H.P.), GM066232(P.A.I.), HL081400 (D.M.R.), HL46345 (R.S.R.), HL088390(R.S.R.), HL103566 (R.S.R.), and HL105713 (R.C.B.); andVeterans Affairs merit grant BX000783 (D.M.R.). Authorcontributions: H.N.F., Y.K., D.M.R., P.A.I, and H.H.P plannedthe majority of experiments and wrote the paper; S.S.A.,M.W.K., M.Y.M., and A.L.M contributed to the design andinterpretation of EPR and mitochondrial assays and per-formed calcium swelling assays; M.P. and M.J performed invivo ischemia reperfusion and preconditioning experimentsand cell-based ROS assays; I.R.N. performed all electron andimmunoelectron microscopy; H.N.F., J.C.F., and S.K. createdpurified mitochondria and performed mitochondrial assays,membrane fluidity EPR studies, and C. elegans studies; Y.K.and I.R.N. performed all studies with HT29 and HCT116cells; Y.K., H.O., and A.M designed, generated, and per-formed experiments with the AAV vector; R.C.B., R.S.R., andP.M.P. contributed to the discussion; Q.C. and E.J.L. per-formed SSM and IFM experiments and helped revise drafts ofthe manuscripts; B.P.H, P.A.I, and H.H.P made the initialconnection and analysis of the association of caveolae andmitochondria.

4647CAVEOLIN AND MITOCHONDRIA IN STRESS

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Received for publication June 21, 2012.Accepted for publication July 24, 2012.

4649CAVEOLIN AND MITOCHONDRIA IN STRESS