HAX-1 regulates cyclophilin-D levels and mitochondriapermeability transition pore in the heartChi Keung Lam1, Wen Zhao1, Guan-Sheng Liu, Wen-Feng Cai, George Gardner, George Adly, and Evangelia G. Kranias2

Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575

Edited by Andrew R. Marks, Columbia University College of Physicians & Surgeons, New York, NY, and approved October 21, 2015 (received for reviewMay 5, 2015)

The major underpinning of massive cell death associated withmyocardial infarction involves opening of the mitochondrialpermeability transition pore (mPTP), resulting in disruption ofmitochondria membrane integrity and programmed necrosis.Studies in human lymphocytes suggested that the hemato-poietic-substrate-1 associated protein X-1 (HAX-1) is linked toregulation of mitochondrial membrane function, but its role incontrolling mPTP activity remains obscure. Herein we used modelswith altered HAX-1 expression levels in the heart and uncoveredan unexpected role of HAX-1 in regulation of mPTP and cardio-myocyte survival. Cardiac-specific HAX-1 overexpression was asso-ciated with resistance against loss of mitochondrial membranepotential, induced by oxidative stress, whereas HAX-1 heterozygousdeficiency exacerbated vulnerability. The protective effects of HAX-1were attributed to specific down-regulation of cyclophilin-D levelsleading to reduction in mPTP activation. Accordingly, cyclophilin-Dand mPTP were increased in heterozygous hearts, but geneticablation of cyclophilin-D in these hearts significantly alleviated theirsusceptibility to ischemia/reperfusion injury. Mechanistically, alter-ations in cyclophilin-D levels by HAX-1 were contributed by theubiquitin-proteosomal degradation pathway. HAX-1 overexpressionenhanced cyclophilin-D ubiquitination, whereas proteosomal in-hibition restored cyclophilin-D levels. The regulatory effects ofHAX-1 were mediated through interference of cyclophilin-D bindingto heat shock protein-90 (Hsp90) in mitochondria, rendering itsusceptible to degradation. Accordingly, enhanced Hsp90 expres-sion in HAX-1 overexpressing cardiomyocytes increased cyclophilin-Dlevels, as well as mPTP activation upon oxidative stress. Takentogether, our findings reveal the role of HAX-1 in regulatingcyclophilin-D levels via an Hsp90-dependent mechanism, resultingin protection against activation of mPTP and subsequent cell deathresponses.

HAX-1 | necrosis | mitochondrial permeability transition | cyclophilin-D |heat shock protein-90

The dynamics of contraction and relaxation in the heart arehighly regulated by a finely tuned cross-talk between demand

by the periphery and energy production by the mitochondria.The heart relies on mitochondria for fueling ATP on a beat-to-beat basis and for adjusting mitochondrial metabolism to meetthe increases in cardiac output during exercise and flight-or-fightresponses (1, 2). Mitochondria are also essential in controllingcell death through apoptosis, necrosis, and autophagy (3, 4). Indeed,disruption of mitochondrial integrity underlies cardiomyocyte deathassociated with myocardial infarction (5) that attributes to one ofevery six deaths in the United States (6).The mitochondrial hematopoietic-substrate-1 associated pro-

tein X-1 (HAX-1), a 35-kDa protein has recently emerged as acritical regulator of cell survival in various tissues (7–9). Indeed,global ablation of HAX-1 was shown to result in death by 14 wkof age due to neuronal degeneration from excessive cell death(10). In addition, a recent study demonstrated that degradationof HAX-1 triggers cell death in human B-cell lymphoma, furthersupporting the pivotal role of HAX-1 in dictating cell survival(11). Although HAX-1 has been studied in various cell types

since its discovery in 1997 (7), its presence in cardiomyocytes wasonly recently identified. Interestingly, cardiac HAX-1 localizesnot only to mitochondria but also the endo/sarcoplasmic re-ticulum (ER/SR) and it binds to phospholamban, increasinginhibition of the SR calcium transport ATPase and reducingSR calcium cycling. As cardiomyocyte contraction-relaxation iscoupled to calcium oscillation, HAX-1 is an important regulator ofcardiomyocyte contractility (12, 13). HAX-1 can also modulate ERstress responses by inhibiting the inositol requiring enzyme-1 (IRE-1) signaling arm, which diminishes cell death through reduction ofproapoptotic transcription factor expression and caspase activationin an ischemic insult, leading to improved contractile recoveryduring reperfusion (14). However, HAX-1 also localizes to mito-chondria in the heart (13), but its role in this organelle is unknown.Importantly, human mutations in HAX-1 have been linked to mi-tochondrial function. These mutations, resulting in loss of protein oractivity, were discovered in a subgroup of severe neutropenia pa-tients (8, 15, 16) and they were associated with loss of mitochondrialmembrane potential in the neutrophils of the human carriers (16).Thus, HAX-1 deficiency may cause a defect in the mitochondrialmembrane.One of the mechanisms that regulate mitochondrial mem-

brane integrity is the mitochondria permeability transition pore(mPTP), a large conductance channel that spans through theouter and inner membranes of mitochondria and becomes acti-vated upon oxidative stress, mitochondrial calcium overload, oralkaline intracellular environment. The opening of this poreduring an ischemia/reperfusion insult underlies the etiology of

Significance

The massive cell death, associated with a heart attack, is mainlydue to disruption of mitochondrial membrane integrity uponactivation of the mitochondrial permeability transition pore.Thus, it is important to understand how this pore is regulatedto prevent cardiac cell death. In this study, we reported thathematopoietic-substrate-1 associated protein X-1 (HAX-1) is aninhibitor of the pore and promotes cell survival. HAX-1 worksthrough recruitment of a chaperone protein called Hsp90 fromcyclophilin-D, a major component of the pore. Displacement ofHsp90 from cyclophilin-D promotes cyclophilin-D degradation,resulting in inhibition of pore opening and cell death. Given thatthe opening of the mitochondrial permeability transition porecontributes to various diseases, our findings have broaderapplications reaching beyond the heart.

Author contributions: C.K.L., W.Z., and E.G.K. designed research; C.K.L., W.Z., G.-S.L.,W.-F.C., G.G., and G.A. performed research; C.K.L., W.Z., and G.-S.L. contributed newreagents/analytic tools; C.K.L., W.Z., and G.-S.L. analyzed data; and C.K.L. and E.G.K.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1C.K.L. and W.Z. contributed equally to this work.2To whom correspondence should be addressed. Email: litsa.kranias@uc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1508760112/-/DCSupplemental.

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cardiac injury (5), because it allows solutes and ions that shouldbe excluded from mitochondria to infuse into their matrix. Con-comitantly, influx of water results in swelling of the originallycompacted cristae structure, leading to complete mitochondriamembrane failure and subsequent activation of massive necroticcell death and fibrosis (5, 17). This injury is irreversible, posinggreater damage to organs that possess limited regenerativecapacity, such as the heart (5). Although the mitochondrialpermeability transition pore was first described in 1976 (18), thefull identity of its protein components remains obscure. Severalconstituents have been proposed (17), but only cyclophilin-D(Cyp-D), a peptidyl-prolylcis-trans isomerase (PPI) in the mito-chondrial matrix, has been a genetically proven regulator ofmPTP (19). Ablation of this protein by gene targeting or phar-macological inhibition by cyclosporine A can effectively preventthe opening of the pore and inhibit the activation of cell deathduring ischemia/reperfusion injury (17, 19), demonstrating thecrucial role of Cyp-D function in cell survival. Furthermore, Cyp-Dactivity in cancer cells can be regulated by a mitochondrial chap-erone network that involves the cytosolic heat shock protein-90(Hsp90) and its mitochondria analogs, which are crucial in inducingcancer cell death (20).Based on our recent observations that Hsp90 is a newly iden-

tified binding partner of HAX-1 (14), it was intriguing to speculatethat the Hsp90/HAX-1 interaction may be also involved in reg-ulation of Cyp-D function in the heart. Thus, the current studywas designed to address this hypothesis by using mouse modelswith alterations in HAX-1 levels. Our findings indicate thatHAX-1 could specifically reduce Cyp-D levels by enhancing

its ubiquitination and proteosomal degradation, which, in turn,protected mitochondria from oxidative stress and calcium overload.These beneficial effects of HAX-1 were mediated through Hsp90,which appeared to stabilize Cyp-D protein in cardiomyocytes.Taken together, our results reveal a novel mechanism by whichthe HAX-1/Hsp90 complex may inhibit activation of the mPTPvia regulation of Cyp-D levels, and promote cardiomyocyte sur-vival upon oxidative stress and calcium overload in ischemia/reperfusion injury.

ResultsHAX-1 Preserves Mitochondria Membrane Integrity and Inhibits CellDeath. The integrity of the mitochondrial membrane is crucial inmaintaining cardiomyocyte function and survival. Increases inthe generation of reactive oxygen species or mitochondrial cal-cium overload during cardiac stress, such as ischemia/reperfusioninjury, can disrupt mitochondrial membrane integrity, dissipatemembrane potential for ATP production, and cause massivecell death (5). HAX-1 has recently emerged as a regulator ofcardioprotection, and we have shown that it localizes to mito-chondria besides ER/SR (13). To further address its localization,we isolated mitochondria (Fig. S1) and subjected them to pro-tease accession. HAX-1 was present in the inner mitochondrialmembrane (Fig. S2), consistent with previous reports (10, 21).Thus, we sought to investigate whether HAX-1 may regulatemitochondria membrane potential and control activation of celldeath. To examine this hypothesis, cardiomyocytes were isolatedfrom HAX-1 overexpressing (HAX-OE, 2.5-fold expression; Fig.1A) (13), wild-type (WT), and HAX-1 heterozygous knockout

Fig. 1. Overexpression of HAX-1 protects mitochondrial membrane integrity against oxidative stress and calcium overload, whereas heterozygous ablation ofHAX-1 has opposite effects. (A) HAX-1 protein expression in HAX-OE and HAX+/− hearts. CSQ was used as loading control. (B and C) IsolatedWT, HAX-OE, and HAX+/−

cardiomyocytes were loaded with mitochondrial membrane potential fluorescent dye, TMRE. Upon 2 mM H2O2 administration for 20 min, membrane potential wasdiminished in all groups, but this effect was most pronounced in the heterozygous cells. HAX-OE exhibited more than 80% preserved TMRE signals. n = 7 hearts forHAX-OE, 16 hearts for WT, and 9 hearts for HAX+/− (>8 cells per heart); *P < 0.05 vs. WT at 20 min. (D) HAX-1 overexpressing mitochondria demonstrated resistance toswelling induced by 50 μM calcium. n = 3 hearts for each group. (E and F) HAX-1 overexpression in cardiomyocytes prevented cell death due to loss of plasmamembrane integrity (E) and apoptosis (F) after 20 min of 2 mMH2O2 treatment, whereas HAX-1 heterozygous ablation increased cell death. n = 8 hearts for HAX-OE,12 hearts for WT, and 13 hearts for HAX+/− (>10,000 cells per heart); *P < 0.05 vs. WT at 2 mM H2O2. Data are expressed as mean ± SEM.

Lam et al. PNAS | Published online November 9, 2015 | E6467

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(HAX+/−, 40% protein expression; Fig. 1A) (13) mice, and theywere loaded with a mitochondrial localized florescent membranepotential indicator, TMRE, allowing us to monitor the mem-brane potential before and after hydrogen peroxide challenge,using confocal microscopy. Under basal conditions, the per-centage of TMRE-positive cardiomyocytes was similar amongthe three groups (Fig. 1 B and C). However, after administrationof 2 mM hydrogen peroxide to induce oxidative challenge, bothWT and HAX heterozygous cardiomyocytes lost some flo-rescence, and by 20 min, only 30% of the heterozygous cellsmaintained the TMRE signal, compared with 50% of WT.In contrast, more than 70% of the HAX-overexpressing car-diomyocytes were resistant to 20 min of oxidative challenge(Fig. 1 B and C). These findings suggest a protective role ofHAX-1 in maintaining mitochondrial membrane potential duringoxidative stress.The protective effects of HAX-1 on mitochondrial integrity

were also evidenced upon calcium overload, a situation that in-duces the opening of the permeability transition pore (mPTP),leading to failure of osmotic pressure control, water influx, andswelling of mitochondria (17). Isolated WT cardiac mitochondriademonstrated a steady loss of light scattered by the mitochon-drial membrane structure after the addition of 50 mM calcium.However, HAX-1 overexpressing mitochondria were resistantunder the same conditions and showed no sign of swelling (Fig.1D). In contrast, isolated HAX-1 heterozygous-deficient mito-chondria exhibited significant swelling under basal conditions.These findings together with the results from the hydrogenperoxide treatment indicate that HAX-1 may protect the mito-chondrial membrane integrity.

To determine whether the protection of mitochondrial mem-brane by HAX-1 would translate to improved cell survival, we usedisolated cardiomyocytes and assessed the degree of propidiumiodide (PI) inclusion as an indicator of necrotic cell death.Treatment of cells with either 1 or 2 mM hydrogen peroxide for20 min resulted in increases in Propidium iodide-positive cells.Notably, 2 mM hydrogen peroxide, which was associated withloss of mitochondria membrane integrity (Fig. 1C), resulted in a15% increase in PI-positive WT cardiomyocytes, but this in-crease was significantly blunted in HAX-1 overexpressing cells.However, heterozygous loss of HAX-1 exacerbated the increasein cells with propidium iodide inclusion (Fig. 1E), consistent withthe findings in the TMRE experiments. Similar results wereobtained, when the expression of cell surface annexin V wasquantified, as a marker of apoptotic cell death. Increasing HAX-1levels effectively reduced apoptosis, whereas loss of HAX-1 ele-vated its activation under hydrogen peroxide stress (Fig. 1F). Thus,our data suggest that HAX-1 maintains the mitochondrial mem-brane potential and protects membrane integrity, resulting in re-duced apoptosis and necrotic cardiomyocyte death.

HAX-1 Regulates the Opening of Mitochondrial Permeability TransitionPore. It has been demonstrated that mitochondrial membrane in-tegrity highly depends on the function of mPTP. The opening ofthis pore is a key mediator of cardiac ischemia/reperfusion injurybecause it facilitates loss of mitochondrial membrane potential,induces swelling, and leads to the rupture of mitochondrialmembrane and activation of cell death (5). To determine whetherthe HAX-1 regulatory effects in mitochondria are mediatedthrough an mPTP-dependent mechanism, we assessed the effectsof mPTP inhibition in cardiomyocytes. To this end, we treated

Fig. 2. HAX-1 regulates the mitochondrial permeability transition pore. (A and B) Cyclosporine A abrogated the detrimental effect of HAX-1 heterozygous ablationonmitochondrial membrane potential after H2O2 treatment for 20 min. n = 4–8 hearts (>12 cells per heart); *P < 0.05 vs. WTwith vehicle (DMSO) at 20 min; #P < 0.05vs. HAX+/−with vehicle at 20 min. (C) Swelling induced by high calcium concentration in HAX-OE and WT mitochondria. n = 3 hearts for each group. (D) Inhibition ofmitochondrial swelling required a lower cyclosporine A dose in HAX-1 overexpressing mitochondria. n = 3 hearts for each group. (E and F) Cyclosporine A abrogatedthe effect of HAX-1 on necrotic (E) and apoptotic (F) cell death. n = 8–15 hearts (>10,000 cells per heart); *P < 0.05 vs. WT at 2 mM H2O2; #P < 0.05 vs. the respectiveuntreated group at 2 mM H2O2. Data are expressed as mean ± SEM.

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cardiomyocytes with cyclosporine A (CsA), an mPTP inhibitor(17). CsA resulted in preservation of mitochondrial membranepotential in both WT and HAX-1 heterozygous cardiomyocytesexposed to hydrogen peroxide (Fig. 2 A and B). Importantly, thelevels of TMRE in these treated groups were similar to those inHAX-1 overexpressing cells (Fig. 1B), suggesting that the pro-tective effects of HAX-1 may be mediated through the mPTP. Tofurther confirm that the observed protection against loss of mi-tochondrial membrane potential by HAX-1 overexpression wasnot due to impaired function of the mPTP, we used a high cal-cium concentration (375 mM) in the swelling assay to inducemPTP opening in isolated mitochondria. Under these conditions,the swelling response of the HAX-1 overexpressing mitochondriawas similar to that of WTs (Fig. 2C), suggesting that HAX-1regulates the sensitivity of the mPTP opening. To further addressthis hypothesis, we determined the dose of cyclosporine A re-quired for complete inhibition of mitochondrial swelling, inducedby 375 mM calcium. Both WT and HAX-1 overexpressing mito-chondria were resistant to swelling in the presence of 1 μM cy-closporine A. However, upon dose reduction to 0.5 μM, WTmitochondria exhibited swelling in a time-dependent manner,whereas HAX-1 overexpressing mitochondria maintained theirresistance (Fig. 2D). These findings further suggest that over-expression of HAX-1 reduces the sensitivity of mPTP openingin cardiomyocytes.We also applied cyclosporine A before hydrogen peroxide

challenge and assessed the degree of cell death activation inisolated cardiomyocytes to confirm that the prosurvival effects ofHAX-1 under the hydrogen peroxide treatment depended on theopening of mPTP. Indeed, this pretreatment completely abol-ished the differences between the three groups regarding thenumber of cells with reduced plasma membrane integrity (Fig.2E). Accordingly, apoptotic cell death was also reduced in WTand HAX-1 heterozygous cardiomyocytes (Fig. 2F) by CsA in-hibition of mPTP, and the values in the three groups were sim-ilar. Thus, regulation of mPTP activity appears to play a key rolein the cell survival mechanisms mediated by HAX-1.

HAX-1 Regulates Cardiac Cyclophilin-D Protein Levels. Because cy-closporine A is a direct pharmacological inhibitor of cyclophilin-D (17), an important regulator of the permeability transitionpore, we hypothesized that the reduced dose of CsA required forcomplete blockade of swelling in HAX-1 overexpressing mito-chondria may be associated with decreases in Cyp-D level oractivity. Indeed, quantitative immunoblotting indicated that theCyp-D levels in HAX-1 overexpressing hearts were reduced by40% compared with WTs (Fig. 3 A and B). Interestingly, theeffects of HAX-1 appeared specific for Cyp-D, because the ex-pression levels of other proposed mPTP components, namely thevoltage-dependent anion channel (VDAC) and adenine nucle-otide translocase (ANT1), were not altered (Fig. 3A). Further-more, the expression levels of Bax and Bak, which were recentlysuggested to govern the porosity of the mitochondrial outermembrane (22), were not altered by HAX-1 overexpression ei-ther. These findings in cardiac homogenates were also similar tothose obtained in isolated mitochondria (Fig. 3 C and D). Thus,we speculated that if HAX-1 can negatively regulate Cyp-D ex-pression, then loss of HAX-1 should have a positive effect onCyp-D levels. Indeed, heterozygous-deficient hearts with 60%loss of HAX-1 (13) exhibited a 70% increase in Cyp-D levels(Fig. 3 E and F). Similar findings were observed with isolatedmitochondria (Fig. 3 G and H). There were no alterations in thelevels of VDAC, ANT1, Bax, and Bak (Fig. 3 E and G), similarto HAX-1 overexpression. Thus, the protective effect of HAX-1on mPTP opening may be attributed to the reduction of Cyp-Dexpression in the mitochondria.To exclude the possibility that reduction of Cyp-D level was

an indirect compensatory response to chronic HAX-1 over-expression in vivo, we used adenoviral delivery to acutely in-crease HAX-1 levels in cardiomyocytes. At 24 h after infection,HAX-1 overexpression (15-fold increase in HAX-1 levels) wasassociated with a 40% decrease in Cyp-D levels (Fig. S3 A andB). Accordingly, acute down-regulation of HAX-1 by 55%, usingan antisense approach, led to a 60% increase in Cyp-D expres-sion levels in cardiomyocytes (Fig. S3 C and D). The acute al-terations in Cyp-D levels reflected parallel changes in the TMREresponses upon hydrogen peroxide challenge. Cardiomyocytes

Fig. 3. HAX-1 specifically alters cyclophilin-D levels in the heart. (A and B) Cyp-D levels were reduced in HAX-OE cardiac homogenates. n = 4 hearts for eachgroup. (C and D) Cyp-D levels were reduced in HAX-OE cardiac mitochondrial fraction. n = 5 hearts for HAX-OE, 6 hearts for WT. (E and F) Cyp-D levels wereincreased in HAX +/− cardiac homogenates. n = 6 hearts for each group. (G and H) Cyp-D levels were increased in the HAX+/− mitochondrial fraction. n = 9hearts for each group. *P < 0.05 vs. WT. Data are expressed as mean ± SEM.

Lam et al. PNAS | Published online November 9, 2015 | E6469

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with increased HAX-1 levels showed enhanced resistance to lossof mitochondria membrane potential, whereas down-regulationof HAX-1 led to increased dissipation of proton gradient (Fig. S3E and F). These findings coupled with the ones above in animalmodels suggest that HAX-1 may be a regulator of cardiac Cyp-Dexpression levels.To further delineate the contribution of reduced Cyp-D levels

in the protective effects of HAX-1 on mPTP opening andmembrane potential, we used two complementary approachesand examined the effects of altered Cyp-D expression on theTMRE responses upon hydrogen peroxide challenge. First, weinfected adult mouse WT and HAX-1 overexpressing car-diomyocytes with an adenovirus-encoding Cyp-D cDNA. In-creased Cyp-D protein levels to a similar extent between WT andtransgenic cells (Fig. 4A) effectively abolished the protectiveeffects of HAX-1 overexpression on mPTP opening (Fig. 4 Band C). Second, we isolated cardiomyocytes from the Cyp-Dknockout (or PPIF null) mouse and infected them with adeno-viruses carrying HAX-1 sense or antisense cDNA (Ad.HAX-1 orAd.HAX-AS) to alter HAX-1 expression levels (Fig. 4D). In theabsence of Cyp-D, the alterations in HAX-1 levels did not affect theTMRE response to hydrogen peroxide challenge (Fig. 4 E and F).Collectively, the regulatory effects of HAX-1 on mPTP sensitivityappear to be mediated through alterations of Cyp-D levels.

We previously reported that reduction in HAX-1 levels havedetrimental effects in the heart, when subjected to ischemia/reperfusion injury. Because mPTP has also been shown to playan important role in this injury, we examined whether the effectsof HAX-1 may be mediated through Cyp-D. To this end, wecrossed the Cyp-D knockout mouse with the HAX+/− model(CypD-KO/HAX+/−) and obtained HAX-1 heterozygous micethat were also deficient in Cyp-D. Hearts from the WT, HAX+/−,CypD-KO, and the cross model were then subjected to global no-flow ischemia (40 min), followed by reperfusion (60 min) in theLangendorff perfusion mode. This protocol induced a 26% in-farct size in WT hearts (Fig. 4G and H), consistent with previousstudies (14). Heterozygous loss of HAX-1 increased myocardialinfarction to 40%, whereas ablation of Cyp-D significantly di-minished it to 11% (Fig. 4 G and H). Importantly, ablation ofCyp-D in HAX+/− hearts resulted in significant reduction of in-farct size from 40 to 18% (Fig. 4G and H), suggesting that mPTPplays a critical role in the protective effects of HAX-1 in cardiacischemia/reperfusion injury.

HAX-1 Regulates Cyclophilin-D Levels Through Hsp90. To determinewhether the effects of HAX-1 were mediated at the transcriptionallevel, we assessed cyclophilin-D (or ppif) mRNA expression in WT,HAX-OE, and HAX+/− cardiac homogenates (Fig. 5A). There were

Fig. 4. The effects of HAX-1 are mainly mediated by cyclophilin-D. (A) Cyp-D expression was increased to similar levels in WT and HAX-OE cells after adenoviralinfection of adult rat cardiomyocytes. (B and C) Adenoviral delivery of Cyp-D removed the protection mediated by HAX-1 overexpression upon 2 mM hydrogenperoxide administration. CSQ was used as a loading control. n = 4–6 hearts (>12 cells per heart); *P < 0.05 vs. WT GFP at 2 min; #P < 0.05 vs. HAX-OE GFP at 20 min.(D) HAX-1 levels after adenoviral delivery of Ad.GFP, Ad.HAX-1, and Ad.HAX-AS in CypD-KO cells. WT cardiac homogenate was loaded to serve as input. (E and F)Cyp-D ablation abolished the effect of different HAX-1 levels on the maintenance of mitochondrial membrane potential upon 2 mM hydrogen peroxide chal-lenge. CSQ was used as a loading control. n = 4–5 hearts (>16 cells per heart). (G and H) Cyp-D ablation significantly reduced infarct size in HAX-1 heterozygousknockout hearts. n = 4 for each group; *P < 0.05 vs. WT; #P < 0.05 vs. HAX+/−; $P < 0.05 vs. CypD-KO. Data are expressed as mean ± SEM.

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no alterations among the three groups, indicating that HAX-1does not regulate Cyp-D transcription. We then hypothesized thatHAX-1 modulates the Cyp-D protein levels specifically through itsdegradation by the proteosomal pathway. To test this hypothesis, weacutely increased HAX-1 expression in adult rat cardiomyocytesby adenoviral delivery (Ad.HAX-1) and subsequently treated thecells with bortezomib, the well-recognized proteosomal inhibitorin cancer cells (23, 24). HAX-1 overexpression decreased Cyp-Dlevels as expected, but the effect was abolished by bortezomib,suggesting that HAX-1 regulates proteosomal degradation of Cyp-D(Fig. 5B). Because protein degradation is known to be controlled byubiquitination (25), we sought to examine whether HAX-1 mayaffect Cyp-D ubiquitination. Thus, HAX-OE or HAX+/− cardiachomogenates were subjected to immunoprecipitation studies,using either ubiquitin or Cyp-D as a bait. Indeed, HAX-1overexpression markedly increased Cyp-D ubiquitination (Fig. 5C and D), whereas heterozygous ablation of HAX-1 resulted inless ubiquitinated Cyp-D, compared with WTs (Fig. 5 E and F).These findings indicate that HAX-1 regulates the degrada-tion of Cyp-D protein through the ubiquitination-proteosomalpathway.Previous studies in mitochondria isolated from various cell

types have shown that Cyp-D interacts with the heat shockprotein 90 (Hsp90) in the matrix (20). Interestingly, we also re-cently found that Hsp90 is a binding partner of HAX-1 in theheart (14). These findings prompted us to investigate whetherthe observed effects of HAX-1 on Cyp-D levels may involveHsp90. Because the role of Hsp90 on Cyp-D is not known, weacutely overexpressed Hsp90 in adult cardiomyocytes and de-termined the expressions of Cyp-D. We observed a significantincrease in Cyp-D protein levels (Fig. S4) and reduction in itsubiquitination (Fig. S5), suggesting that Hsp90 may serve as achaperone for Cyp-D and prevent its degradation. This notion

was further supported by immunoprecipitation studies in cardiachomogenates from HAX-1 heterozygous-deficient hearts, whichexhibit increases in Cyp-D levels. Using Cyp-D as bait, there wasan increase in the amount of Hsp90 associated with Cyp-D,compared with WTs (Fig. 6A). A similar increase in the Cyp-Dlevels was observed when Hsp90 was used as bait (Fig. 6B). Onthe contrary, overexpression of HAX-1 resulted in reducedHsp90/Cyp-D association, using either Hsp90 or Cyp-D as bait inthe cardiac homogenates (Fig. 6 C and D). These alterationsappear to occur in mitochondria, because similar findings wereobserved when isolated mitochondrial fractions from HAX-OEand HAX+/− hearts were used for immunoprecipitation experi-ments (Figs. S6 and S7). Notably, the changes in Hsp90/Cyp-Dcomplex formation were not associated with alterations in car-diac or mitochondrial Hsp90 levels in either HAX-1 over-expressing or heterozygous-deficient hearts (Figs. S8 and S9).Taken together, these findings suggest that HAX-1 may directlydisplace Cyp-D from binding to Hsp90 in the mitochondria.To urther examine whether HAX-1 physically hinders theinteraction of Cyp-D with Hsp90, we performed an in vitrocompetitive binding ELISA, using recombinant proteins. Weobserved a progressive reduction of the Cyp-D/Hsp90 binding inthe presence of increasing HAX-1 protein levels (Fig. 6E). Thus,increases in HAX-1 appear to reduce the levels of mitochon-drial Hsp90 that bind to Cyp-D, leading to degradation of Cyp-D.To further test whether the effect of HAX-1 overexpression on

decreasing the Cyp-D levels is mediated through reduction of theHsp90/Cyp-D association, we acutely overexpressed Hsp90 torestore complex formation. Indeed, Hsp90 overexpression in thesetting of HAX-1 overexpression resulted in markedly enhancedCyp-D levels (Fig. 6F), abolishing the protective effects ofHAX-1 and exacerbating loss of membrane potential uponhydrogen peroxide treatment (Fig. 6 G and H). These data

Fig. 5. HAX-1 enhances the degradation of cyclophilin-D. (A) Cyp-D mRNA transcript, or ppif levels were similar in WT, HAX-OE, and HAX+/− hearts. n = 4 heartsfor WT, and 5 hearts for each of the HAX-OE and HAX+/−. (B) Proteasomal inhibitor, Bortezomib, restored Cyp-D levels in HAX-1 overexpressing cells. Glycer-aldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. n = 4 hearts for each group; *P < 0.05 vs. Ad.GFP. (C and D) Ubiquitination of Cyp-Dwas enhanced in HAX-OE hearts. Three independent experiments were performed. n = 4 hearts for each group. (E and F) Ubiquitination of Cyp-D was reduced inHAX+/− hearts. Three independent experiments were performed. n = 4 hearts for each group. Data are expressed as mean ± SEM.

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provide further evidence that the cardioprotective effects ofHAX-1 are mediated through regulation of mPTP function andspecifically through recruitment of Hsp90 from the Hsp90/Cyp-Dcomplex, contributing to degradation of Cyp-D protein and in-creased survival.

DiscussionIn this study, we identified HAX-1 as a regulator of mPTP andmitochondrial membrane integrity, impacting cell survival in theheart. Increases in HAX-1 expression protected mitochondrialmembrane from damage associated with oxidative stress andcalcium overload, whereas decreases in HAX-1 levels exacer-bated loss of membrane integrity. The underlying mechanismsinvolved the ability of HAX-1 to sequester Hsp90 from Cyp-D,which then rendered Cyp-D prone to ubiquitination and degra-dation. Thus, HAX-1 inhibits the opening of mPTP and preventsloss of membrane integrity and cell death activation during oxi-dative stress or mitochondrial calcium overload. These findings,coupled with our recent observations, indicating that HAX-1 cansuppress ER stress-induced apoptosis (14), reveal a previouslyunidentified paradigm of cell death regulation by HAX-1 in twodifferent cellular organelles.Growing evidence demonstrates an axiomatic prosurvival role

of HAX-1, and human mutations of this protein have beenshown to associate with diseases characterized of altered cellviability, such as congenital neutropenia (15), neurodevelopmentalretardation (26), and mantle cell lymphoma (11). Although HAX-1has been known to predominantly localize to mitochondria invarious tissues and cell types (8), and mitochondria are knownfor their gate-keeping role in cell death activation, it is surprising

that there are only a few studies addressing the function of HAX-1in regulation of cell death by this organelle. One study reportedthat mitochondrial HAX-1 interacts with the high temperaturerequirement protein-2 (HtrA2) and abrogation of this complexby HAX-1 ablation in mice contributes to Bax accumulation,neurodegeneration, and a short lifespan (14 wk). However,ablation of Bax in HAX-1–deficient mice could only prolongsurvival by 9 wk without fully rescuing the phenotype (10),suggesting that the HtrA2/Bax regulation is not the sole anti-apoptotic mechanism mediated by HAX-1. Our findings hereinsuggest that mitochondrial localized HAX-1 has a direct pro-tective effect on the membrane integrity of this subcellular or-ganelle by regulating the mPTP opening.We previously showed that cardiac ischemia/reperfusion injury

is associated with loss of HAX-1 and overexpression of thisprotein conferred protection by reducing infarct size, limitingtroponin release and inhibiting DNA fragmentation. On thecontrary, heterozygous loss of HAX-1 led to exacerbated cardiacinjury (14). Particularly, leakage of intracellular content, such astroponin proteins, is a sign of plasma membrane rupture andnecrotic cell death (27, 28), which is mainly caused by failure tomaintain plasma membrane potential due to loss of ATP supply(3, 4). The diminished energy production reflects compromisedmitochondrial function associated with mPTP opening duringischemia/reperfusion injury (5). Although we previously showedthat HAX-1 inhibits apoptosis mediated by the ER stress re-sponse, this effect would not account for the reduction in cardiactroponin I release during ischemia/reperfusion injury. Thus, thecurrent identification of HAX-1 as a regulator of Cyp-D levelsand an inhibitor of mPTP activation provides additional insights

Fig. 6. HAX-1 displaces Hsp90 from cyclophilin-D, rendering it susceptible to degradation. (A and B) Immunoprecipitations using Cyp-D (A) or Hsp90 (B) as a bait,demonstrated that the Cyp-D/Hsp90 association was enhanced in HAX+/− hearts. WT cardiac homogenate served as input. (C and D) Immunoprecipitations, usingCyp-D (C) or Hsp90 (D) as a bait, demonstrated that the Cyp-D/Hsp90 association was reduced in HAX-OE hearts. WT cardiac homogenate served as input. (E) TheCyp-D/Hsp90 interaction was examined in the presence of increasing levels of HAX-1, using recombinant proteins. ELISA signals were calculated after subtractionof the mean value of controls, represented by GST-coated wells (n = 25 wells). (F) Increases in Hsp90 levels by adenoviral infection of cultured adult WT andHAX-OE cardiomyocytes enhanced Cyp-D protein expression. CSQ was used as a loading control. (G and H) Hsp90 overexpression exacerbated the loss of mi-tochondrial membrane potential in both WT and HAX-OE cells. n = 4–7 hearts (>10 cells per heart); *P < 0.05 vs. WT GFP at 20 min; #P < 0.05 vs. HAX-OE GFP at20 min. Data are expressed as mean ± SEM.

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to its cardioprotective mechanisms. The role of Cyp-D was furtherelucidated by generation of a cross model with ablation of thisprotein in the HAX-1 heterozygous the deficient hearts, whichwere characterized by increased levels of this protein and mPTPactivity. The cross model exhibited diminished myocardial in-farction, although its infarct size was not reduced to the level ofCyp-D knockout hearts, suggesting that inhibition of bothmPTP and ER stress contributes to the cardioprotective effectsof HAX-1. Given that myocardial infarction is characterizedby a continuum of apoptosis and necrosis, our data demon-strate that HAX-1 orchestrates a dual protective mechanism byinhibiting a mitochondrial-based necrotic activation, in addi-tion to the previously reported ER-based apoptotic cell deathprogram.The activation of mitochondrial permeability transition pore

has been shown to be a main driver in the development ofmyocardial infarction upon ischemia/reperfusion injury (5). Al-though the genetic identity of the pore is unknown, it has beensuggested to consist of two complexes located on the outer andinner mitochondrial membrane. Although it was originally in-dicated that VDAC constitutes the outer membrane portion, arecent report suggested that VDAC is dispensable in mPTP ac-tivation (29) and outer membrane porosity is induced by theoligomerization of Bax/Bak (22). Furthermore, HAX-1 homo-zygous ablation was shown to facilitate Bax accumulation andcause neurodegeneration (10). However, in both HAX-1 over-expressing and heterozygous-deficient hearts, the levels of Baxand Bak were not altered in either the homogenates or mito-chondrial fractions, suggesting that the alterations of mPTP inour models were independent of Bax/Bak accumulation.The only genetically proven regulator of the mPTP is Cyp-D,

which is located within the matrix of the mitochondria. Eitherablation or pharmacological inhibition of this protein results insignificant reduction of infarct size after ischemia/reperfusioninjury (17, 19). However, increasing the expression of Cyp-Dresults in enhanced mPTP activity and mitochondrial swelling(19, 30). Thus, regulation of Cyp-D expression level becomes ofparamount importance in the control of cell survival. To this end,our study presents the first evidence to our knowledge that Cyp-Dlevels can be posttranslationally regulated by HAX-1 and thechaperone protein Hsp90. Chaperone proteins have been wellrecognized to work coordinately with cochaperones and regulateprotein turnover, directing unfolded or unwanted proteins to theubiquitination/proteosomal degradation pathway. We previouslydemonstrated that HAX-1 works with Hsp90 to control the ac-tivity of IRE-1, one of the ER stress response signaling compo-nents, suggesting that HAX-1 may function as a cochaperoneprotein for Hsp90 (14). This chaperone/cochaperone regulationappears to exist also in mitochondria, controlling the ubiquiti-nation and degradation of Cyp-D. Increases in HAX-1 expres-sion can sequester Hsp90 from the Cyp-D/Hsp90 complex,promoting Cyp-D ubiquitination and degradation. In turn, thereduced Cyp-D levels confer resistance to mPTP opening andsubsequent membrane potential loss upon stress stimuli. Thesefindings are consistent with previous observations in breast car-cinoma, colon cancer, and HEK293 cell lines, which showed thatcleavage of HAX-1 by granzyme B leads to loss of mitochondrialmembrane potential, and this loss can be prevented by intro-duction of Cyp-D siRNA (31).Recently, several breakthroughs have been made regarding

regulation of Cyp-D function and necrotic cell death. In tumorcells, it was reported that the Cyp-D activity is inhibited by a mi-tochondrial Hsp90 chaperone network that contains cytosolicHsp90, a mitochondrial Hsp90 analog, the tumor necrosis factorreceptor associated protein-1 (TRAP-1), and several cochaperoneproteins. Inclusion of a mitochondrial-directed inhibitor, tar-geting the ATPase activity of both Hsp90 analogs, was able toincrease the opening of mPTP upon oxidative stress (20). Thus, it

was proposed that both Hsp90 and TRAP-1 serve to antagonizeCyp-D function. Based on this hypothesis, it is expected thatdisplacement of Hsp90 from Cyp-D by HAX-1 overexpressionwould increase Cyp-D activity and promote mPTP opening.However, we did not observe a reduction of mPTP activa-tion when HAX-1 was overexpressed. Moreover, we foundthat overexpression of Hsp90 in either WT or HAX-OE car-diomyocytes resulted in increased Cyp-D expression (Fig. 6F)and exacerbated loss of mitochondrial membrane potential afterhydrogen peroxide challenge (Fig. 6 G and H). These resultssuggest that Hsp90 promotes Cyp-D function in cardiac cells.The apparent discrepancy between our current results and pre-vious reports in tumor cell lines may be due to the high de-pendency of Hsp90 function on the availability and combinationof its cochaperone proteins, which may differ between cell types.It is likely that tumor cells have a remodeled mitochondrialHsp90 chaperone network to enhance their survival and pro-liferation. In support of this notion, we failed to observe anysignificant association between HAX-1/TRAP-1 (Fig. S10),suggesting that the Hsp90 interaction with HAX-1 in cardiaccells may not be associated with TRAP-1. Indeed, mitochon-drial targeted Hsp90 inhibitors have been shown to exhibitexcellent specificity toward tumor cells, but not normal cells (20, 32).These reports support our results and point to a complex regu-lation of Hsp90 function by its cochaperones and the intracellularenvironment.Our findings on the newly discovered function of HAX-1 in

controlling Cyp-D levels and activation of the mitochondrialpermeability transition pore are not only critical in heart functionand survival, but also provide significant insights in other diseases,such as immunodeficiency, tumorgenesis, and neurodegeneration.Studies in patients with HAX-1 mutation, exhibiting severe con-genital neutropenia, showed that their neutrophils had low mito-chondrial membrane potential compared with the healthy controls(16). However, elevated HAX-1 levels are associated with varioustypes of cancer (8), where the mPTP activity can be inhibited forprosurvival purposes (33).In summary, this is the first study to our knowledge to dem-

onstrate that HAX-1 and specifically the HAX-1/Hsp90 complexpromote Cyp-D ubiquitination and degradation. This regulatorymechanism confers resistance to mPTP opening and preventscell death against oxidative stress and mitochondrial calciumoverload (Fig. S11). Thus, HAX-1 and Hsp90, which are ubiq-uitously expressed, serve as common nodal points in bothapoptotic and necrotic cell death. Because mPTP opening isimplicated in the pathogenesis of various diseases, our findingssuggest that the HAX-1/Hsp90 complex may be potentially exploitedas a feasible therapeutic target.

Materials and MethodsAnimal Models. HAX-1 transgenic (HAX-OE, FVB/N) (13), HAX-1 knockoutheterozygous (HAX+/−, FVB/N) (13), Cyp-D knockout (B6129s) (19), the crossof CypD-KO/HAX+/− (B6129s) mice, and their wild-type littermates were usedin this study. To avoid the complication of gender differences, only malemice at 3 mo of age were used for these studies, according to the NationalInstitutes of Health Publication No. 8523: Guide for the Care and Use ofLaboratory Animals (34).

Mouse Cardiomyocyte Isolation and Virus Infection. Mouse myocytes from WTand HAX-1 OE hearts were isolated as described (14). Briefly, adult mousehearts were excised following mouse anesthesia with sodium pentobarbital(70 mg/kg, i.p.) and cannulated on a Langendorff system. Ca-free Tyrodesolution (113 nmol/L NaCl, 4.7 mmol/L KCl, 0.6 mmol/L KH2PO4, 0.6 mmol/LNa2HPO4, 1.2 mmol/L MgSO4·7H2O, 12 mmol/L NaHCO3, 10 mmol/L KHCO3,5 mmol/L Hepes, 3 0 mmol/L taurine, 10 mmol/L 2,3-butanedione monoxime,and 5.5 mmol/L glucose, pH 7.4) was used to perfuse the heart for 3 min at37 °C. The perfusion was then switched to a digestion solution containing0.25 g/L liberase blendzyme (Roche). Following digestion, the left ventriculartissue was excised, minced, and dissociated into a cell suspension. Calcium

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was serially added to the cellular suspension until final calcium concentra-tion in the Tyrode solution was 1.2 mmol/L. Cardiomyocytes were pelleted bygravity, resuspended in plating media [DMEMwith 5% (vol/vol) FBS, 10 mmol/L2,3-butanedione monoxime, 100 units/mL penicillin streptomycin and 2 mLof L-glutamine] and placed on a six-well plate containing laminin-coatedcoverslips for 1 h. Supplementing the media with 5% (vol/vol) FBS allows op-timal cardiomyocyte attachment. Once the cells attached to the coverslip, themedium was replaced with infection medium (plating medium containing noFBS). Adenoviruses (Ad.GFP, Ad.HAX-1, Ad.HAX-AS, Ad.Cyp-D, and Ad.Hsp90)were applied at 500 multiplicity of infection (MOI). After 3 h of incubation at37 °C, medium was removed and replaced with culture medium, which isDMEM containing 5 mg/L ITS (bovine insulin, human transferrin and sodiumselenite; Sigma), 100 units/mL penicillin streptomycin, 2 mmol/L L-glutamine,4 mmol/L NaHCO3, 10 mmol/L Hepes, 0.2% BSA, and 25 μmol/L blebbistatin(Cayman Chemical). The cell phenotype and morphology remained similaramong noninfected and adenoviral-infected groups after 24 h of infection.

Mitochondrial Membrane Potential. At 24 h after adenoviral infection, mousecardiomyocytes were stained with TMRE dye (eBioscience) for 15 min at roomtemperature. Fluorescence signals from TMRE excited at 560 nm were col-lected at emission of 610 nm, using live-cell confocal imaging. To inducemitochondrial membrane potential transition, stainedmouse cardiomyocyteswere treated with 2 mM hydrogen peroxide, in the absence or presence ofcyclosporin A (Sigma). The TMRE signal was recorded every minute for acourse of 20 min to calculate the percentage of cells with preserved mito-chondrial membrane potential before and after oxidative challenge. Therewas no significant loss of mitochondrial membrane observed until the 15th–18th minute of the treatment, suggesting that primary cardiomyocytes areresistant to cell death initiation.

Mitochondria Isolation and Swelling.Mouse heart mitochondria were isolatedby homogenization followed by different centrifugation. Briefly, mousehearts were homogenized in Precellys 24 Lysis and homogenization machineat 5,000 cpm for 10 s with 750 μL of 2.3-nm Zirconia/silica beads and an equalamount of homogenization buffer containing 250 mM sucrose, 10 mM Trisat pH 7.4, and 1 mM EDTA. The homogenates were spun at 1,300 × g for10 min at 4 °C to pellet nuclei and cell debris. The supernatant was then spunat 12,000 × g for 30 min at 4 °C to pellet the mitochondria. After washingtwice in homogenization buffer (minus EDTA), the mitochondria wereresuspended in buffer containing 120 mM KCl, 10 mM Tris at pH 7.4, and5 mM KH2PO4 for the swelling assay. Mitochondrial swelling was induced by50 or 375 mM, where the light-scattering of 250 μg of mitochondria in a 1-mLvolume was measured at 540 nm for 10 min.

Propidium Iodide and Annexin V Assays. Cells were gently washed once andstained with annexin V-specific APC dye (eBioscience) for 20 min. Variousconcentrations of hydrogenperoxidewere applied at the beginning of annexinV treatment. Stained cardiomyocytes were then washed gently once andresuspended in solution containing propidium iodide (eBioscience) for exclu-sion of cells that had lost their membrane integrity. Fluorescence signals fromAPC and propidium iodide, excited at 633 nm and 488 nm, were collected atemission of 660 nm and 610 nm, respectively, in >10,000 cardiomyocytes persample group. FlowJo software (Tree Star) was used to generate the diagramof cell distribution according to fluorescence intensity and to calculate thepercent of Annexin V-positive population as an indication of apoptosis.

Quantitative Immunoblot Analysis. Hearts were snap frozen in liquid nitrogenat the end of the Langendorff perfusion period and homogenized in 1× CellLysis Buffer (Cell Signaling Technology) supplemented with 1 mM PMSF andcomplete protease inhibitor mixture (Roche Applied Science). For eachprotein, equal amounts of samples (5–120 μg) from each heart were an-alyzed by SDS/PAGE, as described (13). After transfer to membranes, im-munoblotting analysis was performed with the corresponding primaryantibodies (Bax, Bak, and ubiquitin from Cell Signaling; Cyp-D, COX4VDAC, ANT1, Hsp90, and TRAP-1 from Abcam; HAX-1 from BD Biosciences;calsequestrin (CSQ) and GAPDH from Thermo Scientific). This step wasfollowed by incubation with secondary antibody conjugated with horse-radish peroxidase at a 1:5,000 dilution. Visualization was achieved byusing SuperSignal West Pico chemiluminescent substrate (Pierce) or ECL-PLUS Western Blotting Detection kit (Amersham Pharmacia Biotech). Theintensities of bands were determined by the AlphaEaseFC software. Foreach protein, the densitometric values from pre-I/R WT controls werearbitrarily converted to 1.0, and the values of samples from the othergroups were normalized accordingly.

Global Ischemia/Reperfusion Injury. Myocardial infarction was induced byusing an isolated perfused heart model, as described (14). Briefly, hearts weremounted on a Langendorff apparatus, and perfused with Krebs–Henseleitbuffer (KHB). Temperature was maintained constant at 37 °C by water-jacketed glassware for the heart chamber, buffer reservoirs, and perfusionlines. In addition, an overhead light source was used to ensure maintenanceof temperature during ischemia, which was monitored by a thermometerplaced close to the perfused heart in the glass chamber. A water-filledballoon made of plastic film was inserted into the left ventricle and adjustedto achieve a left ventricular end-diastolic pressure of 5–10 mmHg. The distalend of the catheter was connected to a Heart Performance Analyzer (Micro-Med) via a pressure transducer. Hearts were paced at 400 bpm except duringischemia, and pacing was reinitiated 2 min after reperfusion. After a 20-minequilibration period, hearts were subjected to 40 min of no flow global is-chemia, followed by 60 min of reperfusion. Maximum rate of contraction(+dP/dt) and maximum rate of relaxation (−dP/dt) were monitored duringthis process. After reperfusion, the heart was perfused with 1% 2,3,5-triphenyltetrazolium chloride and incubated at 37 °C for 10 min. The heartwas perfused and rinsed with PBS solution, followed by 10% (vol/vol) for-malin tissue fixation. Five or six transverse slices were cut and weighed after1 h of fixation. The images of the slices were digitally analyzed for infarctarea and total ventricular area, using NIH image software.

Quantitative Real-Time PCR. Total RNA was extracted and purified from hearttissue with miRNeasy Mini Kit (QIAGEN 133220834). The first-strand cDNAwere generated from total RNA (1 μg) with reverse transcriptase kit (Invitrogencatalog no.11752). PCR was then performed with Bio-Rad real-time ther-mal cycler by using the following specific primer sequences: Mouse mito-chondria cyclophilin-D (Ppif): (Forward) 5′-TGGCTCTCAGTTCTTTATCTGC-3′,(Reverse) 5′-ACATCCATGCCCTCTTTGAC-3′, and mouse GAPDH (Forward)5′-TCAACAGC AACTCCCACTCTT-3′, (Reverse) 5′-ACCCTGTTGCTGTAGCCG-TATTCA-3′. Rat Ppif: Forward: 5′-TGG AGT TAA AGG CAG ATG TCG-3′,Reverse: 5′-CAT TGT GAT TGG TGA AGT CGC-3′; Rat GAPDH Forward:5′- CCTAAATGATACCCCACCGTG-3′, Reverse: 5′-TGT CAC AAG AGA AGGCAG C-3′. The values were normalized to those obtained with GAPDH.

Rat Myocyte Isolation and Virus Infection. Adult male Sprague–Dawley rats(8-10 wk, 250–350 g) were anesthetized by pentobarbital (100 mg/kg) andsupplemented with heparin (5000 U/kg). Hearts were quickly removed andthe aorta was cannulated by the Langendorff mode and perfused withmodified KHB: 118 mM NaCl, 4.8 mM KCl, 25 mM Hepes, 1.25 mM K2HPO4,1.25 mM MgSO4, 11 mM glucose, 5 mM taurine, and 10 mM butanedionemonoxime; pH 7.4) for 5–10 min. Subsequently, hearts were perfused withan enzyme solution [KHB containing 0.7 mg/mL collagenase type II (278 U/mg),0.2 mg/mL hyaluronidase, 0.1% BSA, and 25 μM Ca] for 15 min. Then,25 μM Ca was added to the perfusion buffer, and hearts were perfused foranother 5 min. The Ca concentration in the perfusion buffer was raised to100 μM and perfusion continued for 5 min. Once the hearts became flaccid,the left ventricular tissue was excised, minced, and filtered through a 240-μmscreen. Cells were quickly centrifuged at low speed (200 × g for 5 min),harvested, and resuspended in 25 mL of 100 μM Ca-KHB with 1% BSA. Thecells were allowed to settle automatically, and the Ca concentration wasgradually raised to 1.0 mM. Afterward, the cells were washed two times withDMEM supplemented with ATCC (2 mg/mL BSA, 2 mM L-carnitine, 5 mMcreatine, 5 mM taurine, 100 IU penicillin, and 100 μg/mL streptomycin). Cellswere then counted and plated on laminin-coated dishes. After 2 h of plat-ing, isolated rat cardiomyocytes were infected with Ad.Hsp90, Ad.HAX-1,Ad.HAX-AS viruses, or control virus Ad.GFP at an MOI of 500 for 2 h, beforeaddition of a suitable volume of complete DMEM (DMEM containing 2 mg/mLBSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/mL penicillin and100 μg/mL streptomycin), as described previously (14). The efficiency of ade-noviral gene transfer was evaluated in cultured adult rat myocytes with theexpression of GFP. Nearly 100% of myocytes appeared infected at 500 MOI by24–48 h. The cell phenotype and morphology remained similar among non-infected and adenoviral-infected groups after 24 h of infection. The myocyteswere washed with PBS and harvested for quantitative immunoblotting, orused in the experiments outlined in the results.

Coimmunoprecipitation. Hearts from WT, HAX-OE, and HAX+/− mice werehomogenized with 1× cell lysis buffer (Cell Signaling Technology), supple-mented with protease inhibitor mixture and phosphatase inhibitor mixture.The homogenate was centrifuged at 9,000 × g for 30 min at 4 °C. Thecentrifuged homogenate was diluted to 1 μg/μl (1 mL of final volume) andincubated with either anti-HAX-1 (BD Biosciences), anti-Hsp90 (Abcam), anti-Cyp-D (Abcam), or anti-ubiquitin (Abcam) at 4 °C overnight with rotation.

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One hundred microliters of protein G PLUS agarose beads (Santa Cruz Bio-technology) were added into the mixture and incubated for an additional5 h. Agarose beads were sedimented and washed six times with the cell lysisbuffer. Beads-bound proteins were dissociated in 2× SDS at room temper-ature for 30 min with vortexing at 5-min intervals. The identification of theassociated proteins was detected by Western blots. Homogenates from WThearts were used as positive controls.

Competitive Protein Binding ELISA. Competitive binding assay usingrecombinant proteins was performed as described (35). Briefly, each well ona 96-well high affinity binding plate (BD Falcon catalog no. 351172) wascoated with 200 ng of recombinant Hsp90β protein (StressMarq) or GSTprotein (Novus Biologicals, as control) in 100 μL of coating buffer (85 mmol/LNaHCO3 and 15 mmol/L Na2CO3, pH 9.5) overnight at 4 °C. After five washeswith PBS and 0.05% Tween-20 (PBS-T), blocking solution (PBS containing1% BSA) was added for 1 h at room temperature. Following another five PBS-Twashes, 400 ng of Cyp-D protein (Abnova) was added along with 400 ngof “competition protein mixture.” This mixture contained various amountsof HAX-1 (Abnova) supplemented with GST to reach a total of 400 ng of

protein/reaction. Incubation proceeded for 2 h at room temperature. Eachwell was then washed with PBS-T six times, and the HAX-1 antibody, whichwas dissolved in blocking solution, was applied for 2 h at room temperature.After six washes with PBS-T, the secondary antibody conjugated withhorseradish peroxidase was added for 1 h at room temperature. Followingeight PBS-T washes, TMB substrate reagent (BD Biosciences) was applied todevelop signal in blue color. After color was developed, 1 mol/L HCl wasadded to stop the reaction. The colorimetric intensity of each well was readat 450 nm with a microplate reader.

Statistical Analysis. Data were expressed as the mean ± SEM. Comparisons be-tween the means of two groups were performed by unpaired Student’s t test.Multiple groups were analyzed by using one-way ANOVA with a Bonferroni testfor post hoc analysis. Results were considered statistically significant at P < 0.05.

ACKNOWLEDGMENTS. We thank Dr. J. Molkentin for providing the Ad.Cyp-Dvirus; Dr. W. Sessa for providing the Ad.Hsp90 plasmid; Drs. A. Maloyanand J. Robbins for assistance with the mitochondrial swelling assay; andDrs. J. Molkentin and J. Karch for helpful discussions.

1. Carley AN, Taegtmeyer H, Lewandowski ED (2014) Matrix revisited: Mechanismslinking energy substrate metabolism to the function of the heart. Circ Res 114(4):717–729.

2. Chen L, Knowlton AA (2010) Mitochondria and heart failure: New insights into anenergetic problem. Minerva Cardioangiol 58(2):213–229.

3. Galluzzi L, Kepp O, Trojel-Hansen C, Kroemer G (2012) Mitochondrial control of cel-lular life, stress, and death. Circ Res 111(9):1198–1207.

4. Chiong M, et al. (2011) Cardiomyocyte death: Mechanisms and translational impli-cations. Cell Death Dis 2:e244.

5. Murphy E, Steenbergen C (2008) Mechanisms underlying acute protection from car-diac ischemia-reperfusion injury. Physiol Rev 88(2):581–609.

6. Mozaffarian D, et al.; American Heart Association Statistics Committee and StrokeStatistics Subcommittee (2015) Heart disease and stroke statistics–2015 update: Areport from the American Heart Association. Circulation 131(4):e29–e322.

7. Suzuki Y, et al. (1997) HAX-1, a novel intracellular protein, localized on mitochondria,directly associates with HS1, a substrate of Src family tyrosine kinases. J Immunol158(6):2736–2744.

8. Fadeel B, Grzybowska E (2009) HAX-1: A multifunctional protein with emerging rolesin human disease. Biochim Biophys Acta 1790(10):1139–1148.

9. Yap SV, Koontz JM, Kontrogianni-Konstantopoulos A (2011) HAX-1: A family of ap-optotic regulators in health and disease. J Cell Physiol 226(11):2752–2761.

10. Chao JR, et al. (2008) Hax1-mediated processing of HtrA2 by Parl allows survival oflymphocytes and neurons. Nature 452(7183):98–102.

11. Baumann U, et al. (2014) Disruption of the PRKCD-FBXO25-HAX-1 axis attenuates theapoptotic response and drives lymphomagenesis. Nat Med 20(12):1401–1409.

12. Vafiadaki E, et al. (2007) Phospholamban interacts with HAX-1, a mitochondrialprotein with anti-apoptotic function. J Mol Biol 367(1):65–79.

13. Zhao W, et al. (2009) The anti-apoptotic protein HAX-1 is a regulator of cardiacfunction. Proc Natl Acad Sci USA 106(49):20776–20781.

14. Lam CK, et al. (2013) Novel role of HAX-1 in ischemic injury protection involvement ofheat shock protein 90. Circ Res 112(1):79–89.

15. Klein C, et al. (2007) HAX1 deficiency causes autosomal recessive severe congenitalneutropenia (Kostmann disease). Nat Genet 39(1):86–92.

16. Faiyaz-Ul-Haque M, et al. (2010) A novel HAX1 gene mutation in severe congenitalneutropenia (SCN) associated with neurological manifestations. Eur J Pediatr 169(6):661–666.

17. Elrod JW, Molkentin JD (2013) Physiologic functions of cyclophilin D and the mito-chondrial permeability transition pore. Circ J 77(5):1111–1122.

18. Hunter DR, Haworth RA, Southard JH (1976) Relationship between configuration,function, and permeability in calcium-treated mitochondria. J Biol Chem 251(16):5069–5077.

19. Baines CP, et al. (2005) Loss of cyclophilin D reveals a critical role for mitochondrialpermeability transition in cell death. Nature 434(7033):658–662.

20. Kang BH, et al. (2007) Regulation of tumor cell mitochondrial homeostasis by anorganelle-specific Hsp90 chaperone network. Cell 131(2):257–270.

21. Yap SV, Vafiadaki E, Strong J, Kontrogianni-Konstantopoulos A (2010) HAX-1: Amultifaceted antiapoptotic protein localizing in the mitochondria and the sarco-plasmic reticulum of striated muscle cells. J Mol Cell Cardiol 48(6):1266–1279.

22. Karch J, et al. (2013) Bax and Bak function as the outer membrane component of themitochondrial permeability pore in regulating necrotic cell death in mice. eLife 2:e00772.

23. Chen D, Frezza M, Schmitt S, Kanwar J, Dou QP (2011) Bortezomib as the first pro-teasome inhibitor anticancer drug: Current status and future perspectives. CurrCancer Drug Targets 11(3):239–253.

24. Mato AR, Feldman T, Goy A (2012) Proteasome inhibition and combination therapyfor non-Hodgkin’s lymphoma: From bench to bedside. Oncologist 17(5):694–707.

25. Pagan J, Seto T, Pagano M, Cittadini A (2013) Role of the ubiquitin proteasome systemin the heart. Circ Res 112(7):1046–1058.

26. Lanciotti M, et al. (2010) Novel HAX1 gene mutations associated to neurodevelopmentabnormalities in two Italian patients with severe congenital neutropenia. Haematologica95(1):168–169.

27. Morrow DA, et al.; National Academy of Clinical Biochemistry (2007) NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Clinicalcharacteristics and utilization of biochemical markers in acute coronary syndromes.Clin Chem 53(4):552–574.

28. Kurz K, Giannitsis E, Zehelein J, Katus HA (2008) Highly sensitive cardiac troponin Tvalues remain constant after brief exercise- or pharmacologic-induced reversiblemyocardial ischemia. Clin Chem 54(7):1234–1238.

29. Baines CP, Kaiser RA, Sheiko T, Craigen WJ, Molkentin JD (2007) Voltage-dependentanion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol9(5):550–555.

30. Matas J, et al. (2009) Increased expression and intramitochondrial translocation ofcyclophilin-D associates with increased vulnerability of the permeability transitionpore to stress-induced opening during compensated ventricular hypertrophy. J MolCell Cardiol 46(3):420–430.

31. Han J, et al. (2010) Deregulation of mitochondrial membrane potential by mito-chondrial insertion of granzyme B and direct Hax-1 cleavage. J Biol Chem 285(29):22461–22472.

32. Siegelin MD (2013) Inhibition of the mitochondrial Hsp90 chaperone network: Anovel, efficient treatment strategy for cancer? Cancer Lett 333(2):133–146.

33. Rasola A, Bernardi P (2014) The mitochondrial permeability transition pore and itsadaptive responses in tumor cells. Cell Calcium 56(6):437–445.

34. Committee on Care and Use of Laboratory Animals (1996) Guide for the Care and Useof Laboratory Animals (Natl Inst Health, Bethesda), DHHS Publ No (NIH) 85-23.

35. Zhang X, et al. (2012) Hsp20 functions as a novel cardiokine in promoting angio-genesis via activation of VEGFR2. PLoS One 7(3):e32765.

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ded

by g

uest

on

Feb

ruar

y 13

, 202

1