HAX-1 regulates cyclophilin-D levels and mitochondria PNAS PLUS permeability transition pore in the heart

Chi 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 review May 5, 2015) The major underpinning of massive cell death associated with since its discovery in 1997 (7), its presence in cardiomyocytes was myocardial infarction involves opening of the mitochondrial only recently identified. Interestingly, cardiac HAX-1 localizes permeability transition pore (mPTP), resulting in disruption of not only to mitochondria but also the endo/sarcoplasmic re- mitochondria membrane integrity and programmed necrosis. ticulum (ER/SR) and it binds to , increasing Studies in human lymphocytes suggested that the hemato- inhibition of the SR calcium transport ATPase and reducing poietic-substrate-1 associated protein X-1 (HAX-1) is linked to SR calcium cycling. As cardiomyocyte contraction-relaxation is regulation of mitochondrial membrane function, but its role in coupled to calcium oscillation, HAX-1 is an important regulator of controlling mPTP activity remains obscure. Herein we used models cardiomyocyte contractility (12, 13). HAX-1 can also modulate ER with altered HAX-1 expression levels in the heart and uncovered stress responses by inhibiting the inositol requiring enzyme-1 (IRE- an unexpected role of HAX-1 in regulation of mPTP and cardio- 1) signaling arm, which diminishes cell death through reduction of myocyte survival. Cardiac-specific HAX-1 overexpression was asso- proapoptotic transcription factor expression and caspase activation ciated with resistance against loss of mitochondrial membrane in an ischemic insult, leading to improved contractile recovery potential, induced by oxidative stress, whereas HAX-1 heterozygous during reperfusion (14). However, HAX-1 also localizes to mito- deficiency exacerbated vulnerability. The protective effects of HAX-1 chondria in the heart (13), but its role in this organelle is unknown. were attributed to specific down-regulation of cyclophilin-D levels Importantly, human in HAX-1 have been linked to mi- leading to reduction in mPTP activation. Accordingly, cyclophilin-D

tochondrial function. These mutations, resulting in loss of protein or CELL BIOLOGY and mPTP were increased in heterozygous hearts, but genetic ablation of cyclophilin-D in these hearts significantly alleviated their activity, were discovered in a subgroup of severe neutropenia pa- susceptibility to ischemia/reperfusion injury. Mechanistically, alter- tients (8, 15, 16) and they were associated with loss of mitochondrial ations in cyclophilin-D levels by HAX-1 were contributed by the membrane potential in the neutrophils of the human carriers (16). ubiquitin-proteosomal degradation pathway. HAX-1 overexpression Thus, HAX-1 deficiency may cause a defect in the mitochondrial enhanced cyclophilin-D ubiquitination, whereas proteosomal in- membrane. hibition restored cyclophilin-D levels. The regulatory effects of One of the mechanisms that regulate mitochondrial mem- HAX-1 were mediated through interference of cyclophilin-D binding brane integrity is the mitochondria permeability transition pore to heat shock protein-90 (Hsp90) in mitochondria, rendering it (mPTP), a large conductance channel that spans through the susceptible to degradation. Accordingly, enhanced Hsp90 expres- outer and inner membranes of mitochondria and becomes acti- sion in HAX-1 overexpressing cardiomyocytes increased cyclophilin-D vated upon oxidative stress, mitochondrial calcium overload, or levels, as well as mPTP activation upon oxidative stress. Taken alkaline intracellular environment. The opening of this pore together, our findings reveal the role of HAX-1 in regulating during an ischemia/reperfusion insult underlies the etiology of cyclophilin-D levels via an Hsp90-dependent mechanism, resulting in protection against activation of mPTP and subsequent cell death Significance responses. The massive cell death, associated with a heart attack, is mainly HAX-1 | necrosis | mitochondrial permeability transition | cyclophilin-D | due to disruption of mitochondrial membrane integrity upon heat shock protein-90 activation of the mitochondrial permeability transition pore. Thus, it is important to understand how this pore is regulated he dynamics of contraction and relaxation in the heart are to prevent cardiac cell death. In this study, we reported that Thighly regulated by a finely tuned cross-talk between demand hematopoietic-substrate-1 associated protein X-1 (HAX-1) is an by the periphery and energy production by the mitochondria. inhibitor of the pore and promotes cell survival. HAX-1 works The heart relies on mitochondria for fueling ATP on a beat-to- through recruitment of a protein called Hsp90 from beat basis and for adjusting mitochondrial metabolism to meet cyclophilin-D, a major component of the pore. Displacement of the increases in cardiac output during exercise and flight-or-fight Hsp90 from cyclophilin-D promotes cyclophilin-D degradation, responses (1, 2). Mitochondria are also essential in controlling resulting in inhibition of pore opening and cell death. Given that cell death through apoptosis, necrosis, and autophagy (3, 4). Indeed, the opening of the mitochondrial permeability transition pore disruption of mitochondrial integrity underlies cardiomyocyte death contributes to various diseases, our findings have broader associated with myocardial infarction (5) that attributes to one of applications reaching beyond the heart. every six deaths in the United States (6). The mitochondrial hematopoietic-substrate-1 associated pro- Author contributions: C.K.L., W.Z., and E.G.K. designed research; C.K.L., W.Z., G.-S.L., tein X-1 (HAX-1), a 35-kDa protein has recently emerged as a W.-F.C., G.G., and G.A. performed research; C.K.L., W.Z., and G.-S.L. contributed new reagents/analytic tools; C.K.L., W.Z., and G.-S.L. analyzed data; and C.K.L. and E.G.K. critical regulator of cell survival in various tissues (7–9). Indeed, wrote the paper. global ablation of HAX-1 was shown to result in death by 14 wk The authors declare no conflict of interest. of age due to neuronal degeneration from excessive cell death This article is a PNAS Direct Submission. (10). In addition, a recent study demonstrated that degradation 1C.K.L. and W.Z. contributed equally to this work. of HAX-1 triggers cell death in human B-cell lymphoma, further 2To whom correspondence should be addressed. Email: [email protected]. supporting the pivotal role of HAX-1 in dictating cell survival This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (11). Although HAX-1 has been studied in various cell types 1073/pnas.1508760112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1508760112 PNAS Early Edition | 1of10 Downloaded by guest on September 27, 2021 cardiac injury (5), because it allows solutes and ions that should its ubiquitination and proteosomal degradation, which, in turn, be excluded from mitochondria to infuse into their matrix. Con- protected mitochondria from oxidative stress and calcium overload. comitantly, influx of water results in swelling of the originally These beneficial effects of HAX-1 were mediated through Hsp90, compacted cristae structure, leading to complete mitochondria which appeared to stabilize Cyp-D protein in cardiomyocytes. membrane failure and subsequent activation of massive necrotic Taken together, our results reveal a novel mechanism by which cell death and fibrosis (5, 17). This injury is irreversible, posing the HAX-1/Hsp90 complex may inhibit activation of the mPTP greater damage to organs that possess limited regenerative via regulation of Cyp-D levels, and promote cardiomyocyte sur- capacity, such as the heart (5). Although the mitochondrial vival upon oxidative stress and calcium overload in ischemia/ permeability transition pore was first described in 1976 (18), the reperfusion injury. full identity of its protein components remains obscure. Several constituents have been proposed (17), but only cyclophilin-D Results (Cyp-D), a peptidyl-prolylcis-trans isomerase (PPI) in the mito- HAX-1 Preserves Mitochondria Membrane Integrity and Inhibits Cell chondrial matrix, has been a genetically proven regulator of Death. The integrity of the mitochondrial membrane is crucial in mPTP (19). Ablation of this protein by targeting or phar- maintaining cardiomyocyte function and survival. Increases in macological inhibition by cyclosporine A can effectively prevent the generation of reactive oxygen species or mitochondrial cal- the opening of the pore and inhibit the activation of cell death cium overload during cardiac stress, such as ischemia/reperfusion during ischemia/reperfusion injury (17, 19), demonstrating the injury, can disrupt mitochondrial membrane integrity, dissipate crucial role of Cyp-D function in cell survival. Furthermore, Cyp-D membrane potential for ATP production, and cause massive activity in cancer cells can be regulated by a mitochondrial chap- cell death (5). HAX-1 has recently emerged as a regulator of erone network that involves the cytosolic heat shock protein-90 cardioprotection, and we have shown that it localizes to mito- (Hsp90) and its mitochondria analogs, which are crucial in inducing chondria besides ER/SR (13). To further address its localization, cancer cell death (20). we isolated mitochondria (Fig. S1) and subjected them to pro- Based on our recent observations that Hsp90 is a newly iden- tease accession. HAX-1 was present in the inner mitochondrial tified binding partner of HAX-1 (14), it was intriguing to speculate membrane (Fig. S2), consistent with previous reports (10, 21). that the Hsp90/HAX-1 interaction may be also involved in reg- Thus, we sought to investigate whether HAX-1 may regulate ulation of Cyp-D function in the heart. Thus, the current study mitochondria membrane potential and control activation of cell was designed to address this hypothesis by using mouse models death. To examine this hypothesis, cardiomyocytes were isolated with alterations in HAX-1 levels. Our findings indicate that from HAX-1 overexpressing (HAX-OE, 2.5-fold expression; Fig. HAX-1 could specifically reduce Cyp-D levels by enhancing 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 of + − + − HAX-1 has opposite effects. (A) HAX-1 protein expression in HAX-OE and HAX / hearts. CSQ was used as loading control. (B and C) Isolated WT, HAX-OE, and HAX /

cardiomyocytes were loaded with mitochondrial membrane potential fluorescent dye, TMRE. Upon 2 mM H2O2 administration for 20 min, membrane potential was diminished 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 for HAX-OE, 16 hearts for WT, and 9 hearts for HAX+/− (>8 cells per heart); *P < 0.05vs.WTat20min.(D) HAX-1 overexpressing mitochondria demonstrated resistance to swelling induced by 50 μMcalcium.n = 3 hearts for each group. (E and F) HAX-1 overexpression in cardiomyocytes prevented cell death due to loss of plasma

membrane integrity (E) and apoptosis (F) after 20 min of 2 mM H2O2 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.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1508760112 Lam et al. Downloaded by guest on September 27, 2021 + − PNAS PLUS (HAX / , 40% protein expression; Fig. 1A) (13) mice, and they To determine whether the protection of mitochondrial mem- were loaded with a mitochondrial localized florescent membrane brane by HAX-1 would translate to improved cell survival, we used potential indicator, TMRE, allowing us to monitor the mem- isolated cardiomyocytes and assessed the degree of propidium brane potential before and after hydrogen peroxide challenge, iodide (PI) inclusion as an indicator of necrotic cell death. using confocal microscopy. Under basal conditions, the per- Treatment of cells with either 1 or 2 mM hydrogen peroxide for centage of TMRE-positive cardiomyocytes was similar among 20 min resulted in increases in Propidium iodide-positive cells. the three groups (Fig. 1 B and C). However, after administration Notably, 2 mM hydrogen peroxide, which was associated with of 2 mM hydrogen peroxide to induce oxidative challenge, both loss of mitochondria membrane integrity (Fig. 1C), resulted in a WT and HAX heterozygous cardiomyocytes lost some flo- 15% increase in PI-positive WT cardiomyocytes, but this in- rescence, and by 20 min, only 30% of the heterozygous cells crease was significantly blunted in HAX-1 overexpressing cells. maintained the TMRE signal, compared with 50% of WT. However, heterozygous loss of HAX-1 exacerbated the increase E In contrast, more than 70% of the HAX-overexpressing car- in cells with propidium iodide inclusion (Fig. 1 ), consistent with diomyocytes were resistant to 20 min of oxidative challenge the findings in the TMRE experiments. Similar results were (Fig. 1 B and C). These findings suggest a protective role of obtained, when the expression of cell surface V was HAX-1 in maintaining mitochondrial membrane potential during quantified, as a marker of apoptotic cell death. Increasing HAX-1 levels effectively reduced apoptosis, whereas loss of HAX-1 ele- oxidative stress. vated its activation under hydrogen peroxide stress (Fig. 1F). Thus, The protective effects of HAX-1 on mitochondrial integrity our data suggest that HAX-1 maintains the mitochondrial mem- were also evidenced upon calcium overload, a situation that in- brane potential and protects membrane integrity, resulting in re- duces the opening of the permeability transition pore (mPTP), duced apoptosis and necrotic cardiomyocyte death. leading to failure of osmotic pressure control, water influx, and swelling of mitochondria (17). Isolated WT cardiac mitochondria HAX-1 Regulates the Opening of Mitochondrial Permeability Transition demonstrated a steady loss of light scattered by the mitochon- Pore. It has been demonstrated that mitochondrial membrane in- drial membrane structure after the addition of 50 mM calcium. tegrity highly depends on the function of mPTP. The opening of However, HAX-1 overexpressing mitochondria were resistant this pore is a key mediator of cardiac ischemia/reperfusion injury under the same conditions and showed no sign of swelling (Fig. because it facilitates loss of mitochondrial membrane potential, 1D). In contrast, isolated HAX-1 heterozygous-deficient mito- induces swelling, and leads to the rupture of mitochondrial CELL BIOLOGY chondria exhibited significant swelling under basal conditions. membrane and activation of cell death (5). To determine whether These findings together with the results from the hydrogen the HAX-1 regulatory effects in mitochondria are mediated peroxide treatment indicate that HAX-1 may protect the mito- through an mPTP-dependent mechanism, we assessed the effects chondrial membrane integrity. of 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 ablation

on mitochondrial membrane potential after H2O2 treatment for 20 min. n = 4–8hearts(>12 cells per heart); *P < 0.05 vs. WT with vehicle (DMSO) at 20 min; #P < 0.05 vs. 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 of mitochondrial swelling required a lower cyclosporine A dose in HAX-1 overexpressing mitochondria. n = 3 hearts for each group. (E and F) Cyclosporine A abrogated

the effect of HAX-1 on necrotic (E) and apoptotic (F)celldeath.n = 8–15 hearts (>10,000 cells per heart); *P < 0.05 vs. WT at 2 mM H2O2;#P < 0.05 vs. the respective untreated group at 2 mM H2O2. Data are expressed as mean ± SEM.

Lam et al. PNAS Early Edition | 3of10 Downloaded by guest on September 27, 2021 cardiomyocytes with cyclosporine A (CsA), an mPTP inhibitor HAX-1 Regulates Cardiac Cyclophilin-D Protein Levels. Because cy- (17). CsA resulted in preservation of mitochondrial membrane closporine A is a direct pharmacological inhibitor of cyclophilin- potential in both WT and HAX-1 heterozygous cardiomyocytes D (17), an important regulator of the permeability transition exposed to hydrogen peroxide (Fig. 2 A and B). Importantly, the pore, we hypothesized that the reduced dose of CsA required for levels of TMRE in these treated groups were similar to those in complete blockade of swelling in HAX-1 overexpressing mito- HAX-1 overexpressing cells (Fig. 1B), suggesting that the pro- chondria may be associated with decreases in Cyp-D level or tective effects of HAX-1 may be mediated through the mPTP. To activity. Indeed, quantitative immunoblotting indicated that the Cyp-D levels in HAX-1 overexpressing hearts were reduced by further confirm that the observed protection against loss of mi- 40% compared with WTs (Fig. 3 A and B). Interestingly, the tochondrial membrane potential by HAX-1 overexpression was effects of HAX-1 appeared specific for Cyp-D, because the ex- not due to impaired function of the mPTP, we used a high cal- pression levels of other proposed mPTP components, namely the cium concentration (375 mM) in the swelling assay to induce voltage-dependent anion channel (VDAC) and adenine nucle- mPTP opening in isolated mitochondria. Under these conditions, otide translocase (ANT1), were not altered (Fig. 3A). Further- the swelling response of the HAX-1 overexpressing mitochondria more, the expression levels of Bax and Bak, which were recently was similar to that of WTs (Fig. 2C), suggesting that HAX-1 suggested to govern the porosity of the mitochondrial outer regulates the sensitivity of the mPTP opening. To further address membrane (22), were not altered by HAX-1 overexpression ei- this hypothesis, we determined the dose of cyclosporine A re- ther. These findings in cardiac homogenates were also similar to quired for complete inhibition of mitochondrial swelling, induced those obtained in isolated mitochondria (Fig. 3 C and D). Thus, by 375 mM calcium. Both WT and HAX-1 overexpressing mito- we speculated that if HAX-1 can negatively regulate Cyp-D ex- chondria were resistant to swelling in the presence of 1 μM cy- pression, then loss of HAX-1 should have a positive effect on closporine A. However, upon dose reduction to 0.5 μM, WT Cyp-D levels. Indeed, heterozygous-deficient hearts with 60% mitochondria exhibited swelling in a time-dependent manner, loss of HAX-1 (13) exhibited a 70% increase in Cyp-D levels whereas HAX-1 overexpressing mitochondria maintained their (Fig. 3 E and F). Similar findings were observed with isolated G H resistance (Fig. 2D). These findings further suggest that over- mitochondria (Fig. 3 and ). There were no alterations in the E G expression of HAX-1 reduces the sensitivity of mPTP opening levels of VDAC, ANT1, Bax, and Bak (Fig. 3 and ), similar in cardiomyocytes. to HAX-1 overexpression. Thus, the protective effect of HAX-1 We also applied cyclosporine A before hydrogen peroxide on mPTP opening may be attributed to the reduction of Cyp-D expression in the mitochondria. challenge and assessed the degree of cell death activation in To exclude the possibility that reduction of Cyp-D level was isolated cardiomyocytes to confirm that the prosurvival effects of an indirect compensatory response to chronic HAX-1 over- HAX-1 under the hydrogen peroxide treatment depended on the expression in vivo, we used adenoviral delivery to acutely in- opening of mPTP. Indeed, this pretreatment completely abol- crease HAX-1 levels in cardiomyocytes. At 24 h after infection, ished the differences between the three groups regarding the HAX-1 overexpression (15-fold increase in HAX-1 levels) was number of cells with reduced plasma membrane integrity (Fig. associated with a 40% decrease in Cyp-D levels (Fig. S3 A and 2E). Accordingly, apoptotic cell death was also reduced in WT B). Accordingly, acute down-regulation of HAX-1 by 55%, using and HAX-1 heterozygous cardiomyocytes (Fig. 2F) by CsA in- an antisense approach, led to a 60% increase in Cyp-D expres- hibition of mPTP, and the values in the three groups were sim- sion levels in cardiomyocytes (Fig. S3 C and D). The acute al- ilar. Thus, regulation of mPTP activity appears to play a key role terations in Cyp-D levels reflected parallel changes in the TMRE in the cell survival mechanisms mediated by HAX-1. responses 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 each group. (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 were + − + − increased in HAX / cardiac homogenates. n = 6 hearts for each group. (G and H) Cyp-D levels were increased in the HAX / mitochondrial fraction. n = 9 hearts for each group. *P < 0.05 vs. WT. Data are expressed as mean ± SEM.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1508760112 Lam et al. Downloaded by guest on September 27, 2021 PNAS PLUS CELL BIOLOGY

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 adenoviral infection of adult rat cardiomyocytes. (B and C) Adenoviral delivery of Cyp-D removed the protection mediated by HAX-1 overexpression upon 2 mM hydrogen peroxide administration. CSQ was used as a loading control. n = 4–6hearts(>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 = 4–5hearts(>16 cells per heart). (G and H) Cyp-D ablation significantly reduced infarct size in HAX-1 heterozygous + − knockout 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.

with increased HAX-1 levels showed enhanced resistance to loss We previously reported that reduction in HAX-1 levels have of mitochondria membrane potential, whereas down-regulation detrimental effects in the heart, when subjected to ischemia/ of HAX-1 led to increased dissipation of proton gradient (Fig. S3 reperfusion injury. Because mPTP has also been shown to play E and F). These findings coupled with the ones above in animal an important role in this injury, we examined whether the effects models suggest that HAX-1 may be a regulator of cardiac Cyp-D of HAX-1 may be mediated through Cyp-D. To this end, we + − expression levels. crossed the Cyp-D knockout mouse with the HAX / model + − To further delineate the contribution of reduced Cyp-D levels (CypD-KO/HAX / ) and obtained HAX-1 heterozygous mice + − in the protective effects of HAX-1 on mPTP opening and that were also deficient in Cyp-D. Hearts from the WT, HAX / , membrane potential, we used two complementary approaches CypD-KO, and the cross model were then subjected to global no- and examined the effects of altered Cyp-D expression on the flow ischemia (40 min), followed by reperfusion (60 min) in the TMRE responses upon hydrogen peroxide challenge. First, we Langendorff perfusion mode. This protocol induced a 26% in- infected adult mouse WT and HAX-1 overexpressing car- farct size in WT hearts (Fig. 4 G and H), consistent with previous diomyocytes with an adenovirus-encoding Cyp-D cDNA. In- studies (14). Heterozygous loss of HAX-1 increased myocardial creased Cyp-D protein levels to a similar extent between WT and infarction to 40%, whereas ablation of Cyp-D significantly di- transgenic cells (Fig. 4A) effectively abolished the protective minished it to 11% (Fig. 4 G and H). Importantly, ablation of + − effects of HAX-1 overexpression on mPTP opening (Fig. 4 B Cyp-D in HAX / hearts resulted in significant reduction of in- and C). Second, we isolated cardiomyocytes from the Cyp-D farct size from 40 to 18% (Fig. 4 G and H), suggesting that mPTP knockout (or PPIF null) mouse and infected them with adeno- plays a critical role in the protective effects of HAX-1 in cardiac viruses carrying HAX-1 sense or antisense cDNA (Ad.HAX-1 or ischemia/reperfusion injury. Ad.HAX-AS) to alter HAX-1 expression levels (Fig. 4D). In the absence of Cyp-D, the alterations in HAX-1 levels did not affect the HAX-1 Regulates Cyclophilin-D Levels Through Hsp90. To determine TMRE response to hydrogen peroxide challenge (Fig. 4 E and F). whether the effects of HAX-1 were mediated at the transcriptional Collectively, the regulatory effects of HAX-1 on mPTP sensitivity level, we assessed cyclophilin-D (or ppif) mRNA expression in WT, + − appear to be mediated through alterations of Cyp-D levels. HAX-OE, and HAX / cardiac homogenates (Fig. 5A). There were

Lam et al. PNAS Early Edition | 5of10 Downloaded by guest on September 27, 2021 no alterations among the three groups, indicating that HAX-1 was further supported by immunoprecipitation studies in cardiac does not regulate Cyp-D transcription. We then hypothesized that homogenates from HAX-1 heterozygous-deficient hearts, which HAX-1 modulates the Cyp-D protein levels specifically through its exhibit increases in Cyp-D levels. Using Cyp-D as bait, there was degradation by the proteosomal pathway. To test this hypothesis, we an increase in the amount of Hsp90 associated with Cyp-D, acutely increased HAX-1 expression in adult rat cardiomyocytes compared with WTs (Fig. 6A). A similar increase in the Cyp-D by adenoviral delivery (Ad.HAX-1) and subsequently treated the levels was observed when Hsp90 was used as bait (Fig. 6B). On cells with bortezomib, the well-recognized proteosomal inhibitor the contrary, overexpression of HAX-1 resulted in reduced in cancer cells (23, 24). HAX-1 overexpression decreased Cyp-D Hsp90/Cyp-D association, using either Hsp90 or Cyp-D as bait in levels as expected, but the effect was abolished by bortezomib, the cardiac homogenates (Fig. 6 C and D). These alterations suggesting that HAX-1 regulates proteosomal degradation of Cyp-D appear to occur in mitochondria, because similar findings were (Fig. 5B). Because protein degradation is known to be controlled by observed when isolated mitochondrial fractions from HAX-OE + − ubiquitination (25), we sought to examine whether HAX-1 may and HAX / hearts were used for immunoprecipitation experi- + − affect Cyp-D ubiquitination. Thus, HAX-OE or HAX / cardiac ments (Figs. S6 and S7). Notably, the changes in Hsp90/Cyp-D homogenates were subjected to immunoprecipitation studies, complex formation were not associated with alterations in car- using either ubiquitin or Cyp-D as a bait. Indeed, HAX-1 diac or mitochondrial Hsp90 levels in either HAX-1 over- overexpression markedly increased Cyp-D ubiquitination (Fig. 5 expressing or heterozygous-deficient hearts (Figs. S8 and S9). C and D), whereas heterozygous ablation of HAX-1 resulted in Taken together, these findings suggest that HAX-1 may directly less ubiquitinated Cyp-D, compared with WTs (Fig. 5 E and F). displace Cyp-D from binding to Hsp90 in the mitochondria. These findings indicate that HAX-1 regulates the degrada- To urther examine whether HAX-1 physically hinders the tion of Cyp-D protein through the ubiquitination-proteosomal interaction of Cyp-D with Hsp90, we performed an in vitro pathway. competitive binding ELISA, using recombinant proteins. We Previous studies in mitochondria isolated from various cell observed a progressive reduction of the Cyp-D/Hsp90 binding in types have shown that Cyp-D interacts with the heat shock the presence of increasing HAX-1 protein levels (Fig. 6E). Thus, protein 90 (Hsp90) in the matrix (20). Interestingly, we also re- increases in HAX-1 appear to reduce the levels of mitochon- cently found that Hsp90 is a binding partner of HAX-1 in the drial Hsp90 that bind to Cyp-D, leading to degradation of Cyp-D. heart (14). These findings prompted us to investigate whether To further test whether the effect of HAX-1 overexpression on the observed effects of HAX-1 on Cyp-D levels may involve decreasing the Cyp-D levels is mediated through reduction of the Hsp90. Because the role of Hsp90 on Cyp-D is not known, we Hsp90/Cyp-D association, we acutely overexpressed Hsp90 to acutely overexpressed Hsp90 in adult cardiomyocytes and de- restore complex formation. Indeed, Hsp90 overexpression in the termined the expressions of Cyp-D. We observed a significant setting of HAX-1 overexpression resulted in markedly enhanced increase in Cyp-D protein levels (Fig. S4) and reduction in its Cyp-D levels (Fig. 6F), abolishing the protective effects of ubiquitination (Fig. S5), suggesting that Hsp90 may serve as a HAX-1 and exacerbating loss of membrane potential upon chaperone for Cyp-D and prevent its degradation. This notion hydrogen 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 hearts + − for 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-D was 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 in HAX+/− hearts. Three independent experiments were performed. n = 4 hearts for each group. Data are expressed as mean ± SEM.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1508760112 Lam et al. Downloaded by guest on September 27, 2021 provide further evidence that the cardioprotective effects of that there are only a few studies addressing the function of HAX-1 PNAS PLUS HAX-1 are mediated through regulation of mPTP function and in regulation of cell death by this organelle. One study reported specifically through recruitment of Hsp90 from the Hsp90/Cyp-D that mitochondrial HAX-1 interacts with the high temperature complex, contributing to degradation of Cyp-D protein and in- requirement protein-2 (HtrA2) and abrogation of this complex creased survival. by HAX-1 ablation in mice contributes to Bax accumulation, neurodegeneration, and a short lifespan (14 wk). However, Discussion ablation of Bax in HAX-1–deficient mice could only prolong In this study, we identified HAX-1 as a regulator of mPTP and survival by 9 wk without fully rescuing the phenotype (10), mitochondrial membrane integrity, impacting cell survival in the suggesting that the HtrA2/Bax regulation is not the sole anti- heart. Increases in HAX-1 expression protected mitochondrial apoptotic mechanism mediated by HAX-1. Our findings herein membrane from damage associated with oxidative stress and suggest that mitochondrial localized HAX-1 has a direct pro- calcium overload, whereas decreases in HAX-1 levels exacer- tective effect on the membrane integrity of this subcellular or- bated loss of membrane integrity. The underlying mechanisms ganelle by regulating the mPTP opening. involved the ability of HAX-1 to sequester Hsp90 from Cyp-D, We previously showed that cardiac ischemia/reperfusion injury which then rendered Cyp-D prone to ubiquitination and degra- is associated with loss of HAX-1 and overexpression of this dation. Thus, HAX-1 inhibits the opening of mPTP and prevents protein conferred protection by reducing infarct size, limiting loss of membrane integrity and cell death activation during oxi- troponin release and inhibiting DNA fragmentation. On the dative stress or mitochondrial calcium overload. These findings, contrary, heterozygous loss of HAX-1 led to exacerbated cardiac coupled with our recent observations, indicating that HAX-1 can injury (14). Particularly, leakage of intracellular content, such as suppress ER stress-induced apoptosis (14), reveal a previously troponin proteins, is a sign of plasma membrane rupture and unidentified paradigm of cell death regulation by HAX-1 in two necrotic cell death (27, 28), which is mainly caused by failure to different cellular organelles. maintain plasma membrane potential due to loss of ATP supply Growing evidence demonstrates an axiomatic prosurvival role (3, 4). The diminished energy production reflects compromised of HAX-1, and human mutations of this protein have been mitochondrial function associated with mPTP opening during shown to associate with diseases characterized of altered cell ischemia/reperfusion injury (5). Although we previously showed viability, such as congenital neutropenia (15), neurodevelopmental that HAX-1 inhibits apoptosis mediated by the ER stress re-

retardation (26), and mantle cell lymphoma (11). Although HAX-1 sponse, this effect would not account for the reduction in cardiac CELL BIOLOGY has been known to predominantly localize to mitochondria in troponin I release during ischemia/reperfusion injury. Thus, the various tissues and cell types (8), and mitochondria are known current identification of HAX-1asaregulatorofCyp-Dlevels for their gate-keeping role in cell death activation, it is surprising and 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)orHsp90(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, using Cyp-D (C)orHsp90(D) as a bait, demonstrated that the Cyp-D/Hsp90 association was reduced in HAX-OE hearts. WT cardiac homogenate served as input. (E)The Cyp-D/Hsp90 interaction was examined in the presence of increasing levels of HAX-1, using recombinant proteins. ELISA signals were calculated after subtraction of 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 and HAX-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 = 4–7hearts(>10 cells per heart); *P < 0.05 vs. WT GFP at 20 min; #P < 0.05 vs. HAX-OE GFP at 20 min. Data are expressed as mean ± SEM.

Lam et al. PNAS Early Edition | 7of10 Downloaded by guest on September 27, 2021 to its cardioprotective mechanisms. The role of Cyp-D was further was proposed that both Hsp90 and TRAP-1 serve to antagonize elucidated by generation of a cross model with ablation of this Cyp-D function. Based on this hypothesis, it is expected that protein in the HAX-1 heterozygous the deficient hearts, which displacement of Hsp90 from Cyp-D by HAX-1 overexpression were characterized by increased levels of this protein and mPTP would increase Cyp-D activity and promote mPTP opening. activity. The cross model exhibited diminished myocardial in- However, we did not observe a reduction of mPTP activa- farction, although its infarct size was not reduced to the level of tion when HAX-1 was overexpressed. Moreover, we found Cyp-D knockout hearts, suggesting that inhibition of both that overexpression of Hsp90 in either WT or HAX-OE car- mPTP and ER stress contributes to the cardioprotective effects diomyocytes resulted in increased Cyp-D expression (Fig. 6F) of HAX-1. Given that myocardial infarction is characterized and exacerbated loss of mitochondrial membrane potential after by a continuum of apoptosis and necrosis, our data demon- hydrogen peroxide challenge (Fig. 6 G and H). These results strate that HAX-1 orchestrates a dual protective mechanism by suggest that Hsp90 promotes Cyp-D function in cardiac cells. inhibiting a mitochondrial-based necrotic activation, in addi- The apparent discrepancy between our current results and pre- tion to the previously reported ER-based apoptotic cell death vious reports in tumor cell lines may be due to the high de- program. pendency of Hsp90 function on the availability and combination The activation of mitochondrial permeability transition pore of its cochaperone proteins, which may differ between cell types. has been shown to be a main driver in the development of It is likely that tumor cells have a remodeled mitochondrial myocardial infarction upon ischemia/reperfusion injury (5). Al- Hsp90 chaperone network to enhance their survival and pro- though the genetic identity of the pore is unknown, it has been liferation. In support of this notion, we failed to observe any suggested to consist of two complexes located on the outer and significant association between HAX-1/TRAP-1 (Fig. S10), inner mitochondrial membrane. Although it was originally in- suggesting that the Hsp90 interaction with HAX-1 in cardiac dicated that VDAC constitutes the outer membrane portion, a cells may not be associated with TRAP-1. Indeed, mitochon- recent report suggested that VDAC is dispensable in mPTP ac- drial targeted Hsp90 inhibitors have been shown to exhibit tivation (29) and outer membrane porosity is induced by the excellent specificity toward tumor cells, but not normal cells (20, 32). oligomerization of Bax/Bak (22). Furthermore, HAX-1 homo- These reports support our results and point to a complex regu- zygous ablation was shown to facilitate Bax accumulation and lation of Hsp90 function by its cochaperones and the intracellular cause neurodegeneration (10). However, in both HAX-1 over- environment. expressing and heterozygous-deficient hearts, the levels of Bax Our findings on the newly discovered function of HAX-1 in and Bak were not altered in either the homogenates or mito- controlling Cyp-D levels and activation of the mitochondrial chondrial fractions, suggesting that the alterations of mPTP in permeability transition pore are not only critical in heart function our models were independent of Bax/Bak accumulation. and survival, but also provide significant insights in other diseases, The only genetically proven regulator of the mPTP is Cyp-D, such as immunodeficiency, tumorgenesis, and neurodegeneration. which is located within the matrix of the mitochondria. Either Studies in patients with HAX-1 , exhibiting severe con- ablation or pharmacological inhibition of this protein results in genital neutropenia, showed that their neutrophils had low mito- significant reduction of infarct size after ischemia/reperfusion chondrial membrane potential compared with the healthy controls injury (17, 19). However, increasing the expression of Cyp-D (16). However, elevated HAX-1 levels are associated with various results in enhanced mPTP activity and mitochondrial swelling types of cancer (8), where the mPTP activity can be inhibited for (19, 30). Thus, regulation of Cyp-D expression level becomes of prosurvival purposes (33). paramount importance in the control of cell survival. To this end, In summary, this is the first study to our knowledge to dem- our study presents the first evidence to our knowledge that Cyp-D onstrate that HAX-1 and specifically the HAX-1/Hsp90 complex levels can be posttranslationally regulated by HAX-1 and the promote Cyp-D ubiquitination and degradation. This regulatory chaperone protein Hsp90. Chaperone proteins have been well mechanism confers resistance to mPTP opening and prevents recognized to work coordinately with cochaperones and regulate cell death against oxidative stress and mitochondrial calcium protein turnover, directing unfolded or unwanted proteins to the overload (Fig. S11). Thus, HAX-1 and Hsp90, which are ubiq- ubiquitination/proteosomal degradation pathway. We previously uitously expressed, serve as common nodal points in both demonstrated that HAX-1 works with Hsp90 to control the ac- tivity of IRE-1, one of the ER stress response signaling compo- apoptotic and necrotic cell death. Because mPTP opening is nents, suggesting that HAX-1 may function as a cochaperone implicated in the pathogenesis of various diseases, our findings protein for Hsp90 (14). This chaperone/cochaperone regulation suggest that the HAX-1/Hsp90 complex may be potentially exploited appears to exist also in mitochondria, controlling the ubiquiti- as a feasible therapeutic target. nation and degradation of Cyp-D. Increases in HAX-1 expres- Materials and Methods sion can sequester Hsp90 from the Cyp-D/Hsp90 complex, Animal Models. HAX-1 transgenic (HAX-OE, FVB/N) (13), HAX-1 knockout promoting Cyp-D ubiquitination and degradation. In turn, the heterozygous (HAX+/−, FVB/N) (13), Cyp-D knockout (B6129s) (19), the cross + − reduced Cyp-D levels confer resistance to mPTP opening and of CypD-KO/HAX / (B6129s) mice, and their wild-type littermates were used subsequent membrane potential loss upon stress stimuli. These in this study. To avoid the complication of gender differences, only male findings are consistent with previous observations in breast car- mice at 3 mo of age were used for these studies, according to the National cinoma, colon cancer, and HEK293 cell lines, which showed that Institutes of Health Publication No. 8523: Guide for the Care and Use of cleavage of HAX-1 by granzyme B leads to loss of mitochondrial Laboratory Animals (34). membrane potential, and this loss can be prevented by intro- duction of Cyp-D siRNA (31). Mouse Cardiomyocyte Isolation and Virus Infection. Mouse myocytes from WT Recently, several breakthroughs have been made regarding and HAX-1 OE hearts were isolated as described (14). Briefly, adult mouse regulation of Cyp-D function and necrotic cell death. In tumor hearts were excised following mouse anesthesia with sodium pentobarbital cells, it was reported that the Cyp-D activity is inhibited by a mi- (70 mg/kg, i.p.) and cannulated on a Langendorff system. Ca-free Tyrode tochondrial Hsp90 chaperone network that contains cytosolic solution (113 nmol/L NaCl, 4.7 mmol/L KCl, 0.6 mmol/L KH2PO4, 0.6 mmol/L Na2HPO4, 1.2 mmol/L MgSO4·7H2O, 12 mmol/L NaHCO3, 10 mmol/L KHCO3, Hsp90, a mitochondrial Hsp90 analog, the tumor necrosis factor 5 mmol/L Hepes, 3 0 mmol/L taurine, 10 mmol/L 2,3-butanedione monoxime, receptor associated protein-1 (TRAP-1), and several cochaperone and 5.5 mmol/L glucose, pH 7.4) was used to perfuse the heart for 3 min at proteins. Inclusion of a mitochondrial-directed inhibitor, tar- 37 °C. The perfusion was then switched to a digestion solution containing geting the ATPase activity of both Hsp90 analogs, was able to 0.25 g/L liberase blendzyme (Roche). Following digestion, the left ventricular increase the opening of mPTP upon oxidative stress (20). Thus, it tissue was excised, minced, and dissociated into a cell suspension. Calcium

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1508760112 Lam et al. Downloaded by guest on September 27, 2021 was serially added to the cellular suspension until final calcium concentra- Global Ischemia/Reperfusion Injury. Myocardial infarction was induced by PNAS PLUS tion in the Tyrode solution was 1.2 mmol/L. Cardiomyocytes were pelleted by using an isolated perfused heart model, as described (14). Briefly, hearts were gravity, resuspended in plating media [DMEM with 5% (vol/vol) FBS, 10 mmol/L mounted on a Langendorff apparatus, and perfused with Krebs–Henseleit 2,3-butanedione monoxime, 100 units/mL penicillin streptomycin and 2 mL buffer (KHB). Temperature was maintained constant at 37 °C by water- of L-glutamine] and placed on a six-well plate containing laminin-coated jacketed glassware for the heart chamber, buffer reservoirs, and perfusion coverslips for 1 h. Supplementing the media with 5% (vol/vol) FBS allows op- lines. In addition, an overhead light source was used to ensure maintenance timal cardiomyocyte attachment. Once the cells attached to the coverslip, the of temperature during ischemia, which was monitored by a thermometer medium was replaced with infection medium (plating medium containing no placed close to the perfused heart in the glass chamber. A water-filled FBS). Adenoviruses (Ad.GFP, Ad.HAX-1, Ad.HAX-AS, Ad.Cyp-D, and Ad.Hsp90) balloon made of plastic film was inserted into the left ventricle and adjusted were applied at 500 multiplicity of infection (MOI). After 3 h of incubation at to achieve a left ventricular end-diastolic pressure of 5–10 mmHg. The distal 37 °C, medium was removed and replaced with culture medium, which is end of the catheter was connected to a Heart Performance Analyzer (Micro- DMEM containing 5 mg/L ITS (bovine insulin, human transferrin and sodium Med) via a pressure transducer. Hearts were paced at 400 bpm except during selenite; Sigma), 100 units/mL penicillin streptomycin, 2 mmol/L L-glutamine, ischemia, and pacing was reinitiated 2 min after reperfusion. After a 20-min

4mmol/LNaHCO3, 10 mmol/L Hepes, 0.2% BSA, and 25 μmol/L blebbistatin equilibration period, hearts were subjected to 40 min of no flow global is- (Cayman Chemical). The cell phenotype and morphology remained similar chemia, followed by 60 min of reperfusion. Maximum rate of contraction among noninfected and adenoviral-infected groups after 24 h of infection. (+dP/dt) and maximum rate of relaxation (−dP/dt) were monitored during this process. After reperfusion, the heart was perfused with 1% 2,3,5- Mitochondrial Membrane Potential. At 24 h after adenoviral infection, mouse triphenyltetrazolium chloride and incubated at 37 °C for 10 min. The heart cardiomyocytes were stained with TMRE dye (eBioscience) for 15 min at room was perfused and rinsed with PBS solution, followed by 10% (vol/vol) for- temperature. Fluorescence signals from TMRE excited at 560 nm were col- malin tissue fixation. Five or six transverse slices were cut and weighed after lected at emission of 610 nm, using live-cell confocal imaging. To induce 1 h of fixation. The images of the slices were digitally analyzed for infarct mitochondrial membrane potential transition, stained mouse cardiomyocytes area and total ventricular area, using NIH image software. were treated with 2 mM hydrogen peroxide, in the absence or presence of cyclosporin A (Sigma). The TMRE signal was recorded every minute for a Quantitative Real-Time PCR. Total RNA was extracted and purified from heart course of 20 min to calculate the percentage of cells with preserved mito- tissue with miRNeasy Mini Kit (QIAGEN 133220834). The first-strand cDNA μ chondrial membrane potential before and after oxidative challenge. There were generated from total RNA (1 g) with reverse transcriptase kit (Invitrogen was no significant loss of mitochondrial membrane observed until the 15th– catalog no.11752). PCR was then performed with Bio-Rad real-time ther- 18th minute of the treatment, suggesting that primary cardiomyocytes are mal cycler by using the following specific primer sequences: Mouse mito- ′ ′ resistant to cell death initiation. chondria cyclophilin-D (Ppif): (Forward) 5 -TGGCTCTCAGTTCTTTATCTGC-3 , (Reverse) 5′-ACATCCATGCCCTCTTTGAC-3′, and mouse GAPDH (Forward) 5′-TCAACAGC AACTCCCACTCTT-3′, (Reverse) 5′-ACCCTGTTGCTGTAGCCG- CELL BIOLOGY Mitochondria Isolation and Swelling. Mouse heart mitochondria were isolated TATTCA-3′.RatPpif:Forward:5′-TGG AGT TAA AGG CAG ATG TCG-3′, by homogenization followed by different centrifugation. Briefly, mouse Reverse: 5′-CAT TGT GAT TGG TGA AGT CGC-3′;RatGAPDHForward: hearts were homogenized in Precellys 24 Lysis and homogenization machine 5′- CCTAAATGATACCCCACCGTG-3′,Reverse:5′-TGT CAC AAG AGA AGG at 5,000 cpm for 10 s with 750 μL of 2.3-nm Zirconia/silica beads and an equal CAG C-3′. The values were normalized to those obtained with GAPDH. amount of homogenization buffer containing 250 mM sucrose, 10 mM Tris at pH 7.4, and 1 mM EDTA. The homogenates were spun at 1,300 × g for Rat Myocyte Isolation and Virus Infection. Adult male Sprague–Dawley rats 10 min at 4 °C to pellet nuclei and cell debris. The supernatant was then spun (8-10 wk, 250–350 g) were anesthetized by pentobarbital (100 mg/kg) and at 12,000 × g for 30 min at 4 °C to pellet the mitochondria. After washing supplemented with heparin (5000 U/kg). Hearts were quickly removed and twice in homogenization buffer (minus EDTA), the mitochondria were the aorta was cannulated by the Langendorff mode and perfused with resuspended in buffer containing 120 mM KCl, 10 mM Tris at pH 7.4, and modified KHB: 118 mM NaCl, 4.8 mM KCl, 25 mM Hepes, 1.25 mM K HPO , 5mMKH PO for the swelling assay. Mitochondrial swelling was induced by 2 4 2 4 1.25 mM MgSO , 11 mM glucose, 5 mM taurine, and 10 mM butanedione 50 or 375 mM, where the light-scattering of 250 μgofmitochondriaina1-mL 4 monoxime; pH 7.4) for 5–10 min. Subsequently, hearts were perfused with volume was measured at 540 nm for 10 min. an 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, Propidium Iodide and Annexin V Assays. Cells were gently washed once and 25 μM Ca was added to the perfusion buffer, and hearts were perfused for stained with annexin V-specific APC dye (eBioscience) for 20 min. Various another 5 min. The Ca concentration in the perfusion buffer was raised to concentrations of hydrogen peroxide were applied at the beginning of annexin 100 μM and perfusion continued for 5 min. Once the hearts became flaccid, V treatment. Stained cardiomyocytes were then washed gently once and the left ventricular tissue was excised, minced, and filtered through a 240-μm resuspended in solution containing propidium iodide (eBioscience) for exclu- screen. Cells were quickly centrifuged at low speed (200 × g for 5 min), sion of cells that had lost their membrane integrity. Fluorescence signals from harvested, and resuspended in 25 mL of 100 μM Ca-KHB with 1% BSA. The APC and propidium iodide, excited at 633 nm and 488 nm, were collected at cells were allowed to settle automatically, and the Ca concentration was > emission of 660 nm and 610 nm, respectively, in 10,000 cardiomyocytes per gradually raised to 1.0 mM. Afterward, the cells were washed two times with sample group. FlowJo software (Tree Star) was used to generate the diagram DMEM supplemented with ATCC (2 mg/mL BSA, 2 mM L-carnitine, 5 mM of cell distribution according to fluorescence intensity and to calculate the creatine, 5 mM taurine, 100 IU penicillin, and 100 μg/mL streptomycin). Cells percent of Annexin V-positive population as an indication of apoptosis. were 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, Quantitative Immunoblot Analysis. Hearts were snap frozen in liquid nitrogen Ad.HAX-AS viruses, or control virus Ad.GFP at an MOI of 500 for 2 h, before at the end of the Langendorff perfusion period and homogenized in 1× Cell addition of a suitable volume of complete DMEM (DMEM containing 2 mg/mL Lysis Buffer ( Technology) supplemented with 1 mM PMSF and BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/mL penicillin and complete protease inhibitor mixture (Roche Applied Science). For each 100 μg/mL streptomycin), as described previously (14). The efficiency of ade- protein, equal amounts of samples (5–120 μg) from each heart were an- noviral gene transfer was evaluated in cultured adult rat myocytes with the alyzed by SDS/PAGE, as described (13). After transfer to membranes, im- expression of GFP. Nearly 100% of myocytes appeared infected at 500 MOI by munoblotting analysis was performed with the corresponding primary 24–48 h. The cell phenotype and morphology remained similar among non- antibodies (Bax, Bak, and ubiquitin from Cell Signaling; Cyp-D, COX4 infected and adenoviral-infected groups after 24 h of infection. The myocytes VDAC, ANT1, Hsp90, and TRAP-1 from Abcam; HAX-1 from BD Biosciences; were washed with PBS and harvested for quantitative immunoblotting, or calsequestrin (CSQ) and GAPDH from Thermo Scientific). This step was used in the experiments outlined in the results. followed by incubation with secondary antibody conjugated with horse- radish peroxidase at a 1:5,000 dilution. Visualization was achieved by Coimmunoprecipitation. Hearts from WT, HAX-OE, and HAX+/− mice were using SuperSignal West Pico chemiluminescent substrate (Pierce) or ECL- homogenized with 1× cell lysis buffer (Cell Signaling Technology), supple- PLUS Western Blotting Detection kit (Amersham Pharmacia Biotech). The mented with protease inhibitor mixture and phosphatase inhibitor mixture. intensities of bands were determined by the AlphaEaseFC software. For The homogenate was centrifuged at 9,000 × g for 30 min at 4 °C. The each protein, the densitometric values from pre-I/R WT controls were centrifuged homogenate was diluted to 1 μg/μl (1 mL of final volume) and arbitrarily converted to 1.0, and the values of samples from the other incubated with either anti-HAX-1 (BD Biosciences), anti-Hsp90 (Abcam), anti- groups were normalized accordingly. Cyp-D (Abcam), or anti-ubiquitin (Abcam) at 4 °C overnight with rotation.

Lam et al. PNAS Early Edition | 9of10 Downloaded by guest on September 27, 2021 One hundred microliters of protein G PLUS agarose beads (Santa Cruz Bio- protein/reaction. Incubation proceeded for 2 h at room temperature. Each technology) were added into the mixture and incubated for an additional well was then washed with PBS-T six times, and the HAX-1 antibody, which 5 h. Agarose beads were sedimented and washed six times with the cell lysis was dissolved in blocking solution, was applied for 2 h at room temperature. buffer. Beads-bound proteins were dissociated in 2× SDS at room temper- After six washes with PBS-T, the secondary antibody conjugated with ature for 30 min with vortexing at 5-min intervals. The identification of the horseradish peroxidase was added for 1 h at room temperature. Following associated proteins was detected by Western blots. Homogenates from WT eight PBS-T washes, TMB substrate reagent (BD Biosciences) was applied to hearts were used as positive controls. develop signal in blue color. After color was developed, 1 mol/L HCl was added to stop the reaction. The colorimetric intensity of each well was read Competitive Protein Binding ELISA. Competitive binding assay using at 450 nm with a microplate reader. recombinant proteins was performed as described (35). Briefly, each well on a 96-well high affinity binding plate (BD Falcon catalog no. 351172) was Statistical Analysis. Data were expressed as the mean ± SEM. Comparisons be- coated with 200 ng of recombinant Hsp90β protein (StressMarq) or GST tween the means of two groups were performed by unpaired Student’s t test. protein (Novus Biologicals, as control) in 100 μL of coating buffer (85 mmol/L Multiple groups were analyzed by using one-way ANOVA with a Bonferroni test NaHCO3 and 15 mmol/L Na2CO3, pH 9.5) overnight at 4 °C. After five washes for post hoc analysis. Results were considered statistically significant at P < 0.05. with PBS and 0.05% Tween-20 (PBS-T), blocking solution (PBS containing 1% BSA) was added for 1 h at room temperature. Following another five PBS-T ACKNOWLEDGMENTS. We thank Dr. J. Molkentin for providing the Ad.Cyp-D washes, 400 ng of Cyp-D protein (Abnova) was added along with 400 ng virus; Dr. W. Sessa for providing the Ad.Hsp90 plasmid; Drs. A. Maloyan of “competition protein mixture.” This mixture contained various amounts and J. Robbins for assistance with the mitochondrial swelling assay; and of HAX-1 (Abnova) supplemented with GST to reach a total of 400 ng of Drs. J. Molkentin and J. Karch for helpful discussions.

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