Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death

14 Pages·2015·8.09 MB·English

Washington University School of Medicine
Digital Commons@Becker
Open Access Publications
2012
Enhancing lysosome biogenesis attenuates
BNIP3-induced cardiomyocyte death
Xiucui Ma
Washington University School of Medicine in St. Louis
Rebecca J. Godar
Washington University School of Medicine in St. Louis
Haiyan Liu
Washington University School of Medicine in St. Louis
Abhinav Diwan
Washington University School of Medicine in St. Louis
Follow this and additional works at:http://digitalcommons.wustl.edu/open_access_pubs
Recommended Citation
Ma, Xiucui; Godar, Rebecca J.; Liu, Haiyan; and Diwan, Abhinav, ,"Enhancing lysosome biogenesis attenuates BNIP3-induced
cardiomyocyte death." Autophagy.8,3. 297-309. (2012).
http://digitalcommons.wustl.edu/open_access_pubs/2672
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BASICRESEARCHPAPER
Autophagy8:3,297–309;March2012;G2012LandesBioscience
Enhancing lysosome biogenesis attenuates
BNIP3-induced cardiomyocyte death
Xiucui Ma,1,2 Rebecca J. Godar,1,2 Haiyan Liu1 and Abhinav Diwan1,2,*
1CenterforCardiovascularResearch;DivisionofCardiology;DepartmentofInternalMedicine;WashingtonUniversitySchoolofMedicine;St.Louis,MOUSA;
2JohnCochranVAMedicalCenter;St.Louis,MOUSA
Keywords: BNIP3, autophagy, cardiomyocyte death, lysosomes, TFEB
Abbreviations: BNIP3, BCL2/adenovirus E1B 19 kd-interacting protein; FRET, Forster resonance energy transfer;
NRCM, neonatal rat cardiac myocyte; CQ, chloroquine; 3MA, 3-methyladenine
Hypoxia-induciblepro-deathproteinBNIP3(BCL-2/adenovirusE1B19-kDainteractingprotein3),provokesmitochondrial
permeabilizationcausingcardiomyocytedeathinischemia-reperfusioninjury.InhibitionofautophagyacceleratesBNIP3-
induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating
autophagywillattenuateBNIP3-inducedcardiomyocytedeath.Neonatalratcardiacmyocytes(NRCMs)wereadenovirally
© t2ransduc0ed wit1h BNIP23 (or L acLZ as acontronl; at mdultipliceity ofsinfect ionB= 100i); aond autsophagcy wais setimulanted witch e.
rapamycin(100nM).Celldeathwasassessedat48h.BNIP3expressionincreasedautophagosomeabundance8-foldand
causeda3.6-foldincreaseincardiomyocytedeathascomparedwithcontrol.RapamycintreatmentofBNIP3-expressing
cellsledtofurtherincreaseinautophagosomenumberwithoutaffectingcelldeath.BNIP3expressionledtoaccumula-
tion of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed
withdualfluorescentmCherry-GFP-LC3expression).BNIP3,butnotthetransmembranedeletionvariant,interactedwith
LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular
fractionation,Dprovokeolysos omne permoeabilitzatio ndor alteir lyssosomte prH. Riathber, BNIuP3-indtucedeauto.phagy caused a
decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome
consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in
BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes,
prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude
that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced
expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced
celldeath.
Introduction survival under stress such as nutrient deprivation and hypoxia.15
Induction of autophagy is protective in the ischemic heart,10,12-14
Inmyocardialischemia-reperfusioninjury,programmedcelldeath but has been implicated in causing cardiomyocyte death in
causes substantial cardiac myocyte loss in addition to accidental myocardial reperfusion injury.14 Forced expression of BNIP3
necrosistriggeredbylackofoxygenandnutrientsintheischemic stimulates autophagy in cardiac myocytes,9,10,13,16 with a dose-
core.1 The BCL-2 family of proteins is a key regulator of the dependent increase in autophagosome abundance.16 While
initiation and execution of programmed cell death pathways.2 BNIP3-induced autophagy has been implicated in causing cell
BNIP33,4apro-deathmemberofthisfamily,5-7istranscriptionally deathincancerouscells,17,18inductionofautophagyinthesetting
upregulatedinhypoxiccardiacmyocytes,6,8andcausesmitochon- of BNIP3 expression is protective in cardiac myocytes, as inhibi-
drial permeabilization6,7 and dysfunction9 leading to cell death, tion of autophagosome formation either pharmacologically [with
which is an important determinant of cardiac dysfunction10 and 3-methyladenine (3MA)9] or with co-expression of dominant
post-infarctionremodelingfollowingischemia-reperfusioninjury.11 negative autophagy-related (Atg) protein 5 (Atg5)9,10 increases
In the setting of increased BNIP3 expression, as happens with BNIP3-induced cardiomyocyte death. Conversely, enhancing
cardiac ischemia-reperfusion injury,6 cardiomyocyte autophagy autophagosome formation with forced expression of Atg5 and
is upregulated.10,12-14 Autophagy is an evolutionarily conserved BECN1 appears to attenuate BNIP3-induced cell death in HL-1
lysosomal degradative pathway to remove damaged intracellular cardiac myocytes.10,13 It is not known whether further induction
constituentsthatfacilitatescellularhomeostasis,andpromotescell of protective autophagy in BNIP3-expressing cardiac myocytes is
*Correspondenceto:AbhinavDiwan;Email:[email protected]
Submitted:07/09/11;Revised:10/26/11;Accepted:11/04/11
http://dx.doi.org/10.4161/auto.8.3.18658
www.landesbioscience.com Autophagy 297
limited by the availability of constituents of the autophagic significant increase in punctate GFP-LC3 localization (Fig.1A
machinery, or is actively suppressed, whereby it is unable to fully and B) and increased autophagosome-bound LC3-II abundance
protect cells from BNIP3-induced cell death. (Fig.1C), without a change in LC3 transcription (Fig. S1A).
BNIP3 permeabilizes cardiac mitochondria,19 promotes mito- Importantly, levels of p62, a protein that brings ubiquitinated
chondrial fission,20 and/or renders them dysfunctional,9 and the aggregates into autophagosomes and gets consumed during
damaged mitochondria are removed via macroautophagy,10,20 to autophagy,31 were elevated in BNIP3-expressing cells (Fig.1C),
ensure cellular viability. Indeed, BNIP3 has been proposed as a with a trend toward reduction in p62 transcription (Fig. S1B),
key mediator for autophagic removal of damaged mitochondria suggesting BNIP3-induced impaired clearance of p62. Rapamy-
under hypoxic stress21,22 and in unstressed cardiac myocytes.23 cin added simultaneously (Fig.1A) stimulated autophagy with
This process involves coordinatedaction of multiple Atg proteins increased autophagosome abundance (Fig.1A and B), and
to sequester cargo, such as BNIP3-damaged mitochondria that increased LC3-II expression with a decline in p62 abundance
are targeted for destruction within autophagosomes, which then (Fig.1C) as compared with control. Interestingly, while rapamy-
fuse with lysosomes, wherein degradative enzymes break down cinincreasedautophagosomenumbers(Fig.1AandB)andLC3-
complex organic matter in an intralysosomal acidic environment, II abundance (Fig.1C) in BNIP3-expressing cells, p62 levels did
to recycle amino acids, simple sugars and lipids.24 Rapamycin, a not decline (Fig.1C) indicating lack of p62 clearance despite
potent inducer of cardiomyocyte autophagy,14 also stimulates further induction of autophagosome formation by rapamycin.
selective removal of dysfunctional mitochondria in yeast25 and BNIP3 expression provoked a ~3.6-fold increase in cell death as
neurons,26andattenuatesapoptoticcelldeath.26Inthisstudy,we compared with controls (Fig.1D and E), and further induction
evaluatedwhetherfurtherinductionofautophagosomeformation of autophagy with rapamycin treatment either simultaneously
©with r apa2mycin0treatm1ent att2enuate s BLNIP3a-inducned cellddeathe. ors24 h afBter induictioon of sBNIPc3 expiresseion (Fnig.1Cc), didenot .
Our results implicate lysosomal consumption as a rate-limiting affect BNIP3-induced cell death (Fig.1D and E). Inhibition of
factor in BNIP3-induced autophagy, whereby a strategy for autophagosomeformationwith3MAincreasedcelldeathinboth
enhancingfluxthroughthemacroautophagypathway,ratherthan control and BNIP3-expressing cells, confirming a beneficial role
stimulating autophagosome formation alone, may accelerate forautophagyincardiomyocytehomeostasis andprotection from
removal of BNIP3-damaged mitochondria. BNIP3-induced cell death, as previously described.9
Recent studies have identified a critical role for transcription BNIP3 induces autophagosome accumulation in cardiac
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factor EB (TFEB), in upregulating synthesis of autophagy myocytes. Accumulation of p62 with increased autophagosome
proteins and stimulating lysosomal biogenesis in a coordinated abundanceinBNIP3-expressingcells(Fig.1C)suggestedimpair-
fashion to facilitate starvation-induced autophagy.27,28 We ment of autophagosome processing. Accordingly, we examined
therefore evaluated whether expression of TFEB will enhance autophagosome abundance in the absence and presence of
flux through macroautophagy in the setting of increased BNIP3 chloroquine (CQ), a lysosomal acidification inhibitor, which
expression and protect against BNIP3-induced cell death. Our leads to accumulation of autophagosomes due to impairment in
results demonstrate thatexogenousexpressionofTFEBincreased autophagosome-lysosome fusion and autophagosome removal.32
lysosome biogenesis, alleviating this rate limiting step in BNIP3- Cumulative flux, expressed as a ratio of autophagosome
induced autophagy and significantly attenuated BNIP3-induced abundance in the presence of CQ to autophagosome numbers
cardiomyocyte death. in its absence, was partially impaired with BNIP3 expresssion as
compared with LacZ-expressing adenovirus-transduced control
Results cells (1.3 vs. 4.4 in the control; Fig.2A and B). In contrast,
nutrient deprivation and rapamycin led to autophagy induction
Rapamycin treatment does not attenuate BNIP3-induced with better preserved flux as compared with BNIP3-expressing
cardiomyocyte death. BNIP3-induced autophagy appears pro- cells (ratio of autophagosome abundance with/without CQ: 2.5
tective in cardiac myocytes, as inhibition of autophagy increases with nutrient deprivation and 2.5 with rapamycin; Fig.2B). We
cell death.9,10 In myocardial ischemia-reperfusion injury, induc- next examined the relative abundance of autophagosomes and
tion of BNIP3 is a critical determinant of cell death leading to autolysosomes in BNIP3-expressing NRCMs lentivirally trans-
myocardial dysfunction and post-ischemic ventricular remodel- duced with an mCherry-GFP dual tandem tagged LC3. While
ing.10,11 Rapamycin is a potent inducer of autophagy in autophagosomes are evident as dual fluorescent LC3 puncta (red
cardiomyocytes;14 rapamycin pretreatment attenuates hypoxic +green=yellow),autolysosomesappearred,asGFPfluorescence
cardiomyocyte death,29 and mTOR inhibition with a related is quenched in the acidic intralysosomal environment.33 Control
compound, Everolimus, reduces infarct size and attenuates post- cellsdemonstratedbasalautophagywithapreponderanceofauto-
infarction remodeling in rats subjected to cardiac ischemia- lysosomes (Fig.2C and D). BNIP3 expression led to increased
reperfusion injury.30 Accordingly, to test the hypothesis that autophagosomeabundance,withoutanincreaseinautolysosomes
further induction of autophagy with rapamycin will attenuate (Fig.2C and D). This is in contrast to the predominant increase
BNIP3-induced cell death, we treated neonatal rat cardiac in autolysosomes as compared with controls, observed with
myocytes (NRCMs) with rapamycin, both simultaneously and autophagy induction due to nutrient deprivation and rapamycin
24 h after adenoviral transduction of BNIP3 and assessed cell treatment, indicating intact flux through the macroautophagy
death at 48 h. BNIP3 expression induced autophagy with a pathway under these conditions (Fig.2C and D). Chloroquine
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Figure1.RapamycintreatmentdoesnotprotectagainstBNIP3-inducedcelldeath.(A)Representativeepifluorescenceimages(630(cid:1)magnification)
demonstratingcellularlocalizationofGFP-LC3inNRCMsadenovirallytransducedwithBNIP3orLacZ(ascontrol)for48h,andtreatedwithrapamycin
(100nmol/L)atT(time)=0(simultaneouslyattransduction).Nucleiareblue(DAPI).(B)QuantitationofpunctateGFP-LC3dotsincellsfrom(A)(n=25–
40nuclei/group).pvaluesarebypost-hoctest.(C)ImmunoblotdemonstratingLC3,p62andBNIP3(FLAG)expressioninNRCMsadenovirallytransduced
withBNIP3orLacZ(Con)for48h,andtreatedwithrapamycin(100nmol/L)atT(time)=0or24haftertransduction.Expressionofa-sarcomericactin
(aSA)wasassessedasloadingcontrol.(D)Representativeimages(200(cid:1)magnification)demonstratinglive(green)anddead(red)cellstreatedasin(A).
(E)CelldeathinNRCMsadenovirallytransducedwithBNIP3orLacZ(ascontrol)for48h,andtreatedwithrapamycin(100nmol/L)atT(time)=0or24h
aftertransduction;or3methyl-adenine(7mmol/L)att=24h.n=8/group.*p,0.001vsBnip3-expressingcells.Allpvaluesarebypost-hoctest.
treatment of cells transduced with control (LacZ) adenovirus clearance in BNIP3-expressing cells. In contrast to the effect of
increased autophagosome-bound LC3-II abundance and led to CQ on cell death in control cells, the effect of chloroquine
p62accumulation(Fig.2E),whichwasassociatedwitha1.8-fold treatment on cell death in BNIP3-expressing cells was marginal
increase in cell death (Fig.2F), likely secondary to autophago- (1.2 fold; Fig.2F), correlating with an underlying impairment in
some accumulation (Fig.2A and B) and lack of homeostatic clearanceofautophagosomesandpossiblydamagedmitochondria
clearance of autophagic cargo such as damaged mitochondria. In in BNIP3-expressing cells in the absence of CQ treatment.
contrast, while CQ treatment of BNIP3-expressing cells caused a Interestingly, CQ treatment provoked an accumulation of the
further increase inLC3-II abundance (as comparedwith BNIP3- monomeric, but not the dimeric forms of BNIP3 (Fig.2E) and
expressingcellstreatedwithdiluent;Fig.2E);therewasnofurther rapamycin treatment preferentially reduced monomeric BNIP3
p62 accumulation (Fig.2E), suggesting impaired autophagosome protein levels (Fig.1C), indicating an underlying impairment in
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© 2012 Landes Bioscience.
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Figure2.BNIP3inducesautophagosomeaccumulationinNRCMs.(A)Representativeepifluorescenceimages(630(cid:1)magnification)demonstrating
cellularlocalizationofGFP-LC3inNRCMsadenovirallytransducedwithBNIP3(Red;forFLAG)orLacZ(ascontrol)for48h,andtreatedwithchloroquine
(10mM,blackbars)ordiluent(whitebars)for24hpriortofixation.Nucleiareblue(DAPI).(B)QuantificationofpunctateGFP-LC3dotsincellstreatedas
in(A).andincellssubjectedtonutrientdeprivationortreatedwithrapamycin(100nM)for4hinthepresenceofchloroquine(10mM,blackbars)or
diluent(whitebars).pvaluesarebypost-hoctest.*p,0.05vs.diluent-treatedcontrolgroup.#p,0.05vs.CQ-treatedcontrolgroup(n=15–25nuclei/
group).(C)Representativeepifluorescenceimages(630(cid:1)magnification)demonstratingcellularlocalizationofmCherry-GFP-LC3inNRCMsadenovirally
transducedwithBnip3orLacZ(ascontrol)for48h;subjectedtonutrientdeprivationortreatedwithrapamycin(100nM)for4h.(D)Quantitationof
autophagosomes(green+red;whitebars),autolysosomes(red,blackbars)andboth(graybars)inNRCMstreatedasin(C)(n=20–40nuclei/group).
pvaluesarebypost-hoctest.*p,0.05forautophagosomesvs.control;#p,0.05forautolysosomesvs.controland$p,0.05forbothvs.control
(n=15–25nuclei/group).(E)ImmunoblotdemonstratingLC3,p62andBNIP3(FLAG)expressioninNRCMsadenovirallytransducedwithBNIP3orLacZ
(Con)for48handtreatedwithchloroquine(10mmol/L)ordiluentfor24h.Expressionofa-sarcomericactin(aSA)wasassessedasloadingcontrol.
(F)CelldeathinNRCMstreatedasinE(n=8–24/group).
300 Autophagy Volume8Issue3
clearance of BNIP3-dimer and likely mitochondria that are HEK 293 cells. As previously demonstrated,4 BNIP3DTM
permeabilized only by the dimeric forms of BNIP3, during demonstrated diffuse cellular localization (Fig.3A; Fig. S2), and
BNIP3-induced autophagy.4,5,34,35 did not induce autophagy,10 assessed as increased punctate
The transmembrane domain is essential for the interaction LC3 localization, as compared with controls (Fig.3A; Fig. S2).
of BNIP3 with LC3. The C-terminal transmembrane domain Interestingly, induction of autophagy by rapamycin treatment
of BNIP3 is essential for targeting mitochondria4,5 and the led to markedly increased punctate LC3 localization, but did
endoplasmic reticulum,36 inducing mitochondrial permeabiliza- not alter the subcellular localization of BNIP3DTM (Fig. S2),
tion and causing cell death.4-7,34 Recent studies have identified suggesting that the interaction between BNIP3 and LC3
an interaction between BNIP3 and LC3,9 likely via amino acid requires activation of autophagy with an intact transmembrane
sequences upstream of BNIP3’s C-terminal transmembrane domain. Indeed, assessment of interaction between full-length
domain, based on similarities with a related protein NIX/ BNIP3, and BNIP3DTM with LC3 by FRET (Fig.3A and B)
BNIP3L,37,38 suggesting that BNIP3 acts as a ‘receptor’ for and co-immunoprecipitation as previously described for BNIP3
targeting mitochondria toautophagosomes.9,10,23Interestingly, an and LC39 (Fig.3C), confirmed an obligate role for the trans-
endogenous hypoxia-inducible splice variant of BNIP3 lacking membrane domain in the interaction between BNIP3 and
the transmembrane domain (BNIP3Dex3), but retaining the LC3, potentially involving additional proteins recruited upon
higher affinity LC3 interaction region (LIR; homologous to LIR BNIP3-induced mitochondrial permeabilization and dysfunc-
labeled W35 in BNIP3L37) was recently identified.39 It is con- tion9,16,19,23 as observed with a closely related protein, BNIP3L/
ceivable that at high levels of BNIP3 expression, such as may NIX.37,41 Also, while full-length BNIP3 colocalized both with
occur during hypoxia,6,8 the interaction between concomitantly LC3 and mitochondria in NRCMs, BNIP3DTM did not
©hypox ia-u2pregul0ated B1NIP3D2ex339 anLd LCa3 protnein indvolvinge(Fsig. S3 ); iBndicatinig tohat onsly thecfull-liengeth BNnIP3 pcroteineacts .
the non-transmembrane segment of the BNIP3 protein, such as as a receptor to target damaged mitochondria into autophago-
observed in silico with BNIP3L/NIX,37,38 leads to sequestration somes, as observed with the closely related protein, BNIP3L/
of the available LC3, preventing its role in autophagic removal NIX.37,41 This suggests that increased BNIP3 expression results
of damaged mitochondria. To examine this premise, we assessed in accumulation of autophagosomes containing full-length
the interaction of a transmembrane deletion mutant of BNIP3 BNIP3-targeted mitochondria,7,9,10,13,16 with potential deleterious
(BNIP3DTM; with all putative LIR regions37 intact) and LC3, consequences secondary to impaired removal of these damaged
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each tagged with a FRET compatible fluorophore partner in organelles.
Figure3.ThetransmembranedomainofBNIP3isrequiredforitsinteractionwithLC3totargetmitochondriaintoautophagosomes.(A)Representative
confocalimages(630X)demonstratingFRETinteractionbetweenBNIP3,BNIP3DTM(bothtaggedwithDsRedmonomer)andLC3(taggedwithGFP)in
HEK293cells.CellstransfectedwithDsRed-GFPdualfluorescentconstruct40areshownaspositivecontrol.Nucleiareblue(Hoechstdye).Imageswith
acquiredattheexcitation(Ex)andemission(Em)settingsindicatedandmergedasshown.(B)QuantitationofFRETsignalincellstreatedasin(A)(n=8/
group).CellstransfectedwithconstructsexpressingDsRed-monomerandGFP,separately,areshownasnegativecontrol.pvaluesarebypost-hoctest.
(C)3T3fibroblastswereco-transducedwithadenovirusescodingforFLAG-BNIP3,orHA-BNIP3DTMandGFP-LC3(100MOIeachfor48h)andextracts
subjectedtoco-immunoprecipitationemployingBNIP3,GFPandIgGcontrolantibodies.
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BNIP3 does not target lysosomes or alter lysosome pH. Adenovirally transduced TFEB localized to the nucleus (Fig.5A)
Accumulation of autophagosomes in the setting of increased andexpressionofTFEBincreasedlysosomeabundance(Fig.5A–
BNIP3 expression may occur due to inhibition of subsequent C; Fig. S5A) with increased abundance of LC3 (Fig.5D and F;
autophagosome processing. Additionally, previous studies have Fig. S6A) and LAMP-1 (Fig.5D and H; Fig. S6A), induced
suggested direct targeting of BCL-2 family proteins to lysosomes autophagosome formation as evidenced by punctate GFP-LC3
as a potential mechanism for activation of cell death.42,43 Accord- localization (Fig. S8) and increased the ratio of LC3-II/a-
ingly, we examined whether BNIP3 targets to lysosomes or alters sarcomeric actin (Fig.5D and E), as previously described.28
lysosomal integrity. BNIP3 colocalizes together with both Importantly, TFEB expression restored lysosome abundance in
lysosomes and mitochondria in NRCMs (Fig.4A, see arrows, BNIP3-expressing cells (Fig.5A–C; Figs. S5A and S6A), with
bottom panel). This may indicate that BNIP3 targets to the a reduction in the ratio of LC3-II to LC3-I (Fig.5D; Fig. S6B)
lysosomes independently or via localization on mitochondria and reduced p62 accumulation (Fig.5D and G; Fig. S6A) as
engulfed within autophagosomes that have subsequently fused comparedwithNRCMstreatedwithBNIP3alone.Interestingly,
with lysosomes. Accordingly, to examine the organelle-specific whilelowerexpressionofTFEB(MOI=10)didnotalterBNIP3
localization of BNIP3, we performed subcellular fractionation to protein abundance (Fig. S6A), higher expression of TFEB (MOI
isolate fractions enriched in lysosomes, mitochondria and the = 100) was associated with a decline in BNIP3 protein levels
endoplasmic/sarcoplasmic reticulum in HL-1 cardiac myocytes, a (Fig.5D; Fig. S9) which was prevented by inhibition of auto-
cell line that displays mammalian cardiomyocyte physiology and phagy with 3MA (Fig. S9), suggesting enhanced clearance of
can be easily expanded as necessary.44 As previously demon- BNIP3 by TFEB-induced autophagy. Indeed, exogenous expres-
strated,36 BNIP3 protein segregated to subfractions enriched in sion of TFEB restored autophagosome processing in BNIP3-
©the m itoc2hondr0ial mar1ker CO2X IV , aLnd thae endnoplasmdic retie- exspressin gBcells, wiithoa declsine incprevialenece ofnautophcagosoemes .
culummarkerCALNEXIN,butnotalysosomemarker,LAMP-1 and a commensurate increase in autolysosomes (Fig.6A and B)
(Fig.4B).Takentogether,thesedataindicatethatBNIP3 protein as compared with cells expressing BNIP3 alone. As shown
does not target lysosomes independently of being localized to previously,9,10 BNIP3DTM did not stimulate cardiomyocyte
mitochondria, and suggest that autophagosome-lysosome fusion autophagy(Fig.5DandE)orincreasedeath(Fig.6C).Enhance-
in the setting of BNIP3-induced autophagy is not disrupted. ment of autophagosome clearance by TFEB was associated with
Additionally, BNIP3 did not cause lysosome permeabilization a dose-dependent attenuation in BNIP3-induced cell death
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(Fig. S4) or alter lysosome pH in NRCMs (Fig.4C). (Fig.6C) and in BNIP3-induced TUNEL positivity (Fig.6D
BNIP3-induced autophagososme formation leads to lyso- and E). This was likely secondary to enhanced removal of
some consumption. We next examined whether BNIP3 affects damaged mitochondria by a global induction of the autophagic
lysosome abundance. Expression of full-length BNIP3, but not machinerydrivenbyTFEBinthesettingofBNIP3expression,as
BNIP3DTM, provoked a ~20% reduction in lysosome numbers BNIP3provokedanincreaseingreenfluorescentJC-1monomers
inNRCMsasassessed byuptakeoftwopH-dependentlysosome (Fig.7A and B, top) with a reduction in red fluorescent JC-1
probes, LysoTracker red (Fig.5A–C) and LysoTracker green J-aggregates (Fig.7A and B, bottom) and markedly increased
(Fig. S5A), and cellular levels of LAMP-1 (Fig.5D and H; green to red fluorescence ratio (vs controls, Fig.7C), indicating
Fig. S6A), an abundant lysosomal membrane protein. This increased depolarized mitochondria and reduced numbers of
decline in lysosome numbers closely tracked LC3-II and p62 normally polarized mitochondria,47 and suggesting accumulation
accumulation, and was observed early (within 24 h of BNIP3 of damaged mitochondria48 (Fig.7A–C), which was reversed by
transduction; Fig. S5B and S5C), and at 10-fold lower infective co-expression of TFEB (Fig.7A–C).
viral dose (MOI = 10; FigS5D and S5E). BNIP3-induced
decline in lysosome abundance was prevented by 3MA-mediated Discussion
inhibition of BNIP3-induced autophagy (Fig.5C), suggesting
lysosomal consumption during macroautophagy as the mech- In this study, we demonstrated that BNIP3-induced autophagy
anismforreducedlysosomeabundanceinBNIP3-expressingcells, in cardiac myocytes was rate limited by lysosome consumption,
without requisite replenishment as observed in starvation- and which led to upstream autophagosome accumulation. Expres-
rapamycin-induced autophagy.28,45 Interestingly, a decline in sion of transcription factor EB (TFEB) stimulated lysosome
LAMP-1 levels was also observed in NRCMs subjected to biogenesis and restored processing of autophagosomes. The
prolongedhypoxia,whichprovokesmaximalaccumulationofthe resultant enhanced flux through the macroautophagy pathway
BNIP3 protein8,46 (Fig. S7), suggesting that BNIP3-induced attenuated BNIP3-induced cell death.
lysosomalconsumptionmaybeofpathophysiologicalsignificance The primary stimulus for autophagy induction with increased
in ischemic cardiac injury. BNIP3 expression, as occurs with hypoxic insult,6,8 appears to be
Enhancing lysosomal biogenesis with transcription factor EB BNIP3 targeting to the organelle, in particular the mitochon-
expression rescues BNIP3- induced cell death. To determine dria.4,9,10,36 Indeed, our data confirm an obligate role for the
whether restoring lysosome abundance would promote auto- transmembrane domain of BNIP3 protein, which is essential for
phagosomeclearanceinBNIP3-expressingNRCMs,weexpressed organelle targeting,4,36 in inducing autophagy. BNIP3 induces
thetranscriptionfactorEB(TFEB),whichwasrecentlydescribed mitochondrial damage by multiple mechanisms, such as mito-
topromotebiogenesisoflysosomes27,28andautophagyproteins.28 chondrial outer membrane permeabilization in concert with Bax
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Figure4.BNIP3doesnottargettolysosomesoraffectlysosomalacidification.(A)Representativeconfocalimages(630(cid:1))ofNRCMsadenovirally
transfectedwithLacZ(Control,toppanel)orBNIP3(green,middlepanel,zoomed-inviewinbottompanel),costainedforlysosomes(red;LysoTracker
red)andmitochondria(pink,MitoTrackerdeepred),demonstratingcolocalizationofBNIP3withmitochondriaandlysosomes(whitearrowheads).
(B)HL-1cardiacmyocyteswereadenovirallytransducedwithLacZ(Control)orFLAG-Bnip3for48handsubcellularfractionationperformedtoobtain
fractionsenrichedforlysosomes(Lys),mitochondria(mito),endoplasmicreticulum(ER)andcytoplasm(C).Representativeimmunoblotdemonstrating
distributionofBNIP3(FLAG)infractionsenrichedforLAMP1(lysosomemarker),COXIV(mitochondrialmarker)andcalnexin(ERmarker).
(C)Representativeconfocalimages(630(cid:1))ofNRCMsadenovirallytransducedwithLacZ(Control)andBNIP3(red)for48handstainedwithlysosensor
yellow/bluetoassessacidificationstatusoflysosomes.Imageswereobtainedatexcitationwith360nmandemissionsplitbetween390–460nm
(blue,correspondingtoemissionmaximaatpH9.0)and490–550nm(yellow,correspondingtoemissionmaximaatpH3.0).NRCMstreatedwith
chloroquine(10mmol/Lfor1h)toinhibitlysosomalacidificationareshownascontrols.
andBak,49mitochondrialpermeabilitytransitionbyanovelcyclo- phosphorylation/electron transport chain proteins.9,23 The oblig-
philin D-independent mechanism,19 mitochondrial fragmenta- ateroleofthetransmembranedomanintheinteractionofBNIP3
tion in concert with Opa19,50 and Drp1,20 and mitochondrial with LC3 proteins (see Fig.3) suggests that mitochondrially-
energetic dysfunction via protease-mediated cleavage of oxidative localizedBNIP3interactswithautophagosome-boundLC3-IIasa
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Figure5.BNIP3-inducedautophagyisassociatedwithreducedlysosomeabundance,whichisrestoredbyco-expressionofTFEB.(A)Representative
epiflourescenceimages(630X)demonstratinglysosomedistribution(byLysoTrackerredstaining)incellsadenovirallytransducedwithBnip3,TFEB
(green,at100MOI),BNIP3+TFEB(at100MOIeach)for48h.Nucleiareblue(Hoechstdye).AdenoviruscodingforLacZexpressionwasaddedas
necessarytoresultinequivalentMOIs(attotal200MOIpertreatment).(B)FlowcytometricanalysisofLysoTrackerredstainingincellstreatedasin(A).
Controlisdepictedinblack,BNIP3ingreen,TFEBinredandBNIP3+TFEBinblue.(C)AssessmentofLysotTrackerredexpressionbyflowcytometryin
NRCMsexpressingBNIP3,BNIP3DTM,TFEB,BNIP+TFEBfor48h;andinBNIP3expressingcellstreatedfor24hwith3MA(7mmol/L).(D)Representative
immunoblotsdemonstratingLC3,p62,BNIP3(FLAG),BNIP3DTM(HA),TFEB(bothratTFEBandHAtaggedhumanTFEB)andLAMP1expression,with
a-sarcomericactin(aSA)inNRCMsadenovirallytransducedwithLacZ(control),BNIP3,BNIP3DTM,TFEBandBNIP3+TFEBasin(A)(for48h).(E–H)
QuantitativeassessmentofLC3-II/a-sarcomericactinratio(E),totalLC3(F),p62(G)andLAMP1(H)abundanceinNRCMstreatedasinD(n=3–7/group).
pvaluesarebypost-hoctest.
receptor to facilitate autophagic removal of these damaged macroautophagy pathway in BNIP3-expressing cells, and suggest
mitochondria.9,10,23 that autophagic flux is intact in this setting.9 While a BNIP3-
Recent studies have employed bafilomycin A to inhibit induced increase in autophagosomes is incontrovertible, the ratio
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lysosome acidification and assess cumulative flux through the of autophagosome abundance with bafilomycin A treatment as
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304 Autophagy Volume8Issue3
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Figure6.ForcedexpressionofTFEBrestoresautophagosomeprocessingandattenuatescelldeathinBNIP3-expressingNRCMs.(A)Representative
epifluorescenceimages(630(cid:1)magnification)demonstratingcellularlocalizationofmCherry-GFP-LC3inNRCMsadenovirallytransducedwithLacZ
(ascontrol),BNIP3,TFEBandBNIP3+TFEBfor48h.Nucleiareblue(DAPI).AdenoviruscodingforLacZexpressionwasaddedasnecessarytoresultin
equivalentMOIs(attotal200MOIpertreatment).(B)Quantitationofautophagosomes(green+red;whitebars),autolysosomes(red,blackbars)andboth
(graybars)LC3inNRCMstreatedasin(A)(n=25–40nuclei/group).*p,0.05vs.autophagosomes,$p,0.05vsautolysosomesand#p,0.05vs.bothin
controlgroupbypost-hoctest.(C)AssessmentofcelldeathinNRCMstransducedwithLacZ(control),BNIP3,BNIP3DTM,TFEB(atMOIs=10and100)and
BNIP3+TFEB(atMOIs=10and100)for48h.AdenoviruscodingforLacZexpressionwasaddedasnecessarytoresultinequivalentMOIs(attotal
200MOIpertreatment).*p,0.05vs.controland#p,0.05vs.BNIP3bypost-hoctest.(D)Representativeepifluorescenceimages(200(cid:1)magnification)
demonstratingTUNELstaining(green)inNRCMstreatedasin(A).Nucleiareblue(DAPI).(E)QuantitativeassessmentofTUNELpositivityinNRCMs
treatedasin(A).AdenoviruscodingforLacZexpressionwasaddedasnecessarytoresultinequivalentMOIs(attotal200MOIpertreatment)forboth
(DandE).Treatmentwithstaurosporine(1mmol/Lfor24h)wasemployedaspositivecontrol(n=5–9experiments/group).pvaluesarebypost-hoctest.
compared with no treatment was low in BNIP3-expressing cells Atg59,10) or autophagosome processing (with CQ, see Fig.2; or
when compared with a similar ratio in controls (1.6 in BNIP3- bafilomycinA treatment9),andimpairstheroleforautophagyin
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expressing cells vs. 5 in controls).9 These data are comparable to preventing BNIP3-mediated programmed cell death.
our results with CQ (which also inhibits lysosomal acidification; Previous studies have demonstrated that inducing autophago-
see Fig.2A and B), and taken together, suggest a partial impair- some formation with transfection of Atg510 or Becn113 attenuates
ment in autophagosome processing in BNIP3-expressing cells. BNIP3-induced cell death. We observed that stimulating auto-
Our data also suggest that under conditions of high levels of phagosomeformationwithrapamycindoesnotattenuateBNIP3-
BNIP3 expression as may occur with myocardial ischemia- induced cell death (Fig.1). The observed differences may be
reperfusion injury,6,8 autophagosome processing is impaired, as attributable to level of BNIP3 expression, whereby autophago-
indicated by accumulation of p62, a protein that gets consumed some accumulation is only observed at high levels (as obtained
intheautophagicprocess,31andaccumulationofautophagosomes with adenoviral transduction of Bnip3), but not at lower levels
without a commensurate increase in autolysosomes. Impaired typically achieved with transfection based-methods in cardiac
autophagosome clearance prevents removal of BNIP3-damaged myocytes. Alternatively, the previous studies10,13 may indicate a
mitochondria, similar to that observed with preventing auto- specific impairment in ATG5 and/or BECN1 levels or function
phagosome formation (e.g., with 3MA, or dominant negative in the context of BNIP3-induced autophagy, whereby restoring
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