Characterization of Kcnk3 -Mutated Rat, a Novel Model of Pulmonary Hypertension Mélanie Lambert, Véronique Capuano, Angele Boet, Laurent Tesson, Thomas Bertero, Morad Nakhleh, Séverine Remy, Ignacio Anegon, Christine Péchoux, Aurélie Hautefort, et al.
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Mélanie Lambert, Véronique Capuano, Angele Boet, Laurent Tesson, Thomas Bertero, et al.. Charac- terization of Kcnk3 -Mutated Rat, a Novel Model of Pulmonary Hypertension. Circulation Research, American Heart Association, 2019, 125 (7), pp.678-695. 10.1161/CIRCRESAHA.119.314793. hal- 02346635
HAL Id: hal-02346635 https://hal.archives-ouvertes.fr/hal-02346635 Submitted on 8 May 2020
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ORIGINAL RESEARCH Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension Mélanie Lambert, Véronique Capuano, Angèle Boet, Laurent Tesson, Thomas Bertero, Morad K. Nakhleh, Séverine Remy, Ignacio Anegon, Christine Pechoux, Aurélie Hautefort, Catherine Rucker-Martin, Boris Manoury, Valérie Domergue, Olaf Mercier, Barbara Girerd, David Montani, Frédéric Perros, Marc Humbert, Fabrice Antigny
RATIONALE: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier +K channel, have been identified in pulmonary arterial hypertension patients. OBJECTIVE: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. METHODS AND RESULTS: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3-mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3-mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3-mutation Downloaded from http://ahajournals.org by on May 7, 2020 induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3-mutated rats were characterized by overactivation of ERK1/2 (extracellular signal–regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3-mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil- induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3-mutated rats developed age-dependent PH associated with low serum-albumin concentration. CONCLUSIONS: We established the first Kcnk3-mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH. VISUAL OVERVIEW: An online visual overview is available for this article.
Key Words: echocardiography ◼ exon ◼ extracellular matrix ◼ hypoxia ◼ rats
Editorial, see p 696 | In This Issue, see p 659 | Meet the First Author, see p 660
ulmonary arterial hypertension (PAH) is an uncom- pulmonary vascular resistance >3 Wood units at rest.2 mon, progressive, and severe disease with an esti- PAH results from increased pulmonary vascular resis- Pmated prevalence of 15 to 50 per million.1 PAH tance because of remodeling of the small distal pulmo- has been hemodynamically defined by an elevation of nary arteries (PAs) and arterioles (diameter <500 µm), the mean pulmonary artery pressure >20 mm Hg and causing adaptive right ventricular (RV) hypertrophy and
Correspondence to: Fabrice Antigny, INSERM UMR_S 999, Hôpital Marie Lannelongue, 133, Ave de la Résistance, F-92350 Le Plessis Robinson, France. Email [email protected] The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.119.314793. For Sources of Funding and Disclosures, see page 694. © 2019 American Heart Association, Inc. Circulation Research is available at www.ahajournals.org/journal/res
678 September 13, 2019 Circulation Research. 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793 Original Research 679 KCNK3 8 4 channel + mutation KCNK3 In 2013, whole- 3 participates in regu- identified 2 additional channel member of the channel 9 5 September 13, 2019 + -Mutated Rats -Mutated of Kcnk3 Characterization channel family. Patients with the with Patients family. channel + Finally, in 2017, 2 new mutations in 2017, Finally, 4 over-activation, mitochondrial depo- over-activation, mitochondrial KCNK3 mutations carriers’ patients are
6,7 In 2016, Navas et al channel) or K2P3.1 channel) 4 + channel family, characterized by 4 transmem- characterized family, channel + KCNK3 encodes an outward-K mutated rats are sen- mutated rats arteries from Kcnk3- Pulmonary vaso- less responsive to and are sitized to constriction relaxing stimuli. larization, pulmonary artery vasoconstriction, pulmonarylarization, pulmonary artery vasoconstriction, switch,vessels remodeling, endothelial cell phenotype These and reduction of RV cardiomyocytes excitability. results highlight that KCNK3loss of function is consis- and demonstrate that pathobiology with human PAH tent model This KCNK3-dysfunction in PAH. is a key events the initiat- presents new opportunities for understanding hypertension. of pulmonary ing mechanisms (Twik-related-acid- also known as TASK-1 channel, sensitive-K mutation are younger at diagnosis than idiopathicthan diagnosis at younger are mutation KCNK3 is a hallmark expression of function and Loss patients. PAH levels. pulmonary vascular and right ventricular at of PAH dysfunction linking KCNK3 the mechanisms However, unique Kcnk3-mutated were unknown. Using with PAH cellsmuscle smooth arterial pulmonary human and rats associ- mechanisms cellular the decipher to aimed we ated with KCNK3-dysfunction. Our results demonstrate patho- that KCNK3 loss of function is a key event in PAH mea- genesis. Using a combination of hemodynamic and histological analyses, surements, molecular biology, we show that KCNK3-electrophysiology approaches, depolarization, membrane plasma to leads dysfunction overproliferation,cells muscle smooth arterial pulmonary ERK1/2 and HIF1-α brane domains and 2 pore domains per subunit. 2 pore K associated with an aggressive form of PAH and early associated with an aggressive form of PAH disease development. mutations in PAH patients and described the KCNK3 mutations in PAH homozygous a with patient PAH first when unchecked, right heart failure. when unchecked, Pulmonary arterial hypertension (PAH) is a progressive arterial hypertension (PAH) Pulmonary Since 2013, several loss of functionand severe disease. in select gene have been detected mutations in KCNK3 KCNK3 encodes an outward-K patients. PAH lating resting membrane potential in several cell typescell several in potential membrane resting lating including human pulmonary arterial smooth muscle cells exome sequencing revealed 6 types of heterozygous exome subfamily K mutations in KCNK3 (potassium channel channelopathy identi- member 3), representing the first PAH. fied in were identified in an Asian and an American cohort of patients. PAH member of the 2-pore K the member of • younger at diagnosis than idiopathic PAH patients and younger at diagnosis than idiopathic PAH have a higher mean pulmonary artery pressure.
and ERK1/2 Novelty and SignificanceNovelty and HIF-1and factor-1 [hypoxia-inducible α α-smooth muscle actin hypoxia chronic matrix extracellular endothelium-derived hyperpolarizing factor factor-1 α hypoxia-inducible synthase nitric oxide pulmonary artery pulmonary arterial hypertension pulmonary arterial smooth muscle cell pulmonary hypertension right ventricular systolic pressure total pulmonary resistance factor Willebrand Von wild type
monocrotaline
By CRISPR/Cas9 we generate Kcnk3- technology, mutated rats to inactivate the KCNK3 channel. - 4 months old, Kcnk3-mutated rats develop sponta At pressure, neous elevation of right ventricular systolic hypertension are sensitized to induced pulmonary and develop age- hypoxia), (monocrotaline or chronic months. dependent pulmonary hypertension at 12 vessels Kcnk3 dysfunction leads to distal pulmonary deposi- neomuscularization and perivascular collagen proproliferative overactivation of associated with tion pathways (ERK1/2 signal–regulated [extracellular kinase1-/2] α]) and endothelial phenotype switch. identify that an increase of HIF1-We α to linked events molecular primary the are Kcnk3-dysfunction. (potassium in the KCNK3 (potassium function mutations of Loss K member 3) gene have been subfamily channel (PAH) arterial hypertension in pulmonary described who are carriers of KCNK3 muta- patients and those compared with idiopathic PAH tions are younger as patients. Reduced KCNK3 to is common expression/function experimental to and PAH nonheritable and heritable pulmonary hypertension. KCNK3pulmonary arte- mouse in functional is not cell limiting previous mechanistic rial smooth muscle exploration. Nonstandard Abbreviations and Acronyms Nonstandard Abbreviations α-SMA CH ECM EDHF HIF1-α MCT NOS PA PAH PASMC PH RVSP TPR VWF WT • • • What New Information Does This Article New Information Does This What Contribute? • • • What Is Known? What • 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research pathway asalreadymentioned elsewhere. degradation gesting anabsence of nonsense mRNA deleted of94bpbutnotitsabsence(Figure 1B)sug- RV levels. function is a hallmark of PAH at pulmonary vascular and patients) andidiopathicPAH, dys- indicatingthatKCNK3 to the development of heritable PAH (BMPR2 mutated dysfunctioncontributes recently determinedthatKCNK3 PASMCs does not form a functional channel in mouse KCNK3 as in pulmonary hypertension (PH) the role of KCNK3 a lossoffunctionforallKCNK3mutationsidentified. 680 andCas9mRNA, rKCNK3 systemwithaspecificsgRNA- using CRISPR-Cas9 Kcnk3-mutated Sprague-Dawleyratsweregenerated PASMCs From Rats Kcnk3-Mutated Current IsAbsentinFreshlyKCNK3 isolated RESULTS Supplement. the MaterialsandMethodsispresentedinOnlineData the articleanditsOnlineDataSupplement. The authorsdeclarethatallsupportingdataareavailablewithin METHODS (PASMCs). Lambert etal Kcnk3 mRNA, mature stopcodonmaygenerateadegradationofthe aa (amino acid) sequence (Figure stop codonandthegenerationofacompletelydifferent shift intheopenreadingframethatleadstoapremature bp (Δ rats,adeletionof94 Kcnk3 gene.Inoneofthenewborn therapeutic targetsforPH. mutated ratmodel,providingtoolsfordevelopingspecific iology approaches, wecharacterized thefirstKcnk3- molecular biology, histologicalanalyses,andelectrophys- development. nisms involvedinPH study thetime-dependentcellularandmolecularmecha- PAH pathobiology, representinganinterestingmodelto dysfunctioninthecontext of decipher theroleofKCNK3 fore, wedevelopedthefirstKcnk3-mutatedratlineto mRNA from Kcnk3 sequencing oftheKcnk3mRNA only thefirst14aaincommon withtheWTprotein. The aa ofthewild-type(WT)protein (Figure 1A)andshare proteininsteadofthe411 produce atruncated90-aa could less, aputativetranslationof thetruncatedmRNA vasculature, isfunctionallyexpressed inpulmonary where KCNK3 dysfunction withPAH arestillunknown.Contrarytorats Using acombinationofhemodynamicmeasurements, Because ofspacelimitations,adetaileddescription 94ex1) was found,which originatedanoutofframe September 13,2019 Δ94ex1/Δ94ex1 15 we showed the presence of a mutated mRNA we showed the presence of a mutatedmRNA 12 10,11 and is replaced by KCNK6 channel. andisreplacedbyKCNK6 8 Electrophysiological recording demonstrated Electrophysiologicalrecordingdemonstrated 10 However, themechanisms linkingKCNK3 suitable models to study micearenota suitablemodelstostudy ratsconfirmed thedeletionof94 14 targetingthefirst exon of 1A). Although pre- 15 Neverthe- 13 Circulation Research.2019;125:678–695. 10.1161/CIRCRESAHA.119.314793 DOI: +/+ There- and 4 We We KCNK3 channel inhibitor(A293at200 nmol/L) KCNK3 erozygous by otherK suggesting that isolated fromWTandKcnk3-mutatedrats(notshown), from infreshlyisolatedPASMCsstrongly decreased(60%) Kcnk3 The mRNA expression levels of KCNK2, KCNK6, K KCNK6, expression levelsofKCNK2, The mRNA the A293-insensitiveK defined itastheA293-sensitiveK sure (RVSP) inKcnk3 sure (RVSP) but significantincreaseinright ventricularsystolicpres- catheterization at4months of age.We observed aslight weperformedclosedchest right-heart taneous PH, To determine whetherKcnk3-dysfunctionleadstospon- Female Right Ventricular SystolicPressure inMales and Kcnk3-Dysfunction Leads toanIncrease in level ofK However, weobservedasignificantincrease inthemRNA was unchanged inKcnk3-mutatedratscomparedwithWT. K K KCNK9, of KCNK4, the KCNK3 current(I the KCNK3 WT (Figure 1Dand1E).We thenspecificallymeasured demonstrating thattheratswereeitherKcnk3 analysis, ted totheoffspringasshownbygenotypeDNA crossed withaWTpartner, andthemutationwastransmit- potential prematurestopcodons(OnlineFigureI). bp and an aberrant potential protein sequence with 8 rats and 60% inKcnk3 rats and60% current wassignificantlyreduced:50%inKcnk3 forKCNK3, antibodies (Figure 1C). Unfortunately, thereisnoavailablespecific other lungK to evaluatetheconsequencesofKcnk3-dysfunctionon transcription polymerasechain reaction) (Figure 1H). We performed RT-qPCR (quantitativereverse cantly depolarized compared with PASMCs from WT rats PASMCs isolated from inrestingmembranepotential, with theroleofKCNK3 in KCNK3 currentinmutatedrats.Indeed,theglobal-K in KCNK3 from WTandKcnk3-mutatedratsclearlyshowedadefect patch-clamp measurementonfreshlyisolatedPASMCs protein inKcnk3-mutatedrats.Nevertheless,wholecell rats. Thus, wecould not asserttheabsenceofKCNK3 The sameunspecificstainingoccurredinKcnk3-mutated andIIbis). same signalasintheWTmice(OnlineFigureII knockout mice,theywereeithernonreactiveorgavethe tested wereunusablebecausewheninKcnk3- We antibodies showed that all commercially anti-KCNK3 inKcnk3-mutatedrats. of theproteinexpression ofKCNK3 V 2.1, K The founderanimalwiththeΔ Kcnk3 Δ94ex1/Δ94ex1 V V 9.3, K 1.2 andK + currents. Kcnk3 Δ94ex1/+ + channels (Online Figure III). Noexpression channels (Online Figure III). 1F and 1G). Consistent rats(Figure 1Fand1G).Consistent Ca dysfunction is not compensated Kcnk3-dysfunction is not compensated 1.1, K Δ94ex1/+ rats and was abolished (95%) in (95%)in ratsandwasabolished Ca 3.1 inKcnk3 V 16,17 1.4, orK Δ94ex1/Δ94ex1 , orhomozygousKcnk3 + Ca Kcnk3-mutated rats weresignifi- KCNK3 Δ94ex1/Δ94ex1 Characterization ofKcnk3-MutatedRats -current wassimilarinPASMCs 2.1, K thus avoiding any measurement thusavoidinganymeasurement ) after applying a specific ) afterapplyingaspecific Ca Ca ratscompared withWT 4 channels wasdetected. 2.2, K rats compared with the rats comparedwiththe Δ94ex1/Δ94ex1 94ex1 mutationwas + -current. I Ca 2.3, andK rat lungs.Yet, experiments KCNK3 Δ94ex1/Δ94ex1 +/+ 10 Δ94ex1/+ , het- was was Ca and and V 1.5, 1.5, 4.2 4.2 +
Original Research . 681 +/+ (A293 sensitive- =20 cells, 4 rats). KCNK3 =13 cells, 4 rats). rats, corresponding to September 13, 2019 Δ94Ex1/Δ94Ex1 Δ94Ex1/Δ94Ex1 Δ94ex1/Δ94ex1 -Mutated Rats -Mutated of Kcnk3 Characterization and Kcnk3 Δ94ex1/+ =21 cells, 4 rats, Kcnk3 =10 cells, 4 rats, Kcnk3 , Kcnk3 Δ94Ex1/+ +/+ rats, using 2 oligo combinations rKcnk3-17F/rKcnk3-1146R rats, using 2 oligo combinations rKcnk3-17F/rKcnk3-1146R Δ94Ex1/+ +/+ =35 cells, 4 rats). *P<0.05, **P<0.01, ***P<0.001 vs Kcnk3 allele (400 bp) and Δ94ex1deletion allele (306 bp). D, Current- +/+ Δ94Ex1/Δ94Ex1 rats, and Kcnk3 Δ94ex1/+ rats, Kcnk3 =28 cells, 4 rats, Kcnk3 Δ94Ex1/+ Δ94ex1/Δ9e4x1 -currents, in freshly isolated PASMCs from Kcnk3 -currents, in freshly isolated PASMCs + -current, KCNK3 inhibitor (200 nmol/L, current was isolated by applying a specific A293). I + measured at +60 mV. H, Using a current clamp, we measured the resting membrane potential of freshly isolated measured at +60 mV. KCNK3 -current) was then measured and presented in (F, WT=12 cells, 4 rats, Kcnk3 + voltage relationship of global-K the depolarizing step protocol (+60 mV to −100 mV, holding potential −40 current was normalized by the capacitance mV). E, The of the cell the depolarizing step protocol (+60 mV to −100 mV, and is represented in pA/pF (pico ampere/pico farad; WT=16 cells, 4 rats, Kcnk3 (left; WT: 1148 (right; WT: 304 of the deletion bp/Δ94ex1: 1054 bp/Δ94ex1: bp) and rKcnk3-17F/rKcnk3-303R C, Validation 210 bp). by PCR, showing genomic DNA amplicons from rats carrying the Kcnk3 Figure 1. KCNK3 subfamily K member 3) is no longer functional in pulmonary arterial smooth muscle cells channel (potassium rats. (PASMCs) from Kcnk3-mutated generated Kcnk3-mutated rats with 94Kcnk3-mutant overview of the Kcnk3 gene with wild-type (WT) and bp A, Schematic alleles. We DNA Kcnk3 and Δ94deletion in exon 1 of the Kcnk3 (Δ94ex1). bp DNA Below, (strikethrough letter) and protein sequences in WT and (reverse transcription codon. B, mRNAΔ94ex1 analyzed by RT-PCR levels of Kcnk3 rats. Gray: aberrant/potential protein sequence. *STOP reaction) in Kcnk3 polymerase chain After recording, the global-K K G, Current density of I Experiments were analyzed using 1-way ANOVA followed by Tukey post hoc test for multiple comparisons. ns indicates nonsignificant. followed by Tukey Experiments were analyzed using 1-way ANOVA PASMCs (WT=34 4 rats, Kcnk3 cells, PASMCs 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research Kcnk3 mutated rats,comparedwithWTrats. valuesinKcnk3- andTPR demonstrated byhigherRVSP as PH, function increased the severityofCH-induced 682 (Figure 3B). Moreover, thelevelof fibrillarcollagen nificantly increasednumbers ofmuscularizedvessels percentage ofnonmuscularized vesselsalongwithsig- Kcnk3-mutated ratspresentedasignificantly decreased (Von Willebrand factor, endotheliummarker;Figure 3A). smooth muscleactin,smooth-muscle marker)andVWF µm) by immunostaining against sels (<30 We analyzed muscularization of small pulmonary ves- Collagen Crosslinking Vessel Neomuscularization andIncreases Kcnk3-Dysfunction Pulmonary Leads toDistal O MCT-exposure. under Kcnk3-dysfunction sensitizes rats to develop PH pressure (notshown).These resultsdemonstratethat Figure IVB) with no modification of mean carotidartery (Figure 2C;Online group, associatedwithhigherTPR malesandfemales Kcnk3-mutated inboth higher RVSP the severityof MCT-induced asdemonstrated by PH mg/kg).Kcnk3-dysfunctionincreased (60 rats toMCT Therefore, weexposed 4-month-oldmaleandfemale rats displayedamodestbutsignificantincreaseinRVSP. As shown in Figure (CH)–Induced PH (MCT)-orto Monocrotaline Chronic Hypoxia RatsWereKcnk3-Mutated More Susceptible in weresignificantlyincreased monary resistances(TPR) ure 2A) andinfemales (OnlineFigureIVA). Total pul- output ormeancarotidarterypressuresinmales(Fig- hypertrophy (Fulton index) nor by a change in cardiac However, wasnotaccompaniedbyRV increasedRVSP in males(Figure 2A)andfemales (OnlineFigureIVA). Lambert etal in female (OnlineFigureV). icantly decreased,whereasnovariationswereobserved aldosteronism. measured inKcnk3-mutatedrats(OnlineTable IV). We didnotobservemodificationintheotherparameters rate was significantlyincreasedinKcnk3 indicating ahigherresistivePA profile.Inaddition,heart significantly reducedinKcnk3-mutatedrats(Figure 2B), cardiac function.Pulmonary arteryaccelerationtimewas quences ofKcnk3-dysfunctionat4monthsold(males)on ure 2A) andfemales (OnlineFigureIVA). 2 for3weeks). As showninFigure 2D, Kcnk3 In addition, male rats were exposed (10% to CH In mice,Kcnk3deletioncausesprimaryhyper- Using echocardiography, weexamined theconse- September 13,2019 Δ94ex1/Δ94ex1 Δ94ex1/Δ94ex1 18 , serumaldosteroneconcentrationsignif- Conversely, in4-month-oldmale rats compared with WT males (Fig- 2A, 4-month-old Kcnk3-mutated Δ94ex1/Δ94ex1 -SMA (α- α-SMA Kcnk3-dys- Circulation Research.2019;125:678–695. 10.1161/CIRCRESAHA.119.314793 DOI: rats. alteration oftheendothelialfunctions. suggesting that ecules arecriticaltomaintainingendothelialintegrity, ure 4C), which arealteredinPAH patients. strongly reduced(50%)inPASMCs fromKcnk3 ever, phosphataseactivitywas weshowedthatPP2A proteinswasunchanged. How- andPP2A-C PP2A-B, sented inOnlineFigureVIA,theexpression ofPP2A-A, (proteinphosphatase2A).Aspre- threonine PP2A andAKT aretargets for serine– (Figure 4A). ERK1/2 element–binding protein)inKcnk3 response (cAMP in the phosphorylation of CREB transcription factor 1) family bHLH enchymal (twist (endoMT) transcriptionfactorTWIST1 increase in the expression of the endothelial-to-mes- to beinvolvedinPAH pathogenesis. We observed an (or calcineurin)activitywasunchanged (Figure 4B). α that HIF1- (Figure 4A).Wewithout modificationofP38 found nificantly increasedinlungfromKcnk3-mutatedrats, sig- 1/2,andSRC the phosphorylationstateofERK contribute toPAH pathobiology. ComparedwithWT, proproliferative orantiapoptoticproteinsknownto We analyzedtheexpression andactivationofsome RatLungs Mutated Proliferation PathwaysWere ActivatedinKcnk3- increase inthesurvivinproteinKcnk3 expression (notshown).WemRNA alsomeasuredan mutated rats(Figure 4A),withoutalterationofHIF1-α was alsosignificantlyincreasedinlungsfromKcnk3- lungs fromKcnk3 andsignificantincreasesofVWF expression in CD31 Western blot analysis showed significant decreasesof mutated rats compared with WT (Figure not detectmodificationofmicrovesseldensityinKcnk3- (cluster ofdifferentiation-31) pulmonaryvessels,wedid microvessel density. BycountingthenumberofCD31 also be explained by the modification of pulmonary sels fromKcnk3-mutatedrats(Figure 3C). assembly was abnormallyincreasedinpulmonaryves- Mmp2, andStat3inKcnk3 ofNfatc1, measured anincreaseinthemRNA Thr308 significantlyincreasedinKcnk3 Thr308 (Figure 4A). AKT (proteinkinaseB) phosphorylationat Δ94ex1 -mutated rats at 4 months old (Online Figure VIIIA). Kcnk3-mutated ratsat 4monthsold(OnlineFigure VIIIA). IL-17Ra and Figure VII). was observedinlungsfrom Kcnk3-mutated rats(Online andCaveolin-1proteinexpression phospho-SMAD2/3, phospho-SMAD1/5/8, Novariationfor BMPRII, VIB). We alsomeasuredseveralothermoleculesknown The inKcnk3-mutatedratscould increaseinRVSP The mRNA expression ofIL-6, The mRNA receptor(GP130), comparedwithPASMCs fromWTrats,whilePP2B (hypoxia-inducible factor-1α Timp1 was unchanged in the lung from Kcnk3-dysfunction may result in an Δ94ex1/Δ94ex1 Characterization ofKcnk3-MutatedRats Δ94ex1/ Δ94ex1 rats(Figure 3E).Bothmol- rats(OnlineFigure Δ94ex1/Δ94ex1 and a decrease 3D). However, Δ94ex1/Δ94ex1 Δ94ex1/Δ94ex1 ) expression rats (Fig- 19,20 Notch3, Δ94ex1/ rats rats We We +
Original Research 683 , Kcnk3- 9 and = +/+ and <0.01, +/+ Δ94Ex1/+ <0.05, **P P September 13, 2019 Kcnk3-mutated rats and and =14 rats). * +/+ , Four-month-old male Kcnk3 , Four-month-old -Mutated Rats -Mutated of Kcnk3 Characterization 94Ex1 , At 4-month old, male Kcnk3 C Δ94Ex1/Δ =7 rats). D =17rats). =17rats). 94Ex1 94Ex1 =36, Kcnk3 Δ94Ex1/Δ Δ94Ex1/Δ Δ94Ex1/+ Kcnk3 ) or normoxia (NX). Three weeks later, we measured RVSP, CO, we measured RVSP, (NX). weeks later, ) or normoxia Three 17, 2 = 29, Kcnk3 = Δ94Ex1/+ Δ94Ex1/+ =11, CH; WT=18, Kcnk3 94Ex1
Δ94Ex1/Δ =10, MCT; WT=12, Kcnk3 94Ex1 =16, Kcnk3 Δ94Ex1/Δ Δ94Ex1/+ , Representative pulmonary artery (PA) flow through the pulmonary valve in Kcnk3 flow through , Representative pulmonary artery (PA) Kcnk3 12, = =9 rats). B Δ94Ex1/+ 94Ex1 Kcnk3 Δ94Ex1/Δ <0.001. Experiments were analyzed using 1-way ANOVA followed by Tukey post hoc test and experiments with 2 different categorical followed by Tukey <0.001. Experiments were analyzed using 1-way ANOVA , Quantification of right ventricular systolic pressure (RVSP), Fulton index (weight of right ventricle [RV]/left ventricle [LV]+septum [S]), cardiac , Quantification of right ventricular systolic pressure (RVSP), Fulton index (weight of right ventricle [RV]/left ventricle [LV]+septum output (CO), mean carotid pressure (mCP), and total pulmonary resistance (TPR by RVSP/CO; evaluated wild type [WT]=8 Kcnk3 Kcnk3 (potassium channel subfamily K member 3) dysfunction leads to a significant increase in right ventricular systolic (potassium channel Figure 2. Kcnk3 pressure (RVSP) in males at 4 months of age. A to chronic hypoxia (CH,Kcnk3-mutated hypoxia 10% O rats were exposed 3 weeks to chronic mutated rats were exposed to 60 mg/kg of monocrotaline (MCT) or saline (SHAM). Three weeks later, we measured RVSP, CO, and TPR (SHAM;mutated rats were exposed to 60 mg/kg of monocrotaline RVSP, we measured (MCT) or saline (SHAM). weeks later, Three WT=9, ***P comparisons. ns indicates nonsignificant. post hoc test for multiple completed by Tukey independent variables were analyzed with 2-way ANOVA and TPR (NX; WT=8, Kcnk3 Kcnk3 quantification of the PA acceleration time (PAAT; WT=26, Kcnk3 PA acceleration time (PAAT; quantification of the 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research , Representative WesternE, Representative CD31 andVWFexpression inlungsfromKcnk3 blotsandquantificationof were analyzedusing 1-wayANOVA followed byTukey posthoc testformultiplecomparisons. nsindicatesnonsignificant. wild type[WT]=9,Kcnk3 ofnonmuscularizedvesselsandmuscularized vessels(100countedperrat, on ratlungsections.Scalebar:20 µm.B,Percentage ( α-smooth muscleactin)ingreen,VWF(VonA, Immunolabelingandconfocalimagingofα-SMA Willebrand inblue factor) inred,andDAPI and increasedcollagencrosslinking. Figure 3.Kcnk3 (potassium channel subfamilyKmember 3)-dysfunctionleadstodistalpulmonaryvesselneomuscularization 684 Lambert etal Kcnk3 CD31 ofsmall(<20µm)orlarge(>20 µm) (endotheliummarker)labelinginparaffin-embeddedlungsections, expressedasthepercentage CD31 4 monthsold(WT=17, Kcnk3 µm)pulmonaryarteryfromratsat µm)andmedium(50–200 ratioinsmall(<50 signal, collagencrosslinked,andcrosslinked/collagen total Δ94Ex1/+ + vesselsmeasuredin20fieldsofKcnk3 September 13,2019 =8, Kcnk3 Δ94Ex1/Δ94Ex1 Δ94Ex1/+ Δ94Ex1/+ =13, Kcnk3 =8 rats).β-actinwasusedas theloadingcontrol..* P<0.05, **<0.01,***<0.001vs Kcnk3 =13, Kcnk3 Δ94Ex1/Δ94Ex1 +/+ Δ94Ex1/Δ94Ex1 mutated ratsat4monthsold(WT=6,Kcnk3 and Kcnk3-mutated , Upper, Picrosirius Red staining and below quantification of total collagen =14rats).C,Upper, andbelowquantificationoftotal PicrosiriusRedstaining =13 rats).Scalebar:100µm.D,Pulmonaryvesseldensityquantification basedon Circulation Research.2019;125:678–695. 10.1161/CIRCRESAHA.119.314793 DOI: +/+ Characterization ofKcnk3-MutatedRats mutated rats (WT=8, and Kcnk3-mutated Δ94Ex1/+ ,Kcnk3 =7, Δ94Ex1/Δ94Ex1 +/+ . Experiments =4 rats). Original Research 685 or si- +/+ =6 rats). (WT=3, ), and p-308 94Ex1 <0.01 vs Kcnk3 Δ94Ex1/Δ Δ94ex1/Δ9e4x1 September 13, 2019 Kcnk3 , Upper: left, KCNK3 mRNA level 8, <0.05, **P = -Mutated Rats -Mutated of Kcnk3 Characterization Δ94Ex1/+ =7 rats). D (hypoxia-inducible factor-1 α (hypoxia-inducible 94Ex1 Δ94Ex1/Δ , Survivin, p-ERK 1/2, ERK 1/2, p-AKT308, p-SRC, and , Kcnk3 8 = on hPASMCs ethyl ester) on hPASMCs m) using TMRE (Tetramethylrhodamine, φ Δ94Ex1/+ , Upper, KCNK3, Upper, mRNA 9 days after cell transfection with level in human control PASMCs, Kcnk3-mutated rats (WT=8, Kcnk3 and +/+ , Representative Western blots and quantification of TWIST1 blots and quantification (twist family bHLH, Representative Western transcription factor 1) and p-CREB C , Below: representative Western blots and quantifications of blots and quantifications , Below: representativeHIF1 Western α , confocal imaging of mitochondrial membrane potential (Δ membrane , confocal imaging of mitochondrial =3rats). F D 94Ex1 Δ94Ex1/Δ , Representative Western blots and quantification of p-ERK1/2, blots and quantification , Representative Western p-P38, p-SRC, Survivin, HIF1-α , Quantification of from WT and Kcnk3 PP2A and muscle cells (PASMCs) PP2B activity in pulmonary arterial smooth p-P38 in hPASMCs, 72 h after si-RNA 72 transfection. E p-P38 in hPASMCs, si-RNA (n=4 patients), normalized by 18S mRNA percentage of si-KCNK3 proliferation compared with si-Control expression. Below, hPASMC (n=4patients). treated with si-RNA. Right, quantification of TMRE ns: nonsignificant. *P perimeter. intensity and mitochondria Control. Experiments were analyzed using 2-tailed unpaired Student test. ERK1/2 indicates extracellular signal–regulated kinase1-/2. Kcnk3 Kcnk3 AKT (protein kinase B) expression in lungs from wild-type (WT) and Kcnk3-mutated rats (WT=7, In vivo and in vitro Kcnk3 (potassium channel subfamily K member 3)-dysfunction leads to activation of several subfamily K member 3)-dysfunction leads to activation of (potassium channel Figure 4. In vivo and in vitro Kcnk3 proproliferative and antiapoptotic pathways. A in human control PASMCs transfected 72 h with a si-RNA transfected 72 control (si-Control) or against KCNK3in human control PASMCs (si-KCNK3; by 18S n=8patients), normalized transfected with si-KCNK3mRNA (hPASMCs) compared with Si-Control expression. Right, percentage of proliferation of human PASMCs (n=6patients). expression in lungs from Kcnk3 B 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research 686 UsingiCelligence cle condition(OnlineFigureIXC). decreased proliferation ratecomparedwiththevehi- inhibitor, weobservedsignificantly to theKCNK3 humans andrats(OnlineFigureIXA). enhances theproliferation ofcontrolPASMCs from by24-hourexposure toA293at1µmol/L of KCNK3 incorporation (Figure 4D).Pharmacologicalinhibition hPASMCs asmeasuredbyBrdU proliferation by40% knockdown enhanced (by85%).KCNK3 si-KCNK3 decreasedinhPASMCsKCNK3 mRNA transfected with logical inhibition.Seventytwohoursaftertransfection, or specific pharmaco- using either si-RNA-mediated function lial cells proliferation, KCNK3 we abolished (hPASMCs) andhumanpulmonaryarterialendothe- To in human PASMCs evaluate the role of KCNK3 Proliferation InhibitionIncreases HumanPASMCsKCNK3 In Vitro Knockdown KCNK3 orPharmacological marker) wasalsounchanged (OnlineFigureVIIIB). (pan-leukocyte Likewise, thelungexpression ofCD45 Lambert etal that themitochondria ofPASMCs fromKcnk3 related protein-1;OnlineFigureXA).Likewise,wefound (dynamin DRP1 ulating mitochondrial fissionprotein without modificationofthetotal expression ofthereg- hPASMCsmitochondria fromsi-Control (Figure 4F), rhodamine, ethyl ester) and fragmented compared with fluorescenceintensity, tetramethyl- depolarized (TMRE hPASMCs, wefoundthatmitochondria membranewere α enhances PASMCs proliferation. InadditiontoHIF1- demonstratingthatlossofKCNK3 (Online FigureIXB), α as well as HIF1- mal, hPASMCs proliferation was normalized (Figure transfection, phosphorylation (Figure 4D).Ninedaysaftersi-RNA andAKTSRC, of survivin,withoutmodificationP38, andanonsignificantincrease phorylation ofERK1/2, α increase intheexpression ofHIF1- wefound asignificant tion bytheknockdown ofKCNK3, andIXE). Figure IXD (tumor necrosis factor α) stimulation(5ng/mL; Online α without alteration of endothelial barrier opening by TNF human pulmonaryarterialendothelialcellsadhesion inhibitionenhances we observedthatspecific KCNK3 Δ94ex1 ure XD and XE). ure XD without modificationofmitochondrial web (OnlineFig- mM KCl) stronglydepolarizedmitochondria membrane that plasmamembranedepolarization (inducedby138 and ratPASMC andXC). We (OnlineFigureXB found isinvolvedinmitochondria integrityinhuman KCNK3 mented comparedwithWT. This stronglysuggeststhat and ERK1/2 overactivation in si-KCNK3-treated overactivationinsi-KCNK3-treated andERK1/2 In humanpulmonaryarterialendothelialcellsexposed Consistent toanenhancementofPASMCs prolifera- rats werealsosignificantlydepolarizedandfrag- September 13,2019 KCNK3 expression returnedtothenor- expression and ERK1/2 activation expression and ERK1/2 , anover-phos- Circulation Research.2019;125:678–695. 10.1161/CIRCRESAHA.119.314793 DOI: Δ94ex1/ 4E) TM , Kcnk3 from WTratswhereasthedose-responsecurveofPA from ties, wepretreatedPA withL-NAME (L-N PA activi- relaxationvianitricoxide synthase (NOS) tent withthePASMCs depolarization(Figure 1H). the previouslyobserveddifferences inFigure 5A,consis- enhanced PA vasoconstrictioninKcnk3 sensitivity)maycontributeto expression, reducedEDHF ters (PASMCs depolarization,reduced endothelial-NOS or inefficientinKcnk3-mutatedrats.Alltheseparame- productionisstronglyreduced suggested thatEDHF mutated rats(Figure 5E,middleandright).These data fluconazole hadnoimpacton PA contractioninKcnk3- tion thaninthebasalcondition(Figure 5E,left),whereas fluconazole, WT PA contracted atalower KCl concentra- azole, a cytochrome P450 epoxygenase inhibitor. With inPA tone,weusedflucon- the contributionofEDHF hyperpolarize and relax smooth muscles. To investigate producedbycytochrome P450-2C9that acid metabolite resents afamilyofendothelialfactorsasanarachidonic rep- (endothelium-derived hyperpolarizingfactor).EDHF expression wasunchangedNOS (OnlineFigureXIB). was unchanged inPA fromKcnk3 The contractiledose-responsetoincreasingdosesofKCl was unaltered in as theextracellular Ca quence of WT and on PA contractility. UsingisolatedPA from4-month-old We investigatedtheconsequencesofKcnk3-dysfunction Constriction Kcnk3-Dysfunction Predisposes PA to mimetic (U46619), relaxation was stimulated using mimetic (U46619), After inducing contraction with a thromboxane-A Relaxation Kcnk3-Dysfunction inRatsReducedPA that potentiatesvoltage-sensitivityofL-Type-Ca ure 5C). Interestingly, withL-NAME, inKcnk3 ties andvalidatingtheefficacyof treatment(Fig- L-NAME L-NAME, activi- demonstrating thecontributionofNOS at a lower concentration of KCl than in the absence of inhibitor).Withmethyl ester; NOS L-NAME, PA contracted Importantly, L-Type Ca (Figure 5B). pared withtheconditionwithoutBayK8644 nels, thecontractileresponsewasshiftedtoleftcom- from expression wassignificantlyreducedinlungs thelial-NOS lost (Figure 5C).Western blotanalysesshowedthatendo- the difference intheKCl contractiledose-responsewas shown bythesignificantdecreaseinEC To ruleoutthecontributionofendothelial-dependent Pulmonary vascular endothelium also releases EDHF Pulmonary vascularendotheliumalsoreleasesEDHF When PA adrug werepretreatedwithBayK8644, Kcnk3 Δ94ex1/Δ94ex1 Kcnk3-mutated rats,we first assessedthe conse- Kcnk3-dysfunction on basal PA tone, measured Δ94ex1/Δ rats was significantly shifted to the left as ratswassignificantlyshiftedtotheleftas -mutated rats (Online Figure XIA). Kcnk3-mutated rats (Online Figure XIA). 94ex1 rats(Figure 5D),whereasinducible- 2+ 2+ -dependent tone.The basaltone Characterization ofKcnk3-MutatedRats channel potentiation abolished Δ94ex1/+ comparedwithPA 50 Δ94ex1/ Δ94ex1 ( Figure 5A). G Δ94ex1/Δ94ex1 -nitroarginine -nitroarginine 2+ rats. chan- rats, rats, 2
Original Research
687 =8, Δ94Ex1/+ =5 rats). D, Δ94Ex1/Δ94Ex1 =10 rats). B, In September 13, 2019 , Comparison of PA =5 rats). C, Comparison of PA =7, Kcnk3 -nitroarginine methyl ester) and G Δ94Ex1/Δ94Ex1 Δ94Ex1/+ -Mutated Rats -Mutated of Kcnk3 Characterization Δ94Ex1/Δ94Ex1 =7, Kcnk3 =6, Kcnk3 and Kcnk3-mutated rats (WT=8, Kcnk3 Δ94Ex1/+ +/+ Δ94Ex1/+ . Experiments were analyzed using 1-way ANOVA followed . Experiments were analyzed using 1-way ANOVA +/+ =5 rats). Bottom, normalized dose-response curve established by applying increasing and Kcnk3-mutated rats in presence of 10 μmol/L fluconazole and EC50 quantification +/+ Δ94Ex1/Δ94Ex1 =7 rats) . *P<0.05, **P<0.01 vs Kcnk3 =7, Kcnk3 Δ94Ex1/Δ94Ex1 Δ94Ex1/+ and Kcnk3-mutated rats and quantification of EC50 (WT=6, Kcnk3 +/+ -mutated rats and EC50 quantification (WT=7, Kcnk3 and Kcnk3-mutated rats and EC50 quantification (WT=7, =6, Kcnk3 +/+ =7 rats). β-actin was used as the loading control. E, Normalized dose-response curve established by applying increasing Δ94Ex1/+ Δ94Ex1/Δ94Ex1 concentrations of KCl in the presence of 300concentrations of KCl μmol/L of L-NAME and EC50Kcnk3 quantification (WT=5, Kcnk3 (potassium channel subfamily K member 3)-dysfunction in rats predisposes pulmonary arteries to constriction. subfamily K member 3)-dysfunction in rats predisposes (potassium channel Figure 5. Kcnk3 A, Normalized dose-response curve (normalized to K100) established on isolated pulmonary applying an increasing concentrations of KCl by from Kcnk3 artery (PA) by Tukey post hoc test for multiple comparisons. ns indicates nonsignificant. post hoc test by Tukey Representative Western blots and quantification of eNOSRepresentative expression in lungs from Kcnk3 Western contraction, in response to increasing dose of KCl, between PA pretreated with 300 μmol/LN between PA of L-NAME (L- contraction, in response to increasing dose of KCl, presence of 1 μmol/L of Bay K 8644, normalized dose-response curve established on PA by applying increasing concentrations of KCl isolated from Kcnk3 PA nontreated (WT=5, Kcnk3 PA (WT=8, Kcnk3 concentrations of KCl on isolated PA from Kcnk3 on isolated PA concentrations of KCl 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research mutated rats,withahigherdegreeinKcnk3 sildenafil-induced PA relaxationwasreducedinKcnk3- Figure XID). Hrpt1 inisolatedPA fromKcnk3-mutatedrats(Online 3 688 Kcnk3 Using electronmicroscopyimagingofPA, wefoundin Membrane Caveolae Kcnk3-Dysfunction Leads toLoss ofPlasma andXIIE). group (OnlineFigureXIID in channel contribution inPAs. Inlinewithnormalaortatone (not shown),demonstratingspecificityofthe KCNK3 unchanged inKcnk3-mutatedratscomparedwithWT mediated relaxationinisolatedpulmonaryveinswere the contractile dose-response to KCl and the sildenafil- Finally, antiapoptotic pathways(OnlineFigureXIIC). not associatedtoanoveractivationofMAP-kinaseand Importantly, in isolated aorta, from relaxationwereunchanged inaorta the SNP-mediated Interestingly, thecontractiledose-response toKCl and Pulmonary Veins or Constriction andRelaxationofAorta Kcnk3-Dysfunction DidNotAlterBoth Kcnk3 wassimilarbetweenWTand acetylcholine orSNP inhibitor (sildenafil;Figure 6).PA relaxationinduced by induce endothelium-independentrelaxationandaPDE5 to donor (sodium nitroprusside [SNP]) ation, an NO acetylcholine to induce endothelium-dependent relax- Lambert etal from switch, including phenotype various genesknowntobeinvolvedinSMC inhibition-inducedPAPDE5 relaxation. is involved inthe These resultssuggestthatKCNK3 ization wassimilarinKcnk3-mutatedrats(notshown). anditslocal- (OnlineFigureXIC) The amountofPDE5A reduced comparedwithvehiclecondition(Figure 6D). induced relaxation. Sildenafil-mediated relaxation was andmeasuredthesildenafil- inhibitor (A293,1μmol/L) esis, wepreincubatedPA fromWTratswithaKCNK3 inhibition-mediated PA relaxation.To explore thishypoth- iscriticalforPDE5 (Figure 6C), suggestingthatKCNK3 cells (Figure 6F). than caveolaefromWTpulmonary arterialendothelial Kcnk3-mutated rats revealed that they were shallower analysis ofthedepththese caveolaeinvaginationsin lae wasunchanged (Figure 6Eand6F).Morphometric compared withWT, whilethetotalnumberofcaveo- per micrometer localized into the luminal ECs surface Δ94ex1/Δ94ex1 Kcnk3-mutated rats,LV weightsweresimilarineach In addition, we found a gain of mRNA expression of In addition,wefoundagainofmRNA -mutated rats (Online Figure XIIA and XIIB). andXIIB). Kcnk3-mutated rats (OnlineFigureXIIA Kcnk3 Δ94ex1/+ September 13,2019 Δ94ex1/Δ rats(Figure 6Aand6B).ComparedwithWT, Δ94ex1/Δ94ex1 rats,whereassignificantlyreducedinKcnk 94ex1 a decrease in the number ofcaveolae Hexim1, ratsanddecreaseexpression in MYL6, and Kcnk3-dysfunction was PLN in isolated PA Δ94ex1/Δ94ex1 Circulation Research.2019;125:678–695. 10.1161/CIRCRESAHA.119.314793 DOI: rats (I (Online Figure XIVB). Significanthigher leveloffibrillary (Online Figure XIVB). and in 36% ofKcnk3 and in36% and in 45% of background-inward-K sions remained unchanged (Figure sion of Kv4.3 channels, while Kv4.2, Kv1.5, Kv1.2 expres- level, weobservedasignificantdecreaseinthe expres- inactivation timeconstants(datanotshown).At mRNA was superior to 40 mmHg in20%ofKcnk3 was superiorto40 (Figure nificant increase in TPR significant decrease in the outward-K with increased action potential duration, we measured a early after depolarization events (data not shown). Along mV (Figure 7B).Kcnk3-mutatedratsalsoshowedsome in RVcardiomyocytesfromKcnk3 over, theactionpotentialsweresignificantlyprolonged brane potentialcomparedwithWT(Figure 7A).More- recorded significantdepolarizationofrestingmem- In RVcardiomyocytesfromKcnk3-mutatedrats,we Prolongation inRVCardiomyocytes Kcnk3-Dysfunction InducesActionPotential was superior to 40 mmHg in25%ofKcnk3 was superiorto40 mm superiorto40 elevation ofRVSP is definedbyan ure 8A andOnlineFigureXIVA). PH in WT and dependent, weperformedright-heartcatheterization To developmentcouldbeage- determinewhetherPH (12 Months) Developed anAge-RelatedIncrease inRVSP Both MaleandFemale Rats Kcnk3-Mutated α HIF1- male nonmuscularized pulmonary vesselsin12-month-old of muscularizedpulmonary vessels andadecreasein artery pressurevalues(Figure 8A; OnlineFigureXIVA). andXV) noralterationofmeancarotid Online FiguresXIV ton index) or areductionincardiacoutput(Figure 8A; associated withthedevelopmentofRVhypertrophy(Ful - XIVA). (OnlineFigure significant increaseinTPR Kcnk3 inRVfrom receptor component)andTimp-1 mRNA we measuredasignificant increaseofGp130(IL-6 RVinflammationinKcnk3-mutatedrats, About XIIIA). Myh6, andMyh7werealsounchanged (OnlineFigure RV cardiomyocyteexcitability. The expression ofAtp2a2, contributes to these results demonstrate that KCNK3 expression remainedunchanged (Figure 7H).Together, mRNA Kcnk3-mutated rats(Figure 7G),whileKCNJ12 strongly reducedinisolatedRVcardiomyocytesfrom (I sus to ; Figure 7C)andinanoutward-K In addition,weobservedan increaseinthenumber In malesandfemales, wasnot theelevationofRVSP ; Figure 7E).I Kcnk3-mutated rats(Figure 8B) associatedwith Δ94ex1/Δ94ex1 overexpression inlungfromKcnk3 Kcnk3-mutated rats at 12 months old (Fig- Kcnk3 rats(OnlineFigureXIIIB). to wasreducedwithoutmodificationof Δ94ex1/Δ94ex1 + -rectifier current(I Δ94ex1/Δ94ex1 Characterization ofKcnk3-MutatedRats ratsassociatedwithasig- rats,associatedwitha 8A). In females, RVSP Δ94ex1/ Δ94ex1Δ94ex1/ + Hg. In males, RVSP Hg. Inmales,RVSP -sustained current + 7D and7F). The -transient current Δ94ex1/Δ94ex1 K1 rats at −60 ratsat−60 ) wasalso Δ94ex1/+ Δ94ex1/+ rats rats rats Original Research 689 =6 Δ94Ex1/Δ94Ex1 rats, treated with +/+ =9, Kcnk3 =73 cells, 3 rats; middle). =73 September 13, 2019 =131 caveolae, 2 rats, Kcnk . Experiments were analyzed Δ94Ex1/+ +/+ Δ94Ex1/+ Δ94Ex1/Δ94Ex1 -Mutated Rats -Mutated of Kcnk3 Characterization =7 rats). D, Concentration-relaxation response curves =29 cells, 2 rats, Kcnk3 M of sildenafil (WT=6, Kcnk3 −6 Δ94Ex1/Δ94Ex1 Δ94Ex1/+ =9, Kcnk3 Δ94Ex1/+ =73 cells, 3 rats). Caveolae were counted as omega-shaped membrane profiles open at the cells, 3 rats). Caveolae were counted as omega-shaped =73 Δ94Ex1/Δ94Ex1 =29 cells, 2 rats, Kcnk3 =234 caveolae, 3 rats). Bars= 400 nm. ns: nonsignificant. *P<0.05 **P<0.01, ***P<0.001 vs Kcnk3 Δ94Ex1/+ Δ94Ex1/Δ94Ex1 3 lumen surface (L) and normalized per micrometer; WT=34 cells, 2 rats, Kcnk3 or without KCNK3 inhibitor (A293 1 µmol/L), and percentage of relaxation to 10 established by applying increasing concentrations of sildenafil (1 nmol/LPA segments isolated from Kcnk3 to 10 µmol/L) to Kcnk3 (potassium channel subfamily K member 3)-dysfunction in rats altered the relaxation of pulmonary artery (PA). subfamily K member 3)-dysfunction in rats altered the relaxation of pulmonary artery (PA). (potassium channel Figure 6. Kcnk3 established by applying increasing concentrations of (A) acetylcholine A, Concentration-relaxation response curves of isolated segment of PA 1 nmol/L(Ach; to 10 µmol/L), (B) sodium nitroprusside (SNP; 10 nmol/L to 10 µmol/L) and (C) sildenafil (1 nmol/L to 5 µmol/L). Right, represent graph of the percentage of relaxation (WT=5, Kcnk3 Depths of the lumen caveolae (invagination depths) were quantified in right (WT=86 caveolae, 2 rats, Kcnk3 using 1-way ANOVA followed by Tukey post hoc test for multiple comparisons. Tukey followed by using 1-way ANOVA , Analysis of the ultrastructure of PA by electron microscopy in 4-month-old rats. Endothelial cells (ECs) were based on the basal lamina rats). E, Analysis of the ultrastructure of PA (BL). F, Caveolae density was quantified as the number of caveolae by micrometer square of total EC surface (Left; WT=34 cells, 2 rats, Kcnk3 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research proposed asamarkerofdiseaseseverity. with highmortalityinPAH patients,serum-albuminwas echography signsofleftandrightheartdilatation. 12 months of age, tility asevaluatedbyRVandLV fractionalshortening.At seems tobecompensatedbyhigherRVandLV contrac- ing membranepotential. opening ofL-type Ca PASMCs, plasmamembranedepolarizationcauses 690 tributors to background-K tion withadecreaseinI succeeded in generating a model of Kcnk3-inactiva- ization ofKcnk3-mutatedratline(Δ94ex1). First, we dynamic, histological,cellular, andmolecularcharacter- In this study, we performed electrophysiological, hemo- DISCUSSION opment ofLV dilatationinKcnk3 reduction inRVandLV thickness, demonstratingdevel- significantly increasedinKcnk3-mutatedratsalongwith diastolic andsystolicdiameters(normalizedbyweight) nary artery acceleration time in analyses (OnlineTable V)revealedareductioninpulmo- (Online FigureXIVC). ure 8C), whiletotalcollagendepositionwassimilar medium (50–200 µm) PA in collagen assemblywasobservedinsmall(< Lambert etal PASMCs contraction, and proliferation via activation of ings relatedtohumanPAH pathobiology. summarized inFigure 8Eisconsistentwithrecentfind- vascular alterationsobservedinKcnk3-mutatedratsare The varietyofpulmonary and60%. ing between30% inanage-dependent mannerwithpenetrancerang- PH stimuli. Finally, Kcnk3-mutatedratsdevelopspontaneous to constrictionandarelessresponsivevasorelaxing tion. Fifth,PAs fromKcnk3-mutatedratsaresensitized as primary molecular events linked to Kcnk3-dysfunc- pathway α andoveractivateERK1/2 amount ofHIF1- in humanPASMCs, knockdown increasethe KCNK3 with alterationofendothelialmarkerexpression. Fourth, acterized by overactivation of proproliferative pathways, tion. At themolecularlevel,Kcnk3-mutatedratsarechar - muscularization andperivascularfibrillarcollagendeposi- Kcnk3-dysfunction leadstoincreasedpulmonaryvessel induce to PH increased inKcnk3-mutatedratsandtheyaresensitized mutated rats. Second, at 4 months of age, RVSP development intheseanimals. (Figure 8D), supporting thenotion of age-dependentPH significantly reducedin12-monthKcnk3-mutatedrats old the serum-albuminlevelwasunchanged in4-month- As lowserum-albuminconcentrationswasassociated KCNK3 channels arecommonlyacceptedascon- KCNK3 At 12-month-oldrats(males),echocardiography -mutated rats (Online Figure XIVD) butwas Kcnk3-mutated rats(OnlineFigureXIVD) September 13,2019 by MCT-exposure or Third,CH-exposure. Kcnk3-mutated rats showed chronic 2+ channels leadingtoinfluxofCa KCNK3 8 Inexcitable cellsincluding + -currents that stabilize rest- inPASMCs fromKcnk3- Kcnk3-mutated rats (Fig- Kcnk3-mutated rats. LV Δ94ex1/Δ94ex1 3 21 . LV dilatation Interestingly, 50 µm)and Circulation Research.2019;125:678–695. 10.1161/CIRCRESAHA.119.314793 DOI: are 2+ , alteration of SOD2 expression. alteration ofSOD2 with mitochondria fromhealthyPASMCs becauseofan from PAH PASMCs aremorehyperpolarizedcompared has beeninvestigatedinPAH, demonstratingthatΔ overexpression. Furthermore, mitochondrial dysfunction α depolarization, which couldalsopartlyexplain HIF1- inducesmitochondrialmembrane well assi-KCNK3 aberrant pulmonaryvascularcellproliferation. markers such asCD31. Migrating endothelialcells losespecificendothelial expression inlungsfromKcnk3-mutatedrats. in CD31 tus leading to aberrant cell proliferation, α inducingapseudo-hypoxic cellsta- vation ofHIF1- membrane potentialandΔΨmareintimatelylinked. PH after CH-exposure. PH In mice,hif1-α-dysfunctionreducesthedevelopmentof activation. α andERK1/2 pathobiology possiblyviaHIF1- downregulationisaninitialtriggerforPAHthat KCNK3 promotes PASMCsof KCNK3 proliferation, confirming ent evidencethatpharmacologicalorgeneticinactivation ment. onstrates thatEndMTcontributes toPAH develop- mitochondrial networkisfragmentediniPAH PASMCs (not shown).Finally, previousreportsdemonstratedthat tein expression wasunchanged inKcnk3-mutatedrats thelial cell-celljunctions. ishighlyexpressed atendo- endothelial function.CD31 gesting thatKcnk3-dysfunctionalteralsopulmonary severely increased in lung of VWF expression. We foundthatVWFexpression is also inducedelevatedlung onstrated thatCH-exposure >25%. neuroblastoma cellsincreasesproliferation ratesby with poorsurvivalinPAH patients. we recently showed in iPAH PASMCs that T-type-Ca activity.308AKT, anddecreasesPP2A Indeed, andSRC the phosphorylationofpro-mitogenproteinsERK1/2, the context ofKcnk3-dysfunction. also foundthatmitochondria networkisfragmentedin in associationwiththedevelopmentofPAH. Here,we We foundthatextracellular high-K Ca P38 orERK1/2) P38 be mediatedbyanactivationofspecifickinases(like activation of ERK1/2. nide-4(trifluoromethoxy)phenylhydrazone]) leadstothe cya- [carbonyl by themitochondrial uncoupler(FCCP sion. Mitochondrial membrane depolarization induced α expres - which couldpartlyenhanceHIF1- ERK1/2, found thatKcnk3-dysfunctionleadstooveractivationof activity. 308AKT, anddecreased PP2A phorylation ofERK1/2, eration/cell survivalandapoptosis resistanceviaphos- channels signaling contributestopathologicalhyperprolif- 2+ Higher levelsoflungVWFhavebeenassociated Our resultssuggestthatKcnk3-dysfunctionenhances sensitivepathways. 20 23 31 EndMT is a process by which endothelial cells In vivo chronic inhibition of KCNK3 induces Invivochronic inhibitionofKCNK3 26,27 orphosphatasesinactivation. 28 24 Characterization ofKcnk3-MutatedRats 22 34 Moreover, theresting plasma In PAH, there is normoxic acti- KCNK3 knockdown inmouse KCNK3 35 Here,weshowedadecrease Increasingevidencedem- Kcnk3-mutated rats, sug- 30 However, SOD2 pro- However, SOD2 32 Mojirietal + concentrationas 25 which might 10 We pres- 33 dem- 26 Ψ We We m 2+ 19 29
Original Research and 691 +/+ - + ) sus and Kcnk3- ; WT=8 cells, +/+ K1 -current (I + suggesting Kcnk3- 20 September 13, 2019 =9 cells, 3 rats). D, mRNA -rectifier-current (I + the loss of plasma-membrane Δ94Ex1/Δ94Ex1 -Mutated Rats -Mutated of Kcnk3 Characterization 37 =11 rats). E, Outward-sustained-K . Experiments were analyzed using 1-way ANOVA . Experiments were analyzed using 1-way ANOVA =5 rats). G, Inward-K +/+ and Kcnk3-mutated rats (wild-type [WT]=11 cells, 3 rats, =8 cells, 3 rats, Kcnk3 +/+ Δ94Ex1/Δ94Ex1 As caveolae are demonstrated to mediate albumin tran- Δ94Ex1/Δ94Ex1 Δ94Ex1/+ , mRNA expression of Kv1.5 and Kv2.1 in RV and Kv2.1 =11 cells, 3 rats). F, mRNA expression of Kv1.5 epithelial-mesenchymal-transition, dysfunction contributes to EndMT pathway. scytosis in human ECs, =11, Kcnk3 =5, Kcnk3 Δ94Ex1/Δ94Ex1 Δ94Ex1/+ Δ94Ex1/+ =11 rats). *P<0.05 vs Kcnk3 =9 cells, 3 rats). H, mRNA expression of KCNJ12 in RV tissues from Kcnk3 , We analyzed the current-voltage relationship of the outward-K =11 cells, 3 rats). C, We Δ94Ex1/Δ94Ex1 =11 cells, 3 rats). B, RepresentativeKcnk3 membrane potential of RV cardiomyocytes from Δ94Ex1/Δ94Ex1 Δ94Ex1/Δ94Ex1 =11 cells, 3 rats, Kcnk3 current (WT=8 cells, 3 rats, Kcnk3 + Δ94Ex1/+ Δ94Ex1/Δ94Ex1 =11, Kcnk3 Δ94Ex1/+ and Kcnk3-mutated rats (WT=5, Kcnk3 ) or 4-AP-sensitive-K =11 cells, 3 rats, Kcnk3 =11 cells, 3 rats, Kcnk3 +/+ to We showed an overexpression of TWIST1, showed an overexpression We 36 Δ94Ex1/+ Δ94Ex1/+ =11 cells, 3 rats, Kcnk3 Δ94Ex1/+ Kcnk3 tissues from Kcnk3 (potassium channel subfamily K member 3)-dysfunction leads to action potential prolongation in adult right subfamily K member 3)-dysfunction leads to action potential (potassium channel Kcnk3 Figure 7. ventricular (RV) cardiomyocytes. RV cardiomyocytes isolated from Kcnk3 resting membrane potential in A, Average followed by Tukey post hoc test for multiple comparisons. ns indicates nonsignificant. post hoc test for multiple comparisons. ns indicates followed by Tukey expression of Kv4.2 and Kv4.3 in RV tissues (WT=11, Kcnk3 and Kv4.3 expression of Kv4.2 current (WT=11 cells, 3 rats, Kcnk3 3 rats, Kcnk3 mutated rats (WT=11, Kcnk3 transient current (I Kcnk3-mutated rats and analysis of RV action potential duration (APD) at 0, −20, and −60 repolarization (WT=20 cells, mV of membrane 4 rats, Kcnk3 acquire a mesenchymal phenotype in association with acquire a mesenchymal of SMCexpression marker genes or loss of endothelial expression. mainly defined as a master controller of EndMT and 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research 692 Lambert etal muscularized vessels(100countperrats;WT=6,Kcnk3 (WT=5, Kcnk3 predispose forpulmonary vascularremodelingandpulmonary arterialhypertension(PAH). nsindicatesnonsignificant. α [hypoxia-inducible factor-1α]).Altogether, theseevents increasedphosphorylation andoverexpressionofHIF1- (partly explainedbyERK1/2 pulmonary arterialvasoconstriction, pulmonaryvascularremodeling,andarterialsmoothmuscle cells(PASMCs) proliferation mitochondrial membrane depolarization,endothelialphenotypeswitch, reducesRVcardiomyocytesexcitability. Alltheeventsleadsto by Tukey resultsinplasmamembrane depolarization, posthoctest.E , ProposedeventsarisingfromKcnk3-dysfunction.LossofKCNK3 were assessedat12monthsold(wildtype[WT]=13,Kcnk3 andmeancarotidpressure (mCP) (TPR), pulmonaryresistance total cardiacoutput(CO), A, Rightventricularsystolicpressure(RVSP), Figure 8.Kcnk3 (potassiumchannel subfamily Kmember3)-mutatedratsdevelopage-relatedpulmonaryhypertension(12mo). ratio in small (<50 µm) and medium (50–200 µm)pulmonaryartery(PA) µm)andmedium(50–200 ratio insmall(<50 fromKcnk3 Kcnk3 Δ94Ex1/+ September 13,2019 =7, Kcnk3 Δ94Ex1/+ =5, Kcnk3 Δ94Ex1/Δ94Ex1 Δ94Ex1/Δ94Ex1 =9 rats).D,Serum-albuminconcentrationinplasma from Kcnk3 =6 rats).*P<0.05,**<0.01vsKcnk3 Circulation Research.2019;125:678–695. 10.1161/CIRCRESAHA.119.314793 DOI: Δ94Ex1/+ Δ94Ex1/+ =41, Kcnk3 =11, Kcnk3 Δ94Ex1/Δ94Ex1 +/+ . Experimentswereanalyzedusing 1-wayANOVA followed Δ94Ex1/Δ94Ex1 +/+ -mutated ratsat12monthsold(WT=11, and Kcnk3-mutated , Percentage ofnonmuscularized and =11 rats).B,Percentage , Collagen crosslinked to total collagen =6 rats).C,Collagencrosslinkedtototal +/+ -mutated ratsat12monthsold and Kcnk3-mutated Characterization ofKcnk3-MutatedRats Original Research
693 Δ94ex1/+ September 13, 2019 rats. These results confirm rats. These -Mutated Rats -Mutated of Kcnk3 Characterization Δ94ex1/Δ94ex1 -mutated rats cannot be considered as as Kcnk3-mutated rats cannot be considered the importance of PA tone and mechanical cues at the tone and mechanical PA the importance of early stages of PH model provides development. This - understanding the initiating mech new opportunities for anisms of PHfor testing and represents a relevant tool against PH.therapeutic targets ARTICLE INFORMATIONARTICLE 2019; accepted July 24, 1, 2019; revision received July 17, Received February 2019. Affiliations Le de Médecine, Université Paris-Saclay, Faculté the University Paris–Sud, From M. A.B., M.K.N., A.H., C.R.-M., O.M., B.G., D.M., F.P., V.C., (M.L., Bicêtre, France Kremlin Centre Service de Pneumologie, Hôpitaux de Paris, Assistance Publique H., F.A.); Bicêtre, Kremlin Hôpital Bicêtre, Le de Référence de l’Hypertension Pulmonaire, Inserm M.H., F.A.); A.B., M.K.N., A.H., C.R.-M., O.M., B.G., D.M., F.P., (M.L., V.C., France UMR_S 999, (M.L., V.C., France Plessis Robinson, Hôpital Marie Lannelongue, Le Université Côte d’Azur, M.H., F.A.); A.B., M.K.N., A.H., C.R.-M., O.M., B.G., D.M., F.P., et en Transplantation CNRS, Centre de Recherche IPMC, (T.B.); France Valbonne, Immunologie UMR 1064, INSERM, S.R., I.A.); (L.T., Université de Nantes, France Rat ImmunoPhenomic (TRIP) (L.T., France facility Nantes, Nantes, PTransgenic Jouy-en- 78350 Université Paris-Saclay, S.R., I.A.); GABI, INRA, AgroParisTech, Signalisation et Physiopathologie Cardiovasculaire - UMR_S (C.P.); Josas, France Paris-Sud,1180, INSERM, Univ. Châtenay-Malabry, Université Paris-Saclay, Saclay d’Innovation Thérapeutique Institut Paris (B.M.); Animal Facility, France (UMS IPSIT), Châtenay-Malabry, Université Paris-Sud, Université Paris-Saclay, de l’Institut Universitaire de Cardiologie and Centre de Recherche (V.D.); France Canada (F.P.). de Québec, Laval University, et de Pneumologie in rats leads to pulmonary vascular alteration, thus facili- alteration, thus to pulmonary vascular in rats leads tating PH manner. in an age-dependent development of KCNK3 highlight that loss our results Importantly, in increased compliance resulting on PA function acts Kcnk3 Heterozygous- ways. RVSP via 2 different and perivas- remodeling by PA characterized rats were and remodeling whereas PA cular collagen crosslinking characterize (Online Figure XVI) vasoconstriction PA homozygous-Kcnk3 Limitations Our CRISPR/Cas9 animals. Using knockout we approach, 1 of Kcnk3 gene, induced a deletion of 94 bp in the exon shift in the coding shifting the open reading frame. This and leads sequence results in an aberrant aa sequence premature stop to a premature stop codon (8 potential commercially Unfortunately, I. Figure Online codons; available KCNK3 antibodiesKCNK3 are not specific to of WT or preventing any evaluation of the expression KCNK3truncated translation of putative proteins. The the truncated mRNA should produce a 90-aa protein with only 14-aa (instead of a 411-aa for the WT protein) could partly affect in common with the WT protein, which -mutation. How- a signaling cascade induced by Kcnk3 we show that most of KCNK3-dependent ever, signaling observed in Kcnk3-mutated rats were also detected in KCNK3 in which is targeted by siRNAhPASMC strat- vivo versus in vivo) indicate similar results (ex These egy. the Kcnk3- that the truncated protein negligibly affected mutated phenotype.
3 47 we 45 the abnormalities In PAH, endothelial- In PAH, 41 have suggested that 3 43 Interestingly at 4 months4 at Interestingly As caveolae are shown to shown are caveolae As 38 Our results strongly suggest a 40 -mutated rats, which may which rats, Kcnk3-mutated We showed that Kcnk3-dys- We 43 In human iPSC- (induced pluripo 42 46 KCNK3 contains multiple PKG phos- 44 - that the knock demonstrated in hPASMCs 39 Previous reports showed similar results in reports Previous 11 We found that PA relaxation, induced by PDE5 relaxation, induced inhibi- found that PA We 8E, our findings, summarized in Figure Collectively, Moreover, increasing evidences suggest an impor- suggest increasing evidences Moreover, In addition to PA remodeling and fibrosis, PAH patients remodeling and fibrosis, In addition to PA At cardiac level, we confirmed that KCNK3 cardiac level, we confirmed that At contributes -channels regulate NO-channels secretion in human pulmonary + function predisposes to PA vasoconstriction associated function predisposes to PA with reduced endothelial-NOS and alteration expression of EDHF al Ouyang et pathway. crosslink between KCNK3 and NO or EDHF in PA. tion, is reduced when KCNK3 is not functional. PDE5 thus inhibition increase intracellular levels of cGMP, relaxing consequently and PKGactivity increasing smooth muscles. observed in caveolae shape from pulmonary arterial observed in caveolae shape from endothelial cell Kcnk3-mutated rats could contribute to the decrease in NOS-mediated Although relaxation. EDHF is important to maintain normal vascular tone, the role of EDHFexact Alter- in PH is unclear. pathogenesis ation of EDHF-mediated responses has been reported failure, heart atherosclerosis, hypertension, aging, with and diabetes mellitus. old, perivascular collagen cross-linking is markedly pro- old, perivascular collagen heterozygous nounced in RVSPcontribute to abnormal because perivas- elevation stiffness. is associated to PA cular collagen cross-linking Bertero et al - tent stem cells)-derived cardiomyocytes, KCNK3 knock down was found to prolong action potential duration. phorylation sites. As PKG regulates KCNK3 activity, -knockout mice. kcnk3-knockout Kcnk3-dysfunction should increase the susceptibility for early after-depolarizations and ventricular arrhythmias. underscore the concept that genetic Kcnk3-inactivation caveolae in pulmonary arterial endothelial cells lacking lacking endothelial cells in pulmonary arterial caveolae KCNK3 abnormal serum-albumin explain could partly rats. in Kcnk3-mutated production in PAH. deposition ECM matrix) of role tant (extracellular PH pulmonary experimental Numerous models exhibit deposition. matrix vascular down of KCNK3 of the collagen promotes expression - expres promotes and oxidase) (lysyl crosslinking enzyme sion of miR130/301 ECM that coordinate remodeling. by vasoconstricition mainly explained have aberrant PA the disruption of the NO pathway. K arterial endothelial cells. propose that KCNK3 is the end point of PDE5/ channel GMP/PKG dependent signaling in PASMCs. and action potential repo- to RV cardiomyocyte excitability that phar- we recently demonstrated larization. Moreover, macological inhibition of KCNK3 (by A293) increased the action potential duration in adult RV cardiomyocytes from rats. regulate endothelial-NOS activity, derived NO activity is decreased, promoting vasocon- thrombosis.and striction 2019;125:678–695. DOI: 10.1161/CIRCRESAHA.119.314793Circulation Research. 2019;125:678–695. Lambert et al Lambert
Downloaded from http://ahajournals.org by on May 7, 2020 Downloaded from http://ahajournals.org by on May 7, 2020 7, May on by http://ahajournals.org from Downloaded Original Research 694 REFERENCES boards. The otherauthorsreportnoconflictsofinterest. scientific advisory companies, otherrelationshipsincludeconsultancyservicesandmembershipsto Novartis, and Pfizer.GSK, In addition to being investigators in trials involving these M. Humberthasrelationshipswithdrugcompanies,includingActelion,Bayer, Disclosures Therapeutic InnovationDoctoralSchool (ED569). diseases program to generate the from Fondation maladiesraresintheframeofsmall-animalmodelsandrare F.Funding AgencyforResearch: Perros ANR-18-CE14-0023. receivedfunding and fromtheFondation Legs Poix. HealsoreceivedfundingfromtheNational tion Recherche enSantéRespiratoirefromtheFondation Lefoulon-Delalande F. AntignyreceivesfundingfromtheFondation et duSouffle Fonds deDota- Sources ofFunding form forcaringtheratlines. plat- We wishtothankthestaffatANIMEX d’avenir—ANR-II-INSBS-0014. This workbenefitedfromthefacilitiesand expertise ofTEFOR—Investissement Acknowledgments Lambert etal
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