从兴奋收缩耦联机制看心力衰竭正性肌力药物发展

从兴奋收缩耦联机制看心力衰竭

正性肌力药物发展田野教授哈医大二院心内科提要兴奋-收缩耦联机制正性肌力药的循证研究洋地黄制剂β-肾上腺素能受体激动剂磷酸二酯酶抑制剂钙增敏剂新型正性肌力药的探索亚硝酰氢

兴奋-收缩耦联机制Excitation-contraction(EC)couplingisatermcoinedin1952todescribethephysiologicalprocessofconvertinganelectricalstimulustomechanicalresponse.SandowA(1952)."Excitation-contractioncouplinginmuscularresponse.".YaleJBiolMed25(3):176–201.PMID130159500Excitation-contractioncouplingCardiacexcitation–contractioncouplingistheprocessfromelectricalexcitationofthemyocytetocontractionoftheheart(whichpropelsbloodout).TheubiquitoussecondmessengerCa2+isessentialincardiacelectricalactivityandisthedirectactivatorofthemyofilaments,whichcausecontraction.Bers,D.M.Excitation–ContractionCouplingandCardiacContractileForceedn2(KluwerAcademic,Dordrecht,Netherlands,2001).Cardiacexcitation–contractioncouplingCardiactissue(Guinea-pigventricularcell)Cardiactissue

CardiaccellsTheactionpotentialmovesthroughsarcolemmaTtubeCa2+-inducedCa2+-releaseCa++Ca++Ca++Ca2+PlbCa2+Ca++Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Ca++Ca++Ca++Ca++Ca2+Ca++Ca++Ca++Ca++Ca2+Ca++Ca++Ca2+Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca2+Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca2+Ca2+Ca2+Ca2+Ca2+Ca2+Na+Na+Na+Ca2+SERCASRRyRL-TypeCa2+ChannelNa+/Ca2+ExchangerCa++SarcolemmaCa2+ActinTropomyosinTroponinTitinMyosin

Myosin-binding-proteinC

CapZ

Tropomodulin

Cross-linkingprotein

肌联蛋白（Titin）将粗肌丝与Z-线连接，维持肌原纤维的完整性和稳定性，保持舒张肌肉的静息张力，使粗肌丝处于肌小节的中央位置，使受牵拉的肌肉可恢复初始状态，以保证肌肉收缩时张力的输出。ZZTitin28,000aminoacids(3MDa)thelargestproteinknowninmammals.TitinThemolecularbasisformyocardialcontractionThinfilament(Actin,Tropom-yosin,Troponin)

Thickfilament(Myosin)OtherproteinsChien,K.R.,1999F-actinZ-lineZ-lineThinFilamentProteinsGtoFactin

MW42kDaTheblueandgreymoleculesareactinmonomers(MW42.000)KenC.Holmes:Max-Planck-Institute

G-ActinF-Actin

肌动蛋白以两种形式存在,即单体和多聚体。单体的肌动蛋白是由一条多肽链构成的球形分子,又称球状肌动蛋白(globularactin,G-actin),外形类似花生果。肌动蛋白的多聚体形成肌动蛋白丝,称为纤维状肌动蛋白(fibrosactin,F-actin)。在电子显微镜下,F-肌动蛋白呈双股螺旋状,直径为8nm,螺旋间的距离为37nm。

LorenzmodelofF-actin.AsingleG-actinmonomerwithinter-actincontactsurfacesisshownontheright,theentireF-actinontheleftActinfilamentsaredynamicpolymerswhoseATP-drivenassemblyinthecellcytoplasmdrivesshapechanges,celllocomotionandchemotacticmigration.Actinfilamentsalsoparticipateinmusclecontraction.Thestructureofthefilamentisnotknownatatomicresolution,butseveralmodelswereproducedinthelaboratoryofKenHolmes(MPIformedicalresearch,Heidelberg,Germany)byrefinementagainstX-rayfiberdiffractiondataTroponinHead-to-tailoverlapABTakeda,S.etal.Nature424,35–41,2003

HCTnCHCTnIHCTnTTropomyosinTropomyosinbindingregionHypervariableregionCrystalstructureofhumancardiactroponinTroponinCC-DomainN-DomainCentralHelixEachTnCdomaincontainstwomotifscalledEFhands,anditistheEFhandsthatdirectlybindcalciumions.Thus,theEFhandsareTnC'swayofsensingthecalciumconcentration;at≈100nMcalcium(theusualcellularconcentration)theN-domainEFhandsareempty,butifthelocalconcentrationrisesto1mM,asitdoeswhenthemusclecontracts,alloftheEFhandbindcalcium.KCa=3x105M-1Ca2+-specificKCa=2x107M-1Ca2+-Mg2+sitesEFhandsThickfilamentproteins

MYOSINMW480kDaFormsthickfilamentsHydrolysesATPInteractswithF-actin300-400myosinmoleculesper1filamentS1S1150nmMyosin重链-helicalcoiled-coil轻链160nmS1S1-MolecularMotorofMuscleContractionRLCELCMyosinHead(S1)–molecularmotorofmusclecontractionRLCELCATPBindingSiteActinBindingSiteATP(Myosin)

ADP+Pi+EnergyF-actinCross-bridge–ActinInteractionGordonetal.2001RegulationofthinfilamentincontractionABCDEFromCraigandLehman,2001,JMB311,1027Thereversiblebindingofcalciumtotroponinalterstheconformationofthethinfilament,therebyturningmusclecontractionONandOFFCross-bridgeSTATE: ThinfilamentSTATE:Relaxed(OFF) BLOCKEDCa2+Activated(WeakBinding) CLOSEDCa2+andMyosinActivated(Strongbinding) OPENThreepositionsofTropomyosin

ActivatedFilaments(blue:actinboundendofactivelycyclingcross-bridges)RegulationofMuscleContraction:a/ba/bATPCa2+MuscleContractionPiIntheabsenceofCa2+,theinteractionofmyosinwithactinandconsequentlycontractionisinhibited.UponreleaseofCa2+fromtheSR,theregulatory,Ca2+specificsitesofTnCbindCa2+exposingapatchofhydrophobicresidueslocatedintheN-terminaldomainofTnCandtheinteractionoftheTnCwithTnIandTnTcantakeplace.TheseinternalTninteractionspromotetranslocationoftheTn·Tmcomplexawayfromtheouterdomainoftheactinfilamentsenablingthecyclicinteractionbetweenmyosinheads(S1)andactin.Themyosinhead,anactinactivated-Mg2+-ATPasedependentmolecularmotor,bindstoactinandundergoesapowerstroke,aphenomenonresponsiblefortheinteractionbetweenthethickfilamentandthethinfilamentsandforcegeneration.ATPaseCycle1.A•M+ATP2.A+M•ATP3.A•M•ADP•Pi4.A•M•ADP+Pi5.A•M+ADP

PiADPPireleaserate:10-20s-1MuscleContraction

Pireleaserates:1.NoTm-Tn:10–20s-1;2.+Tm-TnnoCa2+:0.1-0.2s-1;3.+Tm-Tn+Ca2+:10–20s-1Actin-myosininteractionInvitromotilityassayshowingtheslidingofactinfilamentsoveramyosinsurfaceinitiatedbyflashphotolysisofcagedATP(CliveR.Bagshaw)BersDM.Cardiacexcitation-contractioncoupling[J].Nature,2002,415(6868):198-205.Excitation-contractioncouplingHeartfailureRyanodinereceptor(RyR)

PhosphorylationofRYRincreaseCa2+leakATP-dependentpump

Phospholamban(PLB)

InHFExpressionandactivationofSERCA2PhosphorylationofPLBExpressionofβ1ARATPsupplyuptake↓Re-uptake

StoreRelease

MSRSRCa2+sroredecrease,Ca2+transientdelayTheSRCa2+store123451.ReducedCa++triggerthruL-typechannel2.ReducedRyRfunction(CalciumleaksfromSR)3.DecreasedsensitivityofTN-CtoCa++4.ReducedCa++uptakeduetolossofSERCAfunctionandincreasedPlb5.IncreasedNa/CaexchangerfunctionOverviewofE-Ccoupling

changesinthefailing

heart正性肌力药的循证研究Ancienttreatmentofheart

failure洋地黄制剂（﹥200years）

DigilispurpureaPurplefoxgloveWilliamWithering(1741-1799)DigitalisMechanismofActionDIG试验(1997)总死亡率是中性在3.5年的随访中，心衰恶化而死亡的危险性，地高辛组有降低趋势，地高辛显著降低了因心衰住院死亡的危险性28%（P<0.01）。TheEffectofDigoxinonMortalityandMorbidityinPatientswithHeartFailure

NEng1Med,1997;336:525-533总死亡率

PlaceboDigoxinTheEffectofDigoxinonMortalityandMorbidityinPatientswithHeartFailureNEng1Med,1997;336:525-533因心衰住院死亡的发生率28%P<0.01PlaceboDigoxinTheEffectofDigoxinonMortalityandMorbidityinPatientswithHeartFailureNEng1Med,1997;336:525-533"Digitalis"iswithoutquestionthemostvaluablecardiacdrugeverdiscovered

oneofthemostvaluabledrugsintheent-ire

pharmacopoeia.Theintroductionofdigitaliswasoneofthelandmarksinthehistoryofcardiacdisease."Opie,H.L.DrugsfortheHeart.OrlandoFlorida:Grune&Stratton,Inc.1980.TherapeuticUse各种心脏病引起的充血性心力衰竭。快速性室上性心律失常：心房颤动、心房扑动、房性心动过速、阵发性房室交界区心动过速、反复性心动过速。

Sideeffectsactionpotentialrecordingsfrompurkinjefibercells(A)toxicdosesproduceoscillatoryafterdepolorizations(B)leadstoventriculartachycardia(C)β-肾上腺素能受体激动剂

β-受体激动剂与心肌细胞膜上β-受体结合通过G蛋白偶联激活腺苷酸活化酶(AC)催化ATP生成cAMPcAMP促使L型钙通道开放Ca内流增加，细胞内Ca浓度上升，起到正性肌力作用。DirectactingsympathomimeticsDopamineDobutamineTherapeuticUse对维持血压和心输出量具有重要意义，但易引起心率加快、心肌耗氧量增加，诱发心律失常和心肌受体下调,对生存率有不良影响。多用于紧急情况的急性心衰、难治性心衰。DiesF,etal.Circulation1986;74(supplII):II-39.磷酸二酯酶抑制剂

Thedifferentformsorsubtypesofphosphodiesterasewereinitiallyisolatedfrom

ratbrains

byUzunovandWeissin1972andweresoonafterwardsshowntobeselectivelyinhibitedinthebrainandinothertissuesbyavarietyofdrugsThepotentialforselectivephosphodisteraseinhibitorsastherapeuticagentswaspredictedasearlyas

1977byWeissandHait.Thispredictionmeanwhilehasprovedtobetrueinavarietyoffields.Uzunov,P.andWeiss,BBiochim.Biophys.Acta284:220-226,1972Weiss,B.andHait,W.N.:Ann.Rev.Pharmacol.Toxicol.17:441-477,1977.代表药物为氨力农(amrinone)和米力农(milrinone)。增强心肌收缩力，降低后负荷，提高心肌舒张速率PhosphodiesteraseInhibitorsMechanismofActionβ-ADR和PDEI的作用位点(accordingtoLippincott´sPharmacology,2006)PROMISE临床试验（1991）NYHAIII、IV级，EF<35%米力农1000例结果总死亡率↑28%心血管死亡率的危险性↑34%猝死危险↑69%亚组结论：心功能越差，危险性越高,试验提前终止PackerM,etal.Effectofmilrinoneonmortalityinseverechronicheartfailure.NEnglJMed.1991;325:1468-1475.

TherapeuticUse米力农尚不足以作为充血性心衰的首选强心剂和血管扩张剂只是作为重症心衰的辅助用药或洋地黄中毒患者的二次选择药物主要用于急性心衰钙增敏剂MCI-154、左西孟旦（levosimendan）是其中有代表性的药物。作用机制增加心肌TnC对Ca2＋的敏感性稳定Ca2＋-TnC构象直接增强肌球蛋白和肌动蛋白之间的相互作用MechanismofActionActinTropomyosinTnITnTCa2+cTnCMyosinhead(S1fragment)ATPpocketRLCELC左西孟旦REVIVE-2研究(2005)REVIVE-2研究共入选600例心力衰竭患者,在常规治疗的基础上随机加用Levosimendan研究结果应用Levosimendan组心功能改善者比对照组多33%，心功能恶化者比对照组少30%PackerM.AHAScientificSessions,Dallas,USA,November,2005.PrimaryEndpoint(n=600)PackerM.AHAScientificSessions,Dallas,USA,November,2005.33%30%SideEffects研究发现通过增加钙敏感性的药物也可减慢心肌的舒张。这是由于增加了肌纤维对舒张时细胞内Ca2+的敏感性，使Ca2+从TnC的解离速度减慢，从而妨碍心肌的舒张，影响心室的充盈。WhiteJ,LeeJA,ShahN,etal.DifferentialeffectsoftheopticalisomersofEMD53998oncontractionandcytoplasmicCa2+inisolatedferretcardiacmus-cle[J].CircRes,1993,73:61270.LeeJA,AllenDG.EMD53998sensitizesthecontracti