從興奮收縮耦聯(lián)機(jī)制看心力衰竭正性肌力藥物發(fā)展

從興奮收縮耦聯(lián)機(jī)制看心力衰竭正性肌力藥物發(fā)展第1頁/共76頁提要興奮-收縮耦聯(lián)機(jī)制正性肌力藥的循證研究洋地黃制劑β-腎上腺素能受體激動(dòng)劑磷酸二酯酶抑制劑鈣增敏劑新型正性肌力藥的探索亞硝酰氫

第2頁/共76頁興奮-收縮耦聯(lián)機(jī)制第3頁/共76頁第4頁/共76頁Excitation-contraction(EC)couplingisatermcoinedin1952todescribethephysiologicalprocessofconvertinganelectricalstimulustomechanicalresponse.SandowA(1952)."Excitation-contractioncouplinginmuscularresponse.".YaleJBiolMed25(3):176–201.PMID130159500Excitation-contractioncoupling第5頁/共76頁Cardiacexcitation–contractioncouplingistheprocessfromelectricalexcitationofthemyocytetocontractionoftheheart(whichpropelsbloodout).TheubiquitoussecondmessengerCa2+isessentialincardiacelectricalactivityandisthedirectactivatorofthemyofilaments,whichcausecontraction.Bers,D.M.Excitation–ContractionCouplingandCardiacContractileForceedn2(KluwerAcademic,Dordrecht,Netherlands,2001).Cardiacexcitation–contractioncoupling第6頁/共76頁Cardiactissue(Guinea-pigventricularcell)Cardiactissue第7頁/共76頁

Cardiaccells第8頁/共76頁TheactionpotentialmovesthroughsarcolemmaTtube第9頁/共76頁Ca2+-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+第10頁/共76頁ActinTropomyosinTroponinTitinMyosin

Myosin-binding-proteinC

CapZ

Tropomodulin

Cross-linkingprotein

第11頁/共76頁肌聯(lián)蛋白（Titin）將粗肌絲與Z-線連接，維持肌原纖維的完整性和穩(wěn)定性，保持舒張肌肉的靜息張力，使粗肌絲處于肌小節(jié)的中央位置，使受牽拉的肌肉可恢復(fù)初始狀態(tài)，以保證肌肉收縮時(shí)張力的輸出。ZZTitin28,000aminoacids(3MDa)thelargestproteinknowninmammals.第12頁/共76頁Titin第13頁/共76頁ThemolecularbasisformyocardialcontractionThinfilament(Actin,Tropom-yosin,Troponin)

Thickfilament(Myosin)OtherproteinsChien,K.R.,1999第14頁/共76頁F-actinZ-lineZ-lineThinFilamentProteins第15頁/共76頁GtoFactin

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

G-ActinF-Actin

肌動(dòng)蛋白以兩種形式存在,即單體和多聚體。單體的肌動(dòng)蛋白是由一條多肽鏈構(gòu)成的球形分子,又稱球狀肌動(dòng)蛋白(globularactin,G-actin),外形類似花生果。肌動(dòng)蛋白的多聚體形成肌動(dòng)蛋白絲,稱為纖維狀肌動(dòng)蛋白(fibrosactin,F-actin)。在電子顯微鏡下,F-肌動(dòng)蛋白呈雙股螺旋狀,直徑為8nm,螺旋間的距離為37nm。

第16頁/共76頁LorenzmodelofF-actin.AsingleG-actinmonomerwithinter-actincontactsurfacesisshownontheright,theentireF-actinontheleftActinfilamentsaredynamicpolymerswhoseATP-drivenassemblyinthecellcytoplasmdrivesshapechanges,celllocomotionandchemotacticmigration.Actinfilamentsalsoparticipateinmusclecontraction.Thestructureofthefilamentisnotknownatatomicresolution,butseveralmodelswereproducedinthelaboratoryofKenHolmes(MPIformedicalresearch,Heidelberg,Germany)byrefinementagainstX-rayfiberdiffractiondata第17頁/共76頁TroponinHead-to-tailoverlapAB第18頁/共76頁Takeda,S.etal.Nature424,35–41,2003

HCTnCHCTnIHCTnTTropomyosinTropomyosinbindingregionHypervariableregionCrystalstructureofhumancardiactroponin第19頁/共76頁TroponinCC-DomainN-DomainCentralHelixEachTnCdomaincontainstwomotifscalledEFhands,anditistheEFhandsthatdirectlybindcalciumions.Thus,theEFhandsareTnC'swayofsensingthecalciumconcentration;at≈100nMcalcium(theusualcellularconcentration)theN-domainEFhandsareempty,butifthelocalconcentrationrisesto1mM,asitdoeswhenthemusclecontracts,alloftheEFhandbindcalcium.KCa=3x105M-1Ca2+-specificKCa=2x107M-1Ca2+-Mg2+sites第20頁/共76頁EFhands第21頁/共76頁Thickfilamentproteins

MYOSINMW480kDaFormsthickfilamentsHydrolysesATPInteractswithF-actin300-400myosinmoleculesper1filamentS1S1150nm第22頁/共76頁Myosin重鏈-helicalcoiled-coil輕鏈160nmS1S1-MolecularMotorofMuscleContractionRLCELC第23頁/共76頁MyosinHead(S1)–molecularmotorofmusclecontractionRLCELCATPBindingSiteActinBindingSiteATP(Myosin)

ADP+Pi+Energy第24頁/共76頁第25頁/共76頁F-actinCross-bridge–ActinInteraction第26頁/共76頁Gordonetal.2001Regulationofthinfilamentincontraction第27頁/共76頁ABCDEFromCraigandLehman,2001,JMB311,1027Thereversiblebindingofcalciumtotroponinalterstheconformationofthethinfilament,therebyturningmusclecontractionONandOFFCross-bridgeSTATE: ThinfilamentSTATE:Relaxed(OFF) BLOCKEDCa2+Activated(WeakBinding) CLOSEDCa2+andMyosinActivated(Strongbinding) OPENThreepositionsofTropomyosinActivatedFilaments(blue:actinboundendofactivelycyclingcross-bridges)RegulationofMuscleContraction:第28頁/共76頁a/ba/bATPCa2+MuscleContractionPiIntheabsenceofCa2+,theinteractionofmyosinwithactinandconsequentlycontractionisinhibited.UponreleaseofCa2+fromtheSR,theregulatory,Ca2+specificsitesofTnCbindCa2+exposingapatchofhydrophobicresidueslocatedintheN-terminaldomainofTnCandtheinteractionoftheTnCwithTnIandTnTcantakeplace.TheseinternalTninteractionspromotetranslocationoftheTn·Tmcomplexawayfromtheouterdomainoftheactinfilamentsenablingthecyclicinteractionbetweenmyosinheads(S1)andactin.Themyosinhead,anactinactivated-Mg2+-ATPasedependentmolecularmotor,bindstoactinandundergoesapowerstroke,aphenomenonresponsiblefortheinteractionbetweenthethickfilamentandthethinfilamentsandforcegeneration.第29頁/共76頁ATPaseCycle1.A?M+ATP2.A+M?ATP3.A?M?ADP?Pi4.A?M?ADP+Pi5.A?M+ADPPiADPPireleaserate:10-20s-1第30頁/共76頁MuscleContraction

Pireleaserates:1.NoTm-Tn:10–20s-1;2.+Tm-TnnoCa2+:0.1-0.2s-1;3.+Tm-Tn+Ca2+:10–20s-1第31頁/共76頁Actin-myosininteractionInvitromotilityassayshowingtheslidingofactinfilamentsoveramyosinsurfaceinitiatedbyflashphotolysisofcagedATP(CliveR.Bagshaw)第32頁/共76頁BersDM.Cardiacexcitation-contractioncoupling[J].Nature,2002,415(6868):198-205.Excitation-contractioncoupling第33頁/共76頁Heartfailure第34頁/共76頁Ryanodinereceptor(RyR)

PhosphorylationofRYRincreaseCa2+leak第35頁/共76頁ATP-dependentpump

Phospholamban(PLB)

InHFExpressionandactivationofSERCA2PhosphorylationofPLBExpressionofβ1ARATPsupplyuptake↓第36頁/共76頁Re-uptake

StoreRelease

MSRSRCa2+sroredecrease,Ca2+transientdelayTheSRCa2+store第37頁/共76頁123451.ReducedCa++triggerthruL-typechannel2.ReducedRyRfunction(CalciumleaksfromSR)3.DecreasedsensitivityofTN-CtoCa++4.ReducedCa++uptakeduetolossofSERCAfunctionandincreasedPlb5.IncreasedNa/CaexchangerfunctionOverviewofE-Ccoupling

changesinthefailing

heart第38頁/共76頁正性肌力藥的循證研究第39頁/共76頁Ancienttreatmentofheart

failure第40頁/共76頁洋地黃制劑（﹥200years）

DigilispurpureaPurplefoxgloveWilliamWithering

(1741-1799)第41頁/共76頁DigitalisMechanismofAction第42頁/共76頁DIG試驗(yàn)(1997)總死亡率是中性在3.5年的隨訪中，心衰惡化而死亡的危險(xiǎn)性，地高辛組有降低趨勢(shì)，地高辛顯著降低了因心衰住院死亡的危險(xiǎn)性28%（P<0.01）。TheEffectofDigoxinonMortalityandMorbidityinPatientswithHeartFailure

NEng1Med,1997;336:525-533第43頁/共76頁總死亡率

PlaceboDigoxinTheEffectofDigoxinonMortalityandMorbidityinPatientswithHeartFailureNEng1Med,1997;336:525-533第44頁/共76頁因心衰住院死亡的發(fā)生率28%P<0.01PlaceboDigoxinTheEffectofDigoxinonMortalityandMorbidityinPatientswithHeartFailureNEng1Med,1997;336:525-533第45頁/共76頁"Digitalis"iswithoutquestionthemostvaluablecardiacdrugeverdiscovered

oneofthemostvaluabledrugsintheent-ire

pharmacopoeia.Theintroductionofdigitaliswasoneofthelandmarksinthehistoryofcardiacdisease."Opie,H.L.DrugsfortheHeart.OrlandoFlorida:Grune&Stratton,Inc.1980.第46頁/共76頁TherapeuticUse各種心臟病引起的充血性心力衰竭?？焖傩允疑闲孕穆墒С＃盒姆款潉?dòng)、心房撲動(dòng)、房性心動(dòng)過速、陣發(fā)性房室交界區(qū)心動(dòng)過速、反復(fù)性心動(dòng)過速。

第47頁/共76頁Sideeffectsactionpotentialrecordingsfrompurkinjefibercells(A)toxicdosesproduceoscillatoryafterdepolorizations(B)leadstoventriculartachycardia(C)第48頁/共76頁β-腎上腺素能受體激動(dòng)劑

β-受體激動(dòng)劑與心肌細(xì)胞膜上β-受體結(jié)合通過G蛋白偶聯(lián)激活腺苷酸活化酶(AC)催化ATP生成cAMPcAMP促使L型鈣通道開放Ca內(nèi)流增加，細(xì)胞內(nèi)Ca濃度上升，起到正性肌力作用。第49頁/共76頁DirectactingsympathomimeticsDopamineDobutamine第50頁/共76頁TherapeuticUse對(duì)維持血壓和心輸出量具有重要意義，但易引起心率加快、心肌耗氧量增加，誘發(fā)心律失常和心肌受體下調(diào),對(duì)生存率有不良影響。多用于緊急情況的急性心衰、難治性心衰。DiesF,etal.Circulation1986;74(supplII):II-39.第51頁/共76頁磷酸二酯酶抑制劑

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.第52頁/共76頁代表藥物為氨力農(nóng)(amrinone)和米力農(nóng)(milrinone)。增強(qiáng)心肌收縮力，降低后負(fù)荷，提高心肌舒張速率PhosphodiesteraseInhibitors第53頁/共76頁MechanismofActionPDEI為非強(qiáng)心甙非兒茶酚胺類強(qiáng)心藥，通過抑制cAMP在心肌和平滑肌細(xì)胞的降解，而發(fā)揮正性肌力作用。第54頁/共76頁β-ADR和PDEI的作用位點(diǎn)(accordingtoLippincott′sPharmacology,2006)第55頁/共76頁P(yáng)ROMISE臨床試驗(yàn)（1991）NYHAIII、IV級(jí)，EF<35%米力農(nóng)1000例結(jié)果總死亡率↑28%心血管死亡率的危險(xiǎn)性↑34%

猝死危險(xiǎn)↑69%亞組結(jié)論：心功能越差，危險(xiǎn)性越高,試驗(yàn)提前終止PackerM,etal.Effectofmilrinoneonmortalityinseverechronicheartfailure.NEnglJMed.1991;325:1468-1475.

第56頁/共76頁TherapeuticUse米力農(nóng)尚不足以作為充血性心衰的首選強(qiáng)心劑和血管擴(kuò)張劑只是作為重癥心衰的輔助用藥或洋地黃中毒患者的二次選擇藥物主要用于急性心衰第57頁/共76頁鈣增敏劑MCI-154、左西孟旦（levosimendan）是其中有代表性的藥物。作用機(jī)制增加心肌TnC對(duì)Ca2＋的敏感性穩(wěn)定Ca2＋-TnC構(gòu)象直接增強(qiáng)肌球蛋白和肌動(dòng)蛋白之間的相互作用第58頁/共76頁MechanismofActionActinTropomyosinTnITnTCa2+cTnCMyosinhead(S1fragment)ATPpocketRLCELC左西孟旦第59頁/共76頁REVIVE-2研究(2005)REVIVE-2研究共入選600例心力衰竭患者,在常規(guī)治療的基礎(chǔ)上隨機(jī)加用Levosimendan研究結(jié)果應(yīng)用Levosimendan組心功能改善者比對(duì)照組多33%，心功能惡化者比對(duì)照組少30%PackerM.AHAScientificSessions,Dallas,USA,November,2005.第60頁/共76頁P(yáng)rimaryEndpoint(n=600)PackerM.AHAScientificSessions,Dallas,USA,November,2005.33%30%第61頁/共76頁SideEffects研究發(fā)現(xiàn)通過增加鈣敏感性的藥物也可減慢心肌的舒張。這是由于增加了肌纖維對(duì)舒張時(shí)細(xì)胞內(nèi)Ca2+

的敏感性，使Ca2+從TnC的解離速度減慢，從而妨礙心肌的舒張，影響心室的充盈。WhiteJ,LeeJA,ShahN,etal.DifferentialeffectsoftheopticalisomersofEMD53998oncontractionandcytoplasmicCa2+inisolatedferretcardiacmus-cle[J].CircRes,1993,73:61270.LeeJA,AllenDG.EMD53998sensitizesthecontractileproteinstocalciuminintactferretventricularmuscle[J].CircRes,1991,69:9272936.第62頁/共76頁TherapeuticUse失代償性急性心力衰竭，伴心輸出量下降和高灌注壓心臟術(shù)后心力衰竭（頓抑）急性心肌梗死后心力