從興奮收縮耦聯機制看心力衰竭

正性肌力藥物發展田野教授哈醫大二院心內科提要興奮-收縮耦聯機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

興奮-收縮耦聯機制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

肌聯蛋白（Titin）將粗肌絲與Z-線連接，維持肌原纖維的完整性和穩定性，保持舒張肌肉的靜息張力，使粗肌絲處于肌小節的中央位置，使受牽拉的肌肉可恢復初始狀態，以保證肌肉收縮時張力的輸出。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

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

failure洋地黃制劑（﹥200years）

DigilispurpureaPurplefoxgloveWilliamWithering(1741-1799)DigitalisMechanismofActionDIG試驗(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性，地高辛組有降低趨勢，地高辛顯著降低了因心衰住院死亡的危險性28%（P<0.01）。TheEffectofDigoxinonMortalityandMorbidityinPatientswithHeartFailure

NEng1Med,1997;336:525-533總死亡率

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

oneofthemostvaluabledrugsintheent-ire

pharmacopoeia.Theintroductionofdigitaliswasoneofthelandmarksinthehistoryofcardiacdisease."Opie,H.L.DrugsfortheHeart.OrlandoFlorida:Grune&Stratton,Inc.1980.TherapeuticUse各種心臟病引起的充血性心力衰竭。快速性室上性心律失常：心房顫動、心房撲動、房性心動過速、陣發性房室交界區心動過速、反復性心動過速。

Sideeffectsactionpotentialrecordingsfrompurkinjefibercells(A)toxicdosesproduceoscillatoryafterdepolorizations(B)leadstoventriculartachycardia(C)β-腎上腺素能受體激動劑

β-受體激動劑與心肌細胞膜上β-受體結合通過G蛋白偶聯激活腺苷酸活化酶(AC)催化ATP生成cAMPcAMP促使L型鈣通道開放Ca內流增加，細胞內Ca濃度上升，起到正性肌力作用。DirectactingsympathomimeticsDopamineDobutamineTherapeuticUse對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，誘發心律失常和心肌受體下調,對生存率有不良影響。多用于緊急情況的急性心衰、難治性心衰。DiesF,etal.Circulation1986;74(supplII):II-39.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.代表藥物為氨力農(amrinone)和米力農(milrinone)。增強心肌收縮力，降低后負荷，提高心肌舒張速率PhosphodiesteraseInhibitorsMechanismofActionβ-ADR和PDEI的作用位點(accordingtoLippincott′sPharmacology,2006)PROMISE臨床試驗（1991）NYHAIII、IV級，EF<35%米力農1000例結果總死亡率↑28%心血管死亡率的危險性↑34%猝死危險↑69%亞組結論：心功能越差，危險性越高,試驗提前終止PackerM,etal.Effectofmilrinoneonmortalityinseverechronicheartfailure.NEnglJMed.1991;325:1468-1475.

TherapeuticUse米力農尚不足以作為充血性心衰的首選強心劑和血管擴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鈣增敏劑MCI-154、左西孟旦（levosimendan）是其中有代表性的藥物。作用機制增加心肌TnC對Ca2＋的敏感性穩定Ca2＋-TnC構象直接增強肌球蛋白和肌動蛋白之間的相互作用MechanismofActionActinTropomyosinTnITnTCa2+cTnCMyosinhead(S1fragment)ATPpocketRLCELC左西孟旦REVIVE-2研究(2005)REVIVE-2研究共入選600例心力衰竭患者,在常規治療的基礎上隨機加用Levosimendan研究結果應用Levosimendan組心功能改善者比對照組多33%，心功能惡化者比對照組少30%PackerM.AHAScientificSessions,Dallas,USA,November,2005.PrimaryEndpoint(n=600)PackerM.AHAScientificSessions,Dallas,USA,November,2005.33%30%SideEffects研究發現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舒張時細胞內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.EMD53998sensitizesthecontracti