从兴奋收缩耦联机制看心力衰竭正性肌力药物发展.pptx

从兴奋收缩耦联机制看心力衰竭从兴奋收缩耦联机制看心力衰竭正性肌力药物发展正性肌力药物发展田野田野 教授教授哈医大二院心内科哈医大二院心内科提提 要要3兴奋兴奋-收缩耦联机制收缩耦联机制3正性肌力药的循证研究正性肌力药的循证研究-洋地黄制剂-肾上腺素能受体激动剂-磷酸二酯酶抑制剂-钙增敏剂3新型正性肌力药的探索新型正性肌力药的探索-亚硝酰氢兴奋-收缩耦联机制3Excitation-contraction(EC)coupling is a Excitation-contraction(EC)coupling is a term coined in term coined in 19521952 to describe the to describe the physiological process of converting an physiological process of converting an electrical stimulus to mechanical responseelectrical stimulus to mechanical response.Sandow A(1952).Excitation-contraction coupling in muscular response.Yale J Biol Med 25(3):176201.PMID 130159500Excitation-contraction coupling3Cardiac Cardiac excitationcontraction excitationcontraction coupling coupling is is the the process process from from electrical electrical excitation excitation of of the the myocyte myocyte to to contraction contraction of of the the heart heart(which(which propels propels blood blood out).out).The The ubiquitous ubiquitous second second messenger messenger Ca2+Ca2+is is essential essential in in cardiac cardiac electrical electrical activity activity and and is is the the direct direct activator activator of of the the myofilaments,myofilaments,which which cause cause contraction.contraction.Bers,D.M.ExcitationContraction Coupling and Cardiac Contractile Force edn 2(Kluwer Academic,Dordrecht,Netherlands,2001).Cardiac excitationcontraction couplingCardiac tissue(Guinea-pig ventricular cell)Cardiac tissue Cardiac cellsThe action potential moves through sarcolemmaT tubeCa2+-induced Ca2+-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-Type Ca2+ChannelNa+/Ca2+ExchangerCa+SarcolemmaCa2+ActinTropomyosinTroponinTitinMyosin Myosin-binding-protein C CapZ Tropomodulin Cross-linking protein 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。ZZTitin28,000 amino acids(3MDa)the largest protein known in mammals.TitinThe molecular basis for myocardial contractionThin filament(Actin,Tropom-yosin,Troponin)Thick filament(Myosin)Other proteinsChien,K.R.,1999F-actinZ-lineZ-lineThin Filament ProteinsG to F actin MW 42 kDaThe blue and grey molecules are actin monomers(MW 42.000)Ken C.Holmes:Max-Planck-Institute G-Actin F-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(globular actin,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(fibros actin,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。Lorenz model of F-actin.A single G-actin monomer with inter-actin contact surfaces is shown on the right,the entire F-actin on the leftActin filaments are dynamic polymers whose ATP-driven assembly in the cell cytoplasm drives shape changes,cell locomotion and chemotactic migration.Actin filaments also participate in muscle contraction.The structure of the filament is not known at atomic resolution,but several models were produced in the laboratory of Ken Holmes(MPI for medical research,Heidelberg,Germany)by refinement against X-ray fiber diffraction dataTroponinHead-to-tail overlapABTakeda,S.et al.Nature 424,35 41,2003 HCTnCHCTnIHCTnTTropomyosinTropomyosinbinding regionHypervariable regionCrystal structure of human cardiac troponinTroponin CC-DomainN-DomainCentral HelixEach TnC domain contains two motifs called EF hands,and it is the EF hands that directly bind calcium ions.Thus,the EF hands are TnCs way of sensing the calcium concentration;at 100 nM calcium(the usual cellular concentration)the N-domain EF hands are empty,but if the local concentration rises to 1 M,as it does when the muscle contracts,all of the EF hand bind calcium.KCa=3 x 105 M-1Ca2+-specificKCa=2 x 107 M-1Ca2+-Mg2+sitesEF handsThick filament proteins MYOSIN MW 480 kDaForms thick filamentsHydrolyses ATPInteracts with F-actin 300-400 myosin molecules per 1 filamentS1S1150 nmMyosin重链重链-helical coiled-coil轻链轻链160 nmS1S1-Molecular Motor of Muscle ContractionRLCELCMyosin Head(S1)molecular motor of muscle contractionRLCELCATP Binding SiteActin Binding SiteATP(Myosin)ADP+Pi+EnergyF-actinCross-bridge Actin InteractionGordon et al.2001Regulation of thin filament in contraction A B C D EFrom Craig and Lehman,2001,JMB 311,1027The reversible binding of calcium to troponin alters the conformation of the thin filament,thereby turning muscle contraction ON and OFF Cross-bridge STATE:Thin filament STATE:Relaxed(OFF)BLOCKEDCa2+Activated(Weak Binding)CLOSED Ca2+and Myosin Activated(Strong binding)OPEN Three positions of Tropomyosin Activated Filaments(blue:actin bound end of actively cycling cross-bridges)Regulation of Muscle Contraction:a/ba/bATPCa2+Muscle ContractionPiIn the absence of Ca2+,the interaction of myosin with actin and consequently contraction is inhibited.Upon release of Ca2+from the SR,the regulatory,Ca2+specific sites of TnC bind Ca2+exposing a patch of hydrophobic residues located in the N-terminal domain of TnC and the interaction of the TnC with TnI and TnT can take place.These internal Tn interactions promote translocation of the TnTm complex away from the outer domain of the actin filaments enabling the cyclic interaction between myosin heads(S1)and actin.The myosin head,an actin activated-Mg2+-ATPase dependent molecular motor,binds to actin and undergoes a power stroke,a phenomenon responsible for the interaction between the thick filament and the thin filaments and force generation.ATPase Cycle1.A M+ATP2.A+MATP3.A M ADP Pi4.A M ADP+Pi5.A M+ADP PiADPPi release rate:10-20s-1Muscle Contraction Pi release rates:1.No Tm-Tn:10 20 s-1;2.+Tm-Tn no Ca2+:0.1-0.2 s-1;3.+Tm-Tn+Ca2+:10 20 s-1Actin-myosin interactionIn vitro motility assay showing the sliding of actin filaments over a myosin surface initiated by flash photolysis of caged ATP(Clive R.Bagshaw)Bers DM.Cardiac excitation-contraction coupling J.Nature,2002,415(6868):198-205.Excitation-contraction couplingHeart failureRyanodine receptor(RyR)Phosphorylation of RYR increaseCa2+leak ATP-dependent pump Phospholamban(PLB)In HF Expression and activation of SERCA2Phosphorylation of PLB Expression of 1ARATP supplyuptakeRe-uptake StoreRelease MSRSR Ca2+srore decrease,Ca2+transient delayThe SR Ca2+store123451.Reduced Ca+trigger thru L-type channel2.Reduced RyR function(Calcium leaks from SR)3.Decreased sensitivity of TN-C to Ca+4.Reduced Ca+uptake due to loss of SERCA function and increased Plb5.Increased Na/Ca exchanger functionOverview of E-C coupling changes in the failing heart正性肌力药的循证研究正性肌力药的循证研究Ancient treatment of heart failure洋地黄制剂（洋地黄制剂（200 years）Digilis purpureaDigilis purpureaPurple foxglovePurple foxgloveWilliam WitheringWilliam Withering (1741-1799)(1741-1799)DigitalisMechanism of ActionDIG 试验试验(1997)3总死亡率是中性3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%（P0.01）。The Effect of Digoxin on Mortality and Morbidity in Patients with Heart FailureN Eng1 Med,1997;336:525-533总死亡率总死亡率 PlaceboDigoxinThe Effect of Digoxin on Mortality and Morbidity in Patients with Heart Failure N Eng1 Med,1997;336:525-533因心衰住院死亡的发生率因心衰住院死亡的发生率28%P0.01PlaceboDigoxinThe Effect of Digoxin on Mortality and Morbidity in Patients with Heart Failure N Eng1 Med,1997;336:525-5333Digitalis is Digitalis is without question the most without question the most valuable cardiac drug ever discoveredvaluable cardiac drug ever discovered 3one of one of the most valuable drugs in the ent-irethe most valuable drugs in the ent-ire pharmacopoeiapharmacopoeia.3The introduction of digitalis was The introduction of digitalis was one of the one of the landmarkslandmarks in the history of cardiac disease.in the history of cardiac disease.Opie,H.L.Drugs for the Heart.Orlando Florida:Grune&Stratton,Inc.1980.Therapeutic Use3各种心脏病引起的充血性心力衰竭。3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。Side effects3action potential recordings from purkinje fiber cells(A)3toxic doses produce oscillatory after depolorizations(B)3leads to ventricular tachycardia(C)-肾上腺素能受体激动剂肾上腺素能受体激动剂 3-受体激动剂与心肌细胞膜上-受体结合3通过G蛋白偶联激活腺苷酸活化酶(AC)3催化ATP生成cAMP3cAMP促使L型钙通道开放3Ca内流增加，细胞内Ca浓度上升，起到正性肌力作用。Direct acting sympathomimeticsDopamineDobutamineTherapeutic Use3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。3多用于紧急情况的急性心衰、难治性心衰。Dies F,et al.Circulation 1986;74(suppl II):II-39.磷酸二酯酶抑制剂磷酸二酯酶抑制剂 3The different forms or subtypes of phosphodiesterase were initially isolated from rat brains by Uzunov and Weiss in 1972 and were soon afterwards shown to be selectively inhibited in the brain and in other tissues by a variety of drugs3The potential for selective phosphodisterase inhibitors as therapeutic agents was predicted as early as 1977 by Weiss and Hait.This prediction meanwhile has proved to be true in a variety of fields.Uzunov,P.and Weiss,B Biochim.Biophys.Acta 284:220-226,1972 Weiss,B.and Hait,W.N.:Ann.Rev.Pharmacol.Toxicol.17:441-477,1977.3代表药物为氨力农(amrinone)和米力农(milrinone)。3增强心肌收缩力，降低后负荷，提高心肌舒张速率Phosphodiesterase InhibitorsMechanism of Action3PDEI为非强心甙非儿茶酚胺类强心药，通过抑制cAMP在心肌和平滑肌细胞的降解，而发挥正性肌力作用。-ADR和PDEI的作用位点(according to Lippincotts Pharmacology,2006)PROMISE临床试验（临床试验（1991）3NYHA III、IV级，EF35%米力农 1000例3结果结果-总死亡率28%-心血管死亡率的危险性34%-猝死危险69%3亚组结论亚组结论：心功能越差，危险性越高,试验提前终止Packer M,et al.Effect of milrinone on mortality in severe chronic heart failure.N Engl J Med.1991;325:1468-1475.Therapeutic Use3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3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3主要用于急性心衰 钙增敏剂钙增敏剂3MCI-154、左西孟旦（levosimendan）是其中有代表性的药物。3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Mechanism of ActionActinTropomyosinTnITnTCa2+cTnCMyosin head(S1 fragment)ATP pocketRLCELC左西孟旦左西孟旦REVIVE-2 研究研究(2005)3REV IVE-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Levosimendan3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%Packer M.AHA Scientific Sessions,Dallas,USA,November,2005.Primary Endpoint(n=600)Packer M.AHA Scientific Sessions,Dallas,USA,November,2005.33%30%Side Effects3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。White J,Lee JA,Shah N,et al.Differential effects of the optical isomers of EMD53998 on contraction and cytoplasmic Ca2+in isolated ferret cardiac mus-cleJ.Circ Res,1993,73:61270.Lee JA,Allen DG.EMD53998 sensitizes the contractile proteins to calcium in intact ferret ventricular muscleJ.Circ Res,1991,69:9272936.Therapeutic Use3失代偿性急性心力衰竭，伴心输出量下降和高灌注压3心脏术后心力衰竭（顿抑）3急性心肌梗死后心力衰竭新型正性肌力药的探索新型正性肌力药的探索亚硝酰氢亚硝酰氢3HNO是是NO的去单电子产物，的去单电子产物，HNO去质去质子化将产生相应的阴离子子化将产生相应的阴离子NO-。Ribeiro da Silva MD,et al.,Org Biomol Chem.2004,2(17):2507-12.HNO直接修饰肌丝直接修饰肌丝3HNO对对肌肌浆浆网网钙钙离离子子的的重重吸吸收收和和释释放放可可不不依依赖赖于于cAMP/PKA或或cGMP/PKG途途径径，而而是是与与巯巯基基化化合合物物的的修修饰饰密密切切相相关关；事事实上在心肌细胞的细肌丝上也存在巯基。实上在心肌细胞的细肌丝上也存在巯基。3HNO可可能能直直接接修修饰饰肌肌丝丝巯巯基基，而而独独立立于于钙转运系统。钙转运系统。Ingraham RH,et al.Biochemistry,1988 Aug 9;27(16):5891-8.Tocchetti CG,et al.Circulation Res.2007,100(1):96-104.Excitation-contraction coupling in cardiac contractile cellsThin filament proteinsHead-to-tail overlapHNO增加整体心肌的收缩力增加整体心肌的收缩力Pooled data of force development and Ca2+i transients at varied Ca2+o in the absence(open symbols)and presence(closed symbols)of AS(250 mol/L).(P0.001 by one-way ANOVA).N=7 in each group.HNO不影响心肌的舒张功能不影响心肌的舒张功能HNO不依赖心肌钙离子不依赖心肌钙离子HNO不增加肌纤维不增加肌纤维ATP酶活性酶活性The effects are HNO specific.The control drug(1-nitrosocyclohexyl tertbutylacetate)has no obvious effect on force of Skinning myocyte.HNO结构的特异性结构的特异性小小 结结-洋地黄制剂(Digitalis)-肾上腺素能受体激动剂(-ADR)-磷酸二酯酶抑制剂(Phosphodiesterase inhibitor,PDEI)-钙增敏剂（Calcium sensitizers）-亚硝酰氢（HNO）THANKS FOR YOUR ATTENTION!