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

1、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,田野 教授哈醫(yī)大二院心內(nèi)科,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(lián)機(jī)制,Excitation-contraction (EC) coupling is a term coined in 1952 to describe the physiological

2、process of converting an electrical stimulus to mechanical response.,Sandow A (1952). "Excitation-contraction coupling in muscular response.". Yale J Biol Med 25 (3): 176–201. PMID 130159500,Excitation-contrac

3、tion coupling,Cardiac excitation–contraction coupling is the process from electrical excitation of the myocyte to contraction of the heart (which propels blood out). The ubiquitous second messenger Ca2+ is essential in c

4、ardiac electrical activity and is the direct activator of the myofilaments, which cause contraction.,Bers, D. M. Excitation–Contraction Coupling and Cardiac Contractile Force edn 2 (Kluwer Academic,Dordrecht, Netherlands

5、, 2001).,Cardiac excitation–contraction coupling,Cardiac tissue,,(Guinea-pig ventricular cell),Cardiac tissue,Cardiac cells,The action potential moves through sarcolemma,,,T tube,,Ca2+- induced Ca2+- release,Ca++,Ca++,Ca

6、++,Ca2+,,,,Plb,Ca2+,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+

7、+,Ca++,Ca++,Ca++,Ca2+,Ca2+,Ca2+,Ca2+,Ca2+,Ca2+,Na+,Na+,Na+,Ca2+,SERCA,SR,RyR,L-Type Ca2+Channel,Na+/Ca2+ Exchanger,Ca++,Sarcolemma,Ca2+,,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

8、肌肉收縮時張力的輸出。,The molecular basis for myocardial contraction,Thin filament (Actin ,Tropom-yosin, Troponin) Thick filament (Myosin)Other proteins,Chien, K.R., 1999,F-actin,Z-line,Z-line,,Thin Filament Proteins,

9、G to F actin MW 42 kDaThe blue and grey molecules are actin monomers (MW 42.000),Ken C. Holmes: Max-Planck-Institute,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(gòu)成的球形分子, 又稱球狀肌動蛋白(globular actin, G-actin),外形類似花生果。肌動蛋白的多聚體形成肌動蛋

10、白絲, 稱為纖維狀肌動蛋白(fibros actin, 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。,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 left,

11、Actin 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

12、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 data,Tropo

13、nin,Head-to-tail overlap,A,B,Takeda, S. et al. Nature 424, 35 – 41, 2003,,Crystal structure of human cardiac troponin,Troponin C,C-Domain,N-Domain,Central Helix,Each TnC domain contains two motifs called EF hands, and it

14、 is the EF hands that directly bind calcium ions. Thus, the EF hands are TnC's way of sensing the calcium concentration; at ≈ 100 nM calcium (the usual cellular concentration) the N-domain EF hands are empty, but if

15、the local concentration rises to 1 mM, as it does when the muscle contracts, all of the EF hand bind calcium.,KCa=3 x 105 M-1Ca2+-specific,KCa= 2 x 107 M-1Ca2+-Mg2+ sites,EF hands,,Thick filament proteins,MYOSIN

16、 MW 480 kDaForms thick filamentsHydrolyses ATPInteracts with F-actin 300-400 myosin molecules per 1 filament,,,S1,150 nm,,Myosin,重鏈?-helical coiled-coil,輕鏈,,,160 nm,S1,S1 - Molecular Motor of Muscle C

17、ontraction,,,RLC,ELC,Myosin Head (S1) – molecular motor of muscle contraction,RLC,ELC,,ATP Binding Site,Actin Binding Site,,ATP (Myosin) ? ADP + Pi + Energy,,,,F-actin,Cross-bridge – Actin Interaction,Gordon et al. 2001

18、,Regulation of thin filament in contraction,A B C D E,From Craig and Lehman, 2001,JMB 311, 1027,The reversible binding of calcium to troponin alters the conformation

19、of the thin filament, thereby turning muscle contraction ON and OFF,Cross-bridge STATE: Thin filament STATE:Relaxed (OFF) BLOCKEDCa2+ Activated (Weak Binding) CLOSED C

20、a2+ 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:,In the absence of Ca2+, the

21、 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

22、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 Tn·Tm complex away from the outer domain of the actin filaments en

23、abling 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

24、interaction between the thick filament and the thin filaments and force generation.,ATPase Cycle,1. A ? M + ATP,2. A+ M?ATP,3. A ?M ?ADP ?Pi,4. A ?M ?ADP +Pi,5. A ?M +ADP,,,,,?,?,Pi,ADP,Pi release rate:10-20s-1,Muscle

25、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-1,Actin-myosin interaction,In vitro motility assay showing the sliding of actin filaments over a myos

26、in 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 coupling,Heart failure,Ryanodine rec

27、eptor(RyR),Phosphorylation of RYR increase,,,Ca2+ leak,,ATP-dependent pump,Phospholamban(PLB),In HF,Expression and activation of SERCA2Phosphorylation of PLB Expression of β1ARATP supply,,,,,,,,,uptake↓,,,,,,,,,Re-

28、uptake,Store,Release,M,SR,,SR Ca2+ srore decrease, Ca2+ transient delay,The SR Ca2+ store,1,2,3,4,5,1. Reduced Ca++ trigger thru L-type channel,2. Reduced RyR function (Calcium leaks from SR),3. Decreased sensitivity of

29、TN-C to Ca++,4. Reduced Ca++ uptake due to loss of SERCA function and increased Plb,5. Increased Na/Ca exchanger function,Overview of E-C coupling changes in the failing heart,正性肌力藥的循證研究,Ancient treatment of heart failur

30、e,洋地黃制劑（﹥200 years）,Digilis purpureaPurple foxglove,William Withering (1741 - 1799),Mechanism of Action,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）。,The Effect of Digoxin

31、on Mortality and Morbidity in Patients with Heart FailureN Eng1 Med,1997;336:525-533,總死亡率,The Effect of Digoxin on Mortality and Morbidity in Patients with Heart Failure N Eng1 Med,1997;336:525-533,因心衰住院死亡的發(fā)生率,The Effec

32、t of Digoxin on Mortality and Morbidity in Patients with Heart Failure N Eng1 Med,1997;336:525-533,"Digitalis" is without question the most valuable cardiac drug ever discovered one of the most valuable drugs

33、in the ent-ire pharmacopoeia. The introduction of digitalis was one of the landmarks in the history of cardiac disease.",Opie, H. L. Drugs for the Heart. Orlando Florida: Grune & Stratton, Inc. 1980.,Therapeuti

34、c Use,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(fā)性房室交界區(qū)心動過速、反復(fù)性心動過速。,Side effects,action potential recordings from purkinje fiber cells (A)toxic doses produce oscillatory after depolorizations (B)leads to ventricul

35、ar tachycardia (C),β-腎上腺素能受體激動劑,β-受體激動劑與心肌細(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。,Direct acting sympathomimetics,Dopamine,Dobutamine,Therapeutic Use,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

36、肌耗氧量增加，誘發(fā)心律失常和心肌受體下調(diào), 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。,Dies F, et al. Circulation 1986;74(suppl II):II-39.,磷酸二酯酶抑制劑,The different forms or subtypes of phosphodiesterase were initially isolated from rat brains by Uzunov an

37、d Weiss in 1972 and were soon afterwards shown to be selectively inhibited in the brain and in other tissues by a variety of drugsThe potential for selective phosphodisterase inhibitors as therapeutic agents was predic

38、ted 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. Pharmac

39、ol. Toxicol. 17:441-477, 1977.,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,Phosphodiesterase Inhibitors,Mechanism of Action,PDEI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。,β-ADR和PDEI的作用位點(diǎn),(according to L

40、ippincott´s Pharmacology, 2006),PROMISE臨床試驗(yàn)（1991）,NYHA III、IV級，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性↑34% 猝死危險↑69%亞組結(jié)論：心功能越差，危險性越高, 試驗(yàn)提前終止,Packer M, et al. Effect of milrinone on mortality in severe chronic he

41、art failure. N Engl J Med. 1991;325:1468-1475.,Therapeutic Use,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,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)肌球蛋白和肌動蛋

42、白之間的相互作用,,Mechanism of Action,REVIVE-2 研究(2005),REV IVE-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照組多33%，心功能惡化者比對照組少30%,Packer M. AHA Scientific Sessions, Dallas, USA, November, 2005.,Prim

43、ary Endpoint (n=600),Packer M. AHA Scientific Sessions, Dallas, USA, November, 2005.,33%,30%,Side Effects,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舒張時細(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。,White J ,Lee JA , Shah N

44、, et al. Differential effects of the optical isomers of EMD53998 on contraction and cytoplasmic Ca2 + in isolated ferret cardiac mus-cle[J ]. Circ Res ,1993 ,73 :61270.Lee JA ,Allen DG. EMD53998 sensitizes the contract

45、ile proteins to calcium in intact ferret ventricular muscle[J ]. Circ Res ,1991 ,69 :9272936.,Therapeutic Use,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衰竭,新型正性肌力藥的探索,亞硝酰氫,HNO是NO的去單電子產(chǎn)物，HNO去質(zhì)子化將產(chǎn)生相應(yīng)的陰離子NO-。,Ribeiro da

46、 Silva MD, et al., Org Biomol Chem. 2004,2(17):2507-12.,HNO直接修飾肌絲,HNO對肌漿網(wǎng)鈣離子的重吸收和釋放可不依賴于cAMP/PKA或cGMP/PKG途徑，而是與巰基化合物的修飾密切相關(guān)；事實(shí)上在心肌細(xì)胞的細(xì)肌絲上也存在巰基。HNO可能直接修飾肌絲巰基，而獨(dú)立于鈣轉(zhuǎn)運(yùn)系統(tǒng)。,Ingraham RH, et al. Biochemistry,1988 Aug 9;27(16)

47、:5891-8.Tocchetti CG, et al. Circulation Res. 2007,100(1):96-104.,Excitation-contraction coupling in cardiac contractile cells,Thin filament proteins,Head-to-tail overlap,HNO增加整體心肌的收縮力,Pooled data of force development a

48、nd [Ca2+]i transients at varied [Ca2+]o in the absence (open symbols) and presence (closed symbols)of AS (250?mol/L).(P<0.001 by one-way ANOVA).N=7 in each group.,HNO不影響心肌的舒張功能,HNO不依賴心肌鈣離子,HNO不增加肌纖維ATP酶活性,,The effects

49、 are HNO specific. The control drug (1-nitrosocyclohexyl tertbutylacetate) has no obvious effect on force of Skinning myocyte.,,HNO結(jié)構(gòu)的特異性,小 結(jié),洋地黃制劑(Digitalis)β-腎上腺素能受體激動劑(β-ADR)磷酸二酯酶抑制劑 (Phosphodiesterase inhibitor,