从兴奋收缩耦联机制看心力衰竭正性肌力药物发展.ppt

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 -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) coupling is a term coined in 1952 to describe the physiological process of converting an electrical stimulus to mechanical response.,Sandow A (1952). “Excitation-contraction coupling in muscular response.“. Yale J Biol Med 25 (3): 176201. PMID 130159500,Excitation-contraction coupling,Cardiac excitationcontraction 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 cardiac electrical activity and is the direct activator of the myofilaments, which cause contraction.,Bers, D. M. ExcitationContraction Coupling and Cardiac Contractile Force edn 2 (Kluwer Academic,Dordrecht, Netherlands, 2001).,Cardiac excitationcontraction 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+,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+,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。,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,G to F actin MW 42 kDa The blue and grey molecules are actin monomers (MW 42.000),Ken C. Holmes: Max-Planck-Institute,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 left,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 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,Troponin,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 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 mM, as it does when the muscle contracts, all of the EF hand bind calcium.,KCa=3 x 105 M-1 Ca2+-specific,KCa= 2 x 107 M-1 Ca2+-Mg2+ sites,EF hands,Thick filament proteins,MYOSIN MW 480 kDa Forms thick filaments Hydrolyses ATP Interacts with F-actin 300-400 myosin molecules per 1 filament,S1,150 nm,Myosin,重链 -helical coiled-coil,轻链,160 nm,S1,S1 - Molecular Motor of Muscle Contraction,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,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 of the thin filament, thereby turning muscle contraction ON and OFF,Cross-bridge STATE: Thin filament STATE: Relaxed (OFF) BLOCKED Ca2+ 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:,In 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 Cycle,1. A M + ATP,2. A+ MATP,3. A M ADP Pi,4. A M ADP +Pi,5. A M +ADP,Pi,ADP,Pi release rate: 10-20s-1,Muscle 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 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 coupling,Heart failure,Ryanodine receptor(RyR),Phosphorylation of RYR increase,Ca2+ leak,ATP-dependent pump,Phospholamban(PLB),In HF,Expression and activation of SERCA2 Phosphorylation of PLB Expression of 1AR ATP supply,uptake,Re-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 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,正性肌力药的循证研究,Ancient treatment of heart failure,洋地黄制剂（200 years）,Digilis purpurea Purple foxglove,William Withering (1741 - 1799),Mechanism of Action,DIG 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%（P0.01）。,The Effect of Digoxin on Mortality and Morbidity in Patients with Heart Failure N Eng1 Med,1997;336:525-533,总死亡率,The Effect of Digoxin on Mortality and Morbidity in Patients with Heart Failure N Eng1 Med,1997;336:525-533,因心衰住院死亡的发生率,The Effect 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 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.,Therapeutic Use,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。,Side effects,action potential recordings from purkinje fiber cells (A) toxic doses produce oscillatory after depolorizations (B) leads to ventricular tachycardia (C),-肾上腺素能受体激动剂,-受体激动剂与心肌细胞膜上-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cAMP cAMP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。,Direct acting sympathomimetics,Dopamine,Dobutamine,Therapeutic Use,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。,Dies F, et al. Circulation 1986;74(suppl II):II-39.,磷酸二酯酶抑制剂,The 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 drugs The 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.,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,Phosphodiesterase Inhibitors,Mechanism of Action,PDEI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（1991）,NYHA III、IV级，EF35% 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,Packer M, et al. Effect of milrinone on mortality in severe chronic heart failure. N Engl J Med. 1991;325:1468-1475.,Therapeutic Use,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,Mechanism of Action,REVIVE-2 研究(2005),REV 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%,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 Effects,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 U