硫化氢对心肌细胞收缩的作用及其机制

【作者】 孙英刚；

【导师】 朱依纯； 姚泰； 陆利民； 王文伟；

【作者基本信息】 复旦大学 ， 生理学， 2008， 博士

【摘要】 硫化氢（H₂S）过去一直被认为是毒性气体，它的第一个生理作用的证据是1989年获得的。很多种哺乳动物细胞自身能产生H₂S，在正常大鼠的血液中H₂S浓度达到46μmol／L。体内的H₂S主要通过含硫氨基酸的代谢产生，胱硫醚-β-合酶（cystathionineβ-synthase，CBS）和胱硫醚-γ-裂解酶（cystathionineγ-lyase，CSE）是两个能分解L-半胱氨酸产生硫化氢的酶，前者主要存在于脑和神经系统，后者分布于心血管系统中。神经系统中研究发现，生理浓度的H₂S能够增加NMDA受体的敏感性，诱导海马的长时程增强。在心血管系统中，现已证明，硫化氢与高血压、肺动脉高压等关系密切。在肺动脉、主动脉、尾动脉、肠系膜动脉，以及门静脉等血管的管壁组织有H₂S合成酶的表达，可以产生内源性H₂S发挥舒张血管平滑肌的作用。H₂S是重要内源性血管活化因子，并且是第一个被认识的血管平滑肌细胞ATP敏感性钾离子通道(K_ATP通道)开放剂，H₂S的降血压作用可能是通过兴奋K_ATP通道，使细胞膜出现超极化而使平滑肌舒张，最近发现H₂S还能通过作用于有丝分裂活化蛋白激酶途径抑制平滑肌细胞增殖。在离体心脏灌流实验中，H₂S可以抑制心脏的收缩，减慢心率。外源性给予H₂S可在各种心血管疾病中发挥保护作用，例如，H₂S可以减低缺血再灌注后心律失常的发生率，H₂S预处理可以减小心肌梗死面积；还可以减弱高血压大鼠冠状动脉中层增厚和活性氧的产生。H₂S能够自体产生，并且具有多种生理调节功能，这使H₂S成为继NO和CO后的第三个气体信号分子。近年来，其在心血管系统中的调节作用成为研究热点。心肌细胞的收缩和心脏的泵血功能是息息相关的，既然H₂S可以舒张血管平滑肌，在离体的心脏灌流实验中也有负性作用，这种负性作用是因为H₂S作用于血管呢，还是直接作用于心肌细胞呢?我们的实验就是从大鼠乳头肌收缩、单个心肌细胞收缩试验开始进一步证实了硫化氢对心肌细胞收缩的直接抑制作用；并探讨了H₂S的作用机制。心肌细胞内游离钙变化直接影响细胞收缩，我们用激光共聚焦的方法观察到在给予硫氢化钠（H₂S的供体）以后，由电刺激诱发的心肌细胞收缩时的细胞内游离钙减少了，这就说明硫化氢抑制心肌细胞收缩是通过减少细胞内游离钙引起的；细胞内游离钙主要是胞外钙离子内流激发肌浆网内钙释放出大量钙离子，两个来源的钙离子共同组成。咖啡因可以诱发肌浆网内钙的释放，我们的实验也发现H₂S并没有影响咖啡因诱导的肌浆网内钙释放。然后我们利用膜片钳的方法记录心肌细胞动作电位，L型钙通道电流发现H₂S明显缩短了动作电位复极的平台期，抑制了心肌细胞L型钙通道电流。我们还在自发性高血压大鼠（SHR）中观察了H₂S作用，并比较了硫化氢对正常大鼠和SHR大鼠发挥抑制作用的差别。为了研究H₂S抑制心肌细胞L型钙通道电流的信号转导机制，我们测定了心肌细胞cAMP和cGMP在硫化氢预处理后的表达水平，没有发现H₂S预处理组心肌细胞内cAMP和cGMP水平的改变；同时我们也记录了心肌细胞K_ATP通道电流，发现较低浓度的H₂S也不能显著开放ATP敏感的钾通道。总之，我们发现硫化氢可以抑制大鼠心脏乳头肌的收缩，减弱单个心肌细胞的收缩幅度、速度，这种抑制作用可能是通过抑制心肌细胞膜上的L型钙离子通道，使胞外钙离子内流减少，细胞内游离钙浓度降低来发挥抑制心肌细胞收缩的作用的。它的信号转导途径可能和cAMP、cGMP的介导没有关系。较低浓度的H₂S就能抑制心肌细胞收缩可能和其K_ATP通道开放剂的作用无关。硫化氢可能是一个新的L型钙通道抑制剂，对它的研究将为进一步探索高血压等心血管疾病的发病机制和治疗开创新的领域。更多还原

【Abstract】 Hydrogen sulfide （H₂S） is well known as a toxic gas with the characteristic smell of rotten eggs. But it is becoming increasingly clear that mammalian cells also produce H₂S. The H₂S concentration in rat serum is about 46μmol/L. Endogenous H₂S is synthesized naturally in the body from L-cysteine mainly by the activity of two enzymes, cystathionineβ-synthase （CBS） and cystathionineγ-lyase （CSE）. CBS seems to be main H₂S -forming enzyme in the brain and nervous system, whereas CSE is the main H₂S -synthesizing enzyme in the cardiovascular system. It is reported that physiological concentrations of H₂S specifically potentiate the activity of the N-methyl-D-aspartate （NMDA） receptor, facilitate the induction of hippocampal long-term potentiation. In cardiovascular system, exogenous administration of H₂S has been reported to be cardioprotective in various disease models. For example, administration of NaHS significantly decreases the duration and severity of ischemia/reperfusion-induced arrhythmias and increased the viability of cardiomyocytes in isolated perfused rat hearts. NaHS treatment reduces infarct size in a rat model of coronary artery ligation. Chronic treatment with NaHS for 3 months decreases medial thickening of intramyocardial coronary arterioles, interstitial fibrosis and reactive oxygen species （ROS） production in spontaneously hypertensive rats （SHR）. H₂S was important in hypertension , and the first ATP senstive K⁺ channel opener, the decreasing blood pressure effect of H₂S maybe through the magnism. Recently H₂S was found that it can inhibit the contraction of heart in vitro.However, the mechanisms that mediate the observed cardiac effects of H₂S remain unknown.The endogenous metabolism and physiological functions of H₂S made this gas well known in the novel family of gasotransmitters together with nitric oxide （NO） and carbon monoxide （CO）.Calcium homeostasis plays a pivotal role in myocardial physiology and diseases. The influx of Ca²⁺ through the L-type Ca²⁺ channels is a trigger for the ryanodine-dependent calcium release from the sarcoplasmic reticulum, which plays an essential role in the excitation-contraction coupling in cardiomyocytes. To date, there is no information about the potential role of H₂S in regulating calcium channels in the heart. We hypothesize that H₂S may regulate cardiac function and interact with the pathophysiological pathways by regulating calcium channels in cardiomyocytes. Therefore, the present study aimed to investigate the role of H₂S in regulating L-type calcium current (I_Ca,L), intracellular calcium transient ([Ca²⁺]i) and contraction in rat cardiomyocytes. Our results said that H₂S can decrese the [Ca²⁺]i and inhibit the contraction of cardiomyocytes.Since H₂S has been shown to be an opener for the Katp channels in vascular smooth muscle cells（VSMCs）, the potential role of H₂S on the the K_ATP channels in cardiomyocytes was also examined and found lower concention can not open the K_ATP. Additionally, cAMP and cGMP, the well recognized intracellular secondary messengers which regulate I_Ca,L in cardiomyocytes, were also measured. H₂S has no effect on the level of cAMP and cGMP; To assess whether H₂S exerts differential effects in regulating I_Ca,L and contraction in normal and hypertrophied cardiac myocytes, we examined the effects of H₂S in cardiomyocytes isolated from both Wistar-Kyoto （WKY） and spontaneously hypertensive rats （SHR）.In summary, NaHS inhibited the contraction of single cardiomyocytes and isolated papillary muscles. Electric field-induced [Ca²⁺]i transient were reduced by NaHS. In contrast, caffeine induced an increase in [Ca²⁺]i and was not altered by NaHS. Bath application of NaHS significantly reduced the time required for the depolarization of the action potential. Inhibition of the peak I_Ca,L by NaHS was determined to be concentration dependent. NaHS had no effect on the Katp current or the levels of cAMP and cGMP in the current study. H₂S is a novel inhibitor of L-type calcium channels in cardiomyocytes. Moreover, H₂S-induced inhibition of [Ca²⁺]i appears to be a secondary effect due to its initial action towards I_Ca,L. The inhibitory effect of H₂S on I_Ca,L requires further investigation to develop novel therapeutic approaches to treat cardiac diseases.更多还原