桥接整合因子1在心力衰竭中的研究进展

心力衰竭是多种心脏疾病终末期表现,近年来发病率逐渐增加,其发病原因包括心肌病变、心肌代谢障碍、心脏负荷长期过重、心室充盈受限等,这些病因均可导致心脏结构和收缩舒张的功能受损。心肌细胞的兴奋收缩耦联正常进行是保证心脏有效射血的必要条件,异常的兴奋收缩耦联是发生心力衰竭的重要因素。横管是心肌细胞完成收缩功能的关键所在,选择性地富集了离子通道和结构蛋白,如小窝蛋白-3、亲联蛋白和桥接整合因子1,现主要阐述桥接整合因子1与横管间的作用对心脏功能的调节和影响。     

溶血磷脂酸对幼年大鼠心肌细胞收缩和钙瞬变的影响

目的我们前期的研究发现LPA受体在幼年大鼠心脏的表达显著高于成年的表达,提示LPA信号在心脏收缩功能尚未成熟时可能具有更为重要的调节作用。本研究通过观察LPA对此未成熟阶段心肌细胞钙瞬变和收缩力的作用,探讨LPA信号对幼年心肌兴奋-收缩耦联的影响。方法 Langendorff装置逆向灌流胶原酶分离获得不同发育阶段大鼠心肌细胞;采用IonOptix细胞收缩和钙离子浓度同步测定系统进行心肌细胞收缩力和钙瞬变的测定;Western blot检测不同发育阶段心肌细胞LPA受体的表达。结果 LPA对出生后14天和21天大鼠心肌细胞的收缩和钙瞬变均没有显著影响,表明LPA信号并不参与出生后14-21天大鼠心肌细胞的兴奋-收缩耦联过程和心肌细胞收缩。在大鼠出生后发育过程中,LPA受体在心肌细胞的表达在出生后14天已显著下调,不同于在整体心脏表达下调的时间点(P21d),这可能是LPA对此发育阶段心肌细胞的收缩和钙瞬变均不产生影响的原因,提示在出生后14天LPA信号对心肌细胞的发育调节功能可能即已减弱或消失。结论 LPA信号对出生后14天及之后心肌收缩和钙瞬变无显著影响,但尚不能排除LPA对出生后更早期的未成熟心肌细胞收缩和兴奋-收缩耦联的作用。     

心肌细胞钙离子转运与心力衰竭及药物治疗的研究进展

心肌细胞发生收缩是通过兴奋-收缩耦联(excitation-contraction coupling)实现的,钙离子(Ca2 )在其中起着关键性的作用,而心力衰竭就表现为心脏舒缩功能的严重降低,二者存在密切联系。现就心肌细胞钙离子转运与心力衰竭及药物治疗方面的研究进展综述如下。1心肌细胞Ca2 转运Ca2     

心力衰竭的钙循环与室性心律失常

心力衰竭时心肌细胞的Ca2+稳态遭到破坏,主要表现为Ca2+瞬变幅度减小,衰减延迟,舒张期[Ca2+]1升高.这些变化同Ca2+调节的细胞膜通道和转运体的表达和功能的改变有关,并导致收缩功能障碍和心律失常的发生,治疗时需整体考虑细胞内的钙离子变化和兴奋-收缩耦联的改变.     

肌质网钙泵基因在心力衰竭基因治疗中的发展

在心力衰竭中心肌细胞的收缩和舒张与Ca~(2+)密切相关,而肌质网钙泵对心肌内Ca~(2+)循环的稳态有重要作用。实验和临床研究已证实对心力衰竭细胞导入肌质网钙泵基因可以增强心肌细胞的收缩力,减少心律失常。现就肌质网钙泵基因在心力衰竭基因治疗中的发展做一综述。     

Kv4.3通道蛋白在心力衰竭调节中的分子机制

Kv4.3通道蛋白作为瞬时外向钾电流的核心功能结构,在心肌细胞早期复极化中发挥重要作用。Kv4.3通道蛋白下调致使动作电位时程延长,钙离子内流增加,从而影响心肌细胞钙调节和兴奋收缩耦联;增加的胞内钙离子激活钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)和钙调神经磷酸酶,导致心肌肥厚和心力衰竭的发生。此外,Kv4.3通道蛋白下调促使CaMKⅡ从Kv4.3-Ca MKⅡ复合物中分离并激活,加速心力衰竭过程。上调Kv4.3通道蛋白可抑制CaMKⅡ过度活化及其严重后果,可能是治疗心力衰竭的一大靶点。     

心力衰竭的钙循环与室性心律失常

心力衰竭时心肌细胞的Ca2＋稳态遭到破坏,主要表现为Ca2＋瞬变幅度减小,衰减延迟,舒张期[Ca2＋]1升高.这些变化同Ca2＋调节的细胞膜通道和转运体的表达和功能的改变有关,并导致收缩功能障碍和心律失常的发生,治疗时需整体考虑细胞内的钙离子变化和兴奋-收缩耦联的改变.     

肥大和衰竭心肌L型Ca2+通道研究进展

心肌细胞 L 型钙电流 （ IC a· L）触发了 Ca2 +诱导的 Ca2 +释放 （ CICR） ,调节着细胞内瞬时 Ca2 +动力学 ,从而在兴奋—收缩耦联过程中起关键性作用。ICa· L密度和通道功能的改变参与了心力衰竭的发生。作者综述了 :1L型 Ca2 +通道的结构和生理作用 ;2肥大和衰竭的心肌 ICa· L变化及其频率依赖性调节机制 ;3 Ca2 + 通道和心力衰竭的治疗关系 ;4L型 Ca2 +通道未来研究方向     

Junctophilin-2在心脏疾病中的作用及机制的研究进展

Junctophilin-2（JPH2）可以“连接和沟通”心肌细胞横管与肌质网。在心肌肥厚、心衰、心房颤动等心脏疾病中，均存在JPH2表达下降或突变的现象。调控JPH2的途径有3条:calpain，微管密度和miR-24。直接上调JPH2或间接干预JPH2的调控途径均能够保护心肌细胞，有利于心脏功能改善，故JPH2可能成为治疗多种心脏疾病的新靶点。     

左西孟旦治疗急性心力衰竭

急性心力衰竭是心脏科的危重症,传统的血管活性药物治疗作用有限.钙增敏剂通过增加心肌兴奋收缩耦联过程中Ca2 与心肌收缩系统结合力,从而增强心肌收缩力,改善患者血流动力学.     

Mir-24 regulates junctophilin-2 expression in cardiomyocytes

Rationale: Failing cardiomyocytes exhibit decreased efficiency of excitation-contraction (E-C) coupling. The downregulation of junctophilin-2 (JP2), a protein anchoring the sarcoplasmic reticulum to T-tubules, has been identified as a major mechanism underlying the defective E-C coupling. However, the regulatory mechanism of JP2 remains unknown. Objective: To determine whether microRNAs regulate JP2 expression. Methods and Results: Bioinformatic analysis predicted 2 potential binding sites of miR-24 in the 3'-untranslated regions of JP2 mRNA. Luciferase assays confirmed that miR-24 suppressed JP2 expression by binding to either of these sites. In the aortic stenosis model, miR-24 was upregulated in failing cardiomyocytes. Adenovirus-directed overexpression of miR-24 in cardiomyocytes decreased JP2 expression and reduced Ca(2+) transient amplitude and E-C coupling gain. Conclusions: MiR-24-mediated suppression of JP2 expression provides a novel molecular mechanism for E-C coupling regulation in heart cells and suggests a new target against heart failure.     

Excitation-contraction coupling changes during postnatal cardiac development

Cardiac contraction is initiated by the release of Ca²⁺ from intracellular stores in response to an action potential, in a process known as “excitation-contraction coupling” (ECC). Here we investigate the maturation of ECC in the rat heart during postnatal development. We provide new information on how proteins of the sarcoplasmic reticulum (SR) and the t-tubules (TTs) assemble to form the structures that support EC coupling during postnatal development. We show that the surface membrane protein, caveolin-3 (Cav3), is a good protein marker for TTs in ventricular myocytes and compared it quantitatively to junctophilin-2 (JP2), a protein found on the SR at sites of SR-TT junctions, or couplons. Although JP2 and Cav3 associate primarily with the SR and TTs, respectively, we found that they occupy the appropriate sites at maturing structures in synchrony, as visualized with high resolution, quantitative 3-dimensional imaging. We also found the surprising result that while both ryanodine receptor type 2, (RyR2) and JP2 proteins are localized to the same membrane and sub-compartments, they assume their positions at very different rates: RyR2 moves to the SR membrane at the Z-disc very early in development while JP2 only appears in the SR membrane as the TTs mature. Our data suggest that, although RyR2 appears to be prepositioned at the sites ultimately occupied by dyad junctions, JP2 arrives at these sites in synchrony with the development of the TTs at the Z-discs. Finally, we report that EC coupling efficiency changes with development, in concert with these structural changes. Thus we provide the first well-integrated information that links the developing organization of proteins underlying EC coupling (RyR2, DHPR, Cav3 and JP2) to the developing efficacy of EC coupling.     

Sorcin interacts with sarcoplasmic reticulum Ca2+–ATPase and modulates excitation–contraction coupling in the heart

Sorcin is a 21.6-kDa Ca(2+) binding protein of the penta-EF hand family. Several studies have shown that sorcin modulates multiple proteins involved in excitation-contraction (E-C) coupling in the heart, such as the cardiac ryanodine receptor (RyR2), L-type Ca(2+) channel, and Na(+)-Ca(2+) exchanger, while it has also been shown to be phosphorylated by cAMP-dependent protein kinase (PKA). To elucidate the effects of sorcin and its PKA-dependent regulation on E-C coupling in the heart, we identified the PKA-phosphorylation site of sorcin, and found that serine178 was preferentially phosphorylated by PKA and dephosphorylated by protein phosphatase-1. Isoproterenol allowed sorcin to translocate to the sarcoplasmic reticulum (SR). In addition, adenovirus-mediated overexpression of sorcin in adult rat cardiomyocytes significantly increased both the rate of decay of the Ca(2+) transient and the SR Ca(2+) load. An assay of oxalate-facilitated Ca(2+) uptake showed that recombinant sorcin increased Ca(2+) uptake in a dose-dependent manner. These data suggest that sorcin activates the Ca(2+)-uptake function in the SR. In UM-X7. 1 cardiomyopathic hamster hearts, the relative amount of sorcin was significantly increased in the SR fraction, whereas it was significantly decreased in whole-heart homogenates. In failing hearts, PKA-phosphorylated sorcin was markedly increased, as assessed using a back-phosphorylation assay with immunoprecipitated sorcin. Our results suggest that sorcin activates Ca(2+)-ATPase-mediated Ca(2+) uptake and restores SR Ca(2+) content, and may play critical roles in compensatory mechanisms in both Ca(2+) homeostasis and cardiac dysfunction in failing hearts.     

虎杖苷对严重烫伤大鼠心肌细胞钙信号调控的作用

目的：观察虎杖苷（polydatin,PD）对烫伤大鼠心功能损害的钙信号调控,并探讨其对烫伤大鼠心肌细胞保护作用的机制。方法：28只成年SD大鼠随机分为4组,即假手术组、烫伤组、PD组及烫伤＋PD组,每组7只大鼠（n=7）。 各实验组使用Power lab系统进行实时心功能监测。手术后5 min通过静脉给予PD（10 mg/kg）,12 h后分离心室肌细胞,通过激光共聚焦显微镜观测PD对烫伤大鼠心室肌细胞钙火花发放频率、钙库水平、钙瞬变的作用。结果：与烫伤组比较,烫伤＋PD组大鼠左室收缩压、左心室内压力上升的最大速率（±dP/dtmax）均显著增加、钙瞬变和心肌收缩力明显增强、使烫伤引起的钙库容量下降基本恢复到正常水平,烫伤引起的高频钙火花发放显著抑制（均P〈0.01）,钙火花时程的增加也明显抑制（P〈0.05）。结论： PD可以显著抑制烫伤引起的心肌细胞肌浆网钙漏流,恢复肌浆网钙库容量水平,增强收缩期钙瞬变和心肌收缩力,从而改善烫伤引起的心功能障碍。     

K201 modulates excitation-contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes

OBJECTIVES: The drug K201 (JTV-519) increases inotropy and suppresses arrhythmias in failing hearts, but the effects of K201 on normal hearts is unknown. METHODS: The effect of K201 on excitation-contraction (E-C) coupling in normal myocardium was studied by using voltage-clamp and intracellular Ca(2+) measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed using permeabilised cardiomyocytes. RESULTS: Acute application of <1 micromol/L K201 had no significant effect on E-C coupling. K201 at 1 micromol/L decreased Ca(2+) transient amplitude (to 83+/-7%) without affecting I(Ca,L) or the SR Ca(2+) content. At 3 micromol/L K201 caused a larger reduction of Ca(2+) transient amplitude (to 60+/-7%) with accompanying reductions in I(Ca,L) amplitude (to 66+/-8%) and SR Ca(2+) content (74+/-9%). Spontaneous SR Ca(2+) release during diastole was induced by increasing intracellular [Ca(2+)]. At 1 micromol/L K201 reduced the frequency of spontaneous Ca(2+) release. The effect of K201 on SR-mediated Ca(2+) waves and Ca(2+) sparks was examined in beta-escin-permeabilised cardiomyocytes by confocal microscopy. K201 (1 micromol/L) reduced the frequency and velocity of SR Ca(2+) waves despite no change in SR Ca(2+) content. At 3 micromol/L K201 completely abolished Ca(2+) waves and reduced the SR Ca(2+) content (to approximately 73%). K201 at 1 micromol/L reduced Ca(2+) spark amplitude and frequency. Assays specific to SR Ca(2+)-ATPase and RyR2 activity indicated that K201 inhibited both SR Ca(2+) uptake and release. CONCLUSIONS: K201 modifies E-C coupling in normal cardiomyocytes. A dual inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress spontaneous diastolic Ca(2+) release during Ca(2+) overload without significantly affecting Ca(2+) transient amplitude.     

Na(+)--Ca2+ exchange in the regulation of cardiac excitation-contraction coupling

Cardiac sarcolemmal Na(+)--Ca(2+) exchange is a central component of Ca2+ signaling essential for Ca2+ extrusion and contributing to a variable degree to the development of the systolic Ca2+ transient. Reports on differential gene expression of Na(+)--Ca2+ exchange in cardiac disease and the regulation of its thermodynamic equilibrium depending on intracellular gradients of ion concentrations between subcellular compartments have recently put a new complexion on Na(+)--Ca2+ exchange and its implications for excitation-contraction (E-C) coupling. Heart failure models and genetic approaches to regulate expression of the Na(+)--Ca2+ exchanger have improved our knowledge of exchanger function. Modest overexpression of the Na(+)--Ca2+ exchanger in heterozygous transgenic mice had minimal effects on E-C coupling and cardiac function. However, higher levels of Na(+)--Ca2+ exchange expression in homozygotes led to pathological hypertrophy and failure with an increased interaction between the L-type Ca2+ current and Na(+)--Ca2+ exchange and reduced E-C coupling gain. These results suggested that the Na(+)--Ca2+ exchanger is capable of modulating sarcoplasmic Ca2+ handling and at high expression levels may interact with the gating kinetics of the L-type Ca2+ current by means of regulating subsarcolemmal Ca2+ levels. Despite being a central component in the regulation of cardiac E-C coupling, a newly generated mouse model with cardiac-specific conditional knock-out of the Na(+)--Ca2+ exchanger is viable with unchanged Ca2+ dynamics in adult ventricular myocytes. Cardiac myocytes adapt well to knock-out of the exchanger, apparently by reducing transsarcolemmal fluxes of Ca2+ and increasing E-C coupling gain possibly mediated by changes in submembrane Ca2+ levels. For E-C coupling in the murine model, which relies primarily on sarcoplasmic Ca2+ regulation, this led to the suggestion that the role of Na(+)--Ca2+ exchange should be thought of as a Ca2+ buffering function and not as a major Ca2+ transporter in competition with the sarcoplasmic reticulum.     

Effects of calsequestrin over-expression on excitation-contraction coupling in isolated rabbit cardiomyocytes

OBJECTIVE: This study investigated the role of calsequestrin (CSQ) in the control of excitation-contraction (E-C) coupling in the heart. METHODS: CSQ over-expression was induced in isolated rabbit ventricular cardiomyocytes using an adenovirus coding for rabbit CSQ (Ad-CSQ). After 24 h of culture, CSQ protein expression was increased by 58+/-18% (n=10). An adenovirus coding for beta-galactosidase (Ad-LacZ) was used as a control. RESULTS: In voltage-clamped, Fura-2-loaded cardiomyocytes, L-type Ca2+ current (I(Ca,L)) and Ca2+ transient amplitude were both increased in the Ad-CSQ group by approximately 78%. Doubling the external Ca2+ concentration in the control group (Ad-LacZ) increased the LTCC amplitude to a similar degree (85+/-6%), but increased the Ca2+ transient amplitude by 149+/-13%. This suggests that SR Ca2+ release may be inhibited upon CSQ over-expression. Alternatively, nifedipine (0.5 microM) was used to reduce I(Ca,L) in Ad-CSQ-transfected cells to values comparable to control (Ad-LacZ). Under these conditions, Ca2+ transient amplitude was not different from Ad-LacZ, but the SR Ca2+ content was approximately 60% higher as assessed by both the caffeine-induced Ca2+ release and the accompanying Na+/Ca2+ exchanger current (I(NCX)). The cause of the increased I(Ca,L) is unknown. No change in the expression level of the alpha1-subunit of the L-type Ca channel was observed. beta-Escin-permeabilized cardiomyocytes were used to study Ca2+ sparks imaged with Fluo-3 at 145-155 nmol/L [Ca2+]. Spontaneous Ca2+ spark frequency, duration, width, and amplitude were unchanged in the Ad-CSQ group, but SR Ca2+ content was 48% higher than Ad-LacZ. CONCLUSIONS: CSQ over-expression increased SR Ca2+ content but reduced the gain of E-C coupling in rabbit cardiomyocytes.     

Loss of skeletal muscle strength by ablation of the sarcoplasmic reticulum protein JP45

Skeletal muscle constitutes approximately 40% of the human body mass, and alterations in muscle mass and strength may result in physical disability. Therefore, the elucidation of the factors responsible for muscle force development is of paramount importance. Excitation-contraction coupling (ECC) is a process during which the skeletal muscle surface membrane is depolarized, causing a transient release of calcium from the sarcoplasmic reticulum that activates the contractile proteins. The ECC machinery is complex, and the functional role of many of its protein components remains elusive. This study demonstrates that deletion of the gene encoding the sarcoplasmic reticulum protein JP45 results in decreased muscle strength in young mice. Specifically, this loss of muscle strength in JP45 knockout mice is caused by decreased functional expression of the voltage-dependent Ca(2+) channel Ca(v)1.1, which is the molecule that couples membrane depolarization and calcium release from the sarcoplasmic reticulum. These results point to JP45 as one of the molecules involved in the development or maintenance of skeletal muscle strength.     

Cardiac sarcoplasmic reticulum calcium release and load are enhanced by subcellular cAMP elevations in PI3Kgamma-deficient mice

We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma-/-) mice have increased cardiac contractility without changes in heart size compared with control mice (ie, PI3Kgamma+/+ or PI3Kgamma+/-). In this study, we show that PI3Kgamma-/- cardiomyocytes have elevated Ca2+ transient amplitudes with abbreviated decay kinetics compared with control under field-stimulation and voltage-clamp conditions. When Ca2+ transients were eliminated with high Ca2+ buffering, L-type Ca2+ currents (I(Ca,L)), K+ currents, and action potential duration (APD) were not different between the groups, whereas, in the presence of Ca2+ transients, Ca2+-dependent phase of I(Ca,L) inactivation was abbreviated and APD at 90% repolarization was prolonged in PI3Kgamma-/- mice. Excitation-contraction coupling (ECC) gain, sarcoplasmic reticulum (SR) Ca2+ load, and SR Ca(2+) release fluxes measured as Ca2+ spikes, were also increased in PI3Kgamma-/- cardiomyocytes without detectable changes in Ca2+ spikes kinetics. The cAMP inhibitor Rp-cAMP eliminated enhanced ECC and SR Ca2+ load in PI3Kgamma-/- without effects in control myocytes. On the other hand, the beta-adrenergic receptor agonist isoproterenol increased I(Ca,L) and Ca2+ transient equally by approximately 2-fold in both PI3Kgamma-/- and PI3Kgamma+/- cardiomyocytes. Our results establish that PI3Kgamma reduces cardiac contractility in a highly compartmentalized manner by inhibiting cAMP-mediated SR Ca2+ loading without directly affecting other major modulators of ECC, such as AP and I(Ca,L).