Effects of 4-aminopyridine on the excitation-contraction coupling in frog and rat skeletal muscle

The effects of 4-aminopyridine (4-AP) were studied on isolated single muscle fibres of the frog and toe muscles of the rat. In both muscle preparations, 4-AP potentiated the twitch amplitude without significantly affecting the tetanus response. There was an increase of the time to peak tension and, in frog muscle, an increased time to half relaxation. 4-AP produced no change of the resting membrane potential. The rate of decay and, hence, the total duration of the action potential were markedly prolonged. 4-AP did not induce contractures by itself nor did it affect the contracture induced by caffeine. The mechanical threshold was determined by measuring the contracture response to various degrees of depolarization by potassium. This threshold was not affected by 4-AP. Twitch potentiation by 4-AP was independent of the extracellular calcium concentration. It is concluded that 4-AP potentiates the twitch response by increasing the release of activator calcium into the myofibrillar space by prolongation of the action potential. In addition, there may be a more direct inhibitory action of 4-AP on the calcium re-uptake by the sarcoplasmic reticulum in frog muscle.     

Tityusγ toxin,a high affinity effector of the Na+ channel in muscle,with a selectivity for channels in the surface membrane

Toxin from the venom ofTityus serrulatus scorpion produces a partial block of the surface Na⁺ channel in frog muscle. This block occurs with no change in the voltage-dependence or in the kinetics of the remaining surface Na⁺ current. The partial blockade of Na⁺ channel activity occurs with no change in tubular Na⁺ currents nor in twitch tension. The maximum effect of the toxin is attained at concentrations as low as 3×10^–10 M. Hyperpolarization to potentials more negative than the resting potential (E=–90 mV) reduces or abolishes the effect of the toxin.Radioiodinated toxin binds to frog muscle membranes with a very high affinity corresponding to a dissociation constant of about 1×10^–11 M. Data obtained with both rabbit and frog muscle indicate that toxin is specific for Na⁺ channels in surface membranes. Toxin does not seem to bind to Na⁺ channels in T-tubule membranes. The biochemical data are in good agreement with electrophysiological studies and data on contraction. There is oneTityus toxin binding site per tetrodotoxin binding site in surface membranes. Competition experiments have confirmed thatTityus toxin binds to a new toxin receptor site on the Na⁺ channel structure. This site is the same that the toxin II fromCentruroides suffusus binding site, but this toxin has 100 times less affinity for the Na⁺ channel thanTityus toxin.     

衰老骨骼肌中的兴奋-收缩偶联与MG29蛋白

背景：“兴奋-收缩偶联”是神经-肌肉功能的基本功能特点,在衰老过程中肌肉功能的衰退与肌细胞中的兴奋-收缩解偶联存在密切关系。近年来针对衰老肌细胞功能退化的研究有了许多新进展。 目的：综述有关衰老肌细胞钙离子变化的机制以及MG29蛋白在钙离子及与兴奋-收缩偶联过程中的作用的有关文献,为进一步了解衰老骨骼肌功能退化的机制提供理论参考。 方法：应用计算机检索CNKI 期刊全文数据库(2000-01/2009-11)和PubMed数据库(2009-01/2009-12)与衰老肌细胞钙离子变化的机制以及MG29蛋白及兴奋-收缩偶联相关的文献,检索词分别为“兴奋-收缩偶联;肌组织;肌细胞;衰老;抗阻训练;MG29”和“excitation-contraction;muscle;muscle cell;senescence;resistance training;MG29”。纳入和衰老肌细胞钙离子、兴奋-收缩偶联及MG29蛋白相关的研究,排除陈旧的、重复的以及缺乏可信度的文献。 结果与结论：初步检索到中英文文献共43篇,根据本研究的纳入排除标准,排除9篇不符合标准的文献,最终纳入34篇文献进行分析。结果发现,许多研究已经证明MG29蛋白在肌细胞兴奋-收缩偶联中起着重要的作用,而随增龄MG29会出现明显减少的规律,推断其在肌组织衰老中可能扮演重要角色。更为重要的是,新近有研究者发现适宜的抗阻训练可以使肌组织中MG29表达量增加。     

Effects of Magnesium on Cardiac Excitation-Contraction Coupling

Objective: Magnesium regulates a large number of cellular processes. Small changes in intracellular free Mg²⁺ ([Mg²⁺]_i) may have important effects on cardiac excitability and contractility. We investigated the effects of [Mg²⁺]_i on cardiac excitation-contraction coupling.

Methods: We used our ionic-metabolic model that incorporates equations for Ca²⁺ and Mg²⁺ buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na⁺-K⁺ pump, sarcolemmal and sarcoplasmic Ca²⁺ pumps).

Results: Model results indicate that variations in cytosolic Mg²⁺ level might sensitively affect diastolic and systolic Ca²⁺, sarcoplasmic Ca²⁺ content, Ca²⁺ influx through L-type channels, efficiency of the Na⁺/Ca²⁺ exchanger and action potential shape. The analysis suggests that the most important reason for the observed effects is a modified normal function of sarcoplasmic Ca²⁺-ATPase pump by altered diastolic MgATP levels.

Conclusion: The model is able to reproduce qualitatively a sequence of events that correspond well with experimental observations during cardiac excitation-contraction coupling in mammalian ventricular myocytes.     

当药苷调节兴奋-收缩耦联改善心肌缺血再灌注损伤的机制

目的 探索当药苷通过兴奋-收缩耦联信号通路对缺血再灌注损伤后心脏收缩/舒张功能的影响。方法 24只健康雄性SD大鼠随机分为正常组、模型组、10 μmol·L^-1当药苷给药组与1 μmol·L^-1地高辛给药组,并采用Langendorff系统结合左前降支冠状动脉结扎建立缺血再灌注损伤模型（I/R）,2,3,5-氯化三苯基四氮唑（TTC）染色检测各组心脏梗死面积,Powerlab生理记录仪检测血流动力学参数,如左心室舒张压（LVDP）、左心室终末舒张压（LVEDP）、左心室终末收缩压（LVESP）、左心室内压最大上升速率（+dp/dt_max）和左心室内压最大下降速率（-dp/dt_max）;提取分离乳鼠原代心肌细胞（NRCMs）,建立缺氧/复氧（H/R）损伤模型,随机分为正常组、模型组、1 μmol·L^-1当药苷给药组和10 μmol·L^-1当药苷给药组,多功能成像细胞分析仪和激光共聚焦显微镜测定各组心肌细胞活力、心率、收缩幅度、收缩时程、达峰时程和舒张时程及钙瞬变峰值。根据前期转录组学测序结果及文献调研,利用实时荧光定量聚合酶链式反应（Real-time PCR）检测L型钙通道相关基因（Cacnb2）,细胞色素C氧化酶相关基因（Cox6a2）,肌钙蛋白（Tnnc1、Tnni3、Tnnt2）、肌动蛋白（Actc1）、肌球蛋白（Myh6、Myl2、Myl4）等兴奋-收缩耦联通路基因的mRNA表达并对差异基因进行聚类分析。结果 与正常组比较,模型组心肌梗死面积显著增加（P<0.01）、LVDP显著降低（P<0.01）、LVEDP显著上升（P<0.01）、LVESP明显下降（P<0.05）、+dp/dt_max有下降趋势和-dp/dt_max上升趋势及心肌细胞活力降低、心率降低、收缩幅度降低、收缩时程升高、达峰时程升高和舒张时程升高（P<0.01）,而当药苷可以逆转上述指标（P<0.05）。此外,心肌细胞经H/R损伤后,Cacnb2、Cox6a2、Tnnc1、Tnni3、Tnnt2、Actc1、Myh6、Myl2、Myl4等兴奋-收缩耦联通路的基因表达下降（P<0.05、P<0.01）。而使用当药苷预处理后,可以增强上述基因的表达（P<0.05）。结论 当药苷通过调节兴奋-收缩耦联信号通路,从而调节原代乳鼠心肌细胞内钙离子水平,增强心肌收缩功能,对抗I/R损伤。     

[Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity

1. Cytosolic calcium concentration ([Ca²⁺]_i) by indo 1 microspectrofluorimetry in freshly isolated cells and isometric contraction of isolated rings were measured in response to muscarinic cholinoceptor stimulation in rat tracheal smooth muscle.
2. In isolated myocytes, acetylcholine (ACh, 0.031 μM) caused a rapid and graded increase in [Ca²⁺]_i up to a net amplitude of 492±26 nM (n=19) which gradually declined. The EC₅₀ for ACh was 0.13 μM. This first [Ca²⁺]_i peak was followed, when the ACh concentration increased, in approximately 5060% of the cells, by successive peaks of decreased amplitude ([Ca²⁺]_i oscillations) superimposed on the plateau phase. Whereas the percentage of cells exhibiting [Ca²⁺]_i oscillations remained consistent, the frequency of these oscillations increased to up to 10 min⁻¹ with an ACh concentration of 100 μM.
3. Removal of extracellular calcium (in the presence of EGTA, 0.4 mM) or addition of the voltage-dependent Ca²⁺-channel blocker verapamil (10 μM) did not alter the first [Ca²⁺]_i peak, the plateau or the oscillations induced by ACh or carbachol. In contrast, the specific inhibitor of the sarcoplasmic Ca²⁺-ATPase, thapsigargin (1 μM), completely abolished the [Ca²⁺]_i response. Thapsigargin (1 μM) also blocked the caffeine (5 mM)-induced transient rise in [Ca²⁺]_i.
4. Atropine (a non-selective muscarinic cholinoceptor antagonist) and 4-diphenyl acetoxy N-methyl piperidine (4-DAMP, a selective M₃ antagonist) inhibited the [Ca²⁺]_i response to muscarinic cholinoceptor activation with an IC₅₀ of 13 and 20 nM, respectively. Pirenzepine (a selective M₁ antagonist) also totally inhibited the [Ca²⁺]_i response to ACh but with a higher IC₅₀ of 2 μM. Methoctramine (a selective M₂ antagonist) up to a concentration of 10 μM caused only a 40% inhibition. The effect of muscarinic antagonists on cumulative concentration-response curves (CCRC) for carbachol was assessed at the following concentrations: atropine and 4-DAMP at 3, 10 and 30 nM; pirenzepine 0.3, 1 and 3 μM, and methoctramine at 1, 3 and 10 μM. For these concentrations, all of the antagonists produced a rightward shift of the CCRC for carbachol and pA₂ values were 9.2, 8.8, 6.7 and 6.3, respectively.
5. In conclusion, the present study indicates that muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca²⁺]_i oscillations. The occurrence of these oscillations depends on the graded amplitude of the first [Ca²⁺]_i rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca²⁺]_i and the contractile responses are primarily dependent on activation of the M₃ receptor subtype.

     

Inhibitory effects of (±)-propranolol on excitation-contraction coupling in isolated soleus muscles of the rat

1. The effect of a β-adrenoceptor antagonist, propranolol, was investigated on excitation-contraction coupling in small, intact bundles of soleus muscle fibres from the rat.
2. (±)-Propranolol significantly inhibited twitch and tetanic tension with IC₅₀ values of 6.7 μM and 3.5 μM, respectively.
3. (+)-Propranolol (which has 100 times less β-blocking activity than the (±) form) was approximately one third as effective as the (±) form at inhibiting isometric tension.
4. (±)-Propranolol (20 μM) had no significant effect on the amplitude of caffeine contractures, suggesting that it did not directly inhibit Ca²⁺ release from the sarcoplasmic reticulum.
5. The resting membrane potential measured after 15 min perfusion with 20 μM (±)-propranolol was not significantly different from control. However, this concentration of (±)-propranolol significantly reduced both the peak amplitude and the maximum rate of rise of the action potential. Both effects were only partially reversible after extensive washing.
6. (±)-Propranolol perfusion caused a modest reduction in the amplitude of sub-maximal K⁺ contractures at concentrations (5  μM) that markedly depressed tetanic tension.
7. The results indicate that (±)-propranolol can decrease isometric tension independently of β-receptor occupation by (i) reducing the amplitude and rate of rise of the action potential and (ii) by directly inhibiting excitation-contraction coupling. The relatively low IC₅₀ for the ‘membrane-stabilizing'' action of propranolol on tetanic tension (3.5 μM), combined with the ability of the drug to accumulate gradually in biological membranes, may contribute to a peripheral component of the tremorolytic and fatigue-inducing actions of propranolol on skeletal muscle.

     

Characterization and temporal development of cores in a mouse model of malignant hyperthermia

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca²⁺ release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1^Y522S/WT), the mutation causes SR Ca²⁺ leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1^Y522S/WT knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1^Y522S/WT mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2–4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1^Y522S/WT mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca²⁺ sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.