Electrophysiological properties of the propafenone-analogue GE 68 (1-[3-(phenylethyl)-2-benzofuryl]-2-(propylamino)-ethanol) in isolated preparations and ventricular myocytes of guinea-pig hearts

GE 68 ((Rac.)-1-[3-(Phenylethyl)-2-benzofuryl]-2-(propylamino)-ethanol hydrochloride) is structurally related to propafenone, and exerts negative inotropic and negative chronotropic effects similar to the parent drug, but lacks any β-adrenoceptor blocking activity contrary to propafenone. Thus, the electrophysiological effects of GE 68 were studied in papillary muscles, left atria, Purkinje fibres, sinoatrial nodes and ventricular myocytes of the guinea-pig heart with the intracellular microelectrode technique and the patch-clamp technique in the cell-attached mode. The decrease of the maximum upstroke velocity (V˙_max) by GE 68 (1 to 10 μM) was use- and frequency-dependent. V˙_max recovered from the use-dependent block with a time constant of 4.1 ± 0.6 s. In papillary muscles and Purkinje fibres action potential duration was shortened, while it was prolonged in left atria and sinoatrial nodes. Half-maximal steady-state inactivation of the sodium channels was shifted to more negative membrane potentials (control: –91.5 ± 0.8 mV, 10 μM GE 68: –97.9 ± 2.5 mV). The peak of the current-voltage relationship and the reversal potential were not changed by GE 68. The amplitude of the unitary current remained unaltered, while open state probability was decreased. The most striking effect of GE 68 was an increase of the number of sweeps without single channel openings (1 μM: 2 fold, 10 μM: 6 fold). GE 68 also caused a decrease of the mean open times, and an increase of the mean closed times in unmodified and pronase-modified sodium channels. Besides the lack of β-adrenoceptor blocking activity, data present a faster recovery from the use-dependent block by GE 68 and a lower affinity to inactivated sodium channels compared to the reference drug propafenone, as well as differences in the effect on single channel kinetics. Received: 25 July 1996 / Accepted: 14 October 1996     

Pharmacology of KB‐R7943: A Na+–Ca2+ exchange inhibitor

The Na⁺–Ca²⁺ exchange (NCX) system plays a pivotal role in regulating intracellular Ca²⁺ concentration in cardiomyocytes, neuronal cells, kidney and a variety of other cells. It performs a particularly important function in regulating cardiac contractility and electrical activity. One of the leading NCX inhibitors is KB‐R9743 (KBR) that appears to exhibit selectivity for Ca²⁺‐influx‐mode NCX activity (reverse mode of NCX). In this article we reviewed pharmacology of KBR and provide a brief summary of studies with other NCX inhibitors, such as SEA0400 (SEA) and SN‐6 (SN). Potential clinical usefulness of KBR and other NCX inhibitors is still controversial but the reviewed findings may be helpful in designing more selective and clinically useful NCX inhibitors for the treatment of cardiac, neuronal and kidney diseases.     

Adenosine triphosphate-dependent K currents activated by metabolic inhibition in rat ventricular myocytes differ from those elicited by the channel opener rilmakalim

Adenosine triphosphate (ATP) dependent potassium channels (K_ATP channels) in heart ventricular muscle cells can be activated by depletion of intracellular ATP stores as well as by channel openers. In the present study we examined whether properties of K_ATP channels are dependent on the mode of activation. Whole-cell and single-channel currents were investigated by use of the patch-clamp technique in isolated ventricular rat myocytes. The channel opener rilmakalim dose dependency activated whole-cell currents [concentration for half-maximal activation (EC₅₀) = 1.1 M, Hill coefficient = 3.1, saturation concentration 10 M]. Metabolic inhibition with 2-deoxy-d-glucose (10 mmol/l) also activated K_ATP currents after a time lag of several minutes. These currents were about two-fold higher than the rilmakalim-activated currents (rilmakalim-activated current 3.9 ±0.2nA, 2-deoxy-d-glucose-activated current 8.1±0.9 nA; both recorded at 0 mV clamp potential). While the rilmakalim-activated current could be blocked completely and with high affinity by the sulphonylurea glibenclamide [concentration for half-maximal inhibition (IC₅₀) = 8 nM, Hill coefficient = 0.7] the 2-deoxy-d-glucose-activated current could only be blocked partially (by maximally 46%) and higher glibenclamide concentrations were needed (IC₅₀ = 480 nM, Hill coefficient = 0.8). The partial loss of blocking efficiency after metabolic inhibition was not restricted to glibenclamide but was also observed with the sulfonylureas glimepiride and HB 985, as well as with the non-sulfonylureas HOE 511 and 5-hydroxydecanoate. Single-channel studies were in accordance with these whole-cell experiments. Both rilmakalim and metabolic inhibition with the uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) activated single channels in the attached mode, where the number of current levels was significantly higher in the case of FCCP. Rilmakalim-activated channels were completely blocked by 10 M glibenclamide, whereas several single-channel levels appeared in the presence of 100 M glibenclamide after metabolic inhibition. In conclusion, after metabolic inhibition the amplitude of the activated K_ATP current is about twice as high as under saturating concentrations of the opener rilmakalim. Moreover, channels activated by metabolic inhibition lost part of their sensitivity to known channel blockers.     

人类胚胎生殖细胞体外分化为心肌细胞的研究

胚胎生殖（EG）细胞是来源于胚胎原始生殖细胞（PGCs）的多潜能干细胞。采用无饲养层细胞、无细胞因子等的基础培养液（DMEM＋20％NBS＋0．1mM 2Me）培养EG细胞，部分在基础培养液添加10μmol／LRA＋0．75％DMSO或10μmol／L 5-氮胞苷（5-AZA）诱导人类EG细胞向心肌细胞分化，以检测其是否具有向心肌细胞自发分化的能力。9例（8．49％，9／106）胎儿EG细胞在体外分化得到20个节律性心脏跳动样细胞团，其搏动节律为20～120次／min，体外维持节律性搏动最短2d，最长至15d，呈PAS，Myoglobin，α-actin阳性；对K^＋、Ca^＋、肾上腺素等具有与在体心脏相似的反应性；透射电镜观察具有心肌细胞样结构。添加DMSO和RA或5-AZA诱导未得到跳动样心肌细胞，但可提高心肌α-actin免疫组织化学染色阳性率；表明人类胚胎生殖细胞具有向心肌细胞分化的潜能。     

Decay-accelerating factor in the cardiomyocytes of normal individuals and patients with myocardial infarction

Summary The presence of decay-accelerating factor (DAF) was clearly demonstrated on the surface of normal cardiomyocytes. In patients who had died of myocardial infarction (MI) cardiomyocytes displayed different appearances: outside the ischaemically damaged region the myocytes showed no significant variations in DAF expression when compared with controls without MI. Within myocardial zones damaged by ischaemia, however, apparently normal myocytes showed large gaps in surface staining of DAF or formed clusters which were entirely devoid of reactivity with anti-DAF antibodies. The number of DAF-deficient myocytes increased with the extent of necrosis and also with the number of days between onset of MI and death. Even though injury to myocytes is to a large extent related to anoxia and to the presence of free oxygen radicals, the complement system also appears to be involved; DAF may have protective functions against complement-mediated injury. We speculate that phospholipase may be involved in the removal of DAF from the cardiomyocyte surface.This work was supported in part by grant no. 3.157.88 from the Swiss National Foundation for Scientific Research and a contribution from Sandoz Ltd. Pharma Division, Basel     

Pharmacological characterization of A1 adenosine receptors in isolated rat ventricular myocytes

Summary The aim of the present study was the characterization of adenosine receptors in isolated rat ventricular myocytes. The CAMP-levels of rat ventricular myocytes in the presence of 1 mol/l isoprenaline were reduced by up to 48% by adenosine analogues; the rank order of potency was: R-N⁶-phenylisopropyladenosine (IC₅₀ 60 nmol/1), 5-N-ethylcarboxamidoadenosine (IC₅₀ 360 nmol/l) and S-N⁶-phenylisopropyladenosine (IC₅₀ 16 ol/l). The adenosine receptor antagonist XAC (xanthine amine congener) antagonized the effect of R-N⁶-phenylisopropyladenosine in a concentration-dependent manner with a K_i-value of 20 nmol/l. The A₁ receptor-selective radioligand R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine bound to membranes prepared from rat ventricular myocytes in a saturable manner with a B _max of 17.7 fmol/mg protein and a K _D-value of 1.1 nmol/l. Adenosine analogues competed for the binding with the same rank order of potency as for the inhibition of the isoprenaline-induced cAMP-increase. GTP inhibited radioligand binding with an IC₅₀-value of 73 ol/l. These results suggest the presence of A₁ adenosine receptors on rat ventricular myocytes, which mediate an inhibition of adenylate cyclase. The receptors may be responsible for the effects of adenosine and its analogues on the heart.Abbreviations ¹²⁵I-HPIA R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine - PIA N⁶-phenylisopropyladenosine - NECA 5-N-ethyl-carboxamidoadenosine - XAC 8-4-[([(2-aminoethyl)aminocarbonyl]methyl)oxy]phenyl-1,3-dipropylxanthine (xanthine amine congener) - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - ScAMPTME 2-O-monosuccinyladenosine-3,5-cyclic monophosphate tyrosyl methyl ester - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GTP guanosine-5-tri-phosphate Send offprint requests to D. Martens     

参附注射液对缺氧心肌细胞凋亡的抑制效应

目的　探讨参附注射液对缺氧诱导的培养乳鼠心肌细胞凋亡的作用。方法　将 SD乳鼠的培养心肌细胞随机分为三组:对照组、缺氧72 h组和参附注射液组。缺氧 72 h前加入1 mmol参附注射液。应用TUNEL法及流式细胞仪分析心肌细胞凋亡的变化,借助半胱胺酸 天门冬胺酸酶(caspase- 3)荧光分析试剂盒,荧光比色法检测心肌细胞凋亡过程中 caspase -3 活性的变化。结果　TUNEL及流式细胞仪分析显示,参附注射液组 AI值和细胞凋亡百分率分别为(14. 10±2 .56)%和(14. 93±2 .47)%,明显低于缺氧72 h组(46 .49±4 .93)%和(48 .43±4 .18)%(P<0 .01)。caspase -3活性检测显示,参附注射液组的caspase- 3活性比缺氧72 h组降低了 52. 85%(P<0. 01)。结论　参附注射液能明显抑制缺氧诱导的心肌细胞凋亡。caspase- 3蛋白酶活性抑制可能是其机理之一。     

β—内酰胺丝氨酸样蛋白在高脂诱导的心肌细胞损伤中的作用及机制

目的 探讨高脂对心肌细胞的影响及β-内酰胺丝氨酸样蛋白（LACTB）在其中的作用与机制。方法 将小鼠心肌细胞分为正常组（脂肪酸浓度为0μmol/L）和高脂组（HF组,脂肪酸浓度为400μmol/L）,干预18h。采用siRNA敲低心肌细胞LACTB的表达,进一步将HF组分为对照组（仅予以转染试剂处理）、Scra siRNA组（予以转染试剂+非特异阴性siRNA处理）及LACTB siRNA组（予以转染试剂+ LACTB特异性siRNA处理）。为探讨LACTB在高脂诱导的心肌细胞损伤中可能的作用机制,将LACTB siRNA组分为Vehicle组、N-乙酰半胱氨酸（ NAC）组。通过流式细胞仪检测细胞凋亡率,qRT-PCR及Western blot分别检测细胞LACTB mRNA及蛋白表达,DHE染色及ELISA试剂盒检测细胞内活性氧簇（ROS）含量。结果 高脂可增加心肌细胞的凋亡,增加心肌细胞中LACTB的表达,促进ROS生成（均P＜0.05）。与对照组及Scra siRNA组比较,LACTB siRNA组心肌细胞凋亡与ROS生成进一步增加（P＜0.05）;与此相反,在此基础上使用抗氧化剂NAC部分逆转了心肌细胞凋亡（P＜0.05）。结论 LACTB通过抑制氧化应激反应可缓解高脂诱导的小鼠心肌细胞凋亡。     

家兔右室流出道细胞双孔钾通道电流特性的分析研究

目的已知特发性室速主要起源于右室流出道(RVOT),由于技术上的困难,目前对特发性右室流出道室速(RVOT-VT)的离子通道机制研究很少,本实验意在探索右室心室肌(RV)和RVOT的双孔钾通道电流(IK2p)的特性及其在RVOT-VT发生机制中可能参与的作用。方法采用全细胞膜片钳技术记录右室和右室流出道心肌细胞的单细胞电流。结果 RVOT的稳态外向电流较右室的小。对稳态电流进一步研究发现,右室流出道和右室心肌细胞上均存在IK2p。右室流出道细胞的IK2p电流密度明显小于右室细胞。结论首次在电生理水平上,证实了家兔右室心肌细胞上存在IK2p,RVOT心肌细胞的IK2p电流密度小于RV心肌细胞,是构成右室流出道APD离散度增大及外向电流降低的基础,从而易出现EAD,进而促进RVOT-VT的发生。     

Efficient and non-toxic gene transfer to cardiomyocytes using novel generation amplicon vectors derived from HSV-1

OBJECTIVE: The feasibility of gene transfer to myocardial tissue using viral vectors was investigated over the last few years. In this study we report gene transfer using a recently described improved of Herpes simplex virus (HSV-1)-derived amplicon vectors and demonstrate that these vectors are a powerful and potentially very interesting tool for gene transfer into neonatal primary as well as in adult cardiac myocytes. METHODS AND RESULTS: Non-pathogenic HSV-1 amplicon vectors simultaneously expressing GFP and LacZ were constructed using a novel helper system that yields essentially helper-free vector particles. These vectors were used to infect either cultured primary neonatal rat cardiomyocytes or adult cardiac tissue. Transgenic expression was quantified using a FACS (GFP) or X-gal staining (LacZ). Infection of primary cardiomyocytes showed efficient transduction even at very low multiplicity of infection (MOI), and expression increased with the infectious dose. By investigating release of lactate dehydrogenase (LDH) or spontaneous beating of the cells, we failed to detect cytotoxic effects in cardiomyocytes infected at high MOI. Thin slices of adult cardiac tissue placed in medium containing vectors also showed very good levels of transduction, without any evidence of toxic effects. CONCLUSIONS: Helper-free amplicon vectors very efficiently transduce genes into cardiomyocytes. Our results indicate similar or better transduction efficiencies than those reported using other vector systems. Furthermore, the very high transgenic capacity of amplicon vectors (up to 150 kbp) makes these vectors a unique and very suitable system to transduce large genomic sequences into cardiomyocytes.