细胞外液低钾对小鼠心室肌细胞跨膜电位影响的定量分析

目的 研究不同程度细胞外低钾对心肌细胞跨膜电位的效应,阐明低钾对心肌细胞电生理特性的详细影响。方法 分离C57BL/6J小鼠的左心室乳头肌,采用标准玻璃微电极胞内记录技术记录心室肌细胞的跨膜电位,观察细胞外液K⁺浓度由正常的5.4mmol/L分别降为3、2、1和0mmol/L时,心室肌细胞跨膜电位各参数的变化。结果 低钾对心肌细胞的静息电位(RP)有双向影响:细胞外K⁺浓度降为3mmol/L时,RP显著增大(超极化)(P=0.000),而细胞外K⁺浓度降为2、1和0mmol/L时,细胞RP先显著增大后显著减小(P=0.000),这些结果异于传统观点。当细胞外液K⁺浓度为3mmol/L时,动作电位振幅(APA)和0期最大除极速度(Vmax)均增大,动作电位时程APD10、APD20、APD50和APD90均显著缩短(P<0.05),而动作电位复极到APD90后,复极速度减慢,即复极化有拖尾现象。当细胞外K⁺浓度为2mmol/L时,APA极度减小,Vmax明显减慢,AP呈侏儒型,而当细胞外K⁺浓度为1和0mmol/L时,细胞兴奋性丧失,电刺激不能诱发动作电位。此外,低钾可诱发早期后除极以及连串的触发活动,且后者两种形式,也表现为量-效和时-效的特点。结论 细胞外液低钾对心室肌细胞的RP、APA和Vmax有双重影响:中度低钾(K⁺ 3mmol/L)使这3个参数均增大;重度低钾(K⁺ 2mmol/L及以下)使这3个参数均减小;低钾使动作电位早期复极加快,晚期复极减慢;极度低钾(K⁺ 1mmol/L及以下)会导致心室肌细胞的兴奋性丧失;中、重度低钾可导致心室肌细胞发生早期后除极及连串的触发活动,后者相当于细胞水平的心动过速,但不是工作细胞获得了自律性。本研究在一定程度上澄清了以往对低钾的心肌电生理效应的模糊认识。     

Characterisation of postnatal growth of the murine heart

Using a new technique to isolate rod-shaped cardiomyocytes from small tissue pieces we were able to analyse the developmental profile of postnatal cardiomyocyte growth in the mouse. During the first 4 postnatal days the volume of the cardiomyocytes remains relatively constant despite a concomitant increase in heart weight, indicating growth due to cell division of the cardiomyocytes, also called hyperplasia. After postnatal day 5 the volume of the cardiomyocytes increases dramatically until postnatal day 14, when the increment of the volume curve decreases again. The cardiomyocytes reach their adult volume at around 3 months of age. These measurements present the first detailed analysis of the phase of so-called developmental hypertrophy, i.e. normal cardiomyocyte growth in the mouse, and provide an essential base-line for the analysis of growth parameters in mouse models for cardiomyopathies. We used this method to characterise the growth characteristics of cardiomyocytes from MLP (muscle LIM protein) knockout mice, a mouse model for dilated cardiomyopathy. During the first 2 postnatal weeks there is no significant difference in the growth parameters between MLP knockout and wildtype mice. However, in the adult animals cardiomyocytes from MLP knockout mice are not only characterised by a more irregular shape, but also by a high variability in size compared to cardiomyocytes from wildtype animals. This suggests that the alterations in ventricular morphology in the MLP heart are not due to a general elongation of the cardiomyocytes but to myocyte disarray and ventricular wall thinning caused by the heterogeneous volume of the cardiomyocyte population. Accepted: 26 June 2001     

Glutamate-loading Stimulates Metabolic Flux and Improves Cell Recovery Following Chemical Hypoxia in Isolated Cardiomyocyte

The amino acid glutamate is used in cardioplegic solutions, yet evidence is conflicting as to whether or not exogenous glutamate is indeed cardioprotective. This controversy may be because increasing extracellular glutamate does not necessarily lead to an increase in intracellular glutamate. In this study we aimed to determine whether isolation of myocytes in the presence of glutamate resulted in glutamate-loading of the cells, and, if so, whether such loading protected myocytes from simulated (chemical) hypoxia. Single ventricular myocytes were isolated from rat hearts in the presence and absence of 6.4 m glutamate. Levels of glutamate and ATP were determined using HPLC, and NADH/NAD⁺was determined from cell autofluorescence. Chemical hypoxia was induced by superfusion with a solution containing 2.5 m cyanide and no glucose. Intracellular [Ca²⁺] was measured by loading cells with indo-1, and cell length was measured using an edge-tracking device. Isolation of myocytes in the presence of glutamate resulted in increased intracellular glutamate levels compared with cells isolated in the absence of glutamate, 1324±108 v 948±124 pmol/mg protein, respectively (P<0.05). Cells loaded with glutamate showed increased NADH/NAD⁺, (0.384±0.032v 0.281±0.029, P<0.05) and greater ATP levels (36.031±1.633 nmol/mg protein v 19.279±3.327 nmol/mg protein, P<0.005) compared to control cells. When subjected to chemical hypoxia, cells underwent rigor-contracture at various timepoints, and were then reperfused following 5 min in rigor. Cells loaded with glutamate showed better recovery of diastolic [Ca²⁺], Ca²⁺transient amplitude, and improved contractile function compared with cells isolated in absence of glutamate. This study demonstrates an efficient method for loading myocytes with glutamate during cell isolation, and myocytes loaded with glutamate showed increased metabolic flux, as indexed by a higher NADH/NAD⁺and ATP content. Myocytes also exhibited better recovery from chemical hypoxia in terms of both Ca²⁺handling and cell contraction.     

钠尿肽C型受体信号通路与心血管疾病

钠尿肽家族(natriuretic peptides,NPs)主要包括心房钠尿肽(atrial natriuretic peptide, ANP)、脑钠尿肽(brain natriuretic peptide, BNP)和C型钠尿肽(C-typenatriuretic peptide, CNP)3类,新发现的还有曼巴蛇钠尿肽、尿扩张素及在澳大利亚大班蛇毒液中的一类钠尿肽样肽类[1].     

MEF2C介导microRNA-214发挥抑制心肌细胞肥大的作用

目的:研究微小RNA-214(mi R-214)对心肌细胞肥大的调控作用及其可能的作用靶基因。方法:建立血管紧张素Ⅱ(angiotensin-Ⅱ,Ang-Ⅱ)诱导的C57BL/6乳小鼠心室肌细胞肥大模型;双萤光素酶报告基因实验检测mi R-214与潜在靶基因MEF2C 3’端非翻译区(3’UTR)的结合作用;实时荧光定量PCR(RT-q PCR)和Western blot法分别检测MEF2C及肥厚标志物的m RNA和蛋白表达水平。结果:心肌肥厚标志物ANP、ACTA1和β-MHC,以及mi R-214的表达在Ang-II诱导肥大的小鼠心肌细胞中显著增强;双萤光素酶报告基因实验提示mi R-214与MEF2C 3’UTR相互作用,证实mi R-214可在转录水平抑制MEF2C的表达,MEF2C蛋白水平在肥大的心肌细胞中显著上调;过表达mi R-214及沉默MEF2C均能一致性地抑制Ang-Ⅱ诱导的心肌细胞中肥大标志物的表达。结论:MEF2C是mi R-214的靶基因,并介导了mi R-214发挥抑制心肌细胞肥大的作用。     

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     

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.     

Triiodothyronine restricts myofibrillar growth and enhances beating frequency in cultured adult rat cardiomyocytes

Adult rat cardiomyocytes (ARC) isolated from ventricles follow a defined sequence of structural remodeling during culturing for 2–3 weeks. Rod-shaped cells round up, attach to the substratum, and start growing out in all directions until they form contacts with one another and resume rhythmic contractile activity. In general, myofibrils redevelop along the actin scaffold into the periphery. IGF-I enhances this process while bFGF restricts the outgrowing of myofibrils to the central cell area. Presence of T3 in the culture medium also restricts myofibrillar growth like bFGF. At the same time, T3 increases spontaneous beating frequency in a dose-dependent manner. With 10 nM T3 beating frequency is increased three-fold versus control. Addition of isoproterenol or of epinephrine further increases the frequency at all T3 concentrations tested. Propranolol inhibits the fully stimulated beating frequency to about the same extent at all T3 concentrations. Therefore, T3 seems to determine the beating frequency of ARC in culture directly and not by changing the composition of the adrenoceptor population nor by changing their responsiveness. Received: 10 April 1997, Returned for 1. revision: 28 May 1997, 1. Revision received: 18 June 1997, Accepted: 20 June 1997     

Reduction of ventricular M2 muscarinic receptors in cardiomyopathic hamster (CHF 147) at the necrotic stage of the myopathy

We have previously demonstrated that isolated ventricular myocytes from cardiomyopathic hamsters (CHF 147) during the necrotic stage (70–100 days) exhibit an attenuated contractile response to muscarinic stimulation. In the present study we have investigated whether this dysfunction may be related to a change in the density (or affinity) of cardiac muscarinic receptors. Thus, we have characterized and quantified the binding of the muscarinic antagonist [³H]-N-methyl scopolamine (NMS) to M₂ muscarinic receptors in cardiac micropunches and in suspensions of isolated intact cardiomyocytes obtained from cardiomyopathic (CHF 147) and Golden Syrian hamsters. The hamsters were either 70–100 days old, when the cardiomyopathy had reached the cytolytic and necrotic stage or 30 days old, i. e. before the onset of the cardiomyopathy. In both preparations (micropunches and dissociated cardiomyocytes) the specific binding of [³H]-NMS was stereospecific, reversible, saturable, of high affinity and linearly dependent upon increasing amounts of tissue and cells. The binding site also possessed the drug specificity typical of an M₂ muscarinic receptor. Saturation binding analysis revealed that the hearts of the older CHF 147 hamsters contain significantly fewer M₂ muscarinic receptors than the control Golden Syrian hamsters while the affinity (K _d) was not altered. This reduction of M₂ receptor number was not observed in CHF 147 hamsters at 30 days. Further, we found no differences in -adrenergic or in ₁-adrenergic binding in the two strains of hamster at either age. Thus, our results indicate that the parasympathetic regulation of cardiac function in CHF 147 hamsters may be compromised by a decreased number of muscarinic receptors at the necrotic stage of the cardiomyopathy.     

The antiadrenergic effect of neuropeptide Y on the ventricular cardiomyocyte

Summary The effect of neuropeptide Y (NPY) on adenylate cyclase activity was examined in ventricular myocytes isolated from the adult rat heart. In the presence of the phosphodiesterase inhibitor Ro 20-1724 (0.5 mM) and adenosine deaminase (5 U/ml), these intact cells accumulate cyclic AMP when stimulated by isoproterenol. NPY (10^–9 to 10^–6 M) reduced the degree of cAMP accumulation achieved by 10^–7 M isoproterenol in a dose dependend manner by 10 to maximally 48%. The IC ₅₀ value was 3 x 10^–8 M NPY. A maximal concentration (10^–6 M) of N⁶-phenylisopropyladenosine (PIA) decreased cAMP levels by 39%, i.e. to a similar extent. Prior treatment of the myocytes with pertussis toxin (1 g/ml for 6 h) increased the mean stimulated values in the presence of isoproterenol (10^–7 M) by a factor 4.1. In such cells, NPY and PIA were ineffective in antagonizing the stimulation of cAMP production by isoproterenol. These results indicate that the ventricular myocyte has receptors for NPY, similar to the A₁ adenosinereceptor in that they are linked to the adenylate cyclase by an inhibitory guanylate binding protein.Abbreviations NPY neuropeptide Y - PIA N⁶-phenylisopropyl-adenosine - Ro 20-1724 4-(3-butoxy-4-ethoxybenzyl)-2-imidazolidione - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid Send offprint requests to H. M. Piper at the above address