Evidence for functional expression of TRPM7 channels in human atrial myocytes

Transient receptor potential melastatin-7 (TRPM7) channels have been recently reported in human atrial fibroblasts and are believed to mediate fibrogenesis in human atrial fibrillation. The present study investigates whether TRPM7 channels are expressed in human atrial myocytes using whole-cell patch voltage-clamp, RT-PCR and Western blotting analysis. It was found that a gradually activated TRPM7-like current was recorded with a K(+)- and Mg(2+)-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca(2+) and Mg(2+), and the current increase could be inhibited by Ni(2+) or Ba(2+). The TRPM7-like current was potentiated by acidic pH and inhibited by La(3+) and 2-aminoethoxydiphenyl borate. In addition, Ca(2+)-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca(2+) ionophore A23187 in bath solution. RT-PCR and Western immunoblot analysis revealed that in addition to TRPM4, TRPM7 channel current, mRNA and protein expression were evident in human atrial myocytes. Interestingly, TRPM7 channel protein, but not TRPM4 channel protein, was significantly increased in human atrial specimens from the patients with atrial fibrillation. Our results demonstrate for the first time that functional TRPM7 channels are present in human atrial myocytes, and the channel expression is upregulated in the atria with atrial fibrillation.     

Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle

OBJECTIVE: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. METHODS: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting. RESULTS: Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. CONCLUSIONS: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.     

Multiple sequences in the C terminus of MaxiK channels are involved in expression, movement to the cell surface, and apical localization

Apical expression of the large-conductance, calcium- and voltage-activated potassium (MaxiK) channel in the cortical collecting duct is responsible for flow-stimulated potassium secretion. Here, we identify two cytoplasmic regions controlling apical expression of the MaxiK channel. Disruption of the proximal region results in the intracellular retention of the MaxiK channel without affecting channel assembly, thereby reducing surface expression. Coexpression of the WT channel with this mutant results in a reduction of WT MaxiK channel at the cell surface. Our data indicate that this proximal region is necessary for export of the MaxiK channel from the endoplasmic reticulum as a way to assess the final assembly of the channel. Deletion of a more distal region disrupts apical sorting, resulting in a nonpolarized distribution of the channel without impairing its surface delivery. In summary, we have found that sequences of amino acids in the C terminus of the MaxiK channel operate after the channel is assembled into a multimer and play a role in its expression, movement to the cell surface, and apical localization.     

Unitary sodium channels in isolated cardiac myocytes of rabbit

Transmembrane ionic movements are thought to occur through special membrane areas or channels. Until recently, it has not been possible to study the properties of individual ionic channels, directly. We have used the extracellular patch clamp technique to resolve unitary sodium channels in enzyme-dissociated rabbit ventricular myocytes. Depolarizing voltage clamp steps elicited rectangular inward current fluctuations representing open-closed transition of the channels. Probability of channel opening increased with depolarization. Single channel current amplitude ranged from 0.6 to 1.9 pA. Channel open time increased with depolarization. Sodium channels could be distinguished from calcium channels by their insensitivity to cadmium and inactivation voltage range. First-event latency histograms suggest multiple-closed states. The patch clamp technique can be readily applied to the study of sodium channels in adult cardiac myocytes.     

二十二碳六烯酸对心室肌细胞离子通道的影响

近年来,n-3多不饱和脂肪酸(n-3PUFAs)对心血管的有益作用已受到广泛重视.大量研究表明n-3PUFAs具有抗心律失常作用,能降低冠心病的猝死率及心肌梗死后恶性心律失常的发生率[1].基于这些研究结果,在心血管疾病的一级和二级预防中,美国和欧洲等心脏病学会推荐每日摄入小剂量n-3PUFAs[2].目前,n-3PUFAs抗心律失常的机制仍不完全清楚.n-3PUFAs主要包括二十二碳六烯酸(DHA)和二十碳五烯酸(EPA).本文通过膜片钳技术探讨DHA对大鼠心室肌细胞静息电位(RP)、动作电位时限(APD)、延迟整流性钾通道电流(IK)及内向整流性钾通道电流( IKl)的影响,阐述DHA抗心律失常的可能机制. 1.材料和溶液组成:Axopatch 700B膜片钳放大器、Digi-Data 1322型数/模(或模/数)转换器、pClamp 9.0脉冲发放和数据采集软件(美国Axon Instruments公司).DHA(美国Sigma公司)以无水乙醇配制成50 mmol/L的母液,分装避光保存于-80℃备用.使用时无水乙醇的终浓度＜0.4％,以避免无水乙醇对离子通道的影响.牛血清白蛋白(BSA,美国Sigma公司)配制成2 mg/ml,4℃保存.     

Sulfhydryl modulation of K+ channels in rat ventricular myocytes

Oxidative stress markedly alters protein function through redox modification of sulfhydryl groups present in cysteine residues. To explore the role of redox state in modulating cardiac K+ channels, this study examined the effects of sulfhydryl modifiers on the repolarizing transient outward current (Ito) in voltage-clamped myocytes from rat ventricle. Oxidized glutathione (GSSG; 5mM), an endogenous disulfide that specifically reacts with protein sulfhydryls, decreased maximum Ito amplitude from baseline by 49% when added to the external solution (P<0.05) and by 27% during internal dialysis (P<0.05). The membrane-impermeable disulfide, 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) did not alter Ito when added to the external solution, but it decreased current amplitude by 31% during internal dialysis (P<0.05). GSSG-mediated Ito inhibition varied in a frequency- and voltage-dependent manner, consistent with a state-dependent blocking mechanism. This phenomenon was also observed in myocytes internally dialyzed with DTNB or Cd2+, which also covalently binds to free sulfhydryls. Inhibition of Ito by GSSG was not reversed by washout alone, consistent with the stable nature of covalently-modified sulfhydryl groups. However, when myocytes pretreated with GSSG were dialyzed with the reducing agent dithiothreitol, Ito amplitude increased significantly by 42% (P<0.05). These data suggest that alpha-subunits underlying Ito, or associated proteins, have one or more sulfhydryl groups within the cytoplasmic domain that directly modulate channel activity in response to changes in cell redox state. Redox modulation of Ito channels may be an important post-translational mechanism contributing to acute changes in cardiac repolarization under conditions of oxidative stress, such as ischemia and reperfusion.     

Accessory Kvbeta1 subunits differentially modulate the functional expression of voltage-gated K+ channels in mouse ventricular myocytes

Voltage-gated K+ (Kv) channel accessory (beta) subunits associate with pore-forming Kv alpha subunits and modify the properties and/or cell surface expression of Kv channels in heterologous expression systems. There is very little presently known, however, about the functional role(s) of Kv beta subunits in the generation of native cardiac Kv channels. Exploiting mice with a targeted disruption of the Kvbeta1 gene (Kvbeta1-/-), the studies here were undertaken to explore directly the role of Kvbeta1 in the generation of ventricular Kv currents. Action potential waveforms and peak Kv current densities are indistinguishable in myocytes isolated from the left ventricular apex (LVA) of Kvbeta1-/- and wild-type (WT) animals. Analysis of Kv current waveforms, however, revealed that mean+/-SEM I(to,f) density is significantly (P< or =0.01) lower in Kvbeta1-/- (21.0+/-0.9 pA/pF; n=68), than in WT (25.3+/-1.4 pA/pF; n=42), LVA myocytes, and that mean+/-SEM I(K,slow) density is significantly (P< or =0.01) higher in Kvbeta1-/- (19.1+/-0.9 pA/pF; n=68), compared with WT (15.9+/-0.7 pA/pF; n=42), LVA cells. Pharmacological studies demonstrated that the TEA-sensitive component of I(K,slow), I(K,slow2,) is selectively increased in Kvbeta1-/- LVA myocytes. In parallel with the alterations in I(to,f) and I(K,slow2) densities, Kv4.3 expression is decreased and Kv2.1 expression is increased in Kvbeta1-/- ventricles. Taken together, these results demonstrate that Kvbeta1 differentially regulates the functional cell surface expression of myocardial I(to,f) and I(K,slow2) channels.     

Relations of vascular calcium channels with blood pressure and endothelium in hypertension and with aging.

To investigate the relationships between the activity in potential operated Ca2+ channels (POC), blood pressure, and endothelium in hypertension, we tested the contractile responses to a Ca2+ channel agonist Bay K 8644 (BAY K) in aorta from deoxycorticosterone-acetate-saline (DOCA-S) and reduced renal mass-saline (RRM-S) hypertensive rats. The effects of mechanical rubbing, N omega-Nitro-L-Arginine Methyl Ester (l-NAME) and indomethacin were also examined. Sensitivity to BAY K increased in experimental rats before they became hypertensive and contractile responses were enhanced as hypertension developed. Force development to BAY K was correlated with blood pressure levels. Endothelium removal enhanced the contractile response to BAY K. L-NAME, but not indomethacin, potentiated the response to BAY K. Contractile response to BAY K was negatively correlated with relaxation to acetylcholine. An enhanced contractile response to BAY K was observed also in aged rats. Enhanced activation of vascular POC in hypertension results from elevated blood pressure and partly from diminished inhibitory action of endothelium. Senescence also enhances vascular POC activity.     

Two types of calcium channels in guinea pig ventricular myocytes.

In cardiac muscle, Ca2+ plays a key role in regulation of numerous processes, including generation of the action potential and development of tension. The entry of Ca2+ into the cell is regulated primarily by voltage-gated channels in the membrane. Until recently, it was felt that only one type of Ca2+ channel existed in cardiac ventricular muscle. Experiments reported here suggest that in isolated guinea pig ventricular myocytes, there are two distinct types of Ca2+ channels with markedly different activation thresholds, inactivation kinetics, and sensitivities to inorganic and organic Ca2+ channel blockers. The channels were also distinguished based on their response to increased frequency of clamping such that the current through the low-threshold channel decreased while that through the high-threshold channel increased. In a few cells, the current through both channels was enhanced by isoproterenol, a beta-adrenergic agonist, but only the high-threshold channel was enhanced by the Ca2+-channel agonist Bay K 8644. Thus, isolated guinea pig ventricular myocytes appear to have two types of Ca2+ channels distinguished by various criteria.     

睾酮对衰老心肌细胞的干预作用及其机制

目的探讨不同剂量睾酮对衰老心肌细胞的干预作用及其可能机制。方法将心肌细胞随机分为正常组、衰老组、1μmol/L睾酮组、100 nmol/L睾酮组、10 nmol/L睾酮组,检测各组心肌细胞周期分布,去磷酸化视网膜母细胞瘤蛋白(RB)表达,细胞内活性氧水平以及细胞线粒体DNA突变率。结果衰老组心肌细胞G_0/G_1期比例较正常组明显升高,而1μmol/L睾酮组、100 nmol/L睾酮组、10 nmol/L睾酮组G_0/G_1期比例较衰老组明显降低(P<0.05)。除10 nmol/L睾酮组对RB表达无明显影响外,睾酮干预可下调去磷酸化RB表达,降低细胞内活性氧水平,降低线粒体DNA突变率(P<0.05,P<0.01)。结论睾酮可抑制小鼠心肌细胞衰老,这一作用部分是通过下调去磷酸化RB表达,降低细胞内活性氧水平,降低线粒体DNA突变率来实现的。     

Properties of angiotensin-converting enzyme in intact cerebral microvessels

Angiotensin-converting enzyme (ACE) was studied in preparations of microvessels isolated from rabbit cerebral cortex. Activity was determined by measuring the degradation of hippuryl-histidyl-leucine (Hip-His-Leu) by the intact microvessels in a physiological salt solution at pH 7.4. ACE activity was dependent on both substrate and chloride ion concentration and was inhibited by captopril in a manner similar to that observed previously with tissue homogenates. Angiotensin I was rapidly degraded by the intact microvessels, even in the presence of 10(-6)M captopril. An advantage of the methodology employed was the ability to pretreat the microvessels and then assess the effect of pretreatment by transfer to a postincubation assay system. Pretreatment with a hyperosmolar urea solution did not change ACE activity or cause release of ACE from the microvessels, although lactic dehydrogenase and lysosomal enzymes were released. Pretreatment with captopril caused a lag in the subsequent degradation of Hip-His-Leu, presumably reflecting dissociation of inhibitor from the cell-associated enzyme. ACE activity was unaffected by hypoxic or anoxic incubation conditions. The ability to measure ACE activity of the microvessels in vitro provides a unique opportunity to study the properties of the enzyme in intact cerebrovascular endothelial cells.     

Roles of mechano-sensitive ion channels, cytoskeleton, and contractile activity in stretch-induced immediate-early gene expression and hypertrophy of cardiac myocytes.

Mechanical loading of cardiac and skeletal muscles in vivo and in vitro causes rapid activation of a number of immediate-early (IE) genes and hypertrophy of muscle cells. However, little is known as to how muscle cells sense mechanical load and transduce it into intracellular signals of gene regulation. We examined roles of putative cellular mechanotransducers, mechanosensitive ion channels, the cytoskeleton, and contractile activity in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channel that was completely blocked by gadolinium (Gd3+). Inhibition of this channel by Gd3+ did not affect either the stretch-induced expression of IE genes or the increase in protein synthesis. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression and increase in protein synthesis. Arresting contractile activity of myocytes by high K+, tetrodotoxin, or Ba2+ did not affect the stretch-induced IE gene expression. Tetrodotoxin-arrested myocytes could increase protein synthesis in response to stretch. These results suggest that Gd(3+)-sensitive ion channels, microtubules, microfilaments, and contractile activity may not be necessary for transduction of mechanical stretch into the IE gene expression and hypertrophy. The stimulus of membrane stretch may be transmitted to the cell nucleus through some mechanisms other than electrical or direct mechanical transduction in cardiac myocytes.     

Effects of alloimmune injury on contraction and relaxation in cultured myocytes and intact cardiac allografts

Objectives. This study was performed to determine the mechanisms by which allosensitized lymphocytes cause contractile dysfunction in cultured ventricular myocytes and to compare the effects on isolated myocytes with those observed in an intact heart preparation during allograft rejection.Background. Allograft rejection may be associated with reversible abnormalities of both systolic and diastolic function. The immunologic mechanisms that cause ventricular dysfunction are poorly understood.Methods. Vascularized heterotopic abdominal heart transplantation was performed in mice. Contractile function of excised allografts undergoing rejection was assessed using a Langendorff perfusion apparatus and a strain gauge. Spontaneously beating monolayers of cultured ventricular myocytes from donor strain fetal mice were exposed to the allosensitizcd cytotoxic T lymphocytes, and the effects on myocyte motion, intracellular calcium transients, relaxation half-time, membrane potential and myocyte lysis (chromium-51 release) were measured.Results. In intact hearts, histologically mild rejection without myocyte necrosis was associated with decreased systolic function without slowing of relaxation. In cultured fetal myocytes, sensitized lymphocytes induced a progressive decrease in the amplitudes of myocyte motion and calcium transients, with cessation of beating within 40 min. Also, the diastolic membrane potential and amplitude of the action potential decreased. Relaxation half-time, as estimated by measurement of cell motion, was unchanged. The effect was allospecific and was reversible with early removal of lymphocytes from the myocyte monolayer. Pretreatment of lymphocytes with the degranulation inhibitor 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene blocked both the negative inotropic effect and myocyte lysis.Conclusions. We conclude that impaired relaxation is not a prominent feature of contractile dysfunction caused directly in myocytes by alloimmune injury from cytotoxic lymphocytes. Allosensitized lymphocytes can cause reversible systolic dysfunction in myocytes by means of a direct cell-cell interaction. This effect may be in part responsible for the reversible systolic dysfunction associated with allograft rejection.     

成年大鼠心房肌牵张激活钾通道的牵张敏感性和门控机制

目的探讨成年大鼠心房肌细胞牵张激活钾通道（stretch-activated K+-selective channels,SAKCs）的电生理学特性,确定皮质细胞骨架在通道门控机制中的作用,对从通道水平阐明机械-电反馈具有重要的理论和实际意义。方法联合应用单通道膜片钳技术和压力钳技术,在急性分离的成年大鼠心房肌细胞上,采用细胞贴附（cell-atta-ched）方式记录SAKCs的活动。结果实验所记录的通道为SAKCs,通道闪烁样开放,无整流特性。当细胞外液为高K+液（140mmol/L）时,翻转电位为0mV。钳制膜电位+60mV时单通道的电导值为（59±5）pS,-60mV时为（51±8）pS。通道约在负压刺激开始700800ms内被快速激活,刺激解除后,通道快速在500ms内去激活。超过-30mmHg（1mmHg=0.133kPa）的刺激可使多个通道同时开放。实验中未观察到通道活动达到饱和现象。单通道电流幅度不受负压刺激的影响。随膜片钳电极内负压的增加,通道开放概率增大,呈刺激强度依赖性。Cytocha-lasin B不改变SAKCs的电流幅度,但增加SAKCs的开放概率,增强SAKCs的背景活动和对机械刺激的敏感性。结论我们推测生理状态下细胞皮质肌动蛋白内衬于细胞膜,可能作为细胞膜的并联成分承受部分细胞应力,并使脂质膜的应力减少,从而使SAKCs不易被激活。     

Identification and properties of ATP-sensitive potassium channels in myocytes from rabbit Purkinje fibres

OBJECTIVE: Our goal was to identify the ATP-sensitive potassium (KATP) channels in cardiac Purkinje cells and to document the functional properties that might distinguish them from KATP channels in other parts of the heart. METHODS: Single Purkinje cells and ventricular myocytes were isolated from rabbit heart. Standard patch-clamp techniques were used to record action potential waveforms. and whole-cell and single-channel currents. RESULTS: The KATP channel opener levcromakalim (10 microM) caused marked shortening of the Purkinje cell action potential. Under whole-cell voltage-clamp, levcromakalim induced an outward current, which was blocked by glibenclamide (5 microM), in both Purkinje cells and ventricular myocytes. Metabolic poisoning of Purkinje cells with NaCN and 2-deoxyglucose caused a significant shortening of the action potential (control 376 +/- 51 ms; 6 min NaCN/2-deoxyglucose 153 +/- 21 ms). This effect was reversed with the application of glibenclamide. Inside-out membrane patches from Purkinje cells showed unitary current fluctuations which were inhibited by cytoplasmic ATP with an IC50 of 119 microM and a Hill coefficient of 2.1. This reflects approximately five-fold lower sensitivity to ATP inhibition than for KATP channels from ventricular myocytes under the same conditions. The slope conductance of Purkinje cell KATP channels, with symmetric, 140 mM K+, was 60.1 +/- 2.0 pS (mean +/- SEM). Single-channel fluctuations showed mean open and closed times of 3.6 +/- 1.5 ms and 0.41 +/- 0.2 ms, respectively, at -60 mV and approximately 21 degrees C. At positive potentials. KATP channels exhibited weak inward rectification that was dependent on the concentration of internal Mg2+. Computer simulations, based on the above results, predict significant shortening of the Purkinje cell action potential via activation of KATP channels in the range 1-5 mM cytoplasmic ATP. CONCLUSIONS: Purkinje cell KATP channels may represent a molecular isoform distinct from that present in ventricular myocytes. The presence of KATP channels in the Purkinje network suggests that they may have an important influence on cardiac rhythm and conduction during periods of ischemia.