Gene transfer of connexin43 into skeletal muscle

Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.     

The heme oxygenase inducer hemin protects against cardiac dysfunction and ventricular fibrillation in ischaemic/reperfused rat hearts: role of connexin 43

Cardiac expression of cytoprotective gene heme oxygenase-1 (HO-1) is modulated by ischaemia and reperfusion (I/R). We therefore hypothesized that pretreatment with hemin, an inductor of HO-1, would precondition the heart against post-ischaemic dysfunction and ventricular fibrillation (VF). Male Wistar rats were given either hemin or HO enzyme inhibitor zinc protoporphyrin IX (ZnPP IX). Isolated hearts were subjected to 30 min global ischaemia followed by 120 min of reperfusion or were aerobically perfused in a time-matched non-ischaemic protocol. Control animals received no pretreatment. Compared to non-perfused controls, pretreatment with hemin increased HO-1 mRNA 13-fold (p<0.001) and HO-1 protein 3.5-fold (p相似文献   

Slow ventricular conduction in mice heterozygous for a connexin43 null mutation.

To characterize the role of the gap junction protein connexin43 (Cx43) in ventricular conduction, we studied hearts of mice with targeted deletion of the Cx43 gene. Mice homozygous for the Cx43 null mutation (Cx43 -/-) die shortly after birth. Attempts to record electrical activity in neonatal Cx43 -/- hearts (n = 5) were unsuccessful. Ventricular epicardial conduction of paced beats, however, was 30% slower in heterozygous (Cx43 -/+) neonatal hearts (0.14+/-0.04 m/s, n = 27) than in wild-type (Cx43 +/+) hearts (0.20+/-0.07 m/s, n = 32; P < 0.001). This phenotype was even more severe in adult mice; ventricular epicardial conduction was 44% slower in 6-9 mo-old Cx43 -/+ hearts (0.18+/-0.03 m/s, n = 5) than in wild-type hearts (0.32+/-0.07 m/s, n = 7, P < 0.001). Electrocardiograms revealed significant prolongation of the QRS complex in adult Cx43 -/+ mice (13.4+/-1.8 ms, n = 13) compared with Cx43 +/+ mice (11.5+/-1.4 ms, n = 12, P < 0.01). Whole-cell recordings of action potential parameters in cultured disaggregated neonatal ventricular myocytes from Cx43 -/+ and +/+ hearts showed no differences. Thus, reduction in the abundance of a major cardiac gap junction protein through targeted deletion of a Cx43 allele directly leads to slowed ventricular conduction.     

Sodium hydrogen exchange 1 (NHE-1) regulates connexin 43 expression in cardiomyocytes via reverse mode sodium calcium exchange and c-Jun NH2-terminal kinase-dependent pathways

Connexin 43, the major connexin isoform in gap junctions of cardiac ventricular myocytes, undergoes changes in distribution and expression in cardiac diseases. The Na(+)-H(+) exchanger (NHE-1), a key mediator of hypertrophy and heart failure, has been shown to be localized in the cardiomyocyte gap junctional regions; however, whether NHE-1 regulates gap junction proteins in the hypertrophied cardiomyocyte is not known. To address this question, neonatal rat ventricular myocytes were treated with phenylephrine (PE) for 24 h to induce hypertrophy. Increased Cx43 expression observed with PE treatment (132.4 +/- 6.3% compared to control; P < 0.05) was further significantly augmented by the specific NHE-1 inhibitor EMD87580 [N-[2-methyl-4,5-bis(methylsulfonyl)-benzoyl]-guanidine hydrochloride] (173.2 +/- 8.7% increase compared to control; P < 0.05 versus PE), an effect that was mimicked by another NHE-1 inhibitor cariporide [4-isopropyl-3-(methylsulfonyl)benzoyl-guanidine methanesulfonate]. PE-induced hypertrophy was associated with mitogen-activated protein kinase c-Jun NH(2)-terminal kinase (JNK) 1/2 activation, whereas inhibition of JNK1/2 with either SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] or small interfering RNA significantly increased PE-induced up-regulation of Cx43 protein levels. Inhibition of reverse mode Na(+)-Ca(2+) exchange (NCX) with KB-R7943 [2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate] partially reversed JNK1/2 activation (195.2 +/- 21.4 versus 143.7 +/- 14.4% with KB-R7943; P < 0.05) and augmented up-regulation of Cx43 protein (121.1 +/- 8.3 versus 215.9 +/- 25.6% with KB-R7943; P < 0.05) in the presence of PE. Our results demonstrate that NHE-1 negatively regulates Cx43 protein expression in PE-induced cardiomyocyte hypertrophy via a JNK1/2-dependent pathway, which is probably activated by reverse mode NCX activity.     

Sympathoadrenal modulation of stress-activated signaling in burn trauma

Burn injury stimulates stress-responsive components, p38 mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinases (JNK)/nuclear factor (NF)-kappaB. p38 MAPK plays a role in postburn cardiomyocyte tumor necrosis factor-alpha secretion and cardiac dysfunction. Since burn trauma increases circulating catecholamine levels, which in turn modulate inflammatory cytokine production, we hypothesized that increased sympathetic activity after major burn trauma may trigger postburn cardiac p38 MAPK activation via an adrenergic receptor-mediated phenomenon. We examined adrenergic receptor populations involved in burn-activated cardiac stress signaling. Sprague Dawley rats were divided into six groups: 1) control, 2) control plus alpha1-adrenergic agonist phenylephrine (2 microg/kg, intravenous), 3) control plus beta-adrenergic agonist isoproterenol (1 microg/kg, intravenous), 4) burn (fluid resuscitation with lactated Ringer's 4 ml/kg/% burn), 5) burn plus alpha1-adrenergic antagonist prazosin (1 mg/kg, by mouth), and 6) burn plus beta-adrenergic antagonist propranolol (3.3 mg/kg, by mouth). Phenylephrine, but not isoproterenol, increased cardiac p38 MAPK/JNK/NF-kappaB activation. Burn trauma activated p38 MAPK, JNK, and NF-kappaB, and this stress response was blocked by either prazosin or propranolol. Thus, stimulation of the adrenergic pathway may constitute one upstream activator of stress response in burn.     

Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca²⁺ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca²⁺ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca²⁺ entry and coupling to Ca²⁺ release microdomains at the intercalated disc, resulting in enhanced Ca²⁺ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca²⁺ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure.     

Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxia-inducible factor-1alpha and VEGF

Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1alpha (HIF-1alpha) levels and demonstrate increased HIF-1alpha-dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1alpha and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1alpha-mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1alpha-independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.     

Differential effects of bucindolol and carvedilol on noradenaline-induced hypertrophic response in ventricular cardiomyocytes of adult rats

In adult rat ventricular cardiomyocytes, noradrenaline exerts dual effects on protein synthesis: increases via alpha(1)-adrenoceptors and decreases via beta(1)-adrenoceptors. Carvedilol and bucindolol are beta-blockers with additional alpha(1)-adrenoceptor blocking activities. We studied the effects of carvedilol and bucindolol on noradrenaline-induced protein synthesis (assessed by [(3)H]phenylalanine incorporation) in adult rat ventricular cardiomyocytes. Radioligand binding studies with [(125)I]iodocyanopindolol and [(3)H]prazosin revealed that carvedilol had a much higher affinity to alpha(1)-adrenoceptors than bucindolol (beta(1)-/alpha(1)-adrenoceptor ratio for carvedilol, 1:2.7; for bucindolol, 1:43). Noradrenaline-evoked increases in protein synthesis were enhanced by propranolol (1 microM) and beta(1)-adrenoceptor-selective antagonists bisoprolol (1 microM) and CGP 20712A [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)-ethyl-amino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propranol methanesulfonate] (300 nM). Carvedilol (100 pM-10 microM) inhibited 1 microM noradrenaline-induced increase in protein synthesis with monophasic concentration-inhibition curves independent of whether CGP 20712A was present or not; K(i) values for carvedilol were 5 to 6 nM. In contrast, bucindolol (100 pM-10 microM) inhibited l microM noradrenaline-induced increase in protein synthesis with a bell-shaped concentration-inhibition curve; it increased noradrenaline-induced protein synthesis at 10 nM, although at concentrations >100 nM it was inhibited. In the presence of 300 nM CGP 20712A or 1 microM propranolol, however, bucindolol inhibited 1 microM noradrenaline-induced increase in protein synthesis with monophasic concentration-inhibition curves; K(i) values were 40 to 75 nM. On the other hand, both carvedilol and bucindolol inhibited 1 microM phenylephrine-induced protein synthesis with monophasic concentration-inhibition curves; K(i) values were 4 (carvedilol) and 45 nM (bucindolol). These results indicate that, at low (beta-adrenoceptor blocking) concentrations, bucindolol can enhance noradrenaline-induced protein synthesis whereas it is inhibited by carvedilol.     

The heme oxygenase inducer hemin protects against cardiac dysfunction and ventricular fibrillation in ischaemic/reperfused rat hearts: role of connexin 43

Cardiac expression of cytoprotective gene heme oxygenase‐1 (HO‐1) is modulated by ischaemia and reperfusion (I/R). We therefore hypothesized that pretreatment with hemin, an inductor of HO‐1, would precondition the heart against post‐ischaemic dysfunction and ventricular fibrillation (VF). Male Wistar rats were given either hemin or HO enzyme inhibitor zinc protoporphyrin IX (ZnPP IX). Isolated hearts were subjected to 30?min global ischaemia followed by 120?min of reperfusion or were aerobically perfused in a time‐matched non‐ischaemic protocol. Control animals received no pretreatment. Compared to non‐perfused controls, pretreatment with hemin increased HO‐1 mRNA 13‐fold (p<0.001) and HO‐1 protein 3.5‐fold (p?0.001), improved post‐ischaemic aortic flow, coronary flow, LVDP and ?Dp/dt (p<0.01) and decreased LVEDP (p<0.001) and the incidence of VF (p = 0.001). The improved post‐ischaemic cardiac function and reduction of VF were accompanied by a higher total connexin 43 (Cx43) level compared to non‐pretreated and ZnPP IX pretreated hearts, and accumulation of non‐phosphorylated gap junction protein Cx43 in intercalated discs and lateral plasma membrane of cardiomyocytes. Cardioprotection by HO‐1 appeared to be independent of cGMP. Administration of ZnPP IX had no effect on cardiac function or VF. Our results show that pharmacological modulation of HO‐1 pathway may provide a new therapeutic approach to protect the heart against post‐ischaemic dysfunction and I/R‐induced VF possibly by a Cx43 dependent mechanism.     

Oxidant stress derails the cardiac connexon connection

Connexin 43 (Cx43) is the major protein component of gap junctions that electrically couple cardiomyocytes at the intercalated disc. Oxidant stress, reduced Cx43 expression, and altered subcellular localization are present in many forms of structural heart disease. These changes in Cx43 lead to alterations in electrical conduction in the ventricle and predispose to lethal cardiac arrhythmias. In their study in this issue of the JCI, Smyth et al. tested the hypothesis that oxidant stress perturbs connexon forward trafficking along microtubules to gap junctions (see the related article beginning on page 266). Failing human ventricular myocardium exhibited a reduction in Cx43 and the microtubule-capping protein EB1 at intercalated discs. Oxidant stress in the adult mouse heart reduced N-cadherin, EB1, and Cx43 colocalization. In HeLa cells and neonatal mouse ventricular myocytes, peroxide exposure displaced EB1 from the plus ends of microtubules and altered microtubule dynamics. Mutational disruption of the EB1-tubulin interaction mimicked the effects of oxidant stress, including a reduction in surface Cx43 expression. These data provide important new molecular insights into the regulation of Cx43 at gap junctions and may identify targets for preservation of cellular coupling in the diseased heart. Rapid propagation of electrical impulses in excitable tissue is essential to processes as diverse as cognition, movement, and the genesis of the heartbeat. Central to rapid conduction in the heart and other organs are gap junctions. Gap junctions are low-resistance conduits between cells, comprised of proteins called connexins. In the heart, connexins are key mediators of electrical conduction and are thus central to excitation and contraction. Connexins hexamerize to form connexons or hemichannels in the membranes of apposing cells that dock head-to-head to form intact gap junction channels (Figure ​(Figure1). 1). Open in a separate windowFigure 1Structure of the cardiac intercalated disc.(A) A schematic of the intercalated disc under normal conditions shows normal forward trafficking along microtubules of Cx43-containing connexons to the adherens junction for incorporation into gap junction plaques between cardiomyocytes. Connexins mediate electrical conduction between cells and are thus central to cardiomyocyte excitation and contraction. (B) In this issue of the JCI, Smyth et al. (9) show that in the setting of oxidative stress, the microtubule-capping protein EB1 dissociates from the microtubule plus end, impeding connexon trafficking to the adherens junction and reducing the generation of gap junction channels. This results in cellular uncoupling and slowed electrical conduction in the ventricle and may predispose to lethal cardiac arrhythmias. Adapted with permission from Cell (10) and ref. 9. The major connexin of working ventricular myocardium is connexin 43 (Cx43). Cx43 is richly endowed with protein interaction domains and sites of phosphorylation that contribute to regulation of the functional expression of gap junction channels. The carboxyl terminus contains a PDZ-binding domain, multiple consensus serine and tyrosine phosphorylation sites, and binding sites for tubulins. Post-translational modifications and protein-protein interactions are thought to be important for proper formation and localization of clusters of gap junctions into so-called “plaques,” although Cx43 with a truncated carboxyl terminus forms working gap junction channels (1).     

The involvement of gap junctions in the delayed phase of the protection induced by cardiac pacing in dogs

The present study has examined the role of GJ (gap junctions) in the delayed anti-arrhythmic effect of cardiac pacing, with particular reference to the time-course changes in Cx43 (connexin43) expression both after pacing (4×5?min, at a rate of 240 beats/min) and 24?h later, when the dogs were subjected to a 25?min occlusion and reperfusion of the LAD (left anterior descending coronary artery). Compared with the SP (sham-paced) controls (n=20), in dogs paced 24?h previously (n=16) there were reductions in arrhythmia severity [e.g. number of VPB (ventricular premature beats) during occlusion 294±78 compared with 63±25; survival from the combined ischaemia/reperfusion insult 20% compared with 78%], and in other ischaemic changes [epicardial ST-segment, TAT (total activation time) and tissue impedance]. Pacing also prevented the ischaemia-induced structural impairment of the intercalated discs, and preserved GJ permeability and Cx43 phosphorylation, without modifying Cx43 protein content. Following cardiac pacing the membrane and total Cx43 protein contents were unchanged up to 6?h, but were significantly reduced 12?h later (preceded by a down-regulation of Cx43 mRNA at 6?h), and returned to normal by 24?h. Interestingly, dogs that were subjected to ischaemia 12?h after cardiac pacing showed increased arrhythmia generation. We conclude that cardiac pacing results in time-dependent changes in Cx43 expression, which may alter GJ function and influence arrhythmia generation during a subsequent ischaemia/reperfusion insult. This effect is manifested in protection 24?h after pacing, but of potential clinical interest is the finding that there is a time interval after pacing during which an ischaemic event may generate severe ventricular arrhythmias.     

心室纤颤时心肌缝隙连接蛋白Cx43的变化

目的 观察心室纤颤(室颤)发生后缝隙连接蛋白Cx43的表达以及缝隙连接改造剂ZP123对Cx43表达的影响.方法 按照随机数字表法将30只家猪分为假手术组、模型组和ZP123干预组,每组10只.以80 V电压持续刺激动物5 s诱发室颤;致颤前15 min ZP123组给予ZP123 1μg/kg静脉推注+ZP12310μg·kg-1·h-1微量泵泵入;模型组泵入生理盐水50 ml;假手术组动物不致颤也不补液.室颤持续8 min后开胸取左心室游离壁心肌,用免疫荧光结合激光共聚焦显微镜技术检测Cx43的分布及水平,用蛋白质免疫印迹法(Western blotting)定量检测Cx43蛋白表达.结果 假手术组Cx43荧光信号强,分布均匀;模型组Cx43荧光信号弱,呈不均一分布;ZP123干预组Cx43荧光信号增强,不均一分布减轻.与假手术组比较,模型组心室肌组织Cx43荧光信号面积百分比、积分吸光度(A)值及蛋白表达均明显下降[面积百分比:(0.64±0.36)%比(1.27±0.19)%,积分A值:15 201±2 613比30 634±4 975,Cx43蛋白表达:0.72±0.08比0.97±0.07,均P＜0.05];与模型组比较,ZP123干预组Cx43表达[面积百分比(0.96±0.16)%,积分A值22 100±4 404,Cx43蛋白表达0.82±0.04]均明显升高(均P＜0.05).结论 室颤发生时心肌组织Cx43表达减少;应用ZP123可减少或逆转Cx43的降解.     

缝隙连接蛋白Cx43在SD大鼠电点燃癫痫模型中的表达

目的探讨缝隙连接蛋白(Cx)在癫痫形成中的作用。方法将32只雄性SD大鼠随机分为4组,分别造模。观察SD大鼠行为,记录脑电图。通过免疫蛋白印迹的方法检测Cx43在大鼠海马区的表达变化。通过药物对癫痫电点燃模型成模过程的干预来观察癫痫形成与缝隙连接蛋白间的关系。结果模型组大鼠出现癫痫发作。蛋白印迹显示Cx43的表达异常增高。药物干预的2组SD大鼠模型痫性发作滞后,发作频率降低,程度减轻,痫性脑电波波幅降低,蛋白印迹显示Cx43的表达增高受到抑制。结论缝隙连接蛋白是癫痫形成的物质基础。抑制Cx43的过度表达,减少异常缝隙连接的形成,癫痫形成也受到抑制,从而预防癫痫产生。     

Loss of anti-arrhythmic effect of vagal nerve stimulation on ischemia-induced ventricular tachyarrhythmia in aged rats

Reduced vagal activity is associated with increased risk for life-threatening arrhythmia during myocardial ischemia (MI); conversely, the increase in vagal tone may provide protective effect against ventricular arrhythmias. In fact, vagal nerve stimulation (VNS) exerted an anti-arrhythmic effect by preserving connexin 43 (Cx43), a gap junction protein in ventricles, in a rat model of MI. We investigated the effects of VNS on ventricular tachyarrhythmia during acute MI and the expression of Cx43 in aged rats. Both adult (3-4 months) and aged (≥ 24 months) male rats were subjected to ischemia of 30 min. VNS was applied before ischemia either alone or in combination with atropine (0.5 mg/kg) or carbenoxolone, a gap junction inhibitor (10 mg/kg). During the 30-min ischemia, the incidence of ventricular tachycardia (VT) or ventricular fibrillation (VF) was higher in aged rats compared with adult rats. VNS significantly suppressed VT and VF in adult rats and these effects were eliminated by atropine or carbenoxolone. In contrast, VNS did not suppress VT and VF in the aged rats. Moreover, ischemia did not change the expression levels of total Cx43 protein in adult and aged rat ventricles. However, the expression level of total Cx43 protein was two times lower in sham-operated aged rats than that in sham-operated adult rats. Thus, in aged rats, loss of anti-arrhythmic effect of VNS is associated with reduced expression of Cx43 protein. These findings suggest that Cx43 may be an important target for inhibiting ischemia-induced VT in adult patients but not in aged patients.     

Fibroblasts modulate cardiomyocyte excitability: implications for cardiac gene therapy

In an earlier study exploring the potential of gene transfer to repair myocardial conduction defects, we observed that myotubes, generated by forced expression of MyoD, exhibit reduced excitability when also modified to express connexin43 (Cx43). We hypothesized that this effect was caused by gap junction-mediated coupling between myotubes and the underlying fibroblast feeder layer. This intriguing possibility has important implications for ongoing efforts to develop strategies for repairing myocardial conduction defects by gene transfer, and also provides novel insights into the electrophysiological function of naturally occurring heterologous cell coupling within the heart. Although a conductive function for fibroblasts through heterologous coupling has previously been reported, the current study provides novel evidence that fibroblasts can modulate cardiomyocyte excitability in a Cx43-dependent manner. In a co-culture study system, neonatal rat cardiomyocytes were grown on monolayers of mouse fibroblasts with genetically altered Cx43 expression and the effect on intrinsic beat frequency examined. Cardiomyocytes grown on wild-type (WT) fibroblasts expressing native levels of Cx43 beat significantly slower than cells grown on fibroblasts devoid of this molecule (germline knockout) or with dominant-negative functional suppression. Expression of Cx43 in fibroblasts from Cx43 knockout mice restored cardiomyocyte beat frequency, to rates comparable with those observed in co-culture with WT fibroblasts.     

全反式维A酸增强双自杀基因系统对乳腺癌旁观者效应的观察

目的:观察全反式维A酸(ATRA)对腺病毒为载体的双自杀基因系统治疗乳腺癌旁观者效应的增强作用及与Cx43表达的关系.方法:MTT法观察ATRA对双自杀基因系统治疗乳腺癌旁观者效应的影响;RT-PCR和FCM法检测ATRA作用前后乳腺癌MCF-7细胞内连接蛋白Cx43基因和蛋白的表达情况.结果:以不同比例混合MCF-7和MCF-7/CDTK细胞在前药作用下细胞存活率ATRA组较对照组明显降低(P＜0.05).RT-PCR和FCM分析结果表明,经ATRA处理的MCF-7细胞,其Cx43基因和蛋白的表达明显提高.结论:乳腺癌MCF-7细胞中,ATRA具有明显增强CD/TK双自杀基因系统旁观者效应的作用,细胞内Cx43的表达增强可能是旁观者效应增强的机制之一.     

Actions of HSVtk and connexin43 gene delivery on gap junctional communication and drug sensitization in hepatocellular carcinoma.

We have previously demonstrated that transfected hepatocellular carcinoma cells (Hepa1-6) with one copy (pAGO) and two copies (pYED) of the HSVtk gene, using liposomes, induced cell death of untransfected cells in the presence of ganciclovir (GCV). This phenomenon is called the 'bystander effect'. To determine whether an elevated level of connexin43 increases the bystander effect, we have cotransfected Hepa1-6 cells with a plasmid containing the HSVtk gene driven by the alpha-fetoprotein promoter (pFTK) or pAGO or pYED and connexin43. The results showed that, after GCV treatment, the percentage of growth inhibition was higher (25-30%) in cells cotransfected with HSVtk and connexin43 than in cells transfected only with HSVtk gene. The IC50 of GCV on cells transfected with pFTK/Connexin43 was 17.85-fold lower than cells transfected with pFTK alone. To improve these results, stable connexin43 transduced Hepa1-6 cells were transfected with pFTK followed by GCV treatment. In this case, the cell growth was markedly inhibited as compared with parental cells. Furthermore, we have studied the correlation between the expression of the HSVtk and the connexin43 proteins. Using flow cytometric analysis, scrape loading/dye transfer and immunoblotting assay we found that the cells transfected separately by pAGO, pYED, pFTK and pLTR-Cx43 showed an increase of connexin43 protein. This study indicates that transfecting Hepa1-6 cells with both connexin43 and HSVtk genes up-regulates connexin43 expression which enhances the bystander effect and subsequently tumor cell death.