Background Mutations in the lamin A/C gene （LMNA） may cause familial dilated cardiomyopathy （dilated cardiomyopathy） characterized by early onset atrio-ventricular block （A-V block） before the manifestation of d...Background Mutations in the lamin A/C gene （LMNA） may cause familial dilated cardiomyopathy （dilated cardiomyopathy） characterized by early onset atrio-ventricular block （A-V block） before the manifestation of dilated cardiomyopathy and high risk of sudden death due to ventricular arrhythmia, which is very similar to the phenotype of gap junction related heart disease. This study aimed to determine the expression and localization of connexins in neonatal myocytes transfected with wild-type （WT） or mutant LMNA to elucidate how these mutations cause heart diseases. Methods We studied the connexin 43 （Cx43） and connexin 40 （Cx40） expression in cultured neonatal myocytes transfected with wild-type （WT） or mutant LMNA （Glu82Lys （E82K） and Arg644Cys （R644C）） using confocal imaging and Western blotting analysis. Results Cx43 protein expression was reduced by 40% in cells transfected with LMNA E82K than that in cells transfected with WT LMNA cDNA. Confocal imaging showed that the Cx43 located inside the cells by LMNA E82K. By contrast, LMNA E82K mutation had no effect on expression and localization of Cx40. LMNA R644C transfection did not show any significant effects on gap junctions at all. Conclusions Our findings suggest that LMNA E82K significantly reduced the Cx43 expression and altered its localization which may be one of the pathological mechanisms underlying LMNA-related heart disease.展开更多
目的:研究温胆汤对精神分裂症模型大鼠行为学及其海马神经元突触后致密蛋白95(PSD-95)、生长相关蛋白43(GAP-43)、JNK相互作用蛋白3(JIP-3)、连接蛋白43(Cx-43)表达的影响。方法:将SD大鼠随机分为:正常对照组、模型对照组、氯氮平0.02 g...目的:研究温胆汤对精神分裂症模型大鼠行为学及其海马神经元突触后致密蛋白95(PSD-95)、生长相关蛋白43(GAP-43)、JNK相互作用蛋白3(JIP-3)、连接蛋白43(Cx-43)表达的影响。方法:将SD大鼠随机分为:正常对照组、模型对照组、氯氮平0.02 g/kg组、温胆汤10、20、40 g/kg组。正常对照组和模型对照组分别灌胃生理盐水,各组分别灌胃相应药物,1次/d,连续给药21 d。末次给药2 h后,除正常对照组外,其余各组一次性腹腔注射MK-8010.6 mg/kg,以诱发精神分裂症模型。造模后,观察记录各组大鼠的行为学改变和刻板行为评分,旷场实验评价大鼠滞留时间及穿越次数;RT-q PCR法检测大鼠海马组织Psd95、Gap43、Jip3、Cx43 m RNA表达;WB法检测大鼠海马组织PSD-95、GAP-43、JIP-3、Cx-43蛋白表达。结果:与正常对照组比较,模型对照组大鼠刻板行为评分显著升高,在中央区滞留时间及穿越次数显著下降(P<0.01);其海马组织Psd95、Gap43、Jip3、Cx43 m RNA和蛋白表达显著下调(P<0.01);与模型对照组比较,温胆汤10、20、40 g/kg组均能明显降低大鼠刻板行为评分,明显升高中央区滞留时间及穿越频次(P<0.05或P<0.01);能明显上调海马组织Psd95、Gap43、Jip3、Cx43 m RNA和蛋白的表达(P<0.05或P<0.01)。结论:温胆汤可能通过上调PSD-95、GAP-43、JIP-3、Cx-43等突触相关蛋白的表达,从而调控海马神经元突触可塑性,缓解刻板行为和紧张、焦虑等情绪,增强学习和记忆功能,有效改善精神分裂症的认知障碍。展开更多
Connexin 43 （Cx43） is the major structural protein of gap junctions in the ventricular myocardium and a major determinant of myocardial electrical properties. Cx43 expression is decreased in the wild type mice after...Connexin 43 （Cx43） is the major structural protein of gap junctions in the ventricular myocardium and a major determinant of myocardial electrical properties. Cx43 expression is decreased in the wild type mice after myocardial infarction and this effect is attenuated in MMP-7-/- mice. Matrix metalloproteinase expression is regulated at the transcription level by the modulation of the activation of transcription factors such as activator protein （AP）-I and nuclear factor kappa-light-chain enhancer of activated B cells （NF-KB）. Methods Rat myocardial cells （H9c2） were cultured and maintained at 37 ~C and 5% CO2. H9c2 cells in 6-well plates were treated with lipopolysaccharides （LPS）, NF-kB inhibitor （JSH 23, 30 μ, Santa Cruz） ＋ LPS and c-Jun N-terminal kinase （JNK） inhibitor （SP600125, 10μM, Sigma） ＋ LPS for 6, 12 and 24 h. Apoptosis rate of cells was determined by flow cytometry. Cx43 expression was assessed by Western blotting. Results LPS induced a time-dependent apoptosis in all cell line. We have found that the treatment with LPS induced increase of apoptosis in H9c2 cells at 6 h, 12 h and 24 h, but the effect was decreased by the addition of JSH-23 and SP600125 to LPS respectively （P 〈 0.05） . LPS resulted in decreased expression of Cx43 expression at 6 h, 12 h and 24 h. However, JSH-23 and SP600125 attenuated the loss of Cx43 respectively （P 〈 0.05）. Conclusion Transcription factors NF-kB and JNK/AP-1 signaling pathway participates in the regulation of LPS-induced Cx43 expression in the H9c2 cells, and maybe play an important role in regulation of Cx43 expression.展开更多
文摘Background Mutations in the lamin A/C gene （LMNA） may cause familial dilated cardiomyopathy （dilated cardiomyopathy） characterized by early onset atrio-ventricular block （A-V block） before the manifestation of dilated cardiomyopathy and high risk of sudden death due to ventricular arrhythmia, which is very similar to the phenotype of gap junction related heart disease. This study aimed to determine the expression and localization of connexins in neonatal myocytes transfected with wild-type （WT） or mutant LMNA to elucidate how these mutations cause heart diseases. Methods We studied the connexin 43 （Cx43） and connexin 40 （Cx40） expression in cultured neonatal myocytes transfected with wild-type （WT） or mutant LMNA （Glu82Lys （E82K） and Arg644Cys （R644C）） using confocal imaging and Western blotting analysis. Results Cx43 protein expression was reduced by 40% in cells transfected with LMNA E82K than that in cells transfected with WT LMNA cDNA. Confocal imaging showed that the Cx43 located inside the cells by LMNA E82K. By contrast, LMNA E82K mutation had no effect on expression and localization of Cx40. LMNA R644C transfection did not show any significant effects on gap junctions at all. Conclusions Our findings suggest that LMNA E82K significantly reduced the Cx43 expression and altered its localization which may be one of the pathological mechanisms underlying LMNA-related heart disease.
文摘目的:研究温胆汤对精神分裂症模型大鼠行为学及其海马神经元突触后致密蛋白95(PSD-95)、生长相关蛋白43(GAP-43)、JNK相互作用蛋白3(JIP-3)、连接蛋白43(Cx-43)表达的影响。方法:将SD大鼠随机分为:正常对照组、模型对照组、氯氮平0.02 g/kg组、温胆汤10、20、40 g/kg组。正常对照组和模型对照组分别灌胃生理盐水,各组分别灌胃相应药物,1次/d,连续给药21 d。末次给药2 h后,除正常对照组外,其余各组一次性腹腔注射MK-8010.6 mg/kg,以诱发精神分裂症模型。造模后,观察记录各组大鼠的行为学改变和刻板行为评分,旷场实验评价大鼠滞留时间及穿越次数;RT-q PCR法检测大鼠海马组织Psd95、Gap43、Jip3、Cx43 m RNA表达;WB法检测大鼠海马组织PSD-95、GAP-43、JIP-3、Cx-43蛋白表达。结果:与正常对照组比较,模型对照组大鼠刻板行为评分显著升高,在中央区滞留时间及穿越次数显著下降(P<0.01);其海马组织Psd95、Gap43、Jip3、Cx43 m RNA和蛋白表达显著下调(P<0.01);与模型对照组比较,温胆汤10、20、40 g/kg组均能明显降低大鼠刻板行为评分,明显升高中央区滞留时间及穿越频次(P<0.05或P<0.01);能明显上调海马组织Psd95、Gap43、Jip3、Cx43 m RNA和蛋白的表达(P<0.05或P<0.01)。结论:温胆汤可能通过上调PSD-95、GAP-43、JIP-3、Cx-43等突触相关蛋白的表达,从而调控海马神经元突触可塑性,缓解刻板行为和紧张、焦虑等情绪,增强学习和记忆功能,有效改善精神分裂症的认知障碍。
基金supported by the National Nature Science Foundation of China (81160152)
文摘Connexin 43 （Cx43） is the major structural protein of gap junctions in the ventricular myocardium and a major determinant of myocardial electrical properties. Cx43 expression is decreased in the wild type mice after myocardial infarction and this effect is attenuated in MMP-7-/- mice. Matrix metalloproteinase expression is regulated at the transcription level by the modulation of the activation of transcription factors such as activator protein （AP）-I and nuclear factor kappa-light-chain enhancer of activated B cells （NF-KB）. Methods Rat myocardial cells （H9c2） were cultured and maintained at 37 ~C and 5% CO2. H9c2 cells in 6-well plates were treated with lipopolysaccharides （LPS）, NF-kB inhibitor （JSH 23, 30 μ, Santa Cruz） ＋ LPS and c-Jun N-terminal kinase （JNK） inhibitor （SP600125, 10μM, Sigma） ＋ LPS for 6, 12 and 24 h. Apoptosis rate of cells was determined by flow cytometry. Cx43 expression was assessed by Western blotting. Results LPS induced a time-dependent apoptosis in all cell line. We have found that the treatment with LPS induced increase of apoptosis in H9c2 cells at 6 h, 12 h and 24 h, but the effect was decreased by the addition of JSH-23 and SP600125 to LPS respectively （P 〈 0.05） . LPS resulted in decreased expression of Cx43 expression at 6 h, 12 h and 24 h. However, JSH-23 and SP600125 attenuated the loss of Cx43 respectively （P 〈 0.05）. Conclusion Transcription factors NF-kB and JNK/AP-1 signaling pathway participates in the regulation of LPS-induced Cx43 expression in the H9c2 cells, and maybe play an important role in regulation of Cx43 expression.
