| Epidemiological studies have shown a strong correlation between tumor and AF.However,the molecular link between tumor and AF remains unexplored.ECRG4,a newly-discovered tumor suppressor gene that is also highly expressed in the A-V node and in sporadic ventricular myocytes,inhibits tumorigenesis and monitors tissue homeostasis by functioning as a ‘sentinel' molecule gauging inflammatory and cell proliferative responses.To explore the potential physiological function of Ecrg4 in heart,we evaluated its distribution in heart,analyzed its expression in patients with persistent AF and in a canine AF model,and dissected the molecular events downstream of ecrg4 using loss-of-function experiments.Methods: All animal studies are conducted in accordance with the "Guidelines for the Use of Experimental Animals in the People's Republic of China".All research protocols were approved by the Experimental Animal Ethics Committee of Southwest Medical University.Experimental methods together with a concised description of of each method are listed below:(1)Hearts of SD rats were removed under anesthesia,and the sinus node,atrioventricular node,left and right atria,and left and right ventricules were dissected under microscopy,which were then fixed with parformaldehyde and proceeded for microtomy.The slides were processed for IHC with anti-Ecrg4 primary antibody following the vendor's instructions.The Ecrg4 positive brown immune-staining was observed under microscopy,and compared among the sub-anatomical areas.(2)Neonatal rat cardiomyocytes from atria and ventricules were isolated and cultured separately as described in materials and methods,and the expression of Ecrg4 was evaluated by immunofluorence using anti-Ecrg4 primary antibody,followed by a secondary antibody conjugated with FITC.The expression and subcellular localization of Ecrg4 were observed under microscopy.(3)Canine AF model was established in the laboratory by rapid atria pacing.The dogs were euthanized,hearts removed,and different parts of the heart were dissected.Total RNA was extractred,reverse-transcribed,and real-time PCR was employed to evaluate the expression of ECRG4 in atria,which were then compared between the model and control dogs.(4)The atrial appendage of patients with sinus rhythm and persistent AF were obtained,fixed,and proceeded to microtomy.The expression of Ecrg4 was evaluated by immunohistochemistry and compared between the two groups.(5)The expression of ECRG4 was knocked down by si RNA technology in neonatal rat cardiomyocytes,and action potential was recorded,analyzed,and compared between the knockdown and control cardiomyocytes.(6)For gene expression analysis AFter ECRG4 knockdown,total RNA was extracted,reverse transcribed,and reverse transcription-PCR and real-time PCR were performed to evaluate the expression of ECRG4,and proinflammatory and cardiomyocyte remodeling associated genes.Results: The experimental results were analyzed with SPSS19.0.The experimental data are presented as "mean ± SD" and the differences between groups were analyzed by one-way ANOVA with P <0.05 indicating statistical significance.All experiments were repeated at least three times with at least 3 parallel samples per group.The results showed that the level of Ecrg4 expression is homogenously high in atria and the conduction systems and sporadically expressed in ventricular myocytes.Importantly,the expression of Ecrg4 was significantly decreased in atrial appendages of AF patients than patients with SR.Moreover,in rapid pacing canine AF models,the expression of ECRG4 in atria was significantly decreased compared to that of the controls.Mechanistically,knockdown ECRG4 in atrial myocytes significantly shortened the action potential durations,inhibited the expression of Gap junction alpha-1 protein,and activated pro-inflammatory cascades and genes involved in cardiac remodeling.Conclusion:These results suggest that Ecrg4 play a critical role in the pathogenesis of AF. |
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