A Pathway That Contributes To Connexin43 Modulation And A Therapeutic Target In Electrophysiological Diseases

Objectives:To investigate the regulative effects of autophagy on Connexin43 (Cx43) expression in primary culture of neonatal rat ventricular cardiomyocytes. And to establish two electrophysiological diseases in vitro cell model:1, a Human Herpes Simplex Virus-1 (HSV-1) viral myocarditis in vitro model; 2, a short-term ventricular tachycardia in vitro model, then to observe the dynamic Cx43 impairment in the two models, and to confirm the therapeutic effects of autophagy on Cx43 impairment. In addition that, to certify that autophagy could apply as a therapeutic target in treating electrophysiological diseases.Methods:1. Isolation of Neonatal Rat Ventricular Myocytes (NRVM): Primary culture of NRVM were isolated from 1-3 days old SD (Sprague Dawley) neonatal rat, using repeated pancreatin/collagenase digestion, and followed by discontinuous Percoll gradients in order to separate from non-myocytes. Identified by microscopic observation of contractile characteristics, the NRVM were more than 95% pure.2. Establishment of two electrophysiological (EP) disease in vitro models:(1). Establishment of HSV-1 viral myocarditis in vitro model: Wild-type HSV-1 (McKrae strain) was propagated in Vero cells and then infected NRVM with a virus titer of 1.0PFU/cell.24h post infection, the CPE (cytopathic effects, CPE) could be observed under microscope, and Western Blot (WB) analysis was applied to detect the HSV-1 glycoprotein D (gD) expression in NRVM.(2). Establishment of short-term ventricular tachycardia in vitro model: Cultured in 6-well plate for 5 days, NRVM were placed in electrical field for up to 24h continual stimulation. The stimulation frequency was kept as 5Hz with output voltage 5.0V/cm, resulting a much faster contraction rate manifestation in NRVM (nearly 300 contractions per min), similar to tachycardia or fibrillation.3. Autophagy activation/inhibition via different drugs: Rapamycin was applied as an autophagy inducer and activator, and 3-methyladenine (3 MA) was applied as an autophagy inhibitor. The two drugs were used to activate/inhibit the autophagy flux in NRVM in the two EP disease models, thus to investigate the expression and function alteration of Cx43. WB analysis was applied to detect the expression of autophagy related proteins, such as LC31/2, Atg5 and Beclin-1, and WB was also used to display the dynamic expression of Cx43. Immunofluorescence (IF) analysis was used to manifest the Cx43 expression and distribution. Lucifer Yellow scratch test was to assess the signaling transduction function of Cx43. And real-time PCR was applied to evaluate the mRNA expression level of Cx43, L-type calcium channel and HSV-1 gD in NRVM.4. Preliminary investigation on the autophagy upstream signaling pathways:In the two EP disease models, when autophagy activity changed, WB analysis was applied to assess the key molecules in autophagy upstream signaling pathways, such as p-Akt and p-S6K in Akt/mTOR signaling pathway, thus to partly explain the underlying mechanisms involved in the autophagy.Results:1. The neonatal rat ventricular cardiomyocytes were successfully isolated and cultured, with consistently spontaneous beating under the microscope. And the NRVM were identified by microscopic observation of contractile characteristics and a-actin expression by IF.2. In the NRVM, it was proved by WB analysis that autophagy could contribute to Cx43 modulation. Increased autophagy flux might improve Cx43 degradation thus decrease the Cx43 expression, in the opposite that inhibited autophagy might made Cx43 protein accumulation.3. WB results of HSV-1 gD expression and CPE observation under microscope, they both provided convincing evidences that HSV-1 could effectively infect NRVM and replicated actively inside the cells, thus the establishment of HSV-1 viral myocarditis in vitro model was proved to be successful. And Cx43 expression displayed by WB and IF also indicated that HSV-1 infection could decrease the Cx43 expression.4. In the rapid electrical stimulated NRVM, Cx43 displayed a dynamic expression while the expression of L-type calcium channel was decreased at 24h post RES, which indicated the successful establishment of short-term ventricular tachycardia.5. Lucifer Yellow scratch tests showed convincing evidences that rapamycin could reduce the HSV-1 replication in target cells, thus improve the signal transduction function of Cx43, however 3MA could make dysfunctional Cx43 protein accumulation and have no effect on virus replication.6. WB analysis of p-Akt and p-S6K expression suggested that all the drugs above, like rapamycin,3MA and captopril, regulated the intracellular autophagy flux activity via modulation of upstream signaling pathways (Akt/mTOR signaling pathway), and thus regulated the Cx43 expression.Conclusions:1. In the NRVM, autophagy flux contributes to Cx43 degradation, thus modulate Cx43 expression. Improved autophagy flux might decrease Cx43 expression while inhibited autophagy might cause Cx43 protein accumulation.2. In the HSV-1 viral myocarditis in vitro model, increased autophagy flux could inhibit virus replication, thus reverse the HSV-1 induced Cx43 impairment, however, autophagy inhibition might cause dysfunctional Cx43 protein accumulation.3. In the short-term ventricular tachycardia in vitro model, rapid electrical field stimulation could modulate the dynamic expression of Cx43 via autophagy regulation.4. All the factors referred above regulate autophagy activity via regulation of upstream signaling pathways, such as Akt/mTOR signaling pathway.