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Cardiac autonomic nerve remodeling (ANR) is an important mechanism of atrial fibrillation (AF). Our previous study illustrated that miR-206 over-expression exacerbates ANR and AF inducibility by regulating SOD1 expression. GTP cyclohydrolase I (GTPCH-1), encoded by GCH1, is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide (NO) synthesis. Previous studies reported that increased BH4 and NO content negatively regulated nerve regeneration. Besides, decreased BH4 level is related to atrial structural and electrical remodeling. This study investigated effects of GCH1 on ANR via BH4 pathway, regulated by microRNA-206 (miR-206).
Whole blood samples from AF patients and the controls were collected. The lenti-miR-206, lenti-anti-miR-206, lenti-GCH1, lenti-anti-GCH1, microRNA inhibitor N.C lentiviruses and the negative control lentiviruses were synthesized. 24 mongrel dogs were allocated into two groups: the control group (control, n = 6) and the atrial-tachypacing group (A-TP, n = 18). The A-TP dogs were subjected to continuous right A-TP (400 beats/min) for 4 weeks and randomly divided into 3 groups: the A-TP group, the A-TP + lenti-miR-206 group and the A-TP + lenti-anti-miR-206 group. Another 24 dogs were randomly divided into four groups: the lenti-miR-206 group, lenti-anti-miR-206 group, GCH1 over-expression group and lenti-anti-GCH1 group. Cells were plated in 24-well dishes and transfected with different lentivirus, including lenti-miR-206, lenti-anti-miR-206, lenti-GCH1, lenti-anti-GCH1, lenti-control and lenti-RNAi-NC. Atrial effective refractory period (AERP) was measured with programmed electric stimulation(PES) and the S1-S2 intervals started at 150ms, followed by decrements of 5 ms (S1: S2 = 8: 1). Anti-protein gene product 9.5 (PGP9.5) antibody were used for immunocytochemical staining. Myocardial BH4 content was measured using a ELISA Kit. NO content was measured using a NOX Detection Kit.
Patients’ plasma was collected and miR-206 expression was up-regulated in AF patients (n=18) than the controls (n=12). In canines, 4 weeks of atrial tachypacing (A-TP), together with miR-206 over-expression, increased PGP9.5(a nerve marker) level. In contrast, a reduction of PGP9.5 level was observed in the lenti-anti-miR-206 canines (P < 0.01). After infection of GCH1 over-expression lentiviruses into right superior fat pad(RSFP) for two weeks, AERP was found increased than that in the control group (105.8±1.537ms vs 99.17±2.007ms, P < 0.05). In contrast, AERP of canines infected with lenti-anti-GCH1 were found decreased compared with the control group (93.33±1.667ms vs 99.17±2.007ms, P < 0.05). GCH1 was validated to be a direct target of miR-206 by luciferase assays. Meanwhile, miR-206 over-expression inhibited atrial GCH1 expression to ∼40% of the controls and further reduced BH4 content to 73.6% of the control canines. Moreover, GCH1 over-expression lentiviruses infection attenuated canines’ atrial PGP9.5 level to ∼56% of the controls. In myocardial cells, transfection of GCH1 over-expression lentiviruses also decreased PGP9.5 expression to 26% of the control group.
Our results demonstrated that GCH1 over-expression increased AERP and attenuated ANR by increasing atrial BH4 and NO content in canine models of A-TP, indicating that GCH1 may inhibit the initiation of AF through attenuating ANR.