Role Of RhoA/ROCK Pathway On Atrial Connexin 43 Regulation In Intermittent Hypoxia

Objective:Obstructive sleep apnea syndrome(OSAS),which is a major risk factor for developing atrial fibrillation(AF),is characterized by intermittent hypoxia(IH).RhoA/ROCK pathway is upregulated by IH.However,the role of RhoA/ROCK pathway on atrial connexin 43(Cx43)regulation in IH is still unknown.We isolated and cultured neonatal rat atrial myocytes,and mimicked the impact of IH on its physiopathologic change.Morphological characteristics,cell viability,hypoxia-inducible factor(HIF-1α),and cleaved caspase-3 protein level of neonatal rat atrial myocytes under different IH stimulations were detected to evaluate the effectiveness of this model.Molecular biological technology measured protein expression of RhoA,ROCK,protein expression,and mRNA level of Cx43 under separate conditions.Y-27632 was used to inhibit the activity of ROCK,and to find the role of RhoA/ROCK pathway on atrial Cx43 regulation in IH.This finding would implicate RhoA/ROCK pathway as an important mediator of Cx43 remodeling and provide novel insights into the underlying mechanisms.Methods:1.Different stimulated patterns(normoxia,IH 2h,IH 4h,and IH 6h)were used to neonatal rat atrial myocytes.MTS and western blot measured the cell viability and protein expression of cleaved caspase-3,HIF-la respectively to evaluate its effectiveness.2.The protein expression and mRNA level of Cx43 were measured respectively under different IH stimulations.3.Western blot measured the protein expressions of RhoA and ROCK under different conditions.The protein levels of ROCK-1,MYPT,and p-MYPT in different groups were measured after using Y-27632 to inhibit ROCK.The protein and mRNA levels of Cx43 were measured under IH stimulation.Results:1.Atrial cell viability showed a gradual decrease(p<0.05)and the protein expression of HIF-1α increased(p<0.001)as stimulation time went on.The protein level of cleaved caspase-3 raised to its peak at IH 4h(p<0.05)stimulation and dropped gradually.2.The protein and mRNA expression of Cx43 experienced a downward trend and were in time-dependent.3.The protein expressions of RhoA and ROCK increased with the prolongation of IH stimulation duration.Considering the cell viability and other protein expressions’level,4h IH was chosen as the stimulation time after adding Y-27632.And neonatal rat atrial myocytes were divided into 4 groups(normoxia,normoxia+Y-27632,IH 4h,IH 4h+Y-27632).The protein levels of ROCK-1 and p-MYPT/MYPT in the normoxia+Y-27632 group showed no difference compared with the normoxia group.The protein levels of ROCK-1 and p-MYPT/MYPT in IH 4h increased significantly compared with the normoxia group and using Y-27632 blocked this change(p<0.05).Y-27632 mildly raised the protein expression of Cx43 in the IH 4h+Y-27632 group,but it did not reach statistical significance.The mRNA level of Cx43 dropped in the 4h IH group,using Y-27632 reversed this effect in the IH 4h+Y-27632 group(p<0.001)and even exceed its level in normoxia.Conclusions:1.5%O2 30min-20.9%O2 30min as a cycle mimics the physiopathologic change of neonatal rat atrial myocytes in OSAS successfully.2.The decreased protein and mRNA expression of Cx43 under IH stimulation possibly attribute to a mechanism of AF.3.RhoA/ROCK pathway is activated and regulated the mRNA level of Cx43 under IH stimulation.But it is not the only upstream factor to modulate the Cx43 remodeling under this condition.