Experimental study of cardiac biological pacemakers constructed by human hyperpolarization-activated cyclic nucleotide-gated cation 4 channel gene recombinant adenovirus

Abstract

Objective To detect the expression of myocardial cell marker and pacing activities-related human hyperpolarization-activated cyclic nucleotide-gated cation 4 (hHCN4),and conduct cell electrophysiology study.Methods Twenty New Zealand white adult rabbits were divided into control group (10) and experimental group(10).Attentive adventitial injection of adenovirus was injected to the left ventricular apex in rabbits of control group and hHCN4 recombinant adenovirus was injected to rabbits in experimental group respectively.Surface ECG of two groups was traced daily during 3 days postoperation.After 7 days,neck bilateral vagus nerves of rabbits were seperated and stimulated with bilateral or unilateral electrical stimulation.Pacing was established in apical area by mapping electrode (stimulation pulse width was 2-4 ms,stimulating voltage was 5-8 V,stimulation frequency was 20-30/s),and synchronization limb 6-lead ECG was recorded.hHCN4 expression was detected in tissues of heart of rabbits from both groups and tissues of liver,kidney and lung of rabbits from experimental group by immunofluorescence method.If and LCa associated with action potential pacemaker ion flow of single apical myocardial cell was recorded by patch clamp.Results There were no records of ECG ventricular arrhythmia events during postoperative 3 days.When 7 days after electrical stimulation on vagus nerve,frequency of ventricular verapamil escape beats rhythm of the experimental group was significantly faster than that control group [(60±8)/min vs (42±6)/min,P＜0.05].Ventricular verapamil escape beats rhythm originated in the vicinity of the injection site.Overexpression of hHCN4 protein was detected in heart tissue of experimental group rabbits by immunofluorescence assay,while there were no detection of this protein in tissues of liver,kidney and lung in rabbits from experimental group.It was also confirmed that If and LCa existed in action potential pacemaker ion flow of single apical myocardial cell by patch clamp.Conclusions Transduction of hHCN4 into myocardial can lead to overexpression of channel protein in local area and increasement of the frequency of ventricular ectopic pacemaker. Key words: Cyclic nucleotide-gated cation channels ; DNA, recombinant; Adenoviridae; Pacemakers, biological; Bradycardia