The Study Of Biological Pacemaker Using Percutaneous Endomyocardial Inject Catheter As In Vivo Delivery Approach

Objective:Bradyarrhythmia is a common arrhythmia type which severely affected the patient's health. The invitation of cardiac implanted electronic devices in1950s is a marvel, and advances in this field have been impressive. However, it's a relive therapy rather than cure, and important shortcomings exist. With the further development in cardiac pacing mechanism, the concept of biological pacemaker evolved, and the active investigation in the field of biological pacing is rapidly progressed. However, the in vivo animal studies of biological pacemaker are mainly performed through open thoracotomy, which cause large injury and had poor lacation controlling. Therefore, exploring optimal implantation approaches is very important for further clinic trails. This study used bone-marrow mesenchymal stem cells (BM-MSCs) as transfection target cells, and construct lentiviral expressing vectors carrying mouse Hyperpolarization-activated Cyclic Nucleotide-gated channels2(HCN2) gene. The in vitro pacing function of the transfected BM-MSCs was investigated. A self-designed percutaneous inject catheter was used to deliver the biological pacemaker into the left ventricular of canine, its feasibility and safety was explored. Meanwhile, the in vivo efficiency of the biological pacemaker was tested.Methods:Canine BM-MSCs was isolated by density gradient centrifugation and cultured. mHCN2gene fragment was amplified through PCR, then was connected with lentiviral vector. T293cells were con-transfected with lentiviral vector and packaging systems. The titers of lentiviral vectors were tested by real-time PCR. Gradient MOI of5,10,15,20,25,30was performed to determine the optimal MOI for the transfection of canine BM-MSCs. Set experiment group (Lenti-GFP-HCN2transfection), negative control group (Lenti-GFP transfection) and blank control group (with lentivirus transfection). Whole cell patch clamp tested the pacing current and western blot tested the HCN2protein expression of BM-MSCs in each group. NRCs were isolated and cultured, BM-MSCs was co-cultured with NRCs, the contraction frequency was observed and recorded. Catheter was inserted via carotid artery and femoral vein into left and right ventricle separately, diluted gentian violet was muitidot injected into the ventricle to testify the manipulation properties of the inject catheter.16health adult mongrel canine (20-25kg) were randomly divided into experiment group (0.6ml Lenti-GFP-HCN2 transfected MSCs was administrated, n=8), negative control group (0.6ml Lenti-GFP transfected MSCs was administrated, n=4), and blank control group (0.6ml saline was administrated, n=4). Inject catheter was inserted via carotid artery into left ventricle, septum inject was performed under the instruction of X-ray. Telemetering ECG recorder was implanted underneath the skin for heart rhythm monitoring.2weeks later, bilateral vague nerve stimulation was conducted (S1S1100ms/60ms, voltage3-5V, duration1min, interval10min). The escaping rhythm was recorded, its origin and frequency was analyzed. Heart tissue was obtained when end point was reached, frozen sections were sliced for GFP fluorescence, immunoflourescens stain and HE stain.Results:DNA sequencing demonstrated that mHCN2recombinated lentiviral vectors were constructed successfully. Western blot test confirmed that there was mHCN2protein expression in the T293cells, which was transfected with the mHCN2recombinated lentiviral vectors. The optimal lentivirus transfection MOI for canine BM-MSCs was20. Patch clamp showed the production of hyperpolarization-activated inward currents in experiment group while not in negative or blank control group. There was a high HCN2protein expression in experiment group other than negative and blank control groups.3days after co-culture, NRCs in experiment group revealed a significantly higher contraction frequency (P<0.05). The injection efficiency was calculated as91.6%, there was no severe complication associated with endomyocardial injection operation. Telemetering ECG recorder revealed premature ventricular contraction and ventricular tachycardia, and no significant differences were observed between three groups. Bilateral vague nerve stimulation showed that in experiment group, the escape rhythm mainly originated from left ventricle with an average frequency of50.8±3.2bpm, while the escape rhythm of negative and blank control groups from right ventricle, average frequency was42.2±6.1bpm (P<0.05). Histopathological examination proved that the injected BM-MSCs survived in vivo, there were Cx43connected between cells, HE stain and CD68immunoflourescens stain indicated no marked inflammation.Conclusions:In vitro transfection of canine BM-MSCs with Lenti-GFP-HCN2produced pacing current and expressed HCN2protein, indicating a potential of pacing function. The self-designed percutaneous endomyocardial inject catheter was proved to have highly-qualified efficiency and safety, it provided a better delivery for biological pacemaker study. Lenti-GFP-HCN2transfected canine BM-MSCs can be successfully delivered by this catheter, cells survived and reconstructed an ectopic pacing.