| Backgrounds and objections:Sick sinus syndrome(SSS) is a frequent clinical syndrome, which have a great healthhazards to human. The pathological mechanisms of SSS such as degeneration,cardiomyopathy, inflammation of myocardium, and damage of operation is not very cleartill now and there are not very effective prevent and treatment methods with it. Althoughimplantation of permanent artificial electric cardiac pacemaker is still the current maintreatment, that is not good enough. Among their shortcomings are limited battery life, theneed for examination regularly, high cost, possible complications of permanent catheterimplantation into the heart such as electrode breakage, severe infection. So cardiacelectrophysiologists or knowledgeable physicians focus on how to build a biologicalpacemaker with the normal physiological function as the substitute for the electronic heartpacemaker. Some researchers has used the gene therapy and cell transplantation to build aexperimental biological pacemaking platform for bradyarrhythmia Some approaches havebeen attempted to establish biological pacemaker by transplanting gene modifiedmesenchymal stem cells (MSCs) or MSCs alone, embryonic stem cells induced in vitro,andcardiac myocyte into animal hearts. These approaches are not optimal enough because ofethical problems, immature technology of stem csll inducing differentiation, the low pacingrate, the uncertain and short duration of pacing function, the low heart rate variability, poorsource of cardiac myocyte, and so on.Hyperpolarization activated cyclic nucleotide gated cation channel (HCN) is themolecular basis of funny current (If). HCN4as1/4members in the family of Ifchannelsmaybe a optimal biological pacemaking target gene because it is essential for modulation ofIf. Recently, Ifwas shown to play an important role in the spontaneous pacing activity ofcardiomyocytes and neurons. HCN genes are becoming important candidate genes in investigations using biological pacemakers to treat bradyarrhythmia, which were based ongene therapy and cell transplantation techniques. In cloned4HCN gene subtypes, HCN4ismainly expressed in the cardiac specific conduction system. Previously,3atrioventricularblock models by radiofrequency catheter were established on a large animal model likedogs or pigs, and HCN2was the target gene. In this study, we established a rabbitbradyarrhythmia model based on chemical ablation of the sinoatrial node and sequentialbilateral vagus nerve stimulation, and evaluated the integration and pacing function aftermHCN4-modified rabbit BMSC were transplanted into left ventricle epicardium. This studyprovides new insights and methods to explore biological pacing therapy.Methods1. BMSCs were separated and purified with Percoll separating medium by densitygradient centrifugation and adherence method. Bone marrow was obtained from2month-old Japanese white rabbits via intertrochanteric aspiration under aseptic conditionsand washed twice by Percoll density gradient centrifugation. The monocytes in middlewhite cloudy layer were harvested and washed twice with DMEM at1,000rpm for5minutes. Cells were cultured and propagated in DMEM containing10%fetal bovine serumat a concentration of10×108/ml. Rabbit MSCs (p=4) were plated into a24-well plate at2×104/well, a supernatant containing pMSCV-mHCN4-EGFP virus was mixed withmedium at1:1and added into rabbit MSCs together with polybrene at a final concentrationof2μg/ml. Twenty-four hours after transduction puromycin at2μg/ml was added and cellswere selected for10~15days to get transduced rabbit MSCs. Immunofluorescence wasused to determine the gene expression level in the control group, EGFP group, and mHCN4group.The electronic characters of If channels were detected by whole cell patch clamp.2. Establishing a bradyarrhythmia rabbit model by sinus node chemical ablation andbilateral vagus nerve stimulation. Methods of chemical ablation: after drying the junction ofsuperior vena cava and right atrial appendage (SAN area), placed the5mm×5mm×5mmcotton with20%formaldehyde at SAN. Stopped the chemical stimulation if HR decreasedby30%, or sinus arrest occurred, or junctional escape occurred. ECGs are still record for2h. If ECG appeared repeatedly, then use the chemical stimulation at SAN again until HRdecreases by30%, or sinus arrest occurres,or junctional escape occurres. Methods of vagusnerve stimulation: incremental classification method was used to perform the vagus nerve stimulation with electrical rectangular pulses in4~6V stimulating voltage at2.5Hz,5Hz,10Hz,15Hz,20Hz stimulus frequency (1Hz=60beats/min), respectively. Thestimulation was performed with pulses at10Hz at first. And the relative indexes, includingHR, time for reaching the the stimulus endpoint, recovery time and so on, were recorded ifthe rabbit arrived at the stimulus endpoint in60s. If rabbit doesn’t arrive in60s, then thenext stimulation with180s intervals was performed until it reached the stimulus endpoint.180s later after reaching the stimulus endpoint, the rabbit received the stimulation for120sagain.3. At3days,1week,2weeks and4weeks after transplantation, chemical ablation ofthe sinoatrial node was performed and bilateral vagus nerves were sequentially stimulatedto observe premature left ventricular contraction or left ventricular rhythm. Cellmorphology and gap junction were measured by HE and DAB staining, respectively. EGFPand mHCN4expression levels were determined by immunofluorescence..Results1. Rabbit MSCs were successfully transfected with pMSCV-mHCN4-EGFP, whichcan stably expressing mHCN4gene after selected by puromysin with a concentration of2μg/mL.2. Hyperpolarization activated inward current which was time and voltage dependentand sensitive to extrocelluar Cs+could be detected in rabbit MSCs transduced withmHCN4. The average current density was-42.8±3.6pA/pF under a reference voltage of-140mV. Apparent tail current could be detected when reference voltage was+20mV.Meanwhile, this hyperpolarization activated inward current could not be detected in EGFPcontrol group under the same conditions.3. Basic heart rate significantly dropped down in those rabbits after chemical ablationwith20%formaldehyde (307±21beats/min vs126±28beats/min, p <0.01). Heart rate ofeach rabbit slowed down with the right vagus nerve stimulation at different pulsefrequencies. Sinus arrest and junctional escape besides heart rate slowed down occurredwith the increasing-frequency pulse stimulation with the left vagus nerve. Sinus arrest andventricular escape beat occurred with the bilateral vagus nerve stimulation at10Hz or more.Some cases were that3-degree atrioventricular block. It spent less time in reaching the thestimulus endpoint with the increase of pulse frequency for bilateral vagus nerve stimulation. The sinus rhythm of all rabbits restored to normal as before stimulation. But recovery timewas needed with the increase of pulse frequency.4. No significant difference in ventricular escape rhythm was observed after chemicalablation of the sinoatrial node and sequential bilateral vagus nerve stimulation among thethree groups at3days after transplantation. At1week after transplantation, only one rabbitin the mHCN4group had a ventricular rhythm higher than the control group or EGFP group.However, the mHCN4group had significantly higher ventricular rhythm and shorter QRSduration than the control or EGFP group, respectively.5. Morphology of the transplanted cells dynamically changed as observed by HEstaining over time: At3days after transplantation, transplanted cells were round andclustered and had well-defined boundaries separating them from the adjacent normalventricle myocytes. At1week after transplantation, the cells decreased significantly, andshort spindle cells could be observed in the region adjacent to the normal ventriclemyocytes. At2weeks after transplantation, the transplanted cells displayed gradualtransition to the adjacent ventricle myocytes and spindle cells increased. At4weeks aftertransplantation, the surviving transplanted cells were in long-spindle morphology and couldnot be clearly discriminated from adjacent ventricle myocytes. In the control group, mildinflammation was observed in the transplanted region as determined by HE staining aftermedium injection, and no dynamic changes were observed. Cx43and Cx45could bedetected between transplanted cells and host cells by DAB staining, which were brownlinear granulates.6. EGFP and mHCN4could be detected in the transplanted cells at3days aftertransplantation. The cells were round, not spread, and were negative for Cx43and Cx45.One week after transplantation, surviving transplanted cells decreased, a few were short andspindle-like, and positive for EGFP, mHCN4, Cx43and Cx45. Two weeks aftertransplantation, transplanted cells were long and spindle-like, with morphology similar tothe adjacent ventricle myocytes. Cx43and Cx45could be detected between transplantedcells and host cells. Similar results could be observed4weeks after transplantation.Conclusions1. mHCN4gene can be successfully transfected into rabbit MSCs, and can stablyexpress after selected by puromysin. 2. Ifcan be detected from rabbit MSCs transfeced with mHCN4gene which have theelectrophysiological ability of cardiac pacemaker cells.3. A bradyarrhythmia model can be successfully established by chemical ablation ofthe sinoatrial node and sequential bilateral vagus nerve stimulation. This model can be usedto study biological pacing in vivo.4. The mHCN4-modified rabbit MSCs displayed evident dynamic morphologychanges after being transplanted into rabbit left ventricle free wall epicardium. Cx43andCx45could be detected between transplanted cells and host cells, which were brown lineargranulates.5. The MSCs showed pacing function approximately1week after transplantation.The mHCN4-transduced MSC group had a significantly higher ventricular rate and a shorter QRS duration than that of the control and EGFP group at2and4weeks aftertransplantation. The pacing cells worked well at2weeks and persisted stably4weeks aftertransplantation. |
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