| Objective:To study the mechanism underlying the alteration of SK2 channel function by transgenetic manipulation of RyR2 in treating elderly dogs with chronic atrial fibrillation.Methods:1. Experimental animals were divided into groups. Minimally invasive thoracotomy without pacemaker implantation was performed on the animals in the sham operation group (n=7) (Group A), whereas minimally invasive thoracotomy with implantation of a pacemaker with 8W pacing in the left atrial appendage was performed on those in the atrial fibrillation group (n=21). The experimental animals were divided into three groups with five animals in each group. Sham operation group (Group A):A pacemaker was implanted off the rapid atrial pacing (RAP) four weeks after intra-pericardial injection of 500μl saline. AAV1/EGFP + atrial fibrillation group (Group B):The chronic atrial fibrillation model was established four weeks after transthoracic and intra-pericardial injection of 500μl of a suspension of AAV1 harboring the EGFP (green fluorescent protein) gene. AAV1/RyR2-EGFP+ atrial fibrillation group (Group C): The chronic atrial fibrillation model was established four weeks after transthoracic and intra-pericardial injection of a 500μl suspension of AAV1 harboring the RyR2 gene. A dog model with chronic atrial fibrillation was established by persistent rapid pacing of the left atrial appendage. Alterations in the ERP of various portions of the left and right artria, as well as the pulmonary vein, of both groups were determined. In addition, histopathological and micro-structural changes in myocardial tissues were assessed by H&E staining and microscopy.Changes in the expression of RyR2 and SK2 genes and their respective proteins were determined by immunohistochemistry, RT-PCR and Western blot. Results:1. We were successful in establishing a dog model of chronic atrial fibrillation in 100% of animals upon completion of the operation and after eight weeks of pacing. Chest radiology of the dogs showed that the pacemaker electrodes were placed in the left atrial appendage. Electrocardiograms indicated that rapid pacing of the left atrial appendage could induce atrial fibrillation when the pacemaker was switched on. Changes in the ERP of various cardiac regions in both groups of dogs eight weeks after the operation:There were no significant changes in the ERP at any point in time in the sham group. Cardiomyocytes from the atrial fibrillation group were not aligned correctly, and the cells themselves were elongated and corrugated. Connective tissue had also accumulated between the myocardial fibers, resulting in enlarged space between cardiomyocytes. Upon RAP of 8W, atrial myocytes from the atrial fibrillation group exhibited micro-structural changes, including:(1) Depletion of contracting material (myolysis), wherein debris-filled, depleted sarcomeres occurred frequently around the nuclei; (2) lysed myocytes filled with glycogen, even forming a’glycogen lake’; (3) mitochondria with typical alterations in size (elongated) and shape at the sites where sarcomeres were absent; (4) vague and discontinuous intercalated disc.2. After transduction of AAV1/RyR2-EGFP by transthoracic and intra-pericardial injection, cryosections of left and right atria were strongly positive for GFP. Meanwhile, after transduction of AAV1/RyR2-EGFP by transthoracic and intra-pericardial injection of AAV1/RyR2-EGFP, cryosections of other major organs such as lung, liver and spleen showed weak GFP expression. A much larger number of brownish particles were observed in cells from Group C, the atrial fibrillation group. There were a large number of brownish particles in Group C atria. Expression of RyR2 and SK2 at the mRNA and protein levels:The expression of RyR2 in Group C was elevated compared to β-actin.Conclusions:1. The established dog RAP model can mimic early electrophysiological alterations in atrial fibrillation, and is reliable and highly reproducible. This model shows abnormalities in both structure and electrophysiological function, similar to clinical atrial fibrillation.2. Adenovirus-mediated transduction of RyR2 leads to its overexpression and increased activity in cardiomyocytes, elevating expression of RyR2 and SK2 proteins... |
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