Changes Of The MRNA And Protein Expression Of T-type Calcium Channels In PVA Of Canine Model And Human With Chronic Atrial Fibrillation

As the most popular arrhythmia in clinic, atrial fibrillation (AF) manifests over 400 beat-per-minute’s irregular rhythm which causes the fibrillating contraction of atrial myocardium. The effect of AF to public health can not be underestimated. It can increase the disability and mortality, no matter which complications take place. As it is often accompanied by the contractile dysfunction of left ventricle, congestive heart failure and cerebral infarction, AF can deteriorate patients’life quality and expend medical resources. However, there is no efficient treatment to AF, because the mechanism of it is still unclear. So many hypotheses are raised to explained the mechanism of AF, while the hot issue is the hypothesis of "Pulmonary Vein Derived Theory’"Pulmonary vein antrum (PVA) is a region in which pulmonary veins (PV) and left atrium (LA) are connected. It is developed by the absorption of pulmonary veins and left atrium in embryo. The fibers of muscle here are so complicated which make the conduction capacity so heterogeneously in different direct. Electrial pulses are usually speed down or even blocked here. So PV and PVA are related closely to the occurrence and maintainance of AF. It is important to uncover their relationship between AF and PVA to improve the understanding the mechanism and developing treatment of AF.It is considered that PV is the closest vena cava to the occurrence and maintainance of AF, that is because its unique anatomic structure. It is found that, in the region of conecting area of PV and LV, some atrial myocytes spread into the PV, which is so-called cells in myocardial sleeves. Some of them have spontaneous rhythm and have shorter refractory period. And the myocardial fibers in PVA are so heterogeneous that make it easy to form the ectopic focuses of arrhythmia and reentries between PV and LV. It tends to cause fibrillative conduction when rapid electrial pulses are passing through. In clincal experiences, catheter ablation in the area between PV and LV can vanish almost all paroxysmal AF and partial chronic AF.Perez-Lugones etc. discovered the pacemaker cells (P cells) and transitional cells in PV for the first time which indicated that PV may had potential capacity of spontaneous rhythm. In terms of P cell, the slowly depolarization of 0 period is its fundamental of spontaneous pacing. It has been proved that, the slowly depolarization of 0 period in P cell was caused by the slowly activating of voltage dependant T-type calcium channel (VDTTCC) in resting period, so the capacity to have spontaneous rhythm of P cell is connected to the distribution and expression of VDTTCC. On the other hand, VDTTCC still plays an important role in regulation of intra-cellular calcium concentration of myocytes, while intra-cellular calcium overloading has been proved to be the key of occurrence and maintainence of AF. It is so significant to clarify the relationship between VDTTCC and AF.In this research, the expression change of VDTTCC in PVA region is observed. Firstly, animal model of chronic AF is established by rapid atrial pacing (RAP). PVA tissues are obtained from animal model, and the differences of VDTTCC between two groups are observed in molecular biological and pathological level. Based on the results of animal experiment, cautious clinlical experiment is carried out from the specimens obtained from chronic AF patients who had the open-chest surgery.Preliminary study shows that, the expression of VDTTCC in PVA region of chronic AF animals and patients is significantly up-regulated than control. Part 1 The Establishment of Canine Model of Chronic Atrial Fibrillation by the Method of Rapid Right Atrial PacingObjective To establish the canine model of chronic AF by the method of rapid right atrial pacing.Methods To divide the totally 12 Beagles into two groups randomly. In experimental group, animals were placed a electrode on the surface of right atrium by open-chest surgery with high frequency electrical stimulation between 400 to 450 bpm, while animals in control were accepted a stitch in the same region of right atrium. Rapid pace for 8 weeks in experimental group, and record the limb lead EKG each week. Check the UCG before and after experiment, and calculate the area of LA and RA by the longest axis.Results All animals in both groups were survived. After 8-week RAP, all animals in experimental group could evoke AF,3 among them could maintain AF without evoking, the other 3 animals could maintain AF for the average of 52±15min after evoking. All of them met the define of chronic AF. In the experimental group, the area of LA and RA in the end of experiment were becoming larger than the beginning, while there was no significant difference in control group.Conclusions It’s possible to establish the canine model of chronic AF by the method of rapid right atrium pacing. Long-term RAP can enlarge the area of LA and RA.Part 2 Changes of the mRNA and Protein Expression of T-type Calcium Channels in PVA of Canine Model with Chronic Atrial FibrillationObjective To obtain PVA specimen from canine model of chronic AF, and analyse the differences of VDTTCC distribution and expression by semi-quantitive PCR, Western-Blot and immunohistochemistry technique. Methods To execute animals of euthanasia. Obtain PVA tissues and wash away the blood. Divide tissue into two parts, one is kept in-80℃refrigerator, the other is kept in formalin solution. Design primers according to the canine’s cDNA of TTCC alH subunit and analyse the abundance of mRNA of this subunit in two groups by semi-quantitive PCR. Western-Blot technique is used to evaluate the protein expression of alH subunit. H-E and immunohistochemistrical staining are used to evaluate the morphological differences between two groups.Results The mRNA abundance and protein expression of a1H subunit of TTCC are significantly up-regulated in experimental group than control group, which is consistent by Pathological results. Immunohistochemistrical staining locates the channel protein on the surface of myocyte.Conclusions TTCC in PVA in experimental group is significantly up-regulated than control.Part 3 Changes of the mRNA and Protein Expression of T-type Calcium Channels in PVA of Human with Chronic Atrial FibrillationObjective To obtain PVA specimen from patients with chronic AF and sinal rhythm during open-chest cardiac surgery, and analyse the differences of VDTTCC distribution and expression by Real-time qantitive PCR, Western-Blot and immunohistochemistry technique.Methods Before infusing myocardial cardioplegia, to place a wallet suture in the PVA region of right superior pulmonary vein. The PVA tissue (200-400mg) was obtained by hole puncture from two groups of patients during the cardiac surgeries. Divide tissue into two parts, one is kept in-80℃refrigerator, the other is kept in formalin solution. Design primers according to the human cDNA of TTCC alG and alH subunits and analyse the abundance of mRNA of these two subunit in two groups by Real-time quantitive PCR by the 2-ΔΔCt method. Western-Blot technique is used to evaluate the protein expression ofα1G andα1H subunits. H-E and immuno-histochemistrical staining are used to evaluate the morphological differences between two groups.Results The mRNA abundance and protein expression ofα1H subunit of TTCC are significantly up-regulated in AF group than SR group, while there is no significant difference between two groups in alG subunit, which is consistent by Pathological results. Immunohistochemistrical staining locates the channel protein on the surface of myocyte.Conclusions Compared to SR group, the alH subunit of TTCC are significant up-regulated in AF group, while there is no difference inα1G subunit between two groups.