Research On The Expressional And Functional Changes Of Membrane Calcium Channels Of 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.Calcium channels, as a big family constituted by different subtype channel proteins, exsit wildly in almost all kinds of tissues and play a part in the control of growing up and autorhythmicity. By now, only T and L-type calcium channels has been found in the sufface of myocardium. It is reported that the intracellular free calcium ion concentration in rapid atrial pacing animal's atrial myocytes increases, which indicates intracellular calcium overloading may play an important role in AF, and this result has been improved not only in clinic experiments of AF patients atrial myocytes but also in atrial myocytes of rapid atrial pacing animal models.The level of intracellular Ca2+([Ca2+]i) is determined by two pathways:the first is the reduction of Ca2+ efflux when the SERCA/PMCA system and Na+-Ca2+/H+-Na+ exchanger system are abnormal which will reduce the ability of Ca2+ uptaking and excretion; the second is the increasing of the Ca2+ influx which also includes two ways-the calcium channel on the surface of sarcoplasmic reticulum (Ryanodine Receptor and 1,4,5-risphosphate rinositol receptor) and the calcium channel on the surface of cardiomyocytes (T-type and L-type Voltage Dependent Calcium Channels).As an important resource of intracellular calcium, calcium channels on the surface of cardiomyocytes became a hot spot in revealing the mechanism of AF.The aim of this study is to survey and evaluate the expressional and functional changes of calcium channels on the surface of cardiomyocytes in order to explain its mechanism in inducing of intracellular calcium overloading. Atrial myocardial tissues are obtained from different patients who accepted open-chest cardiac operation. Specimens are divided into different groups according to the donor's heart rhythm and underlaying diseases. Using molecular biological technique, we investigate the abundance of mRNA and the expression level of proteins of T and L-type calcium channel, and we also observe the function of these two channels in Ca2+ influx changing with the help of Laser Scanning Confocal Fluorescence Microscopy and calcium imaging technique. Moreover, we try to establish a method in preparing living atrial myocardium slices of adult human hearts.Objective To investigate whether there are changes in the expression, at mRNA and protein level, ofα1 subunits of L-type (α1C) and T-type (α1G andα1H) calcium channels in the human atrial myocardium among normal sinal rhythm (NSR), rheumatic sinal rhythm (RSR) and rheumatic atrial fibrillation (RAF) patients.Methods The right atrial tissue was obtained from three distinct groups of patients during the cardiac surgeries, and the numbers of the patient were 10 for NSR,11 for RSR, and 16 for RAF, respectively. All specimens (about 200mg-400mg) were obtained before the perfusing of cardioplegic solution when extracorporeal circulation has been built. Tissues were divided into two parts, one was kept in liquid nitrogen and the rest was kept in the solution of formaldehyde (concentration:10%) for immunohistochemistry research. Extract the total RNA, design the pimers and Real-time quantative RT-PCR was used to measure the abundance of mRNA of three different calcium channelα1 subunits (CACNA1C, CACNA1G, CACNA1H) by the 2-ΔΔCt method. Get out the total protein and Western Blotting was also used to examine the expression of these three channel proteins by a semi-quantative method. Immunohistochemistry technique was used to compared the location of these three channel proteins among these groups.Results In comparison with the NSR and RSR groups, the mRNA abundance and protein expression level ofα1C andα1H subunits was significantly up-regulated in the RAF group (P<0.05). However, the level ofα1G subunit was not significantly different among the three groups. Between the NSR and RSR groups, there were no significant differences of the expression of all three subunits. Compared with NSR and RSR groups, Stained preparation of RAF shows mesenchymal hyperplasia, enlargement of nucleus and heavy steined of the surface channel proteins.Conclusions The present study demonstrated that the expression ofα1C andα1H subunits, but notα1G subunit, was up-regulated in atrial fibrillation, suggesting rheumatics may not affect the expression level of L-type and T-type calcium channel. Whether the rheumatics might still change calcium channel functions remains elusive.Objective 1. To explore and modify a proper method in isolating human single atrial myocardial cell depending on the traditional isolating methods.2. To evaluate the intracellular changes of calcium level induced by T and L-type calcium channels between normal sinal rhythm (NSR) group and rheumatic atrial fibrillation (RAF) group with calcium imaging technique of Laser Scanning Confocal Fluorescence Microscope (LSCFM).3. To establish a method of preparing multicellular myocardium model in vitro which may preserve the normal structures of myocardium and the connections among myocardial cells for the next electrophysiological studies.Methods 1. To isolate human single atrial cardiomyocytes by modified Bustamante' method:All atrial myocardial tissues were obtained before the perfusing of cardioplegic solution when extracorporeal circulation has been built. Tissues were kept in oxygen saturated calcium-free solution in 37℃and sent to laboratory with 5-10 minutes. Tissues were cut into small pieces (less than 2mm3) and washed by oxygen saturated calcium-free solution in 36℃-37℃for three times. Put these tiny tissue blocks into the oxygen saturated calcium-free solution which contains collagenase typeⅠ(0.4mg/ml) and proteinase typeⅩⅩⅣ(0.2mg/ml), and incubate for 20-30 minutes in 37℃with 4Hz stirring. Wash the tissue blocks with calcium-free solution for 1 minute. Then, incubate the tissue blocks in the oxygen saturated calcium-free solution contained collagenase typeⅠ(0.4mg/ml) with 4Hz stirring. Check the solution every 10 minutes under the invert microscope until the single myocardial cells are found. Remove the tissue blocks into oxygen saturated KB solution and keep blowing slightly with pipette. The suspensions were filtered through a 200-mesh screen and kept in KB solution for 30-60 minutes. Take the suspension in keeping and blow it well-distributed, then filter through a 200-mesh screen. Centrifuge the suspension in the speed of 1000 rpm for 5 minutes, remove the supernatant, then recalcificate gradiently to 1.5mmol/L. Blow it well-distributed and check under the invert microscope. Add the Trypan Blue into the suspension (1 part Trypan Blue solution,4g/L+9 parts suspensions) and count the number of viable cells and dead cells.2. To investigate the differences of Ca2+ influx of two types calcium channels between AF group and NSR group:Isolate the single atrial myocardial cells of both groups. Exhausting the calcium storage of sarcoplasmic reticulum (SR) by Thapsigargin (a blocker of SERCA) with the concentration of 1μmol/L for 10 minutes, then washed by calcium-free solution. Divide the suspension of each specimen into two dishes. Add fluorescence dye of Fluo-4/AM (20μmol/L) and incubate togather for 30 minutes, then wash out the dye with calcium-free solution. Replace the incubating solution with recalcification solution (1mmol/L). Add Verapamil (10mmol/L), a kind of L-type calcium channel blocker, into one dish; and add Mibefradil (1μmol/L), a kind of T-type calcium channel blocker, into the other dish for 20 minutes incubation. Put the specimen under the LSCFM. Using a Argon-Krypton ion laser (excitation wave length:494nm; emission wave length:516nm) to activate the fluorescence dye and record the optical density (OD1) within the cells when the surface calcium channels are resting. Depolarize the myocardial cells with 40mmol/L KCl solution, and record the optical density (OD2) within the cells when the surface calcium channels are activated. Statistic is the value of (OD2/OD1). Analyze the differences between two groups with student t test.3. To explore the perparation and culturing of living atrial myocardium slices of adult human hearts:All atrial myocardial tissues were obtained before the perfusing of cardioplegic solution when extracorporeal circulation has been built. Tissues were kept in oxygen saturated calcium-free solution in 37℃and sent to laboratory with 5-10 minutes. Before the specimen arriving, dissolve the low-melting-point agarose (LMP agarose) in ultrapure water (concentration:4%) in 70℃, and keep it under 45℃in water bath. Wash the tissue with cold calcium-free solution to remove the blood, and put it into the chamber on the surface of ice. Pour the LMP agarose solution into the chamer to cover the tissue, and cover the chamber with flake ice to chill it down quickly. Cut off redundant agarose, and keep the agarose block which embed the cardiac tissue. Fix the vibratome correctly and put the flake ice aroud the cutting chamber. Fill the cutting chamber with ice-water mixture of calcium-free solution saturated by pure oxygen. Stick the agarose block to the object stage with adhesive and start slicing (thickness:150μm-300μm). Remove the slices of myocardial tissue into 4℃cold recalcification solution saturated by pure oxygen to recalcificate gradiently for 30 minutes. Then remove the slices carefully into the DMEM solution with high glucose which is saturated by mixed gas of 5% CO2+95%O2 to culture for 30 minutes. Put the slice into the perfusing chamber and observe under the Infrared Differential Interference Contrast Invert Microscope.Results 1. Using the modified Bustamante's method of two-step enzyme digestion, we can successfully gather qualified adult human single atrial myocardial cells with rod shape, clear transverse striation and intact membrane. After recalcification, some of the single atrial myocardial cells regain the automatic contraction which disappears after complete adherence. Dying by Trypan Blue, the viable cells can not be dyed while the dead cells shows slight blue. Survival rate of cells is 20%-50%.2. After blocking the L-type calcium channel, OD ratio (OD2/OD1) of RAF group is significantly higer than NSR group (1.38±0.20 vs 1.88±0.32); while afer blocking the T-type calcium channel, OD ratio of RAF group is also significantly higer than NSR group (1.43±0.24 vs 2.48±0.40). Compared the OD ratio of each group of blocking L-type calcium channel and T-type calcium channel, it is found that, in NSR group, blocking these two channels makes nearly the same influence of the intracellular calcium changing (1.38±0.20 vs 1.43±0.24), while in RAF group, blocking T-type channel affect the intracellular calcium level much serious than blocking L-type channel (1.88±0.32 vs 2.48±0.40).3. The slices obtained are qualified with clear transverse striation, mesenchyme and intercalated disc structure. After recalcification, some part of the myocardium regain the ablitlity of automatic contraction.Conclusions 1. It is available to obtain qualified adult human single atrial myocardial cells by the modified Bustamante's method of two-step enzyme digestion.2. Compared to NSR group, the calcium influx of T and L-type calcium channel both increase in RAF group which indicates the function of these two calcium channels is upregulated and may play a role in the intracellular calcium overload. Moreover, in AF patient, the calcium influx enhancement of L-type calcium channel is much higer than T-type calcium channel which indicates that L-type calcium channel makes more important effect in intracellular calcium overloading.3. Using this method of LMP agarose embedding and vibratome slicing, it is available to prepare living atrial myocardium slice of adult human with good morphous and normal electrical mechnial movement.