Currently, most studies on AF are carried on patients with AF or in animal models. Although good results have been acquired, there are still some limitations, such as inability to precisely interfere, susceptibility to influence by many factors and ethical concerns . As the maturation of culture of myocardial cells, primary myocardial cells can be used as an in vitro model for the study of pathophysiology of some cardiac diseases. The primary myocardial cells as an in vitro model has the following advantages: ① The enzyme induced damage (damage to proteins in cell membrane including receptors and ion channels) during the separation may restore completely after culture . ② The cell culture can last for a long time. Studies showed that the vitality of myocardial cells undergoing acute separation can only be maintained up to 8–10 h, and that of cultured myocardial cells can be maintained for a few days, or even several weeks . In addition, the in vitro study can avoid the influence of numerous factors in in vivo studies. ③ The molecular biological techniques can be used to detect the expression of proteins in myocardial cells alone. These cells maintain favorable vitality even the expression of these protein changes with the help of molecular biological technology [15, 16]. ④ For Human and primate, the source of myocardial tissue from is limited. Thus, the cultured cells are a good choice. ⑤ The technique of storage of cells is mature. Thus, to establish an in vitro AF model with myocardial cells is very important.
The difficulty in sample collection, small number of collected cells And the technique for Cell separation are the major barriers in primary atrial myocardial cell culture. Mature atrial myocardial cells are regarded as terminally differentiated cells and are susceptible to damage by ischemia, hypoxia, enzymes, pH and mechanical stimulation which may ultimately affect the activity and amount of cells. In addition, the separation of these cells is difficult due to strong physical connection via the intercalated disk and extracellular matrix among cells. Moreover, the atrial myocardial cells are isolated under a calcium-free environment and thus intolerant to calcium. Under the physiological conditions, cells may present with contraction in the presence of calcium, which may result in change in the morphology of these cells. Thus, it is extremely difficult for separation and culture of atrial myocardial cells. In this study, the culture of primary atrial myocardial cells was done according to the method described by Benardeau et al. with modification . In the separation and culture of atrial myocardial cells, we have following experience: ① The age and body weight should be taken into account in selection of rats. Rat aged about 2 weeks and weighing 30 g are preferred. Under this condition, the collection of atrial myocardial cells is relatively easy, the vitality of these cells is improved and the purification is more convenient. ② The trypsin may cause damage to these cells, and thus the trypsin concentration should be as low as possible. According to our experience, the trypsin concentration is usually 0.06-0.08%. The duration of digestion should not be too long. Digestion can be performed with short duration for sever times. ③ Brdu can significantly inhibit the growth of fibroblasts in the skeletal muscle and myocardium, but has no influence on or toxicity to myocardial cells. In addition, culture of myocardial cells with Brdu may increase the purity of myocardial cells from 45-50% to 85-90% . Consequently, differential adherence method and drug intervention (Brdu) are crucial to improve the purity of atrial myocardial cells .
A rapid pacing model is established using primary myocardial cells (atrial myocardial cells or ventricular myocardial cells), which can also mimic the in vivo rapid pacing. In addition, electrophysiological detection, pharmacological intervention and molecular biological intervention are more convenient in primary myocardial cells . In the present study, the separation, purification, culture and identification of atrial myocardial cells were conducted, and then these cells were used to establish a model of rapid pacing with electric field. After 24-h rapid pacing, the cell morphology remained unchanged, and the frequency of cell pacing was slightly faster. MTT chromatometry also demonstrated that the cell viability after rapid pacing was comparable to that before pacing. However, under a transmission electronic microscope, aggregation of glucogen, karyopycnosis, and vacuolar changes were observed. These changes were consistent to those in in vivo rapid pacing models and other in vitro rapid pacing models .
During the electrical remodeling after AF, the shortening of APD and ERP is secondary to the functional feedback of ion channel and becomes a characteristic in the early phase. However, in the late phase, the expression of L-type calcium channels and potassium channel Kv4.3 reduced resulting in changes in structure of ion channels. These are a major cause of shortening of APD and ERP. Therefore, the abnormal expression of ion channels is a material basis in the electrical remodeling of atrial myocardial cells after AF .
Our results showed the mRNA and protein expressions of L-type calcium channels α1c after 6 h rapid pacing had a decreasing trend, and the expressions of L-type calcium channels α1c reduced over time. The mRNA and protein expressions of potassium channel Kv4.3 decreased at 12 h after rapid pacing but thereafter remained stable and did not further reduce over time. In addition, the changes in the expression of L-type calcium channel α1c and potassium channel Kv4.3 were consistent with the findings in other models, but these changes were different from previously reported in the speed of changes. This may be attributed to following factors: ① The frequency of pacing is different in different experiments. In the present study, the pacing frequency was 600 beats/min. In the study of Yue et al. , the pacing frequency was 400 beats/min. The different pacing frequencies may present with differences in induction of ion channel remodeling, and rapid pacing may induce earlier remodeling. ② Different models were used in different experiments. In the studies of Grunnet et al.  and Yue et al. , animal models were used, while myocardial cells were used in the present study. ③ Animals of different species were used in different studies. In the goat AF model, the shortening of effective refractory period was earlier than that in dog AF model . In addition, the occurrence of electrical remodeling in horse was later than that in goats and dogs.
Our results also showed that, after rapid pacing, the protein and mRNA expression of potassium channel Kv4.3 also decreased to different extents, which may not explain the shortening of effective refractory period and action potential cycle, because the decline in expression of potassium channel Kv4.3 and reduction of outward K+ flow will lead to extension of action potential cycle and effective refractory period. One possible hypothesis is that the reduced expression of potassium channel is attributed to the self-adaption of atrium. Brundel et al.  investigated the changes in the expression of potassium channel in AF patients, and results showed the mRNA and protein expression of potassium channel in persistent AF patients had a reducing trend, while only the protein expression of potassium channel reduced in the paroxysmal AF patients which suggests the presence of post-transcriptional regulation. This may be explained that rapid stimulation and increase in intracellular calcium may induce calcium overload as well as structural changes. Meanwhile, the expression of proteolytic enzymes can be increased in the atrial tissues and the neutral protease (such as calpains) be activated. These enzymes can lead to degradation of skeleton proteins, membrane proteins and regulatory protein, and also influence the expression of potassium ion channels.
In addition, to generate the cardiac action potential, in addition to inward sodium and calcium currents, 5 potassium currents are primarily involved: the inward-rectifier background current (IK1), the rapidly activating and inactivating transient outward current (Ito), and the ultrarapid (IKur), rapid (IKr), and slow (IKs) components of delayed rectifier currents . More attention has been paid to IKr (KCNH2 gene expression) and Ito (KCND3 gene expression). KCNH2 encodes the α-subunit of the IKr channel, and membrane depolarization induced by strong inward currents produces a sequence of conformation changes within the channel that allows permeation of potassium ions . The transient outward current (Ito) that mediates early (phase 1) repolarization and is conducted by the Kv4.3 pore-forming α-subunit encoded by KCND3 in humans remains central to the “the repolarization disorder” theory of the electrocardiographic and arrythmogenic manifestations of tachyarrhythmia . Both of them result in shortening of action potential duration and atrial refractory period, facilitating multiple re-entrant circuits in AF.