The present data demonstrated that in the rabbit model of RAP-induced AF, atorvastatin suppressed AERP shortening and atrial interstitial fibrosis induced by RAP, but had no effect on RAP-induced atrial enlargement and dysfunction. In addition, atorvastatin suppressed the down-regulation of Cav1.2 mRNA, and prevented the increase in the levels of collagen I and III, MPO, MMP-2 and MMP-9 induced by RAP.
The main mechanisms contributing to AF initiation and maintenance are atrial electrical and structural remodeling. In recent years, several animal models of AF [
11‐
13,
20,
21] have been developed to investigate the molecular mechanism contributing to atrial remodeling. Among them, the dog [
11,
14,
20] and rabbit [
17,
19,
21] AF models induced by RAP are widely used. However, if atrioventricular block is not performed, dogs will develop significant LV dysfunction induced by RAP [
20], whereas rabbits will not [
21]. It is well known that LV dysfunction will subject the LA to a pressure overload, leading to atrial enlargement and electrical instability [
22]. Therefore, in the present study we chose rabbits to create an AF animal model to avoid the influence of LV dysfunction on atrial remodeling.
Effects of atorvastatin on atrial structural remodeling
Atrial structural remodeling is characterized by atrial enlargement and interstitial fibrosis [
4,
23], and has been considered as a major contributor to AF [
23]. LA enlargement has been identified as an independent risk factor for AF. For example, patients are more prone to paroxysmal AF if they have an increased LAD [
24]. Larger LA volume before cardioversion is associated with higher risks of AF recurrence [
25]. LA enlargement also significantly correlates with atrial fibrosis, which serves as a crucial substrate in the formation of AF and is difficult to reverse [
26]. Increased fibrosis has been observed in the atrium of animal models [
20,
21] and patients with AF [
27]. It is characterized by enhanced deposition of matrix collagen proteins, leads to inhomogeneous atrial electrical conduction, and gives rise to electrical reentry circuits which result in AF [
6].
In our study, after 3 weeks of RAP, rabbits showed significant atrial structural remodeling. The pacing time of our rabbit AF model is relatively short compared with the previous canine AF model [
14,
20], but rabbits have already had obvious atrial enlargement and interstitial fibrosis. In the previous canine AF model, after 4–6 weeks of RAP, LA volumes were nearly 2 times that at baseline [
14,
28], and atrial fibrosis of the control group was nearly 9–10 times that of the sham group [
20,
28]. In our model, after 3 weeks of RAP, LA volumes were nearly 4 times that at baseline, and atrial fibrosis of the control group was nearly 7 times that of the sham group. The obvious atrial structural remodeling contributed to a marked increase in AF inducibility. After 3 weeks of RAP, although all rabbits in the control group remained sinus rhythm when pacemakers were deactivated, atrial burst pacing induced sustained AF almost in all rabbits.
Our study showed that atorvastatin treatment could not prevent AF susceptibility and atrial enlargement and dysfunction, but could prevent atrial interstitial fibrosis and collagen protein expression levels. These results are not completely consistent with the previous research [
14], which showed that statins could not prevent AF susceptibility, but could prevent atrial dilatation and fibrosis in a canine AF model induced by 6 weeks of RAP. The different effects of statins on atrial dilatation may be attributed to different AF animal models and drug intervention time. In addition, LA volumes after RAP were only 2 times that at baseline in the previous research, while in our study these were nearly 4 times that at baseline, which may predict more serious LA enlargement, so is hard to reverse.
The metabolism of extracellular matrix is regulated by MMPs and their inhibitors, the TIMPs [
29]. Among many kinds of MMPs and TIMPs, MMP-2 and MMP-9 are key factors leading to atrial fibrosis in AF [
5,
30,
31], while TIMP-1 is a major inhibitor of MMP activity in AF tissues [
31]. MPO, a major contributor to inflammatory oxidative stress, also has an important role in AF. It could promote MMP expression and activation by catalyzing the generation of reactive species, and subsequently resulted in atrial fibrosis and AF [
5,
6]. Previous research showed that patients with AF had higher plasma and atrial MPO levels compared with individuals in sinus rhythm [
5], and high MPO levels predicted an increased risk of AF recurrence after catheter ablation [
7]. In addition, MPO-deficient mice were protected from atrial fibrosis and AF vulnerability induced by angiotensin II, and atrial MMP-2 and MMP-9 levels were profoundly reduced. However, if administrated with recombinant MPO, these MPO-deficient mice would develop a similar degree of atrial fibrosis as that observed in MPO-infused wild type mice [
5].
Many studies showed that statins, by their anti-inflammatory and antioxidant properties, could reduce the levels of plasma MPO in patients with cardiovascular diseases [
15,
16], and inhibit MPO mRNA expression in macrophages [
32]. In addition, statins also could inhibit secretion of MMP-2 and MMP-9 [
33], and down-regulate their expression levels [
34,
35]. In our study, the levels of MPO, MMP-2, MMP-9 and TIMP-1 were significantly increased after RAP. Atorvastatin treatment could suppress the increased levels of MPO, MMP-2 and MMP-9, especially MPO and MMP-9, but could not suppress the increased level of TIMP-1. These may be the potential mechanisms by which statins prevent atrial structural remodeling of AF.
In addition, peroxisome proliferator-activated receptor-gamma (PPARγ) is also involved in atrial remodeling and AF. Recent studies showed that PPARγ was decreased in elderly AF patients [
36] and hypertensive AF patients [
37], while PPARγ agonists could inhibit atrial remodeling in AF models [
38,
39] and prevent new onset AF in patients with non-insulin dependent diabetes [
40]. Statins could activate PPARγ and enhance its expression [
41,
42] by their anti-inflammatory and antioxidant properties. Therefore, whether the modulation of statins on PPARγ is involved in the molecular mechanisms of the prevention of statins against atrial remodeling in our rabbit model of AF is still a question and would be investigated in our future study.
Effects of atorvastatin on atrial electrical remodeling
Atrial electrical remodeling is characterized by ion channel dysfunction [
4], which creates a re-entry-prone substrate. In our study, 3 weeks of RAP caused AERP shortening and down-regulation of Cav1.2 and Kv4.3 mRNA. This is consistent with previous research using dog AF models [
11,
20,
21]. In the present study, atorvastatin treatment could partially suppress AERP shortening and Cav1.2 mRNA down-regulation, but had no effect on Kv4.3 mRNA down-regulation. Many studies have proved that atrial electrical remodeling was promoted by inflammation and oxidative stress, while could be reversed by statins [
8,
11,
13,
43]. As mentioned above, atorvastatin treatment suppressed the increased level of MPO, which is a major contributor to inflammatory oxidative stress. Therefore, our study suggests that statins may prevent electrical remodeling, and the reduced atrial MPO level may contribute to the prevention of statins on this process.