Effects Of Atorvastatin On Atrial Remodeling In A Rabbit Model Of Atrial Fibrillation Produced By Rapid Atrial Pacing

Atrial fibrillation(AF) is the most common arrhythmia in clinical practice. It results in serious potential complications, especially stroke and heart failure, and increased mortality, which seriously affect the quality of life of patients and increase medical costs and social burden. Current treatments for AF include rhythm control and rate control. Rhythm control refers to maintain sinus rhythm and prevent AF recurrence by antiarrhythmic drugs, electrical conversion or catheter ablation, while heart-rate control is to control the rapid ventricular rate and its associated symptoms, and meanwhile give anticoagulant drugs to prevent thromboembolic complications. Despite recent advances in the pharmacological strategy and radiofrequency ablation, how to administrate after cardioversion or catheter ablation to prevent AF recurrence, and how to prevent associated complications of AF by effective anticoagulation, these problems still remain. Therefore, we still need to improve the understanding of the pathophysiology underling AF and explore the novel therapeutic approaches of AF.The main pathophysiological mechanism contributing to the initiation and maintenance of AF is atrial remodeling, which includes electrical and structural remodeling. Atrial electrical remodeling is characterized by ion channel dysfunction, which induces the shortening of atrial effective refractory period(AERP), reduction of adaptability to the frequency of AERP, and increased disproportion of atrial conduction. Atrial structural remodeling is characterized by atrial enlargement and interstitial fibrosis. Increasing evidence supports that inflammation and oxidative stress play important roles in AF. On the one hand, inflammation can alter atrial electrophysiology and structural substrates, thereby leading to increased vulnerability to AF. On the other hand, AF itself can induce inflammation and atrial remodeling, which perpetuates the arrhythmia. This is the so-called ‘AF begets AF’ phenomenon. So anti-inflammatory and antioxidant treatments may become the new methods for treatment of AF.Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors,are widely used in clinical as lipid-lowering agents. In addition, statins also have anti-inflammatory and antioxidant properties. In recent years, some clinical and experimental studies suggested that the use of statins could protect against AF. However, insufficient data are available at this time, and the molecular mechanism by which statins may prevent AF has not been elucidated. Myeloperoxidase(MPO), a heme enzyme released from activated neutrophils, is not only associated with a variety of cardiovascular disease, but has also been shown to be mechanistically linked to AF. MPO catalyzes the generation of reactive species, which promote atrial electrical and structural remodeling resulting in the initiation and perpetuation of AF, by affecting intracellular signaling cascades in various cells. Recent studies showed that patients with AF had high levels of MPO in the atria, and statins could reduce the plasma levels of MPO in patients with cardiovascular diseases. Therefore, statins may prevent atrial remodeling and AF by reducing atrial MPO level.This study was designed to investigate the potential effects and molecular mechanism of atorvastatin on atrial electrical and structural remodeling in a rabbit AF model induced by 3 weeks of rapid atrial pacing(RAP). Part one The establishment of rabbit AF model induced by RAPObjective: To evaluate the feasibility of the establishment of rabbit AF model induced by RAP, and investigate the changes of cardiac structure and function of the AF model.Methods:20 New Zealand white rabbits were randomly divided into 2 groups: the RAP group(n=10) and the sham group(n=10). Rabbits in the RAP group were subjected to 3 weeks of RAP.1 The changes of the general situation before and after RAP in the 2 groups were observed, including weight, respiratory rate, heart rate, and so on.2 Echocardiography was performed before and after RAP in the 2 groups to evaluate the structure and function of atria and ventricle.3 AF inducibility was tested before and after RAP in the 2 groups by atrial burst stimulation.4 Masson trichrome-staining of the left atria was performed to evaluate the degree of the atrial fibrosis after RAP in the 2 groups.Results:1 The general situation: After 3 weeks of RAP, 7 rabbits were eligible in the RAP group and 7 in the sham group. The weight, respiratory and heart rate had no significant difference(P > 0.05) in the 2 groups before RAP. After 3 weeks of RAP, the respiratory and heart rate were increased in the RAP group compared with the sham group(P < 0.05), while the weight still had no significant difference(P > 0.05) in the 2 groups.2 Changes of echocardiographic indices: The echocardiographic indices of atria and ventricle had no significant difference(P > 0.05) in the 2 groups before RAP. After 3 weeks of RAP, compared with the sham group, left atrial diameter(LAD), left atrial maximal volume(LAVmax) and left atrial minimal volume(LAVmin) significantly increased(P < 0.05), whereas left atrial ejection fraction(LAEF) dramatically decreased(P < 0.05) in the RAP group; left ventricular end systolic diameter(LVESD) slightly increased(P < 0.05), and left ventricular ejection fraction(LVEF) slightly decreased(P < 0.05) in the RAP group. However, left ventricular end diastolic diameter(LVEDD) had no significant difference(P > 0.05) in the 2 groups after RAP.3 Changes of inducibility of AF: Electrocardiogram(ECG) confirmed sinus rhythm, and atrial burst stimulation did not induce AF in each rabbit in the RAP and sham groups before RAP. After 3 weeks of RAP, ECG recordings showed that rabbits were still in normal sinus rhythm in the sham and RAP groups when the pacemakers were deactivated; atrial burst stimulation induced sustained AF(AF lasted more than 30 minutes) in 6 out of 7 in the RAP group(86%), and none in the sham groups.4 Change of the degree of atrial fibrosis: After 3 weeks of RAP, masson trichrome-staining of left atrial myocardium showed that the percentage of areas of interstitial fibrous tissue was significantly increased in the RAP group compared to the sham group(P < 0.05).Conclusion: 3 weeks of RAP could induce sustained AF and obvious atrial remodeling of the rabbits, while the structure and function of left ventricle was not changed. Therefore, RAP can establish successful rabbit AF model. Part two Effects of atorvastatin on atrial electrical remodeling in a rabbitmodel of AF produced by RAPObjective: To evaluate the effects of atorvastatin on atrial electrical remodeling in a rabbit model of AF produced by 3 weeks of RAP.Methods:18 New Zealand white rabbits were randomly divided into the sham(n=6), control(n=6) and atorvastatin(n=6) groups. Rabbits in the control and atorvastatin groups were subjected to RAP at 600 bpm for 3 weeks, and treated with placebo or atorvastatin(2.5 mg/kg/d), respectively. Rabbits in the sham group did not receive RAP.1 AERP was tested before and after RAP in the 3 groups by atrial S1S2 diminishing stimulation.2 AF inducibility was tested before and after RAP in the 3 groups by atrial burst stimulation.3 Atrial m RNA levels of Cav1.2 and Kv4.3 were measured in the 3 groups by PCR.4 Atrial protein levels of Cav1.2 and Kv4.3 were measured in the 3 groups by western blot.Results:1 AERP: Before RAP, no significant difference of the AERP was observed among the 3 groups(P > 0.05). After 3 weeks of RAP, the AERP of the control group was significantly shorter than that of the sham group(P < 0.05). The AERP of the atorvastatin group was also shortened compare with that of the sham group(P < 0.05), but its RAP-induced reduction was reversed to some extent compared with that of the control group(P < 0.05).2 AF inducibility: Electrocardiogram(ECG) confirmed sinus rhythm, and atrial burst stimulation did not induce AF in each rabbit in the 3 groups before RAP. After 3 weeks of RAP, ECG recordings showed that rabbits were still in normal sinus rhythm in the sham, control and atorvastatin groups when the pacemakers were deactivated; Atrial burst stimulation induced sustained AF in 5, 4, and no rabbits in the control(83%), atorvastatin(67%), and sham groups, respectively.3 Cav1.2 and Kv4.3 m RNA levels in the left atria: Cav1.2 m RNA significantly decreased in the control and atorvastatin groups compared with the sham group(P < 0.05), but it down-regulation was prevented in the atorvastatin group compared with the control group(P < 0.05). The Kv4.3 m RNA was also significantly decreased in the control and atorvastatin groups compared to the sham group(P < 0.05), but it down-regulation was not prevented in the atorvastatin group compared with the control group(P > 0.05).4 Cav1.2 and Kv4.3 protein levels in the left atria: The protein level of Cav1.2 was significantly decreased in the control and atorvastatin groups compared with the sham group(P < 0.05), but this change was prevented in the atorvastatin group compared with the control group(P < 0.05). The protein level of Kv4.3 was also significantly decreased in the control and atorvastatin groups compared to the sham group(P < 0.05), and these changes in the control and atorvastatin groups had no significant difference(P > 0.05).Conclusion: Atorvastatin could prevent atrial electrical remodeling in a rabbit model of AF produced by RAP, by suppressing the down-regulation of atrial Cav1.2 level and the shortening of AERP. Part three Effects of atorvastatin on atrial structural remodeling in arabbit model of AF produced by RAPObjective: To evaluate the effects of atorvastatin on atrial structural remodeling in a rabbit model of AF produced by 3 weeks of RAP.Methods:18 New Zealand white rabbits were randomly divided into the sham(n=6), control(n=6) and atorvastatin(n=6) groups. Rabbits in the control and atorvastatin groups were subjected to RAP at 600 bpm for 3 weeks, and treated with placebo or atorvastatin(2.5 mg/kg/d), respectively. Rabbits in the sham group did not receive RAP.1 Echocardiography was performed before and after RAP in the 3 groups to evaluate the structure and function of atria and ventricle.2 Masson trichrome-staining of the left atria was performed to evaluate the degrees of the atrial fibrosis in the 3 groups.3 Atrial m RNA levels of collagen Ⅰ and Ⅲ were measured in the 3 groups by PCR.4 Atrial protein levels of collagen Ⅰ and Ⅲ were measured in the 3 groups by western blot.Results:1 Changes of echocardiographic indices: The echocardiographic indices of atria and ventricle had no significant difference(P > 0.05) in the 3 groups before RAP. After 3 weeks of RAP, compared with the sham group, LAD, LAVmax and LAVmin significantly increased(P < 0.05), whereas LAEF dramatically decreased(P < 0.05) in the control and atorvastatin groups. Compared with the control group, these echocardiographic indices of left atria did not differ in the atorvastatin group(P > 0.05). However, LVESD, LVEDD and LVEF still had no significant difference(P > 0.05) in the 3 groups after RAP.2 Change of the degree of atrial fibrosis: 3 weeks of RAP caused a marked atrial fibrosis as estimated by masson trichrome-staining. The degrees of atrial fibrosis in the control and atorvastatin groups were significantly higher than that in the sham group(4%, P < 0.05). Compared with the control group(33%), the atrial fibrosis was partial suppressed in the atorvastatin group(24%, P < 0.05).3 Collagen Ⅰ and Ⅲ m RNA levels in the left atria: The m RNA levels of collagen Ⅰ and Ⅲ were significantly increased in the control group in comparison with the sham group(P < 0.05). These changes in the m RNA levels of these 2 types of collagen were suppressed by atorvastatin(P < 0.05).4 Collagen Ⅰ and Ⅲ protein levels in the left atria: The protein levels of collagen Ⅰ and Ⅲ were significantly increased in the control group in comparison with the sham group(P < 0.05). These changes in the protein levels of these 2 types of collagen were suppressed by atorvastatin(P < 0.05).Conclusion: Atorvastatin could prevent atrial structural remodeling in a rabbit model of AF produced by RAP, by suppressing atrial interstitial fibrosis and the deposition of collagen Ⅰ and Ⅲ. Part four Discussion of the potential mechanisms by which atorvastatinprevented atrial remodeling in a rabbit model of AF producedby RAPObjective: To discuss the potential mechanisms by which atorvastatin prevented atrial remodeling in a rabbit model of AF produced by 3 weeks of RAP.Methods:18 New Zealand white rabbits were randomly divided into the sham(n=6), control(n=6) and atorvastatin(n=6) groups. Rabbits in the control and atorvastatin groups were subjected to RAP at 600 bpm for 3 weeks, and treated with placebo or atorvastatin(2.5 mg/kg/d), respectively. Rabbits in the sham group did not receive RAP.1 Atrial m RNA levels of MPO、matrix metalloproteinase(MMP)-2、MMP-9 and tissue inhibitors of metalloproteinase(TIMP)-1 were measured in the 3 groups by PCR.2 Atrial protein levels of MPO、MMP-2、MMP-9 and TIMP-1 were measured in the 3 groups by western blot.Results:1 The m RNA levels of MPO、MMP-2、MMP-9 and TIMP-1 in the left atria: The m RNA levels of MPO, MMP-2 and MMP-9 were significantly increased in the control and atorvastatin groups compared with the sham group(P < 0.05), but these changes were prevented in the atorvastatin group compared with the control group(P < 0.05).The level of TIMP-1 was also increased in the control and atorvastatin groups compared to the sham group(P < 0.05), and these changes in the control and atorvastatin groups had no significant difference(P > 0.05).2 The protein levels of MPO、MMP-2、MMP-9 and TIMP-1 in the left atria: The protein levels of MPO, MMP-2 and MMP-9 were significantly increased in the control and atorvastatin groups compared with the sham group(P < 0.05), but these changes were prevented in the atorvastatin group compared with the control group(P < 0.05).The level of TIMP-1 was also increased in the control and atorvastatin groups compared to the sham group(P < 0.05), and these changes in the control and atorvastatin groups had no significant difference(P > 0.05).Conclusion: Atorvastatin prevented atrial remodeling in a rabbit model of AF produced by RAP, perhaps by reducing atrial MPO level and subsequently reducing the levels of MMP-2 and MMP-9.