| Background:Atrial fibrillation, or AF, is an abnormal heart rhythm characterised by rapid and irregular beating. AF is the most common arrhythmia. Patients with AF have no symptoms in most episodes. Occasionally they may be have heart palpitations, chest pain, shortness of breath, or fainting. The disease increases the risk of stroke, heart failure and death and it is a serious threat to human health. In addition to ventricular arrhythmias, the atrial arrhythmia, especially the AF is common in post-myocardial infarction. Yet despite its importance and more than 100 years of basic and clinical research, we still do not fully understand its fundamental mechanisms and have not learned how to treat it effectively. It has been demonstrated that atrial structural remodeling, electrical remodeling, and neural remodeling all play important roles in the development, recurrence and persistence of AF. Moreover, atrial structural remodeling may play a more crucial role than electrical remodeling and neural remodeling. Atrial structural remodeling includes cellular and extracellular matrix changes, and its main performance is the atrial interstitial fibrosis. The interstitial fibrosis occurs mainly in the extracellular matrix, the main component of which is collagen.Mitogen-activated protein kinase(MAPK) pathway is one of the most important regulators of cell growth signaling pathways, including three important members of enzymes:ERK, p38, and JNK. The p38 MAPK is one of the major members of the MAPK family. P38 MAPK is involved in various complex biological processes, including cell proliferation, cell differentiation, cell death, and cell migration and invasion. A previous study has shown that inhibition of the p38 MAPK signaling pathway reduces ventricular fibroblast proliferation and collagen synthesis in rats. Another study has also shown that lycopene improves cardiac function and attenuates ventricular remodeling by inhibiting p38 MAPK activation. Phosphorylated p38 (P-p38) MAPK is the active form of p38 MAPK. However, it is currently unclear whether P-p38 MAPK plays a role in atrial structural remodeling after myocardial infarction (MI).Statin,3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, is currently the most widely used lipid-lowering drugs, has become a cornerstone of the treatment of hyperlipidemia and prevention of atherosclerotic disease. It is well known that statins exert lipid-lowering effects. In addition to the lipid-lowering function, a series of beneficial biological effects of statins, commonly termed the "pleiotropic effects", are now being widely recognized, which include the prevention and treatment of AF, the improvement of left ventricular ejection fraction (LVEF) in patients with dilated cardiomyopathy, and the attenuation of left ventricular remodeling and enhancement of left ventricular function in rats with MI. Therefore, statin drugs may attenuate left atrial structural remodeling indirectly by improving the left ventricular function, enhancing LVEF, and reducing left atrial pressure. Research has shown that torasemide significantly decreases left ventricular end-diastolic pressure (LVEDP) in rats suffering from heart failure. Because LVEDP equals left atrial pressure in the absence of valvular heart disease, it is speculated that torasemide may reduce left atrial pressure in rats suffering from heart failure and subsequently attenuate left atrial structural remodeling.In our previous study, rats with MI received daily intragastric administration of normal saline or rosuvastatin, for a total of 8 weeks. The results showed that the structure and morphology of the damaged heart cells was significantly improved, indicating that rosuvastatin can improve atrial structure remodeling after MI. But statins probably improve atrial structural remodeling indirectly by improving ventricular function and lowering atrial pressure after MI.Whether statin drugs directly attenuate atrial structural remodeling after MI and their mechanisms of action remain unclear. To investigate the direct effect of rosuvastatin on the atrial structural remodeling after MI, torasemide was selected on the basis of our preliminary experiments as a positive control. The purpose of using torasemide was to eliminate the indirect effects of rosuvastatin on atrial structural remodeling after MI and to verify whether rosuvastatin attenuates the atrial structural remodeling after MI. In addition, the role of P-p38 MAPK in the rosuvastatin-induced attenuation of left atrial structural remodeling was investigated.Objective:To observe whether statins directly attenuate atrial structural remodeling after MI, analyze the role of P-p38 MAPK in atrial structural remodeling, and to explore the role of P-p38 MAPK in the rosuvastatin-induced attenuation of atrial structural remodeling.Methods:1. Development of animal model of MI and animal groupingA total of 80 male Wistar rats weighing 250-300 g were purchased. To establish a model of MI,66 randomly selected male Wistar rats underwent surgical ligation of the left anterior descending coronary artery (LAD) after anesthesia. Endotracheal intubation and mechanical ventilation were also performed during the surgical process. The 14 rats in the sham-operated group underwent the exact same procedure except that the suture was pulled through under the LAD, and the LAD was not ligated.Ten rats suffering from MI died within 24 hours of the surgery. The remaining 56 rats that survived the surgery were randomly divided into the following 4 groups: the control group (C group, n=14), the rosuvastatin group (R group, n=14), the low-dose torasemide group (T1 group, n=14), and the high-dose torasemide group (T2 group, n=14). These 4 groups of rats received daily intragastric administration of normal saline, rosuvastatin (10 mg/kg body weight), and torasemide (T1:1 mg/kg body weight; T2:2 mg/kg body weight), respectively, for a total of 4 weeks. All rats in the sham-operated group (n=14) survived the surgery and were given normal saline daily by intragastric administration for 4 weeks. Among the 5 groups of rats, only one rat in the C group died during the 4-week period.2. Echocardiographic studyAt the end of the 4-week treatment period, the 5 groups of rats were anesthetized and then underwent endotracheal intubation and mechanical ventilation. Transthoracic echocardiography was performed using an HP Sonos 5500 (S12 probe, frequency,5-12 MHz). The left atrial diameter (LAD), left ventricular end-diastolic dimension (LVDd), left ventricular end-systolic dimension(LVDs), left ventricular fractional shortening(LVFS) and left ventricular ejection fraction (LVEF) were calculated by M-mode tracing.3. LVEDP measurementAfter echocardiographic parameters were determined, a pressure-volume conductance catheter with a diameter of 2F (SPR-838, Millar Instruments, Inc., Houston TX, USA) was inserted into the left ventricle of the rat through the right carotid artery. The other end of the catheter was connected to a 16-channel physiological recorder (MP150, BIOPAC systems, Inc., USA) for hemodynamic monitoring. The LVEDP was measured and considered to represent the left atrial pressure.4. Sample collection and processingAfter the determination of the LVEDP, the rats were sacrificed. The chest of the rats was opened, and the left atrium and left ventricle plus septum were isolated. The left atrium was divided into two parts. One part was fixed in 10% formalin, embedded in paraffin, sectioned (the thickness of the sections was approximately 4 μn), and subjected to Masson’s trichrome staining and immunohistochemical staining. The other part was snap-frozen in liquid nitrogen and used in western blot analysis. After being isolated, the left ventricle was frozen in liquid nitrogen.5. Histological analysisThe rat atrial sections were stained with Masson’s trichrome, mounted with neutral gum, and examined under a microscope. The results showed that the collagen fibers were stained blue while the myocardial fibers were stained red. Collagen volume fraction (CVF) refers to the percentage of the area stained positive for collagen relative to the total area in a field of view [10]. Eight fields of view were randomly selected for each section, and the CVFs were calculated using the ImagePro Plus software (version 6.0, USA) to represent the degree of fibrosis in the left atrium. The areas surrounding the blood vessels were excluded when calculating CVFs. The ventricular sections were stained with triphenyltetrazolium chloride (TTC) and the infarct size was determined as described previously. 6. Immunohistochemical stainingAfter dewaxing in xylene and rehydration in graded alcohol, the tissue sections were subjected to antigen retrieval in sodium citrate buffer (0.01M, PH7.4). The endogenous peroxidase activities were eliminated with 0.3% hydrogen peroxide (H2O2) in methanol. The sections were blocked with normal goat serum for 30 min, covered with drops of diluted primary antibody (rabbit anti-rat P-p38 MAPK antibody, 1:1600 dilution, Cell Signaling Technology, Inc., Massachusetts, USA), and incubated at 4℃ overnight. After washing, the sections were first incubated with secondary antibody (biotinylated anti-rabbit IgG,1:200 dilution, AK 5004, Vectastain, USA) for 30 min and then with the avidin-biotin complex (ABC) at room temperature for 30 min. The sections were subsequently subjected to chromogenic staining (1-3 minutes) using the chromogenic substrate 3,3’-diaminobenzidine (DAB) and 0.005% H2O2. The sections were counterstained with Harris hematoxylin for 2-5 minutes, dehydrated in graded ethanol and xylene, and mounted with the Entellan new mounting medium (Merck, Germany). The sections were observed under a Nikon 180 microscope. Eight fields of view were randomly selected for each section, and the positive cell index was calculated.7. Western blotThe excised left atrial tissue samples were homogenized on ice and were then centrifuged. The supernatant was then collected. All of the protein samples were degenerated at 100 ℃, separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and later transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked by 5% nonfat milk and then incubated overnight with an anti-P-p38 MAPK antibody (1:200 dilution). The membranes were incubated with a biotinylated goat anti-rabbit antibody (1:5000 dilution) for 1 h at room temperature. The proteins were detected by enhanced chemiluminescence (Millipore, Billerica, MA, USA). The results were normalized to GAPDH, and the samples were imaged using an LAS-4000 Mini Luminescent Image Analyzer (Fujifilm, Tokyo, Japan).8. Statistical analysisOne-way analysis of variance (ANOVA), performed using SPSS 19.0 software, was used for statistical analysis.Results:1. Effects of rosuvastatin on cardiac functional parameters and infarct size4 MI groups(C, R, T1 and T2) showed cardiac dilatation and systolic dysfunction, as evidenced by increased LAD, LVDd and LVDs, and decreased LVEF and LVFS, when compared with sham group. LVEF and LVFS were elevated in the R group in comparison to the C group. Rosuvastatin exhibited cardiac protective effects by improving LV function. There is no statistically significant difference in the infarct size among the 4 MI groups (C, R, T1, and T2).2. Hemodynamic assessmentLVEDPs were drastically elevated in the 4 MI groups (the C, R, T1, and T2 groups) compared with the sham-operated group. Among the MI groups, the LVEDP was markedly reduced in the R, T1, and T2 groups by comparison to the C group. No significant difference in the LVEDP was detected between the R group and the T1 group (P=0.37). In addition, the LVEDP was significantly lower in the T2 group than the R group (P<0.05). Therefore, the T1 group was selected as the positive control group in this study to examine the effects of rosuvastatin on atrial structural remodeling after MI.3. Effects of rosuvastatin on atrial fibrosisThe left atrial myocardial tissues isolated from the 5 groups of rats were subjected to Masson’s trichrome staining. The cardiomyocytes were stained red, and the collagen fibers were stained blue. After MI, a proportion of the left atrial cardiomyocytes underwent degeneration and apoptosis, reducing the size of the myocardial area. Meanwhile, increased collagen synthesis led to an enlarged collagen network area. Therefore, the CVF was increased, and myocardial interstitial fibrosis was developed. As shown in Figure 3, the CVFs were significantly elevated in the 4 MI groups (C, R, T1, and T2) compared with the sham-operated group. The CVFs were markedly reduced in the R, T1, and T2 groups in comparison to the C group. Moreover, the CVF was lower in the R group compared to the T1 group or the T2 group, and the differences were statistically significant.4. Rosuvastatin suppressed P-p38 MAPK as demonstrated by immunohistochemical stainingThe positive cell index refers to the percentage of positive cells within the total cells in a field of view. The nuclei of the P-p38 MAPK positive cells were stained brownish-yellow. The positive cell indices were drastically increased in the 4 MI groups (C, R, T1 and T2) compared with the sham-operated group. Compared to the C group, the indices of the P-p38 MAPK positive cells were markedly reduced in the T1 and T2 groups (reduced by 20.4% and 29.6% respectively), while the index was decreased even more profoundly in the R group (decreased by 44.6%). The differences in the indices of the P-p38 MAPK positive cells among the R, T1, and T2 groups were statistically significant.5. Inhibition of P-p38 MAPK by rosuvastatin according to western blot analysisThe expression of P-p38 MAPK was significantly elevated in the 4 MI groups (C, R, T1, and T2) compared with the sham-operated group. The P-p38 MAPK expression was markedly reduced in the R, T1, and T2 groups in comparison with the C group. In addition, the P-p38 MAPK expression in the R group was significantly lower than in the T1 group. There is no statistically significant difference in the P-p38 MAPK level between the R group and the T2 group. No statistically significant difference was detected in the expression of p38 MAPK among all of the 5 groups (S, C,R,T1,and T2).Conclusions:1. Intragastric administration of rosuvastatin (10 mg/kg body weight/day) and low-dose torasemide (1 mg/kg body weight/day) in rats with MI resulted in similar degrees of reduction in LVEDP.2. Atrial CVF was elevated in rats with MI, indicating the occurrence of atrial structural remodeling and the development of atrial fibrosis. In addition, the level of P-p38 MAPK distributed primarily in the nuclei of the atrial cardiomyocytes was increased, which was most likely related to atrial structural remodeling after MI.3. Rosuvastatin inhibited atrial structural remodeling in rats with MI. The mechanism underlying this phenomenon may be associated with the downregulation of P-p38 MAPK by rosuvastatin.Significance:To investigate the direct effect of rosuvastatin on the atrial structural remodeling after MI, torasemide was selected on the basis of our preliminary experiments as a positive control. The purpose of using torasemide was to eliminate the indirect effects of rosuvastatin on atrial structural remodeling after MI. The results showed that P-p38 MAPK played an important role in the atrial structural remodeling and rosuvastatin attenuated atrial structural remodeling in rats with MI through the inhibition of the p38MAPK signaling pathway. Our study may provide new ideas and targets for the prevention of atrial arrhythmias. |
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