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| ORIGINAL ARTICLE |
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| Year : 2017 | Volume
: 2
| Issue : 2 | Page : 81-85 |
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Rapamycin attenuates atrial fibrosis in 5/6 nephrectomized rats by inhibiting mammalian target of rapamycin and profibrotic signaling
Yajuan Yang1, Mengqi Gong1, Hongliang Li1, Xue Liang1, Zhiwei Zhang1, Meng Yuan1, Yue Zhang1, Zhanquan Jiao1, Gary Tse2, Guangping Li1, Tong Liu1
1 Tianjin Key Laboratory of Ionic-Molecular Function of Cardiovascular Disease, Department of Cardiology, Tianjin Institute of Cardiology, Second Hospital of Tianjin Medical University, Tianjin 300211, China
2 Department of Medicine and Therapeutics, Chinese University of Hong Kong; Li Ka Shing Institute of Health Sciences, 30-32 Ngan Shing St, Hong Kong 999077, China
| Date of Web Publication | 31-Jan-2018 |
Correspondence Address:
Tong Liu
Department of Cardiology, Tianjin Key Laboratory of Ionic-Molecular Function of Cardiovascular Disease, Tianjin Institute of Cardiology, Second Hospital of Tianjin Medical University, Tianjin 300211
China
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/IJHR.IJHR_1_17
Background: Atrial fibrosis plays a vital role in the pathogenesis of atrial fibrillation. However, the complex interplay between inflammation and remodeling remains incompletely understood. In this study, we examined the potential beneficial effects of the immunosuppressant rapamycin on reverse atrial remodeling in a 5/6 nephrectomized (5/6Nx) rat model of chronic kidney disease (CKD). Materials and Methods: Sprague-Dawley male rats were housed under controlled conditions with constant temperature and humidity for 1 week before the operation. They were assigned randomly to the following groups: (1) sham procedure with vehicle treatment, (2) 5/6Nx group with vehicle treatment, and (3) 5/6Nx with rapamycin treatment. The 5/6Nx group underwent nephrectomy by resection of the upper and lower thirds of the left kidney, followed by right nephrectomy. The rapamycin group received daily rapamycin (1 mg/kg/day) from the 4th week to the 8th after operation. Results: A significant increase in the protein expression levels of mammalian target of rapamycin (mTOR), p38, and extracellular signal-regulated kinase was observed in the 5/6Nx + vehicle group (1.56 ± 0.12 vs. 0.72 ± 0.06; 2.64 ± 0.40 vs. 1.20 ± 0.20; and 3.02 ± 0.71 vs. 1.42 ± 0.34; all P < 0.05), which were suppressed by rapamycin treatment (0.88 ± 0.08 vs. 1.56 ± 0.12; 1.96 ± 0.21 vs. 2.64 ± 0.40; and 1.87 ± 1.87 vs. 3.02 ± 0.71; all P < 0.05). Cardiomyocyte hypertrophy and extensive interstitial fibrosis of the atrium were observed in the 5/6Nx + VEH group (P < 0.05). These changes were attenuated in the 5/6Nx + rapamycin group (P < 0.05). Conclusions: In this 5/6Nx CKD rat model, atrial fibrosis was mediated via the mTOR pathway, which was attenuated by rapamycin.
Keywords: Atrial fibrillation, atrial fibrosis, mammalian target of rapamycin, nephrectomy, rapamycin
How to cite this article:
Yang Y, Gong M, Li H, Liang X, Zhang Z, Yuan M, Zhang Y, Jiao Z, Tse G, Li G, Liu T. Rapamycin attenuates atrial fibrosis in 5/6 nephrectomized rats by inhibiting mammalian target of rapamycin and profibrotic signaling. Int J Heart Rhythm 2017;2:81-5 |
How to cite this URL:
Yang Y, Gong M, Li H, Liang X, Zhang Z, Yuan M, Zhang Y, Jiao Z, Tse G, Li G, Liu T. Rapamycin attenuates atrial fibrosis in 5/6 nephrectomized rats by inhibiting mammalian target of rapamycin and profibrotic signaling. Int J Heart Rhythm [serial online] 2017 [cited 2023 Jun 5];2:81-5. Available from: https://www.ijhronline.org/text.asp?2017/2/2/81/224353 |
| Introduction | |  |
Atrial fibrillation (AF) is the most common cardiac arrhythmia observed in clinical practice, increasing the risk of stroke, heart failure, and death. The prevalence of AF is increasing, which is in part due to an aging population.[1] Structural remodeling of the atrium, especially interstitial fibrosis, plays an important role in the pathogenesis and maintenance of AF. Thus, in a canine model using ventricular tachycardia pacing (at 240 bpm), significant atrial interstitial fibrosis was observed, which accompanied an increase in AF inducibility by programmed electrical stimulation.[2] In humans, cardiovascular comorbidities and AF appear to have a common denominator of conditions that predispose to fibrosis, such as hypertension, ischemic heart disease, and diabetes, which are highly predictive of AF occurrence.[3]
The 5/6 nephrectomized (5/6Nx) rat model has been used to study chronic kidney diseases (CKDs). In this system, features such as glomerular hypertrophy, increased expression of proinflammatory and profibrotic cytokines, infiltration of the interstitium by leukocytes, and renal fibrosis are observed, in corroboration with clinical findings of humans. Recent work has demonstrated renal fibrosis in the final common pathway of CKD. Clinical studies have found high incidence and prevalence of AF in patients with CKD.[4],[5],[6],[7],[8],[9] Rapamycin, via inhibition of the mammalian target of rapamycin (mTOR) pathway, has been shown to ameliorate interstitial inflammation, fibrosis, and loss of function in the kidneys in an animal CKD model.[10] This agent is used clinically both as an immunosuppressant for organ transplantation with minimal end-organ toxicity [11] and an antineoplastic drug for renal cancers.[12] Moreover, increased circulating levels of inflammatory biomarkers observed in this model may be attributed to higher levels of oxidative stress, such as indoxyl sulfate, a uremic toxin, which can facilitate atrial fibrosis and AF.[13],[14] In the heart, rapamycin is shown to exert antiproliferative action, leading to a significant reduction of coronary artery restenosis when applied locally with a drug-eluting stent [15] or taken systemically.[16]
The mTOR pathway plays an important role in the progression of CKD in animal models. Although its activation has been shown as an early mediator of fibrosis in the kidneys, whether this mediates a similar process in the atrium is unclear.[17] Since interstitial fibrosis is an important substrate for the pathogenesis of AF,[18] we hypothesized that treatment of rapamycin may reduce atrial interstitial fibrosis in the setting of CKD by inhibiting mTOR and profibrotic signaling in a rat model of CKD induced by 5/6Nx.
| Materials and Methods | | |
Animals
Sprague-Dawley male rats aged 8 weeks (average body weight of 200 ± 20 g) were bred in the animal facility of the Tianjin Institute of Cardiology, according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised in 1996). The rats were housed in individual cages under controlled conditions with a constant temperature of 23°C ± 2°C and a humidity of 45%–65% and were exposed to a 12-h day/night cycle and had free access to normal food and tap water for 1 week before the operation. The protocols adopted in the present study were approved by the Experimental Animal Administration Committee of Tianjin Medical University and Tianjin Municipal Commission for Experimental Animal Control, which follows the guidelines established by the US National Institutes of Health.
Chronic kidney disease rat model and effects of rapamycin treatment
Rats were assigned randomly to the following groups by a random number table (n = 8 per group): sham + vehicle (VEH), 5/6Nx + VEH, and 5/6Nx + rapamycin. After 1 week feeding, all animals were fasted for 12 h. Procedures were performed with the animals under general anesthesia (pentobarbital sodium 50 mg/kg intraperitoneally) using strict hemostasis and aseptic techniques. The 5/6Nx group underwent nephrectomy by resection of the upper and lower thirds of the left kidney, followed by right nephrectomy.[19],[20],[21] The control group underwent a sham procedure (sham group). Rapamycin group was given to the rats by daily lavage of rapamycin (1 mg/kg/day) dissolved in carboxymethyl cellulose buffer from the 4th week to the 8th after operation. After 4 weeks of treatment, the animals were sacrificed under general anesthesia and their hearts were removed for subsequent analysis. Left atrial (LA) tissue was immediately harvested, cleaned, and snap-frozen in liquid nitrogen. Frozen tissues were then stored at −80°C for Western blot analysis. Right atrial (RA) tissues were removed, weighed, fixed in 10% phosphate-buffered formalin, and embedded in paraffin for histological analysis.
Renal function parameters
The blood was collected 0.5 mL from angular vein. Serum creatinine and blood urea nitrogen (BUN) were determined before initiation of operation and subsequently every other week by enzyme-linked immuno sorbent assay (ELISA) following the manufacturer's directions (Wuhan Huamei, China).
Western blot analysis
Frozen LA tissues were homogenized and atrial tissue proteins were extracted by lysis buffer. The lysates were centrifuged at 15,000 g for 15 min, and the supernatants were collected. Protein concentrations were measured by the Bradford method. Each lysate was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred by electrophoresis onto a polyvinylidenedifluoride membrane. For immunoblot experiments, membranes were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated at 4°C overnight with primary antibody. Then, the membrane was washed with TBST and incubated with the horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:5000 and goat anti-mouse, 1:5000) for 1 h. Primary antibody used were: β-actin (1:5000, Sigma, USA, A5316), anti-mTOR (1:2000, Abcam, UK, ab32028), anti-p38 (1:2000, Abcam, UK, ab131186), and anti-extracellular signal-regulated kinases (ERK) (1:2000, Abcam, UK, ab73272). Equal protein loading of the samples was verified by staining monoclonal antibody β-actin and Western Lightning TM Chemiluminescence Reagent (PerkinElmer, USA) was applied to visualize bands (Tanon 5200, China).
Histological studies
After cutting into 5 μm cross-sections, the tissues were stained with hematoxylin and eosin (HE) to observe the morphological changes of cardiomyocytes and Masson's trichrome stain to evaluate interstitial fibrosis. Digital images were scanned onto a personal computer using Image-Pro Plus 6.0 (Scion Corporation, USA) and the extent of fibrosis was determined by dividing the area of collagen deposition by the entire cardiac tissue area. Five randomly selected sections of each group were utilized for quantification. The data of cardiac collagen volume fraction and cross-sectional area were analyzed using SPSS 17.0 (SPSS Inc., Chicago, USA).
Statistical analysis
Data were presented as mean ± standard deviation. Differences between the groups were analyzed for statistical significance using the one-way analysis of variance. All data were analyzed using SPSS 17.0 and P < 0.05 was considered statistically significantly.
| Results | | |
Blood parameters on renal function
Following 5/6Nx-treatment, serum creatinine (76.64 ± 5.41 vs. 51.51 ± 3.80 μmol/l, P < 0.0001) and BUN concentrations (14.04 ± 2.72 vs. 5.28 ± 0.6 mmol/l, P < 0.0001) were higher in the 5/6Nx group when compared to the VEH group. These findings confirm the validity of the 5/6Nx rat model, demonstrating features of renal failure. These rats also had dry hair, thin and sparse fur, and increased water consumption and urine volume following nephrectomy. After 4 weeks of treatment, there was no significant difference in serum creatinine (78.45 ± 5.33 vs. 76.64 ± 5.41 μmol/l, P = 0.51) and BUN (15.04 ± 2.05 vs. 14.04 ± 2.72 mmol/l, P = 0.42) between the 5/6Nx + rapamycin group and the 5/6Nx group, respectively. These findings suggest that rapamycin had no significant effect on renal function.
Protein expression levels of the atrium
The protein expression levels of mTOR, p38, and ERK were significantly upregulated in 5/6Nx + VEH group compared with sham + VEH group (1.56 ± 0.12 vs. 0.72 ± 0.06, P < 0.05; 2.64 ± 0.40 vs. 1.20 ± 0.20, P < 0.05; and 3.02 ± 0.71 vs. 1.42 ± 0.34, P < 0.05) [Figure 1]. Treatment of 5/6Nx-treated rats with rapamycin (5/6Nx + rapamycin) significantly reduced the expression levels of these proteins (0.88 ± 0.08 vs. 1.56 ± 0.12, P < 0.05; 1.96 ± 0.21 vs. 2.64 ± 0.40, P < 0.05; and 1.87 ± 1.87 vs. 3.02 ± 0.71, P < 0.05). | Figure 1: Mammalian target of rapamycin, extracellular signal-regulated kinase, and p38 protein expression in left atrial tissue estimated by Western blot. (a) Representative protein expression. (b) Analysis of Western blot. * means significant different (P < 0.05) compared with sham + VEH group. # means significant different (P < 0.05) compared with 5/6Nx+VEH group
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Histology of atrial tissue
A representative image of the RA histology is shown in [Figure 2]a. In the nephrectomy group, the cardiomyocytes were hypertrophied and showed greater collagen deposition. Thus, RA cardiomyocytes in the 5/6Nx + VEH group had increased mean cross-sectional area compared with sham + VEH group (P< 0.05), which was reduced by rapamycin treatment (P = 0.03), although this was not fully reversible compared to baseline (P< 0.05) [Figure 2]b. Compared with the sham + VEH group, extensive, heterogeneous atrial interstitial fibrosis was observed in the 5.6Nx + VEH group (P< 0.05), which was attenuated by rapamycin (P< 0.05) [Figure 2]c. | Figure 2: Right atrial cardiomyocyte mean cross-sectional area and atrial interstitial fibrosis in 3 groups. (a) Showing the representative of right atrial cardiomyocyte cross-sectional area evaluated from hematoxylin and eosin stained and atrial interstitial fibrosis evaluated from Masson's stain. (b) Illustrates cardiomyocyte cross sectional area in sham + VEH, 5/6Nx + VEH and 5/6Nx + rapamycin groups. (c) Percentage of Masson's stain showing interstitial fibrosis from 3 groups. * means significant different (P < 0.05) compared with sham + VEH group. # means significant different (P < 0.05) compared with 5/6Nx+VEH group
Click here to view |
| Discussion | | |
The main finding of our study is that rapamycin attenuated atrial interstitial fibrosis by inhibition of the mTOR pathway in a rat model of CKD using 5/6Nx. Increased reactive oxygen species generation has been detected in the 5/6Nx model, leading to progressive glomerular injury.[22] In terms of atrial remodeling, higher levels and activities of the enzyme, nicotinamide adenine dinucleotide phosphate oxidase, have been implicated in the pathogenesis of interstitial fibrosis and enhanced AF vulnerability in 5/6Nx rats.[21] Higher levels of oxidative stress may further upregulate ERK expression. ERK is important for many cellular processes such as cell proliferation, differentiation, adhesion, migration, and survival.[23] A second pathway, phosphoinositide 3-kinase (PI3K)/Akt/mTOR, is also involved in these processes,[24] and increased activation of this signaling system has been implicated in both renal [25] and cardiac fibrosis.[26] Recent work has demonstrated cross-talks between these two pathways.[27] Previous experiments have demonstrated its significance in cancer therapeutics, in which growth factors can activate both the Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathways.[26],[28] Furthermore, ERK can regulate tuberous sclerosis complex gene, which is also regulated by mTOR.[28]
In our study, we speculated that an increased expression of ERK may be related to mTOR upregulation via receptor cross-activation. When rapamycin inhibited mammalian target of rapamycin complex 1 (mTORC1), ERK was also decreased and maintained the same trend with mTORC1 via cross-inhibition. Interestingly, combined suppression of mTOR and ERK signaling pathways may have advantages over mTOR inhibition individually in the treatment of tuberous sclerosis.[29] These findings indicate possible mTOR-mediated negative feedback regulation on ERK. In some cases, members of the two pathways, such as p70S6K, will phosphorylate the same molecule in the translation complex, such as ribosomal protein S6. In our experiments, we observed that the atrial fibrosis was attenuated in 5/6Nx + rapamycin group compared with the 5/6Nx group. However, other studies have shown that rapamycin at 10 mg/kg, a concentration that activates autophagy, partially abolishes the protective effect of bFGF in a mouse model of myocardial ischemia/reperfusion.[30] By contrast, in our study, a much smaller dose (1 mg/kg) of rapamycin was chosen. The beneficial effects of this agent at a low dose have also been demonstrated in the lungs (2 mg/kg).[31] Further studies need to investigate the optimal dosage of rapamycin to control the autophagy and inhibit atrial fibrosis. Together, these findings suggest that the mTOR pathway was involved in atrial fibrosis, in which increased oxidative stress is likely to play an important role, as reviewed previously.[32]
Limitations
The aim of this study is to examine the proof-of-concept that the mTOR inhibitor rapamycin can reduce adverse structural remodeling of the atrium by inhibiting fibrosis, and to elucidate the molecular pathways by which this is achieved. To better characterize the molecular pathways, levels of phosphorylated mTOR and p38 can also be assessed by Western blotting. Future studies can use echocardiography and electrophysiological study to assess mechanical and electrical functions of the atria, respectively.
| Conclusions | | |
In our study, rapamycin attenuated atrial fibrosis in 5/6Nx rats, in which mTOR signal pathway played an important role. However, further studies are needed to explore the specific mechanisms through which this is achieved.
Financial support and sponsorship
This work was supported by grants (30900618, 81270245, 81570298 to T.L.) from the National Natural Science Foundation of China and the Tianjin Natural Science Foundation (16JCZDJC34900 to T.L).
Conflicts of interest
There are no conflicts of interest.
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