Eplerenone Reverses Age-Dependent Cardiac Fibrosis Through Downregulating Osteopontin

doi:10.21203/rs.3.rs-1130279/v1

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Eplerenone Reverses Age-Dependent Cardiac Fibrosis Through Downregulating Osteopontin

https://doi.org/10.21203/rs.3.rs-1130279/v1

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Background: The risk of cardiovascular disease dramatically increases with Ageing. Nowadays it is fully revealed that the fibrotic remodelling is the main cause of cardiac structural and functional changes related to the normal aging process, but the related signaling pathways and mechanisms are incompletely understood. Thus finding the new therapeutic approaches targeting cardiac fibrotic remodelling, may be necessary to develop preventive care in geriatric population against cardiovascular diseases. In this study, we evaluated the potential role of osteopontin (OPN) as novel mediators of age-dependent fibrotic pathways in heart as well as the effect of eplerenone on cardiac fibrosis reversal.

Methods: Fischer-344 (F-344) rats has been used as three groups: young rats (2–3months old), aged rats (22-24 months old) without any treatment and aged rats treated with eplerenone (100 mg/kg/day) for 2 weeks. The expression level of OPN has been evaluated using real-time PCR and histological assessments were done to assess cardiac tissue fibrosis.

Results: The expression level of OPN in aged rats was significantly higher than young rats and treatment with eplerenone significantly attenuated the level of OPN as well as cardiac fibrosis compare to untreated aged rats.

Conclusion: Targeting cardiac fibroblast function with eplerenone could decrease expression of OPN marker and cause to reversal age-related cardiac fibrotic changes.

Molecular Biology

Cardiac remodeling

aging

fibrosis

osteopontin

eplerenone

inflammation

The prevalence of atrial fibrillation (AF) is increasing with high morbidity, mortality and health care costs worldwide [1]. Aging of the general population is the main causes of the growing of incidence and prevalence of AF [2, 3]. Since age is an unmodifiable risk factor, the probability of onset of new AF doubles due to each decade of age. The prevalence of AF is 0.12– 0.16% in young people aged ≤ 49 years, 3.7– 4.2% in the population between 60–70 years old, and 10–17% in old people aged ≥ 80 years.[4]. Consequently, increasing the risk of AF with aging is very common even in healthy subjects without history of cardiovascular diseases or risk factors of AF [5]. In this regard, considering of aging in the cardiac remodeling studies is so important and critical to provide new insights for preventing arrhythmogenic atrial remodeling and introducing novel therapies for AF [6]. Nonetheless unfortunately poorly basic and clinical researches have been done on cardiac structural and functional changes related to the normal aging trend. Fibroblast activation is one of the main causes of arrhythmogenic cardiac remodeling ultimate to interrupt coupling between cardio-myocyte and electrical signal propagation[7]. Moreover, the age related fibrotic patterns and the quality of the collagen network may impact myocardial stiffness or signal propagation. Although it is known that reactive fibrosis increases left ventricle stiffness [8], the electrical mapping studies suggest that the architecture of fibrotic remodeling in heart differentially impact electrical propagation and conduction delay [9]. In this regard, considering of aging in the cardiac remodeling studies is so important and critical to provide new insights for preventing arrhythmogenic atrial remodeling and introducing novel therapies for AF. Likewise, rhythm and rate control strategies in geriatric population have low efficacy and high mortality [10]. With this background, we are still at the beginning of understanding the detailed role and function of atrial fibroblasts and their contribution in AF and in line with this, finding novel therapeutic possibilities in this regard is of interest. So, finding a way not only to stop the atrial fibrosis but also to reverse this trend could be lead to innovate novel approaches for preventive care in geriatric population or effective therapy for patients suffering from AF. We think that if we could find a way not only to stop the atrial fibrosis but also to reverse this trend, it could be lead to innovate novel approaches for preventive care in geriatric population or therapy for patients with AF. Recently atrial fibrosis reversal has been proposed as novel therapeutic strategy in experimental studies [11]. To the best of our knowledge, no therapies have been developed to reverse atrial fibrosis effectively and much more mechanistic knowledge is essential for this therapeutic innovation. The aim of this study was to reveal the effects of eplerenone to suppress age-dependent fibrosis that is due to normal aging process and not to specific pathology like diabetes and hypertension with the focus on the role of osteopontin as a novel pro-fibrotic marker. In the heart OPN is associated with the extracellular matrix organization including collagen and fibronectin formation and also is involved in the Angiotensin II–induced fibrosis and could be related to the development of heart failure [12]. Recent studies have been reported that OPN could have a pivotal role in cardiac remodeling and fibrosis [13]. The eplerenone is a novel antagonist of aldosterone receptor which could decrease the coronary arteries damage through the OPN related mechanisms and the Angiotensin II–induced inflammation [14].

2.1. Animal model and grouping

To evaluate the age-induced fibrosis, 22 to 24 month-old Fischer-344 (F-344) rats have been used (Pasteur Institute of Iran), which are hybrid rats and offered as a model of aged-related changes by the National Institute on Aging and compare to inbred rats have more gradual aging rate and live longer [15]. All Experimental procedures with animals confirmed by ethics committee of Rajaie Cardiovascular Medical and Research center (ID: RHC.AC.IR.REC.1396.4) and are in accordance with National Institute of Health (NIH) guide for the care and use of laboratory animals. Overall, 45 animals divided in (n=15 rats per each group) the three groups: 1) young rats (2–3 months old), 2) aged rats (22-24 months old) without any treatment and 3) aged rats treated with eplerenone. The eplerenone has been administered 100 mg/kg/day subcutaneously for two weeks [16]. Fallowing treatment, rats were deeply anesthetized with xylazine (10 mg/kg) and ketamine (50 mg/kg) and after a mid-sternal incision, heart was quickly removed and washed in cold normal saline and atrial tissue was prepared for further studies.

2.2. Histological Assessment

Fallowing 2 weeks treatment, the animals were anesthetized and the atrial tissues were removed and fixed overnight in the paraformaldehyde solution (4%) followed by dehydration and embedding in paraffin. Then the tissues were cut into 5-µm thick sections. The Masson's Trichrome staining is applied for the detection of collagen fibers in atrial tissues. In the stained sections, the collagen fibres, the nuclei, and the background (muscle and cytoplasm) were stained blue, black and red respectively. The digital images of the stained sections were acquired with a light microscope at 400 × magnification (Olympus, Hamburg, Germany). Interstitial and perivascular fibrosis were measured using ImageJ software, (http://rsbweb.nih.gov/ij/), and the percentage of fibrosis was calculated.

2.3. RNA isolation and real-time polymerase chain reaction

Total RNA was isolated from atrial tissues using TRIzol reagent (Invitrogen) according to manufactures instructions and as previously described [17]. The concentration of RNA was measured by Nanodrop spectrophotometer (Thermo Scientific, USA) and the quality of RNAs evaluated by gel electrophoresis. The 1 microgram of total RNA was treated with RNasefree DNaseI (Thermo Scientific, USA) to eradicate any possible traces of DNA contamination. 0.5 micrograms of DNase-treated RNA was then used for reverse transcription, performed by PrimeScriptII TM Reagent kit (Takara, Japan). Quantitative PCR (q-PCR) was performed using SYBR Green chemistry with the BIOFACTTM 2X real-time PCR master mix (for SYBR Green I; BIOFACT, South Korea), in a StepOne PlusTM Real-Time PCR system (Applied Biosystems, Foster city, CA). Briefly, cDNA corresponding to 100 ng of RNA was applied to the SYBRGreen ready mix (0.5 µM of each specific primer, 10 µl of SYBR-Green ready mix, and 0.1 µl of Rox) in a total reaction volume of 20 µl. All real-time PCR reactions were performed in duplicates, and the resultant mean threshold cycles (Ct) were used for further analysis. The thermal profile comprised 95°C for 30s, 40 cycles of denaturation at 95°C for 05 s, and annealing at 60°C for 30 s. The Ct for individual reactions was calculated using StepOne software v2.3. The expression level of osteopontin in atrial tissues was normalized to that of internal control, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and relative gene expressions were calculated with the 2ˆ−ΔCt methodThe sequences of OPN and GAPDH primes were as follows: OPN; 5́-CCG ATG AGG CTA TCA AGG TC-3’ (forward) and 5́-ACT GCT CCA GGC TGT GTG TT-3’(reverse), GAPDH;5́-CAACTCCCTCAAGATTGTCACCAA-3́ (forward) and 5́-GGCATGGACTGTGGTCATGA-3́ (reverse). The primers for the target genes and GAPDH were purchased from Macrogen company (South korea).

2.4. Statistical Analysis

The GraphPad Prism was used to analysis the data, version 6.07 (USA), and presented as mean ± standard error of the mean (SEM). Between-group analyses were conducted using one-way analysis of variance (ANOVA), followed by the post hoc Tukey test and a P value equal to or less than 0.05 was considered as significant difference.

3.1. Eplerenone reverses age-dependent atrial fibrosis

The perivascular and interstitial fibrosis were evaluated by detection of collagen fibers in the atrial tissues in study groups. In the young control group interstitial fibrosis was detected very rarely in the atrial tissues (9.6±1.5%) (Fig. 1A and 1D) compare to aged control group (47.3±2.5%) (Fig. 1B and 1D) and differences between the young and aged rats was statistically significant (P≤0.001). Two weeks treatment with eplerenone significantly decreased the percentage of the atrial interstitial fibrosis in aged-treated rats (17.1±1.9%) (Fig. 1C and 1D) compared with untreated aged rats (P≤0.001).

Perivascular fibrosis in aged control rats (42.4±1.76%) (Fig. 2B and 2D) was significantly higher than young control rats (7.2±0.67%, P≤0.001) (Fig. 2A and 2D). Importantly, eplerenone treatment almost completely abolished the aging induced perivascular fibrosis in aged-treated rats compare to aged control rats (17.3±0.85%, P≤0.001) (Fig. 2C and 2D).

Both interstitial and perivascular fibrosis were reversed in atrial of aged rats after treatment with eplerenon.

3.2. Eplerenone decreases OPN Expression in aged rats

To investigate the role of pro-fibrotic mRNA transcripts for OPN and the therapeutic effects of eplerenone in normal aging process leading to atrial fibrosis, real time PCR analysis was done for OPN mRNA. Expression levels of OPN mRNA were significantly upregulated in aged control rats (0.16±0.02) (Fig. 3) compare to young control rats (0.049±0.01P≤0.01) (Fig. 3). Eplerenone treatment significantly downregulates the aging–induced upregulation of OPN mRNA levels compare to un-treated aged rats (0.069±0.018 P≤0.01) (Fig. 3).

The prevalence of AF is increasing worldwide. Age is a nonmodifiable risk factor of AF. Considering of aging in the cardiac remodeling studies could be helpful to provide novel therapy for AF and insight to prevent arrhythmogenic changes. Cardiac fibrosis causing AF trough interrupting fiber continuity and lead to disturbance of conduction and signal propagation [18] which is an crucial reason to therapeutic resistance in long standing AF [19]. In this study, we demonstrated that normal aging process induced the development of atrial fibrosis in aged rats compare to young rats. Treatment with eplenerone, a novel antagonist of aldosterone receptor, inhibited the development of both interstitial and perivascular fibrosis in atrial tissue. This result could reveal the etiological role of aldosterone receptor in the atrial structural remodeling of aged rats. Based on Masson's Trichrome staining results aged rat atrial tissue, showed substantial interstitial fibrosis compared to young rats, accordingly recent study has been shown that interstitial fibrosis could generate early after depolarizations and ectopic pacemaker which leads to arrhythmias and fibrillation [20]. Our study results also demonstrate that these age-related fibrotic changes could be modifiable and reversible by eplerenone. We showed that the mRNA expression level of OPN in aged rats was significantly more than young rats and eplerenone could effectively downregulate the OPN expression and its expression has been decreased in treated-aged rats compare to untreated-aged rats.

Based on these results, OPN has a crucial role in the progression of age-related cardiac fibrosis and atrial structural remodeling. As both tissue fibrosis and OPN expression decreased fallowing treatment with eplerenone, it could be concluded that age-related changes in atrial structure are reversible, through inhibiting pro-fibrotic component in spite of we know that “age” is the non-modifiable risk factor of cardiovascular disease. For the first time Brian L and et al. have been shown that age-induced atrial fibrotic changes could be reversible [21]. They demonstrated that relaxing has extensive anti fibrotic effects in the atria of aged rats via downregulating of the pro-fibrotic components such as, collagen I, collagen III and TGF-β. The OPN initially was discovered as a member of bone matrix protein [22], while more recently has introduced as an important protein involved in cardiovascular diseases, such as acute heart failure, coronary artery disease and post-MI wound repair [3, 23]. The OPN has low basal level expression and it may be less significant in normal cardiac tissue [24]. While in different cardiovascular diseases, activation of pro-inflammatory and pro-fibrotic factors act as trigger for upregulation of OPN [25, 26] and could lead to collagen remodeling. Previously it has been investigated that cardiac fibrosis could be inhibited by nitric oxide (NO) production [27] whereas upregulation of OPN suppresses the expression of inducible NO synthase (iNOS) and could develop the cardiac fibrosis by decreasing NO production [28]. The OPN also could regulate the renin-angiotensin-aldosterone system which is another contributor of fibrotic remodeling and leads to cardiac hypertrophy, left ventricle dilatation and myocardial compliance reduction [29]. The angiotensin II-induced fibrosis could increase through overexpression of OPN induced by cardiotropic Adeno-Associated Virus (AAV) serotype 9. This effect may be abolished by targeting the miR-21 following treatment with locked nucleic (LNA) [30]. These data demonstrate a crucial role of OPN in collagen remodeling in natural aging process [31]. Based on our obtained results eplerenone significantly decreased atrial fibrosis in aged rats and the effect of eplerenone on inhibition of fibrosis might partially be mediated through the inhibition of OPN expression, because treatment of aged rats with eplerenone decreased the mRNA expression of OPN significantly. Recently, an important relationship between OPN expression and angiotensin II–induced coronary artery damage has been suggested, which eplerenone could effectively inhibit the expression of OPN and ameliorate the inflammatory damages [32]. These results suggested that one of the mechanism involved in the renin-angiotensin-aldosterone system induced cardiac fibrosis is inflammation which is mediated by OPN and the inhibitory effects of eplerenone on the cardiac inflammation might be modulated by downregulating OPN gene expression. The previous study revealed that eplerenone decreased myocardial fibrosis and suppressed left ventricle expression level of TGF-b [33] and also recently it's demonstrated that eplerenone could reversed atrial structural remodeling and reduced AF inducibility, through a downregulation of inflammatory cytokines genes and Reactive oxygen species (ROS) production in the atrium [34]. Besides the previously released data the result of our study introduced a novel concept that eplerenone could be a potent anti-fibrotic drug in aged heart and its anti-fibrotic effects conducted via reduction of pro-fibrotic cytokine, OPN. One of the important points of using eplerenone as a potential therapy for cardiac fibrosis is that eplerenone has been already studied in humans particularly for heart failure and accepted as a safe treatment [35].

Aged rat hearts have more interstitial and perivascular fibrosis compared to young adult rats. Targeting cardiac fibroblast function with anti-fibrotic drug, eplerenone, could be effective to reduce atrial fibrosis through downregulating the pro-fibrotic cytokine, OPN. The eplerenone also may reverse age-related cardiac remodeling through inhibition of pro-fibrotic elements replacement in the cardiac tissue. Our findings suggest a novel potential therapy targeting OPN, to reduce aging-related cardiac fibrosis in geriatric population. Obviously we are not at the threshold of achieving this aim now and future mechanistic studies and more analysis are necessary to confirm these effects at the near future and introduce effective and safe therapy to prevent or reduce AF in the healthy geriatric papulation.

FUNDING: All aspects of this study funded by Research Deputy of Rajaie Cardiovascular and Research Center [grant number: 95137].

AUTHOR CONTRIBUTIONS

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mahshid Malakootian, Majid Maleki, Negar Mohammadian and Maedeh Arabian. The first draft of the manuscript was written by Maedeh Arabian and Mahshid Malakootian and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

CONFILICT OF INETREST

The authors declare that there is no conflict of interest in this study.

ETHICS APPROVAL

All Experimental procedures with animals confirmed by ethics committee of Rajaie Cardiovascular Medical and Research center (ID: RHC.AC.IR.REC.1396.4) and are in accordance with National Institute of Health (NIH) guide for the care and use of laboratory animals.

CONSENT TO PUBLISH

All authors have read and approve of the data being presented in the manuscript

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Eplerenone Reverses Age-Dependent Cardiac Fibrosis Through Downregulating Osteopontin

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Abstract

Figures

1. Introduction

2. Methods

2.1. Animal model and grouping

2.2. Histological Assessment

2.3. RNA isolation and real-time polymerase chain reaction

2.4. Statistical Analysis

3. Results

3.1. Eplerenone reverses age-dependent atrial fibrosis

3.2. Eplerenone decreases OPN Expression in aged rats

4. Disscusion

5. Conclusion

Declarations

References

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