Original article
Serotonin receptor 2B signaling with interstitial cell activation and leaflet remodeling in degenerative mitral regurgitation

https://doi.org/10.1016/j.yjmcc.2017.12.014Get rights and content

Highlights

  • Microarray and Network Reconstruction analysis in MVP patients and controls reveals a role for the 5HT signaling pathway in myxomatous mitral valve pathology.

  • 5HTR2B is upregulated in human MVP leaflets when compared to control as well as in a canine model of myxomatous mitral regurgitation.

  • A 5HTR2B antagonist (LY 272015) reduces MVICs activation under biomechanical stimulation ex vivo.

  • LY 272015 prevents Angiotensin II-mediated heart valve thickening in vivo.

  • We reported for the first time a mechanistic connection between serotonin receptor signaling and one of the most common heart valve diseases, MVP, a disorder affecting millions of patients.

Abstract

Aims

Mitral valve interstitial cells (MVIC) play an important role in the pathogenesis of degenerative mitral regurgitation (MR) due to mitral valve prolapse (MVP). Numerous clinical studies have observed serotonin (5HT) dysregulation in cardiac valvulopathies; however, the impact of 5HT-mediated signaling on MVIC activation and leaflet remodeling in MVP have been investigated to a limited extent. Here we test the hypothesis that 5HT receptors (5HTRs) signaling contributes to MVP pathophysiology.

Methods and results

Diseased human MV leaflets were obtained during cardiac surgery for MVP; normal MV leaflets were obtained from heart transplants. MV RNA was used for microarray analysis of MVP patients versus control, highlighting genes that indicate the involvement of 5HTR pathways and extracellular matrix remodeling in MVP. Human MV leaflets were also studied in vitro and ex vivo with biomechanical testing to assess remodeling in the presence of a 5HTR2B antagonist (LY272015). MVP leaflets from Cavalier King Charles Spaniels were used as a naturally acquired in vivo model of MVP. These canine MVP leaflets (N = 5/group) showed 5HTR2B upregulation. This study also utilized CB57.1ML/6 mice in order to determine the effect of Angiotensin II infusion on MV remodeling. Histological analysis showed that MV thickening due to chronic Angiotensin II remodeling is mitigated by a 5HTR2B antagonist (LY272015) but not by 5HTR2A inhibitors.

Conclusion

In humans, MVP is associated with an upregulation in 5HTR2B expression and increased 5HT receptor signaling in the leaflets. Antagonism of 5HTR2B mitigates MVIC activation in vitro and MV remodeling in vivo. These observations support the view that 5HTR signaling is involved not only in previously reported 5HT-related valvulopathies, but it is also involved in the pathological remodeling of MVP.

Introduction

Mitral Valve Prolapse (MVP) affects approximately 7.2 million individuals in the US, and over 144 million individuals worldwide [1], [2]. MVP is defined echocardiographically as a single or bileaflet prolapse that is at least 2 mm above the annular plane in the long-axis view, with or without leaflet thickening [2], [3]. In contrast to other types of mitral regurgitation (MR), such as ischemic MR – which is driven in large part by a ventricular remodeling – mitral insufficiency in the setting of MVP is primarily a disease of the valve leaflets. Severe MR associated with MVP is most commonly treated with surgery via repair or replacement of the mitral valve. In recent years, percutaneous alternatives have become available for patients who are deemed too high risk for surgical intervention [4].

The pathophysiology of MVP involves myxomatous degeneration, which is defined by the accumulation of mucopolysaccharides and other extracellular matrix components that are responsible for the thickening and “proliferative” aspect of the valve tissue. The center scallop of the posterior leaflet is more commonly involved, as is the area of coaptation between the two leaflets at maximum stress. Heart valve leaflets are dynamic, multi-layered structures that are actively remodeled by the activation of their main cellular component, the valve interstitial cells (VICs) [5], [6], [7], [8]. In healthy adult valves, VICs are typically in a quiescent phenotype (qVICs); however these cells become active in response to altered biomechanical stimuli [6], [9], [10]. Notably, VIC activation is often associated with extracellular matrix (ECM) remodeling. We have previously described some of the biomechanical responses controlling VIC activation, typically with upregulation of alpha-smooth muscle cell actin (αSMA) during congenital and acquired valvulopathies [10], [11], [12], [13], [14], [15], [16], [17]. Thus, switching the VIC phenotype with upregulation of αSMA is a key component in mediating leaflet tissue growth and the overall leaflet's deformation [18], [19].

Numerous clinical and experimental studies have reported serotonin (5HT) dysregulation as a possible element in the development of MR in humans and animals [20], [21], [22], [23], [24], [25]. 5HT is associated with cardiac valvulopathy in several clinical settings, including: carcinoid heart disease due to 5HT secreting chromaffin tumors, which is characterized by fibrotic endocardial plaques and associated right-sided heart valve dysfunction [26]; and the diet drug combination Fenfluramine/Phentermine (Fen/Phen) - resulting in sustained 5HT activity based on Fen inhibition of the serotonin transporter (SERT) and 5HT receptor agonist, and sustained 5HT signaling due to monoamine oxidase inhibition (Phen) - which was withdrawn by the FDA in 1997 because of the relatively large number of valvulopathy cases that affected primarily the mitral and aortic valves [27], [28], [29], [30], [31], [32]. The anti-Parkinson's agent, Pergolide, and other related drugs primarily affect dopamine receptors, however off target effects related to 5HT are most likely responsible for the valvulopathy observed in a small, but significant number of patients [27], [28], [33]. 5HT-valvulopathy has been observed in transgenic mice deficient in the serotonin transporter (SERT), resulting in prolonged 5HT-receptors (5HTRs) signaling, resulting in thickened cardiac valves [20], [34]. Valvulopathies have also been observed in rats which were administered high doses of 5HT [35], [36]. All of these clinical and laboratory results indicate a pivotal role for 5HT homeostasis in normal valve physiology that can be disrupted by aberrant 5HTRs signaling activity. However, 5HTR mediated VIC activation and MV leaflet remodeling is not well understood. The present paper represents the first example of a mechanistic connection between serotonin receptor signaling and MVP, one of the most common heart valve diseases affecting millions of patients.

5HT receptors are encoded by 13 distinct genes and classified into 7 families including the Gq/11-coupled 5HT receptors (5HTR2), which are understood to mediate both the contractile and proliferative effects of 5HT on smooth muscle cells, fibroblasts, and valve interstitial cells [37], [38]. In Xu J, et al., we dissected the role of 5HTR2 receptors in isolated valve cells [23]. Specifically, it was shown that 5HTR2 agonism activates the MAP kinase pathway via ERK [37], [38]. In the present studies, we investigated the hypothesis that 5HT receptor (5HTR) signaling contributes to MVP pathophysiology. We will present evidence that 5HT signaling through 5HTR2B controls human-derived VIC activation in vitro (cell culture) and ex vivo (biomechanical assays). We will demonstrate 5HTR signaling in a canine model of MVP with data showing that a 5HT antagonist abrogates VIC activation via inhibition of ERK phosphorylation. Finally, we will show that 5HT-mediated MV remodeling is associated with a murine model using chronic infusion of Angiotensin II (Ang II), a mechanism described to generate heart valve remodeling via TGF-beta/ROS and was recently proposed as a model of MVP [39].

Section snippets

Patient enrollment and classification

Patients requiring MV surgery were enrolled in this study with informed consent at the time of surgery in the Penn Cardiac Bioregistry (PCB) and the Valley Hospital Cardiothoracic Surgery Biobank, as approved by the IRB of the University of Pennsylvania Perelman School of Medicine (protocol #809349) and Valley Hospital (protocol #11.0009). All MVP patients had clinically indicated cardiac surgery. Each patient had a well-documented history of MR and presented with leaflet thickening and annular

Microarray and network reconstruction analysis in MVP patients and controls reveals a role for the 5HT signaling pathway in MVP pathology

As reported in our previous work, we performed microarray analysis of 4 MVP patients and 4 controls [17]; however, our prior publication did not report results on 5HT related gene expression patterns. Among the transcriptional activities of 19,553 human sequences determined by use of an oligonucleotide microarray, we found a total of 1883 probe sets which fulfilled the criteria for differential expression (GEO access number requested). These transcripts represent genes showing at least a

Discussion

5HT-related mechanisms have been previously reported in specific valvulopathies such as those related to 5HT secreting carcinoid tumors, serotonergic agents, such as dexfenfluramine, and ergot-derived dopaminergic agents used to treat Parkinson's Disease, such as carbergoline and pergolide. The present paper represents the first example of a mechanistic connection between serotonin receptor signaling and one of the most common heart valve diseases, MVP.

This work highlights the important role of

Acknowledgements

This research was supported in part by the following research grants and funds: NIHR01-HL131872 (GF and RJL), R01-HL122805 (GF), AHA GRANT24810002 (GF), The Kibel Fund for Aortic Valve Research (GF and RJL), and The Valley Hospital Foundation “Marjorie C Bunnel” charitable fund (GF and JG), NIHT32-HL007954 (KHD), NIHT32-HL007915 (RJL and KHD), Barth Memorial Mitral Valve Disease Fund (MAO, GF, EB), and both Erin's Fund and the William J. Rashkind Endowment of the Children's Hospital of

Disclosures

None.

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