TGF-β1 affects cell-cell adhesion in the heart in an NCAM1-dependent mechanism

https://doi.org/10.1016/j.yjmcc.2017.08.015Get rights and content

Highlights

  • TGFβ1 affects intercalated disc composition leading to ventricular dysfunction.

  • TGFβ1 alters cardiomyocyte adhesion through NCAM1.

  • NCAM1 in failing human hearts is driven by TGFβ1 activation.

  • Targeting NCAM1 counteracts TGFβ1 deleterious effects in the heart.

Abstract

The contractile property of the myocardium is maintained by cell-cell junctions enabling cardiomyocytes to work as a syncytium. Alterations in cell-cell junctions are observed in heart failure, a disease characterized by the activation of Transforming Growth Factor beta 1 (TGFβ1). While TGFβ1 has been implicated in diverse biologic responses, its molecular function in controlling cell-cell adhesion in the heart has never been investigated. Cardiac-specific transgenic mice expressing active TGFβ1 were generated to model the observed increase in activity in the failing heart. Activation of TGFβ1 in the heart was sufficient to drive ventricular dysfunction. To begin to understand the function of this important molecule we undertook an extensive structural analysis of the myocardium by electron microscopy and immunostaining. This approach revealed that TGFβ1 alters intercalated disc structures and cell-cell adhesion in ventricular myocytes. Mechanistically, we found that TGFβ1 induces the expression of neural adhesion molecule 1 (NCAM1) in cardiomyocytes in a p38-dependent pathway, and that selective targeting of NCAM1 was sufficient to rescue the cell adhesion defect observed when cardiomyocytes were treated with TGFβ1. Importantly, NCAM1 was upregulated in human heart samples from ischemic and non-ischemic cardiomyopathy patients and NCAM1 protein levels correlated with the degree of TGFβ1 activity in the human cardiac ventricle. Overall, we found that TGFβ1 is deleterious to the heart by regulating the adhesion properties of cardiomyocytes in an NCAM1-dependent mechanism. Our results suggest that inhibiting NCAM1 would be cardioprotective, counteract the pathological action of TGFβ1 and reduce heart failure severity.

Introduction

Heart failure is a complex disease that results from the impairment of cardiac muscle function and often develops as a consequence of chronic neurohormonal and mechanical stress. While select heart failure treatments are available to reduce neuroendocrine signaling, such therapies do not prevent death and only mildly extend lifespan [1]. Hence, a better understanding of the molecular mechanisms underlying cardiac dysfunction is needed to aid in the development of new treatments that extend beyond neuroendocrine modulation.

The diseased heart is associated with remodeling of the intercalated discs (ID), structures responsible for transmission of contractile force between cardiomyocytes [2]. Proper organization of intercalated discs is necessary to maintain normal cell-cell interactions between myocytes and therefore preserve cardiac function [3]. Intercalated discs are composed of adherens junctions, desmosomes, and gap junctions [4]. The classical adhesion receptor present in the adherens junctions and desmosomes is composed of cadherins. The extracellular domain of cadherins promotes homophilic interactions that mediate strong myocyte-myocyte adhesion and allow proper maintenance of tissue structure [5]. In addition to cadherins, other receptors are targeted to the intercalated discs and regulate cardiomyocytes' coupling. Specifically, connexin proteins are key components of gap junctions, structures specialized in intercellular electric coupling [6]. In the past years it has become clear that intercalated discs are dynamic structures that are a target of complex signaling events that can alter their composition and compromise their function during disease states. Indeed, mislocalization of intercalated disc proteins is observed in heart failure and contributes to cardiac dysfunction [7]. Also, numerous mutations in intercalated disc proteins have been described, resulting in alterations to intercalated disc structure and consequently increasing susceptibility to deterioration of heart pump function [8].

During chronic stress or following insults (i.e. myocardial infarction or cardiotropic viral infection) complex signaling events perturb the homeostasis of the heart [9]. During these injury conditions secreted molecules coordinate the response of the heart to stress [10]. How this response results in disruption of cell adhesion in cardiomyocytes and overall dysfunction is not clear. A master regulator of tissue stress responses is Transforming Growth Factor beta 1 (TGFβ1) [11]. Activation of TGFβ1 is observed in the injured heart and a direct role of TGFβ1 in affecting cardiomyocytes function post-stress has been proven by Koitabashi and colleagues using genetic mouse models of cardiomyocyte-specific deletion of TGFβ1 receptors [12]. However, attempts to generate gain-of-function mouse models recapitulating the pathologic effects of TGFβ1 in the myocardium have been challenging, likely because TGFβ1 activation is tightly regulated and overexpression per se is typically not sufficient to increase its activity. Indeed, TGFβ1 is secreted as part of a complex with two other polypeptides, the latent TGFβ1 binding protein (LTBP) and the latency-associated peptide (LAP), together making up the latency complex [13], [14]. Attachment of TGFβ1 to the latency complex occurs through disulfide bonds that prevent the release of active TGFβ1 and therefore limit TGFβ1 bioavailability [14]. In 2000, Nakajima and colleagues successfully generated a mouse model expressing a mutant form of TGFβ1 (cysteine-to-serine substitution at residue 33) showing increased TGFβ1 activity in the heart, but not sufficient to uncover a ventricular phenotype [15]. These results have left an unmet need to fully understand the role of TGFβ1 signaling in ventricular cardiomyocyte dysfunction.

Here we adopted a mouse model of active TGFβ1 that mimics the degree of TGFβ1 activation observed in cardiomyopathy and we report a novel mechanism of TGFβ1-mediated ventricular dysfunction through a pathway dependent on Neural Adhesion Molecule 1 (NCAM1) to facilitate defective cardiomyocyte adhesion.

Section snippets

Procurement of human heart samples

The studies on human heart tissue were performed under the guidelines of the Declaration of Helsinki, with oversight by the Institutional Review Boards at The Ohio State University (protocol no. 2012H0197). Failing heart samples were obtained from patients who were diagnosed with end-stage heart failure and were receiving a heart transplant at The Ohio State University Wexner Medical Center. Non-failing hearts were obtained in collaboration with the Lifeline of Ohio Organ Procurement program

TGFβ1 activation induces ventricular dysfunction

TGFβ1 is induced in the injured heart and is considered a central player in cardiac diseases [17], [23]. Indeed, we observed increase in TGFβ1 activity in human failing hearts by ELISA (Fig. 1A). We have also found that cardiomyocytes are a source for TGFβ1 in human failing myocardium (Supplemental Fig. 1A and B). To mimic the TGFβ1 activation occurring during cardiomyopathies and to study the direct mechanistic role of TGFβ1 in cardiomyocytes we have adopted a transgenic mouse model for

Discussion

The TGFβ1 signaling pathway has been recognized as critical in the context of stress responses as well as in the development of multiple organ systems, including the heart [27]. The importance of understanding how TGFβ1 exerts its downstream action is demonstrated by the fact that perturbation of TGFβ1 signaling is known to occur in disease states in the heart and beyond. Also, genetic mutations affecting TGFβ1 activation are a primary cause of disease or act as disease modifiers in both

Acknowledgements

The authors would like to thank: Susan Montgomery, Erin Bumgardner, and Emily Jarvis for their help in consenting the patients for this study; Abraham Zawodni (Lifeline of Ohio) for help with obtaining clinical correlates for the donor hearts; Nathaniel Murphy for help with mouse phenotyping; Dr. Patricia Maness (UNC-Chapel Hill) for providing Ncam1 plasmids.

Sources of funding

This work was supported by grants from the NIHR00HL121284 and R01HL136951 (to F.A.), R00HL116778 (to M.A.A.), R01HL113084 (to P.M.L.J.), T32GM068412 (to J.M.P.).

Disclosures

None.

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