Elsevier

Pharmacological Research

Volume 159, September 2020, 104922
Pharmacological Research

Original articles
Gap-134, a Connexin43 activator, prevents age-related development of ventricular fibrosis in Scn5a+/ mice

https://doi.org/10.1016/j.phrs.2020.104922Get rights and content

Highlights

  • Gap-134 chronic administration prevents ventricular fibrosis.

  • Gap-134 chronic administration increases Cx43 expression and phosphorylation.

  • Gap-134 or GW788388 treatments prevent cardiac fibroblasts abnormal proliferation.

Abstract

Down-regulation of Connexin43 (Cx43) has often been associated with the development of cardiac fibrosis. We showed previously that Scn5a heterozygous knockout mice (Scn5a+/−), which mimic familial progressive cardiac conduction defect, exhibit an age-dependent decrease of Cx43 expression and phosphorylation concomitantly with activation of TGF-β pathway and fibrosis development in the myocardium between 45 and 60 weeks of age. The aim of this study was to investigate whether Gap-134 prevents Cx43 down-regulation with age and fibrosis development in Scn5a+/− mice.

We observed in 60-week-old Scn5a+/− mouse heart a Cx43 expression and localization remodeling correlated with fibrosis. Chronic administration of a potent and selective gap junction modifier, Gap-134 (danegaptide), between 45 and 60 weeks, increased Cx43 expression and phosphorylation on serine 368 and prevented Cx43 delocalization. Furthermore, we found that Gap-134 prevented fibrosis despite the persistence of the conduction defects and the TGF-β canonical pathway activation.

In conclusion, the present study demonstrates that the age-dependent decrease of Cx43 expression is involved in the ventricular fibrotic process occurring in Scn5a+/− mice. Finally, our study suggests that gap junction modifier, such as Gap-134, could be an effective anti-fibrotic agent in the context of age-dependent fibrosis in progressive cardiac conduction disease.

Introduction

Chronic fibroproliferative diseases have been proposed to contribute to nearly 45% of the mortality in developed countries [1]. Among those, many cardiovascular diseases are associated with the development of excessive cardiac fibrosis, which contributes to their pathophysiological mechanisms [2]. This is most likely related to the crucial role played by the extracellular matrix in cardiac structure and function. However, despite its significant impact in cardiac diseases, fibrosis has become a focus for therapeutic development only recently [2]. There is thus a need for identifying novel therapeutic targets.

In the past, we have shown that the ∼50% reduction of NaV1.5 expression in Scn5a heterozygous knockout (Scn5a+/−) mice, which exhibit conduction defects, is associated with Cx43 expression remodeling and activation of the TGF-β canonical pathway leading to extensive ventricular fibrosis during ageing [[3], [4], [5]]. In control senescent mice, cardiac interstitial fibrosis onset is also concomitant with NaV1.5 and Cx43 down-regulation and cellular localization remodeling in ventricles [6]. Furthermore, in a heart failure mouse model, structural remodeling was shown to follow electrical remodeling characterized by a ∼50% decrease of NaV1.5 protein expression and a ∼70% reduction of Cx43 protein expression and phosphorylation [7]. A reduction of Cx43 and NaV1.5 expression has also been shown to precede fibrosis in a mouse model of cardiac hypertrophy [8,9]. Finally, based on studies performed with young hypertrophic or senescent conditional heterozygous Cx43 knockout (Cx43Cre-ER(T)/fl) mice, Jansen and co-workers proposed that a reduction of Cx43 expression might be the trigger for fibrosis development [10]. However, this might be more complex. Indeed, in a recent article, Valls-Lacalle and co-workers have shown that the enhanced fibrotic response seen after angiotensin II treatment in the same model of Cx43Cre-ER(T)/fl mice, expressing 50% of normal Cx43 content, is independent on this reduction in Cx43 expression, as it was not apparent on Cx43+/- mice [11]. On the contrary, a further reduction in Cx43 levels after 4-hydroxytamoxifen administration was associated with attenuation in collagen deposition in the angiotensin II-treated group. Similarly, permanent coronary artery ligature has been associated with a reduction in scar area in Cx43+/- mice [12]. These studies highlight the need to take into account all the changes expression and roles of Cx43 (hemichannel, phosphorylation, localization etc.), which can differently participate to fibrosis depending on the context.

Recently, we have shown that young Scn5a+/− mice are characterized by a higher Cx43 expression and phosphorylation than wild-type (WT) mice while, after the age of 45 weeks, Cx43 expression and phosphorylation decrease concomitantly with the activation of the TGF-β pathway and fibrosis in both WT and Scn5a+/− mice, although the decrease is larger in Scn5a+/− mice [3]. These results suggest that connexin 43 dowregulation might play a key role in cardiac fibrosis development in this model.

In this context, the aim of this study was to determine whether preventing Cx43 down-regulation with age prevents fibrosis development in Scn5a+/− mice. For this purpose, we investigated the effects of a chronic treatment with a gap-junction activator, Gap-134, on fibrosis development in Scn5a+/− mice between 45 and 60 weeks of age. Gap-134, also known as (2S, 4R)-1- (2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid, Danegaptide, or ZP1609, is a dipeptide analog of the antiarrhythmic hexapeptide, Rotigaptide. Gap-134 was shown to increase Cx43 coupling [13], prevents dephosphorylation of Cx43 [14] and prevent conduction abnormalities in models of cardiac disease [15,16].

Section snippets

Ethics statement

Animal experiments were performed in the animal facility of Nantes University Health Research Institute (Unité de Thérapeutique Expérimentale) which has been accredited by the French Ministry of Agriculture. The animal experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH publication No. 86-23, revised 1985) and to the EU Directive 2010/63/EU on the protection of animals used for

Fibrosis development is associated with Cx43 remodeling

We have previously shown that in Scn5a+/− mice, the decrease in Cx43 expression and phosphorylation at the age of 45 weeks, after an initial up-regulation up to 30 weeks of age, precedes fibrosis development between 45 and 60 weeks of age [3]. Present results confirm the presence of extensive ventricular fibrosis in 60-week-old Scn5a+/- mice when compared to WT mice (Fig. 1a). Since fibrosis expands mostly in subendocardium (Fig. 1a), we investigated Cx43 localization and distribution in

Discussion

In previous studies, we have shown that Scn5a heterozygous knockout mice develop extensive ventricular fibrosis after the activation of TGF-β canonical pathway at around 45 weeks of age [3,4]. We have also shown that Cx43 expression and phosphorylation were higher in 20−30-week-old Scn5a+/− mice than in wild-type littermates, and decreased concomitantly with the activation of TGF-β pathway and fibrosis onset [3], suggesting that Cx43 may be implicated in the fibrosis process in these mice.

In

Sources of funding

This work was supported by the European Community’s Seventh Framework Programme FP7/2007–2013 [Grant number HEALTH-F2-2009-241526]; EUTrigTreat (to IB & FC); the Agence Nationale de la Recherche [Grant number ANR-12-BSV1-0013-01] (to FC); the Fédération Française de Cardiologie (FFC, to HLM); the DHU2020 (MD) and the Fondation Genavie (MD).

Author contributions

JL, HLM, IB, FC and MD conceived the research, obtained the funding and assessed the results. JP, CC, MS, AH, AC, AT, AB, AD performed the experiments and analyzed the data. JP, CC, IB, FC and MD wrote the manuscript. All authors reviewed and corrected the manuscript.

Declaration of Competing Interest

The authors indicated no potential conflicts of interest.

Acknowledgments

The authors wish to thank Cynthia Ore-Cerpa and Nathalie Vaillant, l'institut du thorax, Nantes, France, for their technical assistance. Gap-134 was synthetized by CHEM-Symbiose, a core facility devoted to the synthesis of molecules.

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