TEAM 3 – Elise Balse and Sophie Nadaud – Molecular and Cellular Plasticity in Cardiovascular Diseases
Cardiovascular diseases are often intricate diseases that share pathophysiological mechanisms. Remodeling processes are associated with complex rearrangement at the tissue and at the cellular levels. We aim at understanding the drivers of the molecular and cellular plasticity that characterize cardiovascular remodeling during atrial fibrillation (AF), heart failure (HF), senescence and pulmonary hypertension (PH).
Our projects focus on
– the plasticity of cellular composition of cardiovascular tissues. We notably study the capacity of progenitor and stem cells to be recruited, to differentiate in various mesenchymal cell lineages and to contribute to atrial and vascular remodeling.
– the plasticity of macromolecular protein complexes regulating cardiac function and their role in pump dysfunction and arrhythmias. We focus on the regulation of ion channels trafficking and targeting in cardiomyocytes.
– the role of cellular metabolic shifts in regulating myocardial remodeling and atrial electrical properties.
– the role of immune and inflammatory cells during cardiovascular remodeling leading to HF. We study the mechanisms of macrophages protective role during early adaptive cardiac hypertrophy.
– the role of oxidative stress and inflammation during age-associated cardiovascular remodeling and transition to heart failure.
– the role of the GCN2 gene mutation in the development of Pulmonary Veno-Occlusive Disease, a specific form of pulmonary hypertension.
Atrial fibrillation and heart failure are two leading causes of worldwide mortality and morbidity in modern countries and their prevalence will continue to grow up in the next years with the ageing of population. Pulmonary hypertension is a rare and life-limiting cardiovascular disorder, characterized by an occlusive remodeling of the distal pulmonary vasculature that ultimately leads to right heart failure. Our team 3 is interested in deciphering the molecular and cellular players participating in the remodeling processes that lead to these pathologies.
THEME 1 – Myocyte Organization, Ion Channel Sorting and Targeting – Elise Balse
Scientific context and past contributions
The functional expression of ion channels in the myocyte sarcolemma determines the shape and duration of the action potential, and therefore control the effective refractory period of the myocardium. The proper functional expression of ion channels can be disrupted at several levels such as transcriptional, translational and post-translational levels. Over the past years, we have contributed to better understand the dynamics of the surface expression of cardiac ion channels in cardiac myocytes (Balse et al. PNAS 2009, Balse et al., Physiol Rev 2012; Boycott et al., 2013; Eichel et al., Circ Res 2016). First, following our finding of reservoirs of KV channels beneath the sarcolemma quickly recycled ensuing cholesterol depletion (Balse et al., PNAS 2009; Balse et al., Physiol Review 2012), we showed that mechanical constraint triggers the exocytosis of Kv channels from this reservoir through the activation the integrin/PFAK mechanotransduction pathway. Recycling of KV1.5 channels becomes constitutive during atrial hemodynamic overload, supporting action potential shortening and arrhythmogenesis (Boycott et al., PNAS 2013) (Figure 1). In human biopsies from patients in AF, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Predominance of anterograde trafficking activity over retrograde trafficking consequently result in accumulation ok KV1.5 channels in the plasma membrane during atrial remodeling (Melgari et al., JMCC 2020).
Figure 1. The recycling endosome is a major storage compartment of KV1.5 channels in atrial myocytes and ensure rapid delivery of channels upon changes in mechanical constraints
Second, we previously characterized a new partner of the main cardiac sodium channel NaV1.5. Contrarily to other identified NaV1.5 partners, the MAGUK protein CASK negatively regulates lateral membrane pools of NaV1.5 by impeding anterograde trafficking of the channel. CASK is restricted to costameres, the focal adhesion of myocytes, and interact with dystrophin at the lateral membrane (Eichel et al. Circ Res 2016) (Figure 2). We have shown that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. Thus, due to its multi-modular structure, CASK could simultaneously interact with several targets in cardiomyocyte to potentially couple sodium channel trafficking to costameric cell adhesion in cardiomyocyte (Beuriot et al. Heart Rhythm, 2020).
Figure 2. CASK impedes early anterograde trafficking of pools of channels targeted to the lateral membrane
These finding suggest a continuous interplay between membrane electrical properties, myocyte environment and 3-dimentional architecture of the myocardium. Overall, we will continue investigating the spatiotemporal organization of ion channels in cardiac myocytes, in relation with the microarchitecture of the tissue in normal and diseased conditions.
– Test the hypothesis of a sorting hub at early stages of trafficking, where interactions between NaV1.5 channels and regulatory partners orientate the final targeting of the channel in cardiac myocytes membrane subdomains.
– Investigate locally regulated exocytosis/endocytosis of ions channels regarding mechanotransduction sites in cardiac myocytes.
– Characterize the alterations of trafficking machinery in acquired cardiopathies and their role in the formation of the arrhythmogenic substrate.
Beside better understanding of the general mechanisms of ion channel trafficking and targeting in cardiac myocytes, these studies should shed light on cardiac electrical plasticity in the context arrhythmias associated with myocardium structural remodelling as observed in most cardiopathies.
THEME 2 – Mechanisms of Age-Related Cardiac Remodeling: Role of FoxO and NF-κB Pathways – Sophie Besse and Bruno Riou
Our group is interested, since numerous years, in the mechanisms involved in cardiac aging and has contributed to identify structural changes of the myocardial tissue associated with electrical and mechanical remodeling in aged rodents (Figure). The production of reactive oxygen species (ROS), not counterbalanced by anti-oxidant defenses which are deficient with age, has been implicated in this remodeling. ROS, which act as second messengers, modulate the activation/inactivation of various transcription factors such as NF-κBs and FoxOs which in turn regulate many cellular pathways including inflammation, necrosis/necroptosis/apoptosis, autophagy, ionic homeostasis, myocyte hypertrophy/atrophy and remodeling of the extracellular matrix. Different studies suggest that these transcription factors play a central role in the transition from cardiac hypertrophy to heart failure at advanced age.
NF-κB activation induces chronic inflammation, which is a hallmark of heart failure and a predictor of overall prognosis. This activation can be obtained by two different routes : the canonical pathway (classical and alternative) and the non-canonical pathway. Several studies proposed that NF-κB activation stimulated by myocardial damage and/or oxidative stress mainly contributes to aging in different organism models but a cross-talk between NF-κB and FoxO pathways has been recently reported. FoxO3 plays an important role in protection of cells against oxidative stress, increasing lifespan and limiting the age-associated functional decline leading to heart failure. In D. melanogaster, FoxO3 overexpression suppresses the age-associated diastolic dysfunction.
We are now investigating the regulatory pathways of oxidative stress and inflammation, especially the FoxO and NF-κB signaling, in cardiac hypertrophy during aging in rodents to better understand the mechanisms responsible for the transition to heart failure at advanced age. We are also studying these NF-κB and FoxO signaling pathways in human hearts which remains largely unexplored during aging.
THEME 3 – Atrial Cardiomyopathy and Atrial Fibrillation Pathophysiology – Stéphane Hatem
A number of clinical studies have established that adipose tissue accumulation at the atrial surface is a major risk factor for AF, especially during metabolic disorders such as obesity (Hatem JACC 2016). Stephane Hatem’s group was the first to demonstrate a causal link between epicardial adipose tissue and AF. They have identified adipokines secreted by the human atrial epicardial adipose tissue (Activin A) that can induce the fibrosis of neighboring atrial myocardium (Venteclef EHJ 2015). They described how fatty infiltrates become fibrotic in the atrial subepicardium, a remodeling process associated with persistent AF and mediated by an immune response involving T-lymphocytes (Haemers EHJ 2017). They found that adipocytes are derived from epicardial-based progenitor cells).
Epicardium is reactived early during the formation of the atrial cardiomyopathy and the sustrate of atrial fibrillation.
The recruitment of cells derived from epicardial progenitors and pre-engaged in the distinct adipocyte
or fibroblastic lineages can result in the arrythmogenic fibro-fatty infiltration of atrial subepicardial layers.
The natriuretic peptide secreted by atrial myocytes in response to mechanic stress is a powerful adipogenic factor for the epicardial progenitors at low concentration (Suffee PNAS 2017). Recently they identified subpopulation of epicardial progenitors engaged toward adipocyte or fibroblast differentiation and the signaling pathways that govern such a switch (BioRxiv 589705). In collaboration with LIB teams (Biomedical imaging laboratory) and using in-house algorithms, we established that atrial wall strain correlates with the degree of atrial fibro-fatty infiltration which can be used as a biomarker of the AF substrate (Hubert Radiology 2018). They also identified high-field MRI sequences which enables analysis adipose tissue infiltrates of the myocardium (Bouazizi PLoS One 2019).
Fibrotic remodeling (Sirius red staining) of the epicardium in human atrial samples.
Non-fibrotic remodelled epicardium without (A) or with (B) subepicardial adipose tissue
and fibrotic remodelled epicardium with (C) or without (D) subpicardial adipose tissue.
Coimmunostaining for Perilipin-1 (adipogenic marker) and Tomato (marking cells derived from WT1+ epicardial cells)
in atrial cells of mice fed a high fat diet.
Arrows indicate epicardium-derived adipocytes coexpressing Tomato and Perilipin.
THEME 4 – The Role of Immune and Inflammatory Cells During Cardiovascular Remodeling Leading to Heart Failure – Catherine Pavoine
Cardiac hypertrophy (CH) is initially a compensatory process to optimize cardiac pump function. However, CH is progressively associated with structural changes that become pathogenic, with cardiomyocyte death, induction of exacerbated inflammatory responses and interstitial fibrosis. These harmful changes ultimately lead to transition to heart failure (HF). Activation of the sympathetic nervous system plays a determinant role in the induction of early adaptive CH (EACH) and further progression to pathological remodeling. Cardiac remodeling is a complex inflammatory syndrome, and beneficial or detrimental role of inflammatory signaling during EACH is not fully understood. Growing evidence indicates that inflammatory responses emerging in EACH and HF are different, displaying divergent cytokine and chemokine profiling. The pro-inflammatory cytokine TNFα is upregulated in CH and HF. In the 1990’s, the “cytokine hypothesis” argued for the detrimental contribution of an excessive production of TNFα to the pathogenesis of HF suggesting that TNFα neutralization would be beneficial. Surprisingly, large clinical trials failed to demonstrate a benefit of anti-TNFα strategies. There is now evidence that TNFα can also improve remodeling and hypertrophy and alleviate inflammation and fibrosis.
Protective role of macrophages via the CX3CL1/CX3CR1 axis in early adaptative cardiac hypertrophy remodeling.
Chronic β-adrenergic stimulation elicits a CX3CL1/CX3CR1-dependent expansion of CD64+ CCR2- LY6C- MHClIlow Macrophages.
CX3CL1 and TNF-α secreted by cardiac macrophages synergistically trigger cardiomyocyte hypertrophy. This early adaptative cardiac remodeling limits evolution towards heart failure.
In addition to cardiomyocyte hypertrophy, cardiac hypertrophy (CH) is associated with determinant changes of non-myocyte cardiac cell types, including macrophages (Mφ). Cardiac Mφ influence tissue homeostasis, repair and regeneration. They modulate CH and HF, in particular through the regulation of pro- or anti-inflammatory paracrine signaling. However, the functions of cardiac Mφ are not fully understood especially in the context of cardiac hypertrophic remodeling in diseases from non-ischemic origin. Catherine Pavoine’s group recently highlighted that cardiac inflammatory CD11b/c cells exert a protective role in hypertrophied cardiomyocyte by promoting TNFR 2 – and Orai3- dependent signaling (Keck M et al., Sci Rep 2019). Emergence of this novel protective paracrine role of CD11b/c cells during EACH, improves resistance of adult hypertrophied cardiomyocytes to oxidative stress and potentially limits evolution towards HF in response to β-adrenergic stimulation.
Our group is now seeking to identify the origin and role of macrophages selectively amplified in EACH as compared to control and HF hearts in order to better define their protective mechanism of action. Prelimary results identify the crucial role of the CX3CL1/CX3CR1 axis (submitted to publication).
Aim1 – Using flow cytometry, transcriptomic, lipidomic and secretomic approaches, the group will identify typical markers, genes, lipids, proteins and secreted chemokines characterizing EACH Mφ.
Aim2 – We will perform in vitro and in vivo experiments to elucidate the impact of these typical signals on cardiomyocyte hypertrophy and survival, and on EACH and HF transition.
THEME 5 – Cellular and Molecular Plasticity During Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension – Sophie Nadaud
Our group is interested in the cellular and molecular factors involved in regulating vascular remodeling. We are in particular investigating the origin of new smooth muscle cells produced during pulmonary hypertension. This is a rare and devastating disease with no curative options, characterized by an occlusive remodeling of the distal pulmonary vasculature that ultimately leads to right heart failure. Non-muscularized vessels become muscularized and vessels media thickness is increased with in addition, neointima formation. Resident pulmonary progenitors participate in the pulmonary hypertension-associated vascular remodeling by generating new smooth muscle cells. We have identified resident CD34+/PW1+/PDGFRα+ progenitors involved in the early neomuscularization observed during chronic hypoxia (CH: a model for moderate pulmonary hypertension) (Dierick, Circ Res 2016). We have recently demonstrated that the proliferation of these progenitor cells is under the control of the PDGFRα pathway (article in preparation). In collaboration with C. Guignabert (INSERM U999), we have studied pulmonary NG2+ pericytes which are also vascular progenitor cells activated during pulmonary hypertension (article submitted). We develop lineage tracing models to follow the fate of these progenitor cells during pulmonary hypertension associated vascular remodeling. We use transgenic mouse models to induce or suppress PDGFRα activity in vivo following tamoxifen activation to evaluate the role of this pathway in the function of these progenitors and in CH-induced vascular remodeling.
Pulmonary arterioles are mainly non muscularized and neomuscularization is a hallmark of pulmonary hypertension.
Our data show that pulmonary vascular progenitor cells are recruited by PDGFRα signalling activation
and differenciate into new smooth muscle cells following CXCR4 signalling.
We are now investigating the role of metabolic alterations on pulmonary vascular progenitor cells recruitment. In addition, we are studying the role of the PDGFRα pathway and of the vascular progenitor cells in the systemic vascular remodeling during other cardiovascular disorders leading to vascular remodeling.
Progenitor cells in culture differentiate into smooth muscle cells identified by SM-MHC (red) and α-SMA (green) expression.
PW1+ Progenitor cells (green) are clustered around vessels (red) in control human lung.
THEME 6 – Cellular and Molecular Mechanisms Underlying Pulmonary Veno-Occlusive Disease Development – Florent Soubrier
Florent Soubrier’s group, in tight link with the clinical genetics laboratory of the campus hospital (Pitié-Salpêtrière hospital) has identified genes involved in the genetic predisposition to pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease (PVOD). In 2019, this group has identified BMP10 as a new gene for heritable PAH (PMID: 30578383) and has identified two families presenting with heritable PAH and carrying mutations of the KDR gene encoding VEGFR2 (Eyries et al, in revision 2019). One objective of the group is to identify new predisposing genes and whole genome sequencing is the technique used in selected subjects.
Pathology of heritable PVOD.
(a) septal vein and (b) small vein showing intimal fobrosis and thickening of the vascular wall
From Eyries and al. PMID: 24292273
The group is also investigating the cellular and molecular mechanisms underlying the occurrence of the heritable form of pulmonary veno-occlusive disease (PVOD) due to mutation of the EIF2AK4 gene that this group has identified in 2015 (PMID: 24292273). This gene encodes GCN2, a serine-threonine kinase which is one of the four EIF2α kinase and induces the integrated stress response in the cell in response to amino acid deprivation. GCN2 has important interactions with cellular physiological responses such as autophagy, inflammation and oxidative stress and with other signaling systems such as mTOR. The link between complete loss of GCN2 and PVOD is not elucidated and is the goal of intensive research. Three lines of rats carrying deletions of EIF2AK4 have been obtained through CrispR-CAS9 gene targeting and are used for deciphering, in vivo, the modes of initiation of the disease.
Schematic view of the GCN2 induced activation of integrated stress response.
Amino acid deprivation induces GCN2 dimerization and activation, hence inducing phosphorylation of the α subunit of EIF2 (EIF2a)
which in turn inhibits protein translation and increases ATF4 by a translation mechanism.
ATF4 is a transcription factor that has several transcriptional targets.
During inflammation, there is an amino acid consumption by cell proliferation and that causes GCN2 activation.
Figure from Santos-Ribeiro D (PMID: 29499378)
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Alain Castaigne Award – 2018
Jean Valade Award – 2014
Lamonica Cardiology Award – Académie des Sciences – 2017
Marion Elizabeth Brancher Award – 2017