Chronic obstructive pulmonary disease (COPD) is a global burden, leading to chronic respiratory failure and death. Although a severe lung disease, comorbidities are prevalent in this condition. Among them, sarcopenia, defined as a loss of muscle mass and function, significantly impairs exercise capacity and quality of life, and is a major prognostic factor associated with mortality. Restoring muscle function is therefore essential to improving health outcomes in COPD patients. However, the mechanisms of COPD-associated sarcopenia and the role of skeletal muscle stem cell (satellite cells (SCs)) alterations are poorly characterized, hampering adequate management of patients. Here, we aim to determine whether SCs of patients with COPD have intrinsic defects and whether these alterations can be reversed. We will first assess their density and state of activation, proliferation, differentiation and regeneration in muscle samples from COPD patients with and without sarcopenia and in mice exposed to cigarette smoke (WP1). Next, we will examine whether SCs alterations are reversed with exercise training, by performing muscle biopsies before and after pulmonary rehabilitation (WP2). Finally, we will evaluate the therapeutic potential of CXCR4 antagonization in vitro on isolated SCs both in patients and in the mouse model(WP3). The present project should lead to a better understanding of the mechanisms involved in COPD-associated sarcopenia and provide a framework for a potential drug treatment.

The building block of skeletal muscle is the post-mitotic muscle fiber, formed by the fusion of hundreds of specialized mononucleated cells (myoblasts) in which positioning of myonuclei, referred as myonuclei localization and shape, is finely regulated. Myonuclei are located between the plasma membrane of myofibers and the myofibril structures. The peripheral localization of myonuclei induces drastic changes in their shape, mainly due to forces applied on the nuclear envelope. This conformational adaptation of myonuclei is believed to stabilize internal and external mechanical forces, and consequently, to constrain chromatin organization and gene expression. The peripheral myonuclei organization, set by an interplay between the various components of the cytoskeleton (microtubules, actin and intermediate filaments), is thought to guarantee a spatial coordination of the transcriptomic activity, which ultimately contributes to the myofiber functional integrity. This suggests that the maintenance of peripheral myonuclei in mature myofibers is paramount to adjust optimal skeletal muscle homeostasis. In this view, the functional consequences of myonuclei positioning alterations observed in various myopathies remains poorly understood. Indeed, it is still unclear how the positioning of myonuclei regulates gene expression, and how it controls signaling pathways that alter muscle integrity and functionality. Using a large siRNA screen on developing myotubes, we identified a new candidate involved in the long-term organization of myonuclei spreading. Indeed, this protein act as an essential regulator of myonuclear spreading/anchoring in both developing and mature myofibers. Our consortium will use in vitro models of skeletal muscle formation and newly obtained KO mouse models to map the interplay between myonuclei architecture/location and spatial gene expression. This project will help to better understand progression of diseases with affected myonuclei localization. This multidisciplinary approach will provide important information on molecular pathways implicated in skeletal muscle integrity.

Histone variants act through the replacement of conventional histones by dedicated chaperones. They confer novel structural properties to nucleosomes and change the chromatin landscape. The functional and physiological requirement of the replacement of conventional histones by histone variants during organ formation and post-natal life remains poorly described. The incorporation of the histone variant H3.3 into chromatin is DNA-synthesis independent and relies on two different chaperone complexes, HIRA and DAXX/ATRX, which have different genomic deposition domains. While most epigenetic studies are performed in vitro, we intend to study them in an in vivo context where cell behavior can be properly addressed and where consequences for tissue formation, growth, homeostasis and repair can be fully investigated. Skeletal muscle provides the possibility to address yet poorly explored biochemical, cell biology, and developmental aspects of chromatin biology during development and postnatal life. Based on published and preliminary data from the three partners involved in this project, we hypothesize that: (i) HIRA and DAXX play a key role in muscle stem cells identity and muscle fibers organization (ii) H3.3 contributes to genome stability and prevents premature aging in adult muscle fibers (iii) a third H3.3 chaperone exists, which allows H3.3 incorporation into chromatin in the absence of HIRA and DAXX. Therefore, the main objectives of this proposal are defined in three work packages as follows: WP1: Conserved and divergent functions of H3.3 and DAXX-ATRX/HIRA pathways in muscle progenitors: we have recently shown that in the absence of HIRA, the muscle stem cell pool is lost during muscle regeneration. In addition, conditional HIRA inactivation in muscle progenitors during development have reduced myoblast numbers and smaller muscle size. In this context, our investigations will be extended to DAXX and H3.3. Our preliminary results indicate that DAXX is regulates myogenic gene expression via its histone chaperone activity. WP2: Role of H3.3 and DAXX-ATRX/HIRA pathways in adult myofibers structure and function: H2A.Z inactivation in adult muscle causes accelerated aging due to accumulation of DNA damage consecutive defective DNA repair by non-homologous end joining (NHEJ). H3.3 is also required for NHEJ. We therefore predict that H3.3 inactivation in muscle fibers will cause DNA damage and premature aging. Many evidences indicate that H3.3 regulates gene expression. We will determine if similarly to H2A.Z, H3.3 function in muscle fibers is restricted to DNA repair or if it also regulates gene expression. Finally, the roles of H3.3 chaperones have not yet been investigated in post-mitotic muscle fibers. To address these points H3.3, HIRA and DAXX will be inactivated in muscle fibers. We have recently shown that muscle fibers contain several myonuclear domains with specific identity and function defined by nuclei-specific expression profiles. The epigenetic landscape and myonuclei identity will be evaluated by single nuclei RNA seq and ATAC seq in the KO muscles. WP3: characterization of a new H3.3 deposition pathway that can bypass DAXX-ATRX/HIRA: H3.3 Chip-seq in Hira KO and Daxx KO myoblasts show HIRA and DAXX independent H3.3 deposition at specific loci, suggesting the presence of a third chaperone. Like other chaperones, this new chaperone should be part of a large multiprotein complex. We will isolate this complex from myoblasts and identify its composition. The complex will then be reconstituted with recombinant proteins to analyze its deposition properties. We will also invalidate the expression of some of the important components of the new deposition complex in vivo and we will determine the presumably perturbed H3.3 distribution pattern and the resulting cell phenotype at molecular level. Taken collectively, the expected data should shed in depth light on the intimate mechanism of H3.3 deposition and H3.3 function.

Skeletal muscle regenerates after injury, due to the satellite cells (SCs), the muscle stem cells that activate, proliferate, differentiate and fuse to form new myofibers. While SCs are indispensable for regeneration, there is increasing evidence for the need for an adequate cellular environment for SCs to properly execute their myogenic program and achieve muscle regeneration. Among the closest cellular partners of SCs are vascular cells and macrophages. Previous work from both partners highlighted the key role of the reorganizing microvascular network and macrophages as providing important modulatory signals for regeneration. During muscle regeneration, endothelial cells (ECs) stimulate SC differentiation while SCs exhibit pro-angiogenic properties indicating a coupling between angiogenesis and myogenesis. We demonstrated that vascular smooth muscle cells and pericytes regulate SC quiescence during muscle post-natal growth. These studies underline a complex organization of the vascular cells during muscle regeneration: ECs, while detached from peri-ECs (smooth muscle cells and pericytes) at the time of vascular remodeling interact with SCs to promote both angiogenesis and myogenesis. Later on, peri-ECs cover ECs for the stabilization of both vessels and SC quiescence. The specific signaling cues controlling these relationships are still poorly characterized. We propose here to combine the tools and expertise of two teams recognized as world expert in the field of muscle stem cells and the regulation of their environment in order to analyze the functional interactions between vascular cells, SCs and macrophages in physiological conditions and during muscle diseases by combining cellular, molecular and lineage-specific genetic studies in murine preclinical models. Our research program will be organized in 4 interconnected aims: (1) We will investigate the role of hypoxia and of the Hypoxia Inducible Factor (HIF) pathway in the context of skeletal muscle regeneration. (2) We will address the role of pericyte-derived effectors in the return and maintenance of SCs into quiescence during muscle regeneration, and the regulation of pericytes by sympathetic nervous system. (3) We will investigate the involvement of macrophages in the regulation of myo-angiogenesis and of pericyte fate during muscle regeneration. (4) Finally, we will study these cellular interactions in specific pathological contexts inducing myopathies such as limb ischemia or muscular dystrophies. This program will provide the first comprehensive analysis of the cellular and molecular functions of the cells of the vascular bed and their interactions with muscle stem cells during the regeneration of skeletal muscle and in diseased muscle.

Heart failure with preserved ejection fraction (HFpEF) exhibits altered left ventricular (LV) diastolic function but a normal or near normal ejection fraction (>50%). Evidence links aging, metabolic dysregulation with inflammation, coronary microvascular dysfunction (CMD) to HFpEF with up to 75% of patients suffering from impaired coronary flow reserve (CFR) despite no obstructive coronary artery disease. CMD was attributed to both microvascular obstruction and/or rarefaction, endothelial dysfunction and/or vascular smooth muscle dysfunction which may lead to a mismatch between metabolic demand and coronary blood flow in response to stress. Research Hypothesis: Improving coronary microvascular function will reduce the severity of HF-pEF Our aim is to model HFpEF in two preclinical models in order to determine parameters of the microvascular coronary anatomy (vessel diameters, density, tortuosity…) as well as flow velocities which will be quantified in vivo. We will also assess changes in myocardial blood flows distribution during the development of HFpEF and in response to ANGPTL4 treatment. Indeed, we demonstrated that recombinant human (rh)ANGPTL4 protects endothelial cell integrity that limits no-reflow and infarction and thus tissue damage. In contrast, aged angptl4-KO mice display abnormal coronary microvascularization thus offering us the opportunity to i) assess the role of microvascular architectural changes in the pathophysiology of HFpEF and ii) address the role of ANGPTL4. Aim1- Assess the specific role of microvascular architectural and functional changes in HFpEF and in response to ANGPTL4 Aim2- Identify a coronary blood flow distribution pattern to characterize CMD during HFpEF and its modulation by ANGPTL4 via 3D ultrasound imaging. This multidisciplinary project will be a unique opportunity to bring together all three partners in biology and physics and their complementary skills to identify new treatment options urgently needed in HFpEF.