Coronary microcirculation (i.e coronary flow in vessels smaller than 300µm) plays a key role in the control of cardiac perfusion. The importance of coronary microcirculation for relevant pathological conditions including angina in patients with normal or near-normal coronary angiograms is increasingly recognized. Indeed, a large number of patients with anginal symptoms and ischemia on stress testing have a normal coronary angiogram and current evidences suggest that about two third of these patients have coronary microvascular dysfunction (CMD), also known as microvascular angina (MVA). Patients with CMD have poor prognostic with significantly higher rates of cardiovascular events, including hospitalization for heart failure, sudden cardiac death, and myocardial infarction (MI). Another important and frequent alteration of the microcirculation is associated to sustained myocardial hypoperfusion during acute myocardial infarction despite coronary revascularization. This so called no-reflow phenomenon remains largely underdiagnosed, and is associated with adverse outcome. Finally, there are also major evidences for CMD during heart failure with preserved ejection france (HFpEF). Despite the urgent clinical need, there are simply no techniques available routinely in clinic, to directly visualize the coronary microvasculature and assess the local coronary microvascular system. Up to date, only global indirect measurements through functional testing (PET, CMR and contrast echocardiography) or invasive measurements can provide hemodynamic information such as Myocardial Blood Flow (MBF) and Coronary Flow Reserve (CFR) in response to vasodilator effects. In CorUS, a novel ultrasound technology will be developed to image the anatomy and the function of coronary vessels at the microscopic scale using a non-invasive and non-ionizing technology. This approach relies on ultrafast ultrasound imaging of the heart at 5,000 images/s, a breakthrough technology pioneered about twenty years ago by researchers of the laboratory Physics for Medicine Paris and more recently on the new technology of Ultrafast Ultrasound Localization Microscopy (ULM) which was introduced to resolve blood vessels at a micrometer scale in deep organs by tracking ultrasound contrast agents (microbubbles) circulating in the blood flow. Cutting-edge technology will be developed in CorUS for local and direct imaging of the coronary blood flows at the microscopic scale to provide new anatomical and functional markers of the coronary microcirculation. Preliminary proof of concept experiments in perfused porcine hearts and in perfused beating rat hearts have demonstrated the feasibility of 2D and 3D coronary microcirculation imaging. This technology will be translated to large hearts application and the approach will be validated in vivo on preclinical large animal models with alteration of the coronary perfusion. Finally, a clinical proof of concept study will be performed on patients with coronary microcirculation alteration. The main objectives of CORUS are: 1. To develop a new ultrasound technology for coronary microcirculation imaging 2. To validate the technology on large animal preclinical models of coronary microcirculation alteration 3. To perform a proof of concept clinical study on patients with coronary microcirculation disease CorUS will have major impacts in the understanding, the management and the treatment of coronary artery diseases and the non-ionizing, non-invasive imaging technology developed in this project could become a major tool for the clinical investigation of microvascular coronary circulation at the patient’s bedside.

MITOMORT is a basic science project focusing on the role of an endogenous interferon induced gene (ISG) encoding a small protein that targets the mitochondria leading to cell death. The title is a play on mitochondrion and the French word for death, mort. APOBEC3A is an ISG gene encoding a cytidine deaminase that is able to edit C residues in chromosomal DNA. The attack rate can be so high that it causes extensive double stranded breaks and apoptosis. This is referred to as hypermutation. Lower levels of mutation, hypomutation, occur and are associated with oncogenesis. The Molecular Retrovirology Unit at the Pasteur Institute was the first to show that the APOBEC3A enzyme could attack chromosomal DNA. We noted that the initiation codons (AUG) of the two APOBEC3A isoforms used the “adequate” context according to the terminology of Marylin Kozak. Accordingly, we can expect that only 30-40% of ribosomes will settle on these sites, the remainder will continue to scan the mRNA. We asked the question, where will they settle? The next AUG downstream is in an “adequate” context while the following AUG is in a “strong” context. It turns out that initiation at these two sites produce two small proteins isoforms termes A3Ap3 and A3Ap4 (10.5 kDa and 8.6 kDa) that are in the same reading frame but overlapping that of APOBEC3A. They encode transmembrane spanning proteins that target the mitochondrion resulting in apoptosis. We apparently have a unique situation where two pro-apoptotic proteins, APOBEC3A targeting the archive, the genome, and A3Ap3/A3Ap4 targeting the powerhouse, the mitochondrion, are encoded by a single gene – to date called APOBEC3A. Research of A3Ap3/A3Ap4 apparently links apoptosis to the network of stress sensors that constitutes the interferon signalling pathway. It provides a link between the live cell and the death signal. Low levels of APOBEC3A will provide ongoing hypomutation and a weakening the mitochondrial network through sub-lethal doses of A3Ap3/A3Ap4. To compensate this the cell might slowly switch to ATP production via glycolysis as opposed to oxidative phosphorylation, or the Warburg effect. The project seeks to understand the mechanism and biology of this small endogenous pro-apoptotic protein - the singular is used because, so far, the two isoforms appear to have exactly the same function. The MITOMORT team combines the technology to resolve many facets associated with A3Ap3/A3Ap4, notably electron microscopy, confocal imagery and video. As intracellular obligate parasites have to protect them from premature apoptosis it is possible that nature has already developed an antagonist. To explore this hypothesis the consortium includes a laboratory with considerable experience in finding protein interactors, screening of a library of viral orfs as well microbial anti-apoptotic proteins. It is likely that A3Ap3/A3Ap4 is regulated leading to a fine balance between life and death. This would extend considerably the subject and provide new leads. The two Pasteur labs have collaborated in the past while the lab in Tours is already collaborating with the MRU on the mitochondrial localization of A3Ap3/A3Ap4. The lab at Maison-Alfort is an obvious collaborating lab and is well known to the MRU even though they have never collaborated directly before. While MITOMORT is a basic research project, as it concerns apoptosis mediated by an endogenous ISG, we feel that the findings will appeal to a wide audience of cell biologists. It provides a link between inflammation and cell death and so there is the possibility of it shedding some light on some autoimmune diseases like systemic lupus erythematosus. As to patents and the like, it is a little premature to make any predictions.

Bipolar disorder is a severe chronic psychiatric disorder affecting 1% of the population. Lithium is its gold standard treatment. Human MRI and preclinical studies suggest that it may increase neurogenesis, neuroprotection, myelination and modulate synaptic plasticity and neuroinflammation. However, many aspects of its mode of action remain unknown: which cellular effects are associated with the “MRI effects” of lithium in patients? Are its therapeutic cellular effects region specific or brain wide? We will conduct 2 parallel studies (in rats and humans) using similar (longitudinal) designs and methods ([11C]-UCB-J PET and MRI) plus histology and immunochemistry in the rats. We will assess synaptic plasticity, myelination, oligodendrocytes, neurogenesis and neuroinflammatory aspects associated with lithium in the same study. We will thus be able to draw inferences and inter species correspondences between PET/MR findings and immunohistological findings

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.