Periodic Reporting for period 3 - CardHeal (Novel strategies for mammalian cardiac repair) Reporting period: 2021-06-01 to 2022-11-30 Summary of the context and overall objectives of the project The field of cardiac regeneration is in the midst of an exciting period. Recent ground-breaking studies by my team and others demonstrated that latent regeneration machinery can be awakened even in adult mammals. We identified two, apparently different, molecular mechanisms for augmenting cardiac regeneration in adult mice. The first requires transient activation of ErbB2 signalling in cardiomyocytes and the second involves extracellular matrix proteoglycan agrin. Impressively, both mechanisms promote a major regenerative response that enhances cardiac repair. These discoveries and other studies from my lab have positioned me as a leader in the field. Armed with extensive experience in developmental, cell and molecular approaches in vitro and in vivo, we seek in CardHeal to use the two powerful regenerative models we established to obtain a holistic view of cardiac regeneration and repair mechanisms in mammals (mice and pigs). Our second goal is to develop preclinical and translational approaches for novel therapeutic treatment for heart disease. In Aim 1, we will explore the molecular mechanisms underlying ErbB2-mediated cardiomyocyte dedifferentiation and proliferation followed by new vessels formation, scar resolution and functional cardiac repair. Specific objectives include characterising ErbB2-Yap/Hippo signalling during cardiac regeneration; studying transient ErbB2 activation in a chronic heart failure model; investigating an ErbB2- induced regenerative EMT-like process; and characterising cardiomyocyte re-differentiation. In Aim 2, we will investigate the therapeutic effects of agrin, whose administration into injured hearts of mice and pigs elicits a significant regenerative response. Specific objectives include studying matrix-related cardiac regenerative cues, focusing on the role of agrin in modulating the immune response after injury, angiogenesis, and matrix remodelling; developing a preclinical, large animal model to study agrin efficacy for cardiac repair; and, more globally, testing the role of agrin in skeletal muscle regeneration. Interrogating the differences and similarities between our two regenerative models should give us a detailed roadmap for cardiac regenerative medicine by providing deeper knowledge of the regenerative process in the heart and developing novel targets for cardiac repair in human patients. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Current projects related to the ERC are summarized below:1. ERBB2 drives YAP activation and EMT-like processes during cardiac regenerationAlla Aharonov, Avraham Shakked, Kfir Baruch Umansky, Alon Savidor , David Kain, Daria Lendengolts, Or-Yam Revach, Yuka Morikawa, Jiuli Zhou, Jixin Dong, Yishai Levin, Benjamin Geiger, James F. Martin and Eldad Tzahor• NCB 2020 DOI: 10.1038/s41556-020-00588-4Cardiomyocyte (CM) loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. ERBB2-Neuregulin and Hippo-YAP signaling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient over-expression (OE) of activated ERBB2 in CMs promotes cardiac regeneration in a heart failure model. OE CMs present an EMT-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration, and ECM turnover. We identified YAP as a critical mediator of ERBB2 signaling. In OE CMs, YAP interacts with nuclear envelope and cytoskeletal components, reflecting an altered mechanic state elicited by ERBB2. We identified two YAP activating phosphorylation on S352 and S274 in OE CMs, peaking during metaphase and are ERK-dependent and Hippo-independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Taken together, we revealed a potent ERBB2-mediated YAP mechanotransduction signaling, involving EMT-like characteristics, resulting in robust heart regeneration.2. Agrin promotes coordinated therapeutic processes leading to improved cardiac repair in pigsAndrea Baehr†, Kfir-Baruch Umansky†, Elad Bassat, Katharina Klett, Victoria Jurisch, Tarik Bozoglu, Nadja Hornaschewitz, Olga Solyanik, David Kain, Bartolo Ferrero, Renee Cohen-Rabi, Markus Krane, Clemens Cyran, Oliver Soehnlein, Karl Ludwig Laugwitz, Rabea Hinkel, Christian Kupatt,*, Eldad Tzahor*• Circulation 2020 DOI: 10.1161/CIRCULATIONAHA.119.045116Ischemic heart diseases are classified among the leading cause of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a significant number of MI patients develop chronic heart failure over time. We have recently reported that a fragment of the extra cellular matrix (ECM) protein Agrin promotes cardiac regeneration following MI in adult mice. Here, we tested the therapeutic potential of Agrin in a preclinical porcine model, comprising either 3 or 28 days reperfusion period. We first demonstrate that local (antegrade) delivery of recombinant human Agrin (rhAgrin) to the infarcted pig heart can target the affected regions in an efficient and clinically-relevant manner. Single dose of rhAgrin resulted in significant improvement in heart function, infarct size, fibrosis and adverse remodeling parameters 28 days post MI. Short-term MI experiment along with complementary murine MI studies revealed myocardial protection, improved angiogenesis, inflammatory suppression and cell cycle re-entry, as Agrin’s mechanisms of action. We conclude that a single dose of Agrin is capable of reducing ischemia reperfusion injury and improving cardiac function, demonstrating that Agrin could serve as a therapy for patients with acute MI and potentially heart failure. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Two major step stones in both Aims have been achieved with the submission of two manuscripts regarding ErbB2-Yap signalling (Aim 1) and Agrin efficacy in pigs including its MOA (Aim 2). Project aims
