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Novel strategies for mammalian cardiac repair

Periodic Reporting for period 1 - CardHeal (Novel strategies for mammalian cardiac repair)

Reporting period: 2018-06-01 to 2019-11-30

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.
Current projects related to the ERC are summarized below:
1. ErbB2 alters cardiomyocyte mechanic state and Yap activation, resulting in EMT-like regenerative processes in the heart
Alla 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
• In preparation
Cardiomyocyte (CM) loss after injury results in adverse remodeling and fibrosis, which inevitably lead to heart failure. Neuregulin-ErbB2 and Hippo/Yap signaling pathways are key mediators of CM proliferation and regeneration although the crosstalk between these pathways is less clear. Here, we demonstrate in mice that temporal over-expression (OE) of activated ErbB2 in CMs promotes robust cardiac regeneration in a heart failure model. Cellularly, OE CMs present an EMT-like regenerative response involving cytoskeletal reprograming, migration, ECM turnover, and cell-cell separation. Molecularly, we identified Yap as a critical mediator of ErbB2 signaling. In OE CMs, Yap interacts with nuclear envelope and cytoskeletal components, reflective of the altered mechanic state elicited by ErbB2. Hippo-independent activating phosphorylation on Yap at S352 and S274 was enriched in OE CMs, peaking during metaphase. Viral overexpression of Yap phospho-mutants dampened the proliferative competence of OE CMs. Taken together, we report a potent ErbB2-mediated Yap mechanosensory signaling involving EMT-like characteristics at the heart of regeneration.
2. Agrin promotes coordinated therapeutic processes leading to improved cardiac repair in pigs
Andrea 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*
• Under review in Circulation
Ischemic 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 (d) 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.
3. Transient p53-mediated regenerative senescence in the injured heart
Rachel Sarig, Rachel Rimmer, Elad Bassat, Lingling Zhang, Kfir-Baruch Umansky, Daria Lendengolts, Gal Perlmoter, Karina Yaniv, Eldad Tzahor
• Published in Circulation 2019
Senescence is commonly associated with aging and age-related diseases; however, transient senescence has been shown to be essential for proper development and regeneration. To follow senescence in the heart we employed injury models in zebrafish and neonatal P1 mice, which are established models for cardiac regeneration. Cryoinjury of zebrafish hearts and resection of P1 mouse hearts induced high levels of senescence 4 days after injury. Interestingly, 21 days post-injury, senescence was no longer observed in either models, indicating its transient nature. Senescent cells in P1 hearts were localized within the scar region and epicardium overlapping with activated cardiac fibroblasts (CFs).
Recently, we have shown that the ECM molecule Agrin promotes heart regeneration after myocardial infarction (MI) in non-regenerating mouse hearts. To determine whether Agrin affects senescence levels, we compared juvenile and adult hearts that underwent MI, followed by a single injection of either PBS or agrin into the myocardial wall. As observed in zebrafish and P1-injured hearts, Agrin induced robust senescence which was markedly decreased after 3 weeks. Senescent cells co-expressed vimentin, suggesting that Agrin induces senescence of CFs.
The tumor suppressor p53 is a well-known key regulator of senescence. We identified a specific expression of p53-S23 phosphorylated form in senescent cells in the epicardium and scar region of P1 injured hearts, 2 days after injury. p53-S23 was also detected in P7 hearts 2 days after MI. In accordance with the transient nature of the senescence, p53-S23 expression was abolished 5 days post-injury, when the injured area was enriched with myofibroblasts. In adult mice, agrin treatment induced a similar pattern of p53-S23 expression in the epicardium and scar region, which was higher than in PBS-treated hearts. Importantly, expression levels of p53-S23 were dramatically higher in injured P1 compared to adult hearts, as well as in agrin- compared to PBS-treated hearts. Thus, Agrin can induce enhanced p53-S23 activity, accompanied by reactivation of transient regenerative senescence in non-regenerating mouse hearts.
This work sheds light on the important role of CFs in the repair and regeneration processes, and raises new questions regarding the cross-talk between CFs, cardiomyocytes and immune cells after injury. Senescence is an evolutionarily conserved mechanism for cardiac regeneration and repair, and likely occurs in other organs too. Our findings expand the understanding of the regenerative function of recombinant Agrin in the mammalian heart.
4. The small molecule Chicago Sky Blue promotes heart repair following myocardial infarction in mice
Oren Yifa, Karen Weisinger, Elad Bassat, Hanjun Li, David Kain, Haim Barr, Noga Kozer, Alexander Genzelinakh, Dana Rajchman, Tamar Eigler, Kfir-Baruch Umansky, Daria Lendengolts, Ori Brener, Nenad Bursac, and Eldad Tzahor
• Published in JCI Insight 2019
The adult mammalian heart regenerates poorly after injury and, as a result, ischemic heart diseases are among the leading causes of death worldwide. The recovery of the injured heart is dependent on orchestrated repair processes that could be modulated in patients such e.g. inflammation, fibrosis, or cardiomyocyte survival, proliferation and contraction properties. In this work we designed an automated high-throughput screening system for small molecules that induce cardiomyocyte proliferation in vitro and identified the small molecule Chicago Sky Blue 6B (CSB). Following induced myocardial infarction, CSB treatment reduced scar size and improved heart function of adult mice. Mechanistically, we show that although initially identified through in vitro screening for cardiomyocyte proliferation, in the adult mouse CSB promotes heart repair through i) inhibition of CaMKII signaling which improves cardiomyocyte contractility, and ii) inhibition of neutrophil and macrophage activation which attenuates the acute inflammatory response thereby contributing for reduced scar size. In summary, we identified CSB as a novel potential therapeutic agent that enhances cardiac repair and function by suppressing post-injury detrimental processes, with no evidence for cardiomyocyte renewal.
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).