Pseudohypoxia-mediated cardiac regeneration

Informations projet

PhyCaR

N° de convention de subvention: 101027429

DOI

10.3030/101027429

Projet clôturé

Date de signature de la CE 12 Avril 2021

Date de début 14 Février 2022

Date de fin 13 Février 2024

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 160 932,48

Contribution de l’UE

€ 160 932,48

Coordonné par

FUNDACIO INSTITUT D'INVESTIGACIO BIOMEDICA DE BELLVITGE
Spain

Periodic Reporting for period 1 - PhyCaR (Pseudohypoxia-mediated cardiac regeneration)

Période du rapport: 2022-02-14 au 2024-02-13

Résumé du contexte et des objectifs généraux du projet

Acute myocardial infarction (MI) leads to loss of heart tissue, tissue scarring associated with fibrosis, dysfunction, and heart failure. The pathophysiological basis of heart failure after MI in humans lies in the heart's inability to regenerate. Although it has been shown that adult cardiomyocytes (CMs) perform mitosis, their proliferation rate is extremely low for restoring normal cardiac function. It has been demonstrated that the hypoxic microenvironment (low oxygen levels) of fetal CMs drives their proliferation, and that exposure to hypoxia in adult mice with MI promotes cardiac regeneration. In both cases, the effects of hypoxia were mediated by a HIF-1α, a protein that participates in adaptation to low oxygen levels. Although hypoxia has been proposed as a treatment to promote cardiac regeneration, its systemic and adverse effects on other organs limit its clinical use. Interestingly, a condition that mimics the hypoxia (pseudohypoxia) can be pharmacologically achieved using drugs such as Roxadustat (also known as FG-4592). Roxadustat prevents the degradation of HIF-1α and consequently promotes its function. In this context, we hypothesized that the generation of a pseudohypoxic condition induced by Roxadustat in the infarcted zone promotes cardiac regeneration by inducing CMs proliferation. To target and localize the effect of this drug, we proposed implanting a biomaterial in the infarcted heart with the capacity to conjugate Roxadustat and release it gradually. The results of this project would have a significant impact on the field of cardiac regeneration, since it is the first time that pharmacological hypoxia localized in the infarct area would be generated and studied. The validation of our hypothesis also presents a unique opportunity to propose pseudohypoxia as a therapeutic alternative to restore cardiac function after MI.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

First, we wanted to determine if Roxadustat (pseudohypoxia) mimics the cellular effects of hypoxia in CMs, such as increased glycolysis, decreased oxidative stress and induction of proliferation. We use inducible Pluripotent Stem Cells (iPSCs) as a resource for CMs. We established hypoxic cell culture, and we determine the optimal conditions for Roxadustat treatment. Several assays with Roxadustat were conducted to evaluate its effect on energy metabolism and proliferation. We have confirmed that Roxadustat promotes glycolytic metabolism, reduces oxidative stress, and increases the number of CMs in vitro.
The final goal of the project is to put a Roxadustat-loaded biomaterial onto an infarcted heart to promote regeneration. We evaluated if Roxadustat is compatible with fibrin, and if this biomaterial is capable of gradually releasing it. We established the optimal conditions for fibrin polymerization and the concentration of Roxadustat for its adequate conjugation to the biomaterial. By assays with iPSC-CM cultures, we determined that fibrin is non-toxic, it is biodegradable, and can gradually release Roxadustat.
Finally, we studied the effects of Roxadustat on cardiac regeneration in zebrafish, a heart regeneration model. Experiments in zebrafish involved apical resection of the heart, followed by intraperitoneal injection of Roxadustat to evaluate regeneration. We conducted two different experiments: 1) evaluation of CM proliferation and 2) evaluation of cardiac regeneration. We did not observe differences in the number of CMs between the control and Roxadustat groups. These data correlated with the results observed in experiment 2), where we did not see increased regeneration in the treated group. Overall, these results show that under the evaluated conditions, Roxadustat treatment does not promote CMs proliferation and cardiac regeneration in zebrafish. Considering that zebrafish have the intrinsic ability to regenerate their heart after myocardial damage, we cannot rule out an effect of Roxadustat on cardiac regeneration in mammalian models of MI. Indeed, experiments on rats with MI will be carried out in the new ongoing project.
These results were presented at national and international scientific dissemination events, e.g. the international congress "Elucidating Principles of Development with Stem Cells" organized by the International Society for Stem Cell Research, in Vienna, seminars organized by the Program for Advancing the Clinical Translation of Regenerative Medicine of Catalonia, P-CMR[C], and "Fellows’ Seminar" organized by IDIBELL for the scientific and non-scientific community.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

One of the major challenges in the field of cardiac regeneration is to stimulate the proliferation of CMs. Although hypoxia has been documented to induce CMs' reentry into the cell cycle, its clinical application as a treatment is debatable due to the risk of adverse effects. Therefore, the results of this work represent a significant achievement as they demonstrate that it is possible to induce pharmacological hypoxia in CMs and stimulate their proliferation. Although we observed that systemic administration of Roxadustat in zebrafish does not enhance cardiac regeneration, we do not rule out a regenerative effect of the drug through its local administration in the myocardium. In line with the strategy of local delivery of Roxadustat, we also demonstrated that fibrin can be used as a vehicle for Roxadustat. Additionally, Roxadustat is a drug recently approved for clinical use, which would facilitate its implementation in other diseases. The innovative aspect of these findings is that, in addition to being compatible with the drug, the biomaterial is capable to gradually release Roxadustat as it degrades. These characteristics are crucial requirements for proposing a biomaterial as part of a clinical treatment. This work also provides relevant information about Roxadustat-loaded fibrin production. Since fibrin is currently used in surgical procedures (mainly as a sealant), the possibility of conjugating it with Roxadustat is a promising approach to testing it in animal models of heart damage and eventually in humans.
Cardiovascular diseases (CVD) are the leading cause of death worldwide. According to the WHO, 19 million people died from CVD in 2019, representing 32% of all global deaths. Of these deaths, 85% were due to MI and stroke. CVD cost the EU an estimated €282 billion in 2021. Currently, there are no treatments that achieve heart regeneration and restore normal cardiac function after MI, so alternatives aimed at reactivating the proliferation of adult CMs to promote regeneration would have a significant scientific and socioeconomic impact. We have achieved solid results that will allow us to continue with a proof-of-concept test of the Roxadustat-loaded scaffold in an animal model of MI. The next stages will involve implanting this scaffold into an infarcted rat heart to study the effects of pseudohypoxia on regeneration. Considering the results of this project, we anticipate observing the gradual and local release of Roxadustat in the heart. We hope to demonstrate that the generation of targeted pseudohypoxia promotes cardiac regeneration, and that this strategy is feasible to implement in preclinical animal models such as the pig.

Fellows Seminar

ISSCR Congress

In vitro assays