Abstract
Despite the development of new pharmacological and device-based therapies, no approaches currently exist to compensate for the irreversible loss of cardiomyocytes occurring after myocardial infarction, which represents a major cause of heart failure worldwide. One area that has generated considerable interest over the last decade is the possibility of promoting cardiac regeneration through the stimulation of the endogenous capacity of cardiomyocytes to proliferate, which our laboratory has pioneered (Eulalio, Mano et al. 2012, Gabisonia, Prosdocimo et al. 2019).In a search for novel treatments able to induce cardiomyocyte proliferation, we performed a high-content, fluorescence-microscopy-based, high-throughput screening in neonatal rat cardiomyocytes using a library 780 FDA-approved small molecules. The screening, which was based on EdU incorporation as an S-phase marker, identified harmine as the most powerful drug to enhance proliferation in combination with miR-199a-3p and miR-590-3p, the most effective microRNAs that the laboratory had previously identified (Eulalio, Mano et al. 2012).
Harmine belongs to the β-carboline alkaloid family and is a potent inhibitor of the DYRK family of protein kinases, with high specificity for the DYRK1A isoform (Bain, Plater et al. 2007). Unexpectedly, harmine was also found to increase cardiomyocyte proliferation by itself, in the absence of the microRNAs, to a level comparable to that of the powerful miR-199a-3p. Further in vivo models of myocardial infarction (MI) in adult CD1 mice showed an improvement in cardiac function as well as a milder fibrotic area upon harmine treatment compared to controls.
Downregulation of DYRK1a showed increased EdU incorporation, consistent with a negative role on cardiomyocyte proliferation. These data are in line with a previous study showing that inhibition of DYRK1a is a possible strategy to induce human pancreas islet β-cell proliferation (Wang, Alvarez-Perez et al. 2015, Dirice, Walpita et al. 2016). Interestingly, experiments showed an interplay between Dyrk1a kinase and the pro-proliferative transcriptional co-activator YAP. The alteration of Dyrk1a protein levels markedly affected YAP nuclear localisation and subsequent YAP-target gene expression. Vice versa, YAP down-regulation led to a striking decrease in Dyrk1a protein levels, suggesting a negative feedback loop between the two proteins. Moreover, in co-transduction experiments, AAV-driven Dyrk1a over-expression surprisingly resulted in the negative regulation of the hyperactive mutant form of YAP (YAP5SA). The YAP5SA mutant is insensitive to Hippo regulation and proteasome degradation, therefore we assumed that YAP could be directed to an alternative degradation pathway. Chaperone-mediated autophagy (CMA) is a specific form of autophagy and intriguingly, when repeating this experiment with the simultaneous blockade of CMA, there is a rescue of YAP5SA levels. This finding strengthens the hypothesis that Dyrk1a directs YAP to degradation via CMA, acting as a secondary regulatory mechanism after the Hippo pathway. It is not clear whether this is a direct or indirect regulation, however, the experimental work outlined in this thesis contributes to our understanding of DYRK1a’s function in cardiomyocytes, through its regulation of YAP via the CMA degradation pathway, and therefore the impact of Dyrk1a in cardiac biology. The elucidated mechanisms and the consequently gained knowledge can lead to an enrichment of the current strategies based on the stimulation of the endogenous capacity of cardiomyocytes to proliferate, eventually leading to further advancement in cardiac regeneration.
| Date of Award | 1 Sept 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Mauro Giacca (Supervisor) & Mateusz Tomczyk (Supervisor) |