Screening the Ubiquitination Components Essential for Cardiomyocyte Proliferation and Cardiac Regeneration

Vu, Diem My (2021). Screening the Ubiquitination Components Essential for Cardiomyocyte Proliferation and Cardiac Regeneration. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0001244f

Abstract

For several years, the inability to replace the lost myocardium has been studied intensively to define the mechanisms that restrict the regenerative capability of the adult heart. Multiple evidence have pointed out that the turnover of postnatal mammalian cardiomyocytes is affected by different crucial pathways. However, a thorough understanding of the intrinsic molecular mechanisms that regulate this process is still far from being complete.

It has been more than a decade since various laboratories initialised the study of ubiquitination processes in the heart, reaching the unanimous conclusion that it plays a critical role in manipulating virtually all heart functions. However, there is fragmentary information about the molecular function of this system on the regulation of cardiomyocyte proliferation and even less on how this could be exploited for therapeutic purposes. Hence, expanding our knowledge on the ubiquitination might devise new strategies for cardiac regeneration.

In this work, we present the identification of ubiquitination factors that are essential for cardiomyocyte replication, which was identified by harnessing the High Throughput Screening approach. An RNAi-based screening was performed, in which approximately 600 ubiquitination factors were silenced individually in primary neonatal cardiomyocytes. After that, the top siRNAs inhibiting proliferation were selected and investigated further to validate their functions in vitro. The validation identified UBE2G1, an E2 conjugation enzyme, as the most effective factor induced cardiomyocyte proliferation. The depletion of UBE2G1 not only suppressed cell cycle progression but also stimulated hypertrophy and counteracted the effects of pro-proliferative miRNAs. Administration of AAV9-UBE2G1 in neonatal mice promoted the cycling of cardiomyocytes while it preserved the heart function in adult mice at the early time points after myocardial infarction. Activation of GSK-3β, ERK1/2, and STAT3 signalling pathways correlated with UBE2G1 activity, whereas its interacting partners remained to be identified. In summary, we have revealed the implicit potential of ubiquitination factors and highlighted their promising therapeutic use in a heart regeneration scenario.