Cardiovascular diseases remain the leading cause of death worldwide. However, the molecular mechanisms controlling homeostasis and proliferation in cardiomyocytes are still poorly understood. A deeper understanding of cardiac regenerative capacity could enable new therapeutic strategies after myocardial infarction in humans. The zebrafish has emerged as a powerful model organism for studying cardiac development by enabling simple genetic modification. Due to its remarkable regenerative capacity, it is ideal for addressing the objectives of this study. The ENU-induced zebrafish mutant line herzbuckel (hzb) exhibits embryonic lethality in homozygous individuals, due to a mutation in the Traf7 gene (tumor necrosis factor receptor-associated factor 7). TRAF7 is an E3 ubiquitin ligase implicated in regulating the NF-κB pathway, which is assumed to modulate cell cycle activity.
The objectives of this research project were to gain insights in the role of TRAF7 on heart development and function in zebrafish and to further explore TRAF7´s impact on proliferation and proteostasis in mammalian cardiomyocytes (CMs). Therefore, neonatal rat CMs were transfected with AAV1 containing shRNA to knockdown (kd.) Traf7 expression.
In this study we were able to show, that hzb mutants exhibited reduced ventricular contractility, a decreased heart rate and reduction of cardiomyocytes proliferation. In neonatal rat ventricular myocytes (NRVMs) a significantly reduced proliferation rate by 56,38 % was observed, while apoptosis levels remained unchanged after Traf7-kd. Quantitative real-time PCR analysis of multiple cell cycle regulatory genes revealed a significant down regulation of Cyclin- D1 by 64,81 %, a key regulator of the G1/S phase transition. Further cell cycle analysis confirmed that Traf7-kd. results in an increased proportion of cells arrested in the G1 phase, indicating their impaired proliferative activity. Given that Cyclin D1 is transcriptionally activated by NF-κB, these findings imply that TRAF7 may impact cell cycle progression through modulation of NF-κB signaling. Downregulation of genes within the NF κB pathway (nfkb1, nfkb2, map3k3, ikkβ, nemo, rela) was observed in zebrafish hzb mutants supporting these findings.
To clarify TRAF7´s role as an E3 ligase in modulating NF-kB signaling, ongoing analyses are focused on identifying TRAF7-dependent ubiquitination targets, considering the critical role of the NF-κB pathway.
In summary, our data reveal that TRAF7 is an important regulator of cardiomyocyte proliferation in both zebrafish and mammalian systems. These results point to a possible role of TRAF7 in cardiac regeneration, suggesting its potential as a therapeutic target.
