Abstract Body: Introduction: Mammalian cardiomyocytes (CMs) transition from a proliferative fetal state to a terminally differentiated adult phenotype, which is crucial for heart maturation. The balance between proliferation and maturation is poorly understood. Our laboratory discovered that C5ORF51/RIMOC1 (C5x) plays an essential role in perinatal CM proliferation. This study aims to investigate C5x's roles in post-proliferative adult cardiomyocyte, using an inducible cardiomyocyte-specific knockout model.
Hypothesis: C5x is indispensable for adult CM cell cycle arrest and maturation, and its loss results in YAP/TAZ-TEAD1 activation, reactivation of fetal gene program, increased proliferation, and the expansion of immature CMs.
Methods: We generated tamoxifen-inducible CM-specific C5x knockout (C5x-icKO = flox/flox;Myh6CreER/+) mice and induced gene deletion in 8-week-old mice. Controls included age and littermate-matched C5x-flox/flox and Myh6CreER/+ mice receiving tamoxifen. 4-5 weeks post-induction, we analyzed cardiac function, cardiomyocyte proliferation, size, and molecular markers using histological, immunohistochemical, immunoblotting, and echocardiographic techniques.
Results: (1) Echocardiographic analyses show no differences in cardiac function between C5x-icKO and control groups, with comparable ejection fraction, fractional shortening, cardiac output, stroke volume, and strain measurements. (2) Histological and immunohistochemical analyses reveal dramatically increased CM proliferation, as evidenced by significantly elevated Ki67 (0.172% in icKO vs. 0.0725% in controls), pHH3 (0.118% in icKO vs. 0.0595% in controls), and EdU (0.628% in icKO vs. 0.2343% in controls) positive cells, reduced CM size and increased cell numbers in C5x-icKO hearts while compared to controls. These results suggest that C5x deletion in adult CMs leads to cell cycle reentry and bona fide cell division without impacting cardiac function. (3) Mechanistically, we found that YAP/TAZ-TEAD1 signaling is upregulated in C5x-deficient CMs assessed by reporter assays, with increased TEAD1 nuclear localization and elevated nuclei YAP/TAZ levels assessed by immunostaining on heart sections. These results suggest C5x deletion may lead to an activation of Hippo pathway.
Conclusions: Our findings highlight C5x as a novel regulator of postnatal CM homeostasis and suggest that modulating C5x activity could provide new therapeutic avenues for enhancing CM proliferation in regenerative cardiac medicine.