Abstract Body: Adult mammalian hearts exhibit modest cardiomyocyte (CMs) turnover, mediated by proliferation of pre-existing CMs and credited to a rare population of hypoxic, cycling CMs. Using an oxygen-dependent degradation domain (ODD) reporter of HIF-1α stabilization, we identified hypoxic CMs that undergo clonal expansion and contribute to cardiomyocyte renewal. RNA-seq comparing hypoxic and normoxic CMs recognized a previously uncharacterized gene, here referred to as Hypoxic Myocyte-enriched Essential for Life (HMEL), enriched in hypoxic cardiomyocytes. Nevertheless, its function remains unknown. Here, we pursued to define the in vivo function of HMEL and the cellular processes it controls.
A global HMEL KO mouse model was generated, and heterozygous intercrosses were used for embryonic analysis (E4.5 E10.5). Embryos were genotyped and evaluated by histology and immunofluorescence for proliferation (phospho-histone H3) and apoptosis (TUNEL). FLAG-tagged HMEL was expressed in HEK293 cells followed by immunoprecipitation and mass spectrometry. RNA binding and interactions were assessed by UV crosslinking and eCLIP. Statistical analysis used two-tailed Student t-test (p<0.05).
HMEL expression declined from neonatal to adult heart. No homozygous KO pups were retrieved at birth, with reduced litter size indicating embryonic lethality. Timed pregnancies revealed resorbed conceptuses at E10.5 and severely compromised embryos at E8.5 E6.5. At E6.5, KO embryos displayed abnormal morphology, decreased proliferation, and increased apoptosis compared to controls (p<0.05). In contrast, KO blastocysts at E4.5 were comparable to controls, indicating post-implantation lethality. Immunoprecipitation-mass spectrometry identified HMEL-associated proteins enriched in RNA processing and splicing. RNA-binding assays confirmed direct RNA association, and eCLIP demonstrated preferential binding to intronic and splice-associated regions.
HMEL is essential for post-implantation embryonic survival. Its KO impairs proliferation and promotes apoptosis, leading to early lethality. These findings identify HMEL as an RNA-binding regulator linked to splicing and fundamental developmental processes.