Unveiling the mechanism of single nuclear inactivation in multinucleated cells

Abstract

Maintaining genomic integrity is crucial to prevent the activation of tumorigenic mechanisms caused by DNA damage. To achieve this, specialized mechanisms must be timely and coherently activated in response to DNA damage. Collectively known as the DNA damage response (DDR), these cellular mechanisms detect damaged sites and activate downstream effectors to repair the DNA. If repair fails, cells can trigger senescence or apoptosis mechanisms to prevent the propagation of unhealthy cells. Among various forms of DNA damage, DNA double-strand breaks (DSBs) pose the most severe threat. Cells have evolved two core pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ), to repair DSBs. These pathways have notable differences, as HR requires homologous sequences and is limited to specific cell cycle phases with sister chromatids available, while NHEJ operates throughout the entire cell cycle, but with error prone ligation of broken DNA strands. Although proliferating cells' DDR mechanisms are well-characterized, our understanding of such mechanisms in post-mitotic cells, such as skeletal muscle cells, remains limited. Terminally differentiated skeletal muscle cells are among the longest-lived cells in the human body and lack the advantage of proliferative renewal. Thus, they must adapt to stress to ensure vital functions for the organism while handling DNA lesions and preserving cell viability. Differentiated muscle cells, unable to re-enter the cell cycle and employ accurate DNA repair via HR, rely on the more mutagenic NHEJ pathway to repair DSBs. Unfortunately, this mechanism leads to progressive accumulation of mutations, compromising genomic stability. In this study, we examined the DDR in differentiated skeletal muscle cells. Our findings demonstrate that myotubes exhibit a prolonged DDR, yet remain competent in repairing DSBs. Through live-cell microscopy and single-molecule kinetic measurements, we observed that myotubes respond to DNA damage by temporarily suppressing global gene expression and altering the epigenetic landscape of the damaged nucleus. Surprisingly, despite the prolonged activation of the DDR compared to their precursor cells, differentiated skeletal muscle cells show remarkable resistance to cell death. This suggests the existence of a specific pathway that helps these cells avoid the catastrophic consequences of DNA damage. Our study reveals evidence that autophagy, the cellular process responsible for clearing damaged cellular material, plays a vital role in the DDR of skeletal muscle cells. We discovered a novel mechanism employed by these cells, whereby unrepaired DNA is removed from the nucleus and processed through autophagy. This process operates in conjunction with DNA repair proteins and contributes to the apoptotic resistance phenotype observed in skeletal muscle cells. Our findings provide insights into the strategy employed by human skeletal muscle to preserve genetic integrity and the remarkable resistance of these cells to DNA damage-induced apoptosis, ensuring long-term organ function even after DNA damage occurs.