Necroptosis, a form of programmed cell death, is characterized by the loss of membrane integrity and the release of intracellular contents. This process is regulated by kinases RIPK1 and RIPK3, culminating in the activation of MLKL, which disrupts the cell membrane. While often associated with inflammation, the potential beneficial roles of necroptosis in tissue repair are less understood. Skeletal muscle possesses a remarkable capacity for regeneration following injury, a process heavily reliant on muscle stem cells (MuSCs). MuSC activation, proliferation, and differentiation are crucial for muscle repair, and the interplay between intrinsic MuSC factors and the extrinsic microenvironment is tightly controlled. Myofiber degeneration, often morphologically resembling necrosis, is observed during muscle repair, raising questions about its role in the regenerative process. This study explores the involvement of necroptosis in myofiber degeneration and its contribution to muscle regeneration, specifically focusing on the impact on MuSC proliferation and the potential role of secreted factors.

The existing literature establishes the mechanisms of necroptosis, highlighting the RIPK1/RIPK3/MLKL axis. Studies have shown that MLKL-mediated necroptosis drives inflammation by releasing damage-associated molecular patterns (DAMPs). However, the potential beneficial effects of necroptotic cell-released factors under pathophysiological conditions remain unclear. Research on skeletal muscle regeneration emphasizes the role of MuSCs and the dynamic interaction between intrinsic MuSC factors (like Pax7, MyoD, Myogenin) and their microenvironment. The influence of myofiber degeneration on this microenvironment is a subject of ongoing investigation. While myofiber necrosis is observed during regeneration, its precise nature and contribution to tissue repair aren't fully elucidated.

The study employed a multi-faceted approach using genetically modified mice. Necroptosis-deficient mice were generated by knocking out MLKL using CRISPR/Cas9. Muscle injury was induced using cardiotoxin (CTX) injection, and muscle regeneration was assessed through H&E staining, measuring myofiber size and fusion index. Immunofluorescence staining of MYH3 and laminin was used to visualize myofibers. Immunoblotting analyzed the expression of myogenic regulatory factors (MRFs) like MyoD and Myogenin. Flow cytometry (FACS) was used to quantify MuSCs and assess their proliferation using Ki67 staining. Quantitative RT-PCR (qRT-PCR) measured Ccnd1 mRNA levels. Myofiber-specific MLKL knockout mice were also generated using MCK-Cre;Mlklfl/fl mice to confirm the myofiber-specific role of necroptosis. In vitro studies used a Tet-inducible MLKL overexpression system in C2C12 cells to generate necroptosis conditioned medium (NCM), which was used to culture MuSCs and assess their proliferation using BrdU assays and cell counting. Biochemical purification of NCM identified Tenascin-C (TNC) as a key MuSC proliferation-promoting factor. Further experiments using TNC knockout and rescue in C2C12 cells and MCK-Cre;Tncmice confirmed TNC's role. The functional domain of TNC was mapped, and its interaction with EGFR was investigated through GST pull-down assays and immunoblotting. In vivo experiments used antibody neutralization of TNC and EGFR to assess their roles in muscle regeneration. Various statistical tests, including t-tests and ANOVA, were used to analyze data.

Necroptosis-deficient mice (Mlkl-/-) exhibited significant muscle regeneration defects, characterized by reduced myofiber size, decreased fusion index, and fewer MuSCs. These defects were rescued by myofiber-specific MLKL deletion, indicating the crucial role of myofiber necroptosis. Necroptotic myofibers, identified by increased RIPK3 and MLKL expression and MLKL phosphorylation, released factors promoting MuSC proliferation. Tenascin-C (TNC) was identified as a key factor released by necroptotic myofibers. TNC, specifically its N-terminus assembly domain and EGF-like domain, stimulated MuSC proliferation by mimicking EGF and activating the EGFR signaling pathway. Inhibition of necroptosis or TNC, or blocking of EGFR signaling, led to significant impairments in muscle regeneration, demonstrating the essential role of the necroptosis-TNC-EGFR pathway. In vivo studies confirmed the crucial role of myofiber-derived TNC in muscle regeneration.

This study reveals a novel and unexpected beneficial role for necroptosis in muscle regeneration. The findings challenge the traditional view of necroptosis as solely detrimental, demonstrating its active participation in tissue repair. The release of TNC from necroptotic myofibers, acting as an EGF mimic to activate EGFR signaling in MuSCs, provides a mechanistic explanation for the observed effects. This discovery adds to the complexity of muscle regeneration, showcasing the coordinated interplay between different cell death pathways and the regulation of the stem cell niche. The study's findings have implications for understanding and potentially treating muscle regeneration defects.

This research demonstrates that myofiber necroptosis plays a critical role in promoting muscle regeneration by releasing Tenascin-C, which subsequently activates EGFR signaling in muscle stem cells, thus driving their proliferation. The study highlights the complex and multifaceted role of necroptosis in tissue repair. Future studies could investigate the broader implications of necroptosis-mediated stem cell activation in other tissues and explore the potential therapeutic applications of targeting this pathway for enhancing muscle regeneration in various muscle diseases.

The study primarily used a mouse model, and the findings may not directly translate to human muscle regeneration. The exact mechanisms by which necroptosis regulates TNC expression remain to be fully elucidated. Future research should investigate other factors released by necroptotic myofibers that could contribute to muscle regeneration.