Pyruvate Kinase M2 Supports Muscle Progenitor Cell Proliferation but Is Dispensable for Skeletal Muscle Regeneration after Injury

Abstract
Background: Skeletal muscle progenitor cells (MPCs) are essential for repairing muscle tissue after injury. Pyruvate kinase M2 (PKM2), a glycolytic enzyme known for its canonical function, also engages in noncanonical activities by interacting with other proteins, influencing various cellular processes. Recent studies have linked PKM2 to the proliferation of MPCs.

Objectives: This study aimed to investigate the role of PKM2 in MPCs and determine its necessity for muscle regeneration following injury.

Methods: Cultured, proliferating MPCs (C2C12 cells) were treated with short hairpin RNA (shRNA) targeting PKM2 or small molecules that selectively modulate PKM2′s canonical and noncanonical activities (shikonin and TEPP-46). Cell counts were recorded, and RNA sequencing and metabolic assays were conducted in subsequent experiments. Immunoprecipitation combined with proteomics was used to identify PKM2 binding partners. Additionally, an MPC-specific PKM2 knockout mouse model was created and subjected to muscle injury to assess the role of PKM2 in regeneration.

Results: Blocking or impairing PKM2′s noncanonical activity led to increased reactive oxygen species (ROS) levels (1.6-2.0-fold, P < 0.01). Inhibition of noncanonical PKM2 activity also caused elevated lactate excretion (1.2-1.6-fold, P < 0.05) and reduced mitochondrial oxygen consumption (1.3-1.6-fold, P < 0.01). Glutamate dehydrogenase 1 (GLUD1) was identified as a binding partner of PKM2, and its activity was enhanced (1.5-1.6-fold, P < 0.05) when noncanonical PKM2 activity was blocked. However, mice with an MPC-specific PKM2 deletion showed no deficits in muscle regeneration.

Conclusions: The findings indicate that PKM2's noncanonical activity plays a ML265 crucial role in MPC proliferation in vitro and reveal GLUD1 as a PKM2 binding partner. Despite this, the absence of PKM2 did not impair muscle regeneration in the mouse model, suggesting that compensatory mechanisms in the endogenous environment may mitigate the loss of PKM2.