Targeting p21high Senescent Cells Enhances PINK1/Parkin-dependent Mitophagy and Mitigates Oxidative Stress in Obese Skeletal Muscle

INTRODUCTION: Obesity imposes chronic metabolic stress that accelerates cellular senescence and impairs mitochondrial function in skeletal muscle. Recent work demonstrated that p21-highly expressing (p21high) senescent cells accumulate predominantly within the vascular endothelium, while skeletal muscle contains negligible senescent burden in young obese mice. In this context, our previous study demonstrated that selective removal of p21high cells significantly improved endothelium-dependent vasodilation and enhanced mitochondrial function in the skeletal muscle of obese mice. Although senescent cell clearance has shown improvements in both vascular and mitochondrial functions, the direct molecular consequences for mitochondrial quality control, including mitophagy and mitochondrial biogenesis, oxidative stress, and myofiber structure in obese skeletal muscle remain unexplored. To address this gap, this study examined whether targeted elimination of p21high senescent cells alters mitochondrial quality control pathways, redox balance, and muscle morphology in obese p21-Cre db/db mice. METHODS: Obese diabetic mice with a p21-Cre background were generated; control (p21-Cre/+; +/+; db/db, “Pdb”) and treatment (p21-Cre/+; DTA/+; db/db, “PDdb”) groups. Starting at 2 months of age, PDdb mice received monthly tamoxifen injections to induce p21high cells ablation. Skeletal muscle was analyzed by Western blotting for mitophagy markers (PINK1, Parkin, LC3-II, OPTN, BNIP3L, Cathepsin D), mitochondrial biogenesis regulators (PGC-1α, NRF1, TFAM), electron transport chain subunits (OXPHOS complexes II–V), inflammatory cytokines (IL-6, TNF-α), the lipid peroxidation marker 4-HNE, and the mitochondrial antioxidant MnSOD. RT-qPCR assessed mRNA levels of mitophagy-related genes (Pink1, Park2, Map1lc3b, Optn, Bnip3l) and biogenesis genes (Ppargc1a, Nrf1, Tfam). Myofiber cross-sectional area was measured using wheat germ agglutinin (WGA) staining. RESULTS: Clearance of p21high cells significantly increased PINK1 and Parkin protein contents in skeletal muscle. Downstream mitophagy markers including LC3-II, OPTN, BNIP3L, or Cathepsin D were unchanged, as were mitochondrial biogenesis regulators PGC-1α, NRF1, and TFAM and OXPHOS complexes II–V. Transcriptional levels for mitophagy (Pink1, Park2, Map1lc3b, OPTN, Bnip3l) and biogenesis (Ppargc1a, Nrf1, and Tfam) gene profiles also did not differ between groups. Skeletal muscle inflammatory cytokines (IL-6, TNF-α) and MnSOD also remained unchanged. Notably, clearance of p21high cells markedly reduced 4-HNE protein. Furthermore, PDdb mice exhibited significantly larger muscle fiber cross-sectional area compared to controls. CONCLUSION: Together with our earlier observation that mitochondrial respiration improved following p21high cell elimination, the current results suggest that the enhancement of mitochondrial function in obese skeletal muscle is driven primarily by the selective removal of dysfunctional mitochondria through PINK1/Parkin-dependent mitophagy rather than by increased mitochondrial biogenesis. Moreover, the concomitant reduction in 4-HNE protein levels suggests that removal of dysfunctional mitochondria may reduce oxidative stress generation within skeletal muscle. In summary, these findings illustrate that selective elimination of p21high senescent cells enhances PINK1/Parkin-dependent mitophagy and mitigates oxidative stress, thereby promoting mitochondrial homeostasis and preserving muscle structure in obesity. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.