Cdkn1a was identified as one of 36 upregulated genes in aged rat muscle, indicating its potential role in .
Increased Cdkn1a expression is associated with mitochondrial dysfunction and cellular in aged muscle.
C2-ceramide treatment led to increased p21 levels, enhanced senescence markers, decreased myogenesis, and increased apoptosis in myoblasts.
Knockdown of p21 in senescent myoblasts resulted in a reduction of senescent cell markers and restored cell proliferation.
The inhibition of p21 reduced levels of senescence-related cytokines, such as interleukin-6 and tumor necrosis factor-alpha.
Improved differentiation of myoblasts into myotubes was observed with p21 inhibition, indicated by enhanced myosin heavy chain expression and myotube growth.
Simplified
BACKGROUND: , characterized by a progressive loss of skeletal muscle mass and function, is associated with the accumulation of senescent muscle stem cells, which impair muscle regeneration and contributes to the decline in muscle health. Cdkn1a, which encodes p21, is a well-known marker of cellular . However, it remains unclear whether p21 inhibition eliminates senescent myoblasts and restores the differentiation capacity.
METHODS: We performed transcriptomic analysis to identify genes related to aging-induced sarcopenia using 21 month-old Sprague-Dawley rats. To investigate the specific role of Cdkn1a gene in muscle aging, we used an in vitro model of ceramide-induced senescence in myoblasts, which was verified by the upregulation of p21 and increased senescence-associated beta-galactosidase (SA-β-gal) staining. To inhibit p21, we treated myoblasts with small interfering RNA (siRNA) targeting Cdkn1a. Using fluorescence-activated cell sorting, we separated subpopulations of cells with high or low caspase 3/7 activity. Protein expression related to myogenesis, muscle atrophy, protein synthesis, and apoptosis were quantified by western blotting.
RESULTS: In our transcriptomic analysis, we identified Cdkn1a as an upregulated gene in both the soleus and white gastrocnemius muscles of aged rats, among 36 commonly upregulated genes. The upregulation of Cdkn1a appears to be linked to mitochondrial dysfunction and cellular senescence, underscoring its significance in sarcopenia pathogenesis. C2-ceramide treatment effectively induced senescence, as evidenced by increased p21 expression, enhanced SA-β-gal staining, decreased myogenesis, and increased apoptosis. Knockdown of p21 in ceramide-treated myoblasts significantly reduced SA-β-gal-positive cells, restored cell proliferation, reduced the expression of senescence-associated cytokines (i.e., interleukin (IL)-6 and tumor necrosis factor (TNF)-α), and selectively induced apoptosis in the senescent cell population, demonstrating a senolytic effect. Notably, p21 inhibition also improved differentiation of myoblasts into myotubes, as indicated by increased myosin heavy chain expression and improvements in myotube diameter and fusion index.
CONCLUSIONS: Our data suggest that p21 inhibition selectively eliminates senescent cells while simultaneously enhancing the regenerative capacity of healthy myoblasts, which may combine to improve muscle regeneration and promote myogenesis, ultimately improving muscle health and function in aged individuals.
Key numbers
4.5×
Increase in Cdkn1a Expression
In aged soleus muscle compared to young rats.
14.5×
Increase in Cdkn1a Expression
In aged white gastrocnemius muscle compared to young rats.
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Declarations. Ethics approval and consent to participate: Approval for all animal experiments was obtained from the Institutional Animal Care and Use Committee of Inha University (IACUC approval number, INHA-180807-587-5, 2020-01-29, Incheon, Korea). All of the experimental procedures involving animals were conducted in accordance with the Institutional Animal Care guidelines and the information that Local Ethics Committee acts on Animals (Scientific Procedures) Act 1986 in the UK and Directive 2010/63/EU in Europe. Consent for publication: The manuscript has been approved by all the authors. Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
PubMed
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