Cyp27b1 ablation in skeletal muscle impairs muscle regeneration
Invited conference paper presented and published in conference proceedings
CUHK Authors
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AbstractIntroduction: Muscle regeneration involves various cellular and molecular mechanisms. Vitamin D is important in muscle function and repair. Cyp27b1 activates vitamin D into 1,25-dihydroxyvitamin-D3 inside mitochondrial inner membrane, which promotes muscle contraction velocity and strength. (Girgis, Clifton-Bligh, Hamrick, Holick, & Gunton, 2013). The local effects of cyp27b1 in muscle and extra-renal activation of vitamin D are not well understood. Global knockout cyp27b1 mice results in decreased muscle mass due to increased oxidative stress (Yu et al., 2021). Understanding the relationship between cyp27b1 and mitochondrial function may provide insights into underlying mechanisms of muscle degeneration.
Method: In vivo cyp27b1 knockdown was induced in male cyp27b1 floxed mice (3.5 months old) via bilateral gastrocnemius (GAS) intra-muscular injection of AAV9-CMVCRE-GFP virus (100 ul, 2 x 10^12 vg/ml). After 10 days, GAS was collected for fiber type analysis, ex vivo functional testing, and qPCR. (n=3). Muscle injury was induced via I.M. injection of BaCl2 (1.2% w/v, 50 μl) in bilateral gastrocnemius 10 days after virus injection and observed for 7 days to evaluate muscle regeneration by H&E staining and qPCR. C2C12 myoblasts was used in loss-of-function and gain-of-function studies (n=3). One-way ANOVA and two-way ANOVA were used for the animal and cellular studies, respectively. The level of significance was set at P<0.05.
Results: In vivo knockdown of cyp27b1 reach up to 90% efficiency by qPCR. The knockdown resulted in significant changes in muscle fiber type distribution, i.e., type I (WT: 0.36%; KD 11.9%) and type IIA (WT: 23.5 %; KD: 46.4%) /IIB (WT: 76%; KD: 41.6%) and decrease in muscle force, specifically reduction in specific twitch force (-16%) and specific tetanic force (-29%). KD group showed lower mitochondrial biogenesis marker expression than the control, as indicated by qPCR and western blot. Following injury, there was decreased gene expression related to muscle regeneration and mitochondria biogenesis, delayed regeneration and increased immune cell infiltration were also observed. Cyp27b1 knockdown in C2C12 suppressed myogenic and mitochondrial biogenesis marker expression and proliferation, while overexpression had the opposite effect.
Discussion: Our findings suggest cyp27b1 plays a critical role in muscle regeneration after injury, likely due to its extra-renal effect on vitamin D metabolism in muscle tissues. The findings suggest potential for developing new therapeutic strategies for treating muscle damage, including the discovery of novel Cyp27b1 activators that could extend to other muscular disorders.
Method: In vivo cyp27b1 knockdown was induced in male cyp27b1 floxed mice (3.5 months old) via bilateral gastrocnemius (GAS) intra-muscular injection of AAV9-CMVCRE-GFP virus (100 ul, 2 x 10^12 vg/ml). After 10 days, GAS was collected for fiber type analysis, ex vivo functional testing, and qPCR. (n=3). Muscle injury was induced via I.M. injection of BaCl2 (1.2% w/v, 50 μl) in bilateral gastrocnemius 10 days after virus injection and observed for 7 days to evaluate muscle regeneration by H&E staining and qPCR. C2C12 myoblasts was used in loss-of-function and gain-of-function studies (n=3). One-way ANOVA and two-way ANOVA were used for the animal and cellular studies, respectively. The level of significance was set at P<0.05.
Results: In vivo knockdown of cyp27b1 reach up to 90% efficiency by qPCR. The knockdown resulted in significant changes in muscle fiber type distribution, i.e., type I (WT: 0.36%; KD 11.9%) and type IIA (WT: 23.5 %; KD: 46.4%) /IIB (WT: 76%; KD: 41.6%) and decrease in muscle force, specifically reduction in specific twitch force (-16%) and specific tetanic force (-29%). KD group showed lower mitochondrial biogenesis marker expression than the control, as indicated by qPCR and western blot. Following injury, there was decreased gene expression related to muscle regeneration and mitochondria biogenesis, delayed regeneration and increased immune cell infiltration were also observed. Cyp27b1 knockdown in C2C12 suppressed myogenic and mitochondrial biogenesis marker expression and proliferation, while overexpression had the opposite effect.
Discussion: Our findings suggest cyp27b1 plays a critical role in muscle regeneration after injury, likely due to its extra-renal effect on vitamin D metabolism in muscle tissues. The findings suggest potential for developing new therapeutic strategies for treating muscle damage, including the discovery of novel Cyp27b1 activators that could extend to other muscular disorders.
Acceptance Date27/04/2023
All Author(s) ListHiu-tung Jessica Lo, Tszlam Yiu, Daniel Kam-Wah Mok, Man-Sau Wong, Wayne Yuk-wai Lee
Name of Conference16th International Conference on Cachexia, Sarcopenia & Muscle Wasting
Start Date of Conference17/06/2023
End Date of Conference19/06/2023
Place of ConferenceStockholm
Country/Region of ConferenceSweden
Year2023
LanguagesEnglish-United Kingdom
