Abstract
Skeletal muscle has a major role in locomotion and muscle disorders are associated with poor regenerative efficiency. Therefore, a deeper understanding of muscle regeneration is needed to provide a new insight for new therapies. CaMKK2 plays a role in the calcium/calmodulin-dependent kinase cascade; however, its role in skeletal muscle remains unknown. Here, we found that CaMKK2 expression levels were altered under physiological and pathological conditions including postnatal myogensis, freeze or cardiotoxin-induced muscle regeneration, and Duchenne muscular dystrophy. Overexpression of CaMKK2 suppressed C2C12 myoblast proliferation and differentiation, while inhibition of CaMKK2 had opposite effect. We also found that CaMKK2 is able to activate AMPK in C2C12 myocytes. Inhibition of AMPK could attenuate the effect of CaMKK2 overexpression, while AMPK agonist could abrogate the effect of CaMKK2 knockdown on C2C12 cell differentiation and proliferation. These results suggest that CaMKK2 functions as an AMPK kinase in muscle cells and AMPK mediates the effect of CaMKK2 on myoblast proliferation and differentiation. Our data also indicate that CaMKK2 might inhibit myoblast proliferation through AMPK-mediated cell cycle arrest by inducing cdc2-Tyr15 phosphorylation and repress differentiation through affecting PGC1α transcription. Lastly, we show that overexpressing CaMKK2 in the muscle of mice via electroporation impaired the muscle regeneration during freeze-induced injury, indicating that CaMKK2 could serve as a potential target to treat patients with muscle injury or myopathies. Together, our study reveals a new role for CaMKK2 as a negative regulator of myoblast differentiation and proliferation and sheds new light on the molecular regulation of muscle regeneration.
Highlights
Skeletal muscle is an important part of the animal body, and represents nearly half of the total body mass [1]
In the present study, we found that CaMKK2 is down-regulated in skeletal muscle in the cardiotoxin (CTX) or freeze-injury-induced muscle regeneration, indicating that CaMKK2 may play a role in skeletal muscle regeneration through regulating cell proliferation and differentiation
Since the CaMKK2 expression was decreased in postnatal myogenesis (Figure 1A,B) and the expression of CaMKK2 mRNA and protein was negatively correlated with the levels of myogenic markers during myogenic differentiation (Figure 1F,G and Figure S1F,G), we speculated that CaMKK2 might have an effect on C2C12 cell differentiation
Summary
Skeletal muscle is an important part of the animal body, and represents nearly half of the total body mass [1]. The function of CaMKK2 in cell development has been investigated extensively in neurons, adipose and myeloid cells, the role of this kinase in the muscle is largely unknown. Williamson.et al showed that AICAR-induced AMPK phosphorylation reduced the differentiation of myoblasts into myotubes through PGC1α transcription [19]. Based on these findings, we hypothesized that CaMKK2 might act as an upstream regulator of AMPK and play a role in regulating myogenesis and muscle regeneration. In the present study, we found that CaMKK2 is down-regulated in skeletal muscle in the cardiotoxin (CTX) or freeze-injury-induced muscle regeneration, indicating that CaMKK2 may play a role in skeletal muscle regeneration through regulating cell proliferation and differentiation. Further study suggests that AMPK might mediate the action of CaMKK2 on myoblast proliferation and differentiation
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