Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy.

Takehito Sugasawa,Yuichi Aita,Reina Maruyama,Kaoru Takeuchi,Haruna Ueda,Yasuko Yoshida,Yoshinori Takeuchi,Rahul Sharma,Shohei Morita,Kazuhiro Takekoshi,Yasushi Kawamai,Naoya Yahagi,Yoshiya Tome

doi:10.3390/ijms21134588

Takehito Sugasawa, Yuichi Aita + Show 11 more

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https://doi.org/10.3390/ijms21134588

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Abstract

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

Highlights

Local cryotherapy has been widely used in the acute treatment of traumatic injuries, such as fractures, dislocations, sprains, and pulled muscles
Two bands appeared in western blot (WB) analysis of C2C12, 3T3-L1, and human fibroblast (HF) cells that make up muscle tissue, in response to anti phosphorylated CREB1 (p-CREB1) antibody
Thirty minutes after the final cold stimulation, the p-CREB1 bands in all cell lines returned to the same intensity as those of the control (Figure 1a–d)

Summary

Introduction

Local cryotherapy has been widely used in the acute treatment of traumatic injuries, such as fractures, dislocations, sprains, and pulled muscles. It has been applied for recovery after daily training and sports competitions. The use of cryotherapy in the management of sports injuries was first reported in Greece in the 1950s. Knight proposed that cryotherapy retards cellular metabolism and mitigates impairment caused by secondary hypoxia in the injured area [1]. This theory has been accepted as the standard explanation for the mechanism underlying local cryotherapy in sports injury management. There are numerous uncertainties regarding the efficacy and basic mechanism of local cryotherapy, as the previous study described below

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