Cooling-promoted myogenic differentiation of murine bone marrow mesenchymal stem cells through TRPM8 activation in vitro

TRPM8 agonist has been reported to promote osteogenic differentiation of mesenchymal stem cells (MSCs), therefore we evaluated whether cooling-induced activation of TRPM8 promotes myogenic differentiation of MSCs. We used 5-azacytidine as a myogenic differentiation inducer in murine bone marrow-derived MSCs. Addition of menthol, a TRPM8 agonist, to the differentiation induction medium significantly, increased the percentage of MyoD-positive cells, a specific marker of myogenic differentiation. We performed intracellular Ca2+ imaging experiments using fura-2 to confirm TRPM8 activation by cooling stimulation. The results confirmed that intracellular Ca2+ concentration ([Ca2+]i) increases due to TRPM8 activation, and TRPM8 antagonist inhibits increase in [Ca2+]i at medium temperatures below 19 degrees C. We also examined the effect of cooling exposure time on myogenic differentiation of MSCs using an external cooling stimulus set at 17 degrees C. The results showed that 60 min of cooling had an acceleratory effect on differentiation (2.18 +/- 0.27 times). We observed that the TRPM8 antagonist counteracted the differentiation-promoting effect of the cooling. These results suggest that TRPM8 might modulate the multiple differentiation pathways of MSCs, and that cooling is an effective way of activating TRPM8, which regulates MSCs differentiation in vitro.