*3.4. Mitochondrial Ca2*<sup>+</sup> *Level in Single Skeletal Muscle Fibers during Di*ff*erent Hibernation Periods*

Compared with that in the SA or PRE groups, the mitochondrial Ca2<sup>+</sup> level in the single muscle fibers was elevated to varying degrees during hibernation (in PL muscle of the IBA group and in AM muscle of the LT and ET groups) (Figure 5).

**Figure 5.** Changes in the mitochondrial Ca2<sup>+</sup> concentration in PL and AM muscles during different periods. (**A**) Representative fluorescence images of single PL and AM muscle fibers. Scale bar = 100 μm. (**B**) Histogram depicting the mitochondrial Ca2<sup>+</sup> fluorescence intensity in PL and AM muscles during different periods. PL, plantaris; AM, adductor magnus. SA, summer active group; PRE, pre-hibernation group; LT, late torpor group; IBA, inter-bout arousal group; ET, early torpor group; POST, post-hibernation group. Values are means ± SEM, n = 6–8. \* *p* < 0.05 and \*\* *p* < 0.01 compared with SA; # *p* < 0.05 compared with PRE.

We comprehensively analyzed the changes in cytoplasmic, SR, and mitochondrial Ca2<sup>+</sup> levels during different periods. Firstly, the opposite changes in cytoplasmic and SR Ca2<sup>+</sup> suggest that SR Ca2<sup>+</sup> participates in fluctuation of the cytoplasmic Ca2<sup>+</sup> level during hibernation. In addition, the slight elevation in mitochondrial Ca2<sup>+</sup> during hibernation may result from increased cytoplasmic Ca2<sup>+</sup> or SR Ca2<sup>+</sup> leakage. Further studies were subsequently carried out to explore the mechanisms involved in intracellular Ca2<sup>+</sup> fluctuations during hibernation.
