Changes of body surface temperature of ground squirrels during hibernation. The hibernating ground squirrels are in the cycle of torpor - arousal when they enter hibernation. The body temperature is close to the ambient temperature in torpor. The temperature rises to above 35 °C in interbout arousal (Fig. 1).
Body weight, skeletal muscle wet weight (MWW), and ratio of skeletal muscle wet weight to body weight (MWW/BW). Three different muscles (SOL, GAS, and EDL) from Daurian ground squirrels were used in the current research. Compared with the summer group (SA), the MWW of the SOL was 14–24% (P < 0.05) lower in the pre-hibernation (PRE), inter-bout arousal (IBA), early torpor (ET), later torpor (LT), and post-hibernation (POST) groups and the MWW of the EDL was 16–21% (P < 0.05) lower in the IBA, ET, LT, and POST groups. Compared with the SA and PRE groups, the MWW of the GAS was 14–25% (P < 0.05) lower in the IBA, ET, LT, and POST groups (Table 1). Moreover, the MWW/BW ratio in the SOL was 20% (P < 0.05) lower in the PRE and 12–22% (P < 0.05) higher in the other four groups compared with the SA group. The MWW/BW ratio in the GAS and EDL muscles was 14–36% (P < 0.05) higher in the other four groups compared with the SA and PRE groups (Table 2).
Cytoplasm Ca 2+ concentration in single muscle fiber. Figure 2a shows representative two-dimensional confocal images of muscle fibers from the SOL, GAS, and EDL of the SA, PRE, IBA, ET, LT, and POST groups. The uniform green fluorescence mainly distributed in the cytoplasm represents Ca2+ concentration in the muscle fiber. The cytosolic Ca2+ fluorescence in the GAS increased by 46% in the ET group compared with that in the SA group. It is worth noting that, compared with the SA group, the Ca2+ fluorescence in the LT and IBA groups increased significantly by 117% (P < 0.001) and 30% (P < 0.05) in the SOL, by 106% (P < 0.001) and 65% (P < 0.01) in the EDL, and by 213% (P < 0.001) and 153% (P < 0.001) in the GAS, respectively. However, compared to the LT group, the Ca2+ fluorescence in the POST group decreased significantly by 56% (P < 0.001) in the SOL and 64% (P < 0.001) in the GAS. Compared with the ET group, Ca2+ fluorescence in the POST group decreased significantly (23%, P < 0.05) in the GAS but increased significantly (38%, P < 0.05) in the EDL (Fig. 2b).
SR-Ca 2+ concentration in single muscle fiber. Figure 3a shows typical two-dimensional confocal images of muscle fibers showing SR-specific fluorescence and SR-Ca2+ fluorescence, respectively. The SR-Ca2+ fluorescence almost coincided with the SR fluorescence in muscle fibers and was distributed uniformly along the stria of muscle fibers.
Figure 3b shows representative two-dimensional confocal images of muscle fibers from all three muscles in the SA, PRE, LT, IBA, ET, and POST groups. The muscle fibers exhibited a relatively uniform green fluorescence, distributed mainly in the SR. The SR Ca2+ fluorescence in the SOL, EDL, and GAS muscles decreased significantly by 80%, 84%, and 68% (P < 0.001), respectively, in the PRE group compared with the SA group. The Ca2+ fluorescence in the SOL showed a slight decrease (14%, P < 0.05) in the IBA group compared with the SA group. An extremely significant decrease in Ca2+ fluorescence was observed in the EDL (83%, P < 0.001) and GAS muscles (65%, P < 0.001). The SR Ca2+ fluorescence in the EDL increased by 23% (P < 0.01) in the ET group compared with the SA group; however, no significant differences were observed in the SOL and GAS. The SR Ca2+ fluorescence in the SOL and GAS muscles from the LT group decreased and recovered to PRE group levels. The greatest differences among the three different muscles were in the POST group. Compared with the SA group, SR Ca2+ fluorescence in the POST group decreased significantly in the SOL (18%, P < 0.05) and EDL (43%, P < 0.05), but increased significantly in the GAS (40%, P < 0.05) (Figure 3c).
Relative protein expression. The protein contents of RyR1, DHPR, FKBP12, SERCA1, SLN, P-PLB, PLB, β-AR2, P-CaMK2, CaMK2, CaM and CSQ1 were detected by Western blot analysis, as shown in Figure 4a. Representative polyacrylamide gels of total protein are shown in Figure 4b. The protein expression levels of RyR1 in the three muscles in the LT, IBA, and ET groups were higher than levels in the other groups. The increments were 37%–51% (P < 0.01) in the SOL, 43%–50% in the EDL (P < 0.05), and 96%–107% (P < 0.01) in the GAS compared to the SA group; however, no significant differences were observed among the three groups (Figure 4c).
The protein expression levels of DHPR in the three muscles in the LT, IBA, and ET groups were lower than the levels in the SA group, with decrements of 30%–32% (P < 0.05) in the SOL, 33%–40% (P < 0.05) in the EDL, and 23%–29% (P < 0.05) in the GAS (Figure 4d).
Compared to the SA group, FKBP12 protein expression in the SOL increased by 103% in the PRE group (P < 0.01), 82% in the LT group (P < 0.01), and 52% in the POST group (P < 0.05). Compared to the SA group, FKBP12 protein expression in the EDL increased by 22% in the PRE (P < 0.05) and 50% in the LT group (P < 0.01) but decreased by 22% in the ET (P < 0.05). Compared to the SA group, FKBP12 protein expression in the GAS decreased by 23% in the PRE (P < 0.05), 18% in the ET (P < 0.05), and 38% in the LT (P < 0.01) (Figure 4e). Overall, the RyR1 regulation pathway was mainly up-regulated during hibernation, except for DHPR.
Compared with the SA group, the SERCA1 protein level increased significantly in the three different muscles by 43%–98% in the IBA group (P < 0.001), 28%–103% in the ET group (P < 0.05), and 9%–102% in the LT group (P < 0.05), respectively. In the SOL muscle, expression of SERCA1 only decreased (24%, P < 0.05) in the POST group compared with the SA group. The main difference between the GAS and other two muscles was that a significant increase in SERCA1 expression was observed in both the PRE group (96%, P < 0.01) and POST group (145%, P < 0.001) (Figure 4f) compared with the SA group.
The SLN protein expression in the three different muscles showed a common pattern. The protein expression levels in most hibernating groups (PRE, LT, IBA, ET) were not lower than that in the SA group. In the SOL, the increments were 140% (P < 0.001) in the PRE, 136% (P < 0.001) in the LT, and 89% (P < 0.05) in the IBA. In the EDL, the increments were 216% (P < 0.001) in the PRE, 74% (P < 0.05) in the LT, 168% (P < 0.001) in the IBA, and 100% (P < 0.05) in the ET. In the GAS, the increments were 175% (P < 0.001) in the PRE, 53% (P < 0.05) in the LT, and 51% (P < 0.05) in the IBA compared to the SA group, respectively (Figure 4g).
The PLB protein expression in the SOL was 19% and 21% (P < 0.05) higher in the PRE and ET groups than that in the SA group. Expression in the EDL increased significantly by 27% (P < 0.05) in the PRE group and 24% (P < 0.05) in the LT group but decreased by 21% (P < 0.05) in the POST group. Expression in the GAS was higher in all groups compared with that in the SA group, with increases of 34% (P < 0.05) in the PRE, 117% (P < 0.001) in the LT, 172% (P < 0.001) in the IBA, 112% (P < 0.001) in the ET, and 72% (P < 0.05) in the POST (Figure 4h).
The p-PLB/PLB ratio, as an important indicator of Ca2+ pump activities, was analyzed in the SOL, EDL, and GAS muscles. In the SOL muscle, the ratio remained unchanged in all hibernation groups, except the IBA group, which was higher (101%, P < 0.001) than that in the SA group. In the EDL, the ratio was significantly decreased in the LT group (43%, P < 0.01) compared with the SA group. In the GAS muscle, the ratio was significantly elevated in the IBA (29%, P < 0.05), ET (50%, P < 0.01), and POST groups (30%, P < 0.05) compared with that in the SA group (Figure 4i).
The β-AR2 protein expression in the SOL remained unchanged in all groups, except for the POST group (-72% compared to SA, P < 0.001). In the EDL, protein expression in all groups was significantly lower than that in the SA group (P < 0.05). In the GAS, however, protein expression was significantly increased in the PRE (45%, P < 0.05), IBA (42%, P < 0.05), and ET groups (50%, P < 0.05) compared with the SA group (Figure 4j).
Similar CaMK2 protein expression trends were found in the SOL and EDL, with levels in the PRE, IBA, ET, and LT groups higher than that in the SA group. In the SOL and EDL, protein expression increased significantly in the PRE, IBA, ET, and LT groups (33% and 20%, 22% and 38%, 31% and 34%, and 38% and 40% (P < 0.01), respectively compared with the SA group. In the GAS, protein expression decreased significantly by 47% in the LT group (P < 0.01) and 33% in the ET group (P < 0.05) compared with the SA group (Figure 4k).
In the SOL, the P-CaMK2 to CaMK2 ratio only increased by 18% (P < 0.05) in IBA group in SOL, while other groups were all lower than that in SA group in three muscles (Figure 4l). Overall, the SERCA regulation pathway was mainly up-regulated during hibernation.
The CaM protein expression showed similar alternation trends in the EDL and GAS muscles, with the level of CaM in the other five groups higher than that in the SA group. In the EDL muscle, the CaM protein expression levels in the PRE, IBA, ET, LT, and POST groups were 48% (P < 0.05), 126%, 124%, 125%, and 134% (P < 0.001) higher, respectively, than that in the SA group. In the GAS, protein expression significantly increased by 116%, 89%, 87%, 87% (P < 0.05), and 136% (P < 0.001) in the PRE, IBA, ET, LT, and POST groups, respectively, compared with the SA group. In the SOL, however, protein expression decreased significantly by 75% and 73% (P < 0.001) in the LT and POST groups, respectively, compared with the SA group (Figure 4m).
Similar change trends in CSQ1 protein expression were observed in the three different muscles, with levels in the five groups higher than that in the SA group. In the SOL, compared with the SA group, protein expression increased significantly by 205% (P < 0.05) in the PRE, 323% (P < 0.001) in the IBA, 334% (P < 0.001) in the ET, 185% (P < 0.001) in the LT, and 114% (P < 0.05) in the POST. In the EDL, protein expression increased significantly by 85% in the PRE, 81% in the IBA, 83% in the ET, and 103% in the LT (P < 0.05). In the GAS, protein expression increased significantly by 97% in the PRE, 280% in the IBA, 240% in the ET, 235% in the LT, and 242% in the POST (P < 0.05) (Figure 4n). Overall, the expression of both proteins showed an increasing trend during hibernation compared to the SA.
Relative mRNA expression. Relative expression levels of serca1, serca2, plb, and sln are shown in Figures 5a, b, c, and d, respectively. Commonly, the mRNA expression of serca1 in the different muscles was lower in the POST group than in the PRE group. In the SOL, the expression levels in the PRE (38%, P < 0.01), IBA (63, P < 0.001), and POST groups (47%, P < 0.001) were significantly lower than that in the SA group. In the EDL, expression levels in the PRE and POST groups were 23% and 34% (P < 0.05) lower and in the ET group was 21% (P < 0.05) higher than that in the SA group (Figure 5a).
Compared with the SA group, the relative serca2 mRNA expression in most groups increased in the three different muscles. In the SOL, expression increased significantly by 95%–313% (P < 0.05) in the PRE, IBA, ET, and LT groups. In the EDL, expression increased significantly by 500%–900% (P < 0.001) in the PRE, IBA, and ET groups. In the GAS, expression increased significantly by 100%–176% in all groups, except the POST group (Figure 5b).
In the SOL, compared with the SA group, the relative plb mRNA expression reduced by 23% (P < 0.05) in the LT group. In the EDL, expression increased by 38% (P < 0.05) in the ET group and decreased by 35% (P < 0.05) in the LT group compared with the SA group. In the GAS, expression increased significantly by 50% in the PRE group and 29% in the ET group (P < 0.05) compared with the SA group (Figure 5c).
The relative sln mRNA expression levels were comparable in the three muscles, with levels similar in the POST and SA groups, but significantly lower in the other groups. In the SOL, no significant differences were found among the four groups, but expression levels were 36%–42% (P < 0.05) lower than that in the SA group. In the EDL, expression decreased significantly by 54% in the PRE, 81% in the LT, 66% in the IBA, and 74% in the ET (all P < 0.001) compared with the SA group. In the GAS, no significant differences in expression levels were observed among the three hibernation groups, with levels 43%–47% (P < 0.05) lower than that in the SA group (Figure 5d).
Relative expression levels of cam and csq1 are shown in Figures 5e and f, respectively. In the SOL, the relative csq1 mRNA expression was lower in the five groups compared with the SA group (16% in the PRE, 14% in the LT, 27% in the IBA, 22% in the ET (P < 0.05), and 64% in the POST (P < 0.001). In the EDL, only expression levels in the PRE (39%, P < 0.01), IBA (26%, P < 0.05), and POST groups (57%, P < 0.001) were lower than that in the SA group, with no significant differences found among the other groups. In the GAS, except for the PRE group, expression was higher in all hibernating groups than in the SA group, with increases of 26% (P < 0.05) in the LT, 40% (P < 0.05) in the IBA, 63% (P < 0.05) in the ET, and 47% (P < 0.05) in the POST (Figure 5e).
In the SOL, the relative cam1 mRNA expression increased by 41% (P < 0.05) in the PRE group and decreased by 75% (P < 0.05) in the POST group compared with the SA group. In the EDL, expression was significantly higher in the PRE (95%, P < 0.05), IBA (204%, P < 0.01), ET (255%, P < 0.001), and POST groups (55%, P < 0.05) compared with the SA group. In the GAS, expression was 36%–75% (P < 0.05) lower in the ET, LT, and POST groups compared with the SA groups (Figure 5f).
Relative expression levels of ryr1 and fkbp12 are shown in Figures 5g and h, respectively. In the SOL, the relative ryr1 mRNA expression increased by 21% (P < 0.05) in the IBA group but decreased by 29% (P < 0.05) in the ET group and by 33% in the LT group (P < 0.05) compared with the SA group. In the EDL, expression decreased significantly by 52% in the ET group and by 54% in the LT group (P < 0.05) compared with the SA group. In the GAS, expression was reduced by 50% in the PRE, 48% in the ET, and 45% in the LT (P < 0.05) compared with the SA group (Figure 5g).
In the SOL, the relative fkbp12 mRNA expression levels were reduced by 38% (P < 0.05), 61% (P < 0.01), 69% (P < 0.01), 68% (P < 0.01), and 37% (P < 0.05) in the PRE, LT, IBA, ET, and POST groups, respectively, compared with the SA group. In the EDL, expression was reduced by 41%, 43%, and 25% (P < 0.05) in the ET, LT, and POST groups, respectively, compared with the SA group. In the GAS, expression was significantly decreased by 64% (P < 0.05), 62% (P < 0.05), and 69% (P < 0.05) in the PRE, ET, and LT groups compared with the SA group (Figure 5h).
Co-localization of regulatory proteins involved in RyR1. The reticulate subcellular distributions of DHPR and RyR1 fluorescently labeled proteins are shown in Figure 6a. The co-localization levels of DHPR and RyR1 in the LT and ET groups were significantly lower than that in the SA group in all three muscles. In the SOL, levels were decreased in all five groups compared to the SA group, with decrements of 24%, 32%, 28%, 38%, and 19% (P < 0.05) in the PRE, LT, IBA, ET, and POST groups, respectively. In the EDL, levels decreased by 25%, 20%, and 27% (P < 0.05) in the LT, IBA, and ET groups, respectively. In the GAS, levels in the LT group (25%, P < 0.05) and ET group (31%, P < 0.05) were lower than that in the SA group, whereas levels in the POST group increased (22%, P < 0.05) compared to the SA group (Figure 6c).
The reticulate subcellular distributions of CSQ1 and RyR1 fluorescently labeled proteins are shown in Figure 6d. The co-localization levels of CSQ1 and RyR1 in the LT, IBA, and ET groups were significantly lower than that in the PRE group in all three muscles. In the SOL, the decrements were 18%, 15%, and 11% (P < 0.05) in ET, IBA, and LT groups, respectively. In the EDL, the decrements were 12% and 12% (P < 0.05) in the ET and LT groups, respectively. In the GAS, the decrements were 23%, 18%, and 24% (P < 0.05) in the ET, IBA and LT groups, respectively (Figure 6f).
The reticulate subcellular distributions of FKBP12 and RyR1 fluorescently labeled proteins are shown in Figure 6g. In the SOL and EDL, the co-localization levels of FKBP12 and RyR1 showed no significant differences among the six groups. In the GAS, however, the co-localization level decreased by 11% (P < 0.05) in the LT group compared with the SA group (Figure 6i).
Co-localization of regulatory proteins involved in SERCA. The reticulate subcellular distributions of SERCA1 and SLN fluorescently labeled proteins are shown in Figure 7a. Similar change trends in the co-localization levels of SERCA1 and SLN were observed in the three different muscles, with lower levels in all groups compared with that in the SA group. In the SOL, levels decreased by 21%, 26%, 33%, and 31% in the PRE, LT, IBA, and ET groups, respectively (P < 0.05). In the EDL, levels decreased by 15%, 24%, and 20% in the PRE, LT, and IBA groups, respectively (P < 0.05). In the GAS, levels decreased by 13% and 24% (P < 0.05) in PRE and ET groups, respectively, whereas all other groups were at the summer level (Figure 7c).
The reticulate subcellular distributions of SERCA2 and SLN fluorescently labeled proteins are shown in Figure 7d. Similar change trends in the co-localization levels of SERCA2 and SLN were observed in the EDL and GAS, with lower levels in all groups compared with that in the SA group. Compared to the SA group, levels in the EDL decreased by 22% and 12% (P < 0.05) in LT and IBA groups, respectively; whereas, levels in the GAS decreased by 31% and 24% (P < 0.05) in PRE and ET groups, respectively. In the SOL, levels increased by 12% and 14% (P < 0.05) in LT and POST groups, respectively, compared to the SA group (Figure 7f).