Fig 2: Changes in the protein expression of distinct Ca2+ transport and binding proteins in PL and AM muscles during different periods. Histograms depicting (A) representative Western blot images of STIM1, ORAI1, RyR1, LETM1, PMCA3, SERCA1, MCU, MICU1, and CALM in PL and AM muscles during different periods. Histograms depicting (B) STIM1 protein expression, (C) ORAI1 protein expression, (D) RyR1 protein expression, (E) LETM1 protein expression, (F) PMCA3 protein expression, (G) SERCA1 protein expression, (H) MCU protein expression, (I) MICU1 protein expression, and (J) CALM protein expression 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, ** p < 0.01, and *** p < 0.001 compared with SA; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with PRE; & p < 0.05 compared with LT; $ p < 0.05 and $$ p < 0.01 compared with IBA; ++ p < 0.01 compared with ET.

Fig 3: Dihydropyridine receptor alpha 1 (DHPRα1) and ryanodine receptor 1 (RyR1) localization. Cross-sections of the gastrocnemius (a,c,e,g) and soleus (b,d,f,h) muscles were stained with anti-DHPRα1 (a–d) or anti-RyR1 (e–h) antibodies. Asterisks indicate normal immunoreactivity (IR) in the cytoplasm and subsarcolemma of DHPRα1 or RyR1 (a,b,d–f,h). Arrows indicate muscle fiber with ectopic DHPRα1 or RyR1 IR (c,g). Scale bar = 50 μm. The number of muscle fibers with ectopic DHPRα1 or RyR1 IR was measured in the gastrocnemius (i,k) and soleus (j,l) muscles. Data are presented as mean ± standard deviation, n = 8 per group. Significant differences between young and old mice were determined using Student’s t-test. *** p < 0.0001. n.s.: not significant. All data are represented as dots in the graph. GAS, gastrocnemius muscle; SOL, soleus muscle; 3M, 3-month-old mice; 24M, 24-month-old mice.

Fig 4: Protein overlap levels of CSQ1 and RyR1 in three different types of muscle during different periods. (A) Reticulate subcellular distribution of CSQ1 and RyR1 fluorescently labeled proteins in three different types of muscle. DAPI (blue) for nuclei, AF488 (green) for CSQ1, 647 (red) for RyR1. Scale bar = 20 μm. (B) Representative immunofluorescence of protein overlap between CSQ1 and RyR1 in three different types of muscle during different periods. Scale bar = 20 μm. (C) Box-plot representing protein overlap level of CSQ1 and RyR1. Boxes represent upper and lower quartiles, middle horizontal line represents median, hollow circle represents average, lines extending from upper and lower ends represent upper and lower edges, respectively, asterisks represent extreme outliers, and points represent individual sample values. n = 8. SOL, soleus muscle; EDL, extensor digitorum longus; GAS, gastrocnemius muscle; 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. Different letters (such as a and b) indicate differences between period groups (P < 0.01), same letters (including a and ab) indicate no differences between period groups, and no letters indicate no differences among all six period groups.

Fig 5: Relative expression level of Ca2+ transporters located on nuclear membrane and its regulatory proteins. (A) Representations of Calsequestrin 1 (CSQ1), Calnexin (CAX), Lamina protein B1 (LaminB1) in cellular protein fraction and nuclear protein fraction. C: cellular protein fraction; N: nuclear protein fraction. (B) Bands of Ca2+ transporters and its regulatory proteins. (C–I) Relative expression levels of Ryanodine receptor 1 (RyR1), Inositol 1,4,5-tetrakisphosphate receptor 1 (IP3R1), Ca2+-ATPase type 2 (Ca2+-ATPase2), Na+/Ca2+ exchanger 3 (NCX3), Ca2+/calmodulin dependent protein kinase II (CaMK II), Protein kinase A (PKA) and Cyclic ADP ribose hydrolase (CD38), respectively. Each circle represented a value. n = 6–8. Data were analyzed by t-test. Data are shown as Mean ± SEM and considered statistically significant at p < 0.05.