Effects of hibernation on bone marrow transcriptome in thirteen-lined ground squirrels

Abstract

Mammalian hibernators adapt to prolonged periods of immobility, hypometabolism, hypothermia, and oxidative stress, each capable of reducing bone marrow activity. In this study bone marrow transcriptomes were compared among thirteen-lined ground squirrels collected in July, winter torpor, and winter interbout arousal (IBA). The results were consistent with a suppression of acquired immune responses, and a shift to innate immune responses during hibernation through higher complement expression. Consistent with the increase in adipocytes found in bone marrow of hibernators, expression of genes associated with white adipose tissue are higher during hibernation. Genes that should strengthen the bone by increasing extracellular matrix were higher during hibernation, especially the collagen genes. Finally, expression of heat shock proteins were lower, and cold-response genes were higher, during hibernation. No differential expression of hematopoietic genes involved in erythrocyte or megakaryocyte production was observed. This global view of the changes in the bone marrow transcriptome over both short term (torpor vs. IBA) and long term (torpor vs. July) hypothermia can explain several observations made about circulating blood cells and the structure and strength of the bone during hibernation.

Keywords: adipose; erythrocyte; leukocyte; megakaryocyte; osteoblast.

Fig. 1.

Graph showing body temperature tracings (solid line) of a thirteen-lined ground squirrel measured using a surgically implanted transmitter. The dashed orange line represents the ambient (environmental) temperature, which is lowered to 5°C on November 1 and raised back to 23°C in March or April depending on the experiment. Periodic interbout arousals (IBAs) are seen as regular spikes in body temperature despite a constant ambient temperature of 5°C. Characteristic measurements of body temperature (Tb), oxygen consumption (V̇o₂), and heart rate (HR) during torpor (adapted from Refs. 96a, 96b).

Fig. 2.

Bone marrow from femurs isolated from animals at the following time points: nonhibernating (July) (A), entrance into hibernation (October) (B), IBA (January) (C), 1 day post-Spring arousal (D), and 1 wk post-Spring arousal (March) (E). Samples were paraffin embedded, sectioned, and stained with hematoxylin and eosin (A–E). In B, arrows indicate cell types found in A–E. Blue, megakaryocyte; orange, leukocytes; black, adipocyte; green, erythrocyte. F: mature megakaryocyte counts per mm² at each time point: NH, July nonhibernator; EN, October entrance into hibernation; H, January hibernator; 24H PA, 24 h post-Spring arousal; 1WK PA, 1 wk post-Spring arousal. Error bars represent SE for n = 6 animals. Size bar is 100 μm in all panels.

Fig. 3.

Specific collagen genes expressed in bone marrow isolated from July, Torpor, and IBA animals (n = 6 for each condition). Due to the large variation in expression, numbers are graphed on a log scale. Error bars represent SE. The letters above each bar represent post hoc pair-wise comparisons to determine significance between collection points. Any collection point not connected by the same letter is significantly different [false discovery rate (FDR) < 0.05].

Fig. 4.

Femur shafts (top) and heads (bottom) stained with Masson's trichrome and magnified ×200. Collagen and bone will stain blue, muscle and keratin stain red, and cytoplasm is pink. A, B: July nonhibernators. C, D: October entrance into hibernation. E, F: January hibernators. G, H: March animals 1 wk postarousal. Arrows indicate the following structures: Black, chondrocytes in the articular cartilage; yellow, collagen-rich deep matrix; green, adipocyte in bone marrow; purple, osteocyte; blue, skeletal muscle. Size bar is 50 μm in all panels.

Fig. 5.

Extracellular matrix genes expressed in bone marrow isolated from July, Torpor, and IBA animals (n = 6 for each condition). Error bars represent SE. The letters above each bar represent post hoc pair-wise comparisons to determine significance between collection points. Any collection point not connected by the same letter is significantly different (FDR < 0.05). DCN, Decorin; LUM, Lumican; FN1, Fibronectin; LAM, Laminin; NDST2, heparan glucosaminyl N-deacetylase/N-sulfotransferase; HSPG2, heparin sulfate proteoglycan 2.

Fig. 6.

Matrix remodeling metallopeptidase genes expressed in bone marrow isolated from July, Torpor, and IBA animals (n = 6 for each condition). Error bars represent SE. The letters above each bar represent post hoc pair-wise comparisons to determine significance between collection points. Any collection point not connected by the same letter is significantly different (FDR < 0.05). MMP, matrix metallopeptidase; TIMP2, TIMP metallopeptidase inhibitor 2.

Fig. 7.

Acid phosphatase (ACP) genes expressed in bone marrow isolated from July, Torpor, and IBA animals (n = 6 for each condition). Error bars represent SE. The letters above each bar represent post hoc pair-wise comparisons to determine significance between collection points. Any collection point not connected by the same letter is significantly different (FDR < 0.05).

Fig. 8.

Cyclin genes expressed in bone marrow isolated from July, Torpor, and IBA animals (n = 6 for each condition). Error bars represent SE. The letters above each bar represent post hoc pair-wise comparisons to determine significance between collection points. Any collection point not connected by the same letter is significantly different (FDR < 0.05).

Fig. 9.

Hypoxia and hypothermia-sensitive genes expressed in bone marrow isolated from July, Torpor, and IBA animals (n = 6 for each condition). Due to the large variation in expression, numbers are graphed on a log scale. Error bars represent SE. The letters above each bar represent post hoc pair-wise comparisons to determine significance between collection points. Any collection point not connected by the same letter is significantly different (FDR < 0.05). TRPM7, transient receptor potential cation channel; RBM3, RNA binding motif; CIRBP, cold inducible RNA binding protein; EPOR, erythropoietin receptor; COLFA1, collagen, type IV, alpha 1; CYR61, cysteine-rich, angiogenic inducer; HIFIA, hypoxia inducible factor 1; VEGF, vascular endothelial growth factor; PPARG, peroxisome proliferator-activated receptor gamma; ANGPTL4, angiopoietin-like 4; HMOX2, heme oxygenase 2.