M. Effects of hibernation on bone marrow transcriptome in thirteen-lined ground squirrels. Physiol Genomics 48: 513-525, 2016. First published May 20, 2016 doi:10.1152/physiolgenomics.00120.2015.-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. erythrocyte; leukocyte; megakaryocyte; osteoblast; adipose BONE MARROW IS A COMPLEX ORGAN consisting of many different cell types and stem cell pools. These cells express both unique and common sets of genes and likely influence each other's activities. Hematopoietic stem cells can differentiate into lymphoid progenitor cells that produce natural killer cells and lymphocytes, or into myeloid progenitor cells that give rise to erythrocytes, the remaining leukocytes, and megakaryocytes. Megakaryocytes in turn shed anucleated platelets involved in blood clotting and inflammation. Bone marrow resident mesenchymal stem cells can differentiate to produce resident bone cells including osteoblasts, adipocytes, and chondrocytes (64). Osteoblasts can terminally differentiate into osteocytes and are involved in maintaining bone strength and extracellular matrix production. Bone marrow adipose composition is affected by age, diet, and disease states. Bone marrow may also contain skeletal muscle and hepatocyte stem cell pools. Because of the rapid rate of mitosis by multiple resident stem cell pools, bone marrow is sensitive to damage by radiation and chemotherapy, leaving patients immunocompromised and anemic (25). Bone mineral density and collagen are decreased by prolonged disuse such as bed rest or immobilization (45,86). Recent studies have also demonstrated an increase in neutrophils, natural killer cells, and lymphocytes and a decrease in monocytes in patients subjected to 60 days of bed rest (38). Finally, bone marrow adipocytes increase in ...

During torpor in a hibernating mammal, decreased blood flow increases the risk of blood clots such as deep vein thrombi (DVT). In other animal models platelets, neutrophils, monocytes and von Willebrand factor (VWF) have been found in DVT. Previous research has shown that hibernating mammals decrease their levels of platelets and clotting factors VIII (FVIII) and IX (FIX), increasing both bleeding time and activated partial throm-boplastin time. In this study, FVIII, FIX and VWF activities and mRNA levels were measured in torpid and non-hibernating ground squirrels (Ictidomys tridecemlineatus). Here, we show that VWF high molecular weight multimers, collagen-binding activity, lung mRNA and promoter activity decrease during torpor. The VWF multimers reappear in plasma within 2 h of arousal in the spring. Similarly, FIX activity and liver mRNA both dropped threefold during torpor. In contrast, FVIII liver mRNA levels increased twofold while its activity dropped threefold, consistent with a post-transcriptional decrease in FVIII stability in the plasma due to decreased VWF levels. Finally, both neutrophils and monocytes are decreased eightfold during torpor which could slow the formation of DVT. In addition to providing insight in how blood clotting can be regulated to allow mammals to survive in extreme environments, hibernating ground squirrels provide an interesting model for studying.

The objective of this study is to determine how a hibernating mammal avoids the formation of blood clots under periods of low blood flow. Hibernating ground squirrels have dramatically decreased heart rates (3‐5 beats per minute) and blood flow, which should put them at risk of forming deep vein thrombi. In response, they have several adaptations during hibernation that prevent blood clotting, including 3‐fold decreases in Factors VIII and IX, and 10‐fold decreases in platelets. During hibernation, platelets are maintained at temperatures of 4‐8°C for up to six months. Within two hours of arousal, platelet levels return to normal, are not rapidly cleared from circulation, and are still functional. We have shown that in addition to decreased clotting factors, ground squirrels have a 10‐fold decrease in von Willebrand factor (vWF) protein and a 3‐fold decrease in vWF mRNA during hibernation. Platelets adhere to exposed collagen in a wound through binding to vWF, and unlike other mammalian platelets, cold storage of ground squirrel platelets does not increase binding of vWF. Ground squirrel platelets are also atypical in that their microtubules are cold‐resistant and change in shape from a circumferential ring to an equatorial rod once they reach 20ºC. This shape change was originally thought to trap the platelets in the spleen, but splenectomized and sham operated ground squirrels had the same platelet levels during torpor and interbout arousal, suggesting storage in an extrasplenic location. Finally, no evidence of irreversible cardiac ischemic damage was observed during hibernation through either histological staining or blood markers including troponin T, lactate dehydrogenase and creatine kinase. Taken together, these results suggest that ground squirrels avoid deep vein thrombosis by reversibly decreasing platelet levels and activity during hibernation. Grant Funding Source: Supported by NIH grant 2R15HL093680‐02