Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment

Abstract

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca 2+ signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca 2+ was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca 2+ ] and environmental temperatures. Therefore, Ca 2+ signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

Up-regulation of sarcoplasmic reticulum function protects skeletal muscle against cytoplasmic calcium overload during hibernation in ground squirrels

“…Each CaM molecule can bind with four Ca 2+ in the cytoplasm [56,57]; thus, in the present study, the increase in protein expression observed during hibernation undoubtedly helped to reduce the concentration of free Ca 2+ in the cytoplasm. This is similar to results reported in the SOL and GAS muscles of thirteen-lined ground squirrels, which showed increased protein expression during hibernation [28,80]. CSQ molecules can bind with 43 Ca 2+ in the SR [38,55,58,59]; thus, in our study, the increased CSQ1 protein expression observed during hibernation indicated that the ability of skeletal muscle fibers to reduce the free Ca 2+ concentration in the SR was increased.…”

“…Research on mixed skeletal muscles in the hindlimbs of Siberian ground squirrels (Spermophilus undulatus) reported significant decreases in the protein expression levels of SERCA1, RyR1, and CSQ1 during hibernation [27]. Conversely, we previously showed that Ca 2+ pump (SERCA1 and SERCA2) activity increases significantly in the SOL and EDL muscles of ground squirrels during hibernation and inter-bout arousal [60], CaM protein levels increase significantly in mixed hindlimb skeletal muscles of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) [28]. More importantly, the underlying mechanisms of these changes, especially the regulation of RYR1 and calcium pump activity, is not clear.…”

“…The endoplasmic reticulum, called the SR in skeletal muscle, is the most important organelle to maintain intracellular Ca 2+ homeostasis [18][19][20][21]. At present, research on calcium homeostasis in nonhibernating and hibernating animals were mainly focused on SR under the condition of disused skeletal muscle, with few reports on the mitochondria or nuclei [22][23][24][25][26][27][28].…”

Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation

“…Both of these mechanisms are activated by increased cardiac workload due to pressure or volume ( Depre et al, 2006 ). Upregulation of Nuclear Factors of Activated T Cells (NFAT) and Myocyte Enhancer Factor-2 (MEF2) have been implicated in the increased synthesis of numerous proteins during cardiac hypertrophy ( Tessier & Storey, 2012 ; Zhang & Storey, 2015 ; Zhang & Storey, 2016 ). In the postnatal myocardium, these transcription factors represent part of the fetal gene expression profile, which controls pathological cardiac hypertrophy and the development of heart failure ( Frey & Olson, 2003 ; Frey et al, 2004 ).…”

A Temporal Examination of Cytoplasmic Ca2 + Levels, Sarcoplasmic Reticulum Ca2 + Levels, and Ca2 + -Handling-Related Proteins in Different Skeletal Muscles of Hibernating Daurian Ground Squirrels

“…Conversely, our previous study showed that SERCA activity increases in the soleus (SOL) and extensor digitorum longus (EDL) muscles of ground squirrels during inter-bout arousal and torpor compared with that during pre-hibernation (Guo et al, 2017). Furthermore, calmodulin (CaM) protein expression is reported to increase in the mixed hindlimb skeletal muscles of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during torpor (Zhang and Storey, 2016b). Importantly, however, the underlying mechanisms related to the above changes, especially the specific regulation of RyR1 and SERCA activity, are still not clear.…”