**Priority Strategy of Intracellular Ca2**<sup>+</sup> **Homeostasis in Skeletal Muscle Fibers during the Multiple Stresses of Hibernation**

**Jie Zhang 1,2,**†**, Xiaoyu Li 3,**†**, Fazeela Ismail 1,2, Shenhui Xu 1,2, Zhe Wang 1,2, Xin Peng 1,2, Chenxi Yang 4, Hui Chang 1,2,\*, Huiping Wang 1,2 and Yunfang Gao 1,2,\***


Received: 19 November 2019; Accepted: 19 December 2019; Published: 22 December 2019

**Abstract:** Intracellular calcium (Ca2+) homeostasis plays a vital role in the preservation of skeletal muscle. In view of the well-maintained skeletal muscle found in Daurian ground squirrels (*Spermophilus dauricus*) during hibernation, we hypothesized that hibernators possess unique strategies of intracellular Ca2<sup>+</sup> homeostasis. Here, cytoplasmic, sarcoplasmic reticulum (SR), and mitochondrial Ca2<sup>+</sup> levels, as well as the potential Ca2<sup>+</sup> regulatory mechanisms, were investigated in skeletal muscle fibers of Daurian ground squirrels at different stages of hibernation. The results showed that cytoplasmic Ca2<sup>+</sup> levels increased in the skeletal muscle fibers during late torpor (LT) and inter-bout arousal (IBA), and partially recovered when the animals re-entered torpor (early torpor, ET). Furthermore, compared with levels in the summer active or pre-hibernation state, the activity and protein expression levels of six major Ca2<sup>+</sup> channels/proteins were up-regulated during hibernation, including the store-operated Ca2<sup>+</sup> entry (SOCE), ryanodine receptor 1 (RyR1), leucine zipper-EF-hand containing transmembrane protein 1 (LETM1), SR Ca2<sup>+</sup> ATPase 1 (SERCA1), mitochondrial calcium uniporter complex (MCU complex), and calmodulin (CALM). Among these, the increased extracellular Ca2<sup>+</sup> influx mediated by SOCE, SR Ca2<sup>+</sup> release mediated by RyR1, and mitochondrial Ca2<sup>+</sup> extrusion mediated by LETM1 may be triggers for the periodic elevation in cytoplasmic Ca2<sup>+</sup> levels observed during hibernation. Furthermore, the increased SR Ca2<sup>+</sup> uptake through SERCA1, mitochondrial Ca2<sup>+</sup> uptake induced by MCU, and elevated free Ca2<sup>+</sup> binding capacity mediated by CALM may be vital strategies in hibernating ground squirrels to attenuate cytoplasmic Ca2<sup>+</sup> levels and restore Ca2<sup>+</sup> homeostasis during hibernation. Compared with that in LT or IBA, the decreased extracellular Ca2<sup>+</sup> influx mediated by SOCE and elevated mitochondrial Ca2<sup>+</sup> uptake induced by MCU may be important mechanisms for the partial cytoplasmic Ca2<sup>+</sup> recovery in ET. Overall, under extreme conditions, hibernating ground squirrels still possess the ability to maintain intracellular Ca2<sup>+</sup> homeostasis.

**Keywords:** calcium homeostasis; hibernation; mitochondria; sarcoplasmic reticulum; skeletal muscle

#### **1. Introduction**

The maintenance of cytoplasmic calcium (Ca2+) homeostasis is important for the preservation of a normal structure and function of skeletal muscle fibers. Skeletal muscle inactivity can trigger Ca2<sup>+</sup> homeostasis disturbance, often characterized by cytoplasmic Ca2<sup>+</sup> overload [1]. A direct consequence of this overload is the activation of calpain system-mediated protein degradation [2]. In addition, an increased cytoplasmic Ca2<sup>+</sup> concentration can promote cell apoptosis [3]. Increased protein degradation and cell apoptosis are both involved in skeletal muscle loss.

Hibernation is a unique survival strategy exhibited by various mammals in order to cope with adverse environments in winter, during which hibernators not only face the challenge of prolonged skeletal muscle inactivity, but also deal with other stresses, including hypoxia, fasting, and repeated ischemia-reperfusion during the torpor-arousal cycle. However, various studies have reported that skeletal muscle is well-maintained in hibernators during hibernation [4,5]. Therefore, hibernators can be considered typical anti-atrophy models, with their unique skeletal muscle preservation mechanism undoubtedly an attractive and valuable research topic.

Previous findings from our laboratory showed that, under adverse conditions over several months of hibernation, the cytoplasmic Ca2<sup>+</sup> concentration in skeletal muscle fibers of Daurian ground squirrels increased transiently during inter-bout arousal, partially recovered after re-entering torpor, and almost recovered to pre-hibernation levels in the post-hibernation stage, thus exhibiting good Ca2<sup>+</sup> homeostasis during the entire hibernation cycle [6]. During long-term hibernation, the torpor-arousal cycle likely plays an important role in protecting skeletal muscle from atrophy by avoiding or alleviating persistent and excessive cytoplasmic Ca2<sup>+</sup> overload-induced protein degradation. Therefore, exploring the potential mechanisms involved in Ca2<sup>+</sup> homeostasis during hibernation could help reveal the mechanisms against disuse-induced skeletal muscle atrophy of hibernators. To date, however, only one study (from our lab) has reported on sarcoplasmic reticulum Ca2<sup>+</sup> pump (SERCA) expression in skeletal muscles during hibernation [7]. As such, the regulatory mechanisms involved in intracellular Ca2<sup>+</sup> homeostasis in skeletal muscle fibers are far from having been clarified.

The level of intracellular Ca2<sup>+</sup> is closely related to the expression level and activity of Ca2<sup>+</sup> transport proteins or channels located in the plasma membrane and intracellular Ca2<sup>+</sup> storage membrane (mainly sarcoplasmic reticulum (SR) and mitochondria), as well as intracellular Ca2<sup>+</sup> binding proteins. Increased extracellular Ca2<sup>+</sup> influx and intracellular Ca2<sup>+</sup> storage/release (especially in the SR) both contribute to an increase in the intracellular Ca2<sup>+</sup> concentration. Store-operated Ca2<sup>+</sup> entry (SOCE) is the most important channel transporting extracellular Ca2<sup>+</sup> into the cytosol. Stromal interaction molecule-1 (STIM1) located in the endoplasmic reticulum (ER) and Orai1 (also known as calcium-release-activated calcium-modulator, CRACM1) located in the cell membrane are two essential components required for SOCE [8–10]. With external stimulation, Ca2<sup>+</sup> is released from the STIM1 EF-hand domain, which triggers the aggregation and movement of STIM1 to ER/plasma membrane (PM) binding sites, as well as the Orai1 aggregation of STIM1, and leads to the activation of SOCE and Ca2<sup>+</sup> influx [11–14]. The ryanodine receptor (RyR) is a major SR Ca2<sup>+</sup> release channel. Specifically, when sensing cell membrane depolarization, exterior membrane L-type calcium channels (surface membrane and T tubules) and dihydropyridine receptors (DHPR) combine to activate RyR, resulting in substantial SR Ca2<sup>+</sup> release [15,16]. The RyR family is comprised of three isoforms (i.e., RyR1–3), with RyR1 exclusively expressed and particularly enriched in skeletal muscle [17]. Leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) is a Ca2+-H<sup>+</sup> exchanger located in the mitochondrial membrane. When the mitochondrial Ca2<sup>+</sup> concentration is high, LETM1 will extrude excess Ca2<sup>+</sup> from the mitochondria into the cytoplasm [18]. Therefore, LETM1 is another possible contributor to elevated cytoplasmic Ca2<sup>+</sup> levels.

In contrast to the above mechanisms, however, the increase in Ca2<sup>+</sup> efflux, intracellular Ca2<sup>+</sup> uptake of the Ca2<sup>+</sup> pool, and binding capacity of free Ca2<sup>+</sup> binding protein in the cytoplasm all effectively decrease cytoplasmic Ca2<sup>+</sup>. Plasma membrane Ca2<sup>+</sup> ATPase (PMCA) can eject Ca2<sup>+</sup> from the cytosol into the external medium, thereby attenuating the cytoplasmic Ca2<sup>+</sup> concentration. PMCA3

is the major isoform expressed in skeletal muscle [19]. As a primary active transporter located in the SR membrane, SR/ER Ca2<sup>+</sup> ATPase (SERCA) can decrease cytoplasmic Ca2<sup>+</sup> levels by pumping Ca2<sup>+</sup> from the cytosol into the SR, which is one of the key factors attenuating cytoplasmic Ca2<sup>+</sup> overload in skeletal muscle fibers [20]. The mitochondrial calcium uniporter (MCU) complex is considered a major channel for the transportation of Ca2<sup>+</sup> into mitochondria [21]. Mitochondrial calcium uptake 1 and 2 (MICU1 and 2) are two regulatory subunits of MCU [22]. When the Ca2<sup>+</sup> concentration in the intermembrane space is low, the heterodimers of MICU1 and MICU2 block the MCU channel and inhibit the entry of Ca2<sup>+</sup> into the mitochondria. In contrast, when the Ca2<sup>+</sup> level is high upon stimulation, the binding of Ca2<sup>+</sup> to the MICU protein elicits a conformational change, resulting in the opening of the channel and the transportation of Ca2<sup>+</sup> into the mitochondria [21,23]. Calmodulin (CALM), a Ca2<sup>+</sup> binding protein located in the cytoplasm, can directly reduce the concentration of cytoplasmic free Ca2<sup>+</sup> by combining with four Ca2<sup>+</sup> ions [24]. Overall, Ca2<sup>+</sup> uptake channels, extrusion mechanisms, and free Ca2<sup>+</sup> binding proteins all contribute to intracellular Ca2<sup>+</sup> homeostasis.

What, then, is the role of Ca2<sup>+</sup> channels in Ca2<sup>+</sup> fluctuations during the torpor-arousal cycle? To answer this question, we investigated the cytoplasmic, SR, and mitochondrial Ca2<sup>+</sup> levels in the plantaris (PL, calf muscle) and adductor magnus (AM, thigh muscle) muscles of Daurian ground squirrels during different hibernation states (i.e., summer active, pre-hibernation, late torpor (entering a new bout after more than 5 d), inter-bout arousal (arousing spontaneously for less than 12 h), early torpor (entering a new bout for less than 48 h), and post-hibernation). Furthermore, a comprehensive and time-course investigation was carried out to explore the roles of the above major Ca2<sup>+</sup> transport proteins/channels, including SOCE, RyR1, LETM1, PMCA3, SERCA1, and MCU, as well as the major Ca2<sup>+</sup> binding protein CALM, in the fluctuations of Ca2<sup>+</sup> concentration throughout hibernation.

### **2. Materials and Methods**
