Introduction

Sarcolipin is a 31 amino acid transmembrane protein mainly expressed in skeletal and cardiac atria muscles [4, 33]. In skeletal muscle cells, sarcolipin is present in the sarco/endoplasmic reticulum, and interacts with sarco/endoplasmic reticulum calcium ion (Ca2+) ATPase (SERCA) [15]. SERCA is responsible for transporting Ca2+ from the cytosol into the lumen of the sarco/endoplasmic reticulum by hydrolyzing ATP following muscular contraction. When sarcolipin interacts with SERCA, SERCA produces heat instead of Ca2+ transport by hydrolyzing ATP [5, 9]. Indeed, several studies have shown the significance of sarcolipin in vivo as follows. Sarcolipin knockout mice exhibit hypothermia during cold exposure and have a trend toward obesity compared with wild-type mice [3]. On the other hand, skeletal muscle-specific sarcolipin overexpression mice are resistant to obesity under high-fat feeding and show ameliorated metabolic parameters such as levels of blood glucose and serum lipids [17, 27]. These findings suggest that sarcolipin plays pivotal roles in muscle thermogenesis and systemic metabolism.

The endoplasmic reticulum (ER) is the site of synthesis, folding, and maturation of secreted and transmembrane proteins, synthesis of lipids, and storage of Ca2+ [7]. Disruption of ER homeostasis causes ER stress, which activates unfolded protein responses (UPRs). ER stress has been studied in the pathogenesis of obesity, type 2 diabetes, and insulin resistance [22]. Thus, the roles of UPRs have been mainly studied in secretory and metabolic organs such as the liver, pancreas, and adipose tissue. Compared with these organs, little is known about the roles of UPRs in skeletal muscle. However, several reports showed that UPRs in muscles are involved in nutritional insults, skeletal muscle diseases, and hypo/hyper-contraction [10].

Regarding thermogenesis, uncoupling protein 1 (UCP1) is a well-known regulator in brown adipose tissue in rodents. In UCP1 knockout mice, sarcolipin expression in skeletal muscle is upregulated compensatorily [25]. Conversely, UCP1 expression is increased in sarcolipin knockout mice. In short, both UCP1 and sarcolipin are required to maintain optimal thermogenesis in vivo. Because brown adipose tissue decreases with age in humans, skeletal muscle may be more important than brown adipose tissue in human thermogenesis. However, the regulation of sarcolipin in muscles remains elusive. Considering that the site of action of sarcolipin is the sarco/endoplasmic reticulum, we speculated that ER stress affects sarcolipin expression. Therefore, this study aimed to evaluate the effect of ER stress on sarcolipin expression in skeletal muscle cells.

Materials and methods

Cell culture and myotube differentiation

C2C12 mouse myoblasts were purchased from the American Type Culture Collection (CRL-1772™, Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose (Sigma, St. Louis, MO) and 10% fetal bovine serum (Biowest, St. Louis, MO) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. C2C12 myoblasts were cultured and grown to confluence in six-well plates unless otherwise specified. Myotube differentiation was induced by a standard method by reducing the serum concentrations to 5% horse serum (Invitrogen, Carlsbad, CA). For assessing sarcolipin expression during myogenic differentiation, total RNA was extracted from cells at each time point (day −2, 0, 2, or 4). In other experiments, differentiated myotubes were used after 4 days of differentiation.

RNA extraction and qRT–PCR

For evaluating gene expression, total RNA was extracted from the cells using TRIzol Reagents (Invitrogen). Aliquots of the RNA solution were taken for both RNA quantification and qualification using a spectrophotometer (NanoDrop ND-1000, NanoDrop Technologies, Inc., Wilmington, DE). RNA purity was assessed by the ratio of A260/A280 (around 1.9). One microgram of total RNA was subjected to complementary DNA synthesis using the High-Capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instruction. qRT–PCR analysis was performed in a reaction volume of 20 μL per well with a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using validated TaqMan Gene Expression Assays for sarcolipin (Mm00481536_m1) and myogenin (Mm00446194_m1). Amplification was determined using the comparative threshold cycle method with accompanying StepOne™ Software version 2.2.2 (Applied Biosystems). Results were normalized by the expression levels of the eukaryotic 18S rRNA gene (Hs99999901_s1) and then calculated as fold changes relative to control. All experiments were performed at least in triplicate.

Induction of ER stress to C2C12 myotubes

Tunicamycin, thapsigargin, and dimethyl sulfoxide (DMSO) were purchased from Sigma. ER stress was induced into C2C12 myotubes by treatment with tunicamycin or thapsigargin. The dose (tunicamycin 0.3–3.0 μg/mL, thapsigargin 0.1–1.0 μM) and time (0–24 h) of the treatments were determined by literature review and described in each experiment. DMSO was used as a vehicle adjusted at a final concentration of 0.1% in medium.

Detection of UPRs by Western blotting

C2C12 myotubes were treated with tunicamycin (Tun; 3 μg/mL) or thapsigargin (Thap; 1 μM) for 8 h. DMSO was used as a vehicle. Protein extraction and Western blotting were performed as described previously [29]. In brief, equal amounts of extracted protein (20 μg per lane) were resolved on SDS–PAGE for Western blotting using antibodies against mouse binding immunoglobulin protein (BiP), inositol-requiring enzyme 1 (IRE1), phospho-protein kinase R-like ER protein kinase (p-PERK), PERK, and C/EBP homologous protein (CHOP), purchased from Cell Signaling Technology (Danvers, MA). β-Actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a loading control. The bands were visualized with a Light Capture II System (ATTO Co., Tokyo, Japan). The band intensities were quantified using ImageJ software (version 1.50i, National Institutes of Health, Bethesda, MD). Results were normalized by the expression levels of the β-actin or PERK as indicated in the figure and then calculated as fold change over control.

Protein detection by in-cell ELISA

Sarcolipin protein expression was detected using In-Cell ELISA kits according to the manufacturer’s instruction (cat. no. 3440-02; Bioo Scientific Co., Austin, TX). C2C12 myotubes cultured in a 96-well plate were treated with tunicamycin (0.3 μg/mL), thapsigargin (0.3 μM), or vehicle (DMSO) for 24 h. The doses used were determined as the lowest concentration to sufficiently reduce the sarcolipin messenger RNA (mRNA) level for each chemical based on dose-dependent analyses. Primary antibodies used were sarcolipin (Proteintech Group, Rosemont, IL), CHOP (Cell Signaling Technology, Danvers, MA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; inclusion in the kit). The absorbance of each well was read at a wavelength of 450 nm with a microplate reader (MTP-120, Corona Electric, Hitachinaka, Japan). Results were standardized by the absorbance of GAPDH, and the relative protein abundance was determined as fold changes compared with vehicle control. All experiments were performed at least in sextuplicate.

Analysis of RIDD of mRNA

Because activated IRE1, in response to ER stress, degrades a subset of mRNA in a process called regulated IRE1-dependent decay (RIDD), the involvement of IRE1 on the ER stress-induced sarcolipin mRNA reduction was examined. The IRE1 inhibitor 4μ8C (Sigma), which specifically prevents splicing of X-box binding protein-1 (XBP-1) mRNA and inhibits RIDD [8], was employed. C2C12 myotubes were pretreated with 4μ8C (30 μM) or vehicle (DMSO) for 30 min and further treated with tunicamycin (0.3 μg/mL), thapsigargin (0.3 μM), or vehicle for 4 h. At the end of the treatment, RNA was extracted and then synthesized to cDNA as described above for the analyses of XBP-1 and sarcolipin mRNA expression. XBP-1 splicing was assessed as described previously [6]. In brief, XBP-1 mRNA was amplified by PCR using TaKaRa Ex Taq (Takara Bio, Inc., Kusatsu, Japan) with primers 5′-AAACAGAGTAGCAGCGCAGACTGC-3′ and 5′-GGATCTCTAAAACTAGAGGCTTGGTG-3′. The amplified fragment of XBP-1 was digested by endonuclease PstI (Invitrogen) for 1 h at 37 °C, and then the digested fragments were resolved on a 2.5% agarose gel. An aliquot of cDNA was also used for the analysis of sarcolipin mRNA expression by qRT–PCR.

Sarcolipin mRNA stability assay

To assess the stability of sarcolipin mRNA under ER stress, actinomycin D (Sigma), a transcriptional inhibitor, was employed. C2C12 myotubes were treated with tunicamycin (3 μg/mL), actinomycin D (5 μg/mL), or both at times of 0, 3, 6, and 9 h. At the end of each time point, the cells were subjected to total RNA extraction and subsequently cDNA synthesis. Gene expression levels of sarcolipin were measured by qRT–PCR as described above. Results were calculated as fold changes relative to the values at time 0.

Statistical analysis

The results are expressed as means ± SE. The statistical significance in mean values among multiple sample groups was examined with analysis of variance and Bonferroni’s post-hoc test using PRISM software version 6.0 (GraphPad Software Inc., San Diego, CA). p < 0.05 was considered significant.

Results

Sarcolipin gene expression during myogenesis in C2C12 cells

We examined the gene expression levels of sarcolipin during myogenic differentiation in C2C12 cells by qRT–PCR. As shown in Fig. 1a, the expression of sarcolipin mRNA was detected in myoblasts and transiently increased at day 2 of differentiation (2.7-fold compared with the expression at day −2; p < 0.05). We confirmed that the mRNA levels of sarcolipin were maintained during differentiation from myoblasts to myotubes. On the other hand, myogenin, a myogenic marker, was increased (13.3-fold at day 2 and 6.8-fold at day 4, compared with the expression at day −2; p < 0.05) during the differentiation (Fig. 1b). These results suggest that C2C12 myotubes can be used as a model of skeletal muscle cells for the investigation of sarcolipin.

Fig. 1
figure 1

Expression of sarcolipin mRNA during myogenesis in C2C12 cells. The gene expressions of sarcolipin (SLN) (a) and myogenin (b) were determined during myogenesis of C2C12 cells by quantitative reverse transcription–PCR (qRT–PCR). Results were calculated as fold changes relative to the values on day −2 and are expressed as means ± SE. *p < 0.05; n = 3 per group

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ER stress induction in C2C12 myotubes

C2C12 myotubes were treated with tunicamycin and thapsigargin, both of which are well-known chemical ER stress inducers. ER stress was evaluated by the protein expression of UPRs. As shown in Fig. 2, protein expression of BiP, IRE1, phospho-PERK, and CHOP was increased by the treatment with both compounds, thereby inducing UPRs in C2C12 myotubes.

Fig. 2
figure 2

Induction of endoplasmic reticulum (ER) stress in C2C12 myotubes. C2C12 myotubes were treated with tunicamycin (Tun) or thapsigargin (Thap). Protein expression related to ER stress, including BiP, IRE1 (inositol-requiring enzyme 1), phospho-PERK (p-PERK), PERK, CHOP, and β-actin, were evaluated by Western blotting (a) and quantification (b–e). *p < 0.05; n = 3 per group

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Effect of ER stress on the mRNA and protein expression of sarcolipin in C2C12 myotubes

C2C12 myotubes were treated with different concentrations of tunicamycin (Tun) (Fig. 3a) or thapsigargin (Thap) (Fig. 3b) for 8 h. Gene expression levels of sarcolipin were analyzed by qRT–PCR. Tunicamycin significantly reduced sarcolipin mRNA expression levels (−78% at 0.3 μg/mL; p < 0.05). Thapsigargin also significantly reduced sarcolipin mRNA expression levels (−51% at 0.1 μM; p < 0.05). For the time course analysis, the cells were treated with 3.0 μg/mL of tunicamycin (Tun) (Fig. 3c) or 1.0 μM of thapsigargin (Thap) (Fig. 3d) for 4, 8, and 24 h. After 4 h of the treatments, tunicamycin (−64% at 4 h; p < 0.05) and thapsigargin (−57% at 4 h; p < 0.05) significantly reduced sarcolipin mRNA expression levels. Thus, ER stress inducers reduced sarcolipin mRNA expression of the C2C12 myotubes in time- and dose-dependent manners. Next, protein expression levels of sarcolipin (Fig. 3e) and CHOP (Fig. 3f) were evaluated by In-Cell ELISA Kits after the treatment of C2C12 myotubes with 0.3 μg/mL of tunicamycin (Tun) or 0.3 μM of thapsigargin (Thap) for 24 h. The protein expression of sarcolipin (Fig. 3e) was reduced by ER stress at the lowest concentration of each chemical to sufficiently reduce the sarcolipin mRNA level (−44% by tunicamycin; p < 0.05; −39% by thapsigargin; p < 0.05). Simultaneously, the expression levels of CHOP (Fig. 3f), used as a positive control of UPRs, were increased (49% by tunicamycin; p < 0.05; 37% by thapsigargin; p < 0.05). These results demonstrate that ER stress reduces both mRNA and protein expression of sarcolipin in C2C12 myotubes.

Fig. 3
figure 3

Sarcolipin expression under ER stress in C2C12 myotubes. The mRNA expression of sarcolipin was evaluated by qRT–PCR in the myotubes treated with different concentrations of tunicamycin (Tun) (a) or thapsigargin (Thap) (b) and at different times during tunicamycin (Tun) (c) or thapsigargin (Thap) (d) treatment, as indicated in the figures. Protein expression levels of sarcolipin (SLN) (e) and CHOP (f) were evaluated by in-cell ELISA after the treatment of C2C12 myotubes with 0.3 μg/mL of tunicamycin (Tun) or 0.3 μM of thapsigargin (Thap) for 24 h. *p < 0.05; n = 3 or 6 per group

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Involvement of IRE1 in the reduced sarcolipin mRNA expression by ER stress

To clarify the involvement of IRE1 and RIDD in the reduced sarcolipin mRNA levels by ER stress, we used an IRE1 inhibitor, 4μ8C, that specifically inhibits the splicing of XBP-1 mRNA and RIDD in response to ER stress [8]. We first examined the effectiveness of 4μ8C in C2C12 myotubes by the inhibition of ER stress-induced XBP-1 splicing. As shown in Fig. 4a, treatment with tunicamycin or thapsigargin caused XBP-1 splicing, as visualized by spliced bands (sp). However, pretreatment of C2C12 myotubes with 4μ8C yielded unspliced bands (unsp), even in the presence of ER stress inducers. Then, we evaluated the mRNA expression levels of sarcolipin by qRT–PCR in the same conditions as in Fig. 4a. The expression levels of sarcolipin in the pretreatment with 4μ8C were similar to those in either tunicamycin or thapsigargin alone (Fig. 4b). Similar results were obtained with longer treatment (8 h) of ER stress (data not shown). These results suggest that the ER stress-induced sarcolipin mRNA reduction is independent of IRE1 activation.

Fig. 4
figure 4

The involvement of regulated IRE1-dependent decay in ER stress-induced sarcolipin mRNA reduction. a 4μ8C, an IRE1 inhibitor, prevents the X-box binding protein-1 (XBP-1) splicing effectively induced by tunicamycin (Tun) or thapsigargin (Thap). The arrows indicate spliced band (sp) or unspliced bands (unsp). b 4μ8C did not prevent ER stress-induced sarcolipin (SLN) mRNA reduction. NS not significant. n = 3

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Effect of ER stress on sarcolipin mRNA stability

To further investigate the mechanisms by which ER stress reduces mRNA expression of sarcolipin, actinomycin D, an inhibitor of mRNA synthesis, was employed. C2C12 myotubes were treated with actinomycin D (ActD; 5 μg/mL), tunicamycin (Tun; 3 μg/mL), or both (ActD + Tun). As shown in Fig. 5, the decrease in sarcolipin mRNA levels with the combination of ActD + Tun was significantly higher than that with ActD alone (−26% in ActD vs. −63% in ActD + Tun at 3 h; p < 0.05) and was similar to that with Tun after 3 h of the treatments. Thus, the reduction of sarcolipin transcripts by tunicamycin is largely due to the decreased sarcolipin mRNA stability.

Fig. 5
figure 5

The effect of ER stress on sarcolipin mRNA stability in C2C12 myotubes. C2C12 myotubes were treated with actinomycin D (ActD), tunicamycin (Tun), or both (ActD + Tun). Gene expression levels of sarcolipin (SLN) were measured at the times of 3, 6, and 9 h by qRT–PCR. *p < 0.05 ActD + Tun vs. ActD. # p < 0.05 Tun vs. ActD; n = 3

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Discussion

In the present study, we have shown that ER stress suppresses sarcolipin mRNA and protein expression in C2C12 myotubes by altering its mRNA stability. We first confirmed sarcolipin expression in C2C12 cells because changes in sarcolipin mRNA expression levels during myoblast-to-myotube differentiation had not been reported in the cells. The results revealed that the mRNA expression levels of sarcolipin were transiently increased at day 2 of differentiation, but were maintained throughout myogenic differentiation. Also, the validity of the myogenic process was confirmed by the expression of myogenin. Although the significance of the transient increase in sarcolipin mRNA expression remains to be clarified, sarcolipin may play a role in the process of myogenesis in C2C12 cells.

To examine the effect of ER stress on sarcolipin expression in C2C12 myotubes, we employed well-known chemical ER stress inducers, tunicamycin and thapsigargin [22, 34]. The exposure to these chemicals of C2C12 myotubes induced UPRs and reduced sarcolipin mRNA and protein expression. These results provide evidence that ER stress suppresses sarcolipin expression. Indeed, the mechanisms of these two chemicals on ER stress induction are different: tunicamycin inhibits N-glycosylation, and thapsigargin inhibits the function of SERCA [34]. Considering the site of action of thapsigargin, thapsigargin may affect sarcolipin expression through interaction with SERCA. However, the similar suppression levels of sarcolipin mRNA by both inhibitors indicate that the suppression was not induced by the mediation of SERCA but by ER stress.

Several conditions have been reported regarding the induction of UPRs in skeletal muscle. First, nutritional load induces ER stress in skeletal muscle. For instance, mice fed on a high-fat diet exhibit UPRs in skeletal muscle [11]. Also, exposure of the primary myotubes from human subjects to palmitate induces an ER stress reaction [24]. Moreover, high-glucose incubation of skeletal muscle cells induces ER stress [28]. Second, UPRs are detected in muscle diseases such as myotonic dystrophy type 1 [13], sporadic inclusion body myositis [32], and autoimmune myositis [20]. In addition, muscle contractile activity is associated with ER stress. Exercise training leads to UPRs in muscles of mice [35]. Similarly, endurance exercise activates ER stress in human skeletal muscles [14]. In contrast, physical inactivity is associated with increased expression of genes related to UPRs [1]. These lines of evidence suggest that ER stress is involved in muscle pathogenesis. Further studies regarding the involvement of reduced sarcolipin expression by ER stress under these muscle conditions are required.

Because sarcolipin increases energy expenditure by producing heat through a modification of SERCA and eventually modulates systemic metabolism, sarcolipin expression in skeletal muscle has been investigated in the pathogenesis of obesity. Considering that ER stress is recognized as one of the important pathogenic factors in obesity [22], it is reasonable that ER stress may be related to decreased sarcolipin expression. However, reports have shown that the protein expression of sarcolipin in the soleus muscle is increased in high-fat diet-induced obesity in mouse models [3, 5]. Similarly, Paran et al. reported that the mRNA and protein expression of sarcolipin in human skeletal muscle cells isolated from muscle biopsy specimens is increased in severely obese subjects compared with lean controls [23]. Although the mechanisms of increased sarcolipin expression in obesity have not been clarified, it is considered as a protective reaction against obesity. Interestingly, they also found that the efficiency of sarcolipin-dependent respiration is reduced in human skeletal muscle cells [23]. These reports suggest that the evaluation of sarcolipin should be done at both expression and functional levels.

To date, little is known about the regulation of sarcolipin expression in skeletal muscle. In addition to the increased expression of sarcolipin in high-fat diet-induced obesity, protein expression of sarcolipin is also increased in the skeletal muscles of a mouse model of Duchenne muscular dystrophy [26]. Moreover, triamcinolone, one of the corticosteroids, increases sarcolipin mRNA levels in the rat diaphragm [12]. Furthermore, a deficiency of vitamin E, in both α-tocopherol-deficient mice and α-tocopherol transfer protein knockout mice, shows significantly high levels of sarcolipin mRNA expression in the quadriceps and gastrocnemius muscles [31]. As well as these conditions and factors, our study provides evidence that ER stress is a new regulating factor on sarcolipin expression in muscle.

To investigate the precise link between ER stress and reduced sarcolipin mRNA, we focused on IRE1, one of the key factors in UPRs. IRE1 has unique ribonuclease activity, including the unconventional splicing of both XBP-1 mRNA and RIDD [16]. RIDD targets numerous mRNAs as a post-transcriptional regulator. As demonstrated in Fig. 4, the results suggested that the reduced sarcolipin transcripts induced by ER stress are independent of IRE1 activation. Then, we investigated the mechanism by which ER stress reduces sarcolipin expression in C2C12 myotubes. Based on the study in RNA turnover using actinomycin D (Fig. 5), the reduced sarcolipin mRNA induced by ER stress was largely due to the decline of its mRNA stability. Regarding the mechanisms of the regulation of sarcolipin transcripts, one study reported similar findings that sarcolipin mRNA is post-transcriptionally downregulated by triiodothyronine in rat atrial myocytes [18]. Thus, sarcolipin transcripts may be regulated fundamentally at a post-transcriptional level in different tissues.

The tissue distribution of sarcolipin varies widely among muscles and species [30]. In small animals, such as mice and rats, the mRNA and protein expression of sarcolipin is highly expressed in the tongue, diaphragm, soleus, and cardiac muscles, whereas in large animals, such as rabbits, dogs, and pigs, sarcolipin expression is abundant in skeletal muscles [2, 30]. In humans, sarcolipin mRNA is detected at a high level in skeletal muscle compared with cardiac atria [19, 21]. Although our results were obtained from mouse skeletal muscle cells, the fact that human skeletal muscle expresses relatively high levels of sarcolipin supports the concept that sarcolipin plays an important role in human muscle pathophysiology.

In summary, the results presented herein demonstrate that ER stress is a new downregulating factor for sarcolipin expression through the reduction of its mRNA stability in skeletal muscle cells. Further clarification of the mechanisms of sarcolipin expression and functions is needed for a better understanding of both muscle and metabolic disorders, especially in large animals like humans.