Differential regulation of cellular stress responses by the endoplasmic reticulum-resident Selenoprotein S (Seps1) in proliferating myoblasts versus myotubes

Abstract

The antioxidant Selenoprotein S (Seps1, Selenos) is an endoplasmic reticulum (ER)-resident protein associated with metabolic and inflammatory disease. While Seps1 is highly expressed in skeletal muscle, its mechanistic role as an antioxidant in skeletal muscle cells is not well characterized. In C2C12 myotubes treated with palmitate for 24 h, endogenous Seps1 protein expression was upregulated twofold. Two different siRNA constructs were used to investigate whether decreased levels of Seps1 exacerbated lipid-induced oxidative and ER stress in C2C12 myotubes and myoblasts, which differ with regards to cell cycle state and metabolic phenotype. In myoblasts, Seps1 protein knockdown of ~50% or ~75% exacerbated cellular stress responses in the presence of palmitate; as indicated by decreased cell viability and proliferation, higher H₂ O₂ levels, a lower reduced to oxidized glutathione (GSH:GSSG) ratio, and enhanced gene expression of ER and oxidative stress markers. Even in the absence of palmitate, Seps1 knockdown increased oxidative stress in myoblasts. Whereas, in myotubes in the presence of palmitate, a ~50% knockdown of Seps1 was associated with a trend toward a marginal (3-5%) decrease in viability (P = 0.05), decreased cellular ROS levels, and a reduced mRNA transcript abundance of the cellular stress marker thioredoxin inhibitory binding protein (Txnip). Furthermore, no enhancement of gene markers of ER stress was observed in palmitate-treated myotubes in response to Seps1 knockdown. In conclusion, reduced Seps1 levels exacerbate nutrient-induced cellular stress responses to a greater extent in glycolytic, proliferating myoblasts than in oxidative, differentiated myotubes, thus demonstrating the importance of cell phenotype to Seps1 function.

Keywords: ER stress; Selenoprotein S; myoblast; myotube; oxidative stress; palmitate.

Figure 1

Seps1 expression in differentiating, palmitate‐ and siRNA‐treated C2C12 muscle cells. Cells treated with 0.1 mmol/L (myoblasts) or 0.35 mmol/L (myotubes) palmitate (black bars) or the corresponding ethanol/BSA vehicle (white bars). (A) Seps1 protein expression was assessed in proliferating and differentiating myoblasts, with a transient increase observed between 24 h and 48 h postdifferentiation when compared to proliferating myoblasts. C2C12 myoblasts were transiently transfected with one of two Seps1 siRNA constructs or a scramble control construct prior to treatment with 0.1 mmol/L (palmitate; black bars) or ethanol/BSA vehicle (vehicle; white bars) for 21 h. To assess gene knockdown and the effect of palmitate (B) Seps1 mRNA and (C) protein levels were determined. C2C12 myotubes were also transiently transfected with one of two Seps1 siRNA constructs or a scramble control construct prior to treatment with 0.35 mmol/L (palmitate; black bars) or ethanol/BSA vehicle (vehicle; white bars) for 24 h. Followed by measurement of (D) Seps1 gene and (D) protein expression. ^† P < 0.0001 compared to proliferating myoblasts (one‐way GLM‐ANOVA). **P < 0.05 for vehicle compared to palmitate‐treated cells (interaction; two‐way GLM‐ANOVA). ⁺⁺ P < 0.05 for Seps1 siRNA compared to scramble control following vehicle treatment (interaction; two‐way GLM‐ANOVA). ^‡ P < 0.05 for Seps1 siRNA compared to scramble control following palmitate treatment (interaction; two‐way GLM‐ANOVA). *P < 0.05 compared to vehicle‐treated cells (main effect treatment; two‐way GLM‐ANOVA). ⁺ P < 0.05 compared to scramble control (main effect siRNA; two‐way GLM‐ANOVA). For the myoblast differentiation and myotube siRNA experiments, cells were seeded in duplicate with three independent biological replicates and for the myoblast siRNA experiments, cells were seeded in triplicate with two independent biological replicates.

Figure 2

Cell cycle state and viability of C2C12 muscle following knockdown of Seps1 in the presence of palmitate. Cells treated with 0.1 mmol/L (myoblasts) or 0.35 mmol/L (myotubes) palmitate (black bars) or the corresponding ethanol/BSA vehicle (white bars). To assess the effects of Seps1 knockdown and nutrient stress on myoblast proliferation and differentiation, (A) manual cell counts were completed and (B) the percentage of dead and dying cells measured using 7‐AAD incorporation. FITC and PI staining were used to assess cell cycle state; specifically, (C) the proportion of myoblasts in S phase (DNA synthesis) and (D) G₀ (quiescence). (E) The mitochondrial oxidation of MTT was used to assess the viability of myotubes following Seps1 siRNA and/or palmitate treatment. ⁺ P < 0.05 compared to scramble control (main effect siRNA; two‐way GLM‐ANOVA). *P < 0.05 compared to vehicle‐treated cells (main effect treatment; two‐way GLM‐ANOVA). ⁺⁺ P < 0.05 for Seps1 siRNA compared to scramble control following vehicle treatment (interaction; two‐way GLM‐ANOVA). To determine cell number and the proportion of dead or dying myoblasts, cells were analyzed in quadruplicate with three independent biological replicates. To assess cell cycle progression, myoblasts were analyzed in quadruplicate with three independent biological replicates. For the MTT assay in myotubes, an N = 8 wells and two independent biological replicates were analyzed.

Figure 3

Differential oxidative stress responses in palmitate‐treated C2C12 myoblasts and myotubes following Seps1 knockdown indicate the importance of cell phenotype. Myoblasts treated with 0.1 mmol/L palmitate (black bars) or ethanol/BSA vehicle (white bars) or myotubes treated with 0.35 mmol/L palmitate (black bars) or ethanol/BSA vehicle (white bars). To assess the antioxidant properties of Seps1 in proliferating myoblasts, (A) H₂O₂ levels via Amplex^® Red, (B) cellular redox state via the GSH:GSSG ratio and (C) Ho‐1 gene and (D) protein expression were measured in palmitate‐ and vehicle‐treated myoblasts transfected with Seps1 siRNA or scramble control constructs. To assess the antioxidant properties of Seps1 in differentiated myoblasts, (E) cellular ROS levels via the oxidation of DCFDA, (F) H₂O₂ via the oxidation of Amplex^® Red assay, (G) Ho‐1 gene and (H) protein expression were measured in palmitate and vehicle‐treated myotubes transfected with the Seps1 siRNA or scramble control constructs. ⁺ P < 0.05 compared to scramble control (main effect treatment; two‐way GLM‐ANOVA). *P < 0.05 compared to vehicle‐treated cells (main effect siRNA; two‐way GLM‐ANOVA). ^† P < 0.05 compared to scramble control (independent T‐test). **P < 0.05 for vehicle compared to palmitate‐treated cells (interaction; two‐way GLM‐ANOVA). ^‡ P < 0.05 for Seps1 siRNA compared to scramble control following palmitate treatment (interaction; two‐way GLM‐ANOVA). In myoblasts, for the Amplex Red assay cells were seeded in quadruplicate with three independent biological replicates; to assess the GSH:GSSG ratio cells were seeded in triplicate with two independent biological replicates; and to measure Ho‐1 gene and protein expression cells were seeded in triplicate with two independent biological replicates. In myotubes, to assess cellular ROS, cells were cultured in N = 7 wells with two independent biological replicates; for the Amplex Red assay, cells were cultured in quadruplicate with three independent biological replicates; and to measure Ho‐1 gene and protein expression cells were cultured in duplicate with three independent biological replicates.

Figure 4

ER stress response in palmitate‐treated C2C12 myoblasts following Seps1 knockdown. Myoblasts treated with 0.35 mmol/L palmitate (black bars) or ethanol/BSA vehicle (white bars). To assess whether Seps1 is associated with the unfolded protein response and/or the thioredoxin antioxidant system, (A) Chop gene expression, (B) Grp94 gene and (C) protein expression, (D) Grp78 gene and (E) protein expression, (F) Thioredoxin‐1 (Trx‐1) gene expression, and (G) Thioredoxin inhibitor protein (Txnip) gene expression were measured in palmitate‐ and vehicle‐treated myoblasts transfected with the Seps1 siRNA or scramble control constructs. ⁺ P < 0.05 compared to scramble control (main effect siRNA; two‐way GLM‐ANOVA). *P < 0.05 compared to vehicle‐treated cells (main effect treatment; two‐way GLM‐ANOVA). ^‡ P < 0.05 for Seps1 siRNA compared to scramble control following palmitate treatment (interaction; two‐way GLM‐ANOVA). For gene and protein analysis, myoblasts were seeded in triplicate with two independent biological replicates.

Figure 5

ER stress response in palmitate‐treated C2C12 myotubes following Seps1 knockdown. Myotubes treated with 0.35 mmol/L palmitate (black bars) or ethanol/BSA vehicle (white bars). To investigate whether cell cycle state or metabolic phenotype differentially affects the association between Seps1 and the unfolded protein response or the thioredoxin antioxidant system, (A) Chop gene expression, (B) Grp94 gene and (C) protein expression, (D) Grp78 gene and (E) protein expression, (F) Thioredoxin‐1 (Trx‐1) gene expression, and (G) Thioredoxin inhibitor protein (Txnip) gene expression were measured in palmitate‐ and vehicle‐treated myotubes transfected with the Seps1 siRNA or scramble constructs. *P < 0.05 compared to vehicle‐treated cells (main effect treatment; two‐way GLM‐ANOVA). ⁺ P < 0.05 compared to scramble control (main effect siRNA; two‐way GLM‐ANOVA). ‡P < 0.05 for cells treated with siRNA1 and palmitate compared to siRNA1 vehicle control (interaction; two‐way GLM‐ANOVA). For gene and protein analysis, myotubes were grown in duplicate with three independent biological replicates.

Figure 6

Seps1 protects the glycolytic proliferative myoblasts from lipid‐induced cell death. The genetic reduction of Seps1 by siRNA1 or siRNA2 in myoblasts and myotubes, respectively results in a 50% reduction in Seps1 protein expression. In the more glycolytic myoblast, reduction of Seps1 causes increased ROS and ER stress, leading to increased cell death and cell cycle exit (quiescence). However in the more oxidative myotubes, Seps1 reduction reduced ROS and has mild change in ER stress and cell death. Thus, the reduction of Seps1 affects the more glycolytic myoblasts, suggesting Seps1 may protect muscle cells of a glycolytic phenotype.