Structure of the transgene and PERK activation in skeletal muscles of HSA-Fv2E-PERK Tg mice.

<p>(A) Schema of the transgene for skeletal muscle-specific expression of Fv2E-PERK. (B) Representative PCR genotyping results from wild-type (WT) and HSA-Fv2E-PERK Tg mice. (C) Representative immunoblots of phosphorylated Fv2E-PERK (P), Fv2E-PERK (non P), phosphorylated eIF2α (P), and total eIF2α (non P) in the gastrocnemius muscles of WT and HSA-Fv2E-PERK Tg mice 8 h after intraperitoneal injections of vehicle or the artificial ligand AP21087 (AP) at 0.1 mg/kg body weight (BW). (D) RT-qPCR analyses of the expression of mRNA encoding targets of eIF2α phosphorylation in HSA-Fv2E-PERK Tg and WT mice at 8 h after administration of vehicle or AP (0.1 mg/kg BW). Data are presented as means ± standard errors of the mean (SEM; n = 3–5), and differences among AP-treated HSA-Fv2E-PERK Tg mice, vehicle-treated HSA-Fv2E-PERK Tg mice, and AP-treated WT mice were considered significant at *<i>p</i> < 0.05 or **<i>p</i> < 0.01.</p

Acute skeletal muscle atrophy in AP-injected HSA-Fv2E-PERK Tg mice.

<p>(A) Body weight (BW) curves of AP-injected wild-type (WT) male, vehicle-injected HSA-Fv2E-PERK Tg mice, and AP-injected HSA-Fv2E-PERK Tg mice; vehicle or AP (0.1 mg/kg BW) were injected intraperitoneally once a day for 7 days (n = 5). (B) BW changes are expressed as percentages of initial BWs (Fig 2A). (C) Muscle weights of the extensor digitorum longus (EDL), tibialis anterior (TA), gastrocnemius (GC), and soleus (Sol) muscles of AP-injected WT, vehicle-injected Tg and AP-injected Tg mice; vehicle or AP (0.1 mg/kg BW) were intraperitoneally injected once a day for 7 days (n = 5). (D) Representative hematoxylin and eosin (H&E) staining of TA muscles of WT and HSA-Fv2E-PERK Tg mice. Vehicle or AP (0.1 mg/kg BW) was intraperitoneally injected once a day for 7 days. Right graph shows relative muscle cross-sectional area (CSA) of each genotype. Differences among AP-treated HSA-Fv2E-PERK Tg mice, vehicle-treated HSA-Fv2E-PERK Tg mice, and AP-treated WT mice treated were considered significant at *<i>p</i> < 0.05 or **<i>p</i> < 0.01.</p

Schematic representation of muscle wasting in AP-injected HSA-Fv2E-PERK Tg mouse.

<p>(A) In normal condition, hormones, such as insulin and IGF-1, stimulate protein synthesis through enhancing the formation of the 43S and 48S preinitiation complexes, which are essential for muscle maintenance. (B) In muscle wasting conditions, signals regulating the formation of the 43S and 48S preinitiation complex were disturbed. (C) In AP-injected HSA-Fv2E-PERK Tg mouse, inhibition of the formation of 43S preinitiation complex by eIF2 phosphorylation led to muscle atrophy due to reduced protein synthesis, along with a counter-regulatory increase in 48S preinitiation complex.</p

Modulation of intercellular signaling pathways that regulate skeletal muscle function in AP-injected HSA-Fv2E-PERK Tg mice.

<p>(A) Representative immunoblots of phosphorylated Akt, total Akt, phosphorylated S6K, total S6K, phosphorylated 4EBP-1, total 4EBP-1, and GAPDH at 8 h after vehicle or AP injections in gastrocnemius muscles of wild-type (WT) and HSA-Fv2E-PERK Tg mice; vehicle or AP (0.1-mg/kg BW) were intraperitoneally injected once a day for 7 days. Right graphs show the band intensity from images (n = 4–5). (B) RT-qPCR analyses of mRNAs encoding regulators of skeletal muscle function at 8 h after vehicle or AP injection in the gastrocnemius muscles of WT and HSA-Fv2E-PERK Tg mice; vehicle or AP (0.1 mg/kg BW) were intraperitoneally injected once a day for 7 days (n = 5). Differences among AP-treated HSA-Fv2E-PERK Tg mice, vehicle-treated HSA-Fv2E-PERK Tg mice, and AP-treated WT mice were considered significant at *<i>p</i> < 0.05 or **<i>p</i> < 0.01.</p

Altered intercellular amino acid metabolism in AP-injected HSA-Fv2E-PERK Tg mice.

<p>(A) Heatmap representation of amino acid metabolites in gastrocnemius muscles of AP-injected WT and AP-injected Tg mice (n = 5). Colors indicate ratios of determined amino acids contents in vehicle-injected WT mice; the vertical axis is labeled with a continuous scale color bar key. AP (1-μg/kg BW) was injected intraperitoneally. (B) Amino acid concentrations in gastrocnemius muscles of AP-injected WT, vehicle-injected Tg, and AP-injected Tg mice. Vehicle or AP (0.1-mg/kg BW) was intraperitoneally injected once a day for 7 days (n = 5). Differences among AP-treated HSA-Fv2E-PERK Tg mice, vehicle-treated HSA-Fv2E-PERK Tg mice, and AP-treated WT mice were considered significant at *<i>p</i> < 0.05 or **<i>p</i> < 0.01.</p

PCR and RT-qPCR primer sequences.

<p>PCR and RT-qPCR primer sequences.</p