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. 2010 Aug;299(2):E241-8.
doi: 10.1152/ajpendo.00151.2010. Epub 2010 May 18.

Ectopic expression of eIF2Bepsilon in rat skeletal muscle rescues the sepsis-induced reduction in guanine nucleotide exchange activity and protein synthesis

Alexander P Tuckow  1 , Thomas C Vary, Scot R Kimball, Leonard S Jefferson
Affiliations

Ectopic expression of eIF2Bepsilon in rat skeletal muscle rescues the sepsis-induced reduction in guanine nucleotide exchange activity and protein synthesis

Alexander P Tuckow et al. Am J Physiol Endocrinol Metab. 2010 Aug.

Abstract

Eukaryotic initiation factor 2B (eIF2B) is a guanine nucleotide exchange factor (GEF) whose activity is both tightly regulated and rate-controlling with regard to global rates of protein synthesis. Skeletal muscle eIF2B activity and expression of its catalytic epsilon-subunit (eIF2Bepsilon) have been implicated as potential contributors to the altered rates of protein synthesis in a number of physiological conditions and experimental models. The objective of this study was to directly examine the effects of exogenously expressed eIF2Bepsilon in vivo on GEF activity and protein synthetic rates in rat skeletal muscle. A plasmid encoding FLAG-eIF2Bepsilon was transfected into the tibialis anterior (TA) of one leg, while the contralateral TA received a control plasmid. Ectopic expression of eIF2Bepsilon resulted in increased GEF activity in TA homogenates of healthy rats, demonstrating that the expressed protein was catalytically active. In an effort to restore a deficit in eIF2B activity, we utilized an established model of chronic sepsis in which skeletal muscle eIF2B activity is known to be impaired. Ectopic expression of eIF2Bepsilon in the TA rescued the sepsis-induced deficit in GEF activity and muscle protein synthesis. The results demonstrate that modulation of eIF2Bepsilon expression may be sufficient to correct deficits in skeletal muscle protein synthesis associated with sepsis and other muscle-wasting conditions.

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Figures

Fig. 1.
Fig. 1.
Efficient delivery of transgene into rat tibialis anterior (TA) muscle fibers. A–C: fluorescence microscopy of transverse (A and B) and longitudinal (C) sections of rat TA muscles transfected with pmaxGFP and harvested 24 h posttransfection. A small portion of transfected TA was excised, fixed in formalin, embedded in paraffin, and sectioned in a cryostat. Green fluorescence protein (GFP) fluorescence was visualized using a FITC-HYQ filter with a ×4 (A and C) or ×10 (B) objective. In A and C, several fields were concatenated for visualization of the entire muscle section.
Fig. 2.
Fig. 2.
Overexpression of eukaryotic initiation factor (eIF) 2B-ε (eIF2Bε) in rat TA enhances eIF2B guanine nucleotide exchange activity in healthy, freely fed rats. A: representative Western blots and densitometric analyses of eIF2Bε protein abundance in TA muscles [left (L) and right (R)] of rats transfected with pmaxGFP (control) or pFLAG-eIF2Bε. α-Tubulin served as loading control. B and C: eIF2B activity and protein synthetic rates in homogenates of TA muscles transfected with pmaxGFP or pFLAG-eIF2Bε. Values are means ± SE for 18 rats and expressed as percentage of pmaxGFP-transfected TA. *P < 0.001 vs. pmaxGFP TA.
Fig. 3.
Fig. 3.
Chronic sepsis results in decreased total body mass and TA mass. A: body mass on the day of surgery and over 5 days following sterile (control, n = 13; ○, solid line) or septic surgery (n = 14; ●, dashed line). Data were analyzed by 2-way repeated-measures ANOVA; significant interaction (F = 17.78, P < 0.0001). *P < 0.05 control vs. septic by Bonferroni's post tests. B: TA wet weight at the time of harvest (day 5) in control (n = 12, open bars) and septic (solid bars, n = 14) animals. Values are means ± SE. *P < 0.05 vs. control (by unpaired t-test).
Fig. 4.
Fig. 4.
Overexpression of eIF2Bε in TA of septic rats rescues eIF2B guanine nucleotide exchange activity and protein synthesis. A: representative Western blots and densitometric analyses of total eIF2Bε protein abundance in TA muscles of control (open bars) and septic (solid bars) rats transfected with empty vector or pFLAG-eIF2Bε; α-tubulin served as loading control. B and C: eIF2B activity (n = 10 control, n = 8 septic rats) and protein synthetic rates (n = 13 control, n = 12 septic rats) in TA homogenates. Values are means ± SE and expressed as percentage of empty vector-transfected TA muscles of control rats. *P < 0.05 vs. control (for empty vector-transfected TA). †P < 0.05, empty vector vs. pFLAG-eIF2Bε (by paired t-test for comparison of contralateral limbs within septic animals). ***P < 0.0001 vs. empty vector in control and septic animals.
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