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. 2010 Sep 15;5(9):e12707.
doi: 10.1371/journal.pone.0012707.

Akt Deficiency Attenuates Muscle Size and Function but Not the Response to ActRIIB Inhibition

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Free PMC article

Akt Deficiency Attenuates Muscle Size and Function but Not the Response to ActRIIB Inhibition

Marcus D Goncalves et al. PLoS One. .
Free PMC article

Abstract

Background: Akt is a critical mediator of developmental skeletal muscle growth. Treatment with a soluble ActRIIB fusion protein (ActRIIB-mFc) increases skeletal muscle mass and strength by inhibiting myostatin and related peptides. Recent in vitro studies have suggested that Akt signaling is necessary for the ability of ActRIIB inhibition to induce muscle hypertrophy. Thus, we hypothesized that mice deficient in either Akt1 or Akt2 would not respond to in vivo inhibition of ActRIIB with ActRIIB-mFc treatment.

Methodology and principal findings: We analyzed body composition and muscle parameters in wild-type C57BL/6J and Akt1 and Akt2 knockout mice, and compared the responses to blockade of ActRIIB signaling via ActRIIB-mFc treatment. Mice lacking Akt1 or Akt2 had reduced muscle mass, grip strength and contractile force. However, deficiency of Akt1 or Akt2 did not prevent the ability of ActRIIB-mFc treatment to induce muscle hypertrophy, or increase grip strength and contractile force. Akt1 and Akt2 deficient mice responded similarly as wild type mice to ActRIIB-mFc treatment by increasing fiber size.

Conclusions and significance: Akt1 and Akt2 are important for the regulation of skeletal muscle mass and function. However, these Akt isoforms are not essential for the ability of ActRIIB inhibition to regulate muscle size, fiber type, strength or contractile force.

Conflict of interest statement

Competing Interests: Jennifer Lachey is an employee of Acceleron Pharma who provided ActRIIB-mFc. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Effects of ActRIIB inhibition on Akt levels in gastrocnemius muscle.
(A) Phosphorylated Akt (Ser473), total Akt1/2/3, and β-actin levels after 10 weeks of vehicle- (Veh) or ActRIIB-mFc treatment. (B) Total and p-Akt levels relative to β-actin. *p<0.05 vs. vehicle.
Figure 2
Figure 2. Effects of Akt deficiency and ActRIIB inhibition on body composition.
Effects of genotype and ActRIIB-mFc treatment (Rx, black bar) or vehicle (Veh, white bar) in wild-type (WT), Akt1 knockout mice (Akt1 KO), and Akt2 knockout mice (Akt2 KO) on (A) body weight, (B) percentage lean, (C) percentage fat. Data are mean +/− SEM, n = 5. + P<0.05 vs. WT treated with vehicle; ++ P<0.01 vs. WT treated with vehicle; *P<0.05 vs. same genotype treated with vehicle; **P<0.01 vs. same genotype treated vehicle.
Figure 3
Figure 3. Effects of Akt deficiency and ActRIIB inhibition on skeletal muscle weights.
Effects of genotype and ActRIIB-mFc treatment (Rx, black bar) or vehicle (Veh, white bar) in wild-type (WT), Akt1 knockout mice (Akt1 KO), and Akt2 knockout mice (Akt2 KO) on (A) Extensor digitorum longus, (B) Gastrocnemius (C) Quadriceps, (D) Tibialis Anterior, (E) Soleus, and (F) Heart. Data are mean +/− SEM, n = 5; + P<0.05 vs. WT treated with vehicle; ++ P<0.01 vs. WT treated with vehicle; +++P<0.0001 vs. WT treated with vehicle. *P<0.05 vs. same genotype treated with vehicle **P<0.01 vs. same genotype treated with vehicle; ***P<0.0001 vs. same genotype treated with vehicle.
Figure 4
Figure 4. Effects of Akt deficiency and ActRIIB inhibition on EDL fiber size and distribution.
(A) Effects of genotype and ActRIIB-mFc treatment on EDL fiber cross-sectional area distributions. Wild-type (WT, red), Akt1 knockout mice (Akt1 KO, green), Akt2 knockout mice (Akt2 KO, blue), ActRIIB-mFc -treated WT (WT Rx, red-dashed), ActRIIB-mFc -treated Akt1 KO (Akt1 KO Rx, green dashed), and ActRIIB-mFc -treated Akt2 KO (Akt2 KO Rx, blue dashed) EDL fiber distributions; n≥792 fibers per group; (B) Single fiber cross sectional areas per genotype (Veh, white bar) and treatment (Rx, black bar); (C) Number of fibers in the EDL. Data are mean +/− SEM, n = 5; +++P<0.0001 vs. WT treated with vehicle; ***P<0.0001 vs. same genotype treated with vehicle.
Figure 5
Figure 5. Effects of Akt deficiency and ActRIIB inhibition on soleus fiber size and distribution.
(A) Effects of genotype and ActRIIB-mFc treatment on soleus type I and type II fiber cross-sectional area distributions. Wild-type (WT, red), Akt1 knockout mice (Akt1 KO, green), Akt2 knockout mice (Akt2 KO, blue), ActRIIB-mFc -treated WT (WT Rx, red-dashed), ActRIIB-mFc -treated Akt1 KO (Akt1 KO Rx, green dashed), and ActRIIB-mFc -treated Akt2 KO (Akt2 KO Rx, blue dashed) on soleus type I fiber distribution; n≥393 fibers per group. (B) Type I single fiber cross sectional area (CSA) per genotype (Veh, white bar) and treatment (Rx, black bar); (C) Number of type I fibers in the soleus; (D) Soleus type II fiber distribution; n≥458 fibers per group. (E) Type II single fiber CSA per genotype and treatment; (F) Number of type II fibers in the soleus; Data are mean +/− SEM, n = 5; +P<0.05 vs. WT treated with vehicle; *P<0.05 vs. same genotype treated with vehicle.
Figure 6
Figure 6. Effects of Akt deficiency and ActRIIB inhibition on in vivo muscle function.
Effects of genotype (Veh, white bar) and ActRIIB-mFc treatment (Rx, black bar) in wild-type (WT), Akt1 knockout mice (Akt1 KO), and Akt2 knockout mice (Akt2 KO) on (A) forelimb grip strength; (B) distance ran during an endurance exercise protocol; (C) work done during an endurance protocol. Data is mean +/− SEM, n = 5; + P<0.05 vs. WT treated with vehicle; ++ P<0.01 vs. WT treated with vehicle. *P<0.05 vs. same genotype treated with vehicle.
Figure 7
Figure 7. Effects of Akt deficiency and ActRIIB inhibition on EDL contraction.
Effects of genotype (Veh, white bar) and ActRIIB-mFc treatment (Rx, black bar) on EDL ex vivo force generation. (A) Peak twitch force (mN) in wild-type (WT), Akt1 knockout mice (Akt1 KO), and Akt2 knockout mice (Akt2 KO); (B) Representative twitch recordings; (C) Peak tetanic force (mN). (D) Representative tetanic recordings. Data in (A, C) are mean +/− SEM, n = 5; + P<0.05 vs. WT treated with vehicle; ++ P<0.01 vs. WT treated with vehicle. *P<0.05 vs. same genotype treated with vehicle **P<0.01 vs. same genotype treated with vehicle.
Figure 8
Figure 8. Effects of ActRIIB inhibition on Akt signaling and glycogen content.
(A) Immunoblots of vehicle (Veh) and ActRIIB-mFc treated (Rx) wild-type (WT), Akt1 knockout (Akt1ko), and Akt2 knockout (Akt2ko) gastrocnemius lysates. P: phospho-specific antibody (B) Effects of genotype (Veh, white bar) and ActRIIB-mFc treatment (Rx, black bar) on quadriceps glycogen content (mg glycogen per g muscle). +++P<0.0001 vs. WT Veh, *p<0.05 vs. vehicle.

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