p21-Activated Kinase 1 Is Permissive for the Skeletal Muscle Hypertrophy Induced by Myostatin Inhibition

Abstract

Skeletal muscle, the most abundant tissue in the body, plays vital roles in locomotion and metabolism. Understanding the cellular processes that govern regulation of muscle mass and function represents an essential step in the development of therapeutic strategies for muscular disorders. Myostatin, a member of the TGF-β family, has been identified as a negative regulator of muscle development. Indeed, its inhibition induces an extensive skeletal muscle hypertrophy requiring the activation of Smad 1/5/8 and the Insulin/IGF-I signaling pathway, but whether other molecular mechanisms are involved in this process remains to be determined. Using transcriptomic data from various Myostatin inhibition models, we identified Pak1 as a potential mediator of Myostatin action on skeletal muscle mass. Our results show that muscle PAK1 levels are systematically increased in response to Myostatin inhibition, parallel to skeletal muscle mass, regardless of the Myostatin inhibition model. Using Pak1 knockout mice, we investigated the role of Pak1 in the skeletal muscle hypertrophy induced by different approaches of Myostatin inhibition. Our findings show that Pak1 deletion does not impede the skeletal muscle hypertrophy magnitude in response to Myostatin inhibition. Therefore, Pak1 is permissive for the skeletal muscle mass increase caused by Myostatin inhibition.

Keywords: PAK1; follistatin; myostatin; sActRIIB; skeletal muscle hypertrophy.

FIGURE 1

Global comparative transcriptomic analysis of skeletal muscles from mouse models of MSTN inhibition identified Pak1 as a MSTN-responsive gene. (A) Venn diagram comparing the differentially expressed genes into muscles from eight different models of pre- and postnatal MSTN inhibition with Pak1 as the only gene commonly regulated between all the transcriptomes compared. (B) Pak1 mRNA fold-changes for each muscle transcriptome studied. (C) Muscle Pak1 mRNA levels and muscle weight from TA muscles of mTrFS vs. WT mice (n = 11/group); from TA muscles of four times sActRIIB-injected vs. PBS-injected mice (n = 6/group) and from GC muscles of MSTN KO vs. WT mice (n = 7/group). (D) Muscle Pak1 mRNA levels and muscle weight from GC muscles of DEXA-treated vs. saline-treated mice (n = 7/group) and from MSTN-transfected vs. CTRL pcDNA-transfected TA muscles (n = 3/group). Representative western blot image and densitometric analysis of PAK1 protein levels in GC muscles from (E) mTrFS vs. WT C57Bl/6 mice (n = 8/group) and from (F) sActRIIB-injected vs. PBS-injected mice (n = 4/group). Results are expressed as means ± SEM and statistical analysis was performed using unpaired t-test (*P < 0.05; **P < 0.01, and ***P < 0.001 vs. CTRL/WT).

FIGURE 2

Deletion of Pak1 does not alter muscle mass and function. (A) Representative western blot image of muscle PAK1 protein levels in GC muscles from Pak1 KO vs. WT mice. (B) Body weight, and (C) hindlimb muscle mass (including TA, GC, SOL, and EDL muscles) from Pak1 KO vs. WT mice (n = 5–8/group). (D,E) Muscle strength from Pak1 KO vs. WT mice (n = 7–8/group). Mice were lowered on a grid connected to a sensor to measure the muscle force of their forelimbs or of both their forelimb and hindlimb and data were then expressed in gram force relative to body weight (g/gBW). (F) Endurance tolerance from Pak1 KO vs. WT mice (n = 7–8/group). Mice were submitted to an incremental treadmill running exercise (from 5 m/min with added 1 m/min every min until maximum 18 m/min) for a maximum running time of 60 min. Results are expressed as means ± SEM and statistical analysis was performed using unpaired t-test to compare results from Pak1 KO and WT mice.

FIGURE 3

Follistatin induces skeletal muscle hypertrophy despite the absence of Pak1. (A) Representative FS-c-myc immunochemistry image from TA muscle of Pak1 KO and WT littermate mice. Red arrows indicate C-myc positive fibers. Scale bar = 50 μm. (B) Muscle fiber size distribution from pM1-FS288-transfected vs. pM1-transfected TA muscles of Pak1 KO (up panel) and WT (bottom panel) mice (n = 3/group). (C) Muscle fiber CSA assessed 17 days after electroporation of pM1-hFS288 (orange columns) or pM1 (white columns) in TA muscles from Pak1 KO vs. WT littermate mice (n = 3/group). Results are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA test and Bonferroni post-tests (FS effect: **P < 0.01). Fiber CSA distribution statistical analysis was performed using χ² Pearson test (P < 0.001).

FIGURE 4

Muscle Pak1 and Pak2 mRNA levels in response to skeletal muscle hypertrophy induced by MSTN inhibition. (A) TA muscle Pak1 and (B) Pak2 mRNA levels, (C) TA and (D) GC muscle weight after one, 2 or 4 injections of sActRIIB (green columns) or PBS (white columns) in WT mice (n = 4–6/group). Results are expressed as means ± SEM. Statistical analysis was performed using unpaired t-test to compare results from sActRIIB-treated and PBS-treated mice (*P < 0.05; **P < 0.01, and ***P < 0.001 vs. CTRL).

FIGURE 5

sActRIIB induces skeletal muscle hypertrophy despite the absence of Pak1. (A,D) TA and (B,E) GC muscle weight and (C,F) TA muscle PAK2 protein levels after 2 injections (n = 4/group) or 4 injections (n = 4–6/group) of sActRIIB (green columns) or PBS (white columns) in Pak1 KO vs. WT mice. Results are expressed as means ± SEM. Statistical analysis was performed using two-way ANOVA test and Bonferroni post-tests (sActRIIB effect: *P < 0.05; **P < 0.01, and ***P < 0.001).

FIGURE 6

Pak1 deletion tends to increase the sActRIIB-induced 4E-BP1 inactivating phosphorylation. (A) Representative western blot images and densitometric analysis of (B) P-AKT, (C) P-S6, (D) P-P70S6K, and (E) P-4E-BP1 levels in GC muscles from Pak1 KO and WT mice after 2 injections of sActRIIB (green columns) or PBS (white columns) (n = 4/group). The results are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA tests (sActRIIB effect: ^#P < 0.05 and ^##P < 0.01) and Bonferroni post-tests (sActRIIB effect: *P < 0.05).

FIGURE 7

Pak1 deletion does not increase Smad3 phosphorylation nor FBXO32 protein expression in skeletal muscle. (A) GC muscle FBXO32 protein levels, (B) P-FOXO3a, and (C) P-Smad3 levels in GC muscles from Pak1 KO and WT mice (n = 4–6/group). The results are expressed as mean ± SEM. Statistical analysis was performed using unpaired t-test to compare results from Pak1 KO and WT mice.