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. 2022 Nov 8;23(22):13703.
doi: 10.3390/ijms232213703.

Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle

Mingjuan Gu  1   2 , Zhuying Wei  1 , Xueqiao Wang  1 , Yang Gao  1 , Dong Wang  1 , Xuefei Liu  1 , Chunling Bai  1 , Guanghua Su  1 , Lei Yang  1 , Guangpeng Li  1
Affiliations

Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle

Mingjuan Gu et al. Int J Mol Sci. .

Abstract

Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.

Keywords: AMPK/SIRT1/PGC-1α; CRISPR/Cas9; knockout; mitochondrial; myostatin; skeletal muscle.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Production of myostatin knockout (Mstn-KO) mice mediated by CRISPR/Cas9 techniques. (a) gRNA sequence of the MSTN gene for CRISPR/Cas9. (b) Mutant Mstn genotypes of derived progenies, red indicates a missing base. (c) Representative images of muscles of Mstn-KO and WT mice. (d) Images of teased single muscle fibers for muscle fibers in Mstn-KO and WT mice. (e) Expression of MSTN protein in Mstn-KO and WT mice (n = 3).
Figure 2
Figure 2
Comparison of growth performance between Mstn-KO and WT mice. (a) Comparison of body weights between Mstn-KO and WT male mice from 3 to 10 weeks. Body weight was slightly higher in Mstn-KO male mice than WT controls after 6 weeks (n = 3). (b) Comparison of body weights between Mstn-KO and WT female mice from 3 to 10 weeks. Body weight was slightly higher in Mstn-KO female mice than WT controls after 7 weeks (n = 3). (c) Mstn mRNA expression in organs of Mstn-KO and WT mice (n = 3). All data are presented as mean ± SD. * p < 0.05, ** p < 0.01; t-tests were used to calculate the p-values.
Figure 3
Figure 3
Mstn knockout decreases basal metabolic rate and body temperature. (a) Comparison of basal metabolic rate between Mstn-KO and WT mice (n = 3). (b) Body temperature in WT compared with Mstn-KO male and female mice (n = 1). All data are presented as mean ± SD. * p < 0.05; t-tests were used to calculate the p-values.
Figure 4
Figure 4
Mstn knockout reduced ATP content and mitochondria activity. (a) ATP content in Mstn-KO and WT mice muscle (n = 3). (b–f) Mitochondrial complexes I–V activity was analyzed by biochemical detection (n = 3). (g) Measurement of the mitochondrial membrane potential of Mstn-KO and WT mice (n = 3). (h) mRNA levels of mitochondrial activity gene by qPCR (n = 3). All data are presented as mean ± SD. * p < 0.05, ** p < 0.01; t-tests were used to calculate the p-values.
Figure 5
Figure 5
Key enzymes and metabolites in the tricarboxylic acid (TCA) cycle. (a) Citrate acid content of the initial step product in the TCA cycle (n = 3). (b) Citrate synthase activity in Mstn-KO and WT mice (n = 3). (c) α-ketoglutarate content in Mstn-KO and WT mice (n = 3). (d) Isocitrate dehydrogenase activity in Mstn-KO and WT mice (n = 3). All data are presented as mean ± SD. * p < 0.05, ** p < 0.01; t-tests were used to calculate the p-values.
Figure 6
Figure 6
Mstn knockout inhibited the AMPK/SIRT1/PGC1alpha pathway. (a) The expression of SIRT1 and pAMPK at the protein level in Mstn-KO and WT mice. (b) Gray intensity analysis of pAMPK/α-Tubulin (n = 3). (c) Gray intensity analysis of SIRT1/α-Tubulin (n = 3). (d) Acetylation level of PGC1α protein by co-immunoprecipitation in Mstn-KO and WT mice. (e) Gray intensity analysis of acetylation level of PGC1α (n = 3). All data are presented as mean ± SD. ** p < 0.01; t-tests were used to calculate the p-values.
Figure 7
Figure 7
Expression of pAMPK and SIRT1 following treatment with AICAR and Compound C. (a) The expression of SIRT1 and pAMPK with AICAR treatment Mstn-KO and WT cells. (b,c) Quantitative analysis showing the expression of pAMPK and SIRT1 following treatment with AICAR (n = 3). (d) Citrate synthase (CS) activity with AICAR treatment Mstn-KO and WT cells (n = 3). (e) α-ketoglutarate acid dehydrogenase (α-KGDH) activity with AICAR treatment Mstn-KO and WT cells (n = 3). (f) Isocitrate dehydrogenase (ICDHm) activity with AICAR treatment Mstn-KO and WT cells (n = 3). (g) The expression of SIRT1 and pAMPK with Compound C treatment Mstn-KO and WT cells. (h,i) Quantitative analysis showing the expression of pAMPK and SIRT1 following treatment with Compound C (n = 3). (j) Citrate synthase (CS) activity with Compound C treatment Mstn-KO and WT cells (n = 3). (k) α-ketoglutarate acid dehydrogenase (α-KGDH) activity with Compound C treatment Mstn-KO and WT cells (n = 3). (l) Isocitrate dehydrogenase (ICDHm) activity with Compound C treatment Mstn-KO and WT cells (n = 3). (m) Pattern of MSTN knockdown affecting mitochondrial function through the AMPK/SIRT1/PGC1α pathway. All data are presented as mean ± SD. ns, non-significant p > 0.05; * p < 0.05; ** p < 0.01; t-tests were used to calculate the p-values.
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