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.

Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. Herein, the myostatin gene (MSTN), a negative regulator of skeletal muscle development, was knocked out by CRISPR/Cas9 technology. Eight MSTN gene-edited bull calves (MT) were born, and six of them are well-developed. Compared with the control cattle (WT), the growth trait indexes of MT cattle were generally increased, and the hindquarters especially were significantly improved. The biochemical indexes and the semen characteristics demonstrated that MT bulls were healthy and fertile. Consistent with our conjecture, the wobble and beating of MT bull spermatozoa were significantly higher than that of WT. Nine sperm motility-related proteins and nineteen mitochondrial-related proteins were identified by up-regulation in MT bull spermatozoa using FLQ proteomic technique and act to govern sperm flagellum assembly, organization, and beating and provide sufficient energy for sperm motility. The current study confirmed that the MSTN gene-edited Chinese Yellow cattle have improved growth traits and normal fertility, which can be used for beef cattle production and breeding.

Interspecies somatic cell nuclear transfer (iSCNT) has been regarded as a potential alternative for rescuing highly endangered species and can be used as a model for studying nuclear–cytoplasmic interactions. However, iSCNT embryos often fail to produce viable offspring. The alterations in normal molecular mechanisms contributing to extremely poor development are for the most part unknown. Przewalski’s gazelle–bovine iSCNT embryos (PBNT) were produced by transferring Przewalski’s gazelle fibroblast nuclei into enucleated bovine oocytes. The percentages of PBNT embryos that developed to morula/blastocyst stages were extremely low even with the use of various treatments that included different SCNT protocols and treatment of embryos with small molecules. Transcriptional microarray analyses of the cloned embryos showed that the upregulation of reprogramming-associated genes in bovine–bovine SCNT (BBNT) embryos was significantly higher than those observed in PBNT embryos (1527:643). In all, 139 transcripts related to various transcription regulation factors (TFs) were unsuccessfully activated in the iSCNT embryos. Maternal degradation profiles showed that 1515 genes were uniquely downregulated in the BBNT embryos, while 343 genes were downregulated in the PBNT embryos. Incompatibilities between mitochondrial DNA (mtDNA) and nuclear DNA revealed that the TOMM (translocase of outer mitochondrial membrane)/TIMM (translocase of inner mitochondrial membrane) complex-associated genes in BBNT embryos had the highest expression levels, while the PBNT embryos exhibited much lower expression rates. Improper degradation of maternal transcripts, incomplete activation of TFs and abnormal expression of genes associated with mitochondrial function in PBNT embryos likely contributed to incomplete reprogramming of the donor cell nuclei and therefore led to the developmental failure of these cloned embryos.

Myostatin (MSTN) is an important negative regulator of skeletal muscle growth in animals. A lack of MSTN promotes lipolysis and glucose metabolism but inhibits oxidative phosphorylation (OXPHOS). Here, we aimed to investigate the possible mechanism of MSTN regulating the mitochondrial energy homeostasis of skeletal muscle. To this end, MSTN knockout mice were generated by the CRISPR/Cas9 technique. Expectedly, the MSTN null (Mstn^−/−) mouse has a hypermuscular phenotype. The muscle metabolism of the Mstn^−/− mice was detected by an enzyme-linked immunosorbent assay, indirect calorimetry, ChIP-qPCR, and RT-qPCR. The resting metabolic rate and body temperature of the Mstn^−/− mice were significantly reduced. The loss of MSTN not only significantly inhibited the production of ATP by OXPHOS and decreased the activity of respiratory chain complexes, but also inhibited key rate-limiting enzymes related to the TCA cycle and significantly reduced the ratio of NADH/NAD+ in the Mstn^−/− mice, which then greatly reduced the total amount of ATP. Further ChIP-qPCR results confirmed that the lack of MSTN inhibited both the TCA cycle and OXPHOS, resulting in decreased ATP production. The reason may be that Smad2/3 is not sufficiently bound to the promoter region of the rate-limiting enzymes Idh2 and Idh3a of the TCA cycle, thus affecting their transcription.

Myostatin (MSTN) is a major negative regulator of skeletal muscle mass and causes a variety of metabolic changes. However, the effect of MSTN knockout on bile acid metabolism has rarely been reported. In this study, the physiological and biochemical alterations of serum in MSTN^+/− and wild type (WT) cattle were investigated. There were no significant changes in liver and kidney biochemical indexes. However, compared with the WT cattle, lactate dehydrogenase, total bile acid (TBA), cholesterol, and high-density lipoprotein (HDL) in the MSTN^+/− cattle were significantly increased, and glucose, low-density lipoprotein (LDL), and triglycerides (TG) were significantly decreased, indicating that MSTN knockout affected glucose and lipid metabolism and total bile acids content. Targeted metabolomic analysis of the bile acids and their derivatives was performed on serum samples and found that bile acids were significantly increased in the MSTN^+/− cattle compared with the WT cattle. As the only bile acid synthesis organ in the body, we performed metabolomic analysis on the liver to study the effect of MSTN knockout on hepatic metabolism. Metabolic pathway enrichment analysis of differential metabolites showed significant enrichment of the primary bile acid biosynthesis and bile secretion pathway in the MSTN^+/− cattle. Targeted metabolomics data further showed that MSTN knockout significantly increased bile acid content in the liver, which may have resulted from enhanced bile acid synthesis due to the expression of bile acid synthesis genes, cholesterol 7 alpha-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1), and upregulation in the liver of the MSTN^+/− cattle. These results indicate that MSTN knockout does not adversely affect bovine fitness but regulates bile acid metabolism via enhanced bile acid synthesis. This further suggests a role of MSTN in regulating metabolism.

Myostatin (MSTN), a growth and differentiation factor, plays an important role in regulating skeletal muscle growth and development. MSTN knockout (MSTN-KO) leads to skeletal muscle hypertrophy and regulates metabolic homeostasis. Moreover, MSTN is also detected in smooth muscle. However, the effect of MSTN-KO on smooth muscle has not yet been reported. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in esophageal smooth muscles of MSTN-KO Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 608.5 ± 17.62 kg) and wild-type (WT) Chinese Luxi Yellow cattle (n = 5, 24 months, average body weight 528.25 ± 11.03 kg). The transcriptome was sequenced using the Illumina Novaseq™ 6000 sequence platform. In total, 337 significantly up- and 129 significantly down-regulated genes were detected in the MSTN-KO cattle compared with the WT Chinese Luxi Yellow cattle. Functional enrichment analysis indicated that the DEGs were mainly enriched in 67 signaling pathways, including cell adhesion molecules, tight junction, and the cGMP-PKG signaling pathway. Metabolomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 130 differential metabolites between the groups, with 56 up-regulated and 74 down-regulated in MSTN knockout cattle compared with WT cattle. Differential metabolites were significantly enriched in 31 pathways, including glycerophospholipid metabolism, histidine metabolism, glutathione metabolism, and purine metabolism. Transcriptome and metabolome were combined to analyze the significant enrichment pathways, and there were three metabolically related pathways, including histidine metabolism, purine metabolism, and arginine and proline metabolism. These results provide important references for in-depth research on the effect of MSTN knockout on smooth muscle.