Identication of driver genes and biological signaling for alcoholic myopathy

Long-term alcohol consumption contributes to muscle weakness and atrophy. However, the mechanism and biological functions are still not clear. In this study, we aim to identify the signicantly changed genes and potential signaling pathways in the gastrocnemius and plantaris muscle from C57BL/6Hsd mice by analyzing RNA sequence. The GSE183665 dataset was created by using the Illumina NovaSeq 6000 (Mus musculus). The KEGG and GO analyses showed that "cell migration", "cell adhesion", and "apoptosis" are major biological processes in the skeletal muscles. Moreover, we identied a number of genes including POSTN, GNAI2, MMP2, ELN, CCND1, CXCL12, COL6A1, COL6A2, SFRP2, and FSTL1 by using the PPI network and Reactome map. Thus, our study may shed light on the development of drugs on alcohol myopathy.


Introduction
Alcohol regulates the biological function of numerous organs and tissues in the body by interaction with cellular components and relative signaling pathways including oxidative and in ammatory signaling 1 .
The metabolism of alcohol contributes to the production of acetaldehyde and reactive oxygen that damage organs and tissues such as muscles 2 . The oxidative stress origins from various tissues and organs, which depends on the in ammatory and oxidative state and immune levels 3 . Additionally, the degree and duration of stress are dependent on the metabolic state of tissues to clear alcohol and its byproducts 4 . Other oxidative stress origins from the increased release of cytokines by the immune cells responding to alcohol 5 . Alcohol is related to signal transduction through cell membranes, signaling receptors, and ion channels 6 . Modi cation of these receptors and signaling molecules results in changing multiple signaling pathways.
Alcoholism is a major reason for muscle myopathy that is categorized as a decreased skeletal muscle protein synthesis and myo brillary protein contents 7 . The major affected muscles are rich in type II muscle bers including plantaris and gastrocnemius 8 . The muscular weakness caused by alcohol may be due to the downregulation of contractile processes. Moreover, the therapeutic strategies targeting muscles may lead to a deleterious effect on others 9 . Therefore, gaining knowledge on the responses to alcohol in muscles may bene t the clinical work.
In our study, we evaluated the effects of alcohol stress on the muscle tissues by analyzing the RNA-seq data. We identi ed a number of DEGs and signi cant signaling pathways. We also performed the gene function enrichment and constructed the protein-protein interaction (PPI) network and Reactome map to nd the valuable drug targets and pathways. The functional genes and biological processes will guide the clinical work on the treatment of muscle damage caused by alcohol.

Data resources
The data was obtained by using the Illumina NovaSeq 6000 (Mus musculus) (Biomedical Sciences, Florida State University, 1115 W. Call Street, Tallahassee, Florida, US). The analyzed dataset includes ve control groups and ve alcohol-treated groups.

Data acquisition and preprocessing
The raw data were processed by the R package as described 10 . A classical t-test was performed to identify DEGs with P< 0.01 and fold change ≥ 1 as being statistically signi cant.
The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses The KEGG and GO analyses were performed by using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (http://david.ncifcrf.gov/). We set the P<.05 and gene counts >10 as the statistically signi cant cutoff.

Protein-protein interaction (PPI) analysis
The PPI was constructed by using the Molecular Complex Detection (MCODE). The biological processes analysis was performed by Reactome (https://reactome.org/), and P<0.05 was considered as the statistically signi cant cutoff.

Results
Identi cation of DEGs in control and alcohol-treated muscles from C57BL/6Hsd mice To determine the molecular mechanism of alcohol on muscles, we analyzed the DEGs from the control and alcohol-treated muscles from mice. A total of 360 genes were identi ed with the threshold of P<0.01.
The top ten of up-and-down-regulated genes for control and alcohol-treated muscles are shown by the heatmap and volcano plot ( Figure 1). The top ten DEGs were listed in Table 1.

PPI network analysis
To further understand the relationship among the DEGs, we constructed the PPI network by using the 297 nodes and 206 edges (String network). The criterion of combined score > 0.4 was de ned to create the PPI in the Cytoscape software. Table 2 indicated the top ten genes with the highest degree scores. The top two modules showed the functional annotation ( Figure 3). We further analyzed the DEGs and PPI networks by using the Reactome map ( Figure 4). We identi ed the top ten signi cant biological processes including "Extra-nuclear estrogen signaling", "ESR-mediated signaling", "Activation of Matrix Metalloproteinases", "NOTCH3 Intracellular Domain Regulates Transcription", "Activation of SMO", "Molecules associated with elastic bers", "Signal ampli cation", "Signaling by NOTCH3", "RUNX3 regulates WNT signaling", and "RET signaling" (Supplemental Table S1).

Discussion
Alcoholism leads to a kind of myopathic lesion characterized by selective atrophy of Type II bers 11 .
Moreover, alcoholism causes reductions in muscle mass and body mass 12 . Muscle strength may also be impaired by alcohol with long-term intake 13 . Our study mainly focused on the mechanism and impact of alcoholism on the muscles, which may help to develop drugs by targeting myopathy.
In this study, we found alcohol mainly affects cell migration, cell adhesion, and apoptosis by analyzing the KEGG and GO enrichment. Similarly, Tatsuro Kumada et al found that alcohol affects the migration of immature neurons 14 . Moreover, the abnormal migration of neurons by alcohol can be improved by regulating the second-messenger pathways 14 . R Ramanathan et al found that alcohol can completely repress the L1-mediated cell-cell adhesion 15 . Ana Rodriguez et al found that the high alcohol level leads to increased caspase-3 activity and declined contractility, whereas the low alcohol level is related to the decreased apoptosis 16 . It is suggested that the low alcohol level may have the potential to protect the muscles by regulating the cell functions.
By analyzing the PPI network, we also identi ed several potential DEGs that may affect the function of muscles by alcohol intake. The study by Masamitsu Hara et al showed that the periostin enhances the migration of broblast and represses the repair after injury 17 . Another nding showed the periostin is expressed in the cardiac broblasts, which can result in cardiomyocyte maturation and innervation 18 . G-protein-coupled receptors (GPCRs), Regulators of G protein signaling (RGS), and their downstream pathways are involved in various physiological and pathological processes [19][20][21] including in ammation, metabolism, cancer, and pain [22][23][24][25][26][27][28] . As a signi cant heterotrimeric G protein, GNAI2 ameliorates cell proliferation and inhibits apoptosis processes 26 . Elin Hadler-Olsen et al found MMP2 is related to the type II bers through regulating the extracellular substrates 29 . MMP2 plays an important role in skeletal muscle cell migration and differentiation 30 . Circadian gene clocks and their downstream genes are associated with a number of signaling pathways such as metabolism, immune, and aging processes [31][32][33][34][35][36][37][38][39][40] . Interestingly, as a circadian clock-controlled gene, MMP2 shows aberrant functions in clock mutant mice 41

Funding
This work was not supported by any funding.

Declarations of interest
There is no con ict of interest to declare.  Tables   Tables 1-2 are in the supplementary les section. Figure 1 Identi cation of DEGs in control and alcohol treated muscles from C57BL/6Hsd mice (A) Heatmap of DEGs between control and alcohol treated muscles from C57BL/6Hsd mice. Regularized matrix was generated using the R package. (B) Volcano plot for DEGs between control and alcohol treated muscles from C57BL/6Hsd mice. The most signi cant genes are marked with symbols.  The PPI network analysis between control and alcohol-treated muscles from C57BL/6Hsd mice 297 nodes and 206 edges were obtained from the STRING database for creating the PPI network. Cluster 1 (A) and cluster 2 (B) were constructed by Cytoscape.

Figure 4
Reactome map indication of the signi cant biological processes of the protein elements identi ed between control and alcohol-treated muscles from C57BL/6Hsd mice

Supplementary Files
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