Comparative transcriptomics of broad-spectrum and synthetic cannabidiol treated C2C12 skeletal myotubes

Abstract

Cannabidiol (CBD) is widely used in sports for recovery, pain management, and sleep improvement, yet its effects on muscle are not well understood. This study aimed to determine the transcriptional response of murine skeletal muscle myotubes to broad-spectrum CBD and synthetic CBD (sCBD). Differentiated C2C12 myotubes were treated with 10 μM CBD, sCBD, or vehicle control (DMSO) for 24 h before RNA extraction. Poly-A tail-enriched mRNA libraries were constructed and sequenced using 2 × 50 bp paired-end sequencing. CBD and sCBD treatment induced 4489 and 1979 differentially expressed genes (DEGs; p < 0.001, FDR step-up <0.05), respectively, with common upregulation of 857 genes and common downregulation of 648 genes. Common upregulated DEGs were associated with "response to unfolded protein," "cell redox homeostasis," "endoplasmic reticulum stress," "oxidative stress," and "cellular response to hypoxia." Common downregulated DEGs were linked to "sarcomere organization," "skeletal muscle tissue development," "regulation of muscle contraction," and "muscle contraction." CBD treatment induced unique DEGs compared to sCBD. The data indicate CBD may induce mild cellular stress, activating pathways associated with altered redox balance, unfolded protein response, and endoplasmic reticulum stress. We hypothesize that CBD interacts with muscle and may elicit a "mitohormetic" effect that warrants further investigation.

Keywords: cannabidiol; skeletal muscle; transcriptome.

FIGURE 1

Experimental workflow for (a) the determination of dose tolerability. C2C12 myoblasts were differentiated in low serum media for 8 days before 24 h exposure to CBD, sCBD, or vehicle (DMSO) control. MTT and propidium iodide exclusion assays as well as visual inspection were performed to determine the maximal tolerable dose of CBD and sCBD across a range of concentrations from 0.001 to 10 μM. In workflow (b), differentiated myotubes were exposed to 10 μM CBD, sCBD, and vehicle control for 24 h before being lysed for column‐based RNA extraction. Total RNA quantity and quality were determined prior to library preparation and next generation sequencing. Pre‐alignment QC was performed prior to trimming and STAR alignment, followed by quantification to the annotation model, counts were then normalized by median ratio, prior to DSeq2 statistical analysis. Image created using Biorender.

FIGURE 2

Dose tolerability experiments on terminally differentiated myotubes. Metabolic activity and cell viability were determined by the MTT assay and propidium iodide exclusion in CBD‐treated (a, b) and sCBD‐treated (c, d) C2C12 myotubes over a range of concentrations (0.001–10 μM).

FIGURE 3

Principal component analysis plot and summary of differentially expressed genes (p < 0.001, FDR step‐up <0.05) by condition.

FIGURE 4

Volcano plot illustrating the relationship between −log₁₀FDR step‐up and log₂ fold‐change for DEGs. Gray data points represent genes that are not statistically significant (−log₁₀FDR step up (<0.05) and log₂FC > −1.1), whilst blue data points are significantly up regulated and red data points are significantly downregulated. Top 5 up and downregulated DEGs are labeled for comparison.

FIGURE 5

Common upregulated genes; (a) Venn diagram illustrating DEGs uniquely upregulated by CBD and sCBD and common upregulated genes in the overlapping region. (b) The top 5 Enriched KEGG pathways and GO terms for common upregulated genes. BP, biological process; CC, cellular compartment; MF, molecular function. (c) Common up‐regulated genes and their known protein–protein interactions, single nodes are not shown, highest confidence (0.9), clusters are colored individually using K‐means clustering.

FIGURE 6

Common downregulated genes; (a) Venn diagram illustrating DEGs uniquely downregulated by CBD and sCBD and common downregulated genes in the overlapping region. (b) The top 5 Enriched KEGG pathways and GO terms for common downregulated genes. BP, biological process; CC, cellular compartment; MF, molecular function. (c) Common downregulated genes and their known protein–protein interactions, single nodes are not shown, highest confidence (0.9), clusters are colored individually using K‐means clustering.