Transcriptomic analysis of Cr(VI)-induced changes in C2C12 cells during myogenic differentiation

Hexavalent chromium [Cr(VI)] is an environmental toxicant extensively used in a variety of industrial processes including chrome plating, leather tanning, textile manufacturing, aircraft production, and stainless-steel production. Our previous study reported that exposure to Cr(VI) inhibited C2C12 myogenic differentiation in a dose-dependent manner, yet the transcriptional mechanisms underlying Cr(VI)-induced disruption of myogenesis remains poorly understood. This study aimed to characterize the global transcriptional alterations during C2C12 myogenic differentiation and identify molecular pathways disrupted upon Cr(VI) exposure.

C2C12 cells were differentiated in the presence of 0, 2, or 5 μM of Cr(VI) and collected at differentiation days 0, 1, 2, and 4. Whole transcriptome analysis of a total of 30 samples (with 3 biological replicates per condition) was performed using RNA-sequencing followed by differential gene expression analysis, unsupervised fuzzy c-means clustering, GO biological processes functional annotation, and KEGG pathway enrichment analysis.

Cr(VI) exposure resulted in a massive transcriptomic change in differentiating C2C12 cells. Fuzzy c-means clustering identified 12 distinct gene expression patterns, with Clusters 3, 10, and 12 showing significant overlap with Cr(VI)-regulated genes. Functional enrichment analyses revealed Cr(VI) alters genes involved in early-stage cell cycle regulation and DNA repair as well as terminal differentiation processes like sarcomere organization and muscle contraction. Specifically, Cr(VI) suppressed expression of key structural and contractile genes and disrupted pathways essential for myogenic differentiation, cell cycle regulation and DNA damage repair. Furthermore, Cr(VI) disrupted Hippo signaling by downregulating Tead4 and its downstream myogenic targets such as MyoG, Cav3, Mustn1, and Dysf suggesting a mechanism for impaired differentiation.

This study highlights the widespread alterations of Cr(VI) exposure on transcriptional programs - including structural development, genomic stability, and cell cycle withdrawal - governing muscle development and maturation, offering insight into how Cr(VI) exposure affects skeletal muscle health.