CRISPR/Cas9-Mediated MSTN Knockout Alters Muscle Proteome and MSTN–ACVR2B Interaction in Buffalo Myoblasts

Abstract

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth and differentiation. Buffalo myoblasts were isolated from the longissimus dorsi muscle and characterized by binucleate morphology and Pax7/myosin immunostaining. Myoblasts were nucleofected with MSTN guide RNA and Cas9, and three independently derived edited clonal lines were established and validated by Sanger sequencing. Comparative proteomic and computational structural analyses were performed to investigate molecular changes associated with MSTN disruption. Proteomic analysis of edited versus wild-type cells identified 122 differentially expressed proteins (54 upregulated, 68 downregulated). Gene Ontology and KEGG pathway analyses showed enrichment for calcium regulation, actomyosin organisation, muscle contraction, and energy metabolism. Protein–protein interaction analysis identified key hub proteins related to muscle structure and calcium regulation, including MYL1, TPM1, TPM3, TMOD1, MYLK, ATP2A1, and CASQ2. Network clustering further resolved functional modules involved in muscle filament organization (TPM1, TPM3, TMOD1, MYH10), calcium signaling (CACNA1A, CACNA1I, CACNA1F), calcium storage and muscle structure (PDLIM3, CASQ2, ATP2A1), glucose/glycogen metabolism (ENO3, SLC2A4, GYS1), and cellular signaling (OXT, CHRM2, PTGER3, EDN3), indicating that MSTN editing affects processes central to muscle structure, contraction, calcium balance, and metabolism. To assess effects on the MSTN protein itself, sequence analysis, structure prediction, molecular docking, and molecular dynamics simulations were performed. Edited MSTN sequences showed predicted structural changes relative to wild-type, and simulations of MSTN–ACVR2B complexes revealed differences in conformational dynamics, stability, and receptor-interaction energetics between edited and wild-type forms. Overall, CRISPR/Cas9-mediated MSTN editing reshapes muscle-related protein networks and alters the predicted MSTN–ACVR2B interaction, providing molecular insight into MSTN disruption's role in promoting muscle growth in buffalo. Keywords: CRISPR/Cas9; Myostatin; Muscle cells; Molecular dynamics; Docking, ACVR2B