Exploring Cardiovascular Implications of col6a2 Knockout in Zebrafish: A Model for COL6-CMD

Introduction: Cardiovascular aspects of Collagen VI congenital muscular dystrophy (COL6-CMD) remain insufficiently explored, despite the presence of COL6 in numerous extracellular matrices critical for cardiovascular health. COL6-CMD is a rare disorder caused by dominant-negative or recessive mutations in the COL6 genes, namely COL6A1, COL6A2, and COL6A3. While the condition predominantly affects skeletal muscle and connective tissues, collagen VI also contributes to the structural integrity and functional support of various tissues, including skin, tendons, cartilage, intervertebral discs, and crucially, components of the cardiovascular system. Although the roles of COL6 in connective tissues are well-established, its pleiotropic effects on cardiovascular function and potential involvement in cardiovascular pathology remain largely unexplored.

Aim: In this study, we aimed to develop a zebrafish model of COL6-CMD to study tissue-specific pathologies with a focus on cardiovascular effects. As the zebrafish has proven to be a powerful model organism for studying developmental biology and disease pathogenesis, we hypothesised that a zebrafish model for COL6-CMD would provide insight into early manifestations of cardiovascular abnormalities associated with COL6 deficiency.

Methods and results: We utilized CRISPR/Cas9 technology to knock out the col6α2 gene in zebrafish, generating mutant "crispants." Phenotypic assessment was conducted through a combination of high-resolution live imaging (using the Keyence Imaging Machine) and behavioral analysis with the ZebraBox system, which provides automated and quantitative measures of locomotion and other behavioral parameters. In situ hybridization was used to validate gene editing success and assess expression patterns of key genes involved in cardiovascular and muscular development. Consistent with clinical features of COL6-CMD, our col6α2-deficient zebrafish showed distinct alterations in locomotor activity and morphological features, including reduced body size and changes in swim bladder structure. Beyond these expected musculoskeletal phenotypes, we observed significant cardiovascular alterations, such as impaired angiogenesis, changes in chamber-specific heart function, and altered blood flow dynamics. These findings suggest that collagen VI plays a critical role in cardiovascular development, and that its absence may disrupt normal vascular and cardiac function during early growth stages.

Conclusion: Our results suggest that col6α2-deficient zebrafish exhibit both muscular and cardiovascular phenotypes similar to those observed in COL6-CMD, supporting the notion that collagen VI deficiencies affect a broader range of tissues than previously thought. This zebrafish model provides a valuable platform for studying the cardiovascular role of COL6 in vivo and provides insights into the developmental origins of COL6-associated vascular and cardiovascular pathologies. Ultimately, this work may contribute to an expanded clinical understanding of COL6-CMD that includes cardiovascular and vascular phenotypes as integral components of disease pathology.