A Titin Knock-in model for Hereditary Myopathy with Early Respiratory Failure

Titin, the largest muscle protein, plays a key role in the architecture of sarcomeres in both the heart and skeletal muscles. Due to its crucial role, variants in this gene have a critical impact on human health. Titinopathies include severe cardiomyopathies and dominant and recessive skeletal muscle diseases, associated with several pathogenic variants. Among these, titin A150/FN3-119 domain variants are associated with hereditary myopathy with early respiratory failure (HMERF), a life-threatening disorder characterized by respiratory failure and proximodistal muscle weakness. Although murine and fish models have been developed for a wide range of titinopathies, an HMERF model is lacking. Here, we generated and characterized an HMERF knock-in model using Oryzias latipes (medaka fish). Upon the generation of this model, which carries the most common HMERF missense variant (p.C31712R), we found that the mutants had impaired muscle structure, with homozygous larvae exhibiting a more severe phenotype than their heterozygous siblings. Focusing our study on the homozygous larvae, we performed RNA sequencing (RNA-seq) analysis, revealing significant dysregulation of genes with key roles in muscle filament organization and autophagy pathway. This suggests exacerbated muscle damage and dysfunction. These results were corroborated by locomotor analyses and mechanical studies, which revealed that homozygous larvae exhibit limited movement and reduced muscle fiber capability to generate force and shortening at high speed. These results demonstrate that structural abnormalities directly correlate with the impaired function in HMERF mutants. Taken together, the altered muscle structure, impaired locomotor behavior, and dysregulated gene expression underscore the complex pathological mechanisms underlying HMERF disease. Beyond elucidating HMERF-disease mechanisms, our work highlights the value of genome editing in medaka fish, a powerful and versatile model system to dissect the molecular basis of human muscle diseases.