Zasp52 in Drosophila melanogaster indirect flight muscles can serve as a model system to investigate the function of clinical variants causing myopathies.

The sarcomere is the basic contractile unit of muscle fibers, bordered by Z-discs. In Drosophila, Z-disc structure and maintenance rely on actinin, and the Z-band alternatively spliced PDZ-motif protein 52 (Zasp52), a member of the Alp/Enigma family. Zasp52 has been shown to bind actin, actinin, and itself, but its full range of functions remains unclear, as a null mutant has not been previously analyzed. To address this, we generated a CRISPR-based null mutant of Zasp52, deleting most of the ∼50 kb locus. Null mutants are viable but flightless and display severe defects in indirect flight muscle architecture. To test redundancy, we also analyzed a double null of Zasp52 and Zasp67, the latter one being a Zasp member restricted to flight muscles. Both single and double mutants exhibit damaged myofibrils, with the double mutant showing a more severe collapse of sarcomere organization. Given the clinical importance of LIM domain-binding 3 (LDB3) mutations, the human ortholog of Zasp52, we next modeled a pathogenic variant. In humans, the P615L mutation in LDB3 has been linked to cardiomyopathy and skeletal myopathy. We engineered the orthologous substitution, Zasp52-P607L, and expressed it in Drosophila indirect flight muscles. The variant produced enlarged sarcomeres and enhanced binding, consistent with a gain-of-function disease phenotype. Together, these findings indicate that Drosophila indirect flight muscles can be used as a system to dissect Zasp function, reveal redundancy in Z-disc assembly, and provide mechanistic insight into how conserved mutations contribute to human myopathies.