bioRxiv2025
BACKGROUNDMyopalladin (MYPN) is a striated muscle-specific sarcomeric protein belonging to the immunoglobulin (Ig)-domain-containing family of actin regulators. The Ig3 domain of MYPN is indispensable for interacting with and stabilizing actin filaments, and an increasing number of cardiomyopathic (CM)-associated mutations have been identified within this domain, highlighting its importance for normal cardiac function. Despite their clinical significance, the molecular basis of how these variants disrupt MYPN activity remains poorly understood. METHODSTo identify actin-binding sites, we introduced charge-neutralizing alanine substitutions at basic residues in the Ig3 domain and assessed their impact using actin co-sedimentation assays. CM mutations were similarly evaluated for their effect on actin interaction. Wild-type (WT) and mutant MYPN were expressed in Drosophila cardiomyocytes and body wall muscles to assess localization and interaction with actin. The impact of these mutations on protein secondary structure and stability was determined by circular dichroism spectroscopy. In addition, the WT Ig3 domain was further analyzed in co-sedimentation and co-polymerization assays to quantify polymerization of monomeric actin, subsequent bundling and to assess how calcium and salt concentrations modulate MYPN Ig3-actin interactions. RESULTSMYPN-actin binding is electrostatically driven and mediated by conserved basic charge clusters within its Ig3 domain, a mechanism common among many actin-binding proteins. CM mutations disrupted the MYPN-actin interaction, leading to significant loss of actin bundling without broadly altering secondary structure, except in the destabilized P961L variant. Immunostaining and green fluorescent protein (GFP)-tagged constructs revealed that R955W and P961L mutants accumulated into clusters at the Z-discs. WT Ig3 domain assays further showed that MYPN promotes actin polymerization and crosslinking even under non-polymerizing conditions, and actin-binding activity was not modulated by calcium. CONCLUSIONSThese findings demonstrate that CM-linked MYPN Ig3 mutations compromise actin binding and organization, suggesting that disruption of the MYPN-actin interface is a key pathogenic mechanism in cardiomyopathy. What Is Known?O_LIMyopalladin (MYPN) is a striated muscle-specific sarcomeric protein. C_LIO_LIMYPN plays an important role in controlling skeletal muscle growth by regulating actin dynamics and modulating stress-responsive signal transduction and gene expression. C_LIO_LIMutations in MYPN are linked to various inherited cardiomyopathies (CMs), including dilated, hypertrophic, restrictive, and left ventricular noncompaction forms. C_LI What new information does this article contribute?O_LITwo conserved clusters of basic residues on the surface of Ig3 domain were identified as key mediators of actin binding and bundling. C_LIO_LISeveral cardiomyopathy (CM)-associated MYPN mutations localize to this region, where they impair actin interactions and nearly abolish actin bundling activity. C_LIO_LIMutants such as R955W and P961L mislocalize abnormally in Drosophila cardiomyocytes and body wall muscles. C_LI Novelty and SignificanceThe importance of the MYPN gene, which encodes the sarcomeric protein myopalladin (MYPN), was highlighted when 66 mutations in this gene were linked to cardiomyopathies (CMs). MYPN bundles and stabilizes actin filaments and is essential for the organization and maintenance of Z-disc integrity. The C-terminal immunoglobulin (Ig) domains mediate actin interactions, with the Ig3 domain representing the minimal actin-binding unit. Notably, many disease-associated mutations cluster within this actin-binding region and give rise to diverse clinical phenotypes. Despite its clinical relevance, the molecular mechanisms by which different MYPN mutations lead to CM remain poorly understood. Prior mechanistic studies focused mainly on mutations within the cardiac ankyrin repeat protein (CARP) interaction domain of MYPN, which disrupt signaling and nuclear shuttling. In contrast, our study addresses mutations localized to the actin-binding region of MYPN. The effects of these mutations on MYPN-actin interactions had not been previously explored. We show that CM-associated mutations within the Ig3 domain disrupt MYPN-actin interactions, reduce or abolish actin bundling, and induce abnormal subcellular localization patterns in Drosophila cardiac and skeletal muscles. This study provides the first direct evidence that disruption of MYPN-actin interactions represents a distinct and clinically relevant mechanism of cardiomyopathy.