Abstract Body: Background: Integrin adhesion complexes (IACs), called costameres, connect peripheral myofibrils to the cell membrane and the extracellular matrix and transmit the force of contraction. Mutations in IAC proteins, including vinculin, α-actinin-2, and integrin-linked kinase (ILK), result in cardiomyopathy in both mice and humans. Work in our lab on C. elegans demonstrates that the PIX signalling pathway is required for the assembly of IACs in muscle (Moody et al. 2020, 2024). This led us to generate a cardiomyocyte-specific deletion of βPIX in mice that develop dilated cardiomyopathy. The known output of the PIX pathway is activation of PAK kinases, but the substrates for these kinases in muscle are unknown. In mammalian tissue culture experiments, PAK kinases are implicated as upstream of MAP kinase signalling. Our recent genetic epistasis and biochemical experiments in C. elegans suggest that PAK may phosphorylate a MAP kinase kinase, which then phosphorylates a MAP kinase. We have reported that a genetic enhancer of pix-1 is ipmk-1, a conserved enzyme that converts PIP2 to PIP3, implicating a role for these PIPs in IAC assembly (Sagadiev et al. 2026).

Methods: Quantitative western blotting was used to assess the level of phospho-p38, normalizing to the level of total p38. Confocal imaging was used to assess the localization of p38 and phospho-p38 at the costamere, co-staining with the IAC marker vinculin. Confocal imaging was used to determine the localization of PIP3 in cardiomyocytes of the heart.

Results: Both p38 and phospho-p38 co-localized with vinculin at the costameres, indicating that p38 MAPK is present and active at IAC-associated structures in cardiomyocytes along with PIX pathway components. Furthermore, PIP3 was found to be concentrated at costameres.

Conclusions: The MAPK signalling cascade may be downstream of the PIX pathway and lead to costamere assembly. Our findings may provide a mechanism linking βPIX–Rac1–PAK1 activation to p38-dependent regulation of IAC assembly and may offer new therapeutic targets for a structural complex frequently implicated in cardiomyopathy. Genes encoding PIX pathway components, and the enzymes that determine the levels of various PIPs, may be new genes that, when mutant, result in cardiomyopathy.