Abstract Body: Background: Cardiomyopathies represent a substantial burden on the global healthcare system. While many cases are associated with mutations in genes encoding mostly sarcomeric proteins, in many other cases the genes involved are unknown, highlighting a significant need for new genetic factor discovery. Loss-of-function mutations in the C. elegans gene pix-1 result in loss of assembly of integrin-associated complexes (IAC) in striated muscle. PIX-1 is a Rac GEF that activates Rac and thereby activates its protein kinase PAK. The mammalian ortholog, βPIX, has a poorly understood role in mammalian muscle. Because mutations in some IAC components result in cardiomyopathy, we postulated that βPIX may be involved in cardiac muscle regulation and disease via IAC dysregulation.
Methods: We localized βPIX in the mouse heart via immunofluorescence and generated a cardiomyocyte-specific knockout (βPIX-cKO) mouse model. Cardiac function was assessed longitudinally with echocardiography. Intercalated disk morphology was quantified by contour length-to-chord length ratio. IAC protein levels and localization were evaluated at two and eight months of age with western blotting and immunohistochemistry, respectively. Cardiomyocyte adhesion to laminin was assessed with GentleMACS perfusion and RICM microscopy.
Results: βPIX localizes to intercalated disks, costameres, and Z-disks in cardiomyocytes. βPIX-cKO mice develop dilated cardiomyopathy by eight months of age and die between eight and eleven months. Intercalated disks show increased amplitude in βPIX-cKO mice at eight months. No differences in IAC protein levels were detected; however, integrin-linked kinase (ILK) is lost at costameres as early as two months of age, prior to overt cardiomyopathy, and Kindlin-2 forms abnormal aggregates at eight months. Finally, cardiomyocytes show a functional decrease in adhesion to laminin at two months of age.
Conclusions: Loss of βPIX perturbs integrin adhesion complex localization, results in decreased adhesion to laminin, and contributes to dilated cardiomyopathy development, identifying βPIX as a novel genetic factor in cardiac disease.