Protein kinase A (PKA) activation reorganizes microtubules and microtubule-plus-end-binding-protein 1 (EB1) and enriches Nav1.5 at the intercalated discs (ICDs) of adult ventricular myocytes.

Abstract

In cardiomyocytes β-adrenergic stimulation increases Na current (INa) through PKA activation, and EB1 targets Nav1.5 to ICDs where INa mediates impulse propagation through gap-junctions. To understand how persistent PKA activation affects INa and Nav1.5 protein expression/distribution in cardiomyocytes, and the role of EB1 and microtubules. Sprague-Dawley ventricular myocytes cultured without or with 8cpt-cAMP (100 uM) plus serine/threonine phosphatase inhibitor (okadaic acid, 100 nM) for 10-20 hr were subject to immunostaining/imaging, subcellular fractionation/immunoblot, and patch clamp recordings. These were supplemented by HEK293 experiments. Cell-attached patch recordings from ICD areas (≤5 sarcomeres from cell ends) showed 10-fold increase in INa in PKA-activated vs control myocytes, without change in total Nav1.5 protein level. To understand Nav1.5 distribution and relationship to EB1 and microtubules at ICDs, we imaged the 3 with n-cadherin (ICD marker) simultaneously using Airyscan at xyz pixel dimension 50 nm. Image reconstruction allowed us to quantify protein signals in en-face views of ICDs. PKA activation (a) increased %ICD area occupied by Nav1.5, β-tubulin and EB1, and (b) increased correlation between Nav1.5/EB1, Nav1.5/β-tubulin, and EB1/β-tubulin. These were supported by HEK293 data: Nav1.5 co-immunoprecipitated more EB1 and β-tubulin in PKA activated (4-6 hr) than control cells. Although PKA activation increased EB1 capture of microtubules at ICD, it decreased microtubule density in the cytoplasmic area, consistent with biochemical data: PKA activation did not change total α-tubulin level but reduced detyrosinated α-tubulin. Myocytes have >10 fold free β-tubulin in cytosol than in microtubules, and >150 fold free EB1 in cytosol than bound to microtubules/membranes. Spatial/temporal dynamics of microtubules and EB1 modulation and PKA-promoted Nav1.5/tubulin/EB1 interactions enrich Nav1.5 at ICDs, facilitating impulse propagation in β-adrenergic stimulated heart.