Na+/K+ ATPase-Cav1.2 nanodomain differentially regulates intracellular [Na+], [Ca2+] and local adrenergic signaling in cardiac myocytes

Background The intracellular Na+ concentration ([Na+]i) is a crucial but understudied regulator of cardiac myocyte function. The Na+/K+ ATPase (NKA) controls the steady-state [Na+]i and thereby determines the set-point for intracellular Ca2+. Here, we investigate the nanoscopic organization and local adrenergic regulation of the NKA macromolecular complex and how it differentially regulates the intracellular Na+ and Ca2+ homeostases in atrial and ventricular myocytes. Methods Multicolor STORM super-resolution microscopy, Western Blot analyses, and in vivo examination of adrenergic regulation are employed to examine the organization and function of Na+ nanodomains in cardiac myocytes. Quantitative fluorescence microscopy at high spatiotemporal resolution is used in conjunction with cellular electrophysiology to investigate intracellular Na+ homeostasis in atrial and ventricular myocytes. Results The NKAα1 (NKAα1) and the L-type Ca2+-channel (Cav1.2) form a nanodomain with a center-to center distance of ∼65 nm in both ventricular and atrial myocytes. NKAα1 protein expression levels are ∼3 fold higher in atria compared to ventricle. 100% higher atrial INKA, produced by large NKA “superclusters”, underlies the substantially lower Na+concentration in atrial myocytes compared to the benchmark values set in ventricular myocytes. The NKA’s regulatory protein phospholemman (PLM) has similar expression levels across atria and ventricle resulting in a much lower PLM/NKAα1 ratio for atrial compared to ventricular tissue. In addition, a huge PLM phosphorylation reserve in atrial tissue produces a high ß-adrenergic sensitivity of INKA in atrial myocytes. ß-adrenergic regulation of INKA is locally mediated in the NKAα1-Cav1.2 nanodomain via A-kinase anchoring proteins. Conclusions NKAα1, Cav1.2 and their accessory proteins form a structural and regulatory nanodomain at the cardiac dyad. The tissue-specific composition and local adrenergic regulation of this “signaling cloud” is a main regulator of the distinct global intracellular Na+ and Ca2+ concentrations in atrial and ventricular myocytes. ### Competing Interest Statement The authors have declared no competing interest. * AKAP : A-kinase anchoring protein [Ca2+]i : Intracellular Ca2+ concentration Cav1.2 : L-type Ca2+ Channel [Na+]i : Intracellular Na+ concentration NCX : Na+/Ca2+ Exchanger NCLX : Mitochondrial Na+/Ca2+ Exchanger NKA : Na+/K+ ATPase NKAα1 : Na+/K+ ATPase isoform α1 NKAα2 : Na+/K+ ATPase isoform α2 PKA : Protein Kinase A PKC : Protein Kinase C PLM : Phospholemman PLM-Ser63 : Phospholemman Serine 63 PLM-Ser68 : Phospholemman Serine 68 PLM-Thr69 : Phospholemman Threonine69 RyR2 : Ryanodine receptor type 2 SR : Sarcoplasmic Reticulum TATS : Transverse or axial tubule system STORM : Stochastic Optical Reconstruction Microscopy