Na + /K + ATPase-Ca v 1.2 nanodomain differentially regulates intracellular [Na + ], [Ca 2+ ] 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 Ca 2+ . Here, we investigate the nanoscopic organization and local adrenergic regulation of the NKA macromolecular complex and how it differentially regulates the intracellular Na + and Ca 2+ 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 Ca 2+ -channel (Ca v 1.2) 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 I NKA , 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 I NKA in atrial myocytes. ß-adrenergic regulation of I NKA is locally mediated in the NKAα1-Ca v 1.2 nanodomain via A-kinase anchoring proteins. Conclusions: NKAα1, Ca v 1.2 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 Ca 2+ concentrations in atrial and ventricular myocytes.

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes. Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte's sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

Region [corrected] of slowed conduction acts as core for spiral wave reentry in cardiac cell monolayers.

Pathophysiological heterogeneity in cardiac tissue is related to the occurrence of arrhythmias. Of importance are regions of slowed conduction, which have been implicated in the formation of conduction block and reentry. Experimentally, it has been a challenge to produce local heterogeneity in a manner that is both reversible and well controlled. Consequently, we developed a dual-zone superfusion chamber that can dynamically create a small (5 mm) central island of heterogeneity in cultured cardiac cell monolayers. Three different conditions were studied to explore the effect of regionally slowed conduction on wave propagation and reentry: depolarization by elevated extracellular potassium, sodium channel inhibition with lidocaine, and cell-cell decoupling with palmitoleic acid. Using optical mapping of transmembrane voltage, we found that the central region of slowed conduction always served as the core region around which a spiral wave formed and then revolved following a period of rapid pacing. Because of the localized slowing in the core region, we observed experimentally for the first time an S shape of the spiral wave front near its tip. These results indicate that a small region of slowed conduction can play a crucial role in the formation, anchoring, and modulation of reentrant spiral waves.