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

Abstract

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.