Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine; Fischell Department of Bioengineering, University of Maryland.
Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine.
J Vis Exp. 2022 May 27(183). doi: 10.3791/59600.
Intracellular sodium concentration ([Na]i) is an important regulator of intracellular Ca. Its study provides insight into the activation of the sarcolemmal Na/Ca exchanger, the behavior of voltage-gated Na channels and the Na,K-ATPase. Intracellular Ca signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca 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.
细胞内钠离子浓度 ([Na]i) 是细胞内 Ca 的重要调节剂。其研究为肌浆网 Na/Ca 交换体的激活、电压门控 Na 通道和 Na,K-ATP 酶的行为提供了深入了解。心房疾病（如心房颤动）会改变细胞内 Ca 信号。虽然已经描述了许多改变细胞内 Ca 稳态的机制，但 [Na]i 及其在心房病变中的失调作用仍知之甚少。在心房肌细胞中，[Na]i 随着刺激频率的增加而增加。因此，对外场刺激的反应性对于这些细胞中的 [Na]i 测量至关重要。此外，长的准备（染料加载）和实验时间（校准）需要一种分离方案，该方案产生的心房肌细胞质量非常高。由于小鼠心房的体积小和细胞间质的组成，高质量成年小鼠心房肌细胞的分离较为困难。在这里，我们描述了一种优化的基于 Langendorff 灌注的分离方案，该方案始终能提供高产量的高质量心房鼠肌细胞。苯并呋喃异邻苯二甲酸（SBFI）是最常用的荧光 Na 指示剂。SBFI 可以通过玻璃吸管以其盐的形式或作为可以穿透肌细胞膜的乙酰氧甲酯（AM）酯的形式加载到心肌细胞中。在细胞内，SBFI-AM 被细胞质酯酶去酯化。由于膜穿透和细胞质去酯化的变异性，每个细胞都必须在原位校准。通常，使用光电倍增管（PMT）通过 SBFI 全细胞荧光测量来测量 [Na]i。这种实验设置一次只能测量一个细胞。由于肌细胞染料加载和每次实验后的校准时间长，因此数据产量低。因此，我们开发了一种基于 EMCCD 相机的技术来测量 [Na]i。这种方法允许同时测量多个肌细胞中的 [Na]i，从而大大提高了实验产量。
