The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.
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.
In atrial diseases (e.g., atrial fibrillation [AF]) intracellular Ca2+ signaling is profoundly altered1. While many of the underlying mechanisms of ‘remodeled’ intracellular Ca2+ signaling in AF have been well characterized2,3, the role an altered intracellular sodium concentration ([Na+]i) may play is poorly understood. [Na+]i is an important regulator of intracellular Ca2+. The study of [Na+]i can provide insight into the activation of the sarcolemmal Na+/Ca2+ exchanger (NCX), the behavior of Na+ channels and Na+,K+-ATPase (NKA)4. We have previously shown that high atrial activation rates, as occur during AF, lead to a significant reduction in [Na+]i 1. Previous work has shown an increase in NCX current density (INCX) and protein expression levels in AF3. An increase in the late component of the voltage-dependent Na+ current (INa, late) in isolated atrial myocytes from patients with AF was also reported5. Thus, there is evidence of profound changes in intracellular Na+ homeostasis in AF. Reliable and reproducible measurements of [Na+]i in isolated atrial myocytes are therefore needed to further our understanding of AF pathology. Here, we demonstrate how to reproducibly isolate high quality murine atrial myocytes that are suitable for measurements of [Na+]i. We have focused our optimized atrial cell isolation protocol on murine atrial myocytes because transgenic (TG) mouse models of atrial fibrillation have become a vital part of AF research6. These mice are often only available in limited numbers and the atria are often fibrotic leading to challenges for cell isolation.
In general, [Na+]i in viable cells can be measured with fluorescent indicators7,8, or with different types of microelectrodes9. Microelectrode-based techniques require penetration of the sarcolemmal membrane. This technique is therefore limited to larger cells and is unsuitable for small and narrow atrial myocytes whose cell integrity is easily compromised.
Sodium-binding benzofuran isophthalate (SBFI) is a fluorescent indicator, which undergoes a large wavelength shift upon binding Na+ 7. SBFI is alternatingly excited at 340 nm and 380 nm and emitted fluorescence is collected after passing through an emission filter (510 nm). Ratios of signals at the two excitation wavelengths (F340/380) can cancel out the local path length, dye concentration, and wavelength-independent variations in illumination intensity and detection efficiency. When an in situ calibration using solutions with known sodium concentration ([Na+]) is performed in each cell the F340/380 ratio obtained during the experiment yields precise and sensitive measurements of [Na+]i. As all Na+ indicators, SBFI also displays some affinity for K+. Using the calibration method shown here allows to reliably ‘clamp’ [Na+]i and intracellular potassium concentration ([K+]i) during the calibration process so that [Na+]i can be reliably calibrated even when it is <10% of [K+]i 10.
We introduce a novel EMCCD camera based technique for ratiometric measurements of [Na+]i using SBFI. The EMCCD camera allows, for the first time, simultaneous [Na+]i measurements (and calibration) in multiple cells. This is especially beneficial in an experimental setting where animal numbers are limited (e.g., transgenic mouse models). Typically, [Na+]i measurements using SBFI are performed using a photomultiplier tube (PMT) to collect whole cell epifluorescence1,11. While PMTs offer very good temporal resolution of the fluorescence signal, the spatial resolution is very low and experiments are limited to one cell at a time.
Our novel protocol facilitates highly reproducible and sensitive measurements of [Na+]i. It is optimized for the simultaneous acquisition of changes in [Na+]i in multiple murine atrial myocytes, but is adaptable to many other cell types.
Here we introduce a novel EMCCD camera-based technique for the simultaneous quantitative measurement of [Na+]i in multiple viable atrial myocytes using sodium-binding benzofuran isophthalate (SBFI). The approach described here is the first to allow for the simultaneous measurement of [Na+]i in multiple cells. The main advantages this new protocol presenta are (i) the significant increase in experimental yield and (ii) the reduction in illumination intensity and duration due to …
The authors have nothing to disclose.
This work was supported by a Scientist Development Grant from the American Heart Association (14SDG20110054) to MG; the NIH Interdisciplinary Training Grant in Muscle Biology (T32 AR007592) and the NIH Cardiovascular Disease Training Grant (2T32HL007698-22A1) to LG; a Scientist Development Grant from the American Heart Association (15SDG22100002) to LB and by NIH grants R01 HL106056, R01 HL105239 and U01 HL116321 to WJL.
2,3-Butanedione monoxime (BDM) | Sigma-Aldrich | B0753 | |
340 Excitation Filter | Chroma | ET40X | 25 mm |
380 Excitation Filter | Chroma | ET80X | 25 mm |
510 Emission Filter | Chroma | ET510/80m | 25 mm |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A7906 | |
Bubble trap | BD Medical Technologies | 904477 | Custom made from a 5 ml Luer Lok Syringe, which is located in the tubing path from the perfusing solution to the cannula |
CaCl2 solution | Sigma-Aldrich | 21115 | |
Cannula | BD Medical Technologies | 305167 | Custom made from a 22 G x 1 1/2 inch needle. Cut to 1 inch and sand 1mm distal tip. |
Cell Chamber | Custom machined with an opening that can securely hold a 25 mm glass cover slip and with a cover that has an inlet and an outlet port for perfusion. | ||
Circulating Water Bath | VWR | ||
Collagenase II | Worthington | LS004176 | Specific activity 290 U/g |
Creatinine | Sigma-Aldrich | C0780 | |
DG5-plus illuminator | Sutter Instrument | Lambda DG-4/DG-5 Plus | |
DMSO | Thermo Fischer | BP231 | |
EGTA | Sigma-Aldrich | E4378 | |
EMCCD camera | Princeton Instruments | ProEM-HS | |
Fine Hemostats | Fine Science Tools | 130-20 | |
Fine Scissors | Fine Science Tools | 14060-10 | |
Forceps Supergrip | Fine Science Tools | 00632-11 | |
Glass Cover slips | VWR | 4838089 | 25 mm circle |
Glucose | Sigma-Aldrich | G7528 | |
Gramicidin D | Sigma-Aldrich | G5002 | |
HEPES | Sigma-Aldrich | H3375 | |
Inner silicon Tubing | VWR | VWRselect brand silicon tubing | |
Inverted microscope | Nikon Instruments | NikonTE 2000 U | |
Isolation Tools | |||
K Gluconate | Sigma-Aldrich | P1847 | |
KCI | Sigma-Aldrich | P5405 | |
KH2PO4 | Calbiochem | 529568 | |
Langendorff perfusion apparatus | |||
MgCl2.6H2O * | Sigma-Aldrich | M0250 | |
MyoPacer Cell Stimulator | IonOptix | ||
Na Gluconate | Sigma-Aldrich | S2054 | |
NaCl | Sigma-Aldrich | S9888 | |
NaH2PO4 | Sigma-Aldrich | S9390 | |
Natural Mouse Laminin | Thermo Fischer | 23017015 | 0.5-2.0 mg/ml |
Outer tubing | VWR | ||
Petri dish 35X10 mm | Falcon | 351008 | |
PowerLoad | Thermo Fischer | P10020 | |
Protease XXIV | Sigma-Aldrich | P8038 | |
SBFI-AM | Thermo Fischer | S1264 | |
Silk suture | Fine Science Tools | 18020-50 | 0.12 mm diameter |
Small Spring scissors | Fine Science Tools | 15000-03 | |
Standard Pattern Forceps | Fine Science Tools | 11000-12 | |
Strophanthidin | Sigma-Aldrich | G5884 | |
Surgical Scissors Tough Cut | Fine Science Tools | 14054-13 | |
Suture Tying Forceps | Fine Science Tools | 00272-13 | |
Taurine | Sigma-Aldrich | T0625 | |
Trisbase | Sigma-Aldrich | TRIS-RO | |
Trypsin | Sigma-Aldrich | T0303 | |
UVFS Reflective 0.1 ND Filter | Thorlabs | NDUV01B | 25 mm |
UVFS Reflective 0.2 ND Filter | Thorlabs | NDUV02B | 25 mm |
UVFS Reflective 0.3 ND Filter | Thorlabs | NDUV03B | 25 mm |
UVFS Reflective 0.5 ND Filter | Thorlabs | NDUV05B | 25 mm |
UVFS Reflective 1 ND Filter | Thorlabs | NDUV010B | 25 mm |
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