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

Zusammenfassung

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.

Zusammenfassung

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.

Einleitung

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 characterized^2,3, 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 (I_NCX) and protein expression levels in AF³. An increase in the late component of the voltage-dependent Na⁺ current (I_{Na, 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 indicators^7,8, 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 (F_340/380) 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 F_340/380 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 epifluorescence^1,11. 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.

Protokoll

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland, Baltimore.

1. Isolation of atrial myocytes from adult murine hearts

1. Place each mouse in a precision vaporizer and induction chamber gassed with isoflurane in 100% oxygen.
2. Set the isoflurane flow to 1% until the animal is unresponsive before giving an intraperitoneal (IP) heparin injection (1–1.25 U/g) 15 min before euthanasia.
3. Deeply anesthetize the mouse by increasing the isoflurane anesthesia to 5% and confirm the deep plane of anesthesia by foot pinch.
4. Perform the euthanasia by performing a quick thoracotomy¹²; use standard pattern forceps and surgical scissors to open the thorax. Passively mobilize heart and lung, isolate the aortic arch, hold with forceps and cut the aorta to remove the heart.
5. Place the heart in ice-cold nominally Ca²⁺-free cell isolation buffer (CIB, Table 1).
6. Cannulate the aorta with a 22 G cannula and tie using a silk suture under a light microscope with 3x magnification in CIB solution (Figure 1A,B).
7. Confirm that the cannula is well above the aortic sinuses by delivering CIB solution through the cannula connected to a syringe and confirm coronary artery perfusion under the microscope.
   NOTE: This step is important to ensure proper perfusion of the atrial tissue because it has been shown that the coronary artery anatomy is highly variable in mice¹³.
8. Mount the heart on a gravity-based Langendorff set up and perfuse with the Ca²⁺-free CIB solution for 5 min at 37 °C to wash out the remaining blood until the eluate is clear (Figure 1C).
9. Switch to enzymatic solution (Table 2) and perfuse for 3–5 min at 37 °C until the atrial tissue is soft and flaccid.
10. Excise the right and left atrium using super-grip forceps and small spring scissors and transfer to a small culture dish containing CIB enzymatic solution used in step 1.8 but with 0.15 mM CaCl₂ and place in an incubator at 37 °C for 5–8 min (Figure 1D).
11. Transfer the atria into a cell culture dish containing 4 mL prewarmed (37 °C) storage solution (modified Tyrode’s solution (Table 3) containing 15 mM bovine serum albumin and 30 mM 2,3-butanedione monoxime).
12. Cut the atria into small tissue strips (10–20, depending on atrial size) using small spring scissors and super-grip forceps.
13. For mechanical dissociation gently aspirate the tissue suspension using different fire-polished glass Pasteur pipettes with openings between 2–5 mm. Begin with the largest pipette tip and move to the smallest pipette tip, aspirating 5–10 times per pipette.
14. Strain the cell suspension through a 200 µm filter and add CaCl₂ three times every 10 min to achieve a final concentration of 0.3 mM.

2. Evaluation of cell quality

1. Place 200 µL of the cell suspension containing the freshly isolated atrial myocytes on a glass coverslip.
2. Use a 10x objective to count all cells in the field of view and categorize them as rounded or rod-shaped. Determine how many of the rod-shaped cells show clear cross striation (switch to 25x objective if this is difficult to determine, see Figure 2C).
3. Repeat steps 2.1–2.2.
4. Calculate the percentages of rounded, rod-shaped and rod-shaped with clear cross striation cells.
5. Modify the cell isolation procedure until 80% of cells are rod-shaped with clear cross-striation.
6. Place 500 µL of the atrial cell suspension on a laminin-coated glass coverslip and place the cell chamber on the inverted microscope. Let the cells settle for 5 min. Perfuse with Tyrode’s solution containing 1.8 mM Ca²⁺.
7. Use a cell stimulator system to start electrical field stimulation (2 ms bipolar pulse, 30 V) at 0.5 Hz and count the number of cells that contract in the field of view of a 10x objective.
8. Repeat with external field stimulation at 3 Hz.
9. Calculate the percentage of cells responding to 0.5 and 3 Hz stimulation rates (see Figure 2B). Refine cell isolation protocol until 50% of cells respond to 3 Hz field stimulation.

3. Na⁺ indicator loading of freshly isolated murine atrial myocytes

1. Use the cell permeant acetoxymethyl (AM) ester of the fluorescent indicator sodium-binding benzofuran isophthalate (SBFI-AM).
2. To facilitate dye dispersion and to achieve homogeneous cell loading dissolve SBFI in dimethyl sulfoxide (DMSO) and suitable surfactant polyols (e.g., pluronic) to achieve a final concentration of 10 μM SBFI in the cell suspension.
3. Load the cells with SBFI for 60 min protected from light on a rocker at room temperature.
4. Let the cells settle for 30 min, remove the supernatant and re-suspend the pellet in storage solution (1–2 mL).
   NOTE: Experiments should be performed within 4 h of cell isolation. BDM is washed-out by perfusion with Tyrode’s solution prior to the start of experiments¹⁴.

4. Instrumentation, [Na⁺]_i measurements and [Na⁺]_i calibration

NOTE: Figure 3 depicts the light path schematic for the experimental instrumentation.

1. Prepare calibration solutions with increasing Na⁺ concentrations as described in Tables 4–6.
2. Add the permeabilizing agent gramicidin D (10 μM; from a stock solution stored at -20 °C) and the Na⁺, K⁺ ATPase inhibitor strophanthidin (100 μM, from a stock solution stored at -20 °C) to each calibration solution. Vortex well, or use a sonicator to ensure that gramicidin and strophantidin are completely dissolved.
3. Fill a multi-barrel perfusion system with the calibration solutions containing the increasing [Na⁺]_o prepared in steps 4.1 and 4.2. Connect to a cell chamber using slow perfusion rates (~1 mL/min; perfusion rates can vary with volume of the cell chamber).
4. Connect suction to the cell chamber and collect perfusate in an appropriate (glass) container on the floor.
5. Stop perfusion and suction.
6. After allowing for 30 min of SBFI de-esterification, place 100 μL of the concentrated (step 3.5), dye-loaded cell suspension on a laminin-coated glass cover slip above the field of view of the inverted microscope’s 40x objective.
7. Use a rapid switching illuminator with a 300 W xenon light source. Achieve wide field imaging with two excitation wavelengths (340 nm and 380 nm) using fast switching scanning mirrors and narrow bandwidth excitation filters (340 nm ± 10 nm; 380 nm ± 10 nm).
8. Optimize the field of view of the EMCCD camera. A larger observation area requires longer frame times. Longer frame times lead to more bleaching of the indicator. Thus, balance the field of view size and frame time so that no noticeable bleaching of the probe occurs.
9. Determine that there is only minimal intrinsic fluorescence in a subset of atrial cells that are not loaded with SBFI by ensuring that the cells’ intrinsic fluorescence is similar to background fluorescence (as shown in Figure 4).
10. Determine the appropriate sampling rate depending on experimental design (sampling rates can be low because changes in [Na⁺]_i are relatively slow (e.g. one data point acquired every 10–20 s).
11. Attenuate the excitation light by using appropriate neutral density (ND) filters and by appropriately reducing the light source intensity (e.g., by altering intensity in the operating software).
12. Collect the emission light at 510 ± 40 nm using appropriate filters with an EMCCD camera connected to the inverted microscope.
13. Define a region of interest (ROI) for each cell and a background ROI (see Figure 4). Subtract the background from the recorded F₃₄₀ and F₃₈₀ signals (either online or during data analysis).
14. Start the data acquisition.
15. Restart perfusion and suction. Perfuse for 10–15 min with Tyrode’s solution containing 1.8 mM Ca²⁺ and record a stable F_340/380 baseline before starting the experiment.
    NOTE: Make sure to record a stable baseline for 10–15 min. If bleaching occurs (e.g., reduction of the F_340/380 baseline) aim to further attenuate illumination intensity either by increasing ND filter density, decreasing excitation light signal intensity and/or acquisition frame time (see steps 4.10–4.11).
16. Start [Na⁺]_i measurement according to research question.

5. [Na⁺]_i Calibration

1. After the conclusion of the experiment (step 4.16) perform a calibration of the F_340/380 signal in each cell in situ.
2. Calibrate the F_340/380 signal by perfusing the SBFI-loaded myocytes with the calibration solutions (prepared in steps 4.1–4.3).
3. Perfuse stepwise to elevate [Na⁺]_o from 0 to 20 mM. Wait for stable F_340/380 signal before moving to the next concentration (~5 min depending on flow rate; see Figure 3B and Figure 5A).
   NOTE: The relation between the F_340/380 signal and the [Na⁺] of the calibration solutions needs to be linear for a valid calibration of the F_340/380 signal (Figure 5 and Figure 6).

Ergebnisse

Evaluation of Atrial Cell Quality
Freshly isolated atrial myocytes were evaluated based on cell morphology and responsiveness to field stimulation as outlined in the protocol in six consecutive atrial cell isolations. Data shown in Figure 2 show a very high percentage of rod-shaped atrial myocytes that retain clear cross striation. Similarly, about 50% of atrial cells respond to high rates of external field stimulation up to 3 Hz.

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Diskussion

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 ...

Materialien

Name	Company	Catalog Number	Comments
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