Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Podsumowanie

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Streszczenie

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Wprowadzenie

Pacemaker myocytes in the sinoatrial node of the heart (sinoatrial myocytes, "SAMs") generate spontaneous, rhythmic action potentials (APs) that propagate through the myocardium to initiate each heartbeat. Experiments using acutely isolated SAMs from many species have been essential for elucidation of mechanisms that contribute to the generation of pacemaker activity. SAMs are highly specialized cardiomyocytes that differ substantially from their counterparts in the atrial and ventricular myocardium in terms of morphology, function, and protein expression. The hallmark of spontaneous APs in SAMs is a spontaneous depolarization during diastole that drives the membrane potential to threshold to trigger the next AP^1,2. This "pacemaker potential" depends on the coordinated activity of many different membrane currents including the "funny current" (I_f), T- and L-type calcium currents, and the sodium-calcium exchanger current (I_NCX), which is driven by Ca²⁺ release from the sarcoplasmic reticulum^3,4.

While acutely isolated mouse SAMs are an essential experimental preparation for the study of pacemaking, the isolation of SAMs from mice can be a challenging method to adopt because the indistinct anatomy and small size of the mouse SAN requires a nuanced microdissection and the combined enzymatic and mechanical dissociation of the cells requires careful optimization.

We provide here a detailed video demonstration of a protocol that has been successfully used to isolate SAMs from adult mice for patch clamp recordings^5-8. To our knowledge, there is no such visual demonstration available from any other source. In addition, a new method is demonstrated in which isolated SAMs from adult mice can be maintained in vitro for several days, thereby permitting the introduction of proteins, genetically encoded reporter molecules or RNAi via adenoviral infection⁹.

Protokół

All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus. The standard protocol below has been optimized using male C57BL/6J mice of 2-3 months of age.

1. Prepare Solution Stocks and Supplies in Advance of Experiments

NOTE: Refer to Materials Table for necessary equipment and supplies.

1. Prepare 1 L each of the following solutions as indicated in Table 1: Complete Tyrode's, Low Ca²⁺/Mg²⁺ Tyrode's, Modified Kraft-Brühe (KB) Solution, and Bovine Serum Albumin (BSA) Solution. Use ultrapure filtered deionized water for all solutions. Divide each solution into 50 ml aliquots and store at 20 °C for up to six months. Thaw individual aliquots immediately before experiments, and store for up to one week at 4 °C.
2. Prepare 50 ml of 10 mM NaCl and 1.8 mM CaCl₂ Adaptation Solution by dissolving NaCl and CaCl₂ in ultrapure filtered deionized water (Table 1). Store at room temperature for up to six months.
3. Prepare 4.75 enzyme activity unit (U) aliquots of elastase by pipetting into microfuge tubes. Store at 4 °C for up to three months.
4. Prepare 375 μg aliquots (in ultrapure H₂O) of collagenase-protease enzyme blend by pipetting into microfuge tubes. Store at -20 °C for up to three months.
5. Prepare two fire-polished Pasteur pipettes, one for tissue transfer (~1.5 mm final opening diameter; Figure 1Aiv and Figure 1C) and one for dissociation (~2 mm diameter; Figure 1Aiii and Figure 1C).
   1. Score Pasteur pipettes with a glass cutter and snap along the score to produce an opening slightly larger than the desired size. Fire-polish the cut end of each pipette for ~30-60 sec over a low flame on a Bunsen burner to produce a thick polished wall and an opening of the desired diameter. Ensure the fire-polished opening is free of any cracks or rough edges.
6. Prepare two dissection dishes by adding ~25 ml silicone elastomer mixed according to manufacturer's directions to each 100 mm Petri dish (Figure 1Ai and Figure 1B). Allow to cure at room temperature for 48 hr.
7. For culture only: prepare 25-50 ml each Plating Medium and Culture Medium as per Table 2. Store for up to two weeks at 4 °C.

2. Prepare Solutions to Be Used on Day of Cell Isolation

NOTE: The following amounts are for isolation of sinoatrial myocytes from one mouse.

1. Add 2.5 ml of Low Ca²⁺/Mg²⁺ Tyrode's (pH 6.9) to each of three small, round-bottomed culture tubes. Place tubes in 35 ± 1 °C water bath.
2. Add 2.5 ml of Low Ca²⁺/Mg²⁺ Tyrode's to one large round-bottomed culture tube. To this tube, add 1 aliquot (4.75 U) elastase and one aliquot (375 μg) collagenase-protease enzyme blend. Swirl to mix. Place tube in 35 ± 1 °C water bath.
3. Add 2.5 ml of KB Medium to three additional small, round-bottomed culture tubes and one additional large, round-bottomed culture tube. Place tubes in 35 ± 1 °C water bath.
4. Add ~7 ml of BSA solution to one large bottomed culture tube. Keep this tube at room temperature.
5. Place 20-40 ml of Complete Tyrode's solution in a 50 ml beaker (Figure 1Aii). Add 10 USP/ml heparin, swirl to mix, and place the beaker in the 35 ± 1 °C water bath.

3. Prepare Additional Solutions and Materials for Cultured Cells (Skip These Steps for Acutely Isolated Cells)

1. In a sterile tissue culture hood, place two 12 mm round glass coverslips per mouse into individual wells of a 24-well plate.
   NOTE: The 24 well plate is used because the wells are a convenient size, which serves to limit the volume for subsequent viral infections.
2. Pipette approximately 200 µl of a 100 ng/ml solution of mouse laminin diluted in sterile phosphate buffered saline (PBS) onto each coverslip.
3. Incubate coverslips with laminin for the duration of the isolation (at least 1 hr) in an incubator at 37 °C.
4. Pre-warm the Plating Medium and Culture Medium (from Step 1.7 and Table 3) to 37 °C.

4. Sinoatrial Node Isolation

1. In a chemical fume hood, place a mouse in one chamber of a two-chamber box and anesthetize with ~200 μl liquid isoflurane introduced via a cotton swab into the other chamber. Confirm anesthesia (usually within ~30-60 sec) with a toe pinch. Euthanize mouse by cervical dislocation.
   NOTE: An empty 1 ml pipette tip box can be used to form the two-chamber box; turn the rack upside down in the box to create the separate compartment for the isoflurane to prevent the mouse from contacting it directly.
2. Remove fur from chest with scissors and transect rib cage to expose the chest cavity using external tissue forceps (Figure 1Aviii) and dissection scissors (Figure 1Avix). Bathe the chest cavity with ~2 ml warmed Complete Tyrode's with Heparin using a transfer pipette.
   NOTE: Continue bathing chest cavity when necessary, do not allow the preparation to dry out.
3. Under a dissecting microscope, carefully remove the lungs and thymus using internal scissors (Figure 1Avi) and dissection forceps (Figure 1Avii).
4. While gently holding the apex of the heart with the internal dissection forceps, carefully cut the inferior vena cava and the aorta with the internal scissors to remove the heart from the chest cavity. Transfer the heart to one of the silicone dissection dishes and bathe with ~4 ml warmed heparinized Complete Tyrode's using the transfer pipette.
5. Orient the heart such that the posterior vessels are visible and facing up, with the animal's right atrium on the experimenter's right and left atrium on the experimenter's left. Once oriented, immobilize the heart by pinning through the apex into the silicone dissection dish.
6. Locate the groove between the ventricles and the atria (clear ring above the ventricles).
7. Using the internal dissection scissors, make an incision at the groove, keeping closer to the ventricles than the atria. Flush the groove and incision with additional warmed heparinized Complete Tyrode's as needed to allow a clear view of the atria and the valves. Continue to cut along the groove to separate the atria from the ventricles.
8. Transfer the atrial tissue to the second silicone dissection dish and bathe with ~3 ml warmed heparinized Complete Tyrode's.
9. Orient the tissue so that the animal's right atrium is now on the experimenter's left, and the left atrium is on the right.
   NOTE: The right atrium is more transparent, while the left atrium has more of a dark red tone.
10. Pin the tissue through the inferior and superior vena cavae and the right and left atrial appendages, stretching the preparation gently. Remove any remaining fat or other tissue to allow a clear view of the preparation (be careful to not cut into the atrial wall, as the sinoatrial node is quite delicate and can be easily damaged).
11. Open the anterior wall of the atria by cutting through the venae cavae. Re-position the pins as necessary to visualize the interatrial septum.
12. Cut along the interatrial septum to remove the left atrium. Re-pin the preparation, stretching gently.
13. Remove the right atrial appendage and free the sinoatrial node by cutting along the cristae terminalis, which appears as a dark orange streak bordering the atrial appendage.
14. Re-pin the nodal tissue and cut it laterally (perpendicular to the crista terminalis) to produce three equally sized strips.

5. Sinoatrial Node Digestion

1. Using the narrow fire-polished pipette (Figure 1Aiv), transfer the three strips of sinoatrial node tissue into the first of three small, round-bottomed tube containing 2.5 ml of low Ca²⁺/Mg²⁺ Tyrode's in the 35 ± 1 °C water bath. Incubate for 5 min.
2. Transfer tissue strips to the second small, round bottom tube containing 2.5 ml low Ca²⁺/Mg²⁺ Tyrode's in the 35 ± 1 °C water bath, using the same narrow pipette. Wash the tissue strips by gentle swirling the tube or by gently pipetting with the narrow pipette. Do not invert the tube.
3. Transfer tissue strips to the third small, round bottom tube containing 2.5 ml low Ca²⁺/Mg²⁺ Tyrode's, and repeat the washing step described in step 5.2.
4. Transfer strips into the large round-bottomed tube containing 2.5 ml of low Ca²⁺/Mg²⁺ Tyrode's with enzymes (elastase plus collagenase-protease blend) in the 35 °C water bath. Ensure that all three tissue strips are present. Incubate for 10-15 min at 35 ± 1 °C. Mix every 5 min by gently swirling the tube. Do not invert the tube.

6. Sinoatrial Node Myocyte Dissociation

1. Following the enzyme digestion, use the narrow fire-polished pipette to gently transfer the tissue strips to the first small, round-bottomed tube containing 2.5 ml KB solution at 35 ± 1 °C. Wash tissue by gently swirling the tube.
   Note: Tissue strips will appear somewhat translucent and may clump together at this point. Handle very gently after enzymatic digestion to avoid losing cells.
2. Transfer the tissue to the second small round-bottomed tube containing 2.5 ml KB at 35 ± 1 °C. Swirl gently to wash.
3. Transfer the tissue to the third small round-bottomed tube containing 2.5 ml KB at 35 ± 1 °C. Swirl gently to wash.
4. Transfer the tissue to the large, round-bottomed tube containing 2.5 ml KB at 35 ± 1 °C.
5. Using the larger fire-polished pipette (Figure 1Aiii and Figure 1C), dissociate the cells in the large round-bottomed tube by constant trituration at approximately 0.5-1 Hz for 5-10 min, taking care to keep the dissociation tube submerged in the 35 ± 1 °C water bath and to avoid introducing bubbles into the solution.
   Note: Trituration time varies with diameter of the dissociation pipette and force of pipetting. Time should be adjusted so that the tissue pieces remaining at the end of the dissociation appear thin, transparent and wispy. If tissue retains any color, the dissociation is likely to be incomplete. Frequency (0.5-1 Hz) is determined by hand.
6. Remove round-bottomed tube containing dissociated SAMs from the water bath and equilibrate at room temperature for 5 min.

7. Sinoatrial Node Calcium Re-adaptation (Performed at Room Temperature)

NOTE: For SAMs destined for culture experiments, the procedures in the following section should be performed in a sterile tissue culture hood. If SAMs are to be used for acute experiments, there is no need to perform these steps in a sterile environment.

1. Add 75 µl of NaCl/CaCl₂ adaptation solution (Table 2). Swirl gently to mix and incubate for 5 min.
2. Add 160 µl of NaCl/CaCl₂ adaptation solution. Swirl gently to mix and incubate for 5 min.
3. Add 390 µl of BSA solution (Table 2). Swirl gently to mix and incubate for 4 min.
4. Add 1.25 ml of BSA solution. Swirl gently to mix and incubate for 4 min.
5. Add 4.37 ml of BSA solution. Swirl gently to mix and incubate for 4 min.
   Note: The final concentration of calcium will be 1.8 mM.
6. Following calcium re-adaptation, collect SAMs by allowing to settle by gravity for ~10 min or by centrifugation at ~2,000 x g for 3 min.
   1. For acutely isolated cells, gently remove and discard about 5 ml of the supernatant using a glass Pasteur pipette, leaving cells in a volume of ~2 ml. Store these cells at room temperature for up to ~8 hr for patch clamp recordings.
   2. For cultured cells, remove as much of the supernatant as possible using a sterile glass Pasteur pipette. Resuspend cell pellet in 1 ml pre-warmed (37 °C) Plating Medium (Table 2).

8. Plating and Culture of Sinoatrial Myocytes (Skip for Acutely Isolated Cells)

1. Remove laminin solution from coverslips from Step 3.3 with a Pasteur pipette. Immediately seed 500 µl (~50-100 cells) onto each laminin-coated coverslip (from step 3).
   NOTE: The contractile inhibitor 2,3-butanedione monoxime (BDM) is included in the plating and culture media to prevent contraction, which causes attrition of cells⁹.
2. Return the 24-well plate containing newly seeded SAMs to the incubator and maintain at 37 °C in an atmosphere of 95% air/5% CO₂. Allow cells to adhere to coverslips for 4-6 hr in plating media (Table 2).
3. Gently remove Plating Medium using a sterile Pasteur pipette. Replace with 500 µl per well of pre-warmed (37 °C) Culture Medium (Table 2).

9. Adenoviral Transduction of Adult Sinoatrial Myocyte Cultures (Skip for Acutely Isolated Cells)

1. Estimate number of cells per coverslip immediately before applying adenovirus by counting the cells in a field of view under a microscope, adjusting for magnification. Count all cells, not just SAMs.
2. Dilute adenovirus in Medium 199 and adjust the dilution for the viral titer so that application of 1-10 μl is required to achieve a final multiplicity of infection (MOI) of 100 viral units per cell. Add adenoviral solution in a dropwise fashion directly onto plated SAMs.
3. Incubate cells with the virus-containing medium overnight (~12-14 hr). Then exchange with fresh culture medium. Maintain cells in incubator, changing culture medium every 48 hr, until desired protein expression is obtained.

10. Functional Evaluation of Acutely Isolated or Cultured SAMs

NOTE: The following protocol is an example of functional assessments of isolated SAMs using the amphotericin perforated-patch technique to record both spontaneous APs and I_f from the same cell (see reference⁹).

1. Prepare recording solutions as described in Table 3.
2. Prepare a stock solution of 20 mg/ml amphotericin-B in DMSO fresh on the day of recording. Keep stock at room temperature and protect from light. Dilute amphotericin-B stock in intracellular solution to a final concentration of 200 ug/ml just before use. Maintain the final pipette solution on ice and protect from light.
   NOTE: The amphotericin-containing pipette solution for experiments should be prepared fresh hourly by diluting an aliquot of the stock solution into the intracellular solution and vortexing for at least 1 min.
3. Transfer an aliquot of the acutely dissociated SAM cell suspension or a fragment of glass coverslip bearing cultured SAMs to a recording chamber containing Tyrode's solution at 35 ± 1 °C. Perfuse cells with Tyrode's solution for at least 2 min prior to electrophysiological recordings to remove any residual BDM remaining from culture medium.
   NOTE: Spontaneous contractions should be evident immediately upon transfer to the Tyrode's solution. SAMs for recording can be identified by a combination of features including spontaneous contractile activity, characteristic morphology (e.g., Figure 2), lack of striations, expression of HCN4 protein, presence of I_f current, membrane capacitance <50 pF, and spontaneous APs with waveforms that include a diastolic depolarization phase and a slow upstroke.
4. Using borosilicate glass pipettes with resistances of 1.5-3.0 MΩ, fill the tip with intracellular solution lacking amphotericin by dipping for 10-30 sec. Then back-fill the pipette with the amphotericin-containing solution. A GΩ cell-attached seal should be obtained as quickly as possible. If seal formation is difficult, increase the tip-fill time.
   NOTE: The access resistance should be continuously monitored following formation of the cell-attached seal, and recordings should only be commenced after obtaining a stable access resistance of <10 MΩ.
5. To record spontaneous APs, switch the amplifier to fast current-clamp mode with no current injection.
   NOTE: 1 nM isoproterenol is included in the extracellular Tyrode's solution when recording APs to stabilize the firing rate, as previously reported¹⁰.

Wyniki

The protocols described here have been previously employed to isolate spontaneously active SAMs from adult mice that are suitable for a variety of different patch clamp studies^5-8. In addition, the protocols allow for isolated SAMs that can be maintained in culture for up to one week. Gene transfer into the cultured cells can be accomplished via adenoviral infection⁹. The results presented in this section derive from our previous work and are shown here as examples o...

Dyskusje

This paper presents detailed protocols for the isolation and culture of fully differentiated sinoatrial node myocytes from adult mice. The isolation protocol reliably produces spontaneously active mouse SAMs suitable for either immediate electrophysiological analysis or subsequent culture. Similar protocols have been reported by many other groups (for example, see references^{11,12,10,13-17}). However, our protocol for maintaining adult mouse SAMs in vitro preserves the characteristic morphology, spontan...

Materiały

Name	Company	Catalog Number	Comments
Sylgruard/Elastomer Kit	Dow Corning	184 SIL ELAST KIT 0.5KG
Borosilicate 9" pasteur pipettes	Fisher Scientific	13-678-20C
Small, round bottomed culture tubes	Fisher Scientific	352059
Large, round bottomed culture tubes	Corning	14-959-11B
Elastase	Worthington Biochemical	LS002279
Liberase TM	Roche	5401119001	Tissue dissociation solution
Heparin	SAGENT Pharmaceuticals	NDC 25021-400-10
Mouse Laminin	Corning	CB-354232
12 mm round glass coverslips	Fisher	12-545-80
24-well culture plate	Fisher	08-772-1
Ad-mCherry	Vector Biolabs	1767
Ad-eGFP	Vector Biolabs	1060
Plastic, disposable transfer pipette	Fisher Scientific
Micro scissors	Fisher Scientific	17-467-496
Dumont #4 Forceps	Roboz Instruments	RS-4904
Tissue Forceps	Roboz Instruments	RS-8164
Dissecting Iris Scissors	WPI, Inc.	501264
Dissecting Pins	Fine Science Tools	26002-20
NaCl	Sigma	71376
KCl	Sigma	60128
KH2PO4	Sigma	60353
HEPES	Sigma	54457
glucose	Sigma	G0350500
MgCl2	Sigma	M8266
CaCl2	Sigma	C1016
taurine	Sigma	T0625
BSA	Sigma	A2153
K-glutamate	Sigma	G1501
K-aspartate	Sigma	A6558
MgSO4	Sigma	M7506
creatine	Sigma	C0780
EGTA	Sigma	E3889
Mg-ATP	Sigma	A9187
Amphotericin-B	Fisher Scientific	1397-89-3
Isoproterenol	Calbiochem	420355
Media199	Sigma	M4530
2,3-butanedione monoxime (BDM)	Sigma	B0753
Fetal Bovine Serum (FBS)	Sigma	SH30071
Bovine Serum Albumin (BSA)	Sigma	A5611
Insulin	Sigma	I3146
Transferrin	Sigma	I3146
Selenium	Sigma	I3146
Penicillin	GE Healthcare	SV30010
Streptomycin	Hyclone	SV30010