Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Summary

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Abstract

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca²⁺ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca²⁺ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Protocol

1. Animal Anesthesia and Organ Harvesting

1. Anaesthetize the mouse by intraperitoneal (i.p.) injection of Ketamine (200 mg/kg BW) and Xylazine (20 mg/kg BW).
2. To anticoagulate inject 250 IU Heparin i.p. to avoid blood clotting and thrombus formation.
3. Wait until deep narcosis is reached, which is characterized by areflexia. To check for areflexia, test corneal reflex by gently touching the cornea or test flight reflex by tail pinching.
4. Transfer the mouse onto operating table and fix it in supine position.
5. Incise the skin and the abdominal wall below the xiphoid and perform clamshell thoracotomy: Extend the cut to both sides along the costal arch and subsequently cut ribs in the medial axillary line, deflect the rib cage upwards.
6. Open pericardium, locate great vessels. Gently press heart caudal to better display the aorta. Clamp the aorta using forceps.
7. Place the heart in concavity of a pair of scissors and dissect all connecting vessels with one single cut. Make sure to preserve a large enough part of the ascending aorta for Langendorff cannulation.
8. Transfer excised heart immediately to a Petri dish filled with ice cold and pre-oxygenized solution 1 (for solutions, see Table 1).

2. Preparation of Heart and Langendorff Perfusion

1. Cannulate the aorta with a 1.8F steel cannula. Make sure to avoid air embolism.
2. Fixate aorta on the cannula with a surgical suture and flush coronaries with 1 ml of solution 1.
3. Connect cannula with a Langendorff apparatus.
4. Make sure time from thoracotomy to Langendorff cannulation does not exceed 120 sec to avoid extended ischemia/reperfusion injury to the myocardium.
5. Perfuse heart with 10 ml of Ca²⁺ free solution 2 (4 ml/min).
6. Perfuse heart with collagenase solution 3 for 8 min (4 ml/min).

3. Microsurgical Dissociation of Cardiac Chambers

1. Transfer heart into a pre-warmed 100-mm Petri dish containing low Ca²⁺ solution 4.
2. Carefully remove aortic and other non-cardiac tissue with scissors and discard it.
3. Separate atria and ventricles and continue with each chamber separately. Keep cells immersed in solution 4. Use small volumes (less than 5 ml).

4. Further Dissociation of Cardiomyocytes

1. Atrial cardiomyocytes:
   1. To individualize atrial cardiomyocytes transfer the atria into a separate pre-warmed 100-mm culture dish and dissociate the tissue through gently pulling it apart with fine forceps. Ensure an almost complete dissociation of the tissue.
   2. Use a 1 ml pipette with an enlarged fire-polished plastic pipette tip to suspend the cells in 1 ml of solution 5 for 5 min.
   3. Separate the cells from debris by using a cell filter (200 μm mesh size).
   4. Add 5 ml solution 5 to the cell suspension and centrifuge for 2 min at 16 x g at room temperature.
   5. The following steps are operated under a cell culture hood. Discard the supernatant and re-suspended the pellet in 5 ml of solution 6.
   6. After sedimentation by gravity for 10 min in a 15 ml tube, centrifuge for 1 min at 16 x g at room temperature. Remove the supernatant. Re-suspend the cells depending on their quantity in 1-5 ml of solution 6.
2. Ventricular cardiomyocytes:
   1. Dissect the left ventricular region of interest with fine forceps in 5 ml of solution 4.
   2. Suspend cells by gently pipetting until most of the cells are separated. Transfer the cell solution after filtering (200 μm mesh size) into a 50 ml tube, add a volume of 25 ml.
   3. Centrifuge for 2 min at 16 x g at room temperature.
   4. The following steps are operated under a cell culture hood. Remove supernatant, re-suspend the pellet in 25 ml of solution 6 and allow sedimentation of the cells for 10 min.
   5. Count the cells and remove the supernatant, add 25 - 50 ml solution 6 on the cells.

5. Preparation of Cardiomyocytes for Cellular Electrophysiology, Biochemical or IF Studies

1. For electrophysiology studies, keep the myocytes in solution 6 in a 50 ml tube and inhibit sedimentation.
2. For biochemical studies, e.g. calcium imaging, plate myocytes on laminin coated cell culture dish (final concentration 20 μg/ml laminin in PBS).
3. For immunofluorescence staining prepare a cell culture dish plate with glass cover slips and coated with laminin solution (final concentration 50 μg/ml laminin in PBS).
   1. Remove the solution before plating myocytes. Plate the cells and control the cell density by using a microscope.
   2. Let the myocytes adhere to cover slip for 1 hr at 37 °C in 2 % CO₂, remove the solution and start immediately with a standard staining procedure protocol.
   3. Incubate with fixative, e.g. 4% PFA in PBS (pH 7.5) for 10 min at room temperature and follow with three PBS washing steps for 5 min each.
   4. To permeabilize the cells and to inhibit unspecific antibody binding incubate the myocytes with 10 % serum, 0.3% Triton, 0.2% BSA in PBS for 30 min at room temperature.
   5. Incubate with the primary antibody for 1 hr at 37 °C and wash as described before.
   6. Incubate with the secondary antibody for 1 hr at room temperature. To counterstain the nuclei and α-actinin use DAPI and fluorochrome conjugated phalloidin (Alexa Fluor 488, Invitrogen).
   7. After washing, transfer the glass cover slips carefully on silane treated microscope slides and embed cells in fluorescence mounting medium.

6. Cellular Electrophysiology

1. Perform Whole-cell patch-clamp recordings on freshly isolated atrial and ventricular cardiomyocytes at room temperature.
2. Transfer healthy appearing cardiomyocytes into perfusion chamber filled with a defined volume of extracellular bath solution. 117 mM NaCl, 4 mM KCl, 1 mM KH₂PO₄, 4 mM NaHCO₃, 1.7 mM MgCl₂, 3 mM CoCl₂, 10 mM HEPES, 10 mM glucose, and 0.02 mM Tetrodotoxin (TTX), (pH 7.4). Use NaOH for pH value adjustment.
3. Use proper patch clamp equipment (i.e. IX71 inverted microscope, a Multiclamp 700B Amplifier, a Digidata 1440A acquisition system and PC with pClamp10.3 software (Molecular Devices)).
4. Use patch pipettes with resistances of 3-5 MΩ when filled with intracellular solution containing 130 mM KCl, 2 mM MgCl₂, 11 mM HEPES, 11 mM EGTA, 5 mM Na₂ATP, 0.4 mM Na₂GTP, 5 mM Na₂CP and 4.9 mM CaCl₂ (pH 7.2). Use KOH for pH value adjustment.
5. Evoke outward K+ currents during 4.5-sec voltage steps to test potentials between -60 and +50 mV in 10-mV increments from a holding potential of -70 mV after a 20-msec prepulse to -40 mV.
6. Make sure leak currents are always <100 pA.
7. For dissection of different K+ currents use specific inhibitors such as 4-aminopyridine (4-AP; ICN Biomedicals, Irvine, CA, USA), Heteropodatoxin 2 (HpTx2; Alomone) or Tetraethylammonium (TEA; Sigma). Stock solutions should be prepared in extracellular bath solution, and applied directly to the closest possible vicinity of the cell via a microtip after "baseline" recordings. Equilibration should be allowed for 2-3 min before "drug" recordings.
8. For analysis normalize current amplitudes in individual cells to cell size (whole-cell membrane capacitance). Analyze data offline by using pClamp10.3 software (Molecular Devices) or comparable software.

Results

Isolation of adult murine cardiomyocytes from genetically engineered mice to study the function of specific genes of interest in vitro has become a powerful tool to further understand cardiac pathophysiology. This method is currently used by only a small but increasing number of basic science laboratories worldwide. However, isolation of adult ventricular murine cardiomyocytes can be tricky and needs to be done thoroughly and repetitively by experienced hands. Figure 1 shows freshly isolated exe...

Discussion

With the growing development of genetically engineered mouse strains to study cardiac function and cardiac pathology related to gene deletion there is also an increasing interest in specialized methods to study effects of the specific gene deletion in vitro. In our laboratory we study the roles of a family of Kv channel ancillary subunits on cardiac repolarization. The KCNE genes comprise a family of 5 genes (KCNE1-5) that play important roles in human ventricular and atrial repolarization

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tetrodotoxin	Alomone
4-aminopyridine	ICN Biomedicals
Heteropodatoxin 2	Alomone
Tetraethylammonium	Sigma Chemicals
Collagenase Type 2	Worthington