Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Summary

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Abstract

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 °C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 °C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca²⁺-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Introduction

Cardiac arrhythmias are common and one of the current major healthcare challenges since they affect millions of people worldwide. Arrhythmias are associated with high morbidity and mortality^1,2 and represent the underlying cause for the majority of sudden cardiac deaths³. Up to date treatment options have improved patient survival but are still mainly symptomatic treatments rather than targeting the underlying mechanisms. Thus, these treatments have limited efficacy and may frequently cause severe side effects^4,5,6. An improvement of current treatment options requires insight into the underlying pathophysiology, creating the need for suitable models to study. Small animal models - and specifically mouse models - play a crucial role in arrhythmia research as they allow to study key mechanisms of arrhythmogenesis, for example the genetic impact on cellular electrophysiology, ion channel function or calcium handling^7,8.

For this purpose, isolated atrial and ventricular cardiomyocytes of sufficient quantity and viability are required. A broad spectrum of different isolation approaches to obtain atrial and ventricular myocytes has been previously described^{9,10,11,12,13} and some groups have presented data from simultaneous measurements of L-type current and calcium current induced calcium transients from either atrial¹⁴ or ventricular¹⁵ murine cardiomyocytes. However, to our best knowledge there is no data available of atrial as well as ventricular measurements from one animal. Researchers focus on a broad variety of topics ranging from electrophysiology to proteomics, functional studies as cell contractility or protein interactions, mitochondrial function, or genetics – all in need of isolated cardiomyocytes. Many of the published protocols thus have not been specifically developed for patch clamp studies, leading to limited yields and insufficient cell quality for patch clamp studies. Thus, simultaneous patch clamp and calcium transient measurements of atrial and ventricular cells isolated from one animal cannot be performed with established protocols.

Isolation of murine – especially atrial – myocytes for patch clamp experiments remains challenging. This article provides a simple and fast method for the isolation of high-quality murine atrial and ventricular myocytes via retrograde enzyme based Langendorff perfusion, which subsequently allows simultaneous measurements of both net membrane current and current induced calcium transients from one animal. This article elaborates a protocol for the isolation of atrial and ventricular myocytes derived from wild type mice and mice carrying genetic mutations. This protocol can be used for male and female mice alike. The myocyte isolation, images, and representative results described below were obtained from wild type C57Bl/6 mice at the age of 6 (± 1) months. Nevertheless, this protocol has successfully been used for mice at various ages ranging from 2 to 24 months with different genotypes. Figure 1 shows the isolation setup and a close-up of a cannulated heart during enzyme perfusion.

Protocol

All animal procedures were approved by the Lower Saxony Animal Review Board (LAVES, AZ-18/2900) and were conducted in accordance with all institutional, national, and international guidelines for animal welfare.

1. Prearrangements

1. Prepare 1 L of 10x perfusion buffer (Table 1), 500 mL of 1x perfusion buffer (Table 2), 50 mL of digestion buffer (Table 3), 10 mL of stop buffer (Table 4), 1 L of Tyrode solution (Table 5), 10 mL of each calcium step solution (Tyrode solution with glucose and respective amount of calcium as indicated), 1 L of 4-AP solution (Table 5), 100 mL of pipette solution (Table 6) and 5 mL of pluronic acid according to the provided recipes.
   NOTE: Bath solutions (Tyrode and 4-AP solution) can be prepared (without glucose) in advance and stored at +4 °C, glucose is added on the experimental day. Pipette solution can be stored at -20 °C, calcium indicator is added on the experimental day and solution then stored on ice until further use. 10x perfusion buffer can be stored at room temperature, 1x perfusion buffer, digestion buffer and stopping buffer should be freshly prepared on experimental day.
2. Turn on the water bath and roller pump.
3. Prefill Langendorff apparatus with perfusion buffer; make sure it is air-free.
4. Prepare aortic cannula by fixing it under the dissection microscope, connect with a 1 mL syringe filled with perfusion buffer and clear air by rinsing the cannula.
   NOTE: It is crucial to avoid any air inside the perfusion system, as this will directly affect coronary perfusion and thus digestion effectiveness. A bubble trap might be added to the setup if necessary, to safely avoid any air trapping.
5. Prepare Petri dishes with enough perfusion buffer for organ collection and microscopy (buffer should securely cover the entire organ, a few millilitres – depending on the used Petri dish size – should be enough).
6. Prepare 3 Petri dishes with digestion buffer for tissue dissociation and microscopy, buffer should cover the organ for dissection under the microscope within the respective Petri dish, amount depends on the used Petri dish size. For the dissociation use 3 mL for ventricular tissue and 1.5 mL for atrial tissue.

2. Organ harvest

1. Inject mouse with 0.1 mL of heparin (1,000 U/mL) i.p. using a 1 mL syringe with a 27 G cannula and wait for 5-10 min.
2. Place the mouse into an induction chamber along with a small tissue soaked in approximately 500 µL of isoflurane. The animal should not be in contact with the tissue. To avoid that, one can use a plastic biopsy-embedding cassette to cover the tissue. Once the animal is fully anesthetized, check for toe pinch reflex and as soon as it is not present anymore, quickly euthanize the mouse by cervical dislocation.
3. Place the mouse on a platform on its back (e.g., on Styrofoam covered with a paper towel) and fix the paws down with cannulas to hold it in place.
4. Remove fur and skin covering the chest and part of the abdomen with a clear cut from jugulum towards symphysis and open the abdomen right under the xiphoid without injuring any organ structure using scissors. Lift the sternum with surgical forceps and cut the diaphragm with scissors along the edge of the ribs, then cut the ribs in medial axillary line and remove the rib cage to expose the heart.
5. Carefully remove the pericardium using blunt forceps and quickly remove the heart by lifting it with blunt forceps from below and by cutting the large vessels with one single cut using scissors.
6. Put the heart into room temperature perfusion buffer and cannulate the aorta with a blunt end needle under the microscope as quickly as possible.
   NOTE: Remove any lung tissue and fatty tissue attached to the organ without losing too much time on it. While cannulating, make sure that the end of the needle does not extend through the aortic valve, as this will impair results by preventing buffers from entering the coronary arteries.
7. Tie the heart with a piece of suturing silk firmly to the needle and disconnect from syringe.
   NOTE: The entire procedure from obtaining the heart (the moment when the large vessels are cut) until suturing the aorta to the needle should take as little time as possible. It is recommended not taking longer than 90-180 s from removing the heart until start of perfusion.

3. Enzymatic digestion

1. After aortic cannulation, immediately connect the cannulated heart to the Langendorff apparatus avoiding any air entering the system.
   NOTE: It can help to have a hanging drop of perfusion buffer at the bottom of the Langendorff apparatus as well as a drop of perfusion buffer sitting on the top of the needle in order to avoid any air entering the system.
2. Perfuse the heart with perfusion buffer for 1 min at a temperature of exactly 37 °C and a perfusion rate of exactly 4 mL/min.
   NOTE: In order to have a temperature of 37 °C at the tip of the perfusion needle, water bath temperature has to be set slightly above at approximately 40 °C. This should be tested regularly by measuring the temperature at the perfusion tip.
3. Switch perfusion to digestion buffer and perfuse for exactly 9 min at a temperature of exactly 37 °C and a perfusion rate of exactly 4 mL/min.
4. Transfer the digested heart to a Petri dish with enough digestion buffer to keep it fully covered. Then carefully dissect the atria and ventricles under the microscope.
5. Transfer the atria into a Petri dish with 1.5 mL of digestion buffer and the ventricles into another Petri-dish with 3 mL of digestion buffer.
6. Atrial dissection
   1. Carefully, but without loss of time, pull the atria apart into tiny pieces using blunt forceps.
   2. Dissolve the tissue by carefully pipetting up and down using a 1,000 µL pipette tip, which has previously been cut to widen the tip opening.
   3. Transfer the solution to a 15 mL centrifuge tube and add an equivalent amount of stop buffer (1.5 mL) by carefully pipetting down at the side of the tube to end the reaction.
   4. Carefully pass all 3 mL of cell/tissue-solutions through a 200 µm nylon mesh to remove remaining larger tissue pieces that have not been fully digested.
      NOTE: A successful digestion will leave almost no solid chunks.
7. Ventricular dissection
   1. Quickly chop the ventricular tissue into tiny pieces using dissection scissors and pipette up and down to dissolve. Use another 1,000 µL pipette tip for pipetting up and down, it may be shortened to widen the opening.
   2. Transfer cell/tissue-solution into a 15 mL centrifuge tube and add an equivalent amount of stop buffer (3 mL) by carefully pipetting down at the side of the tube to end the reaction.
   3. Carefully pass all 6 mL of cell/tissue-solution through a 200 µm nylon mesh to remove larger tissue pieces that have not been fully digested.
      NOTE: A successful digestion will leave almost no solid chunks.
8. Leave both tubes (atrial and ventricular cell suspension) on the bench at room temperature for 6 min to settle.
9. Centrifuge both 15 mL tubes at 5 x g for 2 min.

4. Calcium reintroduction

NOTE: The following steps are identical for both atrial and ventricular cells (unless otherwise mentioned) and are performed at room temperature.

1. Discard the supernatant using a plastic Pasteur pipette and carefully resuspend pellet in 10 mL of calcium free Tyrode solution.
2. Leave cells for 8 min for sedimentation.
3. Centrifuge at 5 x g for 1 min (only atrial cells, sedimentation is enough for ventricular cells).
4. Discard supernatant and carefully resuspend pellet in 10 mL of Tyrode solution with 100 µM calcium concentration.
5. Leave cells for 8 min for sedimentation.
6. Centrifuge at 5 x g for 1 min (only atrial cells, sedimentation is enough for ventricular cells).
7. Discard the supernatant and carefully resuspend pellet in 10 mL of Tyrode solution with 400 µM calcium concentration.
8. Leave cells for 8 min for sedimentation.
9. Centrifuge at 5 x g for 1 min (only atrial cells, sedimentation is enough for ventricular cells).
10. Discard the supernatant and carefully resuspend the pellet in 1 mL (atrial)/ 5 mL (ventricular) of Tyrode solution with 1 mM calcium concentration.

5. Loading of myocytes with fluorescent calcium-indicator Fluo-3 AM

NOTE: Due to the light sensitivity of the fluorescent calcium indicator, the following steps should be executed protected from light (e.g., by covering tubes with aluminium foil).

1. Prepare Fluo-3 AM stock solution by adding 44 µL of 20% pluronic F-127 in anhydrous DMSO to 50 µg of Fluo-3AM (can be stored at -20 °C protected from light).
2. Add 10 µL of Fluo-3 AM stock solution to 1 mL of cell suspension and incubate for 10 min at room temperature protected from light.
3. Centrifuge at 5 x g for 1 min.
4. Discard the supernatant using a plastic Pasteur pipette and resuspend the pellet in a reasonable amount of bath solution to obtain a good working concentration (1-5 mL of bath solution depending on the cell density).
5. Leave for 30 min for de-esterification before starting with experiments.

6. Simultaneous patch-clamp and epifluorescent Ca²⁺-transient measurements as previously described¹⁶

NOTE: Patch clamp measurements are not the topic of this article, the interested reader may be referred to major publications providing in depth descriptions of this method^{17,18,19,20,21,22}. Nevertheless, for a better overall understanding, a summary on a protocol to measure L-type calcium currents along with current induced calcium transients is provided.

1. Transfer myocytes into a cell chamber and superfuse with bath solution at 37 °C.
2. Block potassium currents by adding 4-aminopyridine and barium chloride to the bath solution as indicated in Table 5.
3. Ensure that borosilicate microelectrodes have a tip resistance of 2-5 MΩ filled with pipette solution (Table 6).
4. Setup measurements to allow for simultaneous recording of both electrical signals and epifluorescence at the same time. Voltage clamp mode is used to measure L-type Ca²⁺-current with a protocol holding the cell at -80 mV and a 600 ms ramp-pulse to -40 mV to inactivate the fast Na⁺-current, followed by a 100 ms test-pulse to +10 mV at 0.5 Hz (Figure 2).
5. Use excitation at 488 nm, emitted light at <520 nm to detected and convert to [Ca²⁺]_I assuming
   
   Where k_d = dissociation constant of Fluo-3 (864 nM), F = Fluo-3 fluorescence; F_max = Ca²⁺-saturated fluorescence obtained at the end of each experiment¹⁹.

Results

Isolation yield is determined after calcium reintroduction by pipetting 10 µL of cell suspension onto a microscope slide. More than 100 viable, rod-shaped, non-contracting cells/10 µL for atrial cell isolation and more than 1,000 viable, rod-shaped, non-contracting cells/10 µL for ventricular cell isolation are considered as sufficient yield and are commonly obtained using this protocol. Atrial cells obtained with this protocol showed a variety of different cell types containing cells of the cardiac conduc...

Discussion

This article provides an easy and functional way to obtain high quality atrial and ventricular myocytes from the same mouse for patch-clamp studies with simultaneous calcium transient recordings. The quality of the obtained data highly depends on the quality of the cell isolation. As mentioned above, many methods to isolate murine cardiomyocytes have been described previously^9,10,11,12. The iso...

Materials

Name	Company	Catalog Number	Comments
2,3-Butanedione monoxime	Sigma-Aldrich	31550
27G cannula	Servoprax	L10220
4-Aminopyridine	Sigma-Aldrich	A78403
Anhydrous DMSO	Sigma-Aldrich	D12345
Aortic cannula	Radnoti	130163-20
BaCl2	Sigma-Aldrich	342920
blunt surgical forceps	Kent Scientific	INS650915-4
Bovine Calf Serum	Sigma-Aldrich	12133C
CaCl2	Sigma-Aldrich	C5080
Circulating heated water bath	Julabo	ME
Collagenase Type II	Worthington	LS994177
disscetion scissors	Kent Scientific	INS600124
DL-aspartat K+-salt	Sigma-Aldrich	A2025
EGTA	Sigma-Aldrich	E4378
Fluo-3	Invitrogen	F3715
Fluo-3 AM	Invitrogen	F1242
Glucose	Sigma-Aldrich	G8270
Guanosine 5′-triphosphate tris salt	Sigma-Aldrich	G9002
Heating coil	Radnoti	158821
Heparin	Ratiopharm	25.000 IE/5ml
HEPES	Sigma-Aldrich	H9136
induction chamber	CWE incorporated	13-40020
Isoflurane	Cp-pharma	1214
Jacketed heart chamber	Radnoti	130160
KCl	Merck	1049360250
KH2PO4	Sigma-Aldrich	P5655
MgCl x 6H2O	Sigma-Aldrich	M0250
MgSO4 x 7H2O	Sigma-Aldrich	M9397
Na2ATP	Sigma-Aldrich	A2383
Na2HPO4 x 2H2O	Sigma-Aldrich	S5136
NaCl	Sigma-Aldrich	S3014
NaHCO3	Sigma-Aldrich	S5761
Nylon mesh (200 µm)	VWR-Germany	510-9527
pasteur pipette	Sigma Aldrich	Z331759
petri-dishes	Thermo Fisher	150318
Pluronic Acid F-127	Sigma-Aldrich	P2443
Probenecid	Sigma-Aldrich	P8761
Roller Pump	Ismatec	ISM597D
surgical forceps	Kent Scientific	INS650908-4
surgical scissors	Kent Scientific	INS700540
suturing silk	Fine Science Tools	NC9416241
syringe	Merck	Z683531-100EA
Taurin	Sigma-Aldrich	86330