Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Summary

Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro.

Abstract

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca²⁺-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 µm are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode's solution with 0.9 mM Ca²⁺ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 °C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode.

Introduction

Thin tissue slices have been used frequently in basic science since Yamamot and Mcllwain showed in 1966 that electrical activity of brain slices is maintained in vitro¹. Since then, electrophysiological and pharmacological studies have been conducted on slices from brain ², liver ³, lung ⁴ and myocardial tissue ^5,6,7. First patch-clamp recordings in ventricular slices from neonatal rat hearts were described in 1990 ⁸, but this technique fell into oblivion for some time. More than one decade later, our group established a new method to prepare murine embryonic ⁹, neonatal ¹⁰ and adult ¹¹ heart slices. These viable tissue slices can be used for acute experiments (adult slices can be cultivated for several hours) or short-term culture experiments (embryonic and neonatal slices can be cultivated for a few days). Slices show in vivo like electrophysiological characteristics and a homogenous excitation spread as assessed by sharp electrode action potential and micro electrode array recordings ¹¹. Due to their "two-dimensional" morphology, they allow direct access of recording electrodes to all regions of the ventricle, which makes them an interesting tool for electrophysiological investigations and raises new experimental options in comparison to Langendorff-perfused whole hearts. Drug response of the slices to ion channel blockers like verapamil (L-type Ca²⁺-channel blocker), lidocain (Na⁺-channel blocker), 4-aminopyridine (unselective voltage dependent K⁺-channel blocker) and linopirdine (KCNQ K⁺-channel blocker) ^9,11 corresponded to known effects on dissociated cardiomyocytes. Isometric force measurements revealed a positive force frequency relationship and strongly suggested intact contractile function ¹⁰. These findings demonstrated that murine ventricular slices are suitable as an in vitro tissue model for physiological and pharmacological studies. Furthermore, ventricular slices of recipient hearts in combination with sharp electrode recordings have proven to be a very helpful tool to characterize electrical and mechanical integration as well as maturation of transplanted fetal ^12,13,14 and stem cell-derived ¹⁵ cardiomyocytes.

In summary, ventricular slices are a valuable and well-establish multicellular tissue model and should be considered complementary to dissociated cardiomyocytes and Langendorff-perfused hearts in cardiovascular research, with the major advantage of providing an in vivo like tissue structure (in contrast to dissociated cells) as well as direct access of measurement technologies like sharp electrode recordings to all regions of the heart (in contrast to whole heart preparations).

Protocol

Animal handling has to be conform to guidelines of the local animal welfare committee and to the Directive 2010/63/EU of the European Parliament.

1. Prepare Solutions

1. Prepare Tyrode's solution without Ca²⁺ (composition in mM): NaCl 136, KCl 5.4, NaH₂PO₄ 0.33, MgCl₂ 1, glucose 10, HEPES 5, 2,3-butanedione monoxime (BDM) 30. Adjust pH to 7.4 with NaOH at 4 °C.
2. Prepare Tyrode's solution with Ca²⁺ (composition in mM): NaCl 136, KCl 5.4, NaH₂PO₄ 0.33, MgCl₂ 1, glucose 10, HEPES 5, BDM 30, CaCl₂ 0.9. Adjust pH to 7.4 with NaOH at 4 °C.
3. Prepare 4% low-melt agarose: Put 0.6 g low-melt agarose in 15 mL Tyrode's solution without Ca²⁺. Heat the mixture in a microwave oven two times at 750 W for 10-15 s until the agarose is dissolved. Keep the solution at a constant temperature of 37 °C and stir continuously.
4. Keep Dulbecco's Modified Eagle Medium (DMEM) without serum at 37 °C bubbled with carbogen (5% CO₂, 95% O₂).

2. Prepare the Microtome

1. Switch on the microtome.
2. Fill the outer microtome chamber with ice.
3. Fill the inner microtome chamber with ice cold Tyrode's solution without Ca²⁺ and continuously oxygenate the solution with 100% O₂.
4. Place a steel blade into the blade-holder of the microtome.

3. Mouse Heart Isolation

1. Inject 2,500 U Heparin subcutaneously. Wait 15 min.
2. Sacrifice the animal by cervical dislocation.
3. Open the chest by sternotomy.
4. Carefully dissect the pericardium using small scissors and a forceps #5.
5. Insert a cannula into the ascending aorta and perfuse the coronary arteries in situ with ice cold Tyrode's solution without Ca²⁺ until remaining blood is removed.
6. Gently resect the heart with a forceps and scissors and transfer the heart in ice cold Tyrode's solution rode without Ca²⁺.
7. Separate the atria from the ventricles with a scalpel or scissors.

4. Embedding of the Ventricles in 4% Low-melt Agarose

1. Place the ventricles with the apex facing upwards in the agarose mold (Figure 1). Place the pin in the middle of the mold in the left ventricular chamber.
2. Fill the mold with 4% low-melt agarose at 37 °C, until the heart is completely covered.
3. Place the mold on ice for faster hardening of the agarose preventing floating of the tissue.
4. Remove the agarose block containing the ventricles from the mold with a scalpel.
5. Turn the block upside down and fill ventricular chambers and the gap on the backside of the agarose block, which is left by the pin of the molt, with 4% low-melt agarose using a syringe with a 20 G needle.
6. Trim the agarose block with a scalpel to achieve a flat bottom of the block and upright position of the cardiac apex.

5. Slicing the Ventricular Tissue

1. Fix the block on the specimen holder of the microtome with a drop of cyanoacrylate glue. Face the cardiac apex upwards.
2. Place the specimen holder into the inner specimen chamber of the microtome, which is filled with ice cold Tyrode without Ca²⁺. Completely cover the agarose block with Tyrode's solution.
3. Prepare short-axis slices at a thickness of 150-400 µm, depending on the further application (for sharp electrode recordings 150-200 µm), keep vibration frequency of the blade at 60-70 Hz and move the blade forward as slowly as possible.
4. Use a fine brush to carefully remove the remaining agarose from the slices.
5. Gently transfer slices with a Pasteur pipette into the Tyrode's solution with 0.9 mM Ca²⁺ aerated with 100% O₂ and store them for at least 30 min on ice to recover from the slicing procedure.
6. Afterwards, keep slices for 30 min in DMEM at 37 °C, aerated with carbogen, to wash out BDM before further use.

6. Preparing the Sharp Electrode Setup

1. For pre-heating, switch on all electric devices 30 min before the recordings start.
2. Put a 3 cm cell culture dish on the heating plate placed on the inverted microscope.
3. Place the custom-made ring electrode (Figure 2) in the dish and connect grounding wires of the pre-amplifier and the stimulation electrode.
4. Connect the flexible tubes of the perfusion system to the dish.
5. Fill the reservoir of the perfusion system with DMEM, aerated with carbogen.
6. Switch on the perfusion pump and set the perfusion rate to 2-3 mL/min.
7. Adjust the temperature of DMEM in the dish to 37 °C by regulating the flow heater and the heating plate.

7. Action Potential Recordings

1. Place a ventricular slice into the DMEM filled dish.
2. Check the structural integrity and viability (based on contractile function) of the tissue with the inverted microscope.
3. Fill a recording glass electrode with 3 M KCL.
4. Fill a stimulation electrode with DMEM.
5. Place the recording electrode and the stimulation electrode on the electrode holders.
6. Place the stimulation electrode carefully on the slice and switch on the electric stimulator. Start with a stimulation frequency of 1-2 Hz.
7. Move the recording electrode with the micromanipulator over the intended recording position.
8. Slowly lower the recording electrode until the tip touches the tissue.
9. Apply a short rectangular electric pulse through the recording electrode to penetrate the cell membrane.
10. Carefully reposition the recording electrode until a stable signal is ensured.
11. Start recording of action potentials.

Results

Myocardial infarction leads to a virtually irreversible loss of cardiomyocytes. Cell replacement therapy using stem cell-derived cardiomyocytes for exogenous cardiac regeneration is a promising therapeutic approach. Electrical integration and maturation of the transplanted cells are crucial for safety and efficiency of cell replacement therapy.

To assess integration and maturation, we transplanted cardiomyocytes derived from ind...

Discussion

Ventricular slices enable electrophysiological, pharmacological and mechanical studies with a preserved in vivo like tissue structure and direct access of the measurement technology to all regions of the heart. Physiological action potential properties have been demonstrated in embryonic, neonatal and adult slices ^9,10,11. Vitality of the slices, except for the surface layers directly damaged by the slicing procedure, h...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leica VT 1000s	Leica Microsystems, Wetzlar, Germany		Microtome with vibrating blade.
Stainless Steel Blades	Campden Instruments, Loughborough, England	7550-1-SS
Pasteur pipettes	Sigma-Aldrich, St. Louise, USA	Z627992
Fine brush, e.g. size 6 (4/32")	VWR, International, Radnor, USA	149-2125
Preparation table			self made
Molt for embedding ventricles in agarose			self made
1 mL Syringe	Becton, Dickinson; Franklin Lakes, USA	300013
27 G x 3/4`` Needles	Braun, Melsungen, Germany	4657705
20 G 11/2`` Needles	4657519
Small scissor	WPI, Sarasota, USA	501263
Tweezers #5, 0.1 x 0.06 mm tip	WPI, Sarasota, USA	500342
Oxygen gas (medical grade O2)	Linde, Munich, Germany
Carbogen gas (95 % O2, 5 % CO2)	Linde, Munich, Germany
NaCl	Sigma-Aldrich, St. Louise, USA	7647-14-5
KCl	Sigma-Aldrich, St. Louise, USA	746436
CaCl2	Sigma-Aldrich, St. Louise, USA	746495
KH2PO4	Sigma-Aldrich, St. Louise, USA	NIST200B
HEPES	Sigma-Aldrich, St. Louise, USA	51558
NaHCO3	Sigma-Aldrich, St. Louise, USA	S5761
D(+)-Glucose	Sigma-Aldrich, St. Louise, USA	G8270
MgSO4	Sigma-Aldrich, St. Louise, USA	M7506
NaOH	Sigma-Aldrich, St. Louise, USA	S8045
Cyanoacrylate glue	Henkel, Düsseldorf, Germany
Low-melt Agarose	Roth, Karlsruhe, Germany	6351.2
Heparin-sodium-25000 I.E./5 mL	Ratiopharm, Ulm, Germany
Dulbecco's Modified Eagle Medium (DMEM), high glucose, GlutaMAX	ThermoScientific, Waltham, USA	10566016
SEC-10LX Amplifier	npi electronic GmbH, Tamm, Germany	SEC-10LX
EPC 9	HEKA Elektronik GmbH, Lambrecht, Germany
Zeiss Axiovert 200	Zeiss, Oberkochen, Germany
Low magnification Micromanipulator	Narashige, Tokyo, Japan	Nm-3
High magnification, three-axis micromanipulator	Narashige, Tokyo, Japan	MHW-3
Peristaltic perfusion pump	Multi Channel Systems, Reutlingen, Germany	PPS2
2-channel temperature controller	Multi Channel Systems, Reutlingen, Germany	TCO02
Square pulse stimulator	Natus Europe GmbH, Planegg, Germany	Grass SD9
Glass capillaries	WPI, Sarasota, USA	1B150F-1