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,

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 sol....

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
Caffeine	Sigma-Aldrich	C0750
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