Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Summary

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Abstract

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.

Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.

The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Introduction

Dissection of the electrophysiological properties of the myocardium has progressed markedly after the development of techniques for single cardiac myocyte isolation. Recent advancements in the understanding of cardiac Excitation Contraction Coupling (EC-Coupling) have also been made possible by the capability of isolating viable single cardiomyocytes that retain all the physiological properties of the intact tissue. Patch clamp methods are routinely employed to study the function and pharmacological modulation of cardiac sarcolemmal ion currents. Recordings of intracellular calcium dynamics with Ca²⁺ sensitive dyes are also regularly performed on single car....

Protocol

The experimental protocols on human tissue were approved by the ethical committee of Careggi University-Hospital (2006/0024713; renewed May 2009). Each patient gave written informed consent.

1. Solutions and Equipment Preparation

Solutions are described in Table 1. A simplified flowchart of the cell isolation procedure is found in Figure 1.

Solution		CP	DB	KB	TB	PS	EB1	EB2

Results

The method described above was employed to characterize the functional abnormalities of cardiomyocytes isolated from the interventricular septum of patients with hypertrophic cardiomyopathy (HCM) who underwent myectomy operation, as compared with non failing non hypertrophic surgical patients²¹. The results contained in this section are derived from that work²¹ and are shown here as an example of how this technique can be used to characterize the alterations of myocardial cell function in cardiac di.......

Discussion

We have described and validated a method to isolate viable myocytes from surgical samples of human ventricular myocardium. Starting from previously described protocols that had been successfully used to isolated cells from atrial surgical samples, the technique to allow separation of single viable myocytes from diseased ventricular myocardium was developed and fine tuned. Early reports showed that isolation of single cardiomyocytes from chunks of atrial and ventricular tissue selectively impaired repolarizing potassium c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P9791
Magnesium sulfate heptahydrate(MgSO4*7H2O)	Sigma-Aldrich	M1880
HEPES	Sigma-Aldrich	H3375
Adenosine	Sigma-Aldrich	A9251
D-(+)-Glucose	Sigma-Aldrich	G8270
Mannitol	Sigma-Aldrich	M4125
Taurine	Sigma-Aldrich	T0625
Potassium hydroxide (KOH)	Sigma-Aldrich	P5958
Sodium chloride (NaCl)	Sigma-Aldrich	S7653
Potassium chloride (KCl)	Sigma-Aldrich	P9333
Sodium phosphate dibasic (Na2HPO4)	Sigma-Aldrich	S7907
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S6297
Potassium bicarbonate (KHCO3)	Sigma-Aldrich	237205
Sodium pyruvate	Sigma-Aldrich	P2256
2,3-Butanedione monoxime	Sigma-Aldrich	B0753
Sodium hydroxide(NaOH)	Sigma-Aldrich	S8045
L-Glutamic acid monopotassium salt monohydrate	Sigma-Aldrich	49601
Pyruvic acid	Sigma-Aldrich	107360
3-Hydroxybutyric acid	Sigma-Aldrich	166898
Adenosine 5′-triphosphate dipotassium salt dihydrate (K2-ATP)	Sigma-Aldrich	A8937
Creatine	Sigma-Aldrich	C0780
Succinic Acid	Sigma-Aldrich	S3674
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)	Sigma-Aldrich	E0396
Albumin from bovine serum	Sigma-Aldrich	A0281
Magnesium chloride (MgCl2)	Sigma-Aldrich	M8266
Collagenase from Clostridium histolyticum, Type V	Sigma-Aldrich	C9263
Proteinase, Bacterial, Type XXIV	Sigma-Aldrich	P8038
Calcium chloride solution, ~1 M in H2O	Sigma-Aldrich	21115
Calcium chloride 0.1 M solution	Sigma-Aldrich	53704
Potassium methanesulfonate	Sigma-Aldrich	83000
FluoForte Reagent	Enzo Life Sciences	ENZ-52015
Powerload concentrate, 100X	Life Technologies	P10020
Perfusion Fast-Step System	Warner Instruments	VC-77SP
Amphotericin B solubilized	Sigma-Aldrich	A9528
Multiclamp 700B patch-clamp amplifier	Molecular Devices
Digidata 1440A	Molecular Devices
pClamp10.0	Molecular Devices
Digestion Device	CUSTOM	CUSTOM	The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Figure 1C-1D and in Figure7. We can provide further details if requested.
Silicone elastomer for the digestion device's brushes	Dow Corning	SYLGARD® 184
Variable speed rotating motor for the digestion device	Crouzet	Crouzet 178-4765
Mold for brushes casting	N.A.	N.A.	The mold is custom made from standard PTFE 2.5 cm diameter rods.