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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 cardiac myocytes from a variety of healthy and diseased models, providing vital data on the physiology of EC-Coupling as well as on the pathological alterations of intracellular Ca²⁺ homeostasis leading to mechanical impairment and increased arrhythmogenic burden in cardiac diseases. Information from these studies is critical for understanding the electrophysiological and mechanical effects of drugs in the clinical setting. However, there are species specific differences in the transmembrane currents and in the EC-Coupling proteins that account for specific features of cardiac action potential and cardiac mechanics. Thus, while studies of myocytes isolated from non human mammals have elucidated the biophysical properties and physiological roles of specific transmembrane ion channels and EC-Coupling proteins, they do not necessarily provide relevant models of human cardiac myocytes. Isolation of viable myocytes from human myocardium is therefore essential to fully understand the pathophysiology of cardiac diseases and validate novel therapeutic approaches.

Human atrial tissue is readily available as atrial appendages are often discarded during surgical procedures. Initial quantitative studies of adult human cardiac action potentials and ionic currents employed enzymatically isolated atrial cells^1-4. Recordings of action potentials or currents from isolated adult human ventricular cells have been subsequently reported^3,5-10. Most of these studies have used cells obtained from explanted hearts and utilized either collagenase perfusion of a coronary artery segment or exposure of relatively large quantities of excised tissue to collagenase to obtain isolated cells. These studies allowed a detailed characterization of a number of transmembrane ion currents from human ventricular cardiomyocytes from healthy hearts and from patients with terminal heart failure. Recordings of L-type Ca²⁺ current (I_Ca-L)^5-7, transient outward potassium current (I_to)⁸, inward rectifier potassium current (I_κ1)⁸, the different components of delayed rectifier potassium current (I_κ)⁹ have been reported. Advances and refining of the isolation procedure¹⁰, allowed a clear characterization of the ionic basis of the increased arrhythmogenic potential in terminal heart failure, comprising action potential prolongation¹¹, delayed after depolarizations¹² and increased funny current¹³ leading to diastolic depolarization and premature beats.

Adult cardiac myocytes are normally isolated from small animals by retrograde perfusion of the whole heart with various enzyme mixtures, a technique that produces high yields of Ca²⁺-tolerant cells¹⁴. Isolation of cardiac myocytes from fragments of tissue is inherently less successful probably because of the limited access of enzymes to individual myocytes compared with that achieved by perfusion of coronary arteries. Because of the very limited availability of unused donor hearts, the only practical way to obtain normal human ventricular cells on a regular basis is by enzymatic digestion of the often very small tissue fragments excised during elective surgical procedures. The only human disease model that has been thoroughly characterized at cell level is terminal heart failure, due to the accessibility to transplanted hearts. However, terminal heart failure occurs in a minority of patients and often involves a common pathway of severe remodeling of myocardial cells, which is relatively independent of the underlying cause¹⁵. The ability to assess the function of single cardiomyocytes from patients at an earlier non failing stage of disease is crucial to understand the specific pathophysiology of different inherited or acquired conditions. Hypertrophic cardiomyopathy (HCM) is a telling example. HCM is a common (1/500 individuals) inheritable cardiac condition characterized by cardiac hypertrophy, increased arrhythmogenic risk and contractile alterations due to outflow tract obstruction and diastolic dysfunction¹⁶. Cardiomyocytes from HCM hearts undergo a complex remodeling processes involving changes in cell structure (hypertrophy, myofibrillar disarray) and EC-Coupling¹⁷. However, most information of myocyte dysfunction in HCM has come from transgenic animal models. Since only a minority of HCM patients evolves toward terminal heart failure and requires cardiac transplantation, HCM hearts are very rarely available for cell isolation with standard methods. However, at least 30% of HCM patients develop obstructive symptoms due to massive septal hypertrophy altering outflow tract blood flow during systole (HCM)¹⁸. The most effective available therapeutic option for the relief of obstruction in HCM is surgical septal myectomy: during this surgical procedure, a variable sized portion of upper septum is removed by trans aortic approach. This portion of hypertrophied septum is therefore available for cell isolation from the fresh tissue.

A method for the isolation of human ventricular myocytes from single, small transvenous endomyocardial biopsy specimens has been previously developed and published¹⁹. We implemented a method to isolate single septal myocytes from ventricular myocardium samples from patients undergoing cardiac surgery, including patients with HCM undergoing septal myectomy and patients undergoing valve replacement procedures. In addition to a detailed description of the isolation protocol, representative electrophysiological and Ca²⁺ fluorescence measurements are presented, demonstrating the viability of the isolated human ventricular myocytes and the feasibility of patch clamp and intracellular Ca²⁺ studies.

Protokół

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
Reagent (mM)	KH2PO4	50
	MgSO4	8	1.2	5			1.2	1.2
	HEPES	10			10	10
	adenosine	5
	glucose	140	10	20	10		10	10
	mannitol	100
	taurine	10	20	5			20	20
	NaCl		113		136		113	113
	KCl		4.7	85	5.4	25	4.7	4.7
	MgCl2				1.2	5
	KH2PO4		0.6	30			0.6	0.6
	Na2HPO4		0.6				0.6	0.6
	NaHCO3		12				12	12
	KHCO3		10				10	10
	Na-pyruvate		4				4	4
	BDM		10				10	10
	BHBA			5
	succinic acid			5
	EGTA			0.5
	K2-ATP			2
	pyruvic acid			5
	creatine			5
	KMES					115
Enzymes (U/ml)	Collagenase Type V						250	250
	Protease Type XXIV						4
pH		7.4 KOH	7.3 NaOH	7.1 KOH	7.35 NaOH	7.2 KOH	7.3 NaOH	7.3 NaOH

Table 1. Solutions used for specimen collection, cell isolation and functional characterization of myocytes. CP= cardioplegic solution; DB=dissociation buffer; KB= Kraft-Bruhe solution; TB=Tyrode buffer; PS=pipette solution; EB1= enzyme buffer 1; EB2= enzyme buffer 2.

1. Prepare cardioplegic (CP) solution. CP solution can be stored at 4 °C for up to 1 week.
2. Prepare Ca²⁺-free dissociation buffer (DB). This solution should be used within the day.
3. Prepare Kraft-Bruhe (KB) solution. KB solution can be stored at 4 °C for up to 1 week.
4. Prepare Ca²⁺-free Tyrode buffer (TB). This solution should be used within the day.
5. Filter all solutions using syringe filters prior to use.
6. Prepare the digestion device (Figure 2), a scraping container made of two facing brushes of silicone elastomer, one of which rotated by an electric motor. The digestion device is custom made. Details on the digestion device are in Figure 8; images of the device are in Figures 2C and 2D. Wash the tissue chamber with 70% ethanol and water.

2. Collection and Processing of Myocardial Samples

1. Pour 40 ml of cardiplegic (CP) solution in a 50 ml tube and store it in ice for specimen transportation from the operative room to the cell isolation lab.
2. Collect the ventricular myocardial specimen from the operative room immediately after excision, wash it with ice cold CP solution and store it in the tube. Use endocardial specimens excised from the upper inter ventricular septum during open heart surgery, weighting >100 mg.
3. Rapidly transfer the specimen to the lab area; start specimen processing within 10 min from specimen excision.
4. While keeping the specimen in ice cold CP buffer, carefully remove the endocardial fibrotic layer using fine scissors under a stereomicroscope; afterwards, cut the myocardial tissue to small pieces (2-3 mm long). Depending on tissue sample size, cut a total amount of ventricular myocardium between 100 mg and 1 g for each isolation.
5. Upon completion of tissue mincing, transfer the myocardial chunks into the digestion device, with clean ice cold CP solution. Avoid filling the whole volume between the two silicon brushes (3-4 ml) with myocardial chunks, using no more than 1 g of total tissue.

3. Washing and Digestion of Myocardial Chunks

1. After the chunks are transferred into the scraping chamber of the digestion device, change the CP buffer in the chamber with cold Ca²⁺-free dissociation buffer (DB).
2. Place the digestion device in a thermostatic bath, in order for the chamber to be in contact with the heated water in the bath (Figure 1). Set the bath to 37.5 °C and turn it on, in order to slowly raise the temperature of the tissue chamber. Turn on the motor of the digestion device, setting the rotation speed to 1 revolution/second.
3. Perform 3 washing cycles with DB, changing the solution in the chamber with clean DB every 8 min. The DB is warmed (37 °C) and oxygen saturated before getting in contact with the myocardial chunks.
4. Prepare enzyme buffer 1 (EB1) by adding 250 U/ml of Collagenase Type V and 4 U/ml Protease Type XXIV to DB solution. Prepare enzyme buffer 2 (EB2) by adding 250 U/ml Collagenase Type V to DB solution. Warm up (37 °C) and oxygenate EB1 and EB2.
5. Perform two 12 min cycles of digestion in the rotating digestion device with 100% oxygenated EB1 (at 37 °C). At each cycle, use ~3 ml of EB1. Remove the solution by pipette aspiration and discard it after each cycle.
6. Prepare 6 15 ml tubes for cell collection and ~80 ml of cold (4 °C) KB solution for eluting the buffers.
7. Perform a first 15 min digestion cycle with 3 ml 100% oxygenated EB2 at 37 °C. After the digestion cycle, collect the solution containing the first dissociated myocytes in a 15 ml tube and dilute the cell suspension with 12 ml cold KB solution. Store the tube flat at room temperature.
8. Dilute the remaining EB2 solution with an equal amount of DB in order to halve the concentration of collagenase V for the following digestion cycles.
9. Perform other 5 12 min digestion cycles with 3 ml EB2 at 37 °C; after each of them collect of myocyte containing buffer in a 15 ml conical tube and dilute it with 12 ml KB solution. Store the 6 cell containing tubes at room temperature for 30 min.

4. Cell Resuspension and Ca²⁺ Readaptation

1. Add 1 mg/ml bovine serum albumin (BSA) to 20 ml Ca²⁺-free Tyrode buffer (TB). Filter the solution.
2. Centrifuge the six myocyte containing conical tubes at 100 x g for 5 min to force myocytes to settle. Remove the supernatant and resuspend the cells in each tube with a variable amount (1-3 ml, depending on the yield) of BSA containing TB at RT.
3. Gradually increase Ca²⁺ concentration in the cell containing buffer by adding small aliquots of 100 mmol/L CaCl₂ solution. In the first and second steps Ca²⁺ concentration is raised up to 50 μmol/L and 100 μmol/L, respectively. The following Ca²⁺ addition steps are performed every 5 min and the concentration is raised by 100 μmol/L at each step to a final concentration of 0.9 mmol/L.
4. Assess the yield of the isolation procedure. Transfer 0.5 ml of myocyte containing solution onto the glass bottom chamber of a microscope. Evaluate 15 microscope fields at 10x objective magnification and calculate the percentage of healthy myocytes (e.g. rod shaped cells with clear striations and no significant inclusions, Figure 2). The expected yield is around 20 %.

5. Functional Evaluation of Isolated Cardiomyocytes.

The following protocol is an example of human cardiomyocyte functional assessment including simultaneous recordings of action potentials and intracellular Ca²⁺ fluxes.

1. Prepare pipette solution (PS) for patch clamp experiments in perforated patch configuration. The solution can be stored at -20 °C for up to 3 months.
2. Add 1.8 mmol/L CaCl₂ to Ca²⁺-free Tyrode buffer (TB). Use this solution for superfusion of cardiomyocytes during patch clamp/fluorescence experiments.
3. Transfer 1 ml of cell suspension to a 1.5 ml tube and add 10 μmol/L Fluoforte and 10 μl Powerload Concentrate. Incubate for 30 min at RT. Afterwards, set the tube in vertical position and leave the cell to settle for 5 min; resuspend the cells in Ca²⁺ containing TB.
4. Transfer 0.25 ml of cell suspension to a small (0.5 ml), temperature controlled microscope mounted recording chamber, superfused by gravity with a heated microperfusor system at a flow rate of 0.3 ml/min (temperature: 37 ± 0.5 °C).
5. Using a micropipette puller, prepare patch clamp pipettes with a tip diameter of 3 to 5 μm and a resistance of 3 to 4.5 M when filled with PS.
6. Add amphotericin B to a batch of PS (250 μg/ml) and use it to fill the electrodes. 
7. Select a rod shaped cell with clear striations, devoid of inclusions, form the giga seal and wait 5 to 10 min, until access resistance drops below 20 MΩ.
8. Elicit action potentials in current clamp mode using short pulses (< 3 msec) at different frequencies of stimulation (0.2 Hz, 0.5 Hz and 1 Hz, 1 min at each frequency). During the recording phase, turn on bright field illumination at 492 ± 3 nm and detect Fluoforte fluorescence at 505-520 nm. Acquire fluorescence and membrane potential signals using Digidata 1440A and pClamp 10.0 software. Repeat the recording sequence multiple times if needed; however, keep the total recording time below 15 min for each cell.

Wyniki

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...

Dyskusje

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...

Materiały

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.