Name: Isolamento e caratterizzazione funzionale di Human ventricolare Cardiomyocytes da campioni freschi chirurgici (Video) | JoVE Journal
Uploaded: 2014-04-21T15:45:00.000+00:00
Duration: 14 min 39 s
Description: Impara a isolare cardiomiociti ventricolari umani da campioni chirurgici freschi per studi elettrofisiologici e test farmacologici. Questo metodo consente di caratterizzare alterazioni funzionali nelle malattie cardiache.

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This work was supported by the E.U. (STREP Project 241577 "BIG HEART," 7th European Framework Program, CP), Menarini International Operations Luxembourg (AM), Telethon GGP07133 (CP) and Gilead Sciences (AM).

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.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="2">Solution</td>
			<td>CP</td>
			<td>DB</td>
			<td>KB</td>
			<td>TB</td>
			<td>PS</td>
			<td>EB1</td>
			<td>EB2</td>
		</tr>
		<tr>
			<td rowspan="23">Reagent (mM)</td>
			<td>KH2PO4</td>
			<td>50</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>8</td>
			<td>1.2</td>
			<td>5</td>
			<td></td>
			<td></td>
			<td>1.2</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td>10</td>
			<td>10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>adenosine</td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>140</td>
			<td>10</td>
			<td>20</td>
			<td>10</td>
			<td></td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>mannitol</td>
			<td>100</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>taurine</td>
			<td>10</td>
			<td>20</td>
			<td>5</td>
			<td></td>
			<td></td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td>113</td>
			<td></td>
			<td>136</td>
			<td></td>
			<td>113</td>
			<td>113</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td>4.7</td>
			<td>85</td>
			<td>5.4</td>
			<td>25</td>
			<td>4.7</td>
			<td>4.7</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td></td>
			<td>1.2</td>
			<td>5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td>0.6</td>
			<td>30</td>
			<td></td>
			<td></td>
			<td>0.6</td>
			<td>0.6</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td></td>
			<td>0.6</td>
			<td></td>
			<td></td>
			<td></td>
			<td>0.6</td>
			<td>0.6</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td></td>
			<td>12</td>
			<td></td>
			<td></td>
			<td></td>
			<td>12</td>
			<td>12</td>
		</tr>
		<tr>
			<td>KHCO3</td>
			<td></td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td></td>
			<td>4</td>
			<td></td>
			<td></td>
			<td></td>
			<td>4</td>
			<td>4</td>
		</tr>
		<tr>
			<td>BDM</td>
			<td></td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>BHBA</td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>succinic acid</td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td></td>
			<td></td>
			<td>0.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K2-ATP</td>
			<td></td>
			<td></td>
			<td>2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pyruvic acid</td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>creatine</td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KMES</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>115</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Enzymes (U/ml)</td>
			<td>Collagenase Type V</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>250</td>
			<td>250</td>
		</tr>
		<tr>
			<td>Protease Type XXIV</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>pH</td>
			<td></td>
			<td>7.4 KOH</td>
			<td>7.3 NaOH</td>
			<td>7.1 KOH</td>
			<td>7.35 NaOH</td>
			<td>7.2 KOH</td>
			<td>7.3 NaOH</td>
			<td>7.3 NaOH</td>
		</tr>
	</tbody>
</table>
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.
<ol>
	<li>Prepare cardioplegic (CP) solution. CP solution can be stored at 4 &#176;C for up to 1 week.</li>
	<li>Prepare Ca2+-free dissociation buffer (DB). This solution should be used within the day.</li>
	<li>Prepare Kraft-Bruhe (KB) solution. KB solution can be stored at 4 &#176;C for up to 1 week.</li>
	<li>Prepare Ca2+-free Tyrode buffer (TB). This solution should be used within the day.</li>
	<li>Filter all solutions using syringe filters prior to use.</li>
	<li>Prepare the digestion device (Figure&#160;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&#160;8; images of the device are in Figures 2C and 2D. Wash the tissue chamber with 70% ethanol and water.</li>
</ol>
2. Collection and Processing of Myocardial Samples
<ol>
	<li>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.</li>
	<li>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 &#62;100 mg.</li>
	<li>Rapidly transfer the specimen to the lab area; start specimen processing within 10 min&#160;from specimen excision.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Washing and Digestion of Myocardial Chunks
<ol>
	<li>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).</li>
	<li>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 &#176;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.</li>
	<li>Perform 3 washing cycles with DB, changing the solution in the chamber with clean DB every 8 min. The DB is warmed (37 &#176;C) and oxygen saturated before getting in contact with the myocardial chunks.</li>
	<li>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 &#176;C) and oxygenate EB1 and EB2.</li>
	<li>Perform two 12 min cycles of digestion in the rotating digestion device with 100% oxygenated EB1 (at 37 &#176;C). At each cycle, use ~3 ml of EB1. Remove the solution by pipette aspiration and discard it after each cycle.</li>
	<li>Prepare 6 15 ml tubes for cell collection and ~80 ml of cold (4 &#176;C) KB solution for eluting the buffers.</li>
	<li>Perform a first 15 min&#160;digestion cycle with 3 ml 100% oxygenated EB2 at 37 &#176;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.</li>
	<li>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.</li>
	<li>Perform other 5 12 min&#160;digestion cycles with 3 ml EB2 at 37 &#176;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.</li>
</ol>
4. Cell Resuspension and Ca2+ Readaptation
<ol>
	<li>Add 1 mg/ml&#160;bovine serum albumin (BSA) to 20 ml Ca2+-free Tyrode buffer (TB). Filter the solution.</li>
	<li>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.</li>
	<li>Gradually increase Ca2+ concentration in the cell containing buffer by adding small aliquots of 100 mmol/L&#160;CaCl2 solution. In the first and second steps Ca2+ concentration is raised up to 50 &#956;mol/L and 100 &#956;mol/L, respectively. The following Ca2+ addition steps are performed every 5 min&#160;and the concentration is raised by 100 &#956;mol/L at each step to a final concentration of 0.9 mmol/L.</li>
	<li>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&#160;2). The expected yield is around 20 %.</li>
</ol>
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.
<ol>
	<li>Prepare pipette solution (PS) for patch clamp experiments in perforated patch configuration. The solution can be stored at -20 &#176;C for up to 3 months.</li>
	<li>Add 1.8 mmol/L CaCl2 to Ca2+-free Tyrode buffer (TB). Use this solution for superfusion of cardiomyocytes during patch clamp/fluorescence experiments.</li>
	<li>Transfer 1 ml&#160;of cell suspension to a 1.5 ml tube and add 10 &#956;mol/L Fluoforte and 10 &#956;l 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+&#160;containing TB.</li>
	<li>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 &#177; 0.5 &#176;C).</li>
	<li>Using a micropipette puller, prepare patch clamp pipettes with a tip diameter of 3 to 5 &#956;m and a resistance of 3 to 4.5 M when filled with PS.</li>
	<li>Add amphotericin B to a batch of PS (250 &#956;g/ml) and use it to fill the electrodes.&#160;</li>
	<li>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 M&#937;.</li>
	<li>Elicit action potentials in current clamp mode using short pulses (&#60; 3 msec) at different frequencies of stimulation (0.2 Hz, 0.5 Hz and 1 Hz, 1 min&#160;at each frequency). During the recording phase, turn on bright field illumination at 492 &#177; 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&#160;for each cell.</li>
</ol>

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<li>Lippiat, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-cell+recording+using+the+perforated+patch+clamp+technique%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Whole-cell recording using the perforated patch clamp technique.</a> Methods Mol Biol. 491, 141-149 (2008).</li>
<li>Coppini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Late+sodium+current+inhibition+reverses+electromechanical+dysfunction+in+human+hypertrophic+cardiomyopathy%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.</a> Circulation. 127, 575-584 (2013).</li>
<li>Kuusisto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-grade+inflammation+and+the+phenotypic+expression+of+myocardial+fibrosis+in+hypertrophic+cardiomyopathy%5BTitle%5D)+AND+%22Heart%22%5BJournal%5D)">Low-grade inflammation and the phenotypic expression of myocardial fibrosis in hypertrophic cardiomyopathy.</a> Heart. 98, 1007-1013 (2012).</li>
<li>Yan, G. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+2+early+afterdepolarization+as+a+trigger+of+polymorphic+ventricular+tachycardia+in+acquired+long-QT+syndrome+%3A+direct+evidence+from+intracellular+recordings+in+the+intact+left+ventricular+wall%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Phase 2 early afterdepolarization as a trigger of polymorphic ventricular tachycardia in acquired long-QT syndrome : direct evidence from intracellular recordings in the intact left ventricular wall.</a> Circulation. 103, 2851-2856 (2001).</li>
<li>Yue, L., Feng, J., Li, G. R., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+outward+and+delayed+rectifier+currents+in+canine+atrium%3A+properties+and+role+of+isolation+methods%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Transient outward and delayed rectifier currents in canine atrium: properties and role of isolation methods.</a> The American Journal of Physiology. 270, 2157-2168 (1996).</li>
<li>Li, G. R., Feng, J., Yue, L., Carrier, M., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+two+components+of+delayed+rectifier+K%2B+current+in+human+ventricular+myocytes%5BTitle%5D)+AND+%22Circulation+research%22%5BJournal%5D)">Evidence for two components of delayed rectifier K+ current in human ventricular myocytes.</a> Circulation research. 78, 689-696 (1996).</li>
<li>Viswanathan, P. C., Shaw, R. M., Rudy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+IKr+and+IKs+heterogeneity+on+action+potential+duration+and+its+rate+dependence%3A+a+simulation+study%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study.</a> Circulation. 99, 2466-2474 (1999).</li>
<li>Volders, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+the+contribution+of+IKs+to+canine+ventricular+repolarization%3A+key+role+for+beta-adrenergic+receptor+stimulation%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation.</a> Circulation. 107, 2753-2760 (2003).</li>
<li>Sanguinetti, M. C., Jurkiewicz, N. K., Scott, A., Siegl, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isoproterenol+antagonizes+prolongation+of+refractory+period+by+the+class+III+antiarrhythmic+agent+E-4031+in+guinea+pig+myocytes.+Mechanism+of+action%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action.</a> Circulation Research. 68, 77-84 (1991).</li>
<li>Coppini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+translational+approach+to+treatment+of+hypertrophic+cardiomyopathy%3A+pre-clinical+rationale+and+design+of+a+prospective+randomized+pilot+trial+with+ranolazine%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">A translational approach to treatment of hypertrophic cardiomyopathy: pre-clinical rationale and design of a prospective randomized pilot trial with ranolazine.</a> Circulation. 125, 1(2012).</li>
</ol>

The authors declare that they have no competing financial interests.

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...

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 cells1-4. Recordings of action potentials or currents from isolated adult human ventricular cells have been subsequently reported3,5-10. 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 (ICa-L)5-7, transient outward potassium current (Ito)8, inward rectifier potassium current (I&#954;1)8, the different components of delayed rectifier potassium current (I&#954;)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.

<table><tbody><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/p9791?lang=it&amp;amp;region=IT&quot;&gt;P9791 &lt;/a&gt;</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate(MgSO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/m1880?lang=it&amp;amp;region=IT&quot;&gt;M1880 &lt;/a&gt;</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/h3375?lang=it&amp;amp;region=IT&quot;&gt;H3375 &lt;/a&gt;</td><td></td></tr><tr><td>Adenosine</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/a9251?lang=it&amp;amp;region=IT&quot;&gt;A9251 &lt;/a&gt;</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/g8270?lang=it&amp;amp;region=IT&quot;&gt;G8270 &lt;/a&gt;</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/m4125?lang=it&amp;amp;region=IT&quot;&gt;M4125 &lt;/a&gt;</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma-Aldrich</td><td>P5958</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/s7653?lang=it&amp;amp;region=IT&quot;&gt;S7653&lt;/a&gt;</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=it&amp;amp;region=IT&quot;&gt;P9333 &lt;/a&gt;</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/s7907?lang=it&amp;amp;region=IT&quot;&gt;S7907 &lt;/a&gt;</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/s6297?lang=it&amp;amp;region=IT&quot;&gt;S6297 &lt;/a&gt;</td><td></td></tr><tr><td>Potassium bicarbonate (KHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/237205?lang=it&amp;amp;region=IT&quot;&gt;237205&lt;/a&gt;</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/p2256?lang=it&amp;amp;region=IT&quot;&gt;P2256 &lt;/a&gt;</td><td></td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/b0753?lang=it&amp;amp;region=IT&quot;&gt;B0753 &lt;/a&gt;</td><td></td></tr><tr><td>Sodium hydroxide(NaOH)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/s8045?lang=it&amp;amp;region=IT&quot;&gt;S8045 &lt;/a&gt;</td><td></td></tr><tr><td>L-Glutamic acid monopotassium salt monohydrate</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/fluka/49601?lang=it&amp;amp;region=IT&quot;&gt;49601&lt;/a&gt;</td><td></td></tr><tr><td>Pyruvic acid</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/aldrich/107360?lang=it&amp;amp;region=IT&quot;&gt;107360&lt;/a&gt;</td><td></td></tr><tr><td>3-Hydroxybutyric acid</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/aldrich/166898?lang=it&amp;amp;region=IT&quot;&gt;166898&lt;/a&gt;</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate dipotassium salt dihydrate (K&lt;sub&gt;2&lt;/sub&gt;-ATP)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/a8937?lang=it&amp;amp;region=IT&quot;&gt;A8937&lt;/a&gt;</td><td></td></tr><tr><td>Creatine</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/c0780?lang=it&amp;amp;region=IT&quot;&gt;C0780 &lt;/a&gt;</td><td></td></tr><tr><td>Succinic Acid</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/s3674?lang=it&amp;amp;region=IT&quot;&gt;S3674 &lt;/a&gt;</td><td></td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/e0396?lang=it&amp;amp;region=IT&quot;&gt;E0396 &lt;/a&gt;</td><td></td></tr><tr><td>Albumin from bovine serum</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=it&amp;amp;region=IT&quot;&gt;A0281&lt;/a&gt;</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/m8266?lang=it&amp;amp;region=IT&quot;&gt;M8266 &lt;/a&gt;</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum, Type V</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/c9263?lang=it&amp;amp;region=IT&quot;&gt;C9263 &lt;/a&gt;</td><td></td></tr><tr><td>Proteinase, Bacterial, Type XXIV</td><td>Sigma-Aldrich</td><td>P8038</td><td></td></tr><tr><td>Calcium chloride solution, ~1 M in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=it&amp;amp;region=IT&quot;&gt;21115&lt;/a&gt;</td><td></td></tr><tr><td>Calcium chloride 0.1 M solution</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/53704?lang=it&amp;amp;region=IT&quot;&gt;53704&lt;/a&gt;</td><td></td></tr><tr><td>Potassium methanesulfonate</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/aldrich/83000?lang=it&amp;amp;region=IT&quot;&gt;83000&lt;/a&gt;</td><td></td></tr><tr><td>FluoForte Reagent</td><td>Enzo Life Sciences</td><td>ENZ-52015</td><td></td></tr><tr><td>Powerload concentrate, 100X</td><td>Life Technologies</td><td>P10020</td><td></td></tr><tr><td>Perfusion Fast-Step System</td><td>Warner Instruments</td><td>VC-77SP</td><td></td></tr><tr><td>Amphotericin B solubilized</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/a9528?lang=it&amp;amp;region=IT&quot;&gt;A9528 &lt;/a&gt;</td><td></td></tr><tr><td>Multiclamp 700B patch-clamp amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Digidata 1440A</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>pClamp10.0&amp;nbsp;</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Digestion Device</td><td>CUSTOM</td><td>CUSTOM</td><td>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.</td></tr><tr><td>Silicone elastomer for the digestion device&#039;s brushes</td><td>Dow Corning</td><td>SYLGARD&amp;reg; 184</td><td></td></tr><tr><td>Variable speed rotating motor for the digestion device</td><td>Crouzet</td><td>Crouzet 178-4765</td><td></td></tr><tr><td>Mold for brushes casting</td><td>N.A.</td><td>N.A.</td><td>The mold is custom made from standard PTFE 2.5 cm diameter rods.</td></tr></tbody></table>

isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples

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

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&#160;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.

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...

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.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

The overall goal of this procedure is to isolate viable cardiomyocytes from disease human ventricular specimens and characterize their functional alterations. This is accomplished by first rapidly collecting and mincing fresh ventricular samples in ice cold cardioplegic solution in order to avoid excessive tissue degradation and cell damage. The second step is to perform a stepwise digestion of the myocardial chunks using a custom made digestion device and enzymatic solutions.In six cycles, the cell containing buffer is collected in a tube at each cycle and diluted with a cell preserving solution. The final step is to resuspend the cells contained in the six tubes in a lower volume of standard physiological solution with normal calcium concentrations in order to perform the functional characterization. Ultimately, patch clamp recording of action potentials and simultaneous assessment of intercellular calcium using fluorescent dyes is used to show the alterations of action, potential duration, and intracellular calcium cycling in cardiomyocytes from patients with hypertrophic cardiomyopathy.The main advantage of DYS unique over a 16 method, like the standard chunk dejection method i

isolation-functional-characterization-human-ventricular

Research

JoVE Journal

Medicine

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system.  The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression.  Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolation and Physiological Analysis of Mouse Cardiomyocytes

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&lt;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

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.

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

Impara a isolare cardiomiociti ventricolari umani da campioni chirurgici freschi per studi elettrofisiologici e test farmacologici. Questo metodo consente di caratterizzare alterazioni funzionali nelle malattie cardiache.

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

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