Name: isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples
Uploaded: 2014-04-21T15:45:00
Description: Watch this Scientific Journal Video about Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples at JoVE.com

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.
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			<td colspan="2">Solut...

<ol>
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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.

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			<td height="21" style="height:21px;width:495px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:139px;"><a href="http://www.sigmaaldrich.com/catalog/product/sigma/p9791?lang=it&amp;region=IT">P9791&#160;</a></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate heptahydrate(MgSO4 * 7H2O)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m1880?lang=it&amp;region=IT">M1880&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/h3375?lang=it&amp;region=IT">H3375&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9251?lang=it&amp;region=IT">A9251&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g8270?lang=it&amp;region=IT">G8270&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m4125?lang=it&amp;region=IT">M4125&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7653?lang=it&amp;region=IT">S7653</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=it&amp;region=IT">P9333&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7907?lang=it&amp;region=IT">S7907&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s6297?lang=it&amp;region=IT">S6297&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/237205?lang=it&amp;region=IT">237205</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p2256?lang=it&amp;region=IT">P2256&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/b0753?lang=it&amp;region=IT">B0753&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide(NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s8045?lang=it&amp;region=IT">S8045&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid monopotassium salt monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/49601?lang=it&amp;region=IT">49601</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/107360?lang=it&amp;region=IT">107360</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Hydroxybutyric acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/166898?lang=it&amp;region=IT">166898</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate (K2-ATP)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a8937?lang=it&amp;region=IT">A8937</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatine</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c0780?lang=it&amp;region=IT">C0780&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s3674?lang=it&amp;region=IT">S3674&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/e0396?lang=it&amp;region=IT">E0396&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=it&amp;region=IT">A0281</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m8266?lang=it&amp;region=IT">M8266&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from Clostridium histolyticum, Type V</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c9263?lang=it&amp;region=IT">C9263&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase, Bacterial, Type XXIV</td>
			<td>Sigma-Aldrich</td>
			<td>P8038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride solution, ~1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=it&amp;region=IT">21115</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride 0.1 M solution</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/53704?lang=it&amp;region=IT">53704</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/83000?lang=it&amp;region=IT">83000</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoForte Reagent</td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-52015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerload concentrate, 100X</td>
			<td>Life Technologies</td>
			<td>P10020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusion Fast-Step System</td>
			<td>Warner Instruments</td>
			<td>VC-77SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B solubilized</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9528?lang=it&amp;region=IT">A9528&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1440A</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pClamp10.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="108">
			<td height="108" style="height:108px;">Digestion Device</td>
			<td>CUSTOM</td>
			<td>CUSTOM</td>
			<td style="width:255px;">The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Fig 1 C-D and in Fig.7. We can provide further details if requested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone elastomer for the digestion device&#39;s brushes</td>
			<td>Dow Corning</td>
			<td>SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable speed rotating motor for the digestion device</td>
			<td>Crouzet</td>
			<td>Crouzet 178-4765&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mold for brushes casting</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td style="width:255px;">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

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...

Watch this Scientific Journal Video about Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples at JoVE.com

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

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 ...

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