This research was supported by the DZHK (German Centre for Cardiovascular Research, D.B.), the EKFS (Else-Kr&#246;ner-Fresenius Stiftung, F.H.), and by the BMBF (German Ministry of Education and Research), as well as the BIH-Charit&#233; clinical scientist program funded by the Charit&#233; - Universit&#228;tsmedizin Berlin and the Berlin Institute of Health (F.H.).

<ol>
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IDF Consensus Worldwide Definition of the Metabolic Syndrome Available from: <a target="_blank" href="https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html">https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html</a> (2006)</li><li>Melenovsky, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+atrial+remodeling+and+function+in+advanced+heart+failure+with+preserved+or+reduced+ejection+fraction.">Left atrial remodeling and function in advanced heart failure with preserved or reduced ejection fraction.</a> Circulation: Heart Failure. 8 (2), 295-303 (2015).</li><li>Goette, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EHRA/HRS/APHRS/SOLAECE+expert+consensus+on+atrial+cardiomyopathies:+definition,+characterization,+and+clinical+implication.">EHRA/HRS/APHRS/SOLAECE expert consensus on atrial cardiomyopathies: definition, characterization, and clinical implication.</a> EP Europace. 18 (10), 1455-1490 (2016).</li><li>Schotten, U., Verheule, S., Kirchhof, P., Goette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+mechanisms+of+atrial+fibrillation:+a+translational+appraisal.">Pathophysiological mechanisms of atrial fibrillation: a translational appraisal.</a> Physiological Reviews. 91 (1), 265-325 (2011).</li><li>Hohendanner, F., DeSantiago, J., Heinzel, F. 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The authors have nothing to disclose.

Here, we first described a protocol for the isolation of single-cell ACMs from a rat model of MetS-related HFpEF that shows marked atrial remodeling22. The procedure is uniquely challenging as excessive fatty tissue can make the surgical preparation, as well as the cannulation of the aorta, increasingly difficult. The troubleshooting guide provided in Table 2 addresses the most common issues of the isolation procedure.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Possible Cause</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>No flow through butterfly hose</td>
			<td>Improper closure of the aorta with the cannulation knots</td>
			<td>Remove fatty tissue before tightening</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Add 3rd cannulation knot with respective indentation</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Pull knot with hand for increased force</td>
		</tr>
		<tr>
			<td></td>
			<td>Blocked needle</td>
			<td>Readjust position into the lumen</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Unblock by gentle pull with syringe</td>
		</tr>
		<tr>
			<td>Right atrium / coronary sinus not inflated</td>
			<td>Improper closure of the heart base</td>
			<td>Block flow with additional knots</td>
		</tr>
		<tr>
			<td></td>
			<td>Right atrium damaged during preparation</td>
			<td>Close leakage with clips/knots</td>
		</tr>
		<tr>
			<td>Poor digestion / atrium does not soften</td>
			<td>Reduced enzyme activity through wrong temperature</td>
			<td>Adjust temperature to 37 &#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td>Inactive/degraded enzyme</td>
			<td>Replace</td>
		</tr>
		<tr>
			<td></td>
			<td>Old/overly fibrotic atrium</td>
			<td>Increase digestion time (in steps of +50%)</td>
		</tr>
		<tr>
			<td></td>
			<td>Damaged aortic valve</td>
			<td>Shallow penetration during cannulation</td>
		</tr>
		<tr>
			<td>Poor cell yield</td>
			<td>Atrium over-digested</td>
			<td>Decrease digestion time (in steps of +25%)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Reduce intraluminal pressure by lowering the butterfly hose</td>
		</tr>
		<tr>
			<td></td>
			<td>Atrium under-digested</td>
			<td>Increase digestion time (in steps of 25%)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Increase intraluminal pressure by raising the butterfly hose</td>
		</tr>
		<tr>
			<td></td>
			<td>Cell dispersion too aggressive</td>
			<td>Be more gentle</td>
		</tr>
	</tbody>
</table>
Table 2: Troubleshooting guide. This table displays common issues of the isolation procedure and their respective solutions.
In a recent study, we have shown that the ZFS-1 obese rat model exhibits extensive atrial remodeling with an increased atrial size22. An increase in the left atrial size has been recognized as an important prognostic marker for diastolic dysfunction26 and causes a rarefication of the microvascular vessels relative to the tissue. This leads to a decreased distribution of the digestive buffer through the retrograde perfusion of the aorta during the isolation procedure, rendering the single-cell isolation in this model especially challenging. Other hallmark features of atrial remodeling, atrial fibrosis, and increased fibrosis have been shown for ZDF rats&#8212;a rat model for metabolic diabetes and one parent strain of the ZFS1 rats27. Collagen deposits impair the efficacy of the digestive enzymes to liberate the cardiomyocytes from the extracellular matrix and, therefore, require further adjustments of the cell isolation procedure28.
The mechanism by which the pressure device facilitates the improved isolation results in this model of atrial remodeling is most likely related to a localized attenuation of the coronary blood flow of the left atrium. One major component of the coronary blood flow is coronary perfusion pressure, which is defined as the gradient between the coronary artery pressure and the end-diastolic pressure of the respective cavity29. During the described procedure, a blockage of the heart base leads to a global increase in coronary artery pressure and intraluminal pressure throughout the heart. The subsequent puncture of the left atrium facilitates a local, selective drop of intraluminal pressure in the left atrial cavity. Thus, not only a large coronary perfusion pressure gradient is established, but the perfusion volume is also increased by the diverted digestion solution from the congested left ventricle to the left atrium.
In addition, the choice of digestion enzymes is crucial for the ACM isolation: purified enzyme blends of collagenase I and II have been shown to be superior to less targeted and less pure enzymes like collagenases30. This enzyme does not only allow for a higher yield of morphologically and functionally intact cardiomyocytes but also minimizes any clumping of single cells after their isolation31. Purified enzyme blends of collagenases with an additional high dispase or medium thermolysin content are most commonly used for rat cardiomyocyte isolations. While VCMs are best isolated at higher concentrations, the best results of atrial myocytes were acquired with a concentration of 0.195 W&#252;nsch Units/mL using this protocol32.
As the prevalence of MetS-related HFpEF is rising2 and atrial cardiomyopathies leading to atrial remodeling and atrial fibrillation are clinically highly relevant, research in this field is of pivotal interest. Many new animal models for HFpEF are emerging33,34 and atrial remodeling in line with an increased incidence of atrial rhythm disorders are hallmark features of the disease. The described method allows researchers to isolate viable single cardiomyocytes from rat models with atrial remodeling for a further study with an exceptionally high yield and preserved mechanical and electrical function.

MetS describes a cluster of risk factors for diabetes mellitus type-2 and cardiovascular disease and includes an increased arterial blood pressure, dyslipidemia (raised triglycerides and lowered high-density lipoprotein cholesterol), increased fasting glucose, and central obesity1. The worldwide prevalence of MetS is estimated to be 25&#8211;30% and constantly rising2. HFpEF is a heterogenous clinical syndrome often associated with MetS. The cardiac remodeling during HFpEF and its preceding phases (i.e., hypertensive heart disease) is also accompanied by a remodeling of the atria3. Reduced contractile function and structural changes of the left atrium have been associated with increased mortality, atrial fibrillation, and new-onset heart failure4. Atrial remodeling is characterized by changes in the ion channel function, Ca2+ homeostasis, atrial structure, fibroblast activation, and tissue fibrosis5. Left atrial remodeling in MetS-related HFpEF and its underlying pathological mechanisms are still poorly understood and require a further in-depth investigation. Animal models have proven to be a valuable tool and lead to many advances in the field of atrial cardiomyopathies6,7,8,9.
Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. An isolation, and the potential subsequent cell culture, allow for the investigation of signaling pathways, ionic channel currents, and excitation-contraction-coupling. Under physiologic conditions, cardiomyocytes do not proliferate. The fusion between the transcriptional regulatory sequences of an atrial natriuretic factor and a simian virus 40 large T antigen in transgenic mice led to the creation of the first immortalized ACMs, named AT-110. The further development of AT-1 cells gave rise to HL-1 cells, which cannot only be serially passaged but also contract spontaneously11. They do, however, show structural and functional differences compared to freshly isolated cells, such as a less organized ultrastructure, a high occurrence of developing myofibrils11, and a hyperpolarization-activated inward current12. The isolation of ventricular cardiomyocytes (VCM) in rats and mice from a variety of models is well established13,14,15,16,17,18,19. Generally, the excised heart is mounted to a Langendorff apparatus and retrogradely perfused with a Ca2+-free buffer containing digestive enzymes, such as collagenases and proteases. Calcium is then reintroduced in a stepwise manner to the physiological conditions. However, even though protocols dedicated to the isolation of ACMs are available20,21, due to increased fibrosis and pressure-related differences, their usefulness in disease models with atrial remodeling is limited.
In this article, we have implemented a protocol for the isolation of atrial single-cell cardiomyocytes from animals that show atrial remodeling (i.e., in particular for the ZFS1 rat model for MetS-related HFpEF)22. Existing isolation protocols were optimized and complemented by a simple, custom-made device to control and modify the intraluminal pressure of the cardiac cavities, leading to higher yields of morphologically and functionally intact cardiomyocytes. The following protocol provides the researcher with a step-by-step guide, a detailed description of the custom-made equipment, a list of solutions, as well as a comprehensive troubleshooting guide.

<table><tbody><tr><td>ZSF-1 Obese rat</td><td>Charles River Laboratories, Inc.</td><td></td><td>21 weeks old</td></tr><tr><td>Fine Iris Scissors</td><td>Fine Science Tools GmbH</td><td>14094-11</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools GmbH</td><td>14001-18</td><td></td></tr><tr><td>Micro Dressing Forceps (curved, serrated)</td><td>Aesculap, Inc.</td><td>BD312R</td><td></td></tr><tr><td>Tissue Forceps (straight, 1 x 2 teeth)</td><td>Aesculap, Inc.</td><td>BD537R</td><td></td></tr><tr><td>Tying Forceps (angled)</td><td>Aesculap, Inc.</td><td>MA624R</td><td></td></tr><tr><td>Rodent and Small Animal Guillotine</td><td>Kent Scientific Corp.</td><td>DCAP</td><td></td></tr><tr><td>Low Cost Induction Chamber 3.0 L</td><td>Kent Scientific Corp.</td><td>SOMNO-0730&amp;nbsp;</td><td></td></tr><tr><td>Butterfly Winged Infusion Set 21 G</td><td>Hospira, Inc.</td><td>181106101</td><td></td></tr><tr><td>Abbocath 16 G</td><td>Hospira, Inc.</td><td>0G7149702</td><td></td></tr><tr><td>Microlance Hypodermic Needle</td><td>Becton Dickinson GmbH</td><td>301300</td><td>modify needle to make cannula</td></tr><tr><td>Braun Original Perfusor Syringe 50 ml</td><td>B. Braun Melsungen AG</td><td>8728810F</td><td></td></tr><tr><td>Braun Inject Solo Syringe 10 ml</td><td>B. Braun Melsungen AG</td><td>2057926</td><td></td></tr><tr><td>Beaker 50ml</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 106 17</td><td></td></tr><tr><td>Duroplan petri dish (100 x 20 mm)</td><td>Duran Group (DWK Life Sciences GmbH)</td><td>21 755 48</td><td></td></tr><tr><td>Seraflex Suture USP 3/0</td><td>SERAG-WIESSNER GmbH &amp;amp; Co. KG</td><td>IC208000</td><td></td></tr><tr><td>VWR disposable Square Weighin Boats 100ml</td><td>VWR, Inc.</td><td>10803-148</td><td></td></tr><tr><td>Styrofoam surface</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, Inc.</td><td>71380</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich, Inc.</td><td>P4504</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich, Inc.</td><td>P5379</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich, Inc.</td><td>S0876</td><td></td></tr><tr><td>Magensium sulfate heptahydrate</td><td>Sigma-Aldrich, Inc.</td><td>230391</td><td></td></tr><tr><td>Magensium chloride</td><td>Sigma-Aldrich, Inc.</td><td>M8266</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, Inc.</td><td>H3375</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich, Inc.</td><td>T0625</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, Inc.</td><td>G7528</td><td></td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich, Inc.</td><td>B0753</td><td></td></tr><tr><td>Calcium chloride solution (1 M)</td><td>Sigma-Aldrich, Inc.</td><td>21115</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich, Inc.</td><td>A9647</td><td></td></tr><tr><td>Liberase</td><td>Roche (Sigma-Aldrich, Inc.)</td><td>LIBTM-RO</td><td></td></tr><tr><td>Heparin</td><td>Rotexmedica GmbH</td><td>3862357</td><td></td></tr><tr><td>Forene (Isoflurane)</td><td>Abbvie Deutschland GmbH &amp;amp; Co. KG</td><td>10182054</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma-Aldrich, Inc.</td><td>L2020</td><td></td></tr><tr><td>WillCo glass-bottom dish 500&amp;micro;l 0.005mm</td><td>WillCo Wells B.V.</td><td>HBST-3522</td><td></td></tr><tr><td>Fluo4 AM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>F14201</td><td>5&amp;micro;M for 20min at RT</td></tr><tr><td>Di-8-ANNEPS</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>D3167</td><td>10&amp;micro;M for 45 min at 37&amp;deg; C&amp;nbsp;</td></tr><tr><td>Mitotracker RED FM</td><td>Invitrogen (Thermo Fisher Scientific, Inc.)</td><td>M22425</td><td>20nM for 30 min at 37&amp;deg; C</td></tr><tr><td>Jacketed reaction vessel 500 ml</td><td>Gebr. Rettberg GmbH</td><td>107024414</td><td></td></tr><tr><td>Jacketed reaction vessel 1000 ml</td><td>Gebr. Rettberg GmbH</td><td>107025414</td><td></td></tr><tr><td>Jacketed bubble trap</td><td>Gebr. Rettberg GmbH</td><td>134720001</td><td></td></tr><tr><td>ED heating immersion circulator</td><td>Julabo GmbH</td><td>9116000</td><td></td></tr><tr><td>Reglo Digital MS-2/6 peristaltic pump</td><td>Ismatec (Cole-Parmer Gmbh)</td><td>ISM 831</td><td></td></tr><tr><td>Voltcraft Thermometer 302 K/J</td><td>Conrad Electronic SE</td><td>030300546</td><td></td></tr><tr><td>Tubing</td><td></td><td></td><td></td></tr><tr><td>LSM 700 microscope</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>ZEN 2.3 imaging software</td><td>Carl Zeiss, Inc.</td><td>410135-1011-240&amp;nbsp;</td><td></td></tr><tr><td>Single channel heater controller TC-324B</td><td>Warner Instruments, LLC</td><td>64-2400</td><td></td></tr><tr><td>8 channel perfusion system</td><td>Warner Instruments, LLC</td><td>64-0185</td><td></td></tr><tr><td>8 channel Multi-Line In-Line Solution Heaters</td><td>Warner Instruments, LLC</td><td>64-0105</td><td></td></tr></tbody></table>

isolation of atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

At 21 weeks of age, 60–90% of viable ACMs (estimated as described in step 6.1), after the calcium re-adaptation (step 5.4–5.7), can be isolated from ZSF-1 obese rats by this method (Figure 4A). In rats, ACMs are characterized by a different and more heterogenous phenotype compared to VCMs24,25. Figure 4B shows an individual ACM with preserved membranes and sarcomere structure, both strong indicators of a functionally integral cell.
The acquired ACMs can be processed in a variety of ways. As exemplified in Figure 5, the cells might be loaded with fluorescent dyes used to study morphology and/or function. For instance, di-8-ANEPPS was used to delineate the tubular system in an atrial rat cell (Figure 5A). In another set of experiments, mitochondria-staining far red-fluorescence dye (e.g., mitotracker-Red-FM) is employed to detect cytosolic mitochondria (Figure 5B) in atrial remodeling. As shown in Figure 5C, ACMs isolated with this protocol are also suitable for live-cell Ca2+ imaging and show intact excitation-contraction coupling with a 1 Hz field-stimulation. All images can be used for further analysis using a variety of algorithms available to the researcher6,7 (Figure 5D).
<img alt="Microscopic images showing bacterial colony morphology at 10x and fiber structure at 50 µm scale." class="xfigimg" src="/files/ftp_upload/57953/57953fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Respective yield of viable, single-cell AM. (A) This panel shows the yield of an isolation after the re-adaptation of ACMs to 1 mM Ca2+ in NT. (B) This panel shows an isolated, single-cell atrial cardiomyocyte. The sarcomere structure, cell membrane, and nucleus are clearly visible. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57953/57953fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy images and graphs showing calcium dynamics in muscle fibers using fluorescence analysis." class="xfigimg" src="/files/ftp_upload/57953/57953fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Staining of rat ACMs with fluorescent dyes. (A) This panel shows the staining of a cell membrane and tubular network with the fluorescent dye Di8ANNEPS. (B) This panel shows the staining of mitochondria with the fluorescent dye mitotracker-Red-FM. (C) This panel shows the transversal line scan of a Ca2+-excitation with the fluorescent dye Fluo4-AM over a single cell. The arrows indicate the electrical stimulation, conducted at 1 Hz. (D) This panel shows the longitudenal Ca2+ transients derived from panel C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57953/57953fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction at JoVE.com

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

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.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

isolation-atrial-cardiomyocytes-from-rat-model-metabolic-syndrome

Nanyang Technological University

Research

JoVE Journal

Medicine

10.1K Views.  Charité University Medicine. Hier beschrijven we een geoptimaliseerd, Langendorff gebaseerde procedure voor de isolatie van eencellige atriale cardiomyocytes van een rat model van metabole syndroom-gerelateerde hartfalen met bewaarde ejectie Fractie. Een handmatige regulering van de intraluminale druk van cardiale Holten is geïmplementeerd om de opbrengst van functioneel intact myocytes geschikt voor excitatie-contractie-koppeling studies.

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

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Biology

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.

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 ...

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

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

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

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

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

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 (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

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

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

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.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

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.

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

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