Name: isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of ca2+ transients and l-type calcium current
Uploaded: 2020-11-03T15:00:00.000+00:00
Duration: 6 min 22 s
Description: Watch this Scientific Journal Video about Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current at JoVE.com

This work was supported by German Research Foundation (DFG; Clinician Scientist Program In Vascular Medicine (PRIME), MA 2186/14-1 to P. Tomsits and D. Sch&#252;ttler; VO1568/3-1, IRTG1816, and SFB1002 project A13 to N. Voigt), German Research Foundation under Germany&#8217;s Excellence Strategy (EXC 2067/1- 390729940 to N. Voigt), German Centre for Cardiovascular Research (DZHK; 81X2600255 to S. Clauss and N. Voigt; 81Z0600206 to S. K&#228;&#228;b), the Corona Foundation (S199/10079/2019 to S. Clauss), the ERA-NET on Cardiovascular Diseases (ERA-CVD; 01KL1910 to S. Clauss), the Heinrich-and-Lotte-M&#252;hlfenzl Stiftung (to S. Clauss) and the Else-Kr&#246;ner-Fresenius Foundation (EKFS 2016_A20 to N. Voigt). The funders had no role in manuscript preparation.

All animal procedures were approved by the Lower Saxony Animal Review Board (LAVES, AZ-18/2900) and were conducted in accordance with all institutional, national, and international guidelines for animal welfare.
1. Prearrangements
<ol>
	<li>Prepare 1 L of 10x perfusion buffer (Table 1), 500 mL of 1x perfusion buffer (Table 2), 50 mL of digestion buffer (Table 3), 10 mL of stop buffer (Table 4), 1 L of Tyrode solution (Table 5), 10 mL of each calcium step solution (Tyrode solution with glucose and respective amount of calcium as indicated), 1 L of 4-AP solution (Table 5), 100 mL of pipette solution (Table 6) and 5 mL of pluronic acid&#160;according to the provided recipes. 
	NOTE: Bath solutions (Tyrode and 4-AP solution) can be prepared (without glucose) in advance and stored at +4 &#176;C, glucose is added on the experimental day. Pipette solution can be stored at -20 &#176;C, calcium indicator is added on the experimental day and solution then stored on ice until further use. 10x perfusion buffer can be stored at room temperature, 1x perfusion buffer, digestion buffer and stopping buffer should be freshly prepared on experimental day.</li>
	<li>Turn on the water bath and roller pump.</li>
	<li>Prefill Langendorff apparatus with perfusion buffer; make sure it is air-free.</li>
	<li>Prepare aortic cannula by fixing it under the dissection microscope, connect with a 1 mL syringe filled with perfusion buffer and clear air by rinsing the cannula. 
	NOTE: It is crucial to avoid any air inside the perfusion system, as this will directly affect coronary perfusion and thus digestion effectiveness. A bubble trap might be added to the setup if necessary, to safely avoid any air trapping.</li>
	<li>Prepare Petri dishes with enough perfusion buffer for organ collection and microscopy (buffer should securely cover the entire organ, a few millilitres &#8211; depending on the used Petri dish size &#8211; should be enough).</li>
	<li>Prepare 3 Petri dishes with digestion buffer for tissue dissociation and microscopy, buffer should cover the organ for dissection under the microscope within the respective Petri dish, amount depends on the used Petri dish size. For the dissociation use 3 mL for ventricular tissue and 1.5 mL for atrial tissue.</li>
</ol>
2. Organ harvest
<ol>
	<li>Inject mouse with 0.1 mL of heparin (1,000 U/mL) i.p. using a 1 mL syringe with a 27 G cannula and wait for 5-10 min.</li>
	<li>Place the mouse into an induction chamber along with a small tissue soaked in approximately 500 &#181;L of isoflurane. The animal should not be in contact with the tissue. To avoid that, one can use a plastic biopsy-embedding cassette to cover the tissue. Once the animal is fully anesthetized, check for toe pinch reflex and as soon as it is not present anymore, quickly euthanize the mouse by cervical dislocation.</li>
	<li>Place the mouse on a platform on its back (e.g., on Styrofoam covered with a paper towel) and fix the paws down with cannulas to hold it in place.</li>
	<li>Remove fur and skin covering the chest and part of the abdomen with a clear cut from jugulum towards symphysis and open the abdomen right under the xiphoid without injuring any organ structure using scissors. Lift the sternum with surgical forceps and cut the diaphragm with scissors along the edge of the ribs, then cut the ribs in medial axillary line and remove the rib cage to expose the heart.</li>
	<li>Carefully remove the pericardium using blunt forceps and quickly remove the heart by lifting it with blunt forceps from below and by cutting the large vessels with one single cut using scissors.</li>
	<li>Put the heart into room temperature perfusion buffer and cannulate the aorta with a blunt end needle under the microscope as quickly as possible. 
	NOTE: Remove any lung tissue and fatty tissue attached to the organ without losing too much time on it. While cannulating, make sure that the end of the needle does not extend through the aortic valve, as this will impair results by preventing buffers from entering the coronary arteries.</li>
	<li>Tie the heart with a piece of suturing silk firmly to the needle and disconnect from syringe. 
	NOTE: The entire procedure from obtaining the heart (the moment when the large vessels are cut) until suturing the aorta to the needle should take as little time as possible. It is recommended not taking longer than 90-180 s from removing the heart until start of perfusion.</li>
</ol>
3. Enzymatic digestion
<ol>
	<li>After aortic cannulation, immediately connect the cannulated heart to the Langendorff apparatus avoiding any air entering the system. 
	NOTE: It can help to have a hanging drop of perfusion buffer at the bottom of the Langendorff apparatus as well as a drop of perfusion buffer sitting on the top of the needle in order to avoid any air entering the system.</li>
	<li>Perfuse the heart with perfusion buffer for 1 min at a temperature of exactly 37 &#176;C and a perfusion rate of exactly 4 mL/min. 
	NOTE: In order to have a temperature of 37 &#176;C at the tip of the perfusion needle, water bath temperature has to be set slightly above at approximately 40 &#176;C. This should be tested regularly by measuring the temperature at the perfusion tip.</li>
	<li>Switch perfusion to digestion buffer and perfuse for exactly 9 min at a temperature of exactly 37 &#176;C and a perfusion rate of exactly 4 mL/min.</li>
	<li>Transfer the digested heart to a Petri dish with enough digestion buffer to keep it fully covered. Then carefully dissect the atria and ventricles under the microscope.</li>
	<li>Transfer the atria into a Petri dish with 1.5 mL of digestion buffer and the ventricles into another Petri-dish with 3 mL of digestion buffer.</li>
	<li>Atrial dissection
	<ol>
		<li>Carefully, but without loss of time, pull the atria apart into tiny pieces using blunt forceps.</li>
		<li>Dissolve the tissue by carefully pipetting up and down using a 1,000 &#181;L pipette tip, which has previously been cut to widen the tip opening.</li>
		<li>Transfer the solution to a 15 mL centrifuge tube and add an equivalent amount of stop buffer (1.5 mL) by carefully pipetting down at the side of the tube to end the reaction.</li>
		<li>Carefully pass all 3 mL of cell/tissue-solutions through a 200 &#181;m nylon mesh to remove remaining larger tissue pieces that have not been fully digested. 
		NOTE: A successful digestion will leave almost no solid chunks.</li>
	</ol>
	</li>
	<li>Ventricular dissection
	<ol>
		<li>Quickly chop the ventricular tissue into tiny pieces using dissection scissors and pipette up and down to dissolve. Use another 1,000 &#181;L pipette tip for pipetting up and down, it may be shortened to widen the opening.</li>
		<li>Transfer cell/tissue-solution into a 15 mL centrifuge tube and add an equivalent amount of stop buffer (3 mL) by carefully pipetting down at the side of the tube to end the reaction.</li>
		<li>Carefully pass all 6 mL of cell/tissue-solution through a 200 &#181;m nylon mesh to remove larger tissue pieces that have not been fully digested. 
		NOTE: A successful digestion will leave almost no solid chunks.</li>
	</ol>
	</li>
	<li>Leave both tubes (atrial and ventricular cell suspension) on the bench at room temperature for 6 min to settle.</li>
	<li>Centrifuge both 15 mL tubes at 5 x g for 2 min.</li>
</ol>
4. Calcium reintroduction
NOTE: The following steps are identical for both atrial and ventricular cells (unless otherwise mentioned) and are performed at room temperature.
<ol>
	<li>Discard the supernatant using a plastic Pasteur pipette and carefully resuspend pellet in 10 mL of calcium free Tyrode solution.</li>
	<li>Leave cells for 8 min for sedimentation.</li>
	<li>Centrifuge at 5 x g for 1 min (only atrial cells, sedimentation is enough for ventricular cells).</li>
	<li>Discard supernatant and carefully resuspend pellet in 10 mL of Tyrode solution with 100 &#181;M calcium concentration.</li>
	<li>Leave cells for 8 min for sedimentation.</li>
	<li>Centrifuge at 5 x g for 1 min (only atrial cells, sedimentation is enough for ventricular cells).</li>
	<li>Discard the supernatant and carefully resuspend pellet in 10 mL of Tyrode solution with 400 &#181;M calcium concentration.</li>
	<li>Leave cells for 8 min for sedimentation.</li>
	<li>Centrifuge at 5 x g for 1 min (only atrial cells, sedimentation is enough for ventricular cells).</li>
	<li>Discard the supernatant and carefully resuspend the pellet in 1 mL (atrial)/ 5 mL (ventricular) of Tyrode solution with 1 mM calcium concentration.</li>
</ol>
5. Loading of myocytes with fluorescent calcium-indicator Fluo-3 AM
NOTE: Due to the light sensitivity of the fluorescent calcium indicator, the following steps should be executed protected from light (e.g., by covering tubes with aluminium foil).
<ol>
	<li>Prepare Fluo-3 AM stock solution by adding 44 &#181;L of 20% pluronic F-127 in anhydrous DMSO to 50 &#181;g of Fluo-3AM (can be stored at -20 &#176;C protected from light).</li>
	<li>Add 10 &#181;L of Fluo-3 AM stock solution to 1 mL of cell suspension and incubate for 10 min at room temperature protected from light.</li>
	<li>Centrifuge at 5 x g for 1 min.</li>
	<li>Discard the supernatant using a plastic Pasteur pipette and resuspend the pellet in a reasonable amount of bath solution to obtain a good working concentration (1-5 mL of bath solution depending on the cell density).</li>
	<li>Leave for 30 min for de-esterification before starting with experiments.</li>
</ol>
6. Simultaneous patch-clamp and epifluorescent Ca2+-transient measurements as previously described16
NOTE: Patch clamp measurements are not the topic of this article, the interested reader may be referred to major publications providing in depth descriptions of this method17,18,19,20,21,22. Nevertheless, for a better overall understanding, a summary on a protocol to measure L-type calcium currents along with current induced calcium transients is provided.
<ol>
	<li>Transfer myocytes into a cell chamber and superfuse with bath solution at 37 &#176;C.</li>
	<li>Block potassium currents by adding 4-aminopyridine and barium chloride to the bath solution as indicated in Table 5.</li>
	<li>Ensure that borosilicate microelectrodes have a tip resistance of 2-5 M&#8486; filled with pipette solution (Table 6).</li>
	<li>Setup measurements to allow for simultaneous recording of both electrical signals and epifluorescence at the same time. Voltage clamp mode is used to measure L-type Ca2+-current with a protocol holding the cell at -80 mV and a 600 ms ramp-pulse to -40 mV to inactivate the fast Na+-current, followed by a 100 ms test-pulse to +10 mV at 0.5 Hz (Figure 2).</li>
	<li>Use excitation at 488 nm, emitted light at &#60;520 nm to detected and convert to [Ca2+]I assuming 
	<img alt="Equation 1" src="/files/ftp_upload/61964/61964equ01.jpg" /> 
	Where kd = dissociation constant of Fluo-3 (864 nM), F = Fluo-3 fluorescence; Fmax = Ca2+-saturated fluorescence obtained at the end of each experiment19.</li>
</ol>

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This article provides an easy and functional way to obtain high quality atrial and ventricular myocytes from the same mouse for patch-clamp studies with simultaneous calcium transient recordings. The quality of the obtained data highly depends on the quality of the cell isolation. As mentioned above, many methods to isolate murine cardiomyocytes have been described previously9,10,11,12. The isolated cells are used for various analyses ranging from patch clamp experiments to contractility studies, morphological studies, proteomics, mitochondrial function research and many more. Each individual purpose requires isolated cells optimized differently. Due to this, none of the protocols led to high quality isolations suitable for patch-clamp experiments with simultaneous measurement of calcium handling properties of atrial and ventricular myocytes from one mouse. To change this, the adaption of numerous protocols described before resulted in the development of this protocol. Some steps are mechanically challenging, some are to be done in a specific fashion and some are simply crucial to isolation quality. These steps will be discussed below.
Yield and quality of cell isolation decrease with animal age, possibly due to increased tissue fibrosis11,23,24 making individual adjustments of digestion time and enzyme amount necessary. The older an animal, the more fibrotic tissue is to be expected. The indicated digestion times and enzyme amounts are a good starting point for normal sized, healthy mice at the age of 6 months, but will need adaption to individual needs. It is suggested to apply more enzyme and longer digestion times when using older mice or transgenic mice favouring fibrosis. As previous work has emphasized, rapid and air-free cannulation as well as immediate perfusion of the heart are absolutely critical for good cell quality11. It is recommended not taking longer than 90-180 seconds from obtaining the heart and putting it into room temperature perfusion buffer to start of perfusion &#8211; the faster, the better. It is helpful to set up all the needed equipment in one room to avoid long distances. If further shortening of the ischemic interval is desired, it is possible to use another method of narcosis and subsequent euthanization. Fully anesthetize the animal as described above, apply a sufficient amount of fentanyl (or an equivalent analgesic medication in accordance to your guidelines for animal welfare) i.p. and move the animal to a platform with an isoflurane vaporizer and an anaesthesia mask suitable for mice, providing constant isoflurane and oxygen flow. Afterwards proceed as described above. Since the animal has a normal circulation until the large vessels are disconnected with a single cut, this method will reduce the no-flow time significantly and can thus improve isolation quality if needed.
After harvesting the heart, it is critical to place the aortic cannula at the right depth to secure coronary perfusion. It is, therefore, necessary to cut the aorta at the aortic arch or at the descendent aorta when removing the heart to leave at least 5 mm of the aorta for cannulation allowing to tie the suturing silk distal from the coronary arteries but before the first aortic side-branches.
During the isolation process, multiple cell transfers are necessary and pipetting with small pipette tips will cause high shear stress to the cells. Possible solutions to reduce shear stress are pipetting slowly and carefully, as well as cutting pipette tips by some millimetres in a 45&#176; angle to widen the tip opening. Further reduction of shear stress can be achieved by melting the cut pipette tips to soften the edges. Isolation yield of the ventricular isolation usually is so high that shear stress reduction is not obligatory. Depending on one&#8217;s individual goal, it can even be more useful to expose ventricular cells to shear stress to select for the &#8217;fittest&#8217; cells. However, for the atrial isolation this procedure is critical, as atrial cells are particularly vulnerable after digestion. Whenever adding solutions, it is recommended to avoid pipetting directly onto the cells, but rather trying to pipet slowly at the tube wall.
The choice of collagenase will significantly affect isolation results. Since there are not only differences in enzyme preparations but also a relevant batch-to-batch variation in enzyme activity, titration of collagenase amount and digestion time are necessary when starting to work with a new batch of enzyme or a new strain of mice. However, the amount and time indicated in the tables above mark a good starting point for individual optimization9. Most companies provide small enzyme samples to test individual batches, which makes batch selection much more convenient.
Calcium transients with typical amplitudes and normal monophasic decay can be obtained if cells are isolated without the use of EGTA25 as previously described by Voigt et al.9,16. This protocol, therefore, uses low calcium concentrations and avoids EGTA during isolation process. To protect cells from the Ca2+ paradox phenomenon26, calcium is reintroduced stepwise and slowly until a final concentration of 1 mM. Due to higher intracellular sodium concentrations, rodent cardiomyocytes are especially prone to calcium overload and slow and stepwise reintroduction is crucial27. The need of calcium free solutions represents one of the major limitations of all Langendorff-based rodent cardiomyocyte isolations. Calcium reintroduction always leads to a significant loss of viable cells. Slow and stepwise reintroduction as described here nevertheless, leads to sufficient yield with good quality to obtain measurements from each animal reliably.
It is important to assess cell yield and quality right after calcium reintroduction. Although final evaluation takes place while patching the isolated cells, one can already judge cell quantity and quality by having a look under the microscope prior to loading cells with calcium sensitive dye. Good quality cells are clearly rod-shaped, have a homogenous membrane and are non-contracting. Contraction is a sign of low cell quality as it indicates a loss of membrane integrity and might be caused by overdigestion or application of high shear stress. Another sign of overdigestion is &#8211; besides many cell shadows in relation to viable cells &#8211; an overly nice-looking membrane that has been cleared of many surface proteins and is extremely vulnerable when one tries to approach the cell with the pipette tip for seal formation. Exemplary healthy, good quality cells are shown in Figure 3. (Panel A: atrial myocyte, Panel B: ventricular myocyte). Isolations with high yields can lead to cells immune to seal formation and vice versa. In the end, the ultimate quality test regardless of cell looks and yield, is the answer to a simple question: &#8216;Is it possible to perform the desired measurements with high quality results in the isolated cells of each isolation?&#8217;. The protocol provided above makes a positive answer possible.
Many isolation protocols use the uncoupling agent Blebbistatin to obtain higher yields and better-quality cells. This protocol purposely avoids Blebbistatin, taking into consideration the published evidence of its influence on cardiac electrophysiology28,29 in optical mapping experiments. The use of uncoupling agents in electrophysiologic studies must be treated with caution and should be limited to circumstances where uncoupling is mandatory.
Cells obtained with this protocol can be used for approximately six hours after loading with Fluo-3 with atrial cells being more vulnerable to time. Consequently, it is recommended to study atrial cells before ventricular cells if the same researcher processes them.
One limitation of this protocol &#8211; as true for most Langendorff-based isolation protocols &#8211; is that it is technically difficult. Clearly, mouse hearts are small, and all technical aspects require a lot of exercise. This means that an experienced researcher will obtain better results. Some protocols use a constant pressure to perfuse the heart. This has the advantage that due to digestion and consecutive loss of tissue resistance the perfusion will accelerate, and this helps to define the optimal digestion time, nevertheless the acceleration can harm the tissue und decrease cell quality significantly. In a constant flow approach as used here, there are no clear benchmarks for digestion times and it can often be hard to stop the digestion at an optimal time, which marks a relevant limitation. Another limitation is that in this protocol isolated cells were only tested for suitability to the experiments described above. It is likely that these cells can be used for different analysis as well, but this still must be proven. A first step into that direction was usage of this cells to visualize action potentials by loading them with VF2.1CI, a recently derived voltage sensitivity dye with superior kinetics to previously used voltage sensitive dyes according to a protocol published by Seibertz et al.30. To help with troubleshooting, Table 7 shows common isolation problems and how to approach them.
Taken together, the described isolation protocol offers a working approach to the simultaneous isolation of murine atrial and ventricular cardiomyocytes, enabling the researcher to obtain an atrial and ventricular electrophysiological phenotype of a single mouse. This removes statistical obstacles posed by previous isolation protocols only isolating either atrial or ventricular cells at high quality and helps to reduce the number of animals needed for a specific study. This can be especially important if interventions as for example the implantation of osmotic minipumps filled with expensive agents are part of the experiments and it can be a vital factor in animal welfare considerations.

Cardiac arrhythmias are common and one of the current major healthcare challenges since they affect millions of people worldwide. Arrhythmias are associated with high morbidity and mortality1,2 and represent the underlying cause for the majority of sudden cardiac deaths3. Up to date treatment options have improved patient survival but are still mainly symptomatic treatments rather than targeting the underlying mechanisms. Thus, these treatments have limited efficacy and may frequently cause severe side effects4,5,6. An improvement of current treatment options requires insight into the underlying pathophysiology, creating the need for suitable models to study. Small animal models - and specifically mouse models - play a crucial role in arrhythmia research as they allow to study key mechanisms of arrhythmogenesis, for example the genetic impact on cellular electrophysiology, ion channel function or calcium handling7,8.
For this purpose, isolated atrial and ventricular cardiomyocytes of sufficient quantity and viability are required. A broad spectrum of different isolation approaches to obtain atrial and ventricular myocytes has been previously described9,10,11,12,13 and some groups have presented data from simultaneous measurements of L-type current and calcium current induced calcium transients from either atrial14 or ventricular15 murine cardiomyocytes. However, to our best knowledge there is no data available of atrial as well as ventricular measurements from one animal. Researchers focus on a broad variety of topics ranging from electrophysiology to proteomics, functional studies as cell contractility or protein interactions, mitochondrial function, or genetics &#8211; all in need of isolated cardiomyocytes. Many of the published protocols thus have not been specifically developed for patch clamp studies, leading to limited yields and insufficient cell quality for patch clamp studies. Thus, simultaneous patch clamp and calcium transient measurements of atrial and ventricular cells isolated from one animal cannot be performed with established protocols.
Isolation of murine &#8211; especially atrial &#8211; myocytes for patch clamp experiments remains challenging. This article provides a simple and fast method for the isolation of high-quality murine atrial and ventricular myocytes via retrograde enzyme based Langendorff perfusion, which subsequently allows simultaneous measurements of both net membrane current and current induced calcium transients from one animal. This article elaborates a protocol for the isolation of atrial and ventricular myocytes derived from wild type mice and mice carrying genetic mutations. This protocol can be used for male and female mice alike. The myocyte isolation, images, and representative results described below were obtained from wild type C57Bl/6 mice at the age of 6 (&#177; 1) months. Nevertheless, this protocol has successfully been used for mice at various ages ranging from 2 to 24 months with different genotypes. Figure 1 shows the isolation setup and a close-up of a cannulated heart during enzyme perfusion.

<table><tbody><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich</td><td>31550</td><td></td></tr><tr><td>27G cannula</td><td>Servoprax</td><td>L10220</td><td></td></tr><tr><td>4-Aminopyridine</td><td>Sigma-Aldrich</td><td>A78403</td><td></td></tr><tr><td>Anhydrous DMSO</td><td>Sigma-Aldrich</td><td>D12345</td><td></td></tr><tr><td>Aortic cannula</td><td>Radnoti</td><td>130163-20</td><td></td></tr><tr><td>BaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>342920</td><td></td></tr><tr><td>blunt surgical forceps</td><td>Kent Scientific</td><td>INS650915-4</td><td></td></tr><tr><td>Bovine Calf Serum</td><td>Sigma-Aldrich</td><td>12133C</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5080</td><td></td></tr><tr><td>Circulating heated water bath</td><td>Julabo</td><td>ME</td><td></td></tr><tr><td>Collagenase Type II</td><td>Worthington</td><td>LS994177</td><td></td></tr><tr><td>disscetion scissors</td><td>Kent Scientific</td><td>INS600124</td><td></td></tr><tr><td>DL-aspartat K&lt;sup&gt;+&lt;/sup&gt;-salt</td><td>Sigma-Aldrich</td><td>A2025</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E4378</td><td></td></tr><tr><td>Fluo-3</td><td>Invitrogen</td><td>F3715</td><td></td></tr><tr><td>Fluo-3 AM</td><td>Invitrogen</td><td>F1242</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Guanosine 5&amp;prime;-triphosphate tris salt</td><td>Sigma-Aldrich</td><td>G9002</td><td></td></tr><tr><td>Heating coil</td><td>Radnoti</td><td>158821</td><td></td></tr><tr><td>Heparin</td><td>Ratiopharm</td><td>25.000 IE/5ml</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H9136</td><td></td></tr><tr><td>induction chamber</td><td>CWE incorporated</td><td>13-40020</td><td></td></tr><tr><td>Isoflurane</td><td>Cp-pharma</td><td>1214</td><td></td></tr><tr><td>Jacketed heart chamber</td><td>Radnoti</td><td>130160</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>1049360250</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>MgCl x 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; x 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M9397</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;ATP</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S5136</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Nylon mesh (200 &amp;micro;m)</td><td>VWR-Germany</td><td>510-9527</td><td></td></tr><tr><td>pasteur pipette</td><td>Sigma Aldrich</td><td>Z331759</td><td></td></tr><tr><td>petri-dishes</td><td>Thermo Fisher</td><td>150318</td><td></td></tr><tr><td>Pluronic Acid F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>Probenecid</td><td>Sigma-Aldrich</td><td>P8761</td><td></td></tr><tr><td>Roller Pump</td><td>Ismatec</td><td>ISM597D</td><td></td></tr><tr><td>surgical forceps</td><td>Kent Scientific</td><td>INS650908-4</td><td></td></tr><tr><td>surgical scissors</td><td>Kent Scientific</td><td>INS700540</td><td></td></tr><tr><td>suturing silk</td><td>Fine Science Tools</td><td>NC9416241</td><td></td></tr><tr><td>syringe</td><td>Merck</td><td>Z683531-100EA</td><td></td></tr><tr><td>Taurin</td><td>Sigma-Aldrich</td><td>86330</td><td></td></tr></tbody></table>

isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of ca2+ transients and l-type calcium current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation yield is determined after calcium reintroduction by pipetting 10 &#181;L of cell suspension onto a microscope slide. More than 100 viable, rod-shaped, non-contracting cells/10 &#181;L for atrial cell isolation and more than 1,000 viable, rod-shaped, non-contracting cells/10 &#181;L for ventricular cell isolation are considered as sufficient yield and are commonly obtained using this protocol. Atrial cells obtained with this protocol showed a variety of different cell types containing cells of the cardiac conduction system, especially the sinus node, as well as different myocytes from the right and left atrium as well as the atrial appendages. Since these regions are not dissected separately, various cell morphologies are the result. All atrial cells are small, with cell capacitances ranging from approximately 35 pF&#8211; 100 pF, some are more filiform than others. Figure 3 panel A shows a typical cell from the atrial working myocardium loaded with calcium sensitive dye, ready for measurements obtained with this protocol. Ventricular myocytes are more rod-shaped and larger with cell capacitances ranging from 100 to around 400 pF, Figure 3 panel B shows a typical ventricular cell from the working myocardium loaded with calcium sensitive dye, ready for measurements obtained with this protocol. Figure 4 shows representative examples of L-type calcium current measurements with simultaneous cytosolic calcium transients from one atrial myocyte (panel B and C) and one ventricular myocyte (panel E and F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61964/61964fig01.jpg" /> 
Figure 1: Langendorff-apparatus.&#160;(A) Photograph of the isolation setup with view of the custom designed heat exchanger and jacketed heart chamber. (B) Close-up of jacketed heart chamber with cannulated heart in place. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61964/61964fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61964/61964fig02.jpg" /> 
Figure 2: Stimulation protocol. Voltage-clamp protocol (0.5 Hz) used to measure total net membrane current (IM), predominantly reflecting L-type Ca2+ current and Ca2+ current induced Ca2+ transient. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61964/61964fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61964/61964fig03.jpg" /> 
Figure 3: Isolated murine cardiomyocytes.&#160;(A) An example of an atrial cardiomyocyte loaded with Fluo-3, ready for patch-clamp experiments. (B) An example of a ventricular cardiomyocyte loaded with Fluo-3, ready for patch-clamp experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61964/61964fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61964/61964fig04.jpg" /> 
Figure 4: Representative recordings.&#160;(A) Voltage-clamp protocol (0.5 Hz). (B) Total net membrane current (IM), predominantly reflecting L-type Ca2+ current in a murine atrial myocyte. (C) L-type Ca2+ current triggered Ca2+ transient in a murine atrial myocyte. (D) voltage-clamp protocol (0.5 Hz). (E) Total net membrane current (IM), predominantly reflecting L-type Ca2+ current in a murine ventricular myocyte. (F) L-type Ca2+ current triggered Ca2+ transient in a murine ventricular myocyte. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61964/61964fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Final concentration (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>120.4</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>14.7</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.6</td>
		</tr>
		<tr>
			<td>Na2HPO4 x 2H2O</td>
			<td>0.6</td>
		</tr>
		<tr>
			<td>MgSO4 x 7H2O</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 1: 10x perfusion buffer (1,000 mL).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Final concentration (mM)</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>4.6</td>
		</tr>
		<tr>
			<td>Taurin</td>
			<td>30</td>
		</tr>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>10x perfusion buffer</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>double destilled H2O (18,2 M&#8486;-cm)</td>
			<td>500 ml</td>
		</tr>
	</tbody>
</table>
Table 2: 1x perfusion buffer (500 mL).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>~600 U/ml</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>40 &#181;M</td>
		</tr>
		<tr>
			<td>1x perfusion buffer</td>
			<td>50 ml</td>
		</tr>
	</tbody>
</table>
Table 3: Digestion buffer (50 mL).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Bovine Calf Serum</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>12.5 &#181;M</td>
		</tr>
		<tr>
			<td>1x perfusion buffer</td>
			<td>10 ml</td>
		</tr>
	</tbody>
</table>
Table 4: Stop buffer (10 mL).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td colspan="2">Final concentration (mM)</td>
		</tr>
		<tr>
			<td>Tyrode</td>
			<td>4-AP</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>140</td>
			<td>140</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>4</td>
			<td>4</td>
		</tr>
		<tr>
			<td>MgCl x 6H2O</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>4-Aminopyridine</td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td></td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.35 adjusted with 1 M NaOH at room temperature</td>
			<td>7.35 adjusted with 1 M NaOH at room temperature</td>
		</tr>
	</tbody>
</table>
Table 5: Bath solutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Final concentration (mM)</td>
		</tr>
		<tr>
			<td>DL-aspartat K+-salt</td>
			<td>92</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>48</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>5</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate tris salt</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Fluo-3</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.20 adjusted with 1 M KOH at room temperature</td>
		</tr>
	</tbody>
</table>
Table 6: Pipette solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem observed</td>
			<td>Possible reason</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Low cell yield, tissue chunks left</td>
			<td>Underdigestion</td>
			<td>Increase digestion time in 15 seconds increments</td>
		</tr>
		<tr>
			<td>Low cell yield, tissue chunks left</td>
			<td>Underdigestion</td>
			<td>Increase collagenase amount in 50 U/ml steps</td>
		</tr>
		<tr>
			<td>mix of over- and underdigested cells, tissue chunks left</td>
			<td>No coronary perfusion</td>
			<td>Make sure not to insert aortic cannula into the ventricle</td>
		</tr>
		<tr>
			<td>mix of over- and underdigested cells, tissue chunks left</td>
			<td>Air bubbles</td>
			<td>Make sure not to insert any air into the perfusion system</td>
		</tr>
		<tr>
			<td>Low overall cell quality</td>
			<td>Ischemic time too long</td>
			<td>Decrease ischemic time, consider alternative methods to maintain circulation as long as possible</td>
		</tr>
	</tbody>
</table>
Table 7: Common problems and how to solve them.

Watch this Scientific Journal Video about Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current at JoVE.com

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

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

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

isolation-high-quality-murine-atrial-ventricular-myocytes-for

Nanyang Technological University

Research

JoVE Journal

Biology

3.0K Views.  Ludwig-Maximilians University Munich (LMU). Muismodellen maken het mogelijk om belangrijke mechanismen van aritmogenese te bestuderen. Voor dit doel zijn cardiomyocyten van hoge kwaliteit nodig om patchklemmetingen uit te voeren. Hier wordt een methode beschreven om murine atriale en ventriculaire myocyten te isoleren via retrograde enzymgebaseerde Langendorff-perfusie, die gelijktijdige metingen van calciumtransiënten en L-type calciumstroom mogelijk maakt.

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

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 ...

Medicine

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Bioengineering

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

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.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

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.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

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 ...

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes.
Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte&#8217;s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

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

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