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.
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 °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 °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.
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 – 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 – especially atrial – 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 (± 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.
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
2. Organ harvest
3. Enzymatic digestion
4. Calcium reintroduction
NOTE: The following steps are identical for both atrial and ventricular cells (unless otherwise mentioned) and are performed at room temperature.
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).
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.
Isolation yield is determined after calcium reintroduction by pipetting 10 µL of cell suspension onto a microscope slide. More than 100 viable, rod-shaped, non-contracting cells/10 µL for atrial cell isolation and more than 1,000 viable, rod-shaped, non-contracting cells/10 µL 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– 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).
Figure 1: Langendorff-apparatus. (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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 3: Isolated murine cardiomyocytes. (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. Please click here to view a larger version of this figure.
Figure 4: Representative recordings. (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. Please click here to view a larger version of this figure.
Final concentration (mM) | |
NaCl | 120.4 |
KCl | 14.7 |
KH2PO4 | 0.6 |
Na2HPO4 x 2H2O | 0.6 |
MgSO4 x 7H2O | 1.2 |
HEPES | 10 |
Table 1: 10x perfusion buffer (1,000 mL).
Final concentration (mM) | |
NaHCO3 | 4.6 |
Taurin | 30 |
2,3-Butanedione monoxime | 10 |
Glucose | 5.5 |
10x perfusion buffer | 50 ml |
double destilled H2O (18,2 MΩ-cm) | 500 ml |
Table 2: 1x perfusion buffer (500 mL).
Final concentration | |
Collagenase Type II | ~600 U/ml |
CaCl2 | 40 µM |
1x perfusion buffer | 50 ml |
Table 3: Digestion buffer (50 mL).
Final concentration | |
Bovine Calf Serum | 10% |
CaCl2 | 12.5 µM |
1x perfusion buffer | 10 ml |
Table 4: Stop buffer (10 mL).
Final concentration (mM) | ||
Tyrode | 4-AP | |
NaCl | 140 | 140 |
HEPES | 10 | 10 |
Glucose | 10 | 10 |
KCl | 4 | 4 |
MgCl x 6H2O | 1 | 1 |
Probenecid | 2 | 2 |
4-Aminopyridine | 5 | |
BaCl2 | 0.1 | |
CaCl2 | 1 | 1 |
pH | 7.35 adjusted with 1 M NaOH at room temperature | 7.35 adjusted with 1 M NaOH at room temperature |
Table 5: Bath solutions.
Final concentration (mM) | |
DL-aspartat K+-salt | 92 |
KCl | 48 |
Na2ATP | 5 |
EGTA | 0.02 |
Guanosine 5′-triphosphate tris salt | 0.1 |
HEPES | 10 |
Fluo-3 | 0.1 |
pH | 7.20 adjusted with 1 M KOH at room temperature |
Table 6: Pipette solution.
Problem observed | Possible reason | Solution |
Low cell yield, tissue chunks left | Underdigestion | Increase digestion time in 15 seconds increments |
Low cell yield, tissue chunks left | Underdigestion | Increase collagenase amount in 50 U/ml steps |
mix of over- and underdigested cells, tissue chunks left | No coronary perfusion | Make sure not to insert aortic cannula into the ventricle |
mix of over- and underdigested cells, tissue chunks left | Air bubbles | Make sure not to insert any air into the perfusion system |
Low overall cell quality | Ischemic time too long | Decrease ischemic time, consider alternative methods to maintain circulation as long as possible |
Table 7: Common problems and how to solve them.
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 – 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° 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’s individual goal, it can even be more useful to expose ventricular cells to shear stress to select for the ’fittest’ 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 – besides many cell shadows in relation to viable cells – 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: ‘Is it possible to perform the desired measurements with high quality results in the isolated cells of each isolation?’. 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 – as true for most Langendorff-based isolation protocols – 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.
The authors have nothing to disclose.
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. Schüttler; VO1568/3-1, IRTG1816, and SFB1002 project A13 to N. Voigt), German Research Foundation under Germany’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. Kääb), 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-Mühlfenzl Stiftung (to S. Clauss) and the Else-Kröner-Fresenius Foundation (EKFS 2016_A20 to N. Voigt). The funders had no role in manuscript preparation.
2,3-Butanedione monoxime | Sigma-Aldrich | 31550 | |
27G cannula | Servoprax | L10220 | |
4-Aminopyridine | Sigma-Aldrich | A78403 | |
Anhydrous DMSO | Sigma-Aldrich | D12345 | |
Aortic cannula | Radnoti | 130163-20 | |
BaCl2 | Sigma-Aldrich | 342920 | |
blunt surgical forceps | Kent Scientific | INS650915-4 | |
Bovine Calf Serum | Sigma-Aldrich | 12133C | |
CaCl2 | Sigma-Aldrich | C5080 | |
Caffeine | Sigma-Aldrich | C0750 | |
Circulating heated water bath | Julabo | ME | |
Collagenase Type II | Worthington | LS994177 | |
disscetion scissors | Kent Scientific | INS600124 | |
DL-aspartat K+-salt | Sigma-Aldrich | A2025 | |
EGTA | Sigma-Aldrich | E4378 | |
Fluo-3 | Invitrogen | F3715 | |
Fluo-3 AM | Invitrogen | F1242 | |
Glucose | Sigma-Aldrich | G8270 | |
Guanosine 5′-triphosphate tris salt | Sigma-Aldrich | G9002 | |
Heating coil | Radnoti | 158821 | |
Heparin | Ratiopharm | 25.000 IE/5ml | |
HEPES | Sigma-Aldrich | H9136 | |
induction chamber | CWE incorporated | 13-40020 | |
Isoflurane | Cp-pharma | 1214 | |
Jacketed heart chamber | Radnoti | 130160 | |
KCl | Merck | 1049360250 | |
KH2PO4 | Sigma-Aldrich | P5655 | |
MgCl x 6H2O | Sigma-Aldrich | M0250 | |
MgSO4 x 7H2O | Sigma-Aldrich | M9397 | |
Na2ATP | Sigma-Aldrich | A2383 | |
Na2HPO4 x 2H2O | Sigma-Aldrich | S5136 | |
NaCl | Sigma-Aldrich | S3014 | |
NaHCO3 | Sigma-Aldrich | S5761 | |
Nylon mesh (200 µm) | VWR-Germany | 510-9527 | |
pasteur pipette | Sigma Aldrich | Z331759 | |
petri-dishes | Thermo Fisher | 150318 | |
Pluronic Acid F-127 | Sigma-Aldrich | P2443 | |
Probenecid | Sigma-Aldrich | P8761 | |
Roller Pump | Ismatec | ISM597D | |
surgical forceps | Kent Scientific | INS650908-4 | |
surgical scissors | Kent Scientific | INS700540 | |
suturing silk | Fine Science Tools | NC9416241 | |
syringe | Merck | Z683531-100EA | |
Taurin | Sigma-Aldrich | 86330 |