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 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.
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 25–30% 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.
All experiments were approved by the local Ethics Committee (TVA T0060/15 and T0003-15) and performed in agreement with the Guidelines for the Care and Use of Laboratory Animals (National Institute of Health, U.S.A.).
NOTE: A simplified flowchart of the procedure is shown in Figure 1.
1. Prearrangements
Solution | PB | CB | DB | SB | S1 | S2 | S3 | NT | |
Reagent (mM) | |||||||||
NaCl | 135 | 135 | 135 | 135 | 135 | 135 | 135 | 135 | |
KCl | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | 4.7 | 4 | |
KH2PO4 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | ||
Na2HPO4 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | ||
MgSO4 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | ||
MgCl | 1 | ||||||||
HEPES | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
Taurine | 30 | 30 | 30 | 30 | 30 | 30 | 30 | ||
Glucose | 10 | 10 | 10 | 10 | 10 | 10 | 10 | ||
BDM | 10 | 10 | 10 | 10 | 10 | 10 | 10 | ||
CaCl2 | 1 | 0.01 | 0.125 | 0.25 | 0.5 | 1 | |||
BSA | 150 | 70 | 70 | 70 | |||||
Purified enzyme blend (medium Thermolysin) | 0.195 Wünsch units/mL | ||||||||
pH adjusted to | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 | |
pH adjusted at | 37 °C | 4 °C | 37 °C | 37 °C | 37 °C | 37 °C | 37 °C | 37 °C | |
pH adjusted with | NaOH | NaOH | NaOH | NaOH | NaOH | NaOH | NaOH | NaOH |
Table 1: List of Buffers. PB: perfusion buffer, which can be stored for 3 days at 4 °C (300 mL per animal). CB: cannulation buffer, which can be stored for 3 days at 4 °C (200 mL per animal). DB: digestion buffer, which has to be used within the day (40 mL per animal). SB: stopping buffer, which has to be used within the day (2 mL per animal). S1: Step 1 buffer, which has to be used within the day (2 mL per animal). S2: Step 2 buffer, which has to be used within the day (2 mL per animal). S3: Step 3 buffer, which has to be used within the day (2 mL per animal). NT: normal Tyrode, which has to be used within the day (50 mL per animal).
2. Heart Preparation
3. Cannulation
4. Pressure Manipulation and Digestion
5. Cell Processing and Calcium Re-adaptation
6. Functional Evaluation of Excitation-contraction-coupling
Figure 1: Simplified flowchart of the isolation procedure. The procedure is highly time-sensitive until the enzymatic digestion of the myocytes is completed. Please click here to view a larger version of this figure.
Figure 2: Preparation of equipment prior to the isolation. (A) This panel shows a self-made Langendorff apparatus: (1) a jacketed reaction vessel with PB; (2) a jacketed reaction vessel with CB; (3) a 3-way stopcock; (4) a syringe; (5) a peristaltic pump; (6) a heating immersion; (7) a jacketed bubble trap; and (8) the cannula and heart. (B) This panel shows the set-up for the cannulation and organ excision for an optimized work flow: (1) a 100 mL beaker with 50 mL of ice-cold CB; (2) a 10 mL syringe with ice-cold CB; (3) a Petri dish with ice-cold CB; (4) a light source; (5) the custom-made cannula with a cannulation knot (see also Figure 3A and 3B); (6) fine, curved forceps; (7) tissue forceps; (8) fine forceps, angled 45°; (9) abdominal surgical scissors; (10) fine surgical scissors; (11) 4 x 30 G needles; and (12) a 15 G needle. (C) This panel shows the microscope-mounted equipment for the confocal imaging: (1) electric stimulator electrodes; (2) a superfusion pen; (3) a glass-bottom dish with the ACMs; and (4) immersed platinum electric stimulator electrodes. (D) This panel shows the microscope set-up for the confocal imaging: (1) the confocal microscope; (2) a superfusion pen; (3) a superfusion buffer reservoir; (4) a superfusion flow regulator; (5) a superfusion heating module; (6) a computer workstation; and (7) an electric stimulator. Please click here to view a larger version of this figure.
Figure 3: Custom-made equipment. (A) This panel shows the manipulated 15 G cannula with a Luer lock. The arrows indicate two indentations for the cannulation knots. (B) These are the cannulation knots, two double overhand knots placed on top of each other for rapid tightening. The arrows indicate where and in which order the knot needs to be tightened. (C) This panel shows the assembled pressure control device. A 21 G butterfly needle is hooked into a tripod clamp. The hose is kept at the same height as the needle. The screw top is opened. The elevation of the butterfly hose can be altered as indicated by the arrows in order to change the intraluminal pressure of the left atrium. (D) The left atrium is punctured with the pressure control device. This picture shows an ideally inflated left atrium. The ellipse marks the placement of the overhand knot. (E) The left atrium is punctured with the pressure control device. This picture shows an over-inflated left atrium, which will result in a lower yield of viable ACMs. The ellipse marks the placement of the overhand knot. Please click here to view a larger version of this figure.
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).
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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.
Problem | Possible Cause | Solution |
No flow through butterfly hose | Improper closure of the aorta with the cannulation knots | Remove fatty tissue before tightening |
Add 3rd cannulation knot with respective indentation | ||
Pull knot with hand for increased force | ||
Blocked needle | Readjust position into the lumen | |
Unblock by gentle pull with syringe | ||
Right atrium / coronary sinus not inflated | Improper closure of the heart base | Block flow with additional knots |
Right atrium damaged during preparation | Close leakage with clips/knots | |
Poor digestion / atrium does not soften | Reduced enzyme activity through wrong temperature | Adjust temperature to 37 °C |
Inactive/degraded enzyme | Replace | |
Old/overly fibrotic atrium | Increase digestion time (in steps of +50%) | |
Damaged aortic valve | Shallow penetration during cannulation | |
Poor cell yield | Atrium over-digested | Decrease digestion time (in steps of +25%) |
Reduce intraluminal pressure by lowering the butterfly hose | ||
Atrium under-digested | Increase digestion time (in steps of 25%) | |
Increase intraluminal pressure by raising the butterfly hose | ||
Cell dispersion too aggressive | Be more gentle |
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—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 Wünsch 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.
The authors have nothing to disclose.
This research was supported by the DZHK (German Centre for Cardiovascular Research, D.B.), the EKFS (Else-Kröner-Fresenius Stiftung, F.H.), and by the BMBF (German Ministry of Education and Research), as well as the BIH-Charité clinical scientist program funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health (F.H.).
ZSF-1 Obese rat | Charles River Laboratories, Inc. | 21 weeks old | |
Fine Iris Scissors | Fine Science Tools GmbH | 14094-11 | |
Surgical Scissors | Fine Science Tools GmbH | 14001-18 | |
Micro Dressing Forceps (curved, serrated) | Aesculap, Inc. | BD312R | |
Tissue Forceps (straight, 1 x 2 teeth) | Aesculap, Inc. | BD537R | |
Tying Forceps (angled) | Aesculap, Inc. | MA624R | |
Rodent and Small Animal Guillotine | Kent Scientific Corp. | DCAP | |
Low Cost Induction Chamber 3.0 L | Kent Scientific Corp. | SOMNO-0730 | |
Butterfly Winged Infusion Set 21 G | Hospira, Inc. | 181106101 | |
Abbocath 16 G | Hospira, Inc. | 0G7149702 | |
Microlance Hypodermic Needle | Becton Dickinson GmbH | 301300 | modify needle to make cannula |
Braun Original Perfusor Syringe 50 ml | B. Braun Melsungen AG | 8728810F | |
Braun Inject Solo Syringe 10 ml | B. Braun Melsungen AG | 2057926 | |
Beaker 50ml | Duran Group (DWK Life Sciences GmbH) | 21 106 17 | |
Duroplan petri dish (100 x 20 mm) | Duran Group (DWK Life Sciences GmbH) | 21 755 48 | |
Seraflex Suture USP 3/0 | SERAG-WIESSNER GmbH & Co. KG | IC208000 | |
VWR disposable Square Weighin Boats 100ml | VWR, Inc. | 10803-148 | |
Styrofoam surface | |||
Sodium chloride | Sigma-Aldrich, Inc. | 71380 | |
Potassium chloride | Sigma-Aldrich, Inc. | P4504 | |
Potassium phosphate monobasic | Sigma-Aldrich, Inc. | P5379 | |
Sodium phosphate dibasic | Sigma-Aldrich, Inc. | S0876 | |
Magensium sulfate heptahydrate | Sigma-Aldrich, Inc. | 230391 | |
Magensium chloride | Sigma-Aldrich, Inc. | M8266 | |
HEPES | Sigma-Aldrich, Inc. | H3375 | |
Taurine | Sigma-Aldrich, Inc. | T0625 | |
Glucose | Sigma-Aldrich, Inc. | G7528 | |
2,3-Butanedione monoxime | Sigma-Aldrich, Inc. | B0753 | |
Calcium chloride solution (1 M) | Sigma-Aldrich, Inc. | 21115 | |
Bovine Serum Albumin | Sigma-Aldrich, Inc. | A9647 | |
Liberase | Roche (Sigma-Aldrich, Inc.) | LIBTM-RO | |
Heparin | Rotexmedica GmbH | 3862357 | |
Forene (Isoflurane) | Abbvie Deutschland GmbH & Co. KG | 10182054 | |
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane | Sigma-Aldrich, Inc. | L2020 | |
WillCo glass-bottom dish 500µl 0.005mm | WillCo Wells B.V. | HBST-3522 | |
Fluo4 AM | Invitrogen (Thermo Fisher Scientific, Inc.) | F14201 | 5µM for 20min at RT |
Di-8-ANNEPS | Invitrogen (Thermo Fisher Scientific, Inc.) | D3167 | 10µM for 45 min at 37° C |
Mitotracker RED FM | Invitrogen (Thermo Fisher Scientific, Inc.) | M22425 | 20nM for 30 min at 37° C |
Jacketed reaction vessel 500 ml | Gebr. Rettberg GmbH | 107024414 | |
Jacketed reaction vessel 1000 ml | Gebr. Rettberg GmbH | 107025414 | |
Jacketed bubble trap | Gebr. Rettberg GmbH | 134720001 | |
ED heating immersion circulator | Julabo GmbH | 9116000 | |
Reglo Digital MS-2/6 peristaltic pump | Ismatec (Cole-Parmer Gmbh) | ISM 831 | |
Voltcraft Thermometer 302 K/J | Conrad Electronic SE | 030300546 | |
Tubing | |||
LSM 700 microscope | Carl Zeiss, Inc. | ||
ZEN 2.3 imaging software | Carl Zeiss, Inc. | 410135-1011-240 | |
Single channel heater controller TC-324B | Warner Instruments, LLC | 64-2400 | |
8 channel perfusion system | Warner Instruments, LLC | 64-0185 | |
8 channel Multi-Line In-Line Solution Heaters | Warner Instruments, LLC | 64-0105 |