The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.
Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.
Zebrafish are small teleost fish that have long been used as a model vertebrate organism1 and have recently come to prominence as a viable vertebrate system for high throughput screening of genes and drugs2,3. However, physiological analysis of zebrafish tissues is not well developed. In the cardiovascular system, methods have been described for dissection of zebrafish hearts4 and isolation of ventricular cardiac myocytes5,6,7. There are few detailed descriptions of the effective isolation of atrial myocytes and no reports of vascular smooth muscle (VSM) preparations for patch-clamp studies.
The current work describes methodology for the isolation of zebrafish cardiac and vascular myocytes, viable for electrophysiological and functional studies. This approach includes modifications of previously reported protocols for zebrafish ventricular myocyte isolation5,6 and adapts methods from mammalian VSM cell isolations8, allowing for the isolation of zebrafish vascular smooth muscle cells from the bulbous arteriosus (BA). The protocols result in efficient yields of isolated atrial, ventricular, and VSM cells from zebrafish that can be reliably used in patch-clamp studies for up to 8 h9.
Despite their nearly transparent larvae that develop entirely outside the parental organism, exploring their promised ontogenetic potential in studying cardiovascular development has been limited by challenges in extracting and analyzing tissues at a young age. The current article addresses this limitation by demonstrating patch-clamp experiments on zebrafish hearts isolated as early as 3 days post-fertilization (dpf), using an adapted, published extraction method10.
All zebrafish (wild type strain AB, both male and female) were raised, maintained, and handled for the experiments following the guidelines of the Washington University Institutional Animal Care and Use Committee (IACUC).
1. Isolation of atrium, ventricle, and bulbous arteriosus from adult, juvenile, and larval zebrafish
2. Dissociation of cardiomyocytes from adult, juvenile, and larval zebrafish for electrophysiological studies
3. Dissociation of vascular smooth muscle (VSM) cells from adult and juvenile zebrafish for electrophysiological studies
4. Isolation of hearts from embryonic zebrafish for electrophysiological studies
5. Patch-clamp studies on isolated cardiovascular cells
NOTE: Inside-out and whole-cell patch-clamp recordings from the isolated cells can be obtained as previously reported for KATP channel currents in zebrafish cardiovasculature9.
The above protocols reliably and routinely provide sufficient cardiac and vascular myocytes of consistent quality amenable for patch-clamp studies as recently reported in extensive studies of ATP-sensitive potassium (KATP) channels in wild-type and mutant zebrafish cardiovasculature9. Representative traces of recordings of such KATP channel activity from isolated cardiomyocytes are shown in Figure 3A-C. In the case of cells isolated from bulbous arteriosus, the vascular smooth muscle cells were confirmed by Tg(tagln:egfp) expression11,12 (Figure 1). Successful single-channel recordings were obtained (n = 8 recordings from N = 5 preparations) in patches excised from the embryonic hearts (Figure 3D). Action potentials were recorded from isolated adult zebrafish ventricular and atrial myocytes in whole-cell, current-clamp mode, using a patch-clamp amplifier along with a compatible digitizer (see Table of Materials). The extracellular recording solution for the action potential (AP) measurements contained NaCl (140 mM), KCl (4 mM), CaCl2 (1.8 mM), MgCl2 (1 mM), Glucose (10 mM), HEPES (10 mM), and Blebbistatin (0.01 mM, pH 7.4); the intracellular recording solution contained KCl (120 mM), EGTA (5 mM), K2ATP (5 mM), MgCl2 (5 mM), and HEPES (10 mM, pH 7.2). Patch pipettes were pulled from soda-lime glass and had resistances of 3 – 5 MΩ when filled with intracellular solution. Ventricular myocytes exhibit stable hyperpolarized membrane potentials, and action potentials were stimulated via current injection through the patch pipette (Figure 3E), whereas atrial myocytes exhibited spontaneous action potential firing (Figure 3F). Action potential properties are summarized in Table 3.
Figure 1: Isolation of atrial, ventricular, and bulbous arteriosus cells. Schematic to illustrate the isolation of atrial (A), ventricular (B), and bulbous arteriosus (C) cells. The images depicting each isolated cell type (bottom) have scale bars = 50 µm in each case. BA cells isolated from smooth muscle cell transgenic reporter zebrafish lines (Tg(tagln:egfp)11,12) are positive for green fluorescence. Please click here to view a larger version of this figure.
Figure 2: Schematic to illustrate the isolation of embryonic hearts. (A) Fertilized embryos are removed from the breeding tank and placed in a Petri dish containing E3 water and maintained at 28 °C for up to 5 days. (B) At desired age, embryos are anesthetized in situ using tricaine and transferred into PB in 5 mL centrifuge tubes for dissociation. (C) Embryonic heart isolation apparatus consists of a 10 mL syringe attached to a 19 G needle mounted above the dissociation tube on a bench stand, allowing the hands to perform the trituration. (D) Image of the isolated heart (day 4) attached to a patch-clamp pipette. Please click here to view a larger version of this figure.
Figure 3: Representative patch-clamp recordings from isolated zebrafish cardiovascular tissues. (A, B) ATP-sensitive KATP channels from inside-out excised patch-clamp recordings of atrial (A) and ventricular (B) cardiomyocytes isolated from adult zebrafish. (C) KATP channel conductance was recorded from whole-cell patch-clamping of VSM cells isolated from adult zebrafish. (D) Single K+ channel activity in membrane patches excised from zebrafish embryonic hearts (4 dpf). All excised patch recordings were made with 140 mM KCl on both sides of the membrane, at -50 mV membrane potential. Whole-cell currents were recorded with the membrane potential clamped at -70 mV. (E) Example current-clamp recording from isolated ventricular myocyte. Action potentials were stimulated by current injection as shown below. (F) Example current-clamp recording from isolated atrial myocyte showing spontaneous action potential firing. Please click here to view a larger version of this figure.
PERFUSION BUFFER (PB) | 10 mM HEPES, 30 mM Taurine, 5.5 mM Glucose, 10 mM BDM. Make the solution in phosphate-buffered saline (PBS), adjust pH to 7.4 | ||
DIGESTION BUFFER (DB) | PB + 12.5 µM CaCl2 + 5 mg/mL Col II + 5 mg/mL Col IV + 5 ng/mL Insulin | ||
STOPPING BUFFER (SB) | PB + 10% FBS + 12.5 µM CaCl2 + 10 mg/ml BSA + 5 ng/mL Insulin |
Table 1: Solutions for isolation of zebrafish cardiomyocytes.
S1 BUFFER | 0.1% BSA (w/v), 145 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM Glucose, 0.05 mM CaCl2, 1 mM MgCl2, adjusted to pH 7.4 using NaOH | ||
S2 BUFFER | 2 mL S1, 4 mg Papain, 2 mg DTT | ||
S3 BUFFER | 2 mL S1, 3 mg Collagenase Type H, 2 mg Trypsin Inhibitor, 1 mg Elastase |
Table 2: Solutions for isolation of zebrafish VSM cells.
Ventricular Myocytes (n = 3 Recordings) | |||
Vm (mV) | APA (mV) | APD50 (ms) | |
-75.7 ± 3.9 | -119.6 ± 3.8 | 329 ± 163 | |
Atrial Myocytes (n = 3 Recordings) | |||
AP Rate (min-1) | MDP (mV) | APA (mV) | APD50 (ms) |
107.3 ± 32.6 | -71.2 ± 5.1 | 98.4 ± 4.7 | 141 ± 12 |
Table 3: Action potential properties from isolated zebrafish cardiomyocytes. Recorded in current clamp mode. Data shown as mean ± S.D. Vm = membrane potential; APA = action potential amplitude; APD50 = duration of action potential to 50% repolarization; MDP = maximal diastolic potential.
Previous methods for isolating zebrafish ventricular myocytes5,6, aimed at generating myocytes for culture or electrophysiological studies, provided cells of lower yield and involved lengthy steps of multiple centrifugations that adversely affected the cell quality and viability. The protocols described here are reliable, cover each of the significant cardiovascular tissues (ventricle, atria, and VSM), and importantly are quite practical for acute isolation of live cells. Isolation approaches involving cannulation of the ventricle via the BA13 can be a sophisticated alternative for ventricular cardiomyocytes but require a higher degree of dexterity and may negatively affect atrial isolation. The approach detailed in the current protocol provides a simple and efficient alternative.
Additional considerations for larval cardiac tissue isolation are: (1) In step 1.4, depending on the age of the fish and magnification available, the tissues might be visible even before removing the pectoral muscles, in which case the tissues can be directly "plucked" out of the fish without prior surgery, and then the non-CV tissues can be removed using superfine forceps; (2) In step 2.2, for larvae younger than 14 days, the hearts can be used directly for electrophysiological studies without need for enzymatic dissociation.
As noted above, and typically true of enzymatic dissociation methods in general, it has proven important to use fresh buffers and enzymes each time. However, Perfusion Buffer (PB) and S1 Buffer can be prepared in advance and stored at 4-8 °C for 1 month.
Successful tissue isolation time should not exceed ~90 s per fish, and tissues should not be exposed to air. When dissociation takes longer, the cell quality (i.e. survival) is reduced. When triturating tissues, care should be taken to avoid generating air bubbles, which also reduces cell quality. VSM cell isolation is sensitive to the digestion by collagenase in S3 buffer. After the first 3 min of digestion, the tube should be taken out of the thermoshaker every minute and examined for floating dilated and translucent tissues. Once tissue fragmentation is apparent, the digestion step should be ceased.
It is important to note that these protocols are not extensively optimized for isolating calcium-tolerant cardiomyocytes, as would be required for contractility studies. However, as shown, reliable recording of cardiomyocyte action potentials can be achieved in the presence of physiological extracellular calcium concentrations7,14. Blebbistatin, included to decouple excitation and contraction, and improve giga-Ohm seal formation and recording efficiency in these experiments, has previously been shown to not significantly affect the AP parameters in intact zebrafish hearts14. For electrophysiology, the absolute yield of cells is also not routinely rate-limiting. This protocol has not been optimized for yield, as might be necessary for biochemical studies of isolated cells. Still, the yield achieved with these methods is well suited for electrophysiological studies and likely multiple other approaches.
The authors have nothing to disclose.
This work was supported by NIH grants HL140024 to CGN and HL150277 to CMC. Figure 1 and Figure 2 were created with BioRender.com.
1.5 mL Centrifuge Tubes | Eppendorf | 22364111 | |
10 mL Syringe | Fisher Scientific | 14-955-459 | |
19 Guage Needle | BD | 305187 | |
2,3-Butanedione Monoxime (BDM) | Sigma-Aldrich | B0753 | |
5 mL Centrifuge Tubes | Sigma-Aldrich | EP0030119479 | For embryonic heart isolation |
Axopatch 200B amplifier and Digidata 1200 digitizer | Molecular Devices | Used for action potential recordings | |
Benchtop Mini Centrifuge | Southern Labware | MLX-106 | |
Blebbistatin | Sigma-Aldrich | 203390 | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A9418 | |
Calcium Chloride | Sigma-Aldrich | C4901 | |
Cell-Strainer Sieve | Cole-Parmer | EW-06336-71 | 100 μm sieve for embryonic heart isolation |
Collagenase Type H | Sigma-Aldrich | C8051 | |
Collagenase Type II | Worthington | LS004176 | |
Collagenase Type IV | Worthington | LS004188 | |
Curved Forceps | Fisher Scientific | 16-100-110 | |
DTT | Sigma-Aldrich | D0632 | |
EGTA | Sigma-Aldrich | 324626 | |
Elastase | Worthington | LS003118 | |
Fetal Bovine Serum (FBS) | Sigma-Aldrich | F2442 | |
Fine Forceps | Dumont | Style #5 | Ceramic-coated forceps for adult and juvenile CV tissue isolation (Need two) |
Glucose | Sigma-Aldrich | G8270 | |
HEPES | Sigma-Aldrich | H3375 | |
Insulin | Sigma-Aldrich | I2643 | |
K2ATP | Sigma-Aldrich | A8937 | |
Large Petri Dish | Sigma-Aldrich | P5981 | For dissociation |
Magnesium Chloride | Sigma-Aldrich | M8266 | |
Micro-Hematocrit Capillary Tubes | Kimble Chase | 41A2502 | Soda lime glass for patch pipettes |
Papain | Worthington | LS003118 | |
Pasteur Pipettes | Fisher Scientific | 13-678-6A | |
Petri Dish | Sigma-Aldrich | P5606 | 100 mm x 20 mm, for embryonic heart isolation |
Phosphate-Buffered Saline (PBS) | Sigma-Aldrich | 806552 | |
Potassium Chloride | Sigma-Aldrich | P3911 | |
Scissors | Fine Science Tools | 14090-09 | For adult and juvenile zebrafish decapitation |
Sodium Chloride | Sigma-Aldrich | S9888 | |
Sodium Hydroxide | Sigma-Aldrich | S8045 | |
Super Fine Forceps | Dumont | Style #SF | For isolating larval CV tissues (Need two) |
Taurine | Sigma-Aldrich | T0625 | |
Thermoshaker | ThermoFisher Scientific | 13687711 | |
Tricaine Methanesulfonate (MS222) | For anaesthetizing zebrafish larvae | ||
Trypsin Inhibitor | Sigma-Aldrich | T6522 |