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 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.
In order to study the heart during healthy and pathological states, it is often useful to examine the phenotype at the single cell level. While scientific advances have permitted the robust measurement of single cell calcium dynamics, single cell optical voltage measurements have remained scarce1. Arguably, this is because of the low signal to noise ratio (SNR), phototoxicity, and photobleaching of traditional potentiometric dyes2,3. Nonetheless, isolated myocyte optical action potentials have been obtained2,3,4. Further, with advances in the chemistry and the physics of voltage sensitive dyes, the SNR has improved5. Newer membrane potential probes (Table of Materials) respond to changes in membrane potential in sub-milliseconds and have a fluorogenic response range of approximately 25% per 100 mV. Further, the excitation/emission of the membrane potential kit (e.g., FluoVolt; Table of Materials) used in this protocol works with standard fluorescein isothiocyanate (FITC) or green fluorescent protein (GFP) settings6.
The FITC and GFP excitation/emission spectra overlap with the fluo-4 calcium bound spectra7. Simultaneous acquisition of fluorescence photometry with digital cell geometry measurements traditionally has been used for the simultaneous acquisition of calcium and cellular shortening measurements8. This protocol describes in detail how to isolate murine myocytes and how to record calcium or voltage signals using standard FITC settings. Additionally, it describes how a simple switch in excitation/emission filters on the imaging workstation can be used to obtain calcium and shortening measurements using the ratio metric calcium dye fura-2. Compared to fluo-4, fura-2 has a higher affinity for calcium and is relatively resistant to photobleaching9. Consequently, using a single workstation this protocol allows for a thorough examination of singly myocyte excitation-contraction coupling.
All methods and procedures described in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University.
1. Preparation of Solutions, Instruments, and Coverslips
NOTE: 1x solutions can be used for up to a month.
2. Preparation of the Langendorff Apparatus
NOTE: The individual components of the Langendorff apparatus used in this protocol are listed in Table of Materials.
3. Myocyte Isolation
4. Fura-2 Dye Loading
5. Fluo-4 Dye Loading
6. Membrane Potential Dye Loading
7. Photometry and Charge Coupled Device Recordings
Figure 1A shows the Langendorff apparatus. The oxygenator is in the KHB-HB reservoir. The collagenase solution is in the middle 60 mL syringe reservoir. The degassing line is connected to the empty 60 mL syringe reservoir. After a successful isolation, most of the cells should be rod shaped and striated. Under a 40x objective, most myocytes should have clear striations visible. Figure 1B,C shows examples of healthy rat myocytes. Once isolated, cells can be cultured up to 4 days while maintaining their morphology and electrical properties.
To measure excitation-contraction coupling, the cells are then placed in a heated pacing chamber. Because myocytes are sensitive to changes in temperature, it is important to allow the coverslip to equilibrate for 15 min in the chamber before recording. For fluorescence recordings, the excitation wavelength is generated by a 75 W xenon-arc bulb. Xenon-arc bulbs produce a light spectrum that mimics natural sunlight. The intensity of the light and the wavelength are controlled by neutral density/emission filers. The excitation light then passes through the objective to the myocyte. The emission wavelength is then collected by a photomultiplier tube. Using the system described here, both the excitation and emission filters need to be changed manually.
Shortening on the other hand is obtained by a charge coupled device sensor. Measuring in real time up to 1,000 times per second, the acquisition software performs an average of the lines within an area of interest to create a well resolved striation pattern. A fast Fourier transform (FFT) is then calculated. The peak within the power spectrum represents the average sarcomere spacing. Changes in the sarcomere spacing during pacing are then plotted and subsequently quantified.
Figure 2 shows calcium and shortening traces recorded from a C57/B6 mouse myocyte loaded with the calcium dye fura-2. The pacing protocol is a modification of pacing protocols described previously10,11. Healthy mouse myocytes should be able to be paced at their resting heart rate 10 Hz. Figure 3 is quantification of ensembled averaged data obtained from a C57/B6 mice and their transgenic (TG) littermates who had a point mutation introduced into a potassium channel. Notice there is no difference between the groups except for the relaxation time at 10 Hz pacing.
Unlike fura-2 which is a dual excitation dye, the voltage dye and fluo-4 are single wavelength excitation dyes whose excitation/emission work with standard FITC excitation and emission spectrum (494/506 nm). Therefore, recordings of calcium and sarcomere shortening or voltage and sarcomere shortening can be obtained using this filter set.
Figure 4A shows a voltage tracing recorded from a C57/B6 mouse myocyte paced at 10 Hz. Compared to calcium signals, single cell voltage tracings are smaller in amplitude and need post-processing to obtain a useable signal. Figure 4B shows an ensembled averaged action potential (AP) made from the APs in Figure 4A. Figure 4C,D shows an ensembled average AP after a low pass Butterworth or a Savitzky-Golay digital filter was applied. Care must be taken when filtering the signal as not to distort the real data. Notice the subtle differences in the shape of the APs in Figure 4B-D.
Figure 5 shows traces recorded from rat myocytes paced at 1 Hz. In addition to the voltage signal being lower than the calcium signal, the contraction kinetics are different as well. This is because calcium dyes buffer calcium while voltage dyes do not.
As with the calcium transient (Figure 3), myocytes demonstrated pacing dependent changes in their optical action potential duration (APD) as well (Figure 6). While the fura-2 traces were ensembled averaged before being quantified, the voltage traces were filtered with a Savitzky-Golay polynomial smoothing filter (width 5, order 2) before being ensembled averaged and quantified.
As quantified in Figure 6 and Figure 7, in addition to demonstrating pacing induced changes in APD, they also demonstrated drug induced prolongation of the AP. At 4 Hz pacing, concentration dependent blockade of the transient outward current (Ia) with 4-aminopyridine resulted in prolongation of the APD.
Finally, care must be taken to avoid cytotoxicity. Figure 8 is the last 11 s of a 20 s recording. Indicated by the red arrows in Figure 8, prolonged exposure of myocytes to blue light leads to triggered activity.
Figure 1: Constant pressure Langendorff apparatus. (A) The Langendorff Apparatus with each component labeled in white lettering. (B) Isolated Sprague-Dawley rat myocytes viewed through a 10x objective. (C) Isolated rat myocytes viewed through a 40x objective. Please click here to view a larger version of this figure.
Figure 2: Representative calcium and sarcomere shortening traces recorded from C57/B6 myoyctes using fura-2. Calcium and sarcomere shortening traces recorded at 1, 2, 4, 10, 0.5 and 0.75 Hz. Please click here to view a larger version of this figure.
Figure 3: Quantification of sarcomere shortening, peak calcium, relaxation time, and reuptake time recorded from a C57/B6 wild type (WT) and transgenic (TG) mice. (A) Sarcomere shortening. (B) Peak calcium. (C) Relaxation time defined as 90% return to baseline of the shortening trace. (D) Reuptake time defined as 90% return to baseline of the calcium trace. Please click here to view a larger version of this figure.
Figure 4: Optical action potential recorded from a C57/B6 mouse myocyte paced at 10 Hz. (A) 1 second unfiltered trace. (B) Ensembled averaged optical action potential. (C) Ensembled averaged optical action potential after a lowpass Butterworth filter was applied. (D) Ensembled averaged optical action potential after a Savitzky-Golay polynomial smoothing filter was applied. Please click here to view a larger version of this figure.
Figure 5: Representative calcium, voltage, and sarcomere shortening traces recorded from Sprague-Dawley rat myocytes paced at 1 Hz. (A) Calcium and sarcomere shortening traces recorded at 1 Hz pacing using fluo-4. (B) Voltage and sarcomere shortening traces recorded at 1 Hz pacing using the voltage dye. Please click here to view a larger version of this figure.
Figure 6: Optical action potentials recorded from Sprague-Dawley rat myocytes paced at 1, 2, and 4 Hz pacing. (A) Filtered trace recorded at 1 Hz pacing. (B) Filtered trace recorded at 2 Hz pacing. (C) Filtered trace recorded at 4 Hz pacing. (D) Action potential duration 10, measured as 10% return to baseline. (E) Action potential duration 50, measured as 50% return to baseline. (F) Action potential duration 90, measured as 90% return to baseline. Please click here to view a larger version of this figure.
Figure 7: The Effects of 4-aminopyridine on Sprague-Dawley rat optical action potentials recorded at 4 Hz pacing. (A) Ensembled averaged trace recorded at 4 Hz pacing with no 4-Aminopyridine in the solution. (B) Ensembled averaged trace recorded at 4 Hz pacing with 1 µM 4-Aminopyridine in the solution. (C) Ensembled averaged trace recorded at 4 Hz pacing with 10 µM 4-Aminopyridine in the solution. (D) Action potential duration 10, measured as 10% return to baseline. (E) Action potential duration 50, measured as 50% return to baseline. (F) Action potential duration 90, measured as 90% return to baseline. Please click here to view a larger version of this figure.
Figure 8: Voltage dye induced phototoxicity in Sprague-Dawley rat myocytes after 20 s of continuous light exposure. Red arrows indicate cytotoxic events. Please click here to view a larger version of this figure.
Being able to isolate cardiac myocytes is a powerful method that can be used to understand cardiac physiology, pathology, and toxicology. In the above protocol, we described a method that utilizes a constant gravity pressure Langendorff apparatus to obtain single cardiac myocytes. Afterwards, using the fluorescence photometry system, we describe how to simultaneously acquire either calcium and shortening or voltage and shortening traces.
Because of the different kinetics between calcium dyes, care must be taken on which dye to select. For this protocol, both the fura-2 and fluo-4 used were engineered with AM esters necessitating a wash step to allow for intracellular esterases time to cleave the AM group and trap the dye in the cell. While both fura-2 and fluo-4 are considered high affinity calcium dyes, the Kd for fura-2 is 145 nM compared to the 345 nM for fluo-49. Further, fura-2 is ratiometric. Because of this, it can be used to quantify intracellular calcium levels9,12. Fluo-4 on the other hand is a single wave calcium probe. The advantage of using fluo-4 is it produces a brighter fluorescence signal. Regardless of which calcium dye is used, compared to the calcium dye, membrane voltage probes have a lower SNR.
As shown in Figure 4 and Figure 5, voltage traces compared to calcium traces are smaller in amplitude. Using the software's digital trace filtering, it is possible to increase the SNR and quantify the data (Figure 4 and Figure 7). Once quantified, both calcium transients and optical APDs demonstrate restitution, shortening their duration at faster pacing frequencies (Figure 2, Figure 3, Figure 6, and Figure 7). Shorter APDs during faster pacing cycles are necessary to allow enough time for ventricular filling during diastole. Alterations in this phenomenon is thought to be indicative of an increase in the risk of arrythmias13,14,15,16. While alterations in APD can be caused by disease, they can also be caused by chemicals. As shown in Figure 7, when the predominant murine repolarizing potassium current, Ia, is blocked, the optical APD becomes longer.
Still, as reported previously with voltage sensitive dyes, light intensity and duration can alter the APD2,5,17. This is believed to be the result of the generation of reactive oxidative species (ROS)5. Previously, it has been shown that the addition of antioxidants to the recording solution can prevent voltage sensitive dye cytotoxicity5. As a result, we added the antioxidant L-glutathione (10 mM), to Tyrode's solution. Shown in Figure 8 is the last 11 s of a 20 s recording obtained at 1 Hz pacing. As indicated by the red arrows, alterations in the APD did not occur until 15 s into the recording; therefore, while the modified Tyrode's solution did not prevent phototoxicity it delayed it significantly. Using modified Tyrode's solution, using a low light intensity setting and keeping the duration of the recording to under 5 s, it is possible to avoid any dye induced alterations in APD. This is important because without taking care to avoid phototoxicity, the data could be misinterpreted as causing early or delayed after depolarizations. In addition to limiting the exposure to blue light, there are additional precautions that can be taken to prevent misinterpretation of the data.
The first is to only record from cells that follow one to one pacing and have a resting sarcomere length greater than or equal to 1.75 µm. The 1.75 µm cutoff is taken from the observation by Gordon et al.18 that tension rapidly declines once the sarcomere length is below this amount. Nonetheless, certain pathologies may result in significant alterations in resting sarcomere length. To be sure that the phenotype is real and not an artifact of the isolation, the following trouble shooting approaches should be taken.
If myocytes are consistently not following 1:1 pacing, have sarcomere lengths below 1.75 µm, heavy membrane blebbing, or do not survive the isolation, the first thing to check is the time it took to cannulate the heart. The longer the cannulation time, the lower the yield will be. If a long cannulation time is required, viability can be improved by placing the heart in a cardioplegic solution19. Nonetheless, because the collagenase is an enzyme, the activity and specificity of a specific lot change over time. If the overall yields progressively become worse despite good cannulation times, new lots should be assayed. While our protocol was optimized for 5 s recordings, if longer voltage traces are needed, additional neutral density filters will need to be purchased. The system described in the protocol comes with neutral density filters that reduce the transmitted light by 37%, 50%, 75%, 90%, and 95%.
In summary, we described a methodology that allowed for the isolation of adult murine ventricular myocytes that were used for calcium, voltage, and sarcomere shortening measurements.
The authors have nothing to disclose.
We thank Dana Morgenstern for careful proofreading of the manuscript.
0.25 Liter Water Jacketed Reservoir | Radnoti, LLC | 120142-025 | |
1 liter volumetric flask | Fisher Scientific | 10-205F | |
100 ml beaker | Fisher Scientific | FB-100-100 | |
100 ml graduated cylinder | Fisher Scientific | 08 562 5C | |
1000 ml flask | Fisher Scientific | FB-500-1000 | |
2-Bar Lab Stand with Stabilizer Bar and 24" Stainless Steel Rods | Radnoti, LLC | 159951-2 | |
4-Aminopyridine | Sigma-Aldrich | 275875 | |
40X Oly UApo/340 Non-Immersion Objective (NA 0.9, WD 0.2mm) | IonOptix | MSCP1-40 (b) | |
60-mL syringe, BD Luer-Lok tip | BD | 309650 | |
Aortic Metal Cannulae | Harvard Apparatus | 73-0112 | |
Bovine Serum Albumin | Fisher Scientific | BP9703-100 | |
C-6 Standard Heating Circulator | Chemyx | A30006 | |
CaCl2 | Fisher Scientific | BP510500 | |
Cell framing adapter | IonOptix | CFA300 | |
CellPr0 Vacuum Filtration System, 1 liter, 0.22µm,Cs/12 | Labratory Product Sales, Inc | V100022 | |
CellPro Vacuum Filtration System, 250mL, 0.22µm,Cs/12 | Labratory Product Sales, Inc | V25022 | |
CellPro Vacuum Filtration System, 500mL, 0.22µm,12/CS | Labratory Product Sales, Inc | V50022 | |
CMC (mTCII) Temp Control w/ inline flow heater | IonOptix | TEMPC2 | |
Cole-Parmer Large-bore 3-way, male-lock, stopcocks | Cole-Parmer | EW-30600-23 | |
Cole-Parmer Luer fittings, Large-bore stopcocks, male lock, 4-way | Cole-Parmer | EW-30600-12 | |
Cole-Parmer Stopcocks with Luer Connections; 1-way; male slip | Cole-Parmer | EW-30600-01 | |
Collagenase Type II | Worthington | LS004177 | |
Corning Sterile Cell Strainers | Fisher Scientific | 07-201-432 | |
Dell Optiplex 790 mini-tower, 4G RAM, 250G HD, Windows 7 Pro | IonOptix | CPUD7M | |
DMSO | Fisher Scientific | 50980367 | |
Dumont Tweezers Style 5 | Amazon | B00F70ZDEQ | |
FHD Rapid Change Stimulation Chamber | IonOptix | FHDRCC1 | |
Fluo-3/4 Optics Package | IonOptix | IonOP-Fluo | |
Fluorescence system interface – (w PCI-I/O card) | IonOptix | FSI700 | |
Gibco Penicillin-Streptomycin (10,000 U/mL) | Fisher Scientific | 15-140-122 | |
Glucose | Fisher Scientific | D16-1 | |
Hemostat, Curved 5-1/2" | Amazon | B00GGAAPD0 | |
HEPES | Fisher Scientific | BP310500 | |
HyperSwitch dual excitation light source | IonOptix | HSW400 | |
Inverted Motic Fluorescence Microscope | IonOptix | MSCP1-40 (a) | |
IonWizard Core + Analysis | IonOptix | IONWIZ | |
Iris Scissors, curved | Amazon | B018KRRMY6 | |
K2HPO4 | Fisher Scientific | P288-100 | |
KCl | Fisher Scientific | BP3661 | |
L-Glutathione reduced | Sigma-Aldrich | G4251 | |
LOOK Silk Spool, Black Braided, 4-0, 100yds | SouthernAnesthesiaSurgical Inc. | SP116-EA | |
M199 Media | Fisher Scientific | 12 340 030 | |
MgCl2 | Fisher Scientific | MP021914215 | |
MgSO4 | Fisher Scientific | BP2131 | |
MyoCam-S Digital CCD video system | IonOptix | MCS100 | |
MyoPacer Field Stimulator | IonOptix | MYP100 | |
NaCl | Fisher Scientific | BP358212 | |
NaH2PO4 | Fisher Scientific | 56-754-9250GM | |
Oxygenator Bubbler with Fluid Inlet for 0.25 Liter | Radnoti, LLC | 140143-025 | |
Photomultiplier sub-system | IonOptix | PMT400 | |
PMT Acquisition add-on | IonOptix | PMTACQ | |
Radnoti Heating Coil 5 mL with Degasing Trap | Radnoti, LLC | 158830 | |
Ring Clamp 60 – 80mm Dia. for 250ml Reservoir | Radnoti, LLC | 120141-025 | |
Ring Clamp for Bubble Trap Compliance Chamber | Radnoti, LLC | 120149RC | |
Saint-Gobain ACF000010 5/32 in.9/32 in. | Fisher Scientific | 14-171-214 | |
Saint-Gobain ACF000013 3/16 in.3/8 in. | Fisher Scientific | 14-171-217 | |
Saint-Gobain ACF000016 1/4 in.5/16 in. | Fisher Scientific | 14-171-219 | |
Saint-Gobain ACF000025 5/16 in.5/8 in. | Fisher Scientific | 14-171-226 | |
Saint-Gobain ACF00003 1/16 in.3/16 in. | Fisher Scientific | 14-171-209 | |
Saint-Gobain ACF00005 1/16 in.3/32 in. | Fisher Scientific | 14-171-210 | |
Saint-Gobain ACF00009 5/32 in.7/32 in. | Fisher Scientific | 14-171-213 | |
Sarcomere Length Recording add-on | IonOptix | SARACQ | |
T/C Adson Tissue Platic Surgery Forceps 4.75" | Amazon | B00JDWRBGC | |
VETUS Anti-Static Curved Tip Tweezers | Amazon | B07QMZC94J | |
Vistek 3200 Motic Vibration Isolation Platform | IonOptix | ISO100 |