This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.
The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.
In this experiment we modified the Langendorff perfusion method by adding a small silicon tube, which is especially crucial after the suppression of ventricular contractions with an excitation-contraction uncoupler. The silicon tube is used to prevent solution congestion, acidification of the perfusion solution, and development of ischemia in the left ventricle. The mouse heart is very sensitive to hypothermia; thus, temperature variations across the heart will cause artificial differences in action potential durations. Consequently, a heating system was implemented in the perfusion chamber in order to maintain a constant temperature of 37°C during the entirety of the experiment8. Since a Langendorff model does not retain innervation of the heart, one needs to consider adding neurotransmitters to the perfusate in order to investigate physiological sympathetic and parasympathetic tone9. Besides retrograde perfusion, addition of superfusion of the heart helps to maintain suitable environmental parameters such as pH and temperature. In this method, the Langendorff perfused heart was horizontally placed. A vertical Langendorff perfusion setup can also be used10, but may result in slightly different cardiac mechanics11. In addition to CMOS cameras, alternative detectors are also available and can be applied to map Vm and CaT simultaneously12.
Application of CMOS cameras of high spatio-temporal resolution assures the accuracy of the recordings; however, optical mapping signals are not from a single cell. Rather, each fluorescent signal comes from hundreds or thousands of cells, depending on optical magnification. The much larger ventricular fluorescence can distort atrial signals by optical scattering; therefore, careful interpretation of the optically recorded signals is required. Another limitation of the mouse preparation is the signal distortion and noise induced by the curvature of the surface due to the small size of the heart13. Conduction velocity measurements can be altered not only from the curvature of the mouse heart but also from electrode polarity and virtual electrodes. To achieve accuracy for conduction velocity, activation anisotropy, and repolarization maps, correct focusing of the camera at the surface of the heart is essential.
In this method, real time ECG recordings can supplement the optical investigation of cardiac electrophysiology. Voltage-sensitive dye (RH237) and calcium indicator (Rhod-2AM) are used in the protocol because of their fast response, similar excitation, and distinct emission spectra3,7. There are alternative combinations of dyes that can be used to measure Vm and CaT other than RH237 and Rhod-2AM3. A novel voltage-sensitive dye, PGHI, with a large Stoke’s shift (>200nm) was found to allow better Vm and CaT signals because of the greater separation of the emission wavelengths between PGHI and Rhod-2AM14. Future improvements may focus on exploring novel fluorescent probes, development of new imaging detectors, and improved image processing software. Higher resolution and novel optical imaging modalities for 3D optical mapping are also important future directions of optical mapping5.
The authors have nothing to disclose.
NIH grants R01 HL085369.
Chemical | Company | Catalog Number |
NaCl | Fisher Scientific, Fair Lawn, NJ | S271-1 |
CaCl2 (2H2O) | Fisher Scientific, Fair Lawn, NJ | C79-500 |
KCl | Fisher Scientific, Fair Lawn, NJ | S217-500 |
MgCl2 (6H2O) | Fisher Scientific, Fair Lawn, NJ | M33-500 |
NaH2PO4 (H2O) | Fisher Scientific, Fair Lawn, NJ | S369-500 |
NaHCO3 | Fisher Scientific, Fair Lawn, NJ | S233-3 |
D-Glucose | Fisher Scientific, Fair Lawn, NJ | D16-1 |
Blebbistatin | Tocris Bioscience, Ellisville, MO | 1760 |
RH237 | Invitrogen, Carlsbad, CA | S1109 |
Rhod-2AM | Invitrogen, Carlsbad, CA | R1244 |
Pluronic F127 | Invitrogen, Carlsbad, CA | P3000MP |
Dimethyl sulphoxide (DMSO) | Sigma, St. Louis, MO | D2650 |
Material | Company | Catalog Number |
PowerLab 26T | AD Instruments, Sydney, Australia |