Optical Mapping of Cardiomyocytes in Monolayer Derived from Induced Pluripotent Stem Cells

Figures

• 

  Figure 1. Early cardiac optical mapping: (a) first cinematographic apparatus used by G. Mines to map and record contractions of perfused frog hearts [32]; (b) simultaneous AP recordings from frog hearts using the potentiometric dye merocyanine 540 (Vf) and microelectrodes (Ve) produce faithful recordings of transmembrane potentials using fluorescent signals [42]; (c) merocyanine 540, RH155, Di-4-ANEPPS, and XXV are some of the fluorophores that have been used to record membrane action potentials [43].

• 

  Figure 2. The mechanism of voltage-sensitive electrochromic dyes. (a) Voltage-sensitive dyes (VSDs) adhere to the membrane and orient so that their changing dipole is parallel to the transmembrane electric field. VSDs detect changes in surrounding potential when the generated electric field directly interacts with the chromophore. (b) The unexcited di-4-ANEPPS (ground state) undergoes a large dipole moment change in the presence of the electric field (excited state). (c) Changes in fluorophore energy levels result in small spectral changes in dye emission. (d) The fluorescence intensity of the probe changes in correlation with the change in membrane potential. The shift in emission spectrum is exaggerated for illustrative purpose. The AP trace in (d) is from unpublished data from Dr. M. Chahine’s laboratory.

• 

  Figure 3. Ca2+ transient imaging using fluorescent indicators. (a) Mechanical (i) and luminescent (ii) responses during a contraction of a frog atrium. The first luminescence signal of a Ca2+ transient was obtained by injecting the cardiac muscle with aequorin, a Ca2+-sensitive photoprotein [61]. (b) Ca2+-sensing mechanism of the Rhod-2 AM indicator when loaded into the cell.

• 

  Figure 4. Macroscope configurations for hiPSC-CM monolayer optical mapping. Examples of a lensless (a), single lens (b), and tandem-lens (c) optical setups.

• 

  Figure 5. Dual sensor optical mapping setup. An example of a dual camera macroscope for simultaneous optical mapping of APs and CaTs in hiPSC-CM monolayers using RH237/Rhod2-AM probes combination.

• 

  Figure 6. Blebbistatin, a cardiomyocyte contraction inhibitor: (a) chemical structure of blebbistatin; (b) optically recorded APs of hiPSC-CM monolayers loaded with di-4-ANEPPS and without blebbistatin (in red at the top of the panel). An artifact can be seen after each repolarization due to cardiomyocyte contraction. The APs in black were recorded using blebbistatin. A zoom-in of overlapped AP traces with and without blebbistatin is shown at the bottom of the figure. The traces are unpublished data from Dr. M. Chahine’s laboratory provided for illustrative purposes.

• 

  Figure 7. Optical signal processing and spatial filtering. (a) Representative example of optical mapping raw data (square detector), where the fluorescence intensity of each pixel is converted to a digital value in N matrices of (n × n), where N is the number of frames recorded (N = FPS (s−1) × the recording duration (s)). Fluorescence data are filtered using a mean filter formalism (bottom left). (b) Mean and median filter operations demonstrated by a numerical example. (3 × 3) spatial filters are applied to each pixel to remove high-frequency noise from the APs. (c) Processing of the optical signal. Recorded fluorescence intensity data shown as a heat map. APs are plotted from a selected ROI (blue box). Filters improve the quality of the optical signal by removing high-frequency noise, as shown in the AP traces, which also results in a smoother fluorescence intensity map. The AP baseline usually drifts because of photobleaching. By fitting this baseline drift with a polynomial or exponential equation, the drift is easily subtracted, and the baseline is adjusted closer to zero. The same steps are followed in order to process CaT optical recordings. The traces and images are unpublished…

• 

  Figure 8. Analysis of membrane voltage optical mapping data. (a) Isochronal activation maps are generated using activation times (tAct) of each pixel. APs tAct are measured at the maximum first derivative (dF/dtmax), whereas tAct of CaTs are measured at 50% of the upstroke phase, as shown in (e). Each color in the activation map corresponds to an activation time interval. (b) Macroscopic CV and CaPV calculation. The AP and CaT propagation rates are measured between the points D1 and D2, which were selected in the direction of the wave propagation along a path orthogonal to the propagation isochrones using the simple equation shown in the bottom right. (c) Optical APD measurements. APD50 and APD80 correspond to the duration from tAct to the time at 50% and 80% of AP repolarization. (d) The rising time of AP. tRise was measured from 10% to 90% of the upstroke phase to avoid misleading noise in the optical signal even after spatial filtering. (e) Illustration of a CaT with the calculation methods of the TDs at 50% and 80% of reuptake, the activation half-times between the activation time tAct and the time at the peak tpk, as well as the decay time constants (τ). The traces and images are…