Geometric Understanding of Local Fluctuation Distribution of Conduction Time in Lined-Up Cardiomyocyte Network in Agarose-Microfabrication Multi-Electrode Measurement Assay

Figures

• 

  Figure 1. Conduction time measurement system setup and method. (A) Schematic drawing of multi-electrode array measurement system. Cardiomyocyte networks were cultivated in the rectangular-shaped agarose microstructures on the multi-electrode array (MEA) chip. The external field potentials (FPs) of cardiomyocytes on microelectrodes were amplified and digitally recorded to calculate the conduction time. (B) Setup of optical light pathways of the system. For the preparation of linearly craved MEA chip with collagen coating, a 1480-nm infrared laser beam was focused on the agarose layer of the MEA chip with x10 objective lens for spot heating of a portion of agarose layer for forming the microstructures. Phase-contrast images of cells in the microchambers were also recorded by the charge-coupled device (CCD) camera. (C) Time-course of drug administration. FP waveforms were recorded during 5 min of drug exposure in control medium (Ct), low concentration Quinidine (a1), and high concentration of Quinidine (a2).

• 

  Figure 2. Propagation manner of linear mouse primary cardiomyocyte network and human embryonic stem cell-derived (hES) cardiomyocyte network. (A–C) Mouse primary network in 100 μm width (D), 200 μm width (E), 300 μm width (F). (a) Phase-contrast micrograph of mouse primary cardiomyocytes network with 2100 μm in length. Microelectrodes for measuring the external field potential were set 300 μm interval. The left numbers represent the associated microelectrodes. (b) Fluorescent micrograph of mouse primary cardiomyocytes in the network. The nuclei of each cell were stained by 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI). (c) Examples of Extracellular field potential signals of microelectrodes. The red numbers represent microelectrodes shown in (a). (d) Histograms of propagation time between specific two electrodes. (D–F) hES cardiomyocyte network in 100 μm width (A), 200 μm width (B), 300 μm width (C). (a) Phase contrast micrograph of hES cardiomyocyte network with 2100 μm in length. Microelectrodes for measurering the external field potential were set 300 μm interval. The left numbers represent the associated microelectrodes. (b) Fluorecent microgragh of hES cardiomyocytes in the…

• 

  Figure 3. Characteristics of conduction time distribution in cardiomyocyte networks. (A) Comparison of fluctuations of conduction time in cardiomyocyte networks. (a)–(l) Unit length dependence of mean time (blue circles) and S.D. (orange circles) in 100 μm width (a,d,g,j), 200 μm width (b,e,h,k), and 300 μm width (c,f,i,l). Mouse primary (a–c), 0 μM Quinidine (Control) (d–f), 3 μM Quinidine (g–i), and 9 μM Quinidine (j–l). (m) The short-term variability (STV) are indicated between the neighboring electrodes from both ends of conducting section in 100 μm width (blue open circles and lines), 200 μm width (orange open squares and lines), and 300 μm width (gray open triangles and lines) were plotted. (B) Correlation between inter beat interval (IBI) and conduction time (CT) in 100 μm width hES network (a,d,g), 200 μm width hES network (b,e,h), 300 μm width hES network (c,f,i). The numbers represent those shown in Figure 2D–Fd–f. Zero micrometers Quinidine (Control) (a–c), 3 μM Quinidine(d–f), and 9 μM Quinidine (g–i). R represents correlation coefficient. (C) Cross-correlation of conduction time in 100 μm width hES network (a,d,g), 200 μm width hES network (b,e,h), 300 μm width hES network…

• 

  Figure 4. Cell concentration dependence of conduction velocity in unit lengths. Mean conduction velocities of mouse primary cells (A) and hES cells (B) in 300 μm unit length were plotted. Blue circles for 100 μm width, orange circles for 200 μm width, and green circles for 300 μm width, respectively. Error bars are S.D. of the mean conduction velocities.

• 

  Figure 5. Simulation results of conduction time distribution in the faster firing regulation model. Histogram (A), mean value (B), and S.D. (C) of propagation time acquired from the one way random walk model (n=103, mean propagation time 3.3 μm/s, and S.D. 0.36 μm/s). The distribution of conduction time dispersed, and both the mean time and S.D. increased as the firing signal propagated.