Active force generation contributes to the complexity of spontaneous activity and to the response to stretch of murine cardiomyocyte cultures

Figures

• 

  Figure 1. Murine ventricular cardiomyocyte cultures on stretchable microelectrode arrays and example electrograms A, photograph of a stretchable microelectrode array (sMEA; top), with its schematic layout (middle). Colour code: blue, pacing dipoles; orange, recording electrodes; and red, markers for strain monitoring. The area where cells are seeded is shown as a purple disc. The bottom panel shows a photograph of a culture on an sMEA. Abbreviations: M, marker; P, pacing dipole; R, recording electrode. The borders of the encapsulation layer are visible as circles (black arrows). B, illustration of signal processing for a typical electrogram (Ba), an artefact resulting from an external perturbation (Bb) and a fractionated electrogram with two phases in its downstroke (Bc). Colour code: red, raw signals; magenta, after median filtering; cyan, after AC coupling; and blue, after low‐pass filtering. The bottom panel shows derivatives of the signals before (cyan) and after (blue) low‐pass filtering. Activation time is defined at the minimum of the derivative (dotted line in Ba). Differentiation of the signal in Bc resulted in two negative peaks of the derivative separated by <1 ms, and…

• 

  Figure 2. Analysis of beating variability in an example preparation without pharmacological intervention or mechanical deformation A, interbeat interval (IBI) time series for a 15 min recording. B, x and y components and norm of the slowness vector for every activation. C, tip of the slowness vector (red dots) in the x–y plane. Black lines connect successive vector tips. D, principal components analysis (PCA) of the activation times of every excitation. E, cumulated variance explained by PCA as a function of the number of principal components. F, detrended fluctuation analysis (DFA) plot for the IBI time series. Abbreviations: α, regression slope of DF(n) versus n on a double logarithmic scale; DF(n), detrended fluctuation for segments of length n; and n, length of the detrending segments. G, plot of interbeat slowness differences versus IBIs. The blue lines denote the 0, 0.2, 0.4, 0.6, 0.8 and 1.0 quantiles. H, Spearman correlation analysis between interbeat slowness difference and IBI (same data as in G, but plotted in terms of quantiles). The relative density was computed for every rectangle as the ratio of the effective number of data points to the number that would be expected without…

• 

  Figure 3. Effects of 10 μmol/l blebbistatin on the spatiotemporal characteristics of excitation variability (without external deformation) A, from top to bottom: interbeat interval (IBI) time series, x and y components and norm of the slowness vector for every activation, distribution of the tips of the slowness vectors in the x–y plane (red dots) with two‐dimensional histogram (greyscale), and detrended fluctuation analysis (DFA) plot, for a preparation in control conditions. B, same as A, for the same preparation after application of blebbistatin. C, comparison of mean IBI, entropy of the slowness distribution, DFA exponent α and Spearman's ρ (same analysis as in Fig. 2H ) in control conditions (labelled ‘C’) versus with blebbistatin (labelled ‘B’). * P < 0.05 (Student's paired t test); † P < 0.05 to be positive (Student's t test). n = 11 different monolayer preparations obtained from eight litters.

• 

  Figure 4. Summary analysis of spatiotemporal characteristics of excitation variability (without external deformation) for the experiments with streptomycin (20 μmol/l) Comparison of mean interbeat interval (IBI), entropy of the slowness distribution, detrended fluctuation analysis (DFA) exponent α and Spearman's ρ in control conditions (labelled ‘C’) versus with streptomycin (labelled ‘S’). P > 0.05 for mean IBI, entropy and DFA α (Student's paired t test). † P < 0.05 to be positive (Student's t test). n = 8 different monolayer preparations obtained from six litters. Same layout as Fig. 3C .

• 

  Figure 5. Spontaneous beat rate in an example preparation subjected repeatedly to 5% uniaxial strain A, interbeat interval (IBI) series and corresponding instantaneous beat rate (IBR) for a 15 min recording, during which 5% uniaxial strain was applied repeatedly and held for 1 min in the x direction (protocol on top). The pairs of vertical lines indicate the start and stop of the linear motors upon stretch (blue) and release (red). B, top, raster plot of electrical activations 10 s before and 30 s after each individual stretch (protocol on top), aligned on motor start and end (vertical lines). B, middle, corresponding IBR (black, individual stretches; and cyan, average). The small dots at t = −2.5 s represent the average beat rate during the last 5 s before stretch, and the open circle is the average of these values. The small dots at t = 1.75 s represent the IBR at the end of the stretching phase, and the open circle is the average of these values. B, bottom, same IBR data, but normalized to the average IBR during the last 5 s before stretch. Time is offset to zero at motor start. The time scale is identical for all plots. C, same analysis and representation as in B, but for 10 s before…

• 

  Figure 6. Effects of stretch and release on spontaneous beat rate (without any pharmacological agent) A, normalized average beat rate time series for 25 experiments upon stretch (left) and release (right) for uniaxial 5% strain in the x direction. The black traces represent the average normalized beat rate series in individual preparations. The coloured traces are the averages for the 25 experiments for stretch and release, respectively. Vertical lines indicate the onset and end of the linear stage movement. The open circles at t = −2.5 s represent the average normalized beat rate before stretch and release (defined as one). The coloured dots at t = 1.75 s represent the mean normalized (peak) increase in individual preparations, and the open circle marks the mean of all these values. B, left, ranking of the experiments according to their average beat rate in the undeformed position. The blue dots are the average beat rates immediately before the stretches, and the red dots are the average beat rates immediately before the releases. The black circles are the averages of these two values and served to rank the preparations. B, middle, for the corresponding rank, the graphs report the five…

• 

  Figure 7. Effect of 10 μmol/l blebbistatin on the response of beat rate to stretch and release A, normalized beat rate in an example preparation upon stretch (left) and release (right) in control conditions (top) and after the addition of blebbistatin (bottom); 5% uniaxial strain in the x direction. Same analysis as in Fig. 5. B, comparison of the normalized (fold) change in beat rate upon stretch and release in control conditions (labelled ‘C’) versus with blebbistatin (labelled ‘B’), for the three different target strains. * P < 0.05, Student's paired t test. n = 8 monolayer preparations obtained from seven litters, except for the 2% biaxial strain protocol (n = 7).

• 

  Figure 8. Effect 20 μmol/l streptomycin on the response of beat rate to stretch and release A, normalized beat rate in an example preparation upon stretch (left) and release (right) in control conditions (top) and after the addition of streptomycin (bottom); 5% uniaxial strain in the x direction. Same analysis as in Figs 5 and 7. B, comparison of the normalized (fold) change in beat rate upon stretch and release in control conditions (labelled ‘C’) versus with streptomycin (labelled ‘S’), for the three different strain protocols. n = 6–7 monolayer preparations obtained from five litters. P > 0.05 for all comparisons (Student's paired t test).