Recapitulation of dyssynchrony-associated contractile impairment in asymmetrically paced engineered heart tissue

Figures

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  Fig. 1. Pacing setup. Photographs of the symmetrical and asymmetrical pacing units, the same units with fitted engineered heart tissues (EHTs) and readily assembled in the cell culture dish filled with medium (from top to bottom). Schematic overview with exact dimensions and estimated electrical field strengths below.

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  Fig. 2. Electrical propagation. Visualization and analysis of the Ca2+-wave in human EHT (hEHT) for asymmetrical pacing under different settings. A Original recordings (400 × 32 pixel) with a Hamamatsu ImagEMX-2 fluorescence camera at 614 frames per second (fps). Red boxes (0.7 × 0.7 mm) represent the area for signal averaging at a non-excited (400 ms) and at an excited state (620 ms). Pacing electrodes were placed left of box 1. B Averaged fluorescence intensities in the four boxes plotted over time. C Temporal and spatial propagation of Ca2+-wavefront under different stimulator settings. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3. Acute and long-term effects of pacing on hEHT. A Left, still image of a video-recorded engineered heart tissue. View from above, white glow = illuminating LED. Right, excerpt from original recording of automatic visual tracing of EHT contractions, force over time derived from optical pattern recognition. B Acute effect of pacing on hEHTs. Control (never before paced) hEHTs were subjected to either symmetrical (n = 8) or asymmetrical pacing (n = 7). Forces (≈1 min after the onset of pacing) relative to each EHT's individual pre-pacing force. In a second round symmetrically paced EHTs were asymmetrically paced and vice versa leading to overall n = 15 per group. The order of pacing had no impact on the resulting forces. Parametric one sample tests compared to the control value of 100%. C Force of hEHTs with high forces (cultured on k = 0.80 mN/mm racks) and D Force, contraction and relaxation velocity of low force EHTs (cultured on k = 0.28 mN/mm racks). EHTs were either non-paced or continuously symmetrically or continuously asymmetrically paced from day 4 on (red arrow). For both pacing groups biphasic pulses at 1 Hz, 2 V and 0.6 ms duration per phase were applied. Asterisks…

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  Fig. 4. Afterload enhancement (AE). A Timeline of the experimental procedures. B Forces, C contraction and D relaxation velocities of control and long-term paced hEHTs (symmetrically or asymmetrically) after the AE-procedure (checked bars, 14-fold increased afterload/stiffness) normalized to EHTs without AE (filled bars). Pacing settings as in Fig. 3. Sample sizes n = 10–15 per pacing mode, and n = 4–7 per individual condition, two-way ANOVA followed by Sidak's correction for multiple testing, *p < 0.05.

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  Fig. 5. Protein markers of dyssynchrony. A Western blot of protein markers of dyssynchrony (CaMKII, p38MAPK, and phosphorylated AKT) or a sarcomeric protein (alpha-actinin) at the end of the long-term pacing procedure depicted in Fig. 3. For asymmetrical pacing, hEHTs were divided into two parts, paced (P) and non-paced (NP) end. B Quantification of Western blots for alpha-actinin. Dots in each group represent protein lysates from ≈75,000 input cells. C Quantification of Western blots for CaMKII, p38MAPK and phospho-AKT, all normalized to alpha-actinin values. One-way ANOVA followed by Sidak's correction for multiple testing and n = 3–7 in B–C, *p < 0.05, **p < 0.01, ***p < 0.001.