Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro

Adult cardiac tissue undergoes a rapid process of dedifferentiation when cultured outside the body. The in vivo environment, particularly constant electromechanical stimulation, is fundamental to the regulation of cardiac structure and function. We investigated the role of electromechanical stimulation in preventing culture-induced dedifferentiation of adult cardiac tissue using rat, rabbit and human heart failure myocardial slices. Here we report that the application of a preload equivalent to sarcomere length (SL) = 2.2 μm is optimal for the maintenance of rat myocardial slice structural, functional and transcriptional properties at 24 h. Gene sets associated with the preservation of structure and function are activated, while gene sets involved in dedifferentiation are suppressed. The maximum contractility of human heart failure myocardial slices at 24 h is also optimally maintained at SL = 2.2 μm. Rabbit myocardial slices cultured at SL = 2.2 μm remain stable for 5 days. This approach substantially prolongs the culture of adult cardiac tissue in vitro.


Immunohistochemical staining of α-Actinin to investigate rat myocardial slice sarcomere length
Immunohistochemical staining and confocal microscopy of fresh and cultured rat myocardial slices to investigate average slice sarcomere length. A) Top -Rat myocardial slice stained for α-Actinin. To measure average slice sarcomere length, the distance between 10 consecutive sarcomere was measured. This was repeated in 10 cells per slice and the average of the cells was used. Bottom -Profile plot of 10 consecutive sarcomere. Plot correlates with the yellow line in the image above. B) Assessment of average rat slice sarcomere length using immunohistochemical staining (α-actinin) and confocal microscopy (0 Hr, SL=1.8, 2.2 & 2.4 N=9, Unloaded N=6, SL=2.0 N=8). N = number of myocardial slices. Black dots represent individual data points. Mean ± standard error is shown on graphs. One-way analysis of variance (ANOVA) was used to determine whether there were any statistically significant differences between the means of groups. $, $$, $$$ = p value < 0.05, 0.01 & 0.001 respectively compared to 0Hr. *, **, *** = p value < 0.05, 0.01 & 0.001 respectively between the two groups highlighted by bar. Source data are provided as a Source Data file.

Mechanical load is required to culture myocardial slices
Rat myocardial slices were cultured on stretchers in culture chambers with 1Hz electrical stimulation (width: 10ms, voltage: 30V) for 24 hours. Slices were cultured with a preload equivalent to SL=2.2µm or unloaded (on stretcher with slice length < resting slice length). Unloaded slices had a significantly reduced contractility at 24 hours (Stim + SL=2.2 N=6, Stim + Unloaded N=5). N = number of myocardial slices. Black dots represent individual data points. Mean ± standard error is shown on graphs. An unpaired t-test was used to determine whether there were any statistically significant differences between the means of groups. * = p value < 0.05 between the two groups highlighted by bar. Source data are provided as a Source Data file.

Myocardial slice surface Live/Dead viability assay
A) Whole 0Hr rat myocardial slice (including aligned and non-aligned areas) with the majority of the surface stained with calcein-AM (green). Tiled images of the whole myocardial slice were automatically collected using a x10 objective on a Zeiss AxioObserver equipped with a motorised stage. B) The % of the rat myocardial slice surface that was alive was quantified by measuring the percentage of the slice surface that was stained green using ImageJ. We found 60% of the surface of 0Hr slices were alive and that slices cultured with electromechanical stimulation and physiological level had a similar proportion of live cells on their surface at 24 hours. A small but significant reduction in cardiomyocyte viability was found on the surface of slices cultured in unloaded conditions. However, we have previously demonstrated that the layers of cardiomyocytes below the slice surface remain almost 100% viable 3 and the small reduction on the surface of unloaded slices would only account for ~1% of the total slice cardiomyocyte population (0 Hr N=11, Unloaded, SL=1.8, 2.2 & 2.4 N=12, SL=2.0 N=10). N = number of myocardial slices. Black dots represent individual data points. Mean ± standard error is shown on graphs. One-way analysis of variance (ANOVA) was used to determine whether there were any statistically significant differences between the means of groups. $, $$, $$$ = p value < 0.05, 0.01 & 0.001 respectively compared to 0Hr. *, **, *** = p value < 0.05, 0.01 & 0.001 respectively between the two groups highlighted by bar. Source data are provided as a Source Data file.

Connexin 43 expression heterogeneity of rat myocardial slices
A) Transverse conduction velocity, measured using point stimulation and a multielectrode array, of rat myocardial slices cultured for 24 hours (0 Hr N=14, Unloaded, SL=1.8 N=7, SL=2.0 N=6, SL=2.2 N=9, SL=2.4 N=8). B) Heterogeneity of connexin 43 expression is significantly increased with unloading and SL=2.4μm but preserved at SL=1.8-2.2μm (N=18/6, except 0 Hr & SL=2.4 N=15/6). To assess heterogeneity of Cx43 density within the slices, Z-stacks of 1400x1400μm images were acquired and a minimum threshold for Cx43 signal of 50 applied, as previously described 50 The area of Cx43 signal was measured in 700x700 m quadrants to correlate with the distance between multielectrode array electrodes. The standard deviation of values for Cx43 density in quadrants was calculated as a measure of heterogeneity within each image, as previously described 51 These values were corrected to the mean Cx43 density of each image to allow for comparison of relative heterogeneity between culture conditions with differing overall Cx43 density C) Cx43 lateralisation was investigated using a semi-quantitative technique as previously described 52 . No differences in lateralisation patterns were found between any condition (N=18/6). For conduction velocity, N = number of myocardial slices. Black dots represent individual data points. Mean ± standard error is shown on graphs. For Cx43 lateralisation, N = number of area analysed / number of myocardial slices. One-way analysis of variance (ANOVA) was used to determine whether there were any statistically significant differences between the means of groups. $, $$, $$$ = p value < 0.05, 0.01 & 0.001 respectively compared to 0Hr. *, **, *** = p value < 0.05, 0.01 & 0.001 respectively between the two groups highlighted by bar. Source data are provided as a Source Data file.

Transmission electron microscopy of rat myocardial slices
Transmission electron microscopy was performed to assess the structural properties of rat myocardial slices cultured with electromechanical stimulation for 24 hours. At all SLs, both the architecture of the myocardium and the ultrastructure of cardiomyocytes were preserved. Scale bar = 1μm.