Spatiotemporal stability of neonatal rat cardiomyocyte monolayers spontaneous activity is dependent on the culture substrate

Abstract

In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS) is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.

Fig 1. Spontaneous frequency on glass vs. PDMS substrates.

Example of a spontaneous reentry as observed on glass substrate. A. Normalized calcium transients over time obtained from the pixel marked by a white × in panel B. B. Snapshots of the dF/dt over 1 rotation of reentry with time stamps indicated at the top left of each panel. The white circle in the top left snapshot at 200 ms illustrates a trajectory around the reentry core (scale bar is 3 mm) used for the time/phase plot depicted in panel C. The period of activity in this example was highly stable around 255 ms. C. Propagation along the trajectory depicted as a white circle in panel B shows periodic activity. However, it is clear that the activation delay δ (δ = slope) is not constant with large regions of rapid velocity along the trajectory from 3π/4 to 3π/2, while the region close to 2π illustrates a slow conduction area. The local activation delay was not uniform along the reentrant trajectory and changed from δ₁ = 0.15 second/cm to δ₂ = 0.4 second/cm, δ₃ = 0.18 second/cm, and finally δ₄ = 0.66 second/cm. D. Activation map obtained for the time interval between 0.1 and 0.35 second of reentrant activity. The maps shows a central core of ~0.31 mm² (indicated by the arrow) and highlights the slow region of the pathway of reentry (time near 0.1 to 0.15 second on the top right of the map).

Fig 2. Temporal variation in the period of activity.

A. Left panel shows a sample signal of spontaneous activity obtained by videomicroscopy and right panel shows the calculated interbeat period. The standard deviation of the temporal distribution of activation period (σ_period) was calculated as an estimate of temporal variability. B. Histogram of σ_period obtained for the glass, PDMS 1:20, and PDMS 1:40 groups. The median of the σ_period calculated for each group is: 0.19 second (glass), 0.29 second (PDMS 1:20), and 0.23 second (PDMS 1:40); n = 44, N = 11.

Fig 3. Increased number of initiation sites on PDMS substrate.

A. The mean number of activation sites for cardiomyocyte monolayers cultivated on glass, PDMS 1:20, and PDMS 1:40 were 1.3±0.3, 2.8±0.4, and 2.8±0.5 sites, respectively. The mean number of activation sites is significantly higher on PDMS compared to glass (p<0.05). Examples of activation patterns on different substrates. i) A trace of calcium transients of the spontaneous activity is shown with ii) activation maps of the first beat for each different activation site for glass (B), PDMS 1:20 (C), and PDMS 1:40 substrates (D). Sites are labeled with a number on top of the calcium transient. The corresponding site number is indicated at the time it starts to drive the monolayer (calcium transients without corresponding numbers originated from the same initiation site as the previous one). In these examples, 1 initiation site was observed on glass compared to 3 sites for both PDMS substrates.

Fig 4. Role of the proteins expression on spontaneous activity.

A. Cx43 mRNA expression was 6.24±0.89, 4.55±0.27 and 5.70±0.12. According to our results, Cx43 mRNA expression has a tendency to be lower on PDMS substrates compared to glass, however, significant differences was detected between PDMS 1:20 and PDMS 1:40. B-D. mRNA expression of targeted proteins playing a role on automaticity through the voltage clock could explain the small changes observed in rhythm. Kir2.1 (B) mRNA expression tends to be lowered when cardiomyocytes are cultivated on PDMS compared to glass. There is a tendency towards increased expression of CaV3.1 (C) and HCN2 (D) on PDMS compared to glass (p<0.05, between glass and PDMS 1:40).

Fig 5. Effects of parasympathetic and sympathetic stimulation on the rate of spontaneous activity.

Relative spontaneous frequency was obtained by videomicroscopy and calculated by taking spontaneous frequency recorded at t = 1, 5, 10, 15, and 20 minute(s) and by dividing that value by the spontaneous frequency prior to the addition of the drug (pre-drug at t = 0). A. Sympathetic stimulation by addition of 100 nM of ISO. The rates of contraction at t = 1 minute after the addition of ISO were 1.62±0.28 Hz (glass), 1.64±0.12 Hz (PDMS 1:20), and 1.56±0.13 Hz (PDMS 1:40); consequently, no significant differences were observed at t = 1 minute between the groups (p = 0.53). However, the rates of contraction at t = 20 minutes after the addition of ISO were 1.14±0.15 Hz (glass), 1.84±0.15 Hz (PDMS 1:20), and 1.54±0.11 Hz (PDMS 1:40). Statistical analysis showed significant differences at t = 20 minutes between the groups (p<0.01). B. Effects of parasympathetic stimulation by addition of 1 μM of ACh. The rates of contraction at t = 1 minute after the addition of ACh were 0.56±0.07 Hz (glass), 0.51±0.10 Hz (PDMS 1:20), and 0.63±0.12 Hz (PDMS 1:40); consequently, no significant differences were observed at t = 1 minute between the groups (p = 0.700). The rates of contraction at t = 20 minutes after the addition of ACh were 0.52±0.12 Hz (glass), 0.45±0.11 Hz (PDMS 1:20), and 0.69±0.10 Hz (PDMS 1:40); no significant differences were observed at t = 20 minutes between the groups (p = 0.33). C. No changes in σ_period were observed with time for cardiomyocytes cultivated on glass after the addition of ISO. The σ_period for cardiomyocytes cultivated on PDMS 1:20 was decreased significantly 10, 15, and 20 minutes after the addition of ISO (p<0.05, Mann-Whitney comparison test). There was a tendency towards a decreased σ_period for cardiomyocytes cultivated on PDMS 1:40 in the presence of ISO. D. A significant increase in σ_period at t = 1 minute (p<0.05, Mann-Whitney comparison test) was observed for cardiomyocytes cultivated on glass after the addition of ACh. No significant change was observed for the remaining measurements (t = 5, 10, 15, and 20 minutes). The σ_period for cardiomyocytes cultivated on PDMS 1:20 only increased significantly at t = 10 and 15 minutes after the addition of ISO (p<0.05, Mann-Whitney comparison test). A significant increase in σ_period was observed for PDMS 1:40 at t = 1, 5, 10, 15, and 20 minutes (p<0.05, Mann-Whitney comparison test).

Fig 6. ISO decreases σ_period for cardiomyocytes cultivated on PDMS 1:20 and tends to stabilize the rate of contraction.

Examples of cardiomyocyte activity stabilization by the addition of isoproterenol. i) A trace of the contractile activity is shown with ii) activation maps of the first beat for each different activation site. A. Conditions before the addition of isoproterenol (Pre-ISO) on PDMS 1:20. B. One minute after adding ISO (100 nM) on PDMS 1:20 substrates. Pharmacological sympathetic stimulation appears to decrease the number of activation sites (from 4 sites pre-ISO to 2 sites after ISO).