Examining Cardiomyocyte Dysfunction Using Acute Chemical Induction of an Ageing Phenotype

Figures

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  Figure 1. Effect of hydroxyurea (HU) treatment on spontaneous Ca2⁺ signals in neonatal ventricular myocytes (NRVMs). Panel (A) illustrates the imaging and EFS stimulation protocol used and shows representative Ca2⁺ traces obtained from control NRVMs, and cells incubated with 50 or 500 µM HU for 4 days. Panel (B) quantifies the average frequency of spontaneous Ca2+ signals pre EFS for control NRVMs and for cells incubated with 50 or 500 µM HU for 1, 4 or 7 days. Panel (C) quantifies the average frequency of spontaneous Ca2+ signals post EFS for control NRVMs and for cells incubated with 50 or 500 µM HU for 1, 4 or 7 days. The data are presented as mean ± S.E.M. and were analysed using one-way ANOVA. The data were obtained from nine replicate NRVM coverslips for each condition.

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  Figure 2. Responses to electric field stimulation (EFS) in control or HU-treated NRVMs. Panel A illustrates regular responses during EFS application (Ai) and alternans (Aii). Panel (B) shows the percentage of NRVMs that displayed regular responses to EFS on different days of incubation with HU. Panel (C) illustrates the percentage of NRVMs that responded to EFS with alternans. The data are presented as mean ± S.E.M. and were analysed using one-way ANOVA. * denotes p < 0.05. The data were obtained from nine replicate NRVM coverslips for each condition.

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  Figure 3. Responses to electric field stimulation (EFS) in control or HU-treated NRVMs. Panel (A) shows images of control and HU-treated NRVMs loaded with Cal-520 (day 7 of HU incubation). Images of diastolic (pre EFS) and systolic (peak EFS response) Cal-520 fluorescence are shown. Panel (B) shows quantitation of the peak systolic response to EFS in control and HU-treated cells. Panels (C) and (D) show illustrative responses of control and HU-treated cells to EFS (the EFS was applied during the time shown by the dashed line). The responses shown in Panels (C,D) were obtained by averaging the Cal-520 signal across a whole field of cells (i.e., fields of cells similar to those in Panel (A)). Averaging across a whole field of cells does not allow visualisation of the spontaneous Ca2+ transients that occur within individual cells (due to the asynchronous nature of spontaneous Ca2+ transients between cells). However, spontaneous action potentials that generate simultaneous Ca2+ signals in all the cells in a field of view can be resolved. Examples of spontaneous action potential-evoked Ca2+ transients (marked with *) have the same amplitude as those triggered by EFS. The data were obtained from…

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  Figure 4. Comparison of caffeine-induced Ca2+ transients in control and HU-treated NRVMs. Panel (A) shows representative caffeine-induced Ca2+ transients in control and HU-treated cells. Panel (B) illustrates the parameters quantified to characterise the caffeine-induced Ca2+ signals. Panel (C) shows the quantification of the time constant, tau, derived from fitting mono-exponential decay curves to the caffeine-induced Ca2+ signals. Panel (D) shows the quantification of the transient duration measured as full-width at half-maximal amplitude. Panel (E) shows the quantification of the integrated Ca2+ signal (area under the curve; ‘AUC’). Panel (F) indicates the maximum amplitude (ΔF/F0) of caffeine-induced Ca2+ transients in control and 500 µM HU-treated cells. The data shown were all obtained on day 4 of HU incubation. The data are presented as mean ± S.E.M. and were analysed using unpaired t-tests. * and *** denote p < 0.05 and p < 0.001, respectively. The data were obtained from seven replicate NRVM coverslips for each condition (see Materials and Methods for details).

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  Figure 5. Using the ratiometric fluorescent indicator JC-10 to determine mitochondrial membrane potential in NRVMs. Panel (A) shows the red fluorescence of JC-10 aggregates before (Ai) and after (Aii,Aiii) addition of 10 µM antimycin. Panel (B) shows the green fluorescence of JC-10 monomers before (Bi) and after (Bii,Biii) addition of antimycin. Panel (C) illustrates the fluorescence (a.u.) of JC-10 monomers (green bars) and aggregates (red bars) before (pre) and after (post) addition of antimycin. The ratio of the red/green JC-10 fluorescence is presented in Panel Cii. The data are presented as mean ± S.E.M. and were analysed using one-way ANOVA. **** denotes p < 0.0001. The data were obtained from 16 replicate NRVM coverslips for each condition (see Materials and Methods for details). The scale bar represents 20 µm.

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  Figure 6. Assessment of the mitochondrial membrane potential in control and HU-treated NRVMs using JC-10. Panel (A) shows representative JC-10 and MitoTracker Red fluorescence images obtained using NRVMs on day 7. MitoTracker Red was used to confirm that the JC-10 staining was localised in mitochondria and to choose regions of interest for analysis. The scale bar represents 10 µm. Panel (B) shows a quantitative summary of the JC-10 red:green ratio in Day 7 control and HU-treated NRVMs. The data are presented as mean ± S.E.M. and were analysed using one-way ANOVA. **** denotes p < 0.0001. The data were obtained from nine replicate NRVM coverslips for each condition.

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  Figure 7. ROS production in control and HU-treated NRVMs. Panel (Ai) shows an image of NRVMs stained with MitoTracker Red, which was used to focus on the cells at the start of the experiment and to find unbiased regions of interest to analyse ROS Brite fluorescence. Panels (Aii) and (Aiii) show ROS Brite fluorescence in the same cells pre and 120 s post antimycin treatment, respectively. The scale bar represents 20 µm. Panel (B) shows changes in the ROS Brite fluorescence pre and post antimycin addition in control and 500 µM HU-treated cells on day 4 post-HU treatment. The data are presented as mean ± S.E.M. and were analysed using unpaired t-tests for the individual time points. Panel (C) compares the basal ROS levels in control and 500 µM HU-treated cells. The data are presented as mean ± S.E.M. and were analysed using an unpaired t-test. * denotes p < 0.05. The data were obtained from five replicate NRVM coverslips for each condition.

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  Figure 8. Autophagy in control and HU-treated NRVMs. Panel (A) shows representative images of autophagosomes stained with a CytoID kit in control and HU-treated cells. The scale bar represents 10 µm. Panels (Bi–Biii) show quantitation of autophagosome numbers in control cells and HU-treated NRVMs on days 1, 4 and 7. The data are presented as mean ± S.E.M. and were analysed using one-way ANOVA. **** denotes p < 0.0001, respectively. The data were obtained from nine replicate NRVM coverslips for each condition.

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  Figure 9. Transmission electron microscopy (TEM) images of control and HU-treated cells. Panels (A–C) show representative TEM images of control cells on day 7, illustrating the presence of myofilaments (mf), intercalated discs and mitochondria (mi) with preserved cristae (outlined in orange). Panels (D,E) are representative images from 500 µM HU-treated cells on day 7, showing the presence of mitochondria with disrupted cristae, outlined in green (Panel (D)), ribosome-associated reticular membranes (r-a-RM), membranous cytoplasmic bodies (MCB) and autophagosomes (au). The data were selected from 35 images for each condition.

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  Figure 10. Quantification of ultrastructural aspects of control and HU-treated NRVMs. Panel (A) shows the mitochondrial area. Panel (B) indicates mitochondrial number per 50 µm2. Panel (C) presents the number of mitochondria with disrupted cristae per 50 µm2. Panel (D) shows the percentage of NRVMs containing ribosome-associated membranes in control and HU-treated cells. The data are presented as mean ± S.E.M. and were analysed using unpaired t-tests. * and ** denote p < 0.05 and p < 0.01, respectively. The data are representative of 22 images obtained for each condition.

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  Figure 11. TEM images of control and HU-treated cells. Panel (A) shows a TEM image of control NRVMs on day 7, illustrating the presence of myofilaments (mf) and z-lines. Panel (B) is a representative TEM image from 500 µM HU-treated cells on day 7, which represents the majority of cell sections obtained (25 out of 30 images) showing fewer myofilaments and less obvious z-lines. Panel (C) is an image from an HU-treated cell that displayed some myofibres, evident z-lines, and intercalated discs (ic) (5 out of 30 images). The data are representative of 30 images obtained for each condition.

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  Figure 12. TEM images of control and D-galactose-treated cells. Panels (A–D) show TEM images of D-galactose-treated cells on day 7, illustrating the presence of myofilaments (mf), z-lines, mitochondria (mi) and intercalated discs (ic). The data are representative of 35 images obtained for each condition.