Transthyretin deposition alters cardiomyocyte sarcomeric architecture, calcium transients, and contractile force

Figures

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  FIGURE 1. Cardiomyocytes cultured on transthyretin (TTR)‐coated surfaces have altered calcium‐handling properties and more spontaneous release events. (a) NRVMs grown on 10 kPa hydrogels and loaded with Fluo‐4, AM have increased spontaneous release events as indicated by black arrows when surfaces are coated with TTR fibrils. The number of NRVMs containing at least one spontaneous release event is significantly increased when 10 kPA surfaces are coated with TTR fibrils (b). An increased relative frequency of spontaneous release events per time (c), along with a decreased beat rate (d) is observed on TTR fibrils. Analysis of calcium transients indicates that NRVMs cultured on 10 kPa TTR fibril‐coated surfaces have slower rise time (e), prolonged duration (f), and an increased calcium decay transient tau (g). For all panels (a–g), n = 38 and 33 cells for UT and TTR, respectively. ***p < 0.001, ****p < 0.0001, ns = not significant. Wilcoxon test used for (c), Fisher's Exact Test for (d), and student's two‐tailed t‐test for (e–g), respectively. Consistent with the results from NRVMs, field stimulation (0.5 Hz) induced calcium transients in isolated mouse atrial myocytes exhibited an unaltered…

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  FIGURE 2. Cardiomyocytes cultured on TTR‐coated surfaces have decreased gap junction protein expression and altered intercellular electrical coupling. (a) Immunofluorescent images of NRVMs grown on glass substrates without (UT, left) or with (TTR fibril, right) TTR fibrils and stained for α‐actinin (red), connexin 43 (green), and DAPI (blue). Scale =25 μm. (b) Quantification of immunofluorescence indicates lower connexin 43 area normalized for cardiomyocytes in each field of view. n = 35 images per condition, ****p < 0.0001 (student's t‐test). (c) Example of Gap‐FRAP assay of calcein, AM‐loaded NRVMs grown in microgrooved surfaces. A yellow ROI around one aligned cell is photobleached and fluorescence intensity is allowed to recover, relative to the non‐bleached area. (d) Example FRAP curve of aligned, calcein, AM‐loaded cells. Importantly, single cells without neighboring cells show very slow recovery of fluorescent signal (green line). (e) FRAP constant, k, is decreased for NRVMs grown on TTR fibril‐containing microgrooves but not on TTR tetramer‐containing microgrooves. n = 4–7 experiments per condition, *p < 0.05, **p < 0.01 (One‐way ANOVA)

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  FIGURE 3. Cardiomyocytes cultured on TTR‐coated surfaces have decreased contractile profiles and force generation. (a) Overview of traction force microscopy technique in which displacement of fluorescent microbeads contained in deformable 10 kPa hydrogels is measured. (b) NRVMs on 10 kPa TTR fibril‐coated substrates have slower contraction and relaxation velocities, with a corresponding decrease in total force generation per contraction. n = 32–38 individual cells per condition, *p < 0.05, ***p < 0.001 (student's t‐test). (b) Similarly, hiPSC‐CMs on 10 kPa TTR fibril‐ or tetramer‐coated substrates have slower contraction and relaxation velocities, with a corresponding decrease in total force generation per contraction. n = 12–15 individual cells per condition, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (One‐way ANOVA)

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  FIGURE 4. Cardiomyocytes cultured on TTR‐coated microgrooves have decreased sarcomere organization and decreased mechanical junction content. (a) Immunofluorescent images of NRVMs grown on microgrooved substrates stained for α‐actinin (red), N‐cadherin (green), and DAPI (blue). Scale =25 μm. (b) Corresponding rose plot of sarcomere angles relative to groove axis for each image shown above; 90° indicates perfect longitudinal alignment. Observe a wider distribution of sarcomere angles on TTR fibril‐containing (middle) and TTR tetramer‐containing (right) grooved substrates. (c) Relative to NRVMs grown on untreated microgrooved surfaces, sarcomeres are highly disorganized on surfaces containing TTR fibrils, but to a lesser degree on surfaces containing TTR tetramers. n = 35 images per condition, *p < 0.05, **p < 0.01, ****p < 0.0001 (One‐way ANOVA). (d) N‐cadherin content, normalized to α‐actinin content, is decreased on both TTR‐containing substrates; (e) Formed N‐cadherin positive cell‐cell junctions are decreased on both TTR‐containing substrates. n = 15–30 images per condition, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (One‐way ANOVA)

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  FIGURE 5. Cardiomyocytes cultured on TTR‐coated glass substrates exhibit decreased vinculin intensity. Immunofluorescent images of NRVMs grown on untreated (a) or TTR‐coated glass substrates (b) stained for vinculin (red) and DAPI (blue). Scale =25 μm. (c) Vinculin intensity is decreased for NRVMs grown on TTR‐containing substrates. (d) Focal adhesion area, relative to cell area, is unchanged. n = 15 cells per condition, **p < 0.01, ns = not significant (student's two‐tailed t‐test)

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  FIGURE 6. Cardiomyocytes cultured on TTR‐coated glass substrates have decreased sarcomere content and increased ubiquitination at the Z‐disc. (a) Immunofluorescent images of NRVMs grown on TTR‐coated glass substrates stained for α‐actinin (red), linkage‐specific (K48) ubiquitin (green), and DAPI (blue). Scale =25 μm. (b) Sarcomere intensity, normalized to the cell area, is decreased for NRVMs grown on TTR‐containing substrates. (c) Intensity ratio of ubiquitin (K48 linkage) at the sarcomere to non‐sarcomeric ubiquitin is increased for NRVMs grown on TTR fibrils. n = 35 cells per condition, **p < 0.01, ****p < 0.0001, (student's two‐tailed t‐test)