Sympathetic neurons can modify the intrinsic structural and functional properties of human pluripotent stem cell‐derived cardiomyocytes

Figures

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  Figure 1. Characterization of hiPSC‐CMs differentiated from stem cells Assessment of the efficiency of differentiation into hiPSC‐CMs. A, flow cytometry plots showing expression of key pluripotency markers in IMR90 iPSCs, including SSEA‐4, OCT4, Nanog and SOX2. B, widefield microscopy was used to take a 3 × 3 tile scan of monocultures, staining for nuclei with DAPI (blue), F‐actin with phalloidin (green) and the key CM marker α‐actinin (red). Scale bar = 500 µm. C, the proportion of α‐actinin‐positive cells was calculated as a percentage of α‐actinin‐positive cells within the total number of cells in immunostained cultures as in B. An average of 90.69% α‐actinin‐positive cells was observed. N = 4 differentiations. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Figure 2. Exogenous NGF promotes the expansion of SN neurites Cocultures were maintained in standard medium or treated with 100 ng/ml NGF. Representative images of hiPSC‐CMs and SNs cocultured at three time points stained for α‐actinin (red), tyrosine hydroxylase (TOH, green) and nuclei with DAPI (blue): A, without NGF; or B, with NGF. Maximum projection of Z‐stacks with a 40× objective. Scale bar = 50 µm. C, significant increase in the number of branch points (P < 0.001), path length (P < 0.001) and surface area (P < 0.001) of neurites over time in coculture with NGF between 2–4 days (N/n = 4/25) and 7–10 days (N/n = 4/22), and 2–4 and 13–15 days (N/n = 4/23). Data presented as mean with SD, one‐way ANOVA. D, representative image of cocultures stained for TOH, with arrows indicating varicosities with a 40× objective, and up to 15 days in coculture with NGF. Scale bar = 50 µm. E, comparisons between supplementation with or without NGF show a significant increase in the number of branch points (P = 0.00440, P < 0.001), path length (P < 0.001) and surface area (P < 0.001) at 2–4 and 7–10 days. There was also a significant increase in the number of branch points between 7–10 and 13–15 days of…

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  Figure 3. Functional activity of SNs and morphological characteristics of the neurocardiac junction (NCJ) Functional activity of SNs at sites of contact was investigated with respect to the expression of a select synaptic protein and action potential firing in response to nicotinic stimulation. A, Left: representative immunofluorescence image of cocultured hiPSC‐CMs with 100 ng/ml NGF, stained for a prominent marker of neuroexocytosis, SNAP‐25 (red), along the neurites of SNs. hiPSC‐CMs were stained for cTnT (green), and nuclei stained with DAPI (blue). Right: a 2× zoom showing where the neuronal soma (dashed line) is located, from an above Z‐stack image. Scale bars = 50 µm, 20 µm. B, whole‐cell current clamp recording of a single SN at rest and upon exposure to 10 µM nicotine. C, schematic illustrating the setup for the scanning ion conductance microscope (SICM) to record changes in the NCJ topography over time. D, representative SICM measuring topographical images of 20 × 20 µm (top) and higher resolution 7 × 7 µm scans (bottom) of SNs at the NCJ at three stages in coculture, with no significant difference. E, summary of the volume (P > 0.999), surface area (P > 0.999), surface contact…

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  Figure 4. SNs mature hPSC‐CM cytoarchitecture Monocultures and cocultures were treated with 100 ng/ml NGF for 7–10 days and analysed for changes in cell structural parameters. A, representative immunofluorescence images of hiPSC‐CMs for α‐actinin (red), F‐actin with phalloidin (green), and nuclei with DAPI (blue), in monoculture and coculture. 20× magnification (top) and 40× magnification images (bottom) were taken; with 40× for automated analysis of the structural characteristics of sarcomeres. Scale bar = 100 µm, 50 µm. B, no significant difference in the area of hiPSC‐CMs in coculture (P = 0.740), but a significant reduction in the circularity index (P < 0.001) (monoculture N/n = 4/89, coculture N/n = 4/82). C, significant reduction in sarcomere length (P = 0.00880), and significant increase in sarcomere organization (P = 0.0174), with no significant change in alignment index (P = 0.364) in cocultured (N/n = 8/147) compared to monocultured hiPSC‐CMs (N/n = 8/154). D, representative images of hESC‐CMs with the same staining as in A for comparison. E, in contrast to hiPSC‐CMs, there was no significant change in area in coculture (P = 0.141). However, there was a significant reduction in…

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  Figure 5. Mitochondrial activity of hPSC‐CMs is not affected in coculture Monocultures and cocultures were treated with 100 ng/ml NGF for 7–10 days and analysed for changes in mitochondrial activity. A, representative images of MitoTracker CMXRos (red) used for live immunostaining of mitochondrial networks in hiPSC‐CMs (top) and hESC‐CMs (bottom) prior to fixation and staining with cTNT (green), and nuclei with DAPI (blue). Scale bar = 50 µm. B, no significant change in mitochondrial footprint (P = 0.902) or mean branch length (P = 0.102) of hiPSC‐CMs in monoculture versus coculture (N/n = 6/190 in monoculture, N/n = 5/126 in coculture). C, no significant change in mitochondrial footprint (P = 0.271) or mean branch length (P = 0.138) of hESC‐CMs in monoculture versus coculture (N/n = 2/64 in monoculture, N/n = 2/47 in coculture). Data presented as violin plots showing median with quartile range, Mann–Whitney test. D, flow cytometry analysis in hiPSC‐CMs showed no significant difference in tetramethylrhodamine, methyl ester (TMRM) or MitoTracker Green (MTG), used as a measure of mitochondrial membrane potential and abundance, respectively; normalized to mean fluorescence intensity (MFI).…

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  Figure 6. No significant change in respiration or structural gene expression of CMs in coculture The Seahorse XF test was performed to measure mitochondrial respiration and parameters of oxygen consumption rate (OCR). The effect of NGF on hPSC‐CM respiration was investigated independently. A and B, there was no significant difference in OCR across all three conditions with respect to basal respiration, ATP production or maximal respiration in hiPSC‐CMs. Data presented as mean with SD, one‐way ANOVA (except maximal respiration; Kruskal–Wallis test), N = 4. C and D, there was no significant difference in OCR across all three conditions with respect to basal respiration, ATP production or maximal respiration in hESC‐CMs. Data presented as mean with SD, one‐way ANOVA, N = 3. E, RT‐qPCR confirmed a lack of significant change in the expression of key structural genes involved in hiPSC‐CM contraction. Data presented as mean with SD, Mann–Whitney test, N ≥ 6. N = biological replicate. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Figure 7. cAMP production in cocultured hiPSC‐CMs is larger than monoculture in response to nicotine A, representative traces of FRET response from monoculture and coculture, to 1 and 10 µM nicotine, 100 µM of the PDE inhibitor IBMX, and 10 µM of the adenylyl cyclase activator NKH, to normalize to the maximum cAMP response. Note that upon binding to cAMP, the sensor exhibits a decrease in FRET. YFP, yellow fluorescent protein; CFP, cyan fluorescent protein. B, summary graphs showing cAMP production in monoculture (N/n = 5/58; P < 0.001, P = 0.00930) and coculture (N/n = 5/69; P < 0.001). Data presented as mean with SD, with Friedman test. C, summary graphs comparing monoculture and coculture at each drug application (1 µM nicotine, 10 µM nicotine, IBMX), normalized to the response in monoculture. Data presented as mean with SD, P < 0.001, Mann–Whitney test (except IBMX; unpaired t test). D, concentration–response curves depict the increase in response to isoproterenol (ISO), confirming the presence of functional βARs in hiPSC‐CMs and NRVMs. There was no significant difference in hiPSC‐CMs (monoculture N/n = 6/63; coculture N/n = 7/69) or in NRVMs (monoculture N/n = 3/32; coculture N/n =…

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  Figure 8. Baseline contraction and Ca2+ transient analysis shows larger activity in cocultured hiPSC‐CMs The CytoCypher was used to simultaneously measure Ca2+ and contractile activity of hiPSC‐CMs. Representative traces at baseline of A, pixel correlation and B, Ca2+ transients. A summary of results is shown with monocultures represented in grey, and cocultures in blue. C, pixel correlation peak height (P < 0.001) was significantly larger in coculture (N/n = 15/174) than monoculture (N/n = 12/160), and D, departure velocity (P = 0.0160) and E, return velocity (P = 0.00120) were significantly faster in coculture (N/n = 15/170) than monoculture (N/n = 11/107). F, Ca2+ transient peak height (P = 0.123) was larger in coculture (N/n = 15/250) than monoculture (N/n = 14/242), but not statistically significant. G, departure velocity (P < 0.001) was significantly faster in coculture (N/n = 10/174) than monoculture (N/n = 10/173), whilst H, return velocity (P = 0.359) was not. Data presented as mean with SD, Mann–Whitney test. N/n = biological replicate/cell number. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Figure 9. Baseline contraction and Ca2+ transient analysis shows larger activity in cocultured hESC‐CMs The CytoCypher was used to simultaneously measure Ca2+ and contractile activity of hESC‐CMs. Representative traces at baseline of A, pixel correlation and B, Ca2+ transients. A summary of results is shown with monocultures represented in grey, and cocultures in purple. C, pixel correlation peak height (P = 0.0414) was significantly larger in coculture (N/n = 4/39) than monoculture (N/n = 4/29), and D, departure velocity (P = 0.0109) was significantly faster in coculture, whilst E, return velocity (P = 0.0692) was not significantly changed (N/n = 4/38) compared to monoculture (N/n = 4/29). F, Ca2+ transient peak height (P = 0.517) was not significantly affected between monoculture (N/n = 4/42) and coculture (N/n = 4/53). Similarly, G, departure velocity (P = 0.0718) and H, return velocity (P = 0.880) were not significantly affected in monoculture (N/n = 4/41) and coculture (N/n = 4/54). Data presented as mean with SD, Mann–Whitney test. N/n = biological replicate/cell number. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Figure 10. The pixel correlation response to nicotine is rescued in cocultured hiPSC‐CMs Representative traces show stimulation of cells after 1 and 10 µM nicotine following baseline measurements in A, monoculture and B, coculture. hiPSC‐CMs in monoculture are depicted in grey, and coculture in blue. C, pixel correlation peak height in monoculture (N/n = 12/160) was significantly reduced from baseline after 1 and 10 µM nicotine (P < 0.001), with no significant difference between both nicotine applications (P > 0.999). D, departure velocity (N/n = 11/107) was significantly slower after 1 µM nicotine (P < 0.001), with no change after 10 µM nicotine (P = 0.0502, P = 0.0861). E, return velocity (N/n = 11/107) was significantly slower than baseline after 10 µM nicotine (P = 0.00499), but not after 1 µM nicotine (P = 0.0861), or between both nicotine applications (P > 0.999). F, in coculture, the pixel correlation peak height (N/n = 15/174) was significantly reduced after 1 µM nicotine (P = 0.00959), which recovered after nicotine (P = 0.125, P > 0.999). G, departure velocity (N/n = 15/170) in cocultured hiPSC‐CMs was not significantly affected (P = 0.102, P = 0.760), except after 10 µM nicotine…

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  Figure 11. The pixel correlation response to nicotine is rescued in cocultured hESC‐CMs Representative traces show stimulation of cells after 1 and 10 µM nicotine following baseline measurements in A, monoculture and B, coculture. hiPSC‐CMs in monoculture are depicted in grey, and coculture in purple. C, pixel correlation peak height in monoculture (N/n = 4/29) was significantly reduced from baseline after 1 µM nicotine (P = 0.00758) and 10 µM nicotine (P < 0.001), with no significant difference between both nicotine applications (P = 0.446). D, departure velocity (N/n = 4/29) was significantly slower after 1 µM nicotine (P = 0.00758) and 10 µM nicotine (P < 0.001), with no significant difference between both nicotine applications (P = 0.446). E, return velocity (N/n = 4/29) was significantly slower than baseline after 1 µM nicotine (P = 0.0116) and 10 µM nicotine (P < 0.001), but not significantly affected between both nicotine applications (P > 0.999). F, in coculture, the pixel correlation peak height (N/n = 4/39) in cocultured hiPSC‐CMs was not significantly affected from baseline after nicotine (P > 0.999, P = 0.210, P = 0.639). G, departure velocity (N/n = 4/38) in cocultured…

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  Figure 12. The Ca2+ transient response to nicotine is rescued in cocultured hiPSC‐CMs Representative traces show stimulation of cells after 1 and 10 µM nicotine following baseline measurements in A, monoculture and B, coculture. hiPSC‐CMs in monoculture are depicted in grey, and coculture in blue. C, Ca2+ transient peak height in monoculture (N/n = 14/242) was significantly reduced from baseline after 1 µM nicotine (P = 0.00939) and 10 µM nicotine (P < 0.001), which significantly dropped between both nicotine applications (P < 0.001). D, departure velocity (N/n = 10/173) was not significantly affected after 1 µM nicotine (P = 0.0825), but was significantly slower after 10 µM nicotine (P < 0.001), with no significant change between both applications (P = 0.179). E, return velocity (N/n = 10/173) was not significantly affected after 1 µM nicotine (P = 0.591) or 10 µM nicotine (P = 0.440), nor between both applications (P > 0.999). F, in coculture, the Ca2+ transient peak height (N/n = 15/250) was not significantly affected after 1 µM nicotine (P = 0.132) or 10 µM nicotine (P = 0.322), nor between both applications (P > 0.999). G, departure velocity (N/n = 10/174) in cocultured hiPSC‐CMs was…