Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Formation of mouse primary cardiomyocyte clusters in agarose-coated wells. ( a ) Schematic drawing of the conventional dish cultivation of cardiomyocytes. The dispersed cells were cultured on the bottom of a 35-mm non-agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells attached on the bottom of the 35-mm cultivation dish dispersedly. The cells started to beat 2–3 days after cultivation started. ( b ) A micrograph of dispersed cardiomyocytes in a 35-mm non-agarose-coated dish. ( c ) Schematic drawing of the cultivation of dispersed cells in a 35-mm agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells dispersed on the bottom of the agarose layer in the agarose-coated 35-mm cultivation dish. Even after 2–3 days of cultivation, the cells remained isolated with a round shape, and no clusters formed on the bottom. ( d ) A micrograph of cardiomyocytes in an agarose-coated 35-mm cultivation dish. ( e ) Schematic drawing of the cultivation of dispersed cells in a 15.5-mm agarose-coated cultivation well (in a 24-well cultivation plate). After spread of the 1.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5\times 10^{4}\hbox { cells/mL}$$\end{document} 5 × 10 4 cells/mL isolated single cardiomyocytes, dispersed cells gathered and formed small clusters; finally, they gathered into a single large cluster in the 15.5-mm agarose-coated cultivation well. ( f ) A micrograph of a cardiomyocyte cluster in a 15.5-mm agarose-coated cultivation well.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Cell Culture, Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Micrographs of single cells and clusters of mouse primary and hES-derived cardiomyocytes. ( a ) Mouse primary cardiomyocytes (primary) in a 35-mm non-agarose-coated dish (single cell), ( b ) primary cells in a 24-well agarose-coated plate (cluster), ( c ) hES cardiomyocytes in a 35-mm non-agarose-coated dish (single cell), and ( d ) hES in a 24-well agarose-coated plate (cluster).

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Analysis of interbeat interval (IBI) distribution of single and clustered mouse primary and hES cardiomyocytes. ( a )–( d ): Method of measuring interbeat interval (IBI) of single cardiomyocytes and clusters. Temporal change of luminance in the red square area for single cell ( a ) and cluster ( c ) caused by their beating was recorded, as shown in the time-course intensity profiles ( b ) and ( d ), respectively. IBIs of their beating were acquired from the time intervals between two neighboring peaks in the time-course intensity profiles. ( e ), ( f ): Distribution of IBIs of mouse primary cardiomyocytes. ( e ) The relationship between mean IBIs and fluctuations of beating [coefficient of variability (CV) of IBIs] of single isolated primary cardiomyocytes (blue open circles, n = 73) and primary clusters (red filled triangles, n = 6). ( f ) A histogram of all plots in ( e ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean value and standard deviation (SD) of single-cardiomyocyte IBIs, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters. ( g ), ( h ): Distribution of IBIs in hES cardiomyocytes. ( g ) The relationship between mean IBIs and CV of IBIs in single isolated hES cardiomyocytes (blue open circles, n = 125) and hES clusters (red filled triangles, n = 27). ( h ) A histogram of all plots in ( g ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean values and SD of single cardiomyocytes, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation, Standard Deviation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distribution of IBIs and fluctuation of IBI distribution of the hES cardiomyocyte clusters and their constituent cells. ( a )–( c ): Micrographs of hES cardiomyocyte clusters. ( d )–( f ): Distribution of IBIs and the CV of IBIs in the clusters ( a )–( c ) and isolated constituent cells from each cluster (n=50 from among re-cultivated \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1.0\times 10^{3}\hbox { cells}$$\end{document} 1.0 × 10 3 cells ). These plots ( d )–( f ) correspond to each cluster ( a )–( c ). The red filled triangles indicate the cardiomyocyte clusters, and the blue open circles indicate constituent cardiomyocytes of each cluster. Each cluster was measured 2 days after the beating started. Single cardiomyocytes were isolated from each cluster by trypsinization. IBIs of single constituent cardiomyocytes were measured 3 days after their isolation. Median and 95% confidence interval of single cardiomyocytes were 0.971 s and 0.825–1.25 s ( d ), 1.19 s and 1.00–1.28 s ( e ), and 1.12 s and 0.844–1.22 s ( f ), respectively. ( g )–( i ): Histograms of IBIs of each cluster and its isolated constituent cells. The blue filled bars indicate the ratio of frequency for single constituent cardiomyocytes; the blue arrows and error bars indicate the mean IBIs and SDs, and the red arrows also indicate the mean IBIs of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Influence of trypsinization on interbeat intervals in dispersed individual hES cardiomyocytes. ( a ) Distribution of the IBIs and the CV of IBIs in single hES cardiomyocytes before and after trypsinization. The blue open circles indicate the single hES cardiomyocytes (n = 50) before trypsinization. The orange open circles indicate the single cardiomyocytes (n = 50) after trypsinization. ( b ) Histograms of IBIs of hES single cardiomyocytes before and after trypsinization. The blue filled bars indicate the mean IBIs of single cardiomyocytes before trypsinization; the blue arrow and error bar indicate their mean value and SD. The orange filled bars indicate the frequency of mean IBIs of trypsinized single cardiomyocytes; the orange arrow and error bar indicate their mean value and SD.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distributions of interbeat intervals (IBIs) and fluctuations of the two hES cardiomyocyte clusters before and after their connection and after re-separation. ( a )–( e ): Micrographs of cardiomyocyte clusters. Micrographs of the large cluster ( a ) and small cluster ( b ) before contact. These clusters were measured when they had been cultivated for 7 days. The two hES cardiomyocyte clusters were connected ( c ). The measurement was performed 3 days after the two clusters contacted each other. Micrographs of the large cluster ( d ) and small cluster ( e ) after separation. The measurements were taken within 5 min of separation. ( f ): Distribution of IBIs and fluctuations of two clusters before contact, during contact, and after separation. Blue filled bar and error bar indicate the mean IBIs and SD of the large cluster. Green filled bar and error bar indicate the mean IBIs and SD of the small cluster.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Heart rhythm

Article Title: Disruption of protein quality control of the human ether-à-go-go related gene K + channel results in profound long QT syndrome

doi: 10.1016/j.hrthm.2021.10.005

Figure Lengend Snippet: Colocalization of hERG and RNF207 in guinea pig ventricular cardiomyocytes. A: Confocal images showing colocalization among α-actinin2, hERG K+ channel subunits, and RNF207. Scale bar = 10 μm. The right panels show the corresponding fluorescence intensity profiles perpendicular to the z lines. B: Proximity ligation assay (PLA) for α-actinin2, hERG K+ channel subunits, and RNF207. C: Quantification of PLA signals per cell area (puncta/μm2). n = 15, 11, 15, 10, 9, and 9 cells from left to right bars; *P < .05. D: Auto-ubiquitinylation assay for RNF207WT (lane 3, right) vs negative control (lane 1, left) and MDM2, a known E3 ubiquitin ligase (positive control, lane 2). Transfected HEK 293 cells were immunoprecipitated (IP) for RNF207-FLAG. An auto-ubiquitinylation assay was conducted on isolated protein, followed by SDS-PAGE and Western blot analysis (IB). Proteins were incubated in the presence of E1 and E2 ubiquitin enzymes, ubiquitin, and ATP. E: Ubiquitination assays. Ubiquitinated proteins were absent in the negative control (lane 1). The E3-ubiquitin band appeared for hERGT613M incubated with RNF207WT (lane 3), but not in the presence of RNF207G603fs (lane 5) or with hERGWT subunits (either with RNF207WT [lane 2] or with RNF207G603fs [lane 4]). F: Quantification of the data from panel E. n = 5 independent experiments for each group; *P < .05. Data shown are mean ± SEM. Analyses were performed using 1-way analysis of variance (ANOVA) with Brown-Forsythe post hoc analyses. AU = arbitraty units; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; RNF207 = ring finger protein 207; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM = standard error of the mean; Ub = ubiquitin; WT = wild-type.

Article Snippet: We took advantage of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs, iCell, FUJIFILM Cellular Dynamics, Inc., Madison, WI) as a platform ( – ).

Techniques: Fluorescence, Proximity Ligation Assay, Negative Control, Positive Control, Transfection, Immunoprecipitation, Isolation, SDS Page, Western Blot, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Heart rhythm

Article Title: Disruption of protein quality control of the human ether-à-go-go related gene K + channel results in profound long QT syndrome

doi: 10.1016/j.hrthm.2021.10.005

Figure Lengend Snippet: Regulation of APDs of hiPSC-CMs by RNF207. A: Representative action potential recordings (iCell, Cellular Dynamics) in cells expressing hERGWT:RNF207WT (black trace), hERGWT:hERGT613M:RNF207WT (red trace), and hERGWT:hERGT613M:RNF207WT:RNF207G603fs (blue trace) as well as a nontransfected cell (gray trace). B–F: Summary data for action potential recordings in nontransfected cells (labeled “Non-TF”; gray bar) compared with hERGWT:RNF207WT (labeled “WT”; black bar), hERGWT:hERGT613M:RNF207WT (labeled “Rescue”; red bar), and hERGWT:hERGT613M:RNF207WT:RNF207G603fs (labeled “Mutant”; blue bar) at baseline (solid bars) vs 1 μM E-4031 (striped bars). Data are shown for average diastolic potential (panel B), peak action potential (panel C), action potential amplitude (panel D), action potential duration at 50% repolarization or APD50 (panel E), and action potential duration at 90% repolarization or APD90 (panel F). Data shown represents the average of 5 action potentials per cell, with n = 6–9 cells for baseline recordings and n = 3–5 cells for E-4031 recordings. *P<.05, **P<.01, §P<.001. Data shown are mean ± SEM. Analyses were performed using ANOVA with Tukey’s post hoc analyses. ANOVA = analysis of variance; APD = action potential duration; APD50 and APD90 = APD at 50% and 90% repolarization; hERG = human ether-à-go-go related gene; hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocytes; non-TF = nontransfected cells; RNF207 = ring finger protein 207; SEM = standard error of the mean; WT = wild-type.

Article Snippet: We took advantage of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs, iCell, FUJIFILM Cellular Dynamics, Inc., Madison, WI) as a platform ( – ).

Techniques: Expressing, Labeling, Mutagenesis, Derivative Assay

Journal: Heart rhythm

Article Title: Disruption of protein quality control of the human ether-à-go-go related gene K + channel results in profound long QT syndrome

doi: 10.1016/j.hrthm.2021.10.005

Figure Lengend Snippet: Regulation of hERG currents by RNF207 in hiPSC-CMs and a schematic diagram of RNF207 interaction with hERG-encoded K+ channels in adult ventricular myocytes. A: Representative E-4031–sensitive currents recorded from hiPSC-CMs expressing hERGWT:RNF207WT (black traces), hERGWT:hERGT613M:RNF207WT (red traces), and hERGWT:hERGT613M:RNF207WT:RNF207G603fs (blue traces). B: Summary data of current density for the 3 groups of cells. n = 5–6. C: Summary data for voltage-dependent activation using the peak tail current density fitted using the Boltzmann function (see Online Supplemental Table 1). n = 5–6. In panel B, *P < .05 for hERGWT:RNF207WT compared with hERGWT:hERGT613M:RNF207WT:RNF207G603fs throughout positive voltages and was shown only at the end of the curve for clarity. In panel C, *P < .05 for hERGWT:RNF207WT and hERGWT:hERGT613M:RNF207WT compared with hERGWT:hERGT613M:RNF207WT:RNF207G603fs throughout positive voltages and was shown only at the end of the curves for clarity. Analyses were performed using ANOVA with Tukey’s post hoc analyses. D: Schematic diagram of RNF207 interaction with hERG-encoded K+ channels (Kv11.1) with trafficking and degradation pathways (generated using BioRender, Toronto, Canada). ANOVA 5 analysis of variance; hERG = human ether-à-go-go related gene; hiPSC-CM 5 human induced pluripotent stem cell-derived cardiomyocytes; I = current; RNF207 = ring finger protein 207; SEM = standard error of the mean; V = voltage; WT = wild-type.

Article Snippet: We took advantage of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs, iCell, FUJIFILM Cellular Dynamics, Inc., Madison, WI) as a platform ( – ).

Techniques: Expressing, Activation Assay, Generated, Derivative Assay