maestro pro microelectrode array (mea, axion biosystem)  (Axion BioSystems)

Journal: bioRxiv

Article Title: Plakophilin-2 Coordinates Energy Metabolism and Contractility in Cardiomyocytes, Revealing Its Roles beyond Desmosomes

doi: 10.1101/2025.01.17.633239

Figure Lengend Snippet: a, PKP2 Hom showed increase in lipid granules and no change in lipid uptake compared to WT isogenic cell quantified by confocal imaging using two lipid dyes, QBT Fatty Acid Uptake (Molecular Devices) for measuring real-time fatty acid uptake (FAU) (n = 59-90 technical replicates) and LipidSpot 610 for measuring fatty acid storage in fixed iPSC cardiomyocytes after performing FAU (n = 12-18 technical replicates). b, FIA-TOF mass spectrometry measurement showed reduced acylcarnitines in PKP2 Hom compared to WT isogenic cells (n = 6 technical replicates, 3 wells of 12-well plate were pooled as 1 technical replicate). c, PKP2 Hom showed reduced glycolytic and oxidative metabolism evaluated by the Seahorse MitoStress test (n = 14-43 technical replicates). Top kinetic curves indicated where basal or vehicle/etomoxir-induced ATP rates were calculated. d, Monolayers MEA parameters showed prolonged field potential duration (FPD) along with depressed spike amplitude, conduction velocity, and spike slope using the Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems) 58 (n = 15 technical replicates). e, Monolayers (n = 24 technical replicates) and engineered heart tissues (EHTs) (n = 16 technical replicates) showed reduced contraction force and velocity, and prolonged contraction and relaxation time in the mutant lines using SONY live cell imaging and Pulse video analysis (Curi Bio) 55 and Mantarray (Curi Bio) 57 and Pulse video analysis (Curi Bio), respectively. f, Calcium transient recordings from WT and PKP2 Hom (n = 12 technical replicates each) assessed with CuriBio Nautilai ( https://www.curibio.com/nautilai ). Parameters displayed are: time to peak from 50% to peak (T 50 -Peak), Ca 2+ transient width at 50% (CaD50), and the rate at which [Ca 2+] i reach its peak (Rise Rate). g, Acute PKP2 vs ACADVL silencing showed differential impact on field potential, contractility, and calcium transient of iPSC-CMs (details in Supplementary Fig. 4). Quantified data are presented as mean ± SD. Comparison p values were calculated by Prism unpaired t-test and ordinary One-Way ANOVA (Tukey’s post-hoc test) for c and g : p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = non-significant.

Article Snippet: Perturbed electrophysiological properties of monolayers, determined by an extracellular recording of cardiac field potential using Maestro Pro Microelectrode array (MEA, Axion Biosystem), had been reported in our previous study .

Techniques: Imaging, Mass Spectrometry, Microelectrode Array, Mutagenesis, Live Cell Imaging, Comparison

Journal: bioRxiv

Article Title: Plakophilin-2 Coordinates Energy Metabolism and Contractility in Cardiomyocytes, Revealing Its Roles beyond Desmosomes

doi: 10.1101/2025.01.17.633239

Figure Lengend Snippet: a, Study design to evaluate small-molecule metabolic modulators in isogenic Pkp2 homozygous cell model. Monolayers or engineered heart tissues (EHTs) were treated with isoproterenol or seladelpar for 1 and 48 hours before the measurement on contractility, bioenergetics, electrophysiological properties (field potential), and Ca 2+ transients. b, WT and isogenic PKP2 Hom cells were treated pharmacologically with DMSO, isoproterenol (ISO), AZD7687 (AZD), etomoxir (ETO), bezafibrate (BEZ), seladelpar (SEL), and phenformin (PHE). Contractility was evaluated at 1 and 48 hrs post treatment using SONY live cell imaging and Pulse video analysis (n = 18-48 technical replicates) (Curi Bio) 55 . Average nuclear counts from live cells were used to normalize contraction velocity. c, In response to isoproterenol or seladelpar treatment, glycoATP and mitoATP rates and Max OCR of WT and isogenic PKP2 Hom cells were quantified using the Seahorse MitoStress test (n = 4-18 technical replicates). Statistical significance within the genotypes was determined at 48 hours post post-vehicle control/small molecule administration using Prism one-way ANOVA (Tukey’s post-hoc test). d, Assessment of normalized twitch force, contraction and relaxation velocity in WT and PKP2 Hom generated 3D-EHTs 1 hour or 48 hours post-vehicle control/small molecule administration (n = 8-11 technical replicates) using Mantarray (Curi Bio) 57 and Pulse video analysis (Curi Bio) 55 . e, Assessment of field potential parameters, spike amplitude and spike slope, in WT and PKP2 Hom monolayers at 48-day post-vehicle control/small molecule administration using Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems) 58 . Data were analyzed through AxIS Navigator software and the Cardiac Analysis Tool (Axion Biosystems) (n = 12 technical replicates). f, Calcium transient recordings from WT and PKP2 Hom monolayers (n = 12 technical replicates) or 3D engineered heart tissues (EHTs) (n = 3-4 technical replicates) were evaluated with CuriBio Nautilai ( https://www.curibio.com/nautilai ), multiwell format, and analyzed by Curi Bio’s Pulse platform 55 . Parameters shown are: time to peak from 50% to peak (T 50 -Peak), the rate at which [Ca 2+] i reach its peak (Rise Rate), and half-width/Ca 2+ transient width at 50% (CaD50). Data are presented as mean ± standard deviation (s.d.). Statistical significance was determined using Prism one-way ANOVA (Tukey’s post-hoc test): p values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Perturbed electrophysiological properties of monolayers, determined by an extracellular recording of cardiac field potential using Maestro Pro Microelectrode array (MEA, Axion Biosystem), had been reported in our previous study .

Techniques: Live Cell Imaging, Control, Generated, Microelectrode Array, Software, Standard Deviation

Journal: bioRxiv

Article Title: Plakophilin-2 Coordinates Energy Metabolism and Contractility in Cardiomyocytes, Revealing Its Roles beyond Desmosomes

doi: 10.1101/2025.01.17.633239

Figure Lengend Snippet: a , Representative immunofluorescence images for PKP2 (red) and DSP (green) for the PKP2 Hom iPSC-CM isogenic line 21 days after transduction with TN-401 at the indicated MOIs. Nuclei were counterstained with Hoechst (blue). Scale bars = 200 mm. b, Quantification of PKP2 and DSP protein expression based on immunofluorescence staining for the WT and PKP2 Hom iPSC-CM isogenic lines. Protein expression was normalized to WT MOI 0 expression levels for both proteins; n = 7-9. c, Evaluation of contraction displacement and velocity in WT and PKP2 Hom iPSC-CMs at day 18 following administration of TN-401 at different MOIs: 0, 10000, 30000, 100000, 300000, and 600000 (n = 15-18) using SONY live cell imaging and Pulse video analysis (Curi Bio) 55 . d, Assessment of twitch force, contraction and relaxation velocity in WT and PKP2 Hom generated 3D-EHTs at day 15 (for WT) and 14 (for PKP2 Hom ) post-TN-401 administration, MOI = 100k; n = 12 and 3 for WT and WT + TN-401, respectively; n = 9 and 8, and PKP2 Hom and PKP2 Hom + TN-401, respectively, using Mantarray (Curi Bio) 57 and Pulse video analysis (Curi Bio) 55 . e, Assessment of field potential parameters using Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems) 58 (top row, n = 9-12) and Ca 2+ transient parameters (bottom row, n = 4-6) in WT and PKP2 Hom 11 days and 7 days post-TN-401 administration, respectively, using CuriBio Nautilai ( https://www.curibio.com/nautilai ), multiwell format, and analyzed by Curi Bio’s Pulse platform 55 . Monolayers were paced at 1.5 Hz during MEA recordings and at 1 Hz during Ca 2+ transient measurement. Quantified data are presented as mean ± SD. Comparison p values were calculated by Prism one-way ANOVA (Tukey’s post-hoc test): p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Perturbed electrophysiological properties of monolayers, determined by an extracellular recording of cardiac field potential using Maestro Pro Microelectrode array (MEA, Axion Biosystem), had been reported in our previous study .

Techniques: Immunofluorescence, Transduction, Expressing, Staining, Live Cell Imaging, Generated, Microelectrode Array, Comparison