Journal: Scientific Reports

Article Title: Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of Na V 1.5 sodium channels

doi: 10.1038/s41598-019-47632-4

Figure Lengend Snippet: Inducing CRISPR/Cas9 mediated A735V-Na V 1.5 mutation and cardiac differentiation. ( a ) Scheme of CRISPR/Cas9-mediated introduction of point mutation g.2204C > T in SCN5A . ( b ) Sequences of SCN5A showing mutation g.2204C > T in two derived MUT hiPSC-CMs compared to the isogenic WT hiPSC-CMs. One mutant clone (MUT2) possesses an additional heterozygote point mutation at position g.2197 T > G resulting in p.F733V and thus heterozygous mutant (the relevant sequence position is indicated by an arrowhead). However, this point mutation has not been reported in any cardiac disease and following Supplementary Fig. mutation p.F733V presumably does not influence the channel properties. ( c ) Pluripotency markers (SOX2, OCT4) expression in WT and derived MUT hiPSC-CMs. ( d ) Flow cytometry for the CM-specific markers cardiac Troponin T (cTnT), sarcomeric Actinin (Sarc.Act) and pan-myosin heavy chain (MyHC) showed ~50–70% CMs for WT, MUT1 and MUT2 clones after 14 days of differentiation. Lower bar graphs show qRT-PCR results on MYH6 and SCN5A expression levels for WT, MUT1 and MUT2 clones. ( e ) IF staining of cardiac aggregates with antibodies against SCN5A (red), sarcomeric actinin (Sarc.Act, green) and nuclei (DAPI, blue) suggesting robust Na V 1.5 expression for WT, MUT1 and MUT2 cells, confirmed by a lack of SCN5A staining when adding the Na V 1.5 block peptide. ( f ) Confocal images for IF staining of plated hiPSC-CMs (WT and the two A735V-Na V 1.5 clones MUT1 and MUT2), after 29 days on glass coverslips. Left panel: IF staining specific to sarcomeric actinin (Sarc.Act, green) and SCN5A (red) revealing Na V 1.5 expression in all three hiPSC-CM cell lines. Middle panel: corresponding magnification of framed sections in the left panel displaying a speckled Na V 1.5 distribution pattern in hiPSC-CMs without obvious differences in cells from WT versus mutant clones. Right panel: IF staining for β-MyHC (green) showing organized sarcomere structures and matured cardiac phenotypes in all three hiPSC-CM cell lines. Scale bar for all three panels is 50 µm.

Article Snippet: The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-Na V 1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA).

Techniques: CRISPR, Mutagenesis, Derivative Assay, Sequencing, Expressing, Flow Cytometry, Clone Assay, Quantitative RT-PCR, Staining, Blocking Assay

Journal: Scientific Reports

Article Title: Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of Na V 1.5 sodium channels

doi: 10.1038/s41598-019-47632-4

Figure Lengend Snippet: Mutant A735V-Na V 1.5 channels reduce the upstroke velocity of APs in hiPSC-CMs. ( a ) Distribution of cardiomyocyte phenotypes for all hiPSC-CMs (WT + A735V, left), WT hiPSC-CMs (middle) and A735V hiPSC-CMs (right). Cells were classified according to their AP duration as ventricular-like (APD 50 > 200 ms), atrial-like (APD 50 : 20-200 ms) or atypical (APD 50 <20 ms or no AP). ( b,c ) Representative traces of spontaneous action potentials from ventricular-like hiPSC-CMs expressing WT ( b ) or mutant A735V ( c ) Na V 1.5 channels. ( d–g ) Electrophysiological properties of spontaneous APs (mean values ± s.e.m.) from WT (n = 16) and A735V hiPSC-CMs (n = 41): Maximum diastolic potential MDP ( d ), AP duration at 50% repolarization level APD 50 ( e ), AP amplitude ( f ) and upstroke velocity ( g ). ( h,i ) Representative traces of evoked action potentials from ventricular-like hiPSC-CMs expressing WT ( h ) or mutant A735V ( i ) Na V 1.5 channels. For such recordings CMs were hyperpolarized to physiological resting potentials (-80 mV) and action potentials were elicited by intracellular application of short depolarizing current pulses (≤3 nA, 1 ms). ( j–m ) Electrophysiological properties of evoked APs (mean values ± s.e.m.) from WT (n = 21) and A735V hiPSC-CMs (n = 61): Resting membrane potential RMP ( j ), AP duration at 50% repolarization level APD 50 ( k ), AP amplitude ( l ) and upstroke velocity ( m ). Arrows in ( b,c,h,i ) mark the phase 0 upstroke of action potentials that is significantly decelerated in BrS-mutant A735V-Na V 1.5 hiPSC-CMs.

Article Snippet: The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-Na V 1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA).

Techniques: Mutagenesis, Expressing

Journal: Scientific Reports

Article Title: Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of Na V 1.5 sodium channels

doi: 10.1038/s41598-019-47632-4

Figure Lengend Snippet: Mutation A735V shifts the activation curve of Na V 1.5 channels in hiPSC-CMs. ( a,b ) Voltage protocol for sodium channel activation and representative current traces of cardiac Na V 1.5 channels recorded from WT hiPSC-CMs ( a ) and mutant A735V hiPSC-CMs ( b ). ( c ) Voltage dependence of mean Na V 1.5 current densities (pA/pF) from WT and mutant A735V hiPSC-CMs. Inset: Comparison of the maximum Na V 1.5 current density from WT and mutant A735V hiPSC-CMs. ( d,e ) Voltage protocol for sodium channel inactivation and representative current traces of cardiac Na V 1.5 channels recorded from WT hiPSC-CMs ( d ) and mutant A735V hiPSC-CMs ( e ). ( f ) Sodium channel activation (G/G max ) and inactivation (I/I max ) curves of Na V 1.5 channels from WT and A735V hiPSC-CMs. Solid lines represent Boltzmann functions that were fit to the mean values. ( g,h ) Scatter plots and mean values (±s.e.m.) of mid voltages (V 0.5 ) of activation ( g ) and inactivation ( h ) for WT and A735V-Na V 1.5 channels from hiPSC-CMs. - Data volume for sodium channel activation: WT, n = 20; A735V, n = 57; for sodium channel inactivation: WT, n = 17; A735V, n = 56.

Article Snippet: The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-Na V 1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA).

Techniques: Mutagenesis, Activation Assay

Journal: Scientific Reports

Article Title: Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of Na V 1.5 sodium channels

doi: 10.1038/s41598-019-47632-4

Figure Lengend Snippet: Effects of A735V mutation are reproducible from clone-to-clone and for different approaches of hiPSC-CMs. ( a ) Mean values (±s.e.m.) of upstroke velocities of two WT and two A735V mutant clones. There are no differences among WT clones or A735V clones, but differences between WT and A735V clones are highly significant (one way ANOVA with Tukey’s post-hoc comparison test F(3,78) = 26.32, p < 0.001). ( b ) Inter-experimental variability between different approaches with WT and mutant A735V hiPSC-CMs showing no changes in upstroke velocities among themselves, while WT versus A735V differed considerably from each other (one way ANOVA with Tukey’s post-hoc test F(6,75) = 12.82, p < 0.001). ( c ) Inter-experimental variability of mean maximal sodium current densities for WT and A735V mutant hiPSC-CMs (one way ANOVA with Tukey’s post-hoc test F(6,70) = 7.467, p < 0.001). ( d ) An increase in sodium channel density directly correlates to upstroke velocity acceleration of the same cells throughout the different hiPSC-CM approaches corroborating the effect of A735V sodium channel mutation on upstroke velocity ( d , linear regression with R² = 0.9937 and F(1,5) = 749.9; n = 7-19). Coloured symbols represent mean values of different approaches as depicted in ( b,c ). ( e ) Sodium channel activation (G/G max ) and inactivation (I/I max ) curves of Na V 1.5 channels from WT and two clones of A735V hiPSC-CMs. Solid lines represent fits with Boltzmann functions. ( f ) Mid-voltages V 0.5 of sodium channel activation for two independent A735V clones as compared to WT hiPSC-CMs (one way ANOVA with Tukey’s post-hoc test F(2,74) = 37.76, p < 0.001). ( g ) Mid-voltages V 0.5 of sodium channel inactivation for two independent A735V clones as compared to WT hiPSC-CMs (one way ANOVA with Tukey’s post-hoc test, F(2,70) = 4.261, p < 0.05). Data volume in panels (a–c) and (f–g) is as indicated by numbers. (e) comprises the same cell numbers as in (f) and (g).

Article Snippet: The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-Na V 1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA).

Techniques: Mutagenesis, Clone Assay, Activation Assay

Journal: Scientific Reports

Article Title: Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of Na V 1.5 sodium channels

doi: 10.1038/s41598-019-47632-4

Figure Lengend Snippet: Mutation A735V shifts the activation curve of Na V 1.5 channels heterologously expressed in HEK293T cells. ( a,b ) Voltage protocol for sodium channel activation and representative current traces of WT ( a ) and mutant A735V ( b ) -Na V 1.5 channels heterologously expressed in HEK293T cells. c ) Voltage dependence of mean current densities (pA/pF) from HEK293T cells expressing WT or mutant A735V-Na V 1.5 channels. Inset: Bar graph on comparison of the maximum current density for WT and mutant A735V Na V 1.5 channels. ( d,e ) Voltage protocol for sodium channel inactivation and representative current traces of WT ( d ) and mutant A735V ( e ) Na V 1.5 channels heterologously expressed in HEK293T cells. ( f ) Sodium channel activation (G/G max ) and inactivation (I/I max ) curves of WT and A735V Na V 1.5 channels expressed in HEK293T cells. Solid lines represent fits with Boltzmann functions. ( g,h ) Scatter plots and mean values (±s.e.m.) of mid-voltages (V 0.5 ) of activation ( g ) and inactivation ( h ) for WT and mutant A735V-Na V 1.5 channels. ( i ) Representative western blot analysis for heterologous Na V 1.5 channel expression in HEK293T cells, showing similar intensities for WT and mutant A735V-Na V 1.5 bands. Untransfected HEK293T cells served as negative control (Neg.). GAPDH is visualized as loading control for comparable concentrations of total protein. ( j ) Scatter plot and mean values (±s.e.m.) of relative expression levels of Na V 1.5/GAPDH (WT: n = 7; A735V: n = 7, Student´s t- test, p = 0.27). - Data volume in (c) and (f–h): WT, n = 25; A735V, n = 18.

Article Snippet: The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-Na V 1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA).

Techniques: Mutagenesis, Activation Assay, Expressing, Western Blot, Negative Control

Journal: Scientific Reports

Article Title: Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of Na V 1.5 sodium channels

doi: 10.1038/s41598-019-47632-4

Figure Lengend Snippet: Mutation A735V prolongs the time constant of recovery from inactivation for Na V 1.5 sodium channels expressed in hiPSC-CM and HEK293T cells. ( a ) Two pulse protocol to investigate the recovery from inactivation and representative current responses of WT and mutant A735V Na V 1.5 channels expressed in HEK293T cells or hiPSC-CMs. Inter-pulse interval (Δt) varied from 1 to 1000 ms (inter-pulse voltage: -120 mV). ( b,c ) Time dependent increase of relative current amplitudes (I Na (pulse2) /I Na (pulse1) ) fitted by a bi-exponential function reveal two time constants (τ 1 and τ 2 ) for recovery from inactivation of WT and mutant A735V Na V 1.5 channels expressed in HEK293T cells ( b ) and hiPSC-CMs ( c ; data points < 10 ms are not displayed for hiPSC-CMs, according to improper fast voltage clamp conditions with large cardiomyocytes). ( d – f ) Scatter plot and mean values (±s.e.m.) of fast ( d ) and slow time constants ( e,f ) of recovery from inactivation for WT and mutant A735V channels expressed in HEK293T cells ( d,e ) or hiPSC-CMs ( f ). - Data volume in (b,d) and (e): WT, n = 18; A735V, n = 16. Data volume in (c) and (f): WT, n = 6; A735V, n = 36.

Article Snippet: The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-Na V 1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA).

Techniques: Mutagenesis

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control