Inhibition of Wnt/β‐catenin signaling upregulates Nav1.5 channels in Brugada syndrome iPSC‐derived cardiomyocytes

Figures

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  FIGURE 1. Wnt/β‐catenin signaling reduces SCN5A mRNA, Nav1.5 protein, and I Na in male iPSC‐derived cardiomyocytes. (A) Diagram of the Wnt/β‐catenin pathway which can be activated by CHIR that inhibits GSK‐3β, a key mediator of β‐catenin (β‐cat) degradation; β‐catenin translocates into nucleus and, together with TCF, regulates the transcription of target genes. GSK‐3β, glycogen synthase kinase 3β; TCF, T‐cell factor. (B) qRT‐PCR showing correlation between reductions in SCN5A mRNA and increases in AXIN2 mRNA in male healthy iPSC‐CMs after treatment with CHIR for 48 h (5 μM, n = 8 samples from three differentiations). Data were normalized to values in vehicle (DMSO)‐treated cells (n = 12 samples from three differentiations). The smaller changes in AXIN2 and SCN5A mRNA in experiment #1 are likely due to the reduced activity of the CHIR molecules after prolonged storage (>6 months) in stock solution. Data were analyzed by two‐tailed t‐test. (C) Left panel: representative western blot showing reduced Nav1.5 (using a polyclonal antibody) in male iPSC‐CMs after treatment with CHIR for 48 h. Calnexin was used as a loading control. Right panels show quantification of Nav1.5 higher band, lower…

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  FIGURE 2. Wnt/β‐catenin signaling reduces SCN5A mRNA and Nav1.5 protein in female iPSC‐derived cardiomyocytes. (a) qRT‐PCR showing reduced SCN5A mRNA in female iPSC‐CMs after treatment with CHIR for 48 h (5 μM, n = 8 samples from two cell differentiations). Control group was maintained in culture medium containing equal amount of DMSO (n = 9 samples from two cell differentiations). Data were analyzed by two‐tailed t‐test. (b) Representative western blot showing reduced Nav1.5 (using a polyclonal antibody) in female iPSC‐CMs after treatment with CHIR or DMSO (control) for 48 h. Two Nav1.5 bands were observed with one near 260 kDa and another with a higher molecular weight (arrowhead). (c) Quantification of Nav1.5 band densities in panel B (normalized to calnexin), showing reduced Nav1.5 higher band in CHIR group (n = 5 samples from two cell differentiations) as compared to control, DMSO‐treated group (n = 6 samples from two cell differentiations). Data were analyzed by two‐tailed t‐test. (d) Left panel, diagram showing the binding sites of the two anti‐Nav1.5 antibodies. The polyclonal antibody binds to the intracellular loop between domains I and II and the monoclonal antibody binds to the…

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  FIGURE 3. Regulation of Nav1.5 protein by Wnt/β‐catenin signaling in Brugada syndrome (BrS) iPSC‐CMs. (a) Representative western blot showing reduced Nav1.5 (polyclonal antibody) in BrS iPSC‐CMs after treatment with CHIR (right three lanes) for 48 h, as compared to DMSO‐treated cells (control, middle three lanes). Two samples from healthy iPSC‐CMs (female) were included in the first two lanes for side‐to‐side comparison which showed a lower level of Nav1.5 in BrS iPSC‐CMs. This Brugada syndrome (BrS) patient had a benign variant (R620H) and a disease‐causing variant (R811H) on each of the two SCN5A alleles. (b) Left, western blot showing upregulation of Tbx3 and downregulation of Nav1.5 (monoclonal antibody) in BrS iPSC‐CMs after CHIR treatment. Right, quantification of Nav1.5 band densities (normalized to calnexin) showing reduced Nav1.5 in BrS iPSC‐CMs after treatment with CHIR (n = 9 samples from three cell differentiations) as compared to control, DMSO‐treated cells (n = 8 samples from three cell differentiations). Data were analyzed by two‐tailed t‐test. (c) qRT‐PCR showing reduced AXIN2 and LEF1 mRNA, two known targets of Wnt/β‐catenin signaling, in BrS iPSC‐CMs after treatment with…

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  FIGURE 4. β‐catenin knockdown increases SCN5A mRNA and Nav1.5 protein in Brugada syndrome iPSC‐CMs. (A) qRT‐PCR showing reduced CTNNB1 mRNA (encoding β‐catenin) in BrS iPSC‐CMs expressing β‐catenin shRNA (n = 6 samples) as compared to cells expressing a control, non‐silencing shRNA (n = 7 samples). AXIN2 and LEF1 mRNA, two known targets of Wnt/β‐catenin signaling, were reduced after β‐catenin knockdown in BrS iPSC‐CMs (n = 8 samples for both groups). Cells were from two iPSC differentiations. Data were analyzed by two‐tailed t‐test. (B) qRT‐PCR showing increased SCN5A mRNA (n = 9 samples for control‐shRNA; n = 8 samples for β‐catenin‐shRNA) in BrS iPSC‐CMs expressing β‐catenin shRNA. mRNA of CACNA1C was not affected by β‐catenin shRNA. Cells were from two to three iPSC differentiations. Data were analyzed by two‐tailed t‐test. (C) Western blot showing reduced β‐catenin (n = 15 samples) and increased Nav1.5 (n = 14 samples) in BrS iPSC‐CMs expressing β‐catenin shRNA as compared to control shRNA group. Calnexin was used as a loading control. Cells from a total of six batches of iPSC differentiation were used for this Nav1.5 western blot study (a polyclonal antibody for panels Ce and Cg, and…

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  FIGURE 5. β‐catenin knockdown increases cell surface Nav1.5 and I Na in BrS iPSC‐CMs. (A) Representative confocal images of BrS iPSC‐CMs showing reduced β‐catenin and increased Nav1.5 in β‐catenin shRNA group. Cells were co‐stained with either α‐sarcomeric actinin (α‐SA, a marker of cardiomyocytes) or N‐Cadherin (a plasma membrane protein expressed in cardiomyocytes). The fifth column shows the expanded view of the white box in the fourth column. In the bottom panel, N‐Cadherin was shown in the sixth column to show co‐localization between N‐Cadherin and Nav1.5 (the fifth column). (B) I Na was recorded from BrS iPSC‐CMs with a bath solution containing 100 mM Na+ (E Na = +76 mV) and 0.1 mM Cd2+ (to inhibit L‐type Ca2+ current in whole‐cell recording). (a) Representative I Na recorded from BrS iPSC‐CMs with adenoviral expression of a control non‐silencing shRNA (left) or a shRNA targeting β‐catenin (right). Dashed lines indicate zero current. (b) Current–voltage relationship of I Na in BrS iPSC‐CMs expressing β‐catenin shRNA (n = 9 cells from two batches of cell differentiation, red color) or control shRNA (n = 8 cells from two batches of cell differentiation, black color). *p < 0.05 versus…

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  FIGURE 6. β‐catenin knockdown increases Nav1.5 protein in a second line of Brugada syndrome iPSC‐CMs. (a) Diagram of the genotype of a different line of Brugada syndrome iPSC‐CMs (BrS Line 2). This BrS cell line harbors heterozygous mutation. The mutant allele contains 1‐base pair deletion causing a frame shift mutation, 4190ΔA, in SCN5A gene which is anticipated to generate a truncated non‐functional Nav1.5. The monoclonal Nav1.5 antibody binds to the C‐terminus and will only detect the wild‐type Nav1.5. (b) Western blot showing similar level of α‐sarcomeric actinin (α‐SA, a marker of cardiomyocyte maturation), but reduced level of the wild‐type Nav1.5 (polyclonal antibody) in BrS Line 2 iPSC‐CMs as compared to healthy iPSC‐CMs. (c) Western blot showing upregulation of the full‐length, wild‐type Nav1.5 (the bands above 260 kDa) (using a monoclonal antibody) and unchanged α‐sarcomeric actinin (α‐SA) after shRNA‐mediated β‐catenin knockdown in BrS Line 2 iPSC‐CMs. Right panel: quantification of western blot data. n = 10 samples from two independent differentiations. Data were analyzed by two‐tailed t‐test.