Esophageal Cancer-Related Gene-4 Contributes to Lipopolysaccharide-Induced Ion Channel Dysfunction in hiPSC-Derived Cardiomyocytes

Figures

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  Figure 1. Expression of ECRG4 in hiPSC-CMs (hiPSC-CM-WT1.1) at different differentiation time and immunostaining of hiPSC-CMs for ECRG4. (A) Relative mRNA levels (normalized to GAPDH) of ECRG4 were analyzed by real-time PCR in hiPSC-CMs at different differentiation time as well as human cardiac muscle cell line AC16 and mice cardiomyocytes (MC). (B) Immunostaining of hiPSC-CMs at day 60 after differentiation with α-actinin, cTnT and ECRG4 antibody, showing the expression of ECRG4 in hiPSC-CMs. Nuclear staining was induced with DAPI (blue).

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  Figure 2. Immunostaining of ECRG4 and TLR4, and expression of ECRG4 in hiPSC-CMs after LPS treatment. (A) Immunostaining of non-permeabilized hiPSC-CMs with ECRG4 (red) antibody and WGA (green). Nuclear staining was induced with DAPI (blue). (B) Immunostaining for TLR4 (green) and ECRG4 (red) suggest co-localization of TLR4 and ECRG4, which is supported by merged yellow signaling (red + green). Nuclear staining was induced with DAPI (blue). (C and D) Relative mRNA levels (normalized to GAPDH) of ECRG4 were assessed by real-time PCR in hiPSC-CMs after challenge by LPS at different concentrations for 24 h and different time. Values given are mean ± SEM. ** P<0.01, *** P<0.001.

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  Figure 3. ECRG4 knockdown changes the expression level of TLR4-associated signaling genes, inflammatory cytokines and ion channels in hiPSC-CMs (hiPSC-CM-WT1.1). (A) Relative mRNA (normalized to GAPDH) levels of ECRG4 were analyzed by real-time PCR after infection of ECRG4-siRNA adenovirus and negative control (N.C). (B) The relative mRNA levels of LPS-signaling associated genes after ECRG4 knockdown. (C) The relative mRNA levels of different cytokines after ECRG4 knockdown. (D) The Western blot results of total IKK and NFκB P65 (normalized to GAPDH) as well as phosphorylated IKK and NFκB P65 (normalized to total protein) after transfection of ECRG4-siRNA adenovirus and negative control. (E) Relative mRNA levels (normalized to GAPDH) of ion channels were analyzed by real-time PCR after transfection of ECRG4-siRNA adenovirus and negative control. Values given are mean ± SEM. *P<0.05, **P<0.01, *** P<0.001 versus control (N.C), ns, not significant.

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  Figure 4. ECRG4 knockdown shortened potential duration and intercepted LPS effect on APD in hiPSC-CM-WT1.1. (A) Representative traces of action potentials (AP) in control, LPS, ECRG4 knockdown and ECRG4 knockdown plus LPS-treated hiPSC-CMs. (B) Mean values of APD at 50% repolarization (APD50). (C) Mean values of APD at 90% repolarization (APD90). (D) Mean values of resting potentials (MP). (E) Mean Values of action potential amplitude (APA). (F) Mean values of maximal upstroke velocity of AP (Vmax). Values given are mean ± SEM. *P<0.05, **P<0.01, ns, not significant.

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  Figure 5. ECRG4 knockdown enhanced small conductance calcium-activated K+ channel currents (ISK) and attenuated Na/Ca-exchanger current (INCX) in hiPSC-CM-WT1.1. Membrane currents were recorded in hiPSC-CMs after transfection of ECRG4-siRNA adenovirus and negative control (N.C). 100 nM apamin, a blocker of SK channels was used to isolate ISK from other currents. The apamin-sensitive currents were analyzed as ISK. INCX was recorded by ramp pulses from −30 to +60 mV, then to −100 mV (100 mV/s) at 0.5 hz with the holding potential of −30 mV. NiCl2 (5 mm) was applied to separate INCX from other currents. (A) Representative traces of apamin sensitive ISK in control and ECRG4 knockdown. (B) I–V curves of apamin sensitive ISK from −80 to +80 mV with the holding potential of −50 mV. (C) Averaged values of apamin sensitive ISK at +40 mV. (D) Representative traces of INCX in control and ECRG4 knock-down. (E) Mean values of peak INCX at +60 mV. (F) Mean values of peak INCX at −100 mV. Values given are mean ± SEM. *P<0.05, ns, not significant.

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  Figure 6. ECRG4 knockdown intercepted the LPS effect on ISK and INCX in hiPSC-CM-WT1.1. (A) Current-voltage (I–V) relationship curves of ISK. (B) Mean values of apamin sensitive ISK at +40 mV. (C) Mean values of peak INCX at +60 mV. (D) Mean values of peak INCX at −100 mV. Values given are mean ± SEM. *P<0.05, **P<0.01, ns, not significant.

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  Figure 7. NFκB signaling blockers inhibited the ECRG4 effect on ISK and INCX. ECRG4 was overexpressed in hiPSC-CMs (hiPSC-CM-WT1.1). TPCA-1 (0.5 μM) was applied 1 hour and QNZ (EVP4593) of 100 nM was applied 24 hours before current recordings in hiPSC-CMs infected with ECRG4 overexpression adenovirus or control virus (N.C). (A) Current-voltage (I–V) relationship curves of ISK. (B) Mean values of ISK at +40mV. (C) Mean values of peak INCX at +60 mV. (D) Mean values of peak INCX at −100 mV. Values given are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ns, not significant.