MTMR4 SNVs modulate ion channel degradation and clinical severity in congenital long QT syndrome: insights in the mechanism of action of protective modifier genes

Figures

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  Figure 1. Generation of patient-specific iPSC-CMs. (A) Pedigree of the family segregating the KCNQ1-Y111C mutation. The asterisks indicate the two subjects enrolled in the study: the symptomatic (S) proband is a 16-year-old boy (II-1) with an episode of syncope with sphincter release during a combined emotional and physical stress, while the father (I-4) has never experienced symptoms, i.e. he is an asymptomatic carrier (AS). Other two family members, one uncle (I-5) and one aunt (I-6) of the S proband also carry the same heterozygous mutation, but their QT interval is normal and they have never experienced symptoms. (B) Electrocardiogram tracings. Representative electrocardiograms of the AS individual and of the S patient at age 8 prior to institution of β-blocker therapy. Rate corrected QT interval (QTc, Bazett’s formula) are indicated at the top of the tracings. (C) DNA sequencing. The analysis shows the presence of the heterozygous 332 A>G mutation in the KCNQ1 coding region, resulting, at protein level, in the substitution of the tyrosine (Y) in position 111 with a cysteine (C). (D) iPSC-CMs. Cardiac differentiation was demonstrated by expression of alpha-actinin, cardiac troponin-T,…

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  Figure 2. Expression of endogenous KCNQ1 in Y111C iPSC-CMs. (A) Quantification of KCNQ1 mRNA levels by RT-qPCR and ΔΔCt method. Nkx2.5 was used as reference gene. Data were expressed as fold increase vs. CTR (n = 3, **P < 0.005). (B) Quantification of KCNQ1 protein levels by SDS-PAGE and immunoblot in CTR, S, and AS iPSC-CMs. (C) Immunostaining of the endogenous KCNQ1 in CTR-, AS-, and S-iPSC-CMs. KCNQ1 was stained in green, cardiac troponin-T (cTnT) in red, and the nuclei in blue.

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  Figure 3. Trafficking defect of KCNQ1 in iPSC-CMs. (A) Trafficking defect of the VSV-KCNE1-KCNQ1 (VSV-E1-Q1) Y111C fusion protein. Representative images of CTR-, AS-, and S-iPSC-CMs acquired 72 h after transfection with WT or Y111C VSV-E1-Q1 plasmid. Anti-VSV staining (green) was performed on live cells. Then, the cells were fixed, permeabilized, and co-stained with an anti-Troponin-I (TnI) antibody, used as a marker specific for cardiomyocytes (red). Nuclei were stained with DAPI (blue). Scale bar = 20 μm. (B) Retention of Y111C VSV-E1-Q1-GFP fusion protein in endoplasmic reticulum. Images of CTR-, AS-, and S-iPSC-CMs co-transfected with WT or Y111C VSV-E1-Q1-GFP plasmids (green), and pDsRed2-ER plasmid (red). Cardiac cells were visualized by α-sarcomeric actin (α-SA) staining (purple). Nuclei were stained with DAPI (blue). Hollow and filled arrows indicate membrane and ER localization, respectively. Scale bar = 20 μm.

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  Figure 4. Electrophysiological characterization of LQT-iPSC-CMs. (A) Voltage-clamp recordings of endogenous IKs in CTR- and Y111C-iPSC-CMs. Representative traces (left) and average current–voltage (I–V) relationships (right) of endogenous IKs from CTR- (black), AS- (blue), S-iPSC-CMs (green). (B and C) Voltage-clamp recordings of heterologous IKs current in CTR- and LQT-iPSC-CMs transiently transfected with VSV-E1-Q1-GFP WT or Y111C. Representative patch-clamp recordings (B) and average I–V relationships (C) of heterologous IKs from CTR-, AS-, and S-iPSC-CMs transfected with VSV-E1-Q1-GFP WT or Y111C plasmids. (D) Voltage-clamp recordings of endogenous IKr. Representative IKr recordings (top panel) and average data (bottom panel) for CTR-, AS-, and S-iPSC-CMs. IKr tail currents have been magnified and overlaid in the inset. (E) Response to IKr blocking. Representative field potentials recorded with MEA in CTR-, AS-, and S-iPSC-CMs at baseline (top left, black) and after IKr block with E4031 (top right, grey). Average data (bottom panel) are expressed as delta percentage of the effect vs. the respective baseline (n = 14–19 for patch-clamp data; n = 6–9 for MEA data; *P < 0.05 CTR vs. S; °P…

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  Figure 5. Quantification of Nedd4L in iPSC-CMs and interaction with KCNQ1. (A) KCNQ1–Nedd4L protein interaction. Nedd4L–KCNQ1 protein interactions were investigated in the 293T heterologous system by co-immunoprecipitation. 293T were co-transfected with pcDNA3.1-hNedd4L-WT and pCB6-HA-KCNQ1 expressing the WT or the Y111C KCNQ1. Forty-eight hours after transfection, whole-cell extracts were collected and subjected to immunoprecipitation and western blot with the indicated antibodies (Supplementary material online, Table S2). The upper row represents a western blot of the immunoprecipitated (IP) HA-KCNQ1 proteins and shows depleted level of HA-KCNQ1-Y111C in the presence of Nedd4L over-expression. The middle row represents the immunoblot of phosphorylated-Nedd4L (Ser448) proteins co-immunoprecipitated with HA-KCNQ1-WT or -Y111C and shows dephosphorylation of Nedd4L interacting with HA-KCNQ1-Y111C. The lower row confirms equivalent level of basal Nedd4L interacting either with HA-KCNQ1-WT or -Y111C. The activated Nedd4L in Y111C group ubiquitinated the mutated channel, KCNQ1-Y111C. (B) Nedd4L mRNA levels were quantified by RT-qPCR and ΔΔCt method. Nkx2.5 was used as reference gene (n = 3, **P…

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  Figure 6. Regulation of KCNQ1 and hERG by Nedd4L in Y111C iPSC-CMs. (A) Nedd4L knockdown restores trafficking of KCNQ1-Y111C. CTR-, AS-, and S-iPSC-CMs were co-transfected with WT or mutated VSV-E1-Q1-GFP fusion protein, and with Nedd4L siRNA or scramble. To confirm the localization of KCNQ1, cell membrane was stained with wheat germ agglutinin (WGA). Arrows indicate the cell membrane. (B) Nedd4L knockdown rescued the IKs current in S-iPSC-CMs. Representative patch-clamp recordings (top panels) and average current–voltage (I–V) relationships (bottom panel) of S-iPSC-CMs transfected with the WT VSV-E1-Q1-GFP fusion protein alone (black) or in combination with the Nedd4L siRNA (red) (n = 11, 9, *P < 0.05 vs. KCNE1-Q1 WT). (C) Nedd4L knockdown restored hERG trafficking in S-iPSC-CMs. S-iPSC-CMs were transfected with scramble or Nedd4L siRNA. Expression of endogenous hERG was assessed by immunostaining (green). Surface localization was confirmed by wheat germ agglutinin (WGA)-Texas Red membrane co-staining (red). Arrows indicate the cell membrane.

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  Figure 7. Role of MTMR4. (A) Sequencing results. The analysis identified two interesting SNVs: rs2302189 (V297G), with either wild-type (A/A) or heterozygous variant (A/C) and rs2302189 (L170V), with either wild-type (G/G) or heterozygous variant (G/C). The illustration on the right illustrates the location of the two SNVs V297G and L170V on the conserved phosphatase region of the MTMR4 protein. (B) MTMR4 protein expression revealed by SDS-PAGE and immunoblot in CTR-, S-, and AS iPSC-CMs. Knock-down of MTMR4 by siRNA confirmed the role of MTMR4 on dephosphorylation of Nedd4L in iPSC-CM. (C) Co-localization of MTMR4 and Nedd4L in CTR and S cells. Confocal images of CTR-, AS-, and S-iPSC-CMs visualized with cTnT co-staining (purple). Nedd4L and MTMR4 were shown in red and green channel and the nuclei in blue (DAPI). Scale bar = 20 μm. (D) MTMR4–Nedd4L protein interaction investigated in 293T heterologous system. HEK cells transfected either with pCMV6-XL4-hMTMR4-WT, L170V (rs3744108 G to C single-nucleotide variant), V297G (rs2302189 A to C single-nucleotide variant), or double SNVs together with pcDNA3.1-hNedd4L. Forty-eight hours after transfection, whole-cell extracts were collected and…

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  Figure 8. Isogenic AS-MTMR4Cor-iPSC-CMs. (A) Immunostaining of the endogenous KCNQ1 in AS- and AS-MTMR4Cor-iPSC-CM. KCNQ1 was stained in green, cTnT in red, and nuclei in blue. Scale bar 20 µm. (B) Immunostaining for the hERG channel in AS- and AS-MTMR4Cor-iPSC-CM. hERG was stained in green, cTnT in red, and nuclei in blue. Scale bar =20 µm. (C) Representative field potential recordings of AS- and AS-MTMR4Cor groups. Average values for QT- and RR interval durations recorded in spontaneously beating EBs from AS and AS-MTMR4Cor groups. *P < 0.05 vs. AS (D). Representative IKr recordings and average data from AS and AS-MTMR4Cor groups. *P < 0.05 vs. AS.

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  Figure 9. Schematic illustration of the cellular mechanisms regulated by MTMR4. Cardiomyocytes of normal subject (middle panel): KCNQ1 and hERG are ion channels conducting the IKs and IKr currents, respectively. They are synthetized in the endoplasmic reticulum (ER) from where they traffick to the plasma membrane. Their steady-state expression level at cell surface is physiologically regulated by the ubiquitin ligase Nedd4L. The active form of Nedd4L ubiquitinates ‘old’ KCNQ1 and hERG channels which can then be degraded via the proteasome complex. We have proved that MTMR4 interacts with Nedd4L in iPSC-CMs. Cardiomyocytes of symptomatic patient (left panel): Mutant KCNQ1-Y111C channels are recognized as defective and retained in the ER. This triggers an increased activation of Nedd4L that amplifies the proteasome degradation not only of mutant KCNQ1, but also of WT hERG. Both IKs and IKr are reduced, and repolarization reserve compromised. LQT1 phenotype is manifested. Cardiomyocytes of asymptomatic patient (right panel): SNVs on MTMR4 inhibit Nedd4L activation. The total amount of KCNQ1 and hERG on the plasma membrane is increased compared with cardiomyocytes of the symptomatic patient.…