Severe Cardiac Dysfunction and Death Caused by Arrhythmogenic Right Ventricular Cardiomyopathy Type 5 Are Improved by Inhibition of Glycogen Synthase Kinase-3β

Abstract

Background: Arrhythmogenic cardiomyopathy/arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium, resulting in heart failure and sudden cardiac death. The most aggressive arrhythmogenic cardiomyopathy/ARVC subtype is ARVC type 5 (ARVC5), caused by a p.S358L mutation in TMEM43 (transmembrane protein 43). The function and localization of TMEM43 are unknown, as is the mechanism by which the p.S358L mutation causes the disease. Here, we report the characterization of the first transgenic mouse model of ARVC5.

Methods: We generated transgenic mice overexpressing TMEM43 in either its wild-type or p.S358L mutant (TMEM43-S358L) form in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter.

Results: We found that mice expressing TMEM43-S358L recapitulate the human disease and die at a young age. Mutant TMEM43 causes cardiomyocyte death and severe fibrofatty replacement. We also demonstrate that TMEM43 localizes at the nuclear membrane and interacts with emerin and β-actin. TMEM43-S358L shows partial delocalization to the cytoplasm, reduced interaction with emerin and β-actin, and activation of glycogen synthase kinase-3β (GSK3β). Furthermore, we show that targeting cardiac fibrosis has no beneficial effect, whereas overexpression of the calcineurin splice variant calcineurin Aβ1 results in GSK3β inhibition and improved cardiac function and survival. Similarly, treatment of TMEM43 mutant mice with a GSK3β inhibitor improves cardiac function. Finally, human induced pluripotent stem cells bearing the p.S358L mutation also showed contractile dysfunction that was partially restored after GSK3β inhibition.

Conclusions: Our data provide evidence that TMEM43-S358L leads to sustained cardiomyocyte death and fibrofatty replacement. Overexpression of calcineurin Aβ1 in TMEM43 mutant mice or chemical GSK3β inhibition improves cardiac function and increases mice life span. Our results pave the way toward new therapeutic approaches for ARVC5.

Keywords: GSK3β; TMEM43; arrhythmogenic right ventricular; calcineurin; dysplasia; therapy.

Figures

Figure 1.

TMEM43 (transmembrane protein 43)–S358L expression in cardiomyocytes causes severe cardiac dysfunction.A, Wild-type mice (WT; n=27) and mice overexpressing either WT TMEM43 (TMEM43WT; n=29) or TMEM43-S358L (TMEM43mut; n=84) under the control of the α-myosin heavy chain (αMHC) promoter were monitored for 60 weeks, and their survival rate was determined from a Kaplan-Meier curve. The indicated P value was obtained with a log-rank test. B through D, Left ventricular ejection fraction (LVEF), end-diastolic volume (LVEDV), and tricuspid annular plane systolic excursion (TAPSE) measured by echocardiography at birth (Neon) and at 3 and 5 weeks and 2 and 4 months of age. E, Gross morphology of representative hearts from 4-month-old WT, TMEM43WT, and TMEM43mut mice; bar, 500 μm. Inset, A thrombus in the left atrium of a TMEM43mut mouse; bar, 50 μm. F, Ratio of heart weight to body weight (HW/BW) determined at birth and at 4 months. G, LV posterior wall thickness in diastole (LVPWd) analyzed by echocardiography. H, Ratio of lung weight to body weight (LW/BW) determined at birth and at 4 months. Graphs show mean±SEM. **P<0.01, ***P<0.001, TMEM43mut vs WT. #P<0.05, ##P<0.01, ###P<0.001, TMEM43mut vs TMEM43WT. $P<0.05, $$P<0.01, $$$P<0.001 for different time points vs neonates (B, C, and G) or 3 weeks (D) for each mouse line; 2-way regular (B, C, F, and H) or repeated-measures (D and G) ANOVA was used, followed by Bonferroni post-test; n=5 to 6 per group.

Figure 2.

TMEM43 (transmembrane protein 43) mutant (mut) mice show cardiac conduction defects.A through C, Surface electrocardiographic analysis of cardiac conduction in wild-type (WT), TMEM43WT, and TMEM43mut mice 1 day (A), 5 weeks (B), and 4 months (C) of age. Shown are 0.6-second traces from the V3 lead. D through F, The p-wave duration (D) and QRS duration and amplitude (E and F) assessed at birth (Neon) and at 3 and 5 weeks and 2 and 4 months of age. Graphs show mean±SEM. **P<0.01, ***P<0.001, TMEM43mut vs WT. ###P<0.001, TMEM43mut vs TMEM43WT. $$$P<0.001 for different time points vs neonates for each mouse line; 2-way repeated-measures ANOVA followed by Bonferroni post-test; n=6.

Figure 3.

TMEM43 (transmembrane protein 43)–S358L causes cardiomyocyte death and replacement by fibrofatty tissue.A, Serum troponin I (TnI) determined by ELISA in mice 3 and 5 weeks and 2, 3, and 4 months of age. B and C, Western blot analysis of the presence in myocardium of activated and total caspase 3 (casp-3; B) and beclin1 (Becl; C) at the indicated time points. D through I, Masson trichrome staining in myocardial sections from wild-type (WT; D), TMEM43WT (E), and TMEM43 mutant (mut) mice (F and G). Subepicardial adipocytes were stained with perilipin (H), and the percentage of fibrotic tissue was quantified (I). Bar, 500 μm (D–F), 50 μm (G), and 100 μm (H). Graphs show data points for individual mice and mean±SEM. Vinc indicates vinculin. A and I, **P<0.01, ***P<0.001, TMEM43mut vs WT; #P<0.05, ##P<0.01, ###P<0.001, TMEM43mut vs TMEM43WT; $P<0.05, $$P<0.01, $$$P<0.001 for different time points vs 3 weeks (A) or neonates (I) for each mouse line; 2-way regular ANOVA followed by Bonferroni post-test; n=6 to 18.

Figure 4.

The S358L mutation alters TMEM43 (transmembrane protein 43) conformation and protein interactions.A through F, In silico modeling of the tertiary structure of wild-type (WT; A, C, and E, light blue) and mutant (mut) TMEM43 (B, D, and F, green) as monomers (A and B), dimers (C and D), or in complexes with emerin (EMD; purple molecule; E and F). Orange indicates transmembrane domain 1; red, transmembrane domain 3; dark blue, transmembrane domain 4; and black residue, S358L mutation. G, HA-tagged TMEM43WT and TMEM43mut were expressed in P19 cells and immunoprecipitated (IP) with anti-HA. The presence of TMEM43, EMD, and β-actin in the input and immunoprecipitate was analyzed by Western blot. H through M, Immunofluorescence analysis of TMEM43 localization in myocardial sections from 4-month-old TMEM43WT (H, J, and L) and TMEM43mut mice (I, K, and M) with 3 different anti-TMEM43 antibodies (H and I, Abcam; J and K, Santa Cruz; L and M, antibodies generated by Franke et al). Green indicates TMEM43; red, troponin I (only for H and I); and blue, DAPI. White arrowheads indicate partial TMEM43 localization in the cytoplasm. Bar, 20 μm.

Figure 5.

Inhibition of fibrosis does not improve cardiac function in TMEM43 (transmembrane protein 43) mutant (mut) mice.A, Quantitative reverse-transcribed polymerase chain reaction analysis of myocardial galectin-3 (Lgals3) expression in the mouse lines at the indicated ages. B and C, Western blot analysis (B) and quantification (C) of Gal-3 (galectin-3) protein levels at different time points in the 3 mouse lines at 4 months of age. D, TMEM43mut mice were treated with GM-CT-01 (GM; 120 mg/kg) or saline (Sal), and myocardial sections were stained with Picrosirius red to determine the maturity of the collagen fibers in each ventricle. E, The percentage of fibrotic tissue in the hearts of 4-month-old mice was quantified by Masson trichrome staining. F, Echocardiography-determined left ventricular (LV) ejection fraction (LVEF) in 5-week-, 2-month-, and 4-month-old TMEM43mut mice treated with GM-CT-01 or saline. Graphs show data points for individual mice and mean±SEM. AU indicates arbitrary units; and RV, right ventricle. A, ***P<0.001, TMEM43mut vs WT; ###P<0.001, TMEM43mut vs TMEM43WT; $$$P<0.001 for different ages vs neonates for each mouse line; n=3 to 8; 2-way regular ANOVA followed by Bonferroni post-test. C, **P<0.01, TMEM43mut vs WT; ##P<0.01, TMEM43mut vs TMEM43WT; 1-way ANOVA with Bonferroni correction. D, †††P<0.001, GM-CT-01 vs saline. E and F, ‡‡‡P<0.001 vs 5 weeks; 2-way regular ANOVA followed by Bonferroni post-test; n=7 to 8.

Figure 6.

TMEM43 (transmembrane protein 43) p.S358L promotes cell death by activating glycogen synthase kinase-3β (GSK3β).A through C, TMEM43 mutant (mut) mice were crossed with α-myosin heavy chain–calcineurin Aβ1(CnAβ1) mice overexpressing CnAβ1 in cardiomyocytes (TMEM43mut-CnAβ1 mice). The presence of total and phosphorylated (Ser9) GSK3β, as well as total and phosphorylated (Ser473) AKT, was assessed by Western blot in myocardial samples from wild-type (WT), TMEM43WT, TMEM43mut, and TMEM43mut-CnAβ1 mice (A) and quantified (B and C). Increased phosphorylation of GSK3β indicates inactivation; n=3 to 11. D and E, Neonatal mouse cardiomyocytes were transfected with a reporter plasmid in which luciferase expression is controlled by a β-catenin–dependent TCF multimer, together with expression plasmids for human (h; D and E) or mouse (m; only in D) WT TMEM43, TMEM43-S358L, and CnAβ1. Empty pcDNA3.1 was used as a negative control. When indicated, the GSK3β inhibitor CHIR99021 (Chir; 3 μmol/L) was added. F, Percentage of viable P19 cells transfected with expression plasmids for human WT TMEM43 and TMEM43-S358L grown in the absence of serum for 32 hours. The GSK3β inhibitor CHIR99021 (3 μmol/L) was added when indicated. As a positive control of cell death, P19 cells were incubated with 10% ethanol for 5 hours. G, Sixty-week Kaplan-Meier survival curves for WT (n=27), TMEM43mut (n=84), and TMEM43mut-CnAβ1 mice (n=38). The indicated P value was obtained with a log-rank test. H and I, Serum troponin I (TnI) determined by ELISA in the indicated mice 3 and 5 weeks and 2 and 4 months of age. In I, TMEM43mut mice were treated with the GSK3β inhibitor SB-216763 (SB) or dimethyl sulfoxide (DMSO) as a vehicle control; n=4 to 5. Graphs show mean±SEM. B through D and F, *P<0.05, **P<0.01, ***P<0.001 vs WT animals or control vector; ##P<0.01, ###P<0.001 vs TMEM43WT. B, δδδP<0.001 vs TMEM43mut; δδP<0.01 CHIR99021-treated cells vs untreated. H, **P<0.01, ***P<0.001 vs WT; †††P<0.001, TMEM43mut vs TMEM43mut-CnAβ1. I, ‡‡‡P<0.001, SB vs DMSO. B through F, One-way ANOVA followed by Dunnett posttest; n=3 to 11. H and I, Two-way ANOVA followed by Bonferroni posttest; n=10. Note that WT and TMEM43mut mice data for G and H are those shown in Figures 1A and 3A, respectively, and are repeated here for comparative purposes.

Figure 7.

Calcineurin Aβ1 (CnAβ1) overexpression and chemical inhibition of glycogen synthase kinase-3β (GSK3β) improve cardiac function in TMEM43 (transmembrane protein 43) mutant (mut) mice.A through C, Left ventricular (LV) ejection fraction (EF; A), LV end-diastolic volume (LVEDV; B), and tricuspid annular plane systolic excursion (TAPSE; C) were determined by echocardiography in 4-month-old mice. D through F, Surface electrocardiographic determination of p-wave duration (D) and QRS duration and amplitude (E and F). G and H, Gross heart morphology (G) and ratio of heart weight to body weight (HW/BW; H) in 4-month-old mice; bar, 500 μm. I, Picrosirius red staining analysis of collagen fibers in myocardial sections. J, LVEF was determined by echocardiography in 4-month-old TMEM43mut mice treated with the GSK3β inhibitor SB-216763 (SB) or dimethyl sulfoxide (DMSO) as a control. K and L, The p-wave (K) and QRS (L) durations were analyzed by ECG in TMEM43mut (Tmut) mice treated as in J. M, Ratio of heart weight to body weight (HW/BW; H) in 4-month-old TMEM43mut mice treated as in J. Graphs in A to F, H and J-M show data with mean±SEM. A through F and H, *P<0.05, **P<0.01, ***P<0.001, TMEM43mut or TMEM43-CnAβ1 vs WT; #P<0.05, ###P<0.001, TMEM43mut-CnAβ1 (Tm-Cn) vs TMEM43mut; 1-way ANOVA followed by Bonferroni posttest; n=6 to 8. I, **P<0.01, ***P<0.001, collagen (Col) type III TMEM43mut vs WT; ††P<0.01 Col I TMEM43mut-CnAβ1 vs TMEM43mut; ‡‡‡P<0.001, Col III TMEM43-CnAβ1 vs TMEM43mut; 2-way ANOVA followed by Bonferroni posttest; n=3 to 6. Note that WT and TMEM43mut mice data for A through F and H are those shown in Figures 1B through 1D, 2D through 2F, and 1F and are repeated here for comparative purposes. J through M, $$P<0.01, SB2 vs DMSO, 2-sample ttest; n=7 to 9.

Figure 8.

Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) bearing the p.TMEM43 (transmembrane protein 43)–S358L mutation develop contractile dysfunction.A through C, hiPSC-CMs bearing the wild-type (WT) alleles or a heterozygous p.TMEM43-S358L mutation (Mut) were loaded with 5 µmol/L Fluo 4-AM, and Ca2+ transients were imaged in individual cells under a confocal microscope in the presence or absence of 1 µmol/L isoproterenol (Iso). D through G, WT and mutant hiPSC-CM beating was video recorded, and contraction duration (D), time to peak (E), relaxation time (F), and contraction amplitude (G) were measured. H through K, WT and mutant hiPSC-CMs were treated with the glycogen synthase kinase-3β (GSK3β) inhibitor CHIR99021 (GSK3β in.) or dimethyl sulfoxide (DMSO) as a control and different contraction parameters were measured. *P<0.05, **P<0.01, ***P<0.001, mutant vs WT cells. ###P<0.001 and adjusted P values in H and I refer to GSK3β inhibitor vs DMSO. D through G, Two-sample t test; H through K, regular 2-way ANOVA with Bonferroni posttest; n=10 to 20; (B and C) 46 to 49; and (D–G); n=12-69.