iASPP, a previously unidentified regulator of desmosomes, prevents arrhythmogenic right ventricular cardiomyopathy (ARVC)-induced sudden death

Abstract

Desmosomes are anchoring junctions that exist in cells that endure physical stress such as cardiac myocytes. The importance of desmosomes in maintaining the homeostasis of the myocardium is underscored by frequent mutations of desmosome components found in human patients and animal models. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a phenotype caused by mutations in desmosomal components in ∼ 50% of patients, however, the causes in the remaining 50% of patients still remain unknown. A deficiency of inhibitor of apoptosis-stimulating protein of p53 (iASPP), an evolutionarily conserved inhibitor of p53, caused by spontaneous mutation recently has been associated with a lethal autosomal recessive cardiomyopathy in Poll Hereford calves and Wa3 mice. However, the molecular mechanisms that mediate this putative function of iASPP are completely unknown. Here, we show that iASPP is expressed at intercalated discs in human and mouse postmitotic cardiomyocytes. iASPP interacts with desmoplakin and desmin in cardiomyocytes to maintain the integrity of desmosomes and intermediate filament networks in vitro and in vivo. iASPP deficiency specifically induces right ventricular dilatation in mouse embryos at embryonic day 16.5. iASPP-deficient mice with exon 8 deletion (Ppp1r13l(Δ8/Δ8)) die of sudden cardiac death, displaying features of ARVC. Intercalated discs in cardiomyocytes from four of six human ARVC cases show reduced or loss of iASPP. ARVC-derived desmoplakin mutants DSP-1-V30M and DSP-1-S299R exhibit weaker binding to iASPP. These data demonstrate that by interacting with desmoplakin and desmin, iASPP is an important regulator of desmosomal function both in vitro and in vivo. This newly identified property of iASPP may provide new molecular insight into the pathogenesis of ARVC.

Keywords: ARVC; cell–cell junctions; desmosome; iASPP; sudden death.

Fig. 1.

iASPP anchors desmin and desmoplakin at intercalated discs (IDs) in adult cardiomyocytes in vivo. (A) Ventricular myocardial sections obtained from postmortem human subjects and iASPP^+/+ and iASPP^Δ8/Δ8 adult mouse hearts were stained using anti-iASPP antibodies, pAb-bp18 or LX128.5 as indicated. Specificity of iASPP staining was confirmed by the lack of signal from iASPP^Δ8/Δ8 sections. (B) Myocardial cross-sections obtained from 12-wk-old iASPP^+/+ and iASPP^Δ8/Δ8 hearts were immunostained for desmin. Desmin signal is concentrated at the area composita in control tissue but is absent in iASPP^Δ8/Δ8 hearts (arrowheads). The bar graph shows the reduced percentage of cells expressing desmin at the intercalated discs. iASPP^+/+ n = 204; iASPP^Δ8/Δ8 n = 198. P < 0.01. (C) Double immunofluorescence staining using a monoclonal antibody against desmoplakin and a polyclonal antibody against the adherens junction marker N-cadherin in 12-wk-old iASPP wild-type and deficient mouse myocardial sections. Junctional N-cadherin staining was used as a marker of intercalated discs. We examined an average of 200 cardiomyocytes in each section and compared the number of intercalated discs that express N-cadherin but are negative for desmoplakin in wild-type (n = 3) and iASPP^Δ8/Δ8 (n = 3) myocardial sections. The bar graph shows the fold increase of the intercalated discs lacking desmoplakin signal in iASPP^Δ8/Δ8 myocardial sections. (D) Protein levels of iASPP, desmoplakin, and desmin were assessed by Western blot analyses in wild-type and iASPP^Δ8/Δ8 hearts. GAPDH was used as a loading control. TO-PRO was used as a nuclear stain. (Scale bars in A–C, 20 µm.)

Fig. 2.

iASPP interacts with desmoplakin and desmin in cardiomyocytes. (A) iASPP interacts with desmoplakin and desmin in adult mouse heart extracts. The coprecipitated desmoplakin (DSP), iASPP, and desmin were detected by blotting with corresponding antibodies as indicated. (B, Left) Schematic representation of different desmoplakin truncated mutants relative to full-length desmoplakin protein and the ability to bind (+), bind very well (++), or not bind (−) iASPP. (Right) The ability of iASPP to bind different desmoplakin truncation constructs was measured using in vitro-translated [³⁵S]methionine-labeled iASPP and different desmoplakin truncation mutants. The signals from three independent experiments were quantified using ImageJ software. The percentage of the different desmoplakin truncated constructs binding to iASPP was normalized against the amount of iASPP immunoprecipitated. Representative immunoblots are shown in Fig. S2E. (C) Binding of recombinant N-terminal GST-iASPP(1–240) and GST-iASPP(249–482) and C-terminal His-iASPP(625–828) to in vitro-translated N-terminal desmoplakin was measured using a pulldown assay as indicated. (D) GST-iASPP(1–240) was pulled down with glutathione beads, and its binding to in vitro-translated ARVC-derived desmoplakin mutants DSP-1(1–394)-V30M, DSP-1(1–394)-Q90R, and DSP-1(1–394)-S299R was assessed by probing with a desmoplakin antibody. Ponceau S staining shows similar protein loading in the samples.

Fig. 3.

Junctional iASPP interacts with desmoplakin and increases its solubility. (A) Junctional localization of iASPP (green) and desmoplakin (red) was detected in wild-type primary mouse keratinocytes using high-resolution confocal microscopy (nuclear stain DAPI is blue). Inset shows yellow colocalization between iASPP and desmoplakin. (B) Translocation of iASPP protein to the cell–cell junctions in response to the Ca²⁺ switch was investigated in primary keratinocytes. iASPP can be seen at the cell–cell junctions (arrowheads) at 30 min to 24 h after the switch to 1.2 mM Ca^2+. (C) The total amount of nonionic detergent-soluble (S) and insoluble (I) junctional components was detected in mouse primary keratinocytes under different culture conditions as indicated. (D) The total amount of nonionic detergent-soluble and insoluble fractions from primary keratinocytes isolated from iASPP CreER mice treated with vehicle or tamoxifen to delete iASPP was loaded on a Phos-tag gel to separate phosphorylated (top band) and unphosphorylated (lower bands) desmoplakin. Phosphorylated and unphosphorylated insoluble desmoplakin are indicated by the black arrows. White arrowheads show that phosphorylated soluble desmoplakin is increased in iASPP-deficient keratinocytes compared with vehicle-treated keratinocytes. β-Actin was used as a loading control. (Scale bars in A and B, 20 μm.)

Fig. 4.

iASPP deficiency weakens desmosome function in vivo. Transmission electron microscopy of iASPP^+/+ and iASPP^Δ8/Δ8 hearts shows cross-sections of myocardial intercalated discs. The yellow arrowhead indicates widening of the desmosomal plate. Red arrows indicate the disarrangement and disruption of the intermediate filament network. (Scale bar, 200 nm.)

Fig. 5.

iASPP-deficient embryos exhibit cardiac right ventricular dilatation. Three-dimensional reconstruction of E16.5 iASPP^+/+ and iASPP^Δ8/Δ8 embryonic hearts from HREM shows progressive heart erosion in iASPP^Δ8/Δ8 hearts. Note the thinning of the right ventricular wall (yellow arrowheads), the variegated muscle damage (black arrowheads), and the blood clot accumulation which replaces part of the right ventricular wall (red arrowheads).

Fig. 6.

iASPP-deficient adult mice exhibit cardiac right ventricular dilatation and phenotypic features of ARVC. (A) Representative long-axis MRI images of wild-type and transgenic iASPP hearts show the abnormal size, shape, and position within the chest cavity of the mutant heart. Two midpapillary slices from two different iASPP^+/+ and iASPP^Δ8/Δ8 12-wk-old hearts are shown at diastole. The area covered by the lumen of the left ventricle of the same slice at systole is shown in black. (B) Cross-sections from iASPP^+/+ and iASPP^Δ8/Δ8 hearts, stained using Oil Red O, show that fibrotic deposits are infiltrated with fat droplets and adipocytes in iASPP^Δ8/Δ8 sections only (arrowheads). (Scale bar, 10 µm.) (C, Upper) ECG recordings from iASPP^+/+ and iASPP^Δ8/Δ8 animals demonstrate that rhythm and conduction abnormalities are present in adult iASPP^Δ8/Δ8 mice. Normal sinus rhythm was recorded in all iASPP^+/+ mice (n = 5). In contrast, recordings from iASPP^Δ8/Δ8 mice (n = 5), revealed frequent ventricular ectopy and episodes of spontaneous NSVT. In addition, adult iASPP^Δ8/Δ8 mice displayed other rhythm disturbances, including aberrant junctional rhythm and ventricular conduction. (Lower) Table shows ECG recorded parameters ± SEM.

Fig. 7.

Representative images show iASPP staining in unaffected and ARVC-affected myocardial sections. Note that the ARVC samples showed immunoreactivity to N-cadherin and γ-catenin. (Scale bar, 20 μm.)