Knockout of SORBS2 Protein Disrupts the Structural Integrity of Intercalated Disc and Manifests Features of Arrhythmogenic Cardiomyopathy

Abstract

Background Sorbs2b (sorbin and SH3 domain-containing 2b) was recently identified as a cardiomyopathy gene from a zebrafish mutagenesis screen. However, cardiac functions of its mammalian ortholog remain elusive. Methods and Results We conducted a detailed expression and subcellular localization analysis of Sorbs2 ortholog in mice and a phenotypic characterization in Sorbs2 knockout mice. Sorbs2 is highly expressed in the mouse heart and encodes an adhesion junction/desmosome protein that is mainly localized to the intercalated disc. A mutation with near complete depletion of the Sorbs2 protein in mice results in phenotypes characteristic of human arrhythmogenic cardiomyopathy (ACM), including right ventricular dilation, right ventricular dysfunction, spontaneous ventricular tachycardia, and premature death. Sorbs2 is required to maintain the structural integrity of intercalated disc. Its absence resulted in profound cardiac electrical remodeling with impaired impulse conduction and action potential derangements. Targeted sequencing of human patients with ACM identified 2 rare splicing variants classified as likely pathogenic were in 2 unrelated individuals with ACM from a cohort of 59 patients with ACM. Conclusions The Sorbs2 knockout mouse manifests several key features reminiscent of human ACM. Although the candidacy of SORBS2 as a new ACM-susceptibility gene is supported by preliminary human genetics study, future validation in larger cohorts with ACM is needed.

Keywords: arrhythmogenic cardiomyopathy; intercalated disc; sorbin and SH3 domain‐containing 2; susceptibility gene.

Figure 1. Sorbs2 (sorbin and SH3 domain‐containing 2) is a cardiac‐enriched intercalated disc and desmosome protein in mice.

A, Western blot analysis of Sorbs2 protein expression in different mouse tissues. B, Images of immunostaining using an anti‐Sorbs2 antibody and counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) in sectioned wild‐type mouse hearts. Arrowheads indicate the intercalated disc. The arrows indicate Z‐disc localization. Red arrows indicate the perinucleus. Bar=20 μm. C, Immunostaining and pseudoline analysis of sectioned mouse hearts are shown, indicating that Sorbs2 protein mostly colocalizes with β‐catenin and plakoglobin (Pkg), but less with connexin 43 (Cx43). Bar=2 μm. MW indicates molecular weight.

Figure 2. Targeted deletion of exon 8 in the Sorbs2 (sorbin and SH3 domain‐containing 2) gene led to near null depletion of the whole Sorbs2 transcript and protein in mice.

A, Schematic illustration of targeted deletion of Sorbs2 exon 8 in the Sorbs2^e8/e8 mice and locations of primers used for genotyping polymerase chain reaction (PCR) and transcription analysis. B, Representative DNA gel images of PCR genotyping for identifying wild‐type (WT) (292 bp), Sorbs2^e8/+ heterozygous (hets), and Sorbs2^e8/e8 homozygous (homo) mutant alleles (500 bp). C, Representative DNA gel images and quantitative reverse transcription–PCR analysis of the Sorbs2 transcripts in WT and Sorbs2^e8/e8 mutants. N=3, unpaired 2‐tailed Student t test. D, Western blotting and quantification of Sorbs2 protein expression in each cardiac chamber of WT and Sorbs2^e8/e8 mutants. N=4. One‐way ANOVA. LA indicates left atrium; LV, left ventricle; RA, right atrium; and RV, right ventricle.

Figure 3. Sorbs2 (sorbin and SH3 domain‐containing 2) deficiency leads to right ventricle (RV) dilation, cardiac dysfunction, and premature death.

A, Echocardiography indexes in the RV of sorbs2^e8/− and wild‐type (WT) control mice at 4 weeks postdoxorubicin injection (12 mg/kg body weight). N=14 to 15. Unpaired 2‐tailed Student t test. B, Kaplan‐Meier survival curves of Sorbs2^e8/e8 and WT control mice. N=12 to 17. Log‐rank test. C, long‐axis views of magnetic resonance imaging images of 2‐ and 4‐month‐old mice. RV dilation (arrowheads) is prominent in the Sorbs2^e8/e8 mouse hearts at 4 months old. Note that a septum bending defect (arrows) was also detected. D, Images of dissected whole hearts (top) and cross‐sectional view of hematoxylin and eosin–stained hearts (bottom). Dramatically enlarged RV and protuberant were noted in the Sorbs2^e8/e8 mice. Note that both right atrium (RA) and left atrium (LA) enlargement was also frequently observed. E, Representative images of Masson trichrome staining and quantification analysis demonstrated significant myocardial fibrosis in the RV and septum but not in the LV of Sorbs2^e8/e8 mice. Bar=50 μm. N=4, unpaired 2‐tailed Student t test. FS indicates fractional shortening; and LV, left ventricle.

Figure 4. Sorbs2 (sorbin and SH3 domain‐containing 2) deficiency leads to cardiac arrhythmia.

A, Shown are surface ECGs and quantification analysis in 4‐month‐old wild‐type (WT) and Sorbs2^e8/e8 mice. N=6 to 13. Unpaired 2‐tailed Student t test. *P<0.05. B, Ventricular premature beats and trigeminy (top) and polymorphic ventricular tachycardia (VT) (bottom) were recorded in Sorbs2^e8/e8 mice. C, Induction of nonsustained VT in Langendorff‐perfused WT mouse heart by programmed electrical stimulation (s1‐s1=100 ms, s1‐s2=30 ms) (top); induction of sustained monomorphic VTs in Langendorff‐perfused Sorbs2^e8/e8 mouse heart by programmed stimulation (s1‐s1=100 ms, s1‐s2=80 ms) (bottom). D, Representative action potentials were recorded from the endocardial surface of isolated right ventricles (RVs) of Sorbs2^e8/e8 and WT mice at a pacing cycle length of 200 ms. E, Compared with WT mice, Sorbs2^e8/e8 mice RV action potentials have depolarized resting potentials (RPs), decreased action potential amplitudes (APAs), reduced upstroke velocity at phase 0 (Vmax, maximal velocity), and prolonged action potential durations at 50% (APD50) and 90% (APD90) repolarization, as well as a lengthened ventricular effective refractory period (VERP). N=6. Unpaired 2‐tailed Student t test. Bpm indicates beats per minute; HR, heart rate; P, duration of P waves; P‐R, duration of PR interval; QRS, duration of QRS complex; R+S, QRS amplitude; and S, duration of S wave.

Figure 5. Dual function of Sorbs2 (sorbin and SH3 domain‐containing 2) in maintaining intercalated disc (ICD) integrity and regulating the expression of α‐catenin and gap junction protein connexin 43 (Cx43).

A, Shown are ICD images and represented pseudoline analysis of mouse hearts after immunostaining using an anti–N‐cadherin antibody. The expression of N‐cadherin in ICD manifests as discontinuous dots in wild‐type (WT) mice, which switched to continuous lines in Sorbs2^e8/e8 mice (arrows). Bar=20 μm. The intensity distribution of the N‐cadherin signal along the ICD appears as wavy lines in WT mice, which largely changed to flat lines in the Sorbs2^e8/e8 mice. B, Western blotting and quantification of ICD proteins in WT and Sorbs2^e8/e8 knockout (KO) mice hearts at 4 months old. N=3. Unpaired 2‐tailed Student t test. C, A representative DNA gel and quantitative reverse transcription–polymerase chain reaction (RT‐PCR) analysis of the Cx43/Gja1 mRNA level in the anti‐Sorbs2 antibody and IgG control immunoprecipitated tissue lysates from right ventricle (RV) of WT mice. N=3. Unpaired 2‐tailed Student t test. D, Quantitative RT‐PCR analysis of the mRNA levels of the indicated genes in the RV tissue of Sorbs2^e8/e8 mice compared with WT controls. N=3. Unpaired 2‐tailed Student t test. E, Transmission electron microscopy images and schematic illustration on measuring and quantifying the ICD amplitude in WT and Sorbs2^e8/e8 KO mice hearts at 4 months old. ^#Disrupted myofibril. Bars for top panels=2 μm; bars for bottom panels=500 nm. N=10. Unpaired 2‐tailed Student t test. Dsg indicates desmoglein 1/2; and Pkg, plakoglobin.

Figure 6. Identification and functional validation of an SORBS2 (sorbin and SH3 domain‐containing 2) splicing variant found in a human patient with arrhythmogenic cardiomyopathy (ACM).

A, Chromagraph illustrates c.679+1G>T mutation identified in the SORBS2 gene from a patient with ACM. B, Magnetic resonance images of the patient harboring the c.679+1G>T mutation indicates right ventricle dilation (arrow) and fatty infiltration (arrowhead). Bar=5 cm. C, ECGs from the patient with ACM harboring the c.679+1G>T mutation. Ventricular tachycardia and T‐wave inversion were noted. D, Schematics of the minigene and reverse transcription–polymerase chain reaction (RT‐PCR) analysis to test that the splicing mutant c.679+1G>T leads to exon 11 skip in the HEK 293 cell culture system. E, RT‐PCR assessment of molecular lesions induced by sorbs2a or sorbs2b_ morpholino (MO) in zebrafish embryos. A predicted 352‐bp PCR band was detected in embryos injected with sorbs2a_MO because of an exon skipping effect. A predicted 447‐bp PCR band was detected in embryos injected with sorbs2b MO because of an intron retention effect. F, Fractional shortening (FS) and heart rate analysis in sorbs2a_MO‐, sorbs2b_MO‐, and sorbs2a&2b_MO‐injected morphants and uninjected controls. N=7 to 9. One‐way ANOVA. BPM indicates beats per minute; and WT, wild type.