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. 2014 Mar 11;129(10):1092-103.
doi: 10.1161/CIRCULATIONAHA.113.003077. Epub 2013 Dec 18.

Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype

Marina Cerrone  1 , Xianming Lin, Mingliang Zhang, Esperanza Agullo-Pascual, Anna Pfenniger, Halina Chkourko Gusky, Valeria Novelli, Changsung Kim, Tiara Tirasawadichai, Daniel P Judge, Eli Rothenberg, Huei-Sheng Vincent Chen, Carlo Napolitano, Silvia G Priori, Mario Delmar
Affiliations

Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype

Marina Cerrone et al. Circulation. .

Abstract

Background: Brugada syndrome (BrS) primarily associates with the loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (or arrhythmogenic right ventricular cardiomyopathy). Experimental models showed correlation between the loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced INa. We hypothesized that PKP2 variants that reduce INa could yield a BrS phenotype, even without overt structural features characteristic of arrhythmogenic right ventricular cardiomyopathy.

Methods and results: We searched for PKP2 variants in the genomic DNA of 200 patients with a BrS diagnosis, no signs of arrhythmogenic cardiomyopathy, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L, and MOG1. We identified 5 cases of single amino acid substitutions. Mutations were tested in HL-1-derived cells endogenously expressing NaV1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased INa and NaV1.5 at the site of cell contact. These deficits were restored by the transfection of wild-type PKP2, but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes from a patient with a PKP2 deficit showed drastically reduced INa. The deficit was restored by transfection of wild type, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related INa deficiency to the reduced number of channels at the intercalated disc and increased separation of microtubules from the cell end.

Conclusions: This is the first systematic retrospective analysis of a patient group to define the coexistence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.

Keywords: Brugada syndrome; arrhythmogenic right ventricular dysplasia-cardiomyopathy; desmosomes; plakophilin 2; sodium channels.

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Conflict of interest statement

Conflict of Interest Disclosures: None.

Figures

Figure 1
Figure 1
A: top: ECG showing coved-type ST elevation in leads V1–V2 in patients carriers of PKP2 variants. Q62K, M365V and T526A carriers showed flecainide-induced ECG; S183N carrier showed spontaneous ECG pattern during febrile episode. Bottom: correspondent DNA sequence showing heterozygous missense variants in PKP2. B: left: Spontaneous coved-type ECG in carrier of variant R635Q, and corresponding DNA sequence. Right: pedigree showing co-segregation between genotype and clinical phenotype in this family. Symbols: square, male; circle, female; solid, gene-carrier with clinical symptoms; empty, asymptomatic, negative genotype; half-full left, gene-carrier; half-full right, clinical symptoms.
Figure 2
Figure 2
A: Average peak INa density as a function of voltage command in HL-1 cells WT (black symbols; n=11), treated with PKP2 silencing construct (PKP2-KD; red; n=12), and treated with a non-silencing construct (PKP2-φKD; blue; n=12). For display purposes only, these data are shown as mean +/− standard error of the mean (SEM). Statistical comparisons were carried out by MWW test and limited to peak INa density at −30 mV for PKP2-KD vs. PKP2-φKD. The corresponding dot plot is shown in the inset. **p<0.005. B: NaV1.5 (green in merge) and N-Cadherin (pink in merge) decreased in PKP2-KD and not in PKP2-φKD cells. Right panel: Pearson’s coefficient dot plots, ***p<0.0005 (n=12 for each group). C: Average peak INa density in PKP2-KD cells (red line and symbols; n=12) increased when cells were transfected with PKP2-WT (black; PKP2-KD+PKP2-WT; n=13) but not when PKP2-KD cells were transfected with mCherry (blue; n=11). Inset shows data plot (same format as in A) for comparison of PKP2-KD+mCherry vs PKP2-KD+PKP2-WT; **p<0.005. D: co-localization of NaV1.5 (green) with N-Cadherin (pink) rescued by transfection of PKP2-KD cells with wild-type construct (PKP2-KD+PKP2-WT), but not by transfection of mCherry alone (PKP2-KD+mCherry). Pearson’s coefficient dot plot on the right. ***p<0.0001 when comparing PKP2-KD+mCherry (n=12) against PKP2-KD+PKP2-WT (n=16). Scale bars in B and D: 20 µm.
Figure 3
Figure 3
A: Peak INa as function of voltage in PKP2-KD cells (blue), PKP2-KD cells transfected with wild-type PKP2 (black; PKP2-KD+PKP2-WT) and cells transfected with different PKP2 variants (red). Additional INa properties, supplemental figures 4,5. Data are presented as mean+/− SEM for display purposes only. B: Dot plots of peak INa density at −30 mV measured for each mutant. C: Pearson coefficient values showing loss of NaV1.5 and N-cadherin co-localization for cells transfected with 5 PKP2 mutants, and maintained co-localization in cells transfected with WT or D26N. For B and C: *p<0.05; **p<0.01; *** p<0.001; @p=0.28; #p=0.48. Each variant was compared separately against the WT group. n values in parentheses under each column.
Figure 4
Figure 4
A: Peak INa as function of voltage in PKP2-KD cells (blue; cells transfected with mCherry), PKP2-KD cells transfected with wild-type PKP2 (black) and cells transfected with PKP2WT and a PKP2 variant (red). For co-expression, 1:1 plasmid ratio was used. For WT controls, WT plasmid was equal to the sum of WT+variant in test set. Data are presented as mean+/− SEM for display purposes only. B: For statistical analysis (MWW test), each variant was compared separately against group PKP2-KD+WT (n=14). p values were as follows (n values in parentheses): PKP2-KD+WT+Q62K (n=9): p<0.01; PKP2-KD+WT+S183N (n=7): p<0.005; PKP2-KD+WT+M365V (n=9): p<0.001; PKP2-KD+WT+T526A (n=8): p<0.001; PKP2-KD+WT+R635Q (n=8): p<0.005. PKP2-KD+WT+D26N (n=7): p=0.12 (NS).
Figure 5
Figure 5
Dot plot of INa density for voltage clamp pulse to −20 mV from a holding potential of −120 mV in WT-hESC-CMs (hESC;n=7), AC-hIPSC-CMs (AC;n=8), AC-hIPSC-CMs+PKP2-WT (AC+WT;n=8), and hIPSC-CMs+PKP2-R635Q (AC+R635Q;n=10). AC vs AC-WT, *p<0.05; AC vs AC-R635Q, p=0.85 (NS). Recordings limited to one voltage amplitude, as acceptable recordings (tight and stable gigohm seals and reproducible current traces) were short-lived.
Figure 6
Figure 6
A,B: Peak average INa in macropatches from cell midsection (A; green circle in left inset; n=10 for each group) or from the region previously occupied by the ID (B; yellow circle in left inset; n=7 for WT and 9 for PKP2-Hz). Data are presented as mean+/− SEM for display purposes only. Dot plots comparing INa density at −30 mV in online Figure 12; p<0.05 for ID recordings, and p=0.97 (NS) for M. C: SICM recording of the end of an adult ventricular myocyte. Notice (from bottom to top) the last striations and then a smooth, T-tubule-free region, closer to cell end. D: Single sodium channel data from either WT, or PKP2-Hz cells. Methodological details in.
Figure 7
Figure 7
A: Co-localization of N-Cadherin (red) and NaV1.5 (green) by conventional fluorescence microscopy in adult ventricular tissue from PKP2-Hz mice (right; PKP2+/−) and control littermate (left; WT). Inset in “a” shows brighter signal indicating localization at the ID; long arrows in “b” show staining along striations; arrowheads show decreased staining intensity at ID. B: Dot plot of Pearson’s coefficient showing decreased N-cadherin/NaV1.5 co-localization in PKP2-Hz. n values were 20 for WT and 18 for PKP2-Hz (p<0.001). Same methods as in. For further details see online supplement.
Figure 8
Figure 8
Localization of EB-1 (green) and N-cadherin (purple) in adult ventricular myocytes. A: Image by TIRF (left) or dSTORM (right). Inset enlarged in bottom panels to show increased resolution. B: clusters in WT (left) are closer to each other than in PKP2-Hz cells (right). C: Dot plot of distance from EB-1 to N-cadherin showing increased separation between the proteins in the PKP2-Hz cells. n values for statistical comparison were 10 for WT and 10 for PKP2-Hz. *p<0.005. For further details see and online supplement.
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