Background

Brugada syndrome (BrS) primarily associates with loss of sodium channel function. Previous studies showed features consistent with sodium current (I_Na) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (AC; or arrhythmogenic right ventricular cardiomyopathy, ARVC). Experimental models showed correlation between loss of expression of desmosomal protein plakophilin-2 (PKP2), and reduced I_Na. We hypothesized thatPKP2 variants that reduce I_Na could yield a BrS phenotype, even without overt structural features.

Methods and Results

We searched forPKP2 variants in genomic DNA of 200 patients with BrS diagnosis, no signs of AC, and no mutations in BrS-related genesSCN5A, CACNa1c, GPD1L and MOG1. We identified 5 cases of single amino acid substitutions. Mutations were tested in HL-1-derived cells endogenously expressing Na_V1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased I_Na and Na_V1.5 at site of cell contact. These deficits were restored by transfection of wild-type PKP2 (PKP2-WT), but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) from a patient with PKP2 deficit showed drastically reduced I_Na. The deficit was restored by transfection of WT, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related I_Na deficiency to 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 co-existence of sodium channelopathy and genetic PKP2 variations.PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.

Keywords: plakophilin-2, arrhythmogenic right ventricular dysplasia/cardiomyopathy, Brugada syndrome, sodium channels, desmosomes arrhythmia

Study population and genetic screening

A total of 200 de-identified patients [179 males] from the Registry of the Molecular Cardiology Laboratories, Maugeri Foundation, Pavia, Italy were included in this study. Patients were selected based on clinical definite diagnosis of BrS and absence of mutations on SCN5A, CACNa1c. Genes GPD1L and MOG1 were subsequently screened and no mutation was found. DNA extraction, amplification and direct sequencing of the entire open reading frame/splice junction of PKP2 followed standard techniques.

Experiments in HL-1 cells

hIPSC-CMs

AC-hIPSC-CMs were obtained from line JK#11, derived from a patient with clinical diagnosis of AC and a homozygous c.2484C>T mutation inPKP2 causing a cryptic splicing with a 7-nucleotide deletion in exon 12. Extensive characterization of the cellular/molecular phenotype in^12,13. Details inonline supplement. We performedPKP2 rescue experiments with lentiviral constructs containing WT-PKP2 orPKP2 with a c.1904 G>A mutation (p.R635Q), tagged with green fluorescent proteins (GFP) for verification, according to methods described previously¹².

Experiments in PKP2 heterozygous-null (PKP2-Hz) mice

The PKP2-Hz murine model has been described before⁶. All procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication 58-23, revised 1996). Dissociation of single adult ventricular myocytes, recording by macropatch and immunolocalization of relevant proteins followed standard procedures outlined inonline supplement.

Statistical analysis

Each individual comparison was limited to two data sets (e.g., wild-type versus mutant). Results were statistically analyzed without assuming a defined structure in the data set (non-parametric statistics). Significance was calculated by a Mann-Whitney-Wilcoxon (MWW) test to assess the null hypothesis that two populations were the same, against the alternative hypothesis that a particular population tended to have larger values than the other. No adjustments were made for multiple comparisons due to the nature of the study. Significance was defined by p<0.05. For all data plots involving statistics, a dot plot format was used: open circles represent individual data points, the black circle is the median value, and lower and upper horizontal lines indicate first and third quartiles, respectively.

Genetic Screening

We found five single amino acid substitutions on PKP2 in five unrelated individuals. Variant Q62K (c.185 C>A, on exon 1), was reported in patients with diagnosis of AC, and in 3/12602 (MAF 0.02%)¹⁴ alleles screened in the NHLBI Exome Sequencing Project database (EVS,http://evs.gs.washington.edu/EVS/). It is defined as variant of unknown significance (VUS), because of contrasting data on potential deleterious effect¹⁵: absence of co-segregation with clinical phenotype in some cohorts¹⁶, concomitant presence of additional desmosomal mutations in some affected individuals¹⁷, and detection in individuals without overt clinical phenotype.¹⁸ Four other variants were novel. Three, S183N (c.548G>A); T526A (c.1576A>G) and R635Q (c.1904G>A) are unreported variants, absent in 200 healthy controls screened in our laboratory and 6500 healthy controls reported in EVS. The fifth variant, M365V (c. 1093A>G) is novel, not present in our controls and reported in 2/13004 alleles of the EVS (MAF 0.01%)¹⁴. Amino acid substitution D26N [rs143004808] was found in 7 unrelated patients. This variant is present in healthy individuals and not thought to cause disease¹⁵. It was therefore used as additional control in the cell expression systems.

Clinical characteristics

All patients were evaluated at the Molecular Cardiology Program, Fondazione Maugeri, Pavia, Italy. They were diagnosed with BrS based on clinical history, diagnostic ECG pattern either spontaneous or after flecainide challenge and absence of structural cardiomyopathy at echocardiogram or cardiac magnetic resonance imaging. Detailed clinical data are presented inSupplemental Table 1. Briefly, 3 patients had a saddle-back ECG in baseline with conversion into coved-type diagnostic ECG pattern after flecainide infusion,¹⁹ while two showed an abnormal coved-type pattern at the surface ECG (Figure 1 adSupplemental Figure 1). Three had history of syncope at rest or after large meal, a feature characteristic of the disease¹⁹. In one (mutation carrier S183N), the ECG pattern was elicited by a febrile episode and associated with frequent PVCs¹⁹. One patient (mutation T526A) received an ICD after experiencing 2 syncopal episodes at rest; two months later, he experienced cardiac arrest at rest, with evidence of ventricular fibrillation, treated by the ICD. The carrier of mutation R635Q had 2 syncopal episodes at rest and spontaneous coved-type diagnostic ECG, and received a prophylactic ICD. His family members agreed to undergo cascade genetic screening. The asymptomatic mother did not carry the R635Q variant. His father, who had history of syncopal episodes at rest, and his brother, asymptomatic but with suspect ECG, resulted gene-carriers. The brother underwent flecainide challenge that resulted positive, confirming the genetic diagnosis. The paternal grandfather had history of syncope and died suddenly during sleep, but DNA was not available for testing. Altogether, these data show co-segregation between clinical phenotype and presence of variant R635Q, supporting the hypothesis of a deleterious, disease-causing effect.

The sodium channel complex, and PKP2 in HL-1 cells

Assessment of the relation between PKP2 primary sequence and I_Na required a system to test sequence variations inPKP2 in the setting of stable, endogenous Na_V1.5 expression. As a line of cardiac origin¹⁰, HL-1s present characteristics of differentiated myocytes, including expression of Na_V1.5²⁰. As shown inFigure 2A (black symbols), voltage clamp steps elicited a voltage-dependent, fast inactivating inward current, as previously reported²⁰ (see alsoSupplemental Figure 2A,B). RT-PCR confirmed transcription ofSCN5A. The abundance ofSCN5A was three orders of magnitude higher than that ofSCN1A, SCN3A andSCN8A (Supplemental Figure 2C). Immunolocalization and Western blot experiments showed expression of Na_V1.5 (Supplemental Figure 2D,E). Similarly, we detected expression of PKP2, with undetectable levels of PKP1 or PKP3 by western blot (Supplemental Figure 2E,F). Next, we established the relation between PKP2 expression, and I_Na.

Generation of PKP2-deficient HL-1s. Relation between PKP2 expression, and I_Na

HL-1s were infected with lentivirus coding for PKP2-shRNA, or for a non-silencing oligonucleotide. In both cases, the puromicin-resistant gene was used for selection. The corresponding cell lines were dubbed PKP2-KD, and PKP2-φKD. Western blots showed the expected loss of PKP2 in PKP2-KD but not in PKP2-φKD cells (Supplemental Figure 3A). Interestingly, loss of PKP2 expression led to significant loss of average peak I_Na density (Figure 2A), similar to that reported in neonatal and adult ventricular myocytes⁵. No PKP2-dependent changes in voltage-dependence of steady-state inactivation, or time course of recovery from inactivation were detected (seeSupplemental Figure 3B,C). We observed no change in Na_V1.5 abundance (Supplemental Figure 3A), but decreased co-localization of Na_V1.5 with N-cadherin at site of cell contact in PKP2-deficient cells (Figure 2B).

To confirm the relation between PKP2 expression and I_Na, PKP2-KD cells were transiently transfected with wild-typePKP2 (PKP2-KD+PKP2-WT). To identify transfected cells, PKP2 was concatenated in its C-end with mCherry. Controls were transiently transfected with mCherry alone (PKP2-KD+mCherry). As shown in 2C, peak I_Na density in PKP2-KD+PKP2-WT cells was significantly larger than in PKP2-KD or in PKP2-KD+mCherry cells (see alsoSupplemental Figure 3D,E). Thus, exogenous expression of PKP2-WT rescued I_Na deficiency consequent to loss of expression of endogenousPKP2. Consistent with this observation, the extent of co-localization of Na_V1.5 and N-cadherin was rescued by transfection of the wild-type construct, but not by transfection of mCherry cDNA (2D).

BrS-related PKP2 variants and the sodium channel

The cellular system described above allowed us to determine whether mutations inPKP2 would alter the relation between PKP2 expression, and I_Na. We generated six constructs, all as mCherry concatenants: PKP2-D26N, PKP2-Q62K, PKP2-S183N, PKP2-M365V and PKP2-T526A and PKP2-R635Q. I_Na in cells transfected with PKP2 variants was compared with that in PKP2-KD cells transfected with PKP2-WT or with mCherry. For each experiment, PKP2-KD cells were split from the same flask, and divided for treatment with PKP2-WT, mCherry or the particular PKP2 mutant. I_Na was measured from all three conditions on each test day, to ensure consistency. For each set, data were normalized to the average value of peak I_Na density of the PKP2-KD+PKP2-WT group. In contrast with results from PKP2-WT-transfected cells, mutants PKP2-Q62K, PKP2-S183N, PKP2-M365V, PKP2-T526A and PKP2-R635Q failed to rescue the I_Na deficit observed in PKP2-KD cells (3A); steady-state inactivation and recovery from inactivation were not affected either by loss of PKP2 or by expression of mutants (seeSupplemental Figure 4,5). Yet, variant PKP2-D26N, not thought to be causative of disease¹⁵, behaved as PKP2-WT (Figure 3A; bottom right; cumulative data in 3B). Interestingly, similar trend was observed for N-cadherin/Na_V1.5 co-localization. Transient transfection of PKP2-WT, or PKP2-D26N, enhanced co-localization to levels significantly higher than those for any of five mutant PKP2 constructs (Supplemental Figure 6). Pearson’s coefficient values, in 3C. Western blot showing equivalent levels of PKP2 expression, inSupplemental Figure 7.

Our results show I_Na deficit when PKP2 mutations were expressed in PKP2-null background. BrS patients, on the other hand, were heterozygous for the PKP2 variant. Additional experiments evaluated I_Na properties in HL1s when both, WT and PKP2-variant constructs were co-expressed (transfection at 1:1 ratio). As shown inFig.4 (andSupplemental Figures 8,9,10), I_Na in cells co-expressing PKP2-WT and a BrS-related PKP2 variant was less than that recorded in cells expressing only the wild-type protein, or WT plus variant PKP2-D26N.

AC-hIPSC-CMs showed reduced I_Na, rescued by PKP2-WT but not by a PKP2-BrS variant

Our data show that loss of expression, as well as mutations in PKP2 lead to decreased I_Na. Yet, HL-1s derive from a murine atrial myxoma and as such, their behavior may differ from that of human cardiomyocytes. Therefore, we performed an additional I_Na rescue experiment using a previously characterized hIPSC-CM line from an AC patient with a homozygous loss-of-functionPKP2 mutation¹². As shown inFig.5, I_Na was significantly less in cells from the AC patient (AC-hIPSC-CMs) than in cells from control (H9 human embryonic stem cell-derived cardiomyocytes). More importantly, lentiviral transfection of PKP2WT but not of PKP2-R635Q significantly increased I_Na density. This confirms that I_Na depends on expression/structural integrity of PKP2.

Analysis of sodium channel functional expression in PKP2-Hz cardiomyocytes

Our data so far were obtained in cells in culture, which differ from adult cardiomyocytes in terms of structural organization/function of junctional complexes. To explore the mechanisms of PKP2-dependent I_Na deficiency, we used adult ventricular myocytes from PKP2-Hz mice.

Based on our results, we speculated that PKP2 regulates cell surface expression of functional sodium channels at the ID. As an initial approach, we quantified the amplitude of local I_Na using the macropatch technique²¹. The terms “ID” and “M” refer to recordings from the region previously occupied by the ID, and from the cell midsection, respectively (diagram to left of 6A).Figure 6A shows no difference in average peak I_Na amplitude recorded from the midsection of cells obtained from PKP2-Hz mice (red) or their control littermates (black). However, the amplitude of I_Na measured at the ID (Fig.6B) was significantly reduced in PKP2-Hz when compared to control (additional data inSupplemental Figures 11 and 12). Overall, we show that PKP2 deficit causes selective reduction of I_Na at the ID.

To discard the possibility that reduced I_Na was consequent to decreased single-channel unitary conductance, we recorded sodium channels from a small, highly localized area at the cell end using super-resolution scanning patch clamp⁹. A topology map of the cell end is shown in 6C and the unitary measurements in 6D. The data show that reduced I_Na in PKP2-deficient cells is consequent not to decreased unitary conductance (in fact, a small increase was detected), but to reduced number of available Na_V1.5 channels, specifically at the ID.

The reduced number of available channels could be consequent to decreased open probability of membrane-inserted channels, or to reduced presence of sodium channel-forming proteins. Following on our results in HL1s (Supplemental Figure 6), we assessed the extent of Na_V1.5/N-cadherin co-localization at the ID²². As shown inFig.7, Na_V1.5/N-cadherin co-localization decreased in PKP2-Hz cells when compared to control, though the amount of total Na_V1.5 in the cell lysate was unaffected by PKP2 deficit⁶. Altogether, we propose that decreased I_Na amplitude in PKP2-deficient cells is consequent to decreased cell surface expression of Na_V1.5 at the ID.

PKP2 deficit causes separation of the microtubule plus-end from N-cadherin plaques

The results were consistent with a role for PKP2 in forward trafficking of Na_V1.5 (among other not mutually exclusive possibilities). Previous studies indicated that sodium channels are delivered to the cell membrane via the microtubule network^23,24, and that microtubules at the ID anchor at N-cadherin-rich sites²⁵. We therefore explored whether microtubule arrival to the ID was impaired in PKP2-Hz myocytes. Specifically, we used two-color dSTORM to determine the distance between the plus-end of microtubules (marked by immunoreactive EB-1; see, e.g²⁵) and the cell end (defined by the midline of N-cadherin clusters; seeOS for methods).Fig.8 shows an image of EB-1 and N-Cadherin signals acquired by conventional TIRF (panel A, left) and dSTORM (A, right). The improved resolution of dSTORM (~20 nm, versus ~300 nm in TIRF; see bottom panels in A) allowed us to accurately measure separation distances (B). As shown in C, PKP2 deficiency led to increased average distance between the leading edge of EB-1 clusters, and the N-cadherin plaque midline. These results indicate that PKP2 deficiency, directly or indirectly, affects the ability of microtubules to reach the ID, likely impairing delivery of proteins relevant for sodium channel function.

Limitations

We focused on detection of variants in one desmosomal gene (PKP2). As such, we did not examine the possibility of other desmosomal mutations. We based this choice on a) the body of experimental evidence associating PKP2 with sodium current function^4–7, b) the high frequency of PKP2 mutations when compared to other desmosomal genes¹ and c) the need for an in vitro cell system to test structure-function relation between a specific desmosomal protein, and the sodium channel. Thus, while additional studies have shown that overexpression of a desmoglein-2 mutation also leads to I_Na dysfunction²⁶, we did not expand our study in that direction due to the likely failure to maintain and successfully transfect healthy HL-1 cells in the absence of desmosomal cadherins.

Our patients did not have mutations in eitherSCN5A orCACNA1C, the two genes most commonly associated with BrS and the subject of standard test for clinical diagnosis²⁷. We also confirmed absence of mutations on two additional -though rare- BrS genes coding for sodium channel interacting proteins, MOG1 and GPD1L. While variants in other genes have been found in BrS patients, their occurrence is rare²⁷. As in those cases, we do not know if the PKP2 mutations reported here occur in the context of additional genetic differences. Indeed, there is a large number of known proteins (and likely, a large number of unknown ones) that can affect, directly or indirectly, I_Na. Rather than extending our search to the entire genome of each patient, we focused on one gene while acknowledging that the PKP2 variants found represent part of the substrate and yet, perhaps not the only mechanism, when it comes to the clinical phenomena observed.

Exogenous systems are often used to study ion channel structure-function. In most cases, non-cardiac cells are used. Here we chose a cardiac cell line, so that components of the VGSC complex would follow their native transcription/translation process. A great advantage of this system is that, for the first time, we were able to experimentally assess the relation between single amino acid PKP2 mutations, and VGSC function and localization in a cardiac cell. The system also has limitations, one of which is that, in its most direct application, it does not model heterozygocity (Figure 3). To circumvent this problem, we carried out co-expression experiments (Figure 4). The similarity between results (Figure 3 versus4) may suggest a possible dominant negative effect of the mutant. However, our experimental conditions are too artificial to make such a conclusion. What we do show is that even in the presence of WT-PKP2, an I_Na deficit was detected.

Our results in HL1 cells were confirmed and expanded using the hIPSC-CM system (5). Technical complexities forced us to limit the test to one mutant. We chose mutation R635Q given that co-segregation data were also available (seeFigure 1C). Our data show that the relation between PKP2 expression/primary sequence and I_Na is also present in human cardiac cells.

Because of the more defined compartmentalization of ID molecules, we chose PKP2-Hz cells to assess mechanisms by which PKP2 deficiency leads to I_Na deficit. We recognize that the mechanisms described here may be influenced by expression of a mutant allele. Future experiments will involve generation and characterization of a PKP2-knockin mouse model expressing one of these mutations. Overall, our results show a tight convergence across experimental models demonstrating the importance of PKP2 in the proper function of the sodium channel complex.

PKP2 and BrS

BrS is an inherited channelopathy characterized by ST segment elevation of coved morphology in right precordial leads, increased risk of ventricular tachycardia and ventricular fibrillation and absence of cardiac structural disease¹⁹.SCN5A mutations account for ~20–25% of genotype-positive subjects²⁷, and about 4% of patients carry mutations in theCACNA1c gene. Several other genes have been associated with sporadic cases of BrS, but each one accounts for <2% of patients; as such, current guidelines do not advise to screen for them routinely in the general BrS population²⁷. Overall only 25–30% of patients with clinical diagnosis of BrS have a known genotype, implying that additional, still undiscovered genes may be linked to this disease.

When BrS was initially described, some investigators proposed that this condition shared features with AC, thus opening the possibility that they represent two poles of a common spectrum ultimately leading to increased risk of sudden death²⁸. In fact, on one side, some BrS patients show minor RV structural abnormalities²⁹ while on the other side, desmosomal mutation carriers can experience ventricular fibrillation and sudden death without overt structural disease^30–33. Our study supports the notion that one gene (e.g., PKP2) can be an underlying factor in both ends of this spectrum. There are other cases where the same gene is involved in more than one clinical phenotype (partly depending on whether the mutation leads to loss or gain of function): SCN5A mutations can give origin to different conditions ranging from Long QT Syndrome, to BrS, to progressive cardiac conduction defect, to Dilated Cardiomyopathy³⁴; mutations on potassium channel genes can cause Short or Long QT syndromes³⁵; and several genes for sarcomeric proteins when mutated can cause either Hypertrophic or Dilated Cardiomyopathy³⁶. This “multiplicity” of a clinical phenotype is here proposed for the first time for a desmosomal gene, which can associate with a spectrum that includes the BrS phenotype.

PKP2 and the sodium channel complex

Our previous data demonstrated that PKP2 ablation decreased I_Na and elicited reentrant arrhythmias in monolayers of cardiomyocytes⁵. We also showed that PKP2-Hz mice had reduced I_Na that facilitated flecainide-induced arrhythmias and sudden death⁶. These results support the role of impaired I_Na as a mechanism affecting arrhythmia susceptibility in the “concealed” phase of AC. Additional data showed that the intensity of immunoreactive Na_v1.5 was reduced in most heart sections obtained from AC patients⁷. This finding, consistent with those of Gomes et al³², indicate that a reduction in Na_v1.5 abundance may be a component of the phenotype in subjects with AC.

The role of PKP2 in preserving I_Na may be independent from its function as a component of the desmosome. Indeed, we speculate that some PKP2 mutations may primarily affect one function while not disrupting –or only minimally disrupting- the other. In fact, although AC is associated with fibrofatty replacement of ventricular muscle, our data suggest that cytoskeletal alterations affecting ion channel trafficking may precede larger scale tissue changes and contribute or even dominate the phenotype. We propose that specific PKP2 mutations can lead to a decreased depolarization reserve that manifests as BrS. Future experiments, using whole animal models, will be necessary to better define the role of PKP2 and other “mechanical junction proteins” in establishing the depolarizing reserve of the mammalian heart.

Previous studies have shown that N-cadherin-containing complexes act as anchoring points for microtubules at the intercalated disc²⁵. PKP2 and N-cadherin are both components of the area composita, a “mixed” junction of the adult intercalated disc linked to AC³⁷. We show that PKP2 deficiency increases the distance between the microtubule plus-end and the N-cadherin plaque (Figure 8), suggesting that integrity of the area composita as a whole, rather than only N-cadherin, is relevant to microtubule anchoring. The evidence that Na_V1.5 is delivered to the cell membrane through the microtubular network²³, and our results showing that reduced I_Na is consequent to decreased functional channel expression, lead us to propose that PKP2 is necessary for microtubule anchoring and safe delivery of Na_V1.5 to the ID. Of note, Cx43 is also necessary to preserve I_Na amplitude²², and we recently demonstrated that PKP2 and Cx43 share a subcellular domain³, forming a molecular network (the connexome). Whether this domain (likely at the perinexus³⁸) constitutes an actual point of anchoring and delivery for microtubules, deserves further investigation.

Conclusions

This manuscript represents the first systematic retrospective analysis of a large patient population with diagnosis of BrS to define the co-existence of clinical BrS and genetic variations inPKP2. This is also the first study demonstrating that not only the absence of PKP2, but also single amino acid mutations in its sequence, can alter I_Na. We propose thatPKP2 mutations provide at least part of the molecular substrate of BrS. Whether co-existence ofPKP2 mutations and a positive flecainide test have value in assessing sudden death risk and/or progression toward cardiomyopathy, is unclear. The inclusion of PKP2 as part of routine BrS genetic testing remains premature; yet, the possibility that some patients showing signs of disease may harbor PKP2 variants should be considered when the genotype is negative for other genes associated with BrS.

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