In the ID of cardiomyocytes, desmosomes provide a rigid connection between neighbouring cells, allowing them to withstand mechanical strain. In these structures, the desmosomal cadherins DSG2 and DSC2 homo- and heterodimerize with their extracellular cadherin domains, whereas their intracellular portions bind other desmosomal components, i.e. PG and PKP2, which in turn link to DSP, which provides a link to the desmin cytoskeleton.5
Mutations in these five cardiac desmosomal genes can cause ARVC,1,2
a disease associated with life-threatening arrhythmias and high incidence of sudden cardiac death. Tissue analysis of Individual 55.2 provided insights into potential mechanisms underlying arrhythmias and sudden cardiac death: fibrosis (see Supplementary material online, Figure S5A
) is thought to cause electric isolation of cardiomyocytes and may thereby facilitate reentrant excitation (discussed in Gehmlich et al.13
). In addition, gap junction remodelling (as documented by loss of Cx43 signal from the IDs; see Supplementary material online, Figure S5B
) may contribute to conduction heterogeneity and increase the risk of arrhythmic events.13
Failure of appropriate localization of PG to the ID, irrespective of the underlying genetic mutation, is a recently suggested feature of ARVC.7
We have confirmed this observation for the first time for a DSC2 mutation (Figure A
). This suggests that PG plays a major role in the pathogenesis of ARVC. The mechanisms of how diverse changes in different desmosomal proteins cause the same disease however are poorly understood.
Here, we explore the consequences of ARVC-associated DSC2 mutations at the molecular level. Any mutation resulting in a premature stop codon before the transmembrane domain is predicted not to produce any functional protein. Such null alleles, e.g. the newly identified DSC2 L229X and DSC2 G371fsX378 mutations, as well as previously reported ones,15,17
act primarily through haplo-insufficiency, and such a reduction of one desmosomal component is likely to have deleterious effects on the entire structure, as observed by electron microscopy for ARVC patients and DSC2 knockdown in a zebrafish model.15,28
In particular, 50% reduction of DSC2 at the desmosomes reduces the number of ligands for PG and PKP2 in the structures. DSC2 and DSG2 bind to PG and PKP2 with similar strength (Figure A
), so lack of DSC2 could only be partly compensated by DSG2. In addition, missense mutations in DSC2 can also potentially affect desmosomal structure and integrity.
The two novel missense mutations investigated, DSC2 R203C and DSC2 T275M, are located in the N-terminal cadherin domains. These modules mediate the adhesive properties of the cadherins in a calcium-dependent fashion, and the binding of calcium ions at the interface of neighbouring cadherin domains is a pre-requisite for cadherin function.29,30
Both affected residues (R203 and T275) are evolutionary conserved and structural models suggest crucial roles of both mutated residues for the domain integrity (see Supplementary material online, Figure S4B
Both mutant proteins exhibit defects in processing into the mature form (Figure
; see Supplementary material online, Figure S2
). Normally, this proteolytic process involves endoprotein convertases and results in functional activation of DSC2, because in the precursor form, the pro-protein extension prevents binding to other cadherins.31
In the case of DSC2 R203C, the mutant fails to undergo the complete cleavage, whereas the DSC2 T275M protein can still be processed, but shows a higher pro-protein to mature protein ratio (Figure B
; see Supplementary material online, Figure S2
). As a consequence, the non-functional DSC2 R203C fails to localize at the desmosomes of ID structures, whereas only a proportion of the partly functional DSC2 T275M protein is still incorporated into the desmosomes (Figures
In our study, Patient 35.1 is heterozygous for DSC2 R203C and Patient 37.1 is homozygous for DSC2 T275M. In the former, 50% of DSC2 is functional as there is only one DSC2 WT allele present. In the latter, our experiments show that only two-thirds of the DSC2 are processed into the mature form. Consequently, in both cases, there is a significant reduction of the mature, functional DSC2 protein level at the ID. This further implies that lack of functional DSC2 at the ID is primarily responsible for the disease phenotype rather than a dominant-negative action of the mutant proteins. Partially, abnormal localization of two other ARVC-associated DSC2 missense mutations has also been noted in cardiomyocytes.12
The properties of the DSC2 A897fsX900 protein contrast with the findings with the N-terminal mutations. This C-terminal mutation changes the last five amino acids of the larger DSC2a splice isoform, whereas the DSC2b isoform is unaffected (see Supplementary material online, Figure S1A
). Our findings suggest that this mutant protein is normally processed into its mature form and can be incorporated into the desmosomes of HL-1 cells and cardiomyocytes (Figures
). In contrast to only minor changes in localization observed in our experiments (Figure
), a recent report by De Bortoli et al.18
suggested the mis-localization of this variant in HL-1 cells. The use of a GFP fusion protein construct in their experiments might artificially have obscured the real localization of the mutant protein. We utilized the lack of cross-reactivity of our antibodies with rodent proteins to study the behaviour of non-tagged human constructs (exposing the different carboxy-termini) and found only small changes in cellular localization.
Our GST-pulldown assays clearly demonstrate impaired binding of the DSC2a A897fsX900 variant to PG and DSP (Figure
), while binding to PKP2 was virtually unaffected. This finding was confirmed by co-immunoprecipitation experiments using full-length proteins. As observed before, the DSC2 A897fsX900 protein bound more weakly to PG than DSC2a WT, while binding to PKP2 was normal (Figure C
). However, the effect seen here was not as dramatic as in experiments using only the cytoplasmic portion, and this may be explained by potential homo- or heterodimerization with desmosomal cadherins via the N-terminal cadherin domains.30
The DSC2 A897fsX900 variant has now been found in apparently healthy individuals.18
Although a low expression level of the DSC2a isoform in the myocardium18
may contribute to the lack of an overt phenotype in these individuals, our functional studies demonstrate a pathogenic potential of this variant: its impaired binding to PG cannot directly be compensated by the DSC2b isoform, which does not bind PG (Figure A
). However, the cytoplasmic domain of DSG2 appears to have redundant functions in binding PG in the desmosomes. Currently, it is unclear whether carriers of the variant exhibit subclinical features of disease expression or what the potential is for disease development in individuals who perform endurance training or carry other common variants. In the future, high-throughput genomic analysis of large families and large, well-defined cohorts might help to answer these questions.
The striking observation of our functional studies is that—despite their different individual properties—all the DSC2 mutant proteins appear to affect PG at the IDs: for each of the mutations, the ability of DSC2 to provide an anchor for PG is impaired. As a consequence, PG may not be efficiently targeted to, or retained at, the desmosomes, and in turn might become available for other intracellular pools. This loss of PG from the ID has been reported in ARVC patients7
(including individuals with DSC2 mutations, Figure A
). Beyond being a diagnostic feature, this event may be crucial in disease pathogenesis: data from transgenic animal studies suggest that unbound PG might be translocated into the nucleus and could aberrantly modulate transcriptional pathways, especially by competing with β-catenin in the Wnt-signalling pathways.8,11
Our functional studies on the three DSC2 mutations suggest for the first time a molecular explanation how DSC2 mutations may contribute to the same ‘common final pathway’ of ARVC.
In transfection experiments, we failed to observe a redistribution of PG from the ID in the presence of the mutant DSC2 proteins (Figure D
), neither were changes in desmosomal composition observed (see Supplementary material online, Figure S2
). However, these cellular systems may not be adequate to reflect the in vivo
situation: in patients with heterozygous mutations, one functional WT allele is replaced by a mutant DSC2 form, which affects PG binding. In contrast, the mutant DSC2 proteins were expressed in addition to the endogenous DSC2 in our experiments. Therefore, the abundant endogenous DSC2 protein may compensate any deleterious effects of the DSC2 mutants. As demonstrated for mutations in the cytoplasmic region of DSG2,13
additional indirect effects beyond altered binding properties may contribute to the complexity of the disease.
In the future, the reduction of functional DSC2 protein in a cellular model systems (e.g. by RNA interference) will be a valuable tool to study down-stream pathogenic events, such as the redistribution of PG from the ID and aberrant activation of PG signalling in the nucleus. Moreover, animal models engineered for the cardiac-specific expression of DSC2 mutation forms will help to provide mechanistic insights into ARVC.