A team of international medical researchers have recently discovered that defective desmosomal adhesion causes arrhythmogenic cardiomyopathy, a heart condition affecting many young adults especially athletes.
Individuals with arrhythmogenic cardiomyopathy (ACM) typically present with features ranging from impaired cardiac function and ventricular arrhythmia to sudden cardiac death as a result of Heart Failure.
The study findings were published in the peer reviewed journal: Circulation (A Journal of the American Heart Association)
In this study, we generated a knock-in mouse model with defective binding function of the adhesion molecule DSG2, which demonstrated that impaired desmosomal adhesion is sufficient to induce a phenotype mimicking the characteristics of ACM. Our data suggest a cascade of defective desmosomal adhesion, disrupted ICD structure, and subsequent activation of ITGAV/B6 with profibrotic TGF-β signaling as important underlying mechanisms leading to this phenotype (Figure 7).
Moreover, our pilot study indicates a beneficial effect of ITGAV/B6 inhibition by EMD treatment with regard to fibrosis and several ECG parameters, suggesting that this pathway can be targeted successfully by drug treatment.
Defective Desmosomal Adhesion by DSG2-W2A Mutation Is Inducing an ACM Phenotype
Because of mutations mainly affecting desmosomal genes and evidence of disrupted ICDs, the hypothesis of dysfunctional desmosomes with loss of cell–cell adhesion as a central pathologic step was adopted in the field.2 However, experimental data on this topic are contradictory.6–8
We show that W2 of DSG2 is central for binding and intercellular adhesion in vitro and in vivo. Disrupted desmosomal adhesion is sufficient to induce an ACM phenotype fulfilling the Padua criteria,13 including ventricular fibrosis, depolarization and repolarization abnormalities, arrhythmia, and impaired ventricular function, which are used to establish the diagnosis.
In line with this, 3 mutations were described in patients with ACM directly affecting the DSG2 binding mechanism by exchange of the tryptophan with a serine, leucine, or arginine (ClinVar data bank: VCV000199796, VCV000044282, VCV000420241).10 This indicates a disruption of the tryptophan swap independent from the substituting amino acid and supports defective desmosomal adhesion as important factor in ACM.2,24
Moreover, different published DSG2 mutant mouse models show a phenotype resembling the features of DSG2-W2A mutants. These include a DSG2 mutant in which parts of the 2 outermost DSG2 extracellular domains were deleted (but leaving W2A intact),25 2 mouse lines with loss of DSG2,25,26 and mice overexpressing a patient mutation (DSG2-N271S),27 which also showed that ICD structural aberrations precede functional abnormalities and fibrosis generation.
These data fuel the hypothesis that disruption and rearrangement of the ICD in response to impaired adhesion between cardiomyocytes is a crucial initial step, at least in case of mutations in desmosomal molecules. Other mechanisms described in patients or translational models, such as aberrant WNT or Hippo/YAP signaling, or immune cell infiltrations,2,28 may be secondary responses and in part even represent adaptive attempts to rescue the functional consequences of such severe structural aberrations.
DSG2-W2A Mice as Model to Study ACM
Our results suggest the DSG2-W2A model as a valuable tool to study ACM mechanisms. Within the limitations of a murine model, it reproduces a majority of features found in patients with ACM (Table S2). In homozygous animals, both ventricles are affected from early on in life; heterozygous animals develop a milder phenotype, with fibrosis occurring only in the RV. Whether this milder phenotype would extend to the LV over time or whether a gene dose effect underlies the structural differences is unclear.
LV and RV remodel differently in response to loading and injury29 and it is possible that the RV is less able to compensate a partial reduction of cardiomyocyte cohesion, in contrast to the LV. A fraction of mut/wt animals did not develop the phenotype at all within the observation period of up to 80 weeks. Thus, the model might help clarify why patients with the same mutation (ie, in the same family) develop the disease with variable penetrance.1 Similar to patients,1 male mutant mice appear to experience more arrhythmia and sudden death than do female mice.
We can interpret from the combined histology, echocardiography, and ECG data that the changes in heart function are secondary to fibrosis generation. This is indicated by the notion that functional measures (eg, ejection fraction, QRS interval) worsened over time in homozygous mutants, whereas no increase was detectable for fibrosis. Moreover, functional changes in mut/wt animals were only observed when fibrosis was present.
This is in line with a patient cohort with DSP mutations, in which LV fibrosis preceded systolic dysfunction.30 A different study in DSG2-N271S-expressing ex vivo paced hearts demonstrated conduction velocity impairments before onset of fibrosis.27 More longitudinal studies using different mutational backgrounds and sensitive detection methods (eg, cardiac magnetic resonance imaging and ECG under exercise) are necessary to define the role of fibrosis as cause or consequence of functional changes.
As with all murine models, the DSG2-W2A line has limitations in its ability to fully recapitulate the human disease phenotype. Mutant mice do not show pronounced replacement with adipose tissue, which is a common characteristic in patient heart samples, but in general is not fully modeled in mice.24 Moreover, at least in ECGs under anesthesia, not all mutant animals develop arrhythmia, even if fibrosis is present. However, in contrast to most other ACM models, this knock-in model has the advantage of a single amino acid exchange under the endogenous promotor, thus reducing the possibility of secondary unwanted effects attributable to complete protein absence or overexpression. As in patients, the mutation is present in all cell types and not limited to cardiomyocytes.2,24
DSG2-W2A Leads to Dysfunctional ICDs and ITGAV/B6 Rearrangement
DSG2-W2A led to a severely altered ICD structure consistent with impaired cytoskeletal attachment and ruptured junctions. Whereas these changes provide explanations for compromised intercellular adhesion, we also noted alterations in cell–matrix protein distribution. RNA sequencing before and after onset of the disease phenotype and a comparison with ACM patient datasets identified ITGB6 as commonly deregulated in patients with ACM and DSG2-W2A mice.
Although we noted reduced mRNA levels in mutant hearts, the overall protein content was unaltered. This suggests pronounced posttranslational regulation of ITGB6, which was already demonstrated in skeletal muscle.31 In mutant hearts, ITGB6 was increased at the ICD together with elevated levels of ITGAV and increased heterodimerization of both molecules. By mechanical force exerted through ITGAV/B6, TGF-β is detached from the latency-associated peptide and can induce signaling by means of TGF-β receptors.17,32 In line with higher intracellular forces, talin-2 and vinculin were increased at ICDs, as binding of these molecules activates and stabilizes integrins.
A mutual regulation of both adhesive compartments is well-established. As an example, upon loss of N-cadherin, integrins are activated and induce fibronectin deposition.33 A similar mechanism is conceivable in DSG2-W2A hearts, either by loss of DSG2 or because of reduced N-cadherin anchorage. So far, only limited data are available on the role of integrins in ACM. A recent study showed downregulation of integrin-β1D leading to ventricular arrhythmia.34 Furthermore, knockdown of PKP2 in HL-1 cardiomyocytes was described to deregulate focal adhesions, including integrin-α1.35
Activation of TGF-β Signaling by ITGAV/B6 as Potential Therapeutic Target in ACM
The ITGAV/B6 heterodimer is described as one of the major activators of latent TGF-β1 and TGF-β3.17 Although TGF-β signaling is known as general driver of cardiac fibrosis36 and more specifically was implicated in ACM,2 to our knowledge no data are available on the role of ITGAV/B6 and their regulation of TGF-β signaling in cardiac fibrosis. Uncovering this pathway is of high interest as it offers the possibility to target TGF-β with reduced risk of severe side effects occurring in response to direct inhibition.37
We demonstrate a reduction of profibrotic gene expression under ACM conditions in response to ITGAV/B6 inhibition and our pilot study data suggest that a small molecule blocking ITGAV/B6-dependent TGF-β release diminished the generation of fibrosis. Similar approaches using this small molecule or neutralizing antibodies were shown to be protective in murine models of lung, liver, and biliary fibrosis.23,38,39 However, because of the heterogeneity of the phenotype in heterozygous animals, more detailed studies with larger sample sizes are required to further substantiate this finding.
Whether fibrosis generation is a contributor to the disease or a protective mechanism to ensure integrity of the heart under conditions of compromised adhesion is unclear. In case of the latter, pharmacologic inhibition of fibrosis generation might be detrimental. Nevertheless, fibrosis is a driver of arrhythmia generation,40 which is also supported by our results that ECG abnormalities were reduced by inhibition of fibrosis. Careful studies using different therapeutic approaches to reduce fibrosis in appropriate model organisms need to address this aspect in detail.
In conclusion, we established a new mouse model phenocopying many aspects of ACM, uncovered a novel pathway of fibrosis induction, and identified an approach to target this mechanism with future implication as a potential therapeutic option.