Functional characterization of the novel DES mutation p.L136P associated with dilated cardiomyopathy reveals a dominant filament assembly defect.

BACKGROUND: Dilated cardiomyopathy (DCM) could be caused by mutations in more than 40 different genes. However, the pathogenic impact of specific mutations is in most cases unknown complicating the genetic counseling of affected families. Therefore, functional studies could contribute to distinguish pathogenic mutations and benign variants. Here, we present a novel heterozygous DES missense variant (c.407C>T; p.L136P) identified by next generation sequencing in a DCM patient. DES encodes the cardiac intermediate filament protein desmin, which has important functions in mechanical stabilization and linkage of the cell structures in cardiomyocytes. METHODS AND RESULTS: Cell transfection experiments and assembly assays of recombinant desmin in combination with atomic force microscopy were used to investigate the impact of this novel DES variant on filament formation. Desmin-p.L136P forms cytoplasmic aggregates indicating a severe intrinsic filament assembly defect of this mutant. Co-transfection experiments of wild-type and mutant desmin conjugated to different fluorescence proteins revealed a dominant affect of this mutant on filament assembly. These experiments were complemented by apertureless scanning near-field optical microscopy. CONCLUSION: In vitro analysis demonstrated that desmin-p.L136P is unable to form regular filaments and accumulate instead within the cytoplasm. Therefore, we classified DES-p.L136P as a likely pathogenic mutation. In conclusion, the functional characterization of DES-p.L136P might have relevance for the genetic counseling of affected families with similar DES mutations and could contribute to distinguish pathogenic mutations from benign rare variants.

Rational design of a cytotoxic dinuclear Cu2 complex that binds by molecular recognition at two neighboring phosphates of the DNA backbone.

The mechanism of the cytotoxic function of cisplatin and related anticancer drugs is based on their binding to the nucleobases of DNA. The development of new classes of anticancer drugs requires establishing other binding modes. Therefore, we performed a rational design for complexes that target two neighboring phosphates of the DNA backbone by molecular recognition resulting in a family of dinuclear complexes based on 2,7-disubstituted 1,8-naphthalenediol. This rigid backbone preorganizes the two metal ions for molecular recognition at the distance of two neighboring phosphates in DNA of 6-7 Å. Additionally, bulky chelating pendant arms in the 2,7-position impede nucleobase complexation by steric hindrance. We successfully synthesized the Cu(II)2 complex of the designed family of dinuclear complexes and studied its binding to dsDNA by independent ensemble and single-molecule methods like gel electrophoresis, precipitation, and titration experiments followed by UV-vis spectroscopy, atomic force microscopy (AFM), as well as optical tweezers (OT) and magnetic tweezers (MT) DNA stretching. The observed irreversible binding of our dinuclear Cu(II)2 complex to dsDNA leads to a blocking of DNA synthesis as studied by polymerase chain reactions and cytotoxicity for human cancer cells.

Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.

De novo desmin-mutation N116S is associated with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease, frequently accompanied by sudden cardiac death and terminal heart failure. Genotyping of ARVC patients might be used for palliative treatment of the affected family. We genotyped a cohort of 22 ARVC patients referred to molecular genetic screening in our heart center for mutations in the desmosomal candidate genes JUP, DSG2, DSC2, DSP and PKP2 known to be associated with ARVC. In 43% of the cohort, we found disease-associated sequence variants. In addition, we screened for desmin mutations and found a novel desmin-mutation p.N116S in a patient with ARVC and terminal heart failure, which is located in segment 1A of the desmin rod domain. The mutation leads to the aggresome formation in cardiac and skeletal muscle without signs of an overt clinical myopathy. Cardiac aggresomes appear to be prominent, especially in the right ventricle of the heart. Viscosimetry and atomic force microscopy of the desmin wild-type and N116S mutant isolated from recombinant Escherichia coli revealed severe impairment of the filament formation, which was supported by transfections in SW13 cells. Thus, the gene coding for desmin appears to be a novel ARVC gene, which should be included in molecular genetic screening of ARVC patients.

A novel desmin (DES) indel mutation causes severe atypical cardiomyopathy in combination with atrioventricular block and skeletal myopathy.

BACKGROUND: DES mutations cause different cardiac and skeletal myopathies. Most of them are missense mutations. METHODS: Using a next-generation sequencing cardiac 174 gene panel, we identified a novel heterozygous in-frame indel mutation (DES-c.493_520del28insGCGT, p.Q165_A174delinsAS) in a Caucasian patient with cardiomyopathy in combination with atrioventricular block and skeletal myopathy. This indel mutation is located in the coding region of the first exon. Family anamnesis revealed a history of sudden cardiac death. We performed cell transfection experiments and in vitro assembly experiments to prove the pathogenicity of this novel DES indel mutation. RESULTS: These experiments revealed a severe filament formation defect of mutant desmin supporting the pathogenicity. In addition, we labeled a skeletal muscle biopsy from the mutation carrier revealing cytoplasmic desmin positive protein aggregates. In summary, we identified and functionally characterized a pathogenic DES indel mutation causing cardiac and skeletal myopathy. CONCLUSION: Our study has relevance for the clinical and genetic interpretation of further DES indel mutations causing cardiac or skeletal myopathies and might be helpful for risk stratification.

Arrhythmogenic cardiomyopathy related DSG2 mutations affect desmosomal cadherin binding kinetics.

Cadherins are calcium dependent adhesion proteins that establish the intercellular mechanical contact by bridging the gap to adjacent cells. Desmoglein-2 (Dsg2) is a specific cadherin of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2-gene are regarded to cause arrhythmogenic (right ventricular) cardiomyopathy (ARVC) which is a rare but severe heart muscle disease. The molecular pathomechanisms of the vast majority of DSG2 mutations, however, are unknown. Here, we investigated the homophilic binding of wildtype Dsg2 and two mutations which are associated with ARVC. Using single molecule force spectroscopy and applying Jarzynski's equality we determined the kinetics and thermodynamics of Dsg2 homophilic binding. Notably, the free energy landscape of Dsg2 dimerization exposes a high activation barrier which is in line with the proposed strand-swapping binding motif. Although the binding motif is not directly affected by the mutations the binding kinetics differ significantly from the wildtype. Furthermore, we applied a dispase based cell dissociation assay using HT1080 cell lines over expressing Dsg2 wildtype and mutants, respectively. Our molecular and cellular results consistently demonstrate that Dsg2 mutations can heavily affect homophilic Dsg2 interactions. Furthermore, the full thermodynamic and kinetic description of Dsg2 dimerization provides a consistent model of the so far discussed homophilic cadherin binding.

Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin.

Both fluorescence imaging and atomic force microscopy (AFM) are highly versatile and extensively used in applications ranging from nanotechnology to life sciences. In fluorescence microscopy luminescent dyes serve as position markers. Moreover, they can be used as active reporters of their local vicinity. The dipolar coupling of the tip with the incident light and the fluorophore give rise to a local field and fluorescence enhancement. AFM topographic imaging allows for resolutions down to the atomic scale. It can be operated in vacuum, under ambient conditions and in liquids. This makes it ideal for the investigation of a wide range of different samples. Furthermore an illuminated AFM cantilever tip apex exposes strongly confined non-propagating electromagnetic fields that can serve as a coupling agent for single dye molecules. Thus, combining both techniques by means of apertureless scanning near-field optical microscopy (aSNOM) enables concurrent high resolution topography and fluorescence imaging. Commonly, among the various (apertureless) SNOM approaches metallic or metallized probes are used. Here, we report on our custom-built aSNOM setup, which uses commercially available monolithic silicon AFM cantilevers. The field enhancement confined to the tip apex facilitates an optical resolution down to 20 nm. Furthermore, the use of standard mass-produced AFM cantilevers spares elaborate probe production or modification processes. We investigated tobacco mosaic viruses and the intermediate filament protein desmin. Both are mixed complexes of building blocks, which are fluorescently labeled to a low degree. The simultaneous recording of topography and fluorescence data allows for the exact localization of distinct building blocks within the superordinate structures.

The novel desmin mutant p.A120D impairs filament formation, prevents intercalated disk localization, and causes sudden cardiac death.

BACKGROUND: The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. METHODS AND RESULTS: We identified the 2 novel variants DES-p.A120D (c.359C>A) and DES-p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in cell lines and of the isolated recombinant protein. Model variants of codon 120 indicated that ionic interactions contribute to this filament formation defect. Ex vivo analysis of ventricular tissue slices revealed a loss of desmin staining within the intercalated disk and severe cytoplasmic aggregate formation, whereas z-band localization was not affected. The functional experiments of desmin-p.H326R did not demonstrate any differences from wild type. CONCLUSIONS: Because of the functional in vivo and in vitro characterization, DES-p.A120D has to be regarded as a pathogenic mutation and DES-p.H326R as a rare variant with unknown significance. Presumably, the loss of the desmin-p. A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.