Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy

journal articl

Immunmodulation im Cholesteatom

ZusammenfassungEinleitung Die Ätiopathogenese der chronischen Otitis media epitympanalis bzw. des Cholesteatoms und ihr proliferierender destruierender Verlauf mit möglichen Komplikationen wie Destruktion der knöchernen Strukturen mit Hörverlust, vestibulärer Dysfunktion, Gesichtsnervenlähmung und intrakraniellen Komplikationen sind immer noch ungeklärt. Die Therapie der Wahl ist nach wie vor die operative Sanierung. Aktuelle Studien befassen sich immer mehr mit dem angeborenen Immunsystem.Methoden Unsere Untersuchungen erfolgten im Mausmodell an WT-Mäusen und immundefizienten KO-Mäusen sowie an Gewebeproben vom Cholesteatom, gesunder Gehörgangshaut und gesunder Mittelohrschleimhaut, die während sanierenden Ohroperationen entnommen wurden. Die Expressionsanalysen erfolgten auf Gen- und Proteinebene mit TNF als Major Target zur Therapieevaluation. Mittels TUNEL-Färbung und Immunhistochemie an Kryoschnitten wurde die Apoptose-Rate durch TNF bestimmt.Ergebnisse Das ungerichtet-expansive Cholesteatomwachstum zeigt ein immunmodulatorisches Profil mit Hoch- und Runterregulation von verschiedenen Gen-Netzwerken, vor allem Molekülen der TNF-Down- und -Upstream-Signalwege. Dabei wird TNF sowohl inflammatorisch als auch apoptotisch moduliert und eignet sich als möglicher Therapieansatz in verschiedenen Modellen.Schlussfolgerungen Es gibt Hinweise auf eine immunmodulatorische Regulation im Cholesteatom.</jats:p

Abstract 466: Myopathy Causing Bag3 ^P209L Protein Leads to Restrictive Cardiomyopathy Caused by Aggregate Formation and Sarcomere Disruption in Cardiomyocytes

The co-chaperone BAG3 (Bcl-2 associated athanogene 3) is strongly expressed in cross-striated muscles and plays a key role in the turnover of muscle-proteins as a member of the CASA (chaperone-assisted selected autophagy) complex. An amino acid exchange (P209L) in the human BAG3 gene, caused by a single base mutation, gives rise to a severe dominant childhood muscular dystrophy, restrictive cardiomyopathy, and respiratory insufficiency. To get deeper insights into the pathophysiological mechanisms of the disease, we generated a transgenic mouse model of the human mutation BAG3 P209L , in which a fusion protein consisting of the human BAG3 P209L and the green fluorescent protein eGFP can be conditionally overexpressed. Ubiquitous overexpression of BAG3 P209L -eGFP leads to a severe phenotype between the second and fourth week of life, including decreased body weight, skeletal muscle weakness, and heart failure. Echocardiography revealed that the BAG3 P209L -mice suffer from restrictive cardiomyopathy and Sirius-red-staining of heart tissue showed extensive fibrosis. In cardiomyocytes, isolated from hearts of transgenic mice overexpressing BAG3 wt -eGFP or BAG3 P209L -eGFP, BAG3 wt -eGFP stringently localizes to sarcomeres and intercalated discs, whereas cardiomyocytes from BAG3 P209L -eGFP mice displayed formation of BAG3 containing aggregates and disruption of sarcomeres in vivo . While BAG3 P209L -eGFP binding to á-Hsp70, Filamin C and á-HspB8 was unchanged it was less soluble than BAG3 and had a tendency to aggregate, thereby sequestering BAG3 and its clients. Depletion of the BAG3 pool leads to an impairment of CASA and accumulation of damaged proteins, causing sarcomere disintegration leading to restrictive cardiomyopathy. </jats:p

Hypoxia-Inducible Factor 2α Mutation-Related Paragangliomas Classify as Discrete Pseudohypoxic Subcluster

AbstractRecently, activating mutations of the hypoxia-inducible factor 2α gene (HIF2A/EPAS1) have been recognized to predispose to multiple paragangliomas (PGLs) and duodenal somatostatinomas associated with polycythemia, and ocular abnormalities. Previously, mutations in the SDHA/B/C/D, SDHAF2, VHL, FH, PHD1, and PHD2 genes have been associated with HIF activation and the development of pseudohypoxic (cluster-1) PGLs. These tumors overlap in terms of tumor location, syndromic presentation, and noradrenergic phenotype to a certain extent. However, they also differ especially by clinical outcome and by presence of other tumors or abnormalities. In the present study, we aimed to establish additional molecular differences between HIF2A and non-HIF2A pseudohypoxic PGLs. RNA expression patterns of HIF2A PGLs (n=6) from 2 patients were compared with normal adrenal medullas (n=8) and other hereditary pseudohypoxic PGLs (VHL: n=13, SDHB: n=15, and SDHD: n=14). Unsupervised hierarchical clustering showed that HIF2A PGLs made up a separate cluster from other pseudohypoxic PGLs. Significance analysis of microarray yielded 875 differentially expressed genes between HIF2A and other pseudohypoxic PGLs after normalization to adrenal medulla (false discovery rate 0.01). Prediction analysis of microarray allowed correct classification of all HIF2A samples based on as little as three genes (TRHDE, LRRC63, IGSF10; error rate: 0.02). Genes with the highest expression difference between normal medulla and HIF2A PGLs were selected for confirmatory quantitative reverse transcriptase polymerase chain reaction. In conclusion, HIF2A PGLs show a characteristic expression signature that separates them from non-HIF2A pseudohypoxic PGLs. Unexpectedly, the most significantly differentially expressed genes have not been previously described as HIF target genes

Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy

AbstractAn amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.</jats:p

Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy

An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption

Gold Nanocrystal Labeling Allows Low-Density Lipoprotein Imaging from the Subcellular to Macroscopic Level

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail