Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy

Abstract

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.

Maintaining proteostasis under mechanical stress

“…BAG3 was initially shown to participate in the autophagic degradation of aggregation‐prone, pathologic proteins in neuronal cells and later on also found to be involved in the turnover of the microtubule organizing protein tau in neuronal processes, thus providing an essential proteostasis function in the nervous system (Carra et al , 2008 ; Gamerdinger et al , 2009 , 2011 ; Crippa et al , 2010 ; Lei et al , 2015 ). However, BAG3‐deficient animals and patients carrying BAG3 mutant alleles display a profound pathology in skeletal and cardiac muscle, characterized by a progressive deterioration of sarcomeric structures under mechanical stress (Homma et al , 2006 ; Selcen et al , 2009 ; Arndt et al , 2010 ; Arimura et al , 2011 ; Claeys et al , 2013 ; Ruparelia et al , 2014 ; Konersman et al , 2015 ; Quintana et al , 2016 ; Fang et al , 2017 ; Meister‐Broekema et al , 2018 ; Schänzer et al , 2018 ; Kimura et al , 2021 ). Moreover, a decline in BAG3‐mediated sarcomere turnover contributes to the development of heart failure, the leading cause of mortality in the industrialized world (Martin et al , 2021 ).…”

“…BAG3 and its chaperone partners HSP70 and HSPB8 bind to the mechanosensitive region of filamin comprising Ig domains 19–21 (see above) and direct mechanically unfolded and damaged forms onto the CASA pathway for disposal (Fig 3C ) (Arndt et al , 2010 ; Klimek et al , 2017 ). Indeed, filamin forms aggregates in skeletal and cardiac muscle when the CASA pathway is compromised under pathological conditions (Arimura et al , 2011 ; Ruparelia et al , 2016 ; Fang et al , 2017 ; Kimura et al , 2021 ; Martin et al , 2021 ), illustrating the relevance of CASA for myofilament homeostasis in contracting muscle.…”

“…The inactivation or deregulation of mechanical stress protection factors causes severe pathology in humans. For example, mutations in BAG3 give rise to skeletal muscle myopathies and cardiomyopathies (Claeys et al , 2013 ; Fang et al , 2017 ; Kimura et al , 2021 ), a decline of BAG3 activity contributes to heart failure (Martin et al , 2021 ), and mutation‐induced functional impairment of UNC‐45 leads to skeletal muscle myopathy and ovarian and breast cancer (Bazzaro et al , 2007 ; Janiesch et al , 2007 ; Donkervoort et al , 2020 ). Advancing our knowledge about mechanical stress protection systems in humans will thus be instrumental for the diagnosis of diverse diseases and may pave the road for therapeutic intervention using, for example, proteostasis modulators, such as the autophagy inducing compound rapamycin.…”

Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

“…For this purpose, we have generated mouse G4 ES cells, in which the CAG promoter drives the inducible overexpression of Cx43. The CAG promoter was chosen because of its strong expression in muscle cells [ 34 , 40 , 41 ] at all stages of mouse embryonic development and after birth. The inducibility of the expression cassette is provided by a loxP flanked stop cassette with three SV40 polyadenylation signals that can be removed by Cre protein activity ( Figure 1 a).…”

“…The AAV2.1-Cre virus (pENN.AAV.CMVs.PI.Cre.rBG, Addgene, #105537-AAV1, 1.2 × 10 13 GC/mL) was titrated on a G4 ES cell line (BAG3P209L-eGFPflox3) [ 34 ] expressing eGFP after Cre induction; 5.6 × 10 10 genome copies/mL per 7500 cells were used. Viral transduction was performed in cell culture medium of CAG-floxSTOP-Cx43-P2A-mCherry-Ai6 transfected (Cre − ) lines of ES, HeLa cells, and 3T3-fibroblasts.…”