Background: Idiopathic dilated cardiomyopathy (DCM) is one of the most common forms of heart failure (HF) with left ventricular dilation and dysfunction in absence of an obvious etiology with a significantly reduced life expectancy. Genome-wide association studies (GWAS) identified human BAG3 mutations contributing to either monogenic or sporadic DCM. BAG3 is a central actor of protein quality control system (PQC). The toxic aggregation of misfolded or damaged proteins, referred as proteinopathy, has been identified as a possible pathomechanism in DCM. Therefore, we investigated the molecular mechanism of a novel human BAG3 mutation (V468M) described in patients with familial DCM.

Methods and Results:  To investigate the BAG3 mutation in vivo, ectopic expression of human V468M BAG3 was established in wild-type (WT) zebrafish embryos. BAG3 V468M overexpression induced a DCM phenotype with enlarged ventricular diameter, decreased heart rate and impaired contractility. Immunostaining of zebrafish embryos overexpressing BAG3 V468M showed normal heart chamber differentiation. Whereas, investigation of myocardial ultrastructure and fiber alignment, via transmission electron microscopy (TEM), showed unorganized and disrupted cardiac muscle fibers compared to WT. To investigate the effects of the BAG3 V468M mutation on autophagy, we first evaluated p62 and autophagosome associated protein light chain 3 (LC3) on protein levels. Western Blot analysis of p62 and LC3I-II levels showed no significant difference in zebrafish embryos overexpressing BAG3 V468M compared to WT. The ubiquitin-proteasome system (UPS) is another highly conserved degradation system that mediates ubiquitination and degradation of intracellular proteins. To analyze the effect of the BAG3 V468M mutation on the UPS, a ubiquitin Western Blot was performed showing a significantly increased aggregation of ubiquitin in the zebrafish embryos overexpressing BAG3 V468M.  

Conclusion: Overexpression of BAG3 V468M in zebrafish embryos leads to heart disease phenotype. Impaired heart function and myocardial muscle fiber disruption support a DCM phenotype. Since BAG3 in known to play a crucial role in the PQC system, investigation of the autophagic flux showed no alteration in BAG3 V468M overexpressing embryos, whereas the UPS system revealed to be impaired by the mutation.