Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency.

Mutations in the molecular co-chaperone Bcl2-associated athanogene 3 (BAG3) are found to cause dilated cardiomyopathy (DCM), resulting in systolic dysfunction and heart failure, as well as myofibrillar myopathy (MFM), which is characterized by protein aggregation and myofibrillar disintegration in skeletal muscle cells. Here, we generated a CRISPR/Cas9-induced Bag3 knockout zebrafish line and found the complete preservation of heart and skeletal muscle structure and function during embryonic development, in contrast to morpholino-mediated knockdown of Bag3. Intriguingly, genetic compensation, a process of transcriptional adaptation which acts independent of protein feedback loops, was found to prevent heart and skeletal muscle damage in our Bag3 knockout model. Proteomic profiling and quantitative real-time PCR analyses identified Bag2, another member of the Bag protein family, significantly upregulated on a transcript and protein level in bag3-/- mutants. This implied that the decay of bag3 mutant mRNA in homozygous bag3-/- embryos caused the transcriptional upregulation of bag2 expression. We further demonstrated that morpholino-mediated knockdown of Bag2 in bag3-/- embryos evoked severe functional and structural heart and skeletal muscle defects, which are similar to Bag3 morphants. However, Bag2 knockdown in bag3+/+ or bag3+/- embryos did not result in (cardio-)myopathy. Finally, we found that inhibition of the nonsense-mediated mRNA decay (NMD) machinery by knockdown of upf1, an essential NMD factor, caused severe heart and skeletal muscle defects in bag3-/- mutants due to the blockade of transcriptional adaptation of bag2 expression. Our findings provide evidence that genetic compensation might vitally influence the penetrance of disease-causing bag3 mutations in vivo. Author summary: One form of genetic compensation is described as transcriptional adaptation of gene expression triggered by deleterious gene mutations. Although the precise molecular mechanism that induces genetic compensation needs to be defined, it represents a powerful biological phenomenon that warrants genetic robustness. We find that antisense-mediated knockdown of Bag3 in zebrafish embryos causes heart failure and myopathy. By contrast, CRISPR/Cas9-induced depletion of Bag3 does not result in the abrogation of heart and skeletal muscle function in zebrafish embryos. We find here that transcriptional activation of the Bag family member bag2 is capable of restoring heart and skeletal muscle function in bag3 mutant embryos, whereas this compensatory mechanism is not present in the bag3 morphants. Furthermore, we show that nonsense-mediated decay of bag3 mRNA is the molecular trigger for the compensatory upregulation of bag2. Our study provides evidence that genetic compensation via transcriptional adaptation is a vital modulator of disease peculiarity and penetrance in bag3 mutant zebrafish and that this biological phenomenon might also be active in certain human BAG3 mutation carriers. [ABSTRACT FROM AUTHOR]