Cardiomyopathies are universally characterised by abnormalities in heart structure, but disease severity and progression can be highly variable. This variability sparks even more interest in patients with inherited cardiomyopathies that have mutations in the same causative gene, such as BAG3. One source of variability can be additional mutations in modifier genes that, alone, do not cause a disease but can enhance or dampen its severity. Traditional techniques to assess modifier genes in humans and mice can generate vast amounts of data, so complementary approaches are required to pinpoint and evaluate specific modifiers.
The guest editors of our ‘Moving Heart Failure to Heart Success’ Special Issue – Jeroen Bakkers, Milena Bellin and Ravi Karra – highlight a Resource article by Ding et al. (2022) that adapted a forward genetic screen-based assay to identify modifier genes for an inherited cardiomyopathy in adult zebrafish. The immense advantage of this technique is that it employs CRISPR with single-guide RNA (sgRNA) that induce microhomology-mediated end-joining (MMEJ) rather than traditional non-homologous end-joining (NHEJ). This enables precise and homogenous gene editing in F0 zebrafish for the interrogation of modifier genes – even in adult fish – without time-consuming crossbreeding to generate stable lines with mutations in both the causative gene (e.g. bag3) and the potential modifier gene. Using this technique, the authors found that a known deleterious modifier of anthracycline-induced cardiotoxicity (AIC) – dnajb6b – is also a modifier of inherited dilated cardiomyopathy in bag3e2/e2 KO zebrafish. They validated the technique further by confirming the therapeutic (or salutory) modifier effects of mtor that had previously been associated with bag3 inherited cardiomyopathy. By assessing several parameters of cardiac function in the adult F0 fish, the authors determined that this rapid technique recapitulated most of the phenotypes found in the stable double-mutant fish generated by classic crossbreeding.
Moving forward, the authors designed MMEJ-inducing sgRNA for eight genes that had previously been associated with bag3 cardiomyopathy in zebrafish by transcriptomics. Screening these genes revealed myh9b as a novel modifier gene of bag3 cardiomyopathy, which was then confirmed with the generation of F1 fish with stable mutations in bag3 and myh9b. Importantly, myh9b is a key player in autophagy initiation, which is also regulated by bag3 and mtor.
Overall, the use of MMEJ-inducing sgRNA allows rapid screening of potential modifier genes for inherited cardiomyopathies in adult fish, which can later be validated with stable double mutant lines. This study also highlights the parallels between inherited and chemically induced cardiomyopathies, and expands our knowledge of pathogenic mechanisms, such as autophagy, that could be therapeutically targeted. This innovative technology expands our repertoire of tools to interrogate cardiomyopathies and, potentially, other human diseases modelled in zebrafish.