Noncompaction of the ventricular myocardium (NVM), also known as spongy, fetal, or honeycomb myocardium, is a heterogeneous myocardial disorder characterized by prominent trabeculae and intratrabecular recesses caused by the arrest of normal embryogenesis of the endocardium and myocardium (Towbin et al., 2015; Finsterer et al., 2017). Clinical manifestations are highly variable, ranging from no symptoms to disabling congestive heart failure or lethal arrhythmias, sudden cardiac death, or thromboembolic events (Weiford et al., 2004; Towbin et al., 2015). NVM is reportedly the third most commonly diagnosed cardiomyopathy (Towbin et al., 2015), occurring in infants (0.81/100,000 per year), children (0.12/100,000 per year), and adults (14/100,000 per year) (Oechslin et al., 2000; Risebro and Riley, 2006). The mortality rate of patients with NVM ranges from 5% to 47% (Finsterer et al., 2017). Both patients and their families can be severely burdened by the disease.

Genetic defects play important roles in the development and occurrence of NVM. Both familial and sporadic forms of NVM have been described previously. The inheritance of NVM is most often X-linked recessive or autosomal dominant, although autosomal recessive and mitochondrial inheritances also occur (Ichida et al., 1999; Scaglia et al., 2004; Towbin, 2010). Mutations are significantly more frequent in children than in adults, and most are associated with sarcomeric genes (van Waning et al., 2018). Mutations in >40 genes, such as MYH7, TTN, MYBP3, TPM1, and NKX2.5, as well as chromosomal abnormalities, such as 22q11.2 and 8p23.1 deletion, have been identified (Blinder et al., 2011; Ouyang et al., 2011; Postma et al., 2011; Digilio et al., 2013; van Waning et al., 2018). However, owing to high heterogeneity, the genetic etiology of 40–60% of NVM cases remains unknown (Finsterer et al., 2017).

The zebrafish, Danio rerio, is an excellent model organism to study human cardiovascular diseases because of its almost entirely sequenced genome and high conservation of gene function compared with humans; zebrafish embryos develop externally and transparently to allow direct visual observation of heart function (Shin and Fishman, 2002; Bakkers, 2011). Gene knockdown by the injection of morpholino-modified antisense oligonucleotides or knockout mediated by CRISPR/Cas9 in zebrafish embryos has been confirmed as an elegant and reliable approach for rapidly evaluating the impact of candidate genes on cardiovascular function (Hassel et al., 2009).

Here, we identified the RCAN family member 3 (RCAN3) R223L homozygous variant in two NVM cases using whole-exome sequencing (WES). Furthermore, we demonstrated that rcan3 deficiency induced marked cardiac dysfunction combined with developmental dysplasia of both the endocardial and myocardial layers in a zebrafish model in vivo. Meanwhile, RCAN3 deficiency induced by lentiviral vectors and mediated by short hairpin RNA resulted in reduced proliferation of the human cardiomyocyte cell line AC16, together with an abnormal mitochondrial ultrastructure in vitro. These data suggest that RCAN3 plays an essential role in myocardial development and function and may serve as a susceptibility gene for NVM.