Figure 1. Prioritization of CHCHD6 in HLHS proband and its Drosophila ortholog Chchd3/6.

(A) Pedigree of index family 11 H. The family includes consanguineous parents (denoted by double horizontal lines) without cardiac defects, one son with HLHS (proband), and two siblings without cardiac defects. (B) List of 9 candidate genes derived from proband 11 H with corresponding Drosophila orthologs. Orthology based on DIOPT score. Conserved Drosophila candidate HLHS genes were knocked down individually in the Drosophila heart using the Hand^4.2-Gal4 driver. The functional phenotypes listed were significantly different relative to Control^A or Control^B and were measured in 1-week-old female Drosophila hearts. (C) Schematic of Drosophila highlighting the abdominal region which includes the heart tube and flanking pericardial cells, where the Hand^4.2-Gal4 driver is expressed. Image adapted from Figure 1A of Xie et al., 2013. (D) End-Diastolic diameter (EDD), (E) End-systolic diameter (ESD), and (F) fractional shortening (FS) from 1-week-old female Hand^4.2-Gal4>Chchd3/6 flies.

Figure 1—figure supplement 1. Chchd3/6 KD in the Drosophila heart causes reduced fractional shortening due to systolic dysfunction.

(A) EDD, (B) ESD and (C) FS from 3 week old Hand^4.2>Chchd3/6 female flies. Very low number of Hand^4.2>Chchd3/6^RNAiB flies reaching 3 weeks-of-age indicates lethality possibly due to stronger KD of Chchd3/6. (D–F) 1-week-old Hand^4.2-Gal4, tdtK >Chchd3/6 female flies measured for (D) EDD, (E) ESD and (F) FS. (G) Confocal images of Chchd3/6 mRNA and tinman mRNA inside adult cardiomyocytes (top; nuclei are outlined) and processed images (bottom). (H–I) Chchd3/6 and tinman mRNA in 1-week-old female Drosophila hearts. (H) Chchd3/6 mRNA relative to tinman mRNA is reduced in Hand^4.2>Chchd3/6^RNAiA heart tissue. (I) tinman mRNA % area, used as marker to normalize expression to. Fractional shortening in control and Chchd3/6 KD female flies at 3 weeks of age with a (J) pericardial cell-specific driver (DOT-Gal4), (K) all muscle cell specific driver (Mef2-Gal4), and (L) neuronal driver (elav-Gal4). Note that Mef2 >Chchd3/6^RNAiA was lethal at pupal stages. (M) Fractional shortening measurements from Hand^4.2>Duox^RNAiC 3-week-old flies. Unpaired two-tailed t-test, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001; error bars represent SEM.