A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene

Abstract

Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b(L)) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.

Figure 1. Mutagenesis-based modifier screen identified 4 gene-breaking transposon (GBT) mutants that modified doxorubicin-induced (DOX-induced) adult zebrafish death.

(A) Kaplan-Meier survival curves of adult WT and GBT heterozygous zebrafish injected with a single bolus of 20 μg/gram body mass (gbm) DOX (n = 10–18). GBT0411 mutant was identified that exacerbated DOX-induced fish death. GBT0411 vs. WT, *P < 0.05, log-rank test. (B) The insertional positions of RP2 transposon element in the 4 candidate cardiomyopathy-modifying GBT mutants. (C) qPCR analysis of the percent native transcripts of the tagged genes remaining in the adult fish heart of designated GBT heterozygous and homozygous mutants, respectively. Values represent mean ±SEM.

Figure 2. The GBT0411 mutant was tagged to the dnajb6b(L) isoform that predominantly expresses in the heart.

(A) Northern blot analysis of dnajb6b(S) and dnajb6b(L) isoforms in indicated 3-month-old adult fish tissues demonstrated a predominant expression of dnajb6b(L) in the fish heart and skeletal muscle. (B) Western blot analysis (left) and quantification (right) of Dnajb6(S) and Dnajb6(L) isoforms expression in 1-year-old WT C57BL/6 mouse tissues indicated a predominant expression of Dnajb6(L) in the mouse heart. Values are presented relative to heart expression. Data represent mean ±SEM. (C) Western blot analysis (left) and quantification (right) of DNAJB6(L) and DNAJB6(S) protein expression in human heart and skeletal muscle lysates. Values are presented relative to heart expression. Data represent mean ±SEM. (D) Kaplan-Meier survival curves of indicated fish injected with a single bolus of 20 μg/gbm doxorubicin (DOX). Cardiomyocyte-specific overexpression of zebrafish dnajb6b(L) rescued DOX-induced fish death in the GBT0411 heterozygous mutant. *P < 0.05, log-rank test. Comparison of Tg(cmlc2:dnajb6b(L)-EGFP); GBT0411^+/– vs. GBT0411^+/–.

Figure 3. Dnajb6b(L) localizes to the nuclear envelope and functions as an ER stress inhibitor.

(A) EGFP reporter imaged from Tg(cmlc2:dnajb6b(S)-EGFP) fish heart (left) indicated sarcomeric localization of Dnajb6b(S). EGFP reporter from the Tg(cmlc2:dnajb6b(L)-EGFP) fish heart (right) indicated a primary nuclear localization and an occasional perinuclear localization (arrow) for the Dnajb6b(L). Scale bars: 10 μm. (B and C) Costaining of anti-Dnajb6 with the nuclear envelope protein anti-Lamin A/C antibodies in the HL-1 cell line (B) and in sections of WT C57BL/6 mice heart (C) merged with DAPI (blue), indicating Dnajb6 localization to the nuclear envelope. Scale bar: 10 μm. (D) Costaining of anti-Dnajb6 with the ER-resident protein glucose-regulated protein (Grp78) antibody indicated a partial colocalization (arrows) in the ER of the H9c2 cell line after tunicamycin (TN) treatment (1 μg/mL for 24 hours). Scale bar: 10 μm. (E and F) Western blot (upper) and quantification (lower) analysis of Grp78, an ER stress marker, in the GBT0411^–/– mutant, Tg(cmlc2:dnajb6b(S)-EGFP), Tg(cmlc2:dnajb6b(L)-EGFP), and WT sibling fish hearts at both basal level (E) and upon TN treatment (F). Values in E and F are expressed relative to WT controls. Data represent mean ±SEM; 1-way ANOVA comparisons, *P < 0.05.

Figure 4. Inhibition of ER stress protected against DOX-induced zebrafish death.

(A) Western blot (upper panel) and quantification (lower panel) analysis of glucose-regulated protein (Grp78) levels in adult WT fish heart at indicated time points after a single bolus of 20 μg/gram body mass (gbm) doxorubicin (DOX) injection. DOX injection results in elevation of Grp78 protein since 7 days after injection. Values are expressed relative to WT controls. Data represent mean ±SEM; *P < 0.05, 1-way ANOVA comparisons. (B) Western blot (upper) and quantification (lower) analysis of Grp78 protein levels at 2 months after DOX injection in indicated mutants and/or double-transgenic fish hearts. Grp78 protein level was further elevated in the GBT0411 mutant fish heart after DOX injection. Cardiomyocyte-specific overexpression of dnajb6b(L) inhibited the elevated ER stress in the GBT0411 mutant fish heart at 2 months after DOX injection. Values are expressed relative to WT controls. Data represent mean ±SEM; 1-way ANOVA comparisons, *P < 0.05. (C) Kaplan-Meier survival curves of indicated adult fish injected with a single bolus of 20 μg/gbm DOX with or without incubation with sodium phenylbutyrate (PBA), a chemical chaperone of ER stress inhibitor (250 μM, 10 hours/day, 5 days/week for the entire experiment). PBA administration protected against DOX-induced fish death in both WT and the GBT0411 mutant. *P < 0.05, log-rank test.

Figure 5. Overexpression of Dnajb6(L) in cardiomyocytes protected against DOX-induced cardiomyopathy in mouse.

(A) Schematics of adeno-associated virus serotype-9–Dnajb6(L) (AAV9-Dnajb6[L]) and doxorubicin (DOX) injection protocol in mice. (B) Western blot (upper) and quantification (lower) analysis of Dnajb6, glucose-regulated protein 78 (Grp78), and C/EBP homologous protein (CHOP) protein expression levels at 3 months after 5 × 10¹¹ genomic copy of AAV9-Dnajb6(L) virus preinjection with or without injection of 12 mg/kg body weight of DOX. Values are expressed as relative unit to the GAPDH input. Data represent mean ±SEM. *P < 0.05, 1-way ANOVA comparisons. (C) Kaplan-Meier survival curves of mice preinjected with AAV9-Dnajb6(L) or PBS control and then injected with DOX. Overexpression of Dnajb6(L) through AAV9-mediated gene delivery protected against DOX-induced mice death. Data represent mean ±SEM. *P < 0.05, log-rank test. (D) Percent ejection fraction of mice at 3 months after DOX injection with or without AAV9-Dnajb6(L) preinjection. AAV9-Dnajb6(L) virus preinjected mice had preserved cardiac function. Data represent mean ±SEM. *P < 0.05, 2-way ANOVA comparisons. (E) H&E staining (upper panel) and transmission electron microscopic analysis (lower panel) of mouse myocardium at 3 months after DOX injection with or without AAV9-Dnajb6(L) preinjection. Arrows indicate cytoplasmic vacuoles. The images are representative of n = 3 per treatment. Scale bars: 100 μm (upper) and 2 μm (lower).

Figure 6. Ectopic expression of the human DNAJB6(L)-S316W variant activated ER stress and accelerated DOX-induced zebrafish death.

(A) Illustration of DNAJB6 rare variants identified from patients with dilated cardiomyopathy (DCM) at Mayo Clinic (red line), patients with hypertrophic cardiomyopathy (HCM) at Mayo Clinic (blue line), and patients with DCM from China (green line). (B) Western blot (left) and quantification (right) analysis of protein levels of DNAJB6(L) WT and the 3 variants in zebrafish embryos injected with corresponding mRNA at one-cell stage. hpf, hours postfertilization. Values are expressed as relative percentage to 24 hpf. Data represent mean ±SEM. *P < 0.05. One-way ANOVA comparisons. (C) Western blot analysis of DNAJB6(S), DNAJB6(L) WT, and the 3 variants expressed in zebrafish embryos injected with corresponding mRNAs at 24 hpf. The DNAJB6(L)-S316W variant showed a slower migration rate compared with DNAJB6(L) WT protein. (D) Transient overexpression of DNAJB6(L)-S316W, but not other variants in zebrafish embryos, led to mild activation of Grp78 protein and transcriptional elevation of spliced X-box binding protein (xbp1S) at 48 hpf. (E) qPCR analysis of grp78 and chop, 2 ER stress molecular markers, in indicated mRNA-injected embryos at 48 hpf. Values are expressed as relative units to uninjected controls. Data represent mean ±SEM. *P < 0.05. vOne-way ANOVA comparisons. (F) Western blot (upper panel) and quantification (lower panel) analysis of DNAJB6(L) and Grp78 proteins in stable transgenic fish heart of Tg(ttna:DNAJB6(L)), Tg(ttna:DNAJB(L) p.S316W), and WT sibling controls at both basal level and at 6 hours after i.p. tunicamycin injection of 1 μg/g body weight. Grp78 protein was elevated in the Tg(ttna:DNAJB6(L) p.S316W) but not Tg(ttna:DNAJB6(L)) transgenic fish heart upon tunicamycin (TN) treatment. Values are expressed as relative units to WT siblings. Data represent mean ±SEM. *P < 0.05. One-way ANOVA comparisons. (G) Kaplan-Meier survival curves of Tg(ttna:DNAJB6(L) p.S316W) transgenic fish and WT siblings after a single bolus of 20 μg/gbm doxorubicin (DOX) injection. *P < 0.05, log-rank test.