An F0-based MMEJ screening identified erk1 and mek1 as therapeutic modifiers of ttntv DCM. (A) Quantification of the ejection fraction in adult F0 zebrafish at 3 months via high-frequency echocardiography. (n ≥8 per group). (B) Quantification of representative knock out scores in F0 fish at 3 months. (n ≥6 per group) (C) Quantification of ejection fraction in F1 zebrafish at 3 months via high-frequency echocardiography. (n ≥ 8 per group). (D) Evaluation of nppb gene transcripts expression by quantitative RT-PCR in hearts from 3 months adult zebrafish. n ≥4 per group. One-way ANOVA was used to compare multiple groups for each mutation. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01. WT, wild type, ns, non-significant.

ERK signaling is aberrantly activated in ttntv DCM. (A) Representative western blots of Erk1/2, pErk1/2 and γ-Tubulin from 3 months old zebrafish hearts. (B) and (C) Quantification of pErk and Erk in (A). One-way ANOVA was used to compare multiple groups for each mutation. Data are presented as mean ± SEM, three biological replicates, **p < 0.01. WT, wild type, ns, non-significant (D) Representative images of Erk and pErk in cryosectioned heart tissues of WT, dA/+ and erk1+/−; dA/+ zebrafish mutants co-immunostained using anti-α-actinin antibody with anti-Erk antibody or anti-pErk antibody. α-actinin, green. Erk and pErk, red. DAPI, blue. The open arrowheads and filled arrowheads indicate pErk and Erk, respectively. Erk indicates total Erk1/2; pErk, phosphorylated Erk1/2. Scale bars: 20 μm. (E) and (F) Quantification of pErk and Erk of (D). One-way ANOVA was used to compare multiple groups for each mutation. Data are presented as mean ± SEM, three biological replicates, **p < 0.01. WT, wild type, ns, non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

An F0-based genetic screen of DEGs in Erk signaling pathway identified ppp1r10 as another therapeutic modifier. (A) Transcriptome analysis of ventricles from ttnae201/+ and WT at 6 months identified 1269 DE genes. (B) IPA analysis identified candidate pathways that warrant further investigation. (C) Quantification of ejection fraction in F0 adult zebrafish at 3 months via high-frequency echocardiography. (n ≥ 6 per group). (D) Quantification of representative knock out scores in F0 fish at 3 months. (E) Quantification of ejection fraction in F1 zebrafish at 3 months via high-frequency echocardiography. (n ≥ 6 per group). (F) Evaluation of nppb gene transcripts expression by quantitative RT-PCR in hearts from 6 months adult zebrafish. n ≥ 3. One-way ANOVA was used to compare multiple groups for each mutation. Data are presented as mean ± SEM, **p < 0.01. WT, wild type, ns, non-significant.

ppp1r10 inhibition rescues aberrantly activated Erk signaling in dA/+. (A) Schematics of ppp1r105’UTR generated by MMEJ sgRNA. The underlined sequence indicates sgRNA sequence, red indicates deleted nucleotides. (B) Evaluation of ppp1r10 and ppp2caa gene transcripts expression by quantitative RT-PCR in hearts from heterozygous ppp1r10+/− and ppp2ca+/− adult zebrafish at 3 months old. n = 4. t-test was used to compare 2 groups. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01. (C) Representative western blots of protein Erk, pErk, Rsk, pRsk and Gapdh from 3 months old zebrafish hearts and quantification of Erk, pErk, Rsk, and pRsk. One-way ANOVA was used to compare multiple groups for each mutation. Data are presented as mean ± SEM, three biological replicates, **p < 0.01. WT, wild type, ns, non-significant. (D) Representative images and quantification of pErk in cryosectioned heart tissues of WT, dA/+, ppp2caa+/−; dA/+ and ppp1r10+/−; dA/+ zebrafish mutants co-immunostained using anti-pErk antibody and anti-α-actinin antibody. α-actinin, green. pErk, red. DAPI, blue. The open arrowheads indicate pErk. pErk, phosphorylated Erk1/2. Scale bars: 20 μm. One-way ANOVA was used to compare multiple groups for each mutation. Data are presented as mean ± SEM, three biological replicates, **p < 0.01. WT, wild type, ns, non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Deregulated nutrient response is a pathological event in dA/+ that can be rescued by erk1 inhibition. (A) Representative western blots of protein pS6 and Gapdh from 3 months old zebrafish hearts at 0/4/24 h post feeding and quantification of pS6. One-way ANOVA was used to compare multiple groups. Data are presented as mean ± SEM, three biological replicates, **p < 0.01, WT, wild type, ns, non-significant. (B) Representative images and quantification of pS6 in cryosectioned heart tissues of WT, dA/+, erk1+/−; dA/+ and erk1−/−; dA/+ zebrafish mutants at 0/4/24 h post feeding, co-immunostained using anti-pS6 antibody and anti-α-actinin antibody. α-actinin, green. pS6, red. DAPI, blue. The open arrowheads indicate pS6 signal. pS6 indicates phosphorylated S6 ribosomal protein. Scale bars: 20 μm. One-way ANOVA was used to compare multiple groups. Data are presented as mean ± SEM, three biological replicates, **p < 0.01, WT, wild type, ns, non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ppp1r10, but not ppp2caa inhibition, rescues the deregulated nutrient response in dA/+. Representative images and quantification of pS6 in cryosectioned heart tissues of WT, dA/+, ppp2caa+/−; dA/+ and ppp1r10+/−; dA/+ zebrafish mutants at 0/4 h post feeding, co-immunostained using anti-pS6 antibody and anti-α-actinin antibody. a-α−ctinin, green. pS6, red. DAPI, blue. The open arrowheads indicate pS6 signal. pS6 indicates phosphorylated S6 ribosomal protein. Scale bars: 20 μm. One-way ANOVA was used to compare multiple groups. Data are presented as mean ± SEM, three biological replicates, **p < 0.01. WT, wild type, ns, non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Constitutively activated pERK in ppp2caa−/− manifests deregulated nutrient response. (A) pERK is activated by nutrients. Shown are representative western blots of pErk1/2, Erk1/2 and Gapdh from 3 months old zebrafish hearts and quantification of pErk and Erk. t-test was used to compare 2 groups. Data are presented as mean ± SEM, three biological replicates, **p < 0.01, ns, non-significant. (B) and (C) Representative images of pS6 and pErk in cryosectioned heart tissues of WT, dA/+, mek−/−, erk−/−, ppp1r1+/− and ppp2caa−/− zebrafish mutants at 0/4 h post feeding, co-immunostained using anti-α-actinin antibody with anti-pErk antibody or pS6 antibody. α-actinin, green. pERK and pS6, red. DAPI, blue. The open arrowheads and filled arrowheads indicate pErk and pS6, respectively. pErk, phosphorylated Erk1/2; pS6, phosphorylated S6 ribosomal protein. Scale bars: 20 μm. (D) Quantification of pErk and pS6 in (B) and (C). Data are presented as mean ± SEM, and one-way ANOVA was used to compare multiple groups, three biological replicates, **p < 0.01, WT, wild type, ns, non-significant.