Binding of lncDACH1 to dystrophin and the effects of cardiomyocyte-specific transgenic overexpression of lncDACH1 (lncDACH1-TG) on the expression and function of sodium channel.

(A) Blotting of dystrophin pulled-down by lncDACH1. (B) LncDACH1 precipitated by anti-dystrophin antibody. N=4. * P<0.05 vs IgG by one-way ANOVA, followed by Tukey’s post-hoc analysis. (C, D) The total, membrane and cytoplasm levels of dystrophin and Nav1.5 by Western blot. N-cadherin is the loading control for membrane extracts. N=10-11 for total protein; N= 7-14 for membrane protein; N= 8-14 for cytoplasm protein. *P<0.05 versus WT group. *P<0.05 versus WT group. P-values were determined by unpaired t test. (E) Distribution of lncDACH1, dystrophin and Nav1.5 in isolated cardiomyocytes. (F) The mRNA levels of dystrophin and SCN5A. N=8-11. (G) Peak INa currents, I-V curve and kinetics of INa. N=9-15 cells from 3 or more mice. (H) Conduction velocity of perfused hearts by optical mapping recordings. N=7. * P<0.05 versus WT group. P-values were determined by unpaired t test.

Effects of lncDACH1 overexpression on sodium channel expression and function in cultured neonatal cardiomyocytes.

(A) Verification of the expression of lncDACH1 after transfection of adenovirus carrying lncDACH1. N=7-8 from 3 independent cultures. *P<0.05 versus NC (negative control, empty plasmid). P-values were determined by unpaired t test. (B, C) Peak INa currents, I-V curves and kinetics of INa. N=12-16 cells from 3 independent cultures. (D) Distribution of Nav1.5 and dystrophin by immunofluorescent staining. (E) The mRNA levels of dystrophin and SCN5A. N=8-10 from 3 independent cultures.

Conditional knockout of lncDACH1(lncDACH1-cKO) in cardiomyocytes increased peak sodium current, membrane Nav1.5 expression.

(A) The total, membrane and cytoplasm levels of dystrophin by Western blot and dystrophin mRNA by qRT-PCR. N-cadherin is the loading control for membrane extracts. N=10 for total protein; N= 10 for membrane protein; N= 15 for cytoplasm protein. *P<0.05 versus WT group. P-values were determined by unpaired t test. (B) The total, membrane and cytoplasm levels of Nav1.5 by Western blot and SCN5A mRNA by qRT-PCR. N-cadherin is the loading control for membrane extracts. N=10 for total protein; N= 8 for membrane protein; N= 8 for cytoplasm protein. *P<0.05 versus WT group. P-values were determined by unpaired t test. (C) Distribution of lncDACH1, dystrophin and Nav1.5 in isolated cardiomyocytes. (D) Peak INa currents, I-V curve and kinetics of INa. N=10-15 cells from 3 or more mice. (E) Conduction velocity of perfused hearts by optical mapping recordings. N=7 and 5. * P<0.05 versus WT group. P-values were determined by unpaired t test.

Effects of lncDACH1 knockdown on sodium channel expression and function in cultured neonatal cardiomyocytes.

(A) Verification of the expression of lncDACH1 after infection of adenovirus carrying lncDACH1 siRNAs. N=15 from 3 independent cultures. *P<0.05 versus NC (negative control, empty plasmids). P-values were determined by unpaired t test. (B, C) Peak INa currents, I-V curve and kinetics of INa. N=9-15 from 3 independent cultures. (D) Distribution of Nav1.5 and dystrophin by immunofluorescent staining. (E) The mRNA levels of dystrophin and SCN5A. N=11-15 from 3 independent cultures.

Increased arrhythmia susceptibility in lncDACH1-TG mice.

(A) Ventricular fibrillation (VF) induced by S1S2 pacing in intact mice. N=10-16.( B) Ventricular fibrillation (VF) induced by S1S1 pacing of perfused hearts.(C) Break points during VT of WT and lncDACH1-TG mice by optical mapping. Consecutive phase maps sampled at 10-ms interval during VF from WT and TG mice. Phase singularities (wavebreaks) are indicated by phase maps. Upper panels showed corresponding optical recording of VF at asterisk site. n=8. (D) Representative of dorminant frequency of VF from WT and TG mice. (E) The number of phase singularities and dorminant frequency of WT and TG mice. (F) Ventricular fibrillation (VF) induced by S1S1 pacing in perfused hearts. N=5-7 mice.

The conserved fragment of lncDACH1(cF-lncDACH1) inhibited sodium channel function in mice.

(A) Pulldown of dystrophin by fragments of lncDACH1 as indicated. (B) Verification of the expression of cF-lncDACH1 after injection of adeno-virus carrying cF-lncDACH1. N=5. *P<0.05 versus NC (negative control, Adeno-virus carrying empty plasmid). P-values were determined by unpaired t test. (C) The total, membrane and cytoplasm levels of dystrophin and Nav1.5 by Western blot. N-cadherin is the loading control for membrane extracts. N=11-12 for total protein; N= 10 for membrane protein; N= 5-11 for cytoplasm protein. *P<0.05 versus NC group. P-values were determined by unpaired t test. (D) Distribution of dystrophin and Nav1.5 in isolated cardiomyocytes. (E) Representative traces and I-V curve of peak INa currents. N=12-15 from 3 or more mice. (F) Conduction velocity of perfused hearts by optical mapping recordings. N=9-10. * P<0.05 versus NC group. P-values were determined by unpaired t test. (G) Break points during VT by optical mapping. H. Ventricular (VF) induced by S1S2 pacing in intact mice.

Activation of dystrophin transcription by AAV9 virus carrying dCas9-SAM system (AAV9-Dys-Act) rescued the remodeling of sodium channel in lncDACH1-TG mice.

(A) The mRNA level of dystrophin by real-time PCR (N=12-17) and the total, membrane and cytoplasm protein levels of dystrophin by western blot. N-cadherin is the loading control for membrane extracts. N=7 for total protein; N= 8 for membrane protein; N= 8 for cytoplasm protein. *P<0.05 versus NC group. NC, AAV9 virus carrying dCas9-SAM system with control sgRNA; Dys, AAV9 virus carrying dCas9-SAM system with sgRNA targeting dystrophin promoter. (B) The mRNA level of Nav1.5 by real-time PCR (N=10-12) and the total, membrane and cytoplasm protein levels of Nav1.5 by western blot. N-cadherin is the loading control for membrane extracts. N=10 for total protein; N= 5 for membrane protein; N= 6 for cytoplasm protein. *P<0.05 versus NC group. (C) Representative traces, I-V curves and kinetics of peak INa currents. N=11-20 cells from 3 or more mice. (D) Conduction velocity of perfused hearts by optical mapping recordings. N=6-9. * P<0.05 versus NC group. P-values were determined by unpaired t test. (E) Ventricular fibrillation (VF) induced by S1S1 pacing in perfused hearts. N= 7-10. (F) Ventricular fibrillation (VF) induced by S1S2 pacing in intact mice. N= 10-13. The data are analyzed by one-way ANOVA followed by Tukey’s post-hoc analysis.

Hadhb binds to lncDACH1 and promotes its decay.

(A) The expression level of lncDACH1 in the hearts of TAC mice. N=5-8. * P<0.05 by unpaired t test. (B) The mRNA level of DACH1 in the hearts of TAC mice. N=12-14. (C) Effects of siRNAs for anp32a, eif4a1 and hadhb on the expression of lncDACH1. N=5-12 from 3 independent cultures. * P<0.05 by unpaired t test. (D) The effects of hadhb siRNA on the decay of lncDACH1. N=6-15 from 3 independent cultures. (E) The effects of hadhb siRNA on the protein expression of Nav1.5 and dystrophin. (F) Blotting of hadhb pulled-down by lncDACH1, and precipitation of lncDACH1 by anti-hadhb antibody. N=4. * P<0.05 vs IgG by one-way ANOVA followed by Tukey’s post-hoc analysis. (G) LncDACH1 inhibits Nav1.5 in human iPS differentiated cardiomyocytes. I. Schematic summary of the signaling pathway of hadhb/lncDACH1/dystrophin/Nav1.5.

The conserved fragment of lncDACH1(cF-lncDACH1) inhibited sodium channel function in mice.

(A) The mRNA levels of SCN5A and dystrophin. N=4-5. (B) Kinetics of INa. N=6-17 cells from 3 or more mice.

Effects of cF-lncDACH1 overexpression on sodium channel expression and function in cultured neonatal cardiomyocytes.

(A) Verification of the expression of cF-lncDACH1 after transfection of adenovirus carrying cF-lncDACH1. N=8 from 3 independent cultures. *P<0.05 versus NC (negative control, empty plasmid). P-values were determined by unpaired t test. (B) Peak INa currents, I-V curves and kinetics of INa. N=8-15 cells from 3 independent cultures. (C) Distribution of Nav1.5 and dystrophin by immunofluorescent staining. (D) The mRNA levels of dystrophin and SCN5A. N=8-12 from 3 independent cultures.

Schematic model for the construction of AAV9 virus carrying the dCas9-SAM system (A) to activate the transcription of dystrophin and tail-vein injection to mice (B).

VP64, VP64 transactivator; dCas9, deactivated CRISPR associated protein 9 nuclease. TSS, transcriptional start point.