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 the antibody of dystrophin. N=4. * P<0.05 vs IgG by one-way ANOVA, followed by Tukey’s posthoc 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 vs 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 mice. *P<0.05 vs WT group. h, Conduction velocity of perfused hearts by optical mapping recordings. N=7. *P<0.05 vs 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 vs 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. *P<0.05 vs NC. 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 vs 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 vs 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; N=3 mice of WT; N=4 mice of LncDACH1(cKO). *P<0.05 vs WT group. e, Conduction velocity of perfused hearts by optical mapping recordings. N=7 and 5. * P<0.05 vs 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 shRNA. N=15 from 3 independent cultures. *P<0.05 vs 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. *P<0.05 vs NC. 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. The red lines in the ECG traces indicate VF duration. N=10–16. SN: sinus rhythm 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, The number of phase singularities and dominant frequency of WT and TG mice. e, 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 vs 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 vs 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 cells; N=3 mice of NC; N=4 mice of cF-lncDACH1. *P<0.05 vs NC group. f, Conduction velocity of perfused hearts by optical mapping recordings. N=9–10. *P<0.05 vs 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. The induction rate, average episodes and duration of ventricular (VF) were determined from ECG recordings. The red lines in the ECG traces indicate VF duration. N=11 for NC; N= 18 for cF-lncDACH1. *P<0.05 vs NC group.

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 vs NC group. #P<0.05 vs TG 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 vs NC group. #P<0.05 vs TG group. c, Representative traces, I-V curves and kinetics of peak INa currents. N=11–20 cells; N=3 mice of WT+NC; N=3 mice of WT+Dys; N=3 mice of TG+NC; N=4 mice of TG+Dys. #P<0.05 vs NC group. #P<0.05 vs TG group. d, Conduction velocity of perfused hearts by optical mapping recordings. N=6–9. * P<0.05 vs NC group. #P<0.05 vs TG 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, 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. f, The effects of heart failure on the protein expression of Hadhb. N=9. * P<0.05 vs sham group. P-values were determined by unpaired t test. g, The effects of hadhb siRNA on the protein expression of Nav1.5, N=6. * P<0.05 vs NC group. P-values were determined by unpaired t test. h, LncDACH1 inhibits Nav1.5 in human iPS differentiated cardiomyocytes. i, Schematic summary of the signaling pathway of lncDACH1 and arrhythmia.

Examination of the potential interaction between lncDACH1 and Nav1.5.

a,Pulldown of Nav1.5 with sense and antisense sequences of lncDACH1. b, RNA immunoprecipitation of lncDACH1 with anti-Nav1.5 antibody. N=4.

Fluorescence intensity of lncDACH1, dystrophin and Nav1.5 in isolated cardiomyocytesof lncDACH1-TG mice.

a,b,Membrane levels of dystrophin (dys) and Nav1.5. N=9 for dys. N=8 for Nav1.5.*P<0.05 versus WT group.c,d,Cytoplasm levels of dystrophin and Nav1.5. N=9.*P<0.05 versus WT group.e,Fluorescence in situ hybridization (FISH) images of LncDACH1. N=10. *P<0.05 versus WT group. P-values were determined by unpaired t test.

Overexpression of lncDACH1(oe-lncDACH1) in cultured neonatal cardiomyocytes decreased membrane dystrophin and Nav1.5 expression.

a, The total, membrane and cytoplasm levels of dystrophin by Western blot. N-cadherin is the loading control for membrane extracts. N=10 for total protein; N= 6 for membrane protein; N= 7 for cytoplasm protein. *P<0.05 versus NC group. P-values were determined by unpaired t test. b,The total, membrane and cytoplasm levels of Nav1.5 by Western blot. N-cadherin is the loading control for membrane extracts. N=5 for total protein; N= 5 for membrane protein; N= 9 for cytoplasm protein. *P<0.05 versus WT group. P-values were determined by unpaired t test.

Fluorescence intensity of dystrophin and Nav1.5 in cultured neonatal cardiomyocyte overexpressing lncDACH1.

a,b,Membrane levels of dystrophin and Nav1.5. N=9.*P<0.05 versus NC group.c,d, Cytoplasm levels of dystrophin and Nav1.5. N=9 for dys.N=12 for Nav1.5. *P<0.05 versus NC group.P-values were determined by unpaired t test.

Fluorescence intensity of lncDACH1, dystrophin and Nav1.5 in isolated cardiomyocytes of lncDACH1-cKO mice.

a,b, Membrane levels of dystrophin (dys) and Nav1.5. N=12 for dys. N=8 for Nav1.5.*P<0.05 versus WT group.c,d, Distribution of cytoplasm levels of dystrophin and Nav1.5. N=12.*P<0.05 versus WT group.e,Fluorescence in situ hybridization (FISH) images of LncDACH1 expression. N=8. *P<0.05 versus WT group. P-values were determined by unpaired t test.

Effects of lncDACH1 knockdown with shRNA on the protein expression of dystrophin and Nav1.5 expression in cultured neonatal cardiomyocytes.

a, The total, membrane and cytoplasm levels of dystrophin by Western blot. N-cadherin is the loading control for membrane extracts. N=10 for total protein; N= 7 for membrane protein; N= 5 for cytoplasm protein. *P<0.05 versus NC group. P-values were determined by unpaired t test. b,The total, membrane and cytoplasm levels of Nav1.5 by Western blot. N-cadherin is the loading control for membrane extracts. N=15 for total protein; N= 6 for membrane protein; N= 11 for cytoplasm protein. *P<0.05 versus WT group. P-values were determined by unpaired t test. Dys, dystrophin; NC, negative control; sh-lncDACH1, shRNA for lncDACH1

Fluorescence intensity of dystrophin and Nav1.5 in cultured neonatal cardiomyocytes after knocking down of lncDACH1.

a,b, Distribution of membrane levels of dystrophin and Nav1.5. N=11 for dys.N=8 for Nav1.5.*P<0.05 versus NC group.c,d, Distribution of cytoplasm levels of dystrophin and Nav1.5. N=12 for dys. N=9 for Nav1.5.*P<0.05 versus NC group.P-values were determined by unpaired t test.

The mRNA expression of KLHL1, BORA, MZt1, DIS3 andDach1 in the cardiac tissue of lncDACH1 knockout a, and transgenic overexpression (b) mice.

N=7–12. *P<0.05 versus WT. Data are expressed as mean±SEM. Wt, wild type litter mates; lncDACH1(Tg), lncDACH1 cardiac transgenic overexpression; lncDACH1(CKO), lncDACH1 cardiomyocyte conditional knockout.

Fluorescence intensity of dystrophin and Nav1.5 in isolated cardiomyocytes overexpressingcF-lncDACH1.

a,b,Membrane levels of dystrophin (dys) and Nav1.5. N=9 for dys. N=7 for Nav1.5.*P<0.05 versus NC group.c,d, Cytoplasm levels of dystrophin and Nav1.5. N=6 for dys. N=7 for Nav1.5.*P<0.05 versus NC group.P-values were determined by unpaired t test.

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; N=3 mice of NC; N=4 mice of cF-lncDACH1.

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.*P<0.05 versus NC, P-values were determined by unpaired t test.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.

Fluorescence intensity of dystrophin and Nav1.5 in cultured neonatal cardiomyocytes overexpressing cF-lncDACH1.

a,b,Membrane levels of dystrophin and Nav1.5. N=10 for dys. N=11 for Nav1.5.*P<0.05 versus NC group.c,d,Cytoplasm levels of dystrophin and Nav1.5. N=7 for dys.N=6 for Nav1.5.*P<0.05 versus NC group.P-values were determined by unpaired t test.

Overexpression of cF-lncDACH1(oe-cF-lncDACH1) in cultured neonatal cardiomyocytes decreased membrane dystrophin and Nav1.5 expression.

a, The total, membrane and cytoplasm levels of dystrophin by Western blot. N-cadherin is the loading control for membrane extracts. N=5 for total protein; N= 8 for membrane protein; N= 6 for cytoplasm protein. *P<0.05 versus NC group. P-values were determined by unpaired t test. b,The total, membrane and cytoplasm levels of Nav1.5 by Western blot. N-cadherin is the loading control for membrane extracts. N=8 for total protein; N= 6 for membrane protein; N= 11 for cytoplasm protein. *P<0.05 versus WT group. P-values were determined by unpaired t test.

a,UCSC snapshot of lncDACH1

b,Conservation of lncdach1 between mice and other species

c,Alignment of murine lncDACH1 and human orthologue

d,Alignment of murine lncDACH1 and sheep orthologue

e,Alignment of murine lncDACH1 and Pig orthologue

f, Alignment of murine lncDACH1 and dog orthologue

g, Alignment of murine lncDACH1 and Rat orthologue

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 CRISPRassociated protein 9nuclease. TSS, transcriptional start point.

Fluorescence intensity of Nav1.5 in human iPS differentiated cardiomyocytes overexpressing cF-lncDACH1.

a,Membrane levels of Nav1.5. N=8 for Nav1.5.*P<0.05 versus NC group.b, Cytoplasm levels of Nav1.5. N=10 for Nav1.5.*P<0.05 versus NC group.P-values were determined by unpaired t test.

Effects of cF-lncDACH1 overexpression on sodium current and Vmax of APD inhiPS-CMs.

a,b, Peak INa currents and I-V curve of INa. N=11–12 from 3 independent cultures. *P<0.05 versus NC.c,d,Rising phase diagram of action potential andVmax of ascending branch of action potential phase 0, N=9. *P<0.05 versus NC.

Ubiquitination of Nav1.5 in the Primary cardiomyocytes of lncDACH1 transgenic overexpression and knockout.

a, the ubiquitination of Nav1.5 of overexpression of lncDACH1. b, the ubiquitination of Nav1.5 of Knockdown of lncDACH1.