Generation and characterization of a DYNLT1-knockout mouse model reveals electrophysiological alterations and potential mechanistic contributors to atrial fibrillation

Figures

• 

  Fig. 1.. Generation of KO mice using CRISPR/Cas9 technology. (A) Schematic of the gene KO strategy, consisting of three main steps: (1) WT allele; (2) targeted allele; (3) KO allele. (B) The genotyping results for representative genes: WT mice (644 bp) and homozygous (HO) mice (472 bp) show different bands in PCR detection, while heterozygous (HE) mice exhibit signals at both band positions. (C) Representative gross morphology of KO and WT mice, showing no obvious differences. (D) Kaplan–Meier survival curves of WT (blue, n=40) and KO (red, n=48) mice. No statistically significant difference was observed (ns). Mice euthanized for experimental purposes were excluded from the analysis.

• 

  Fig. 2.. Incidence of AF and electrocardiographic characteristics in KO mice. (A) Representative ECG recordings. Top: normal ECG from WT mice; bottom: typical AF episode recorded in KO mice. Scale bar: 0.2 s. (B) AF incidence in KO mice increased with age, rising from 8.3% at 4 weeks to 83.3% at 28 weeks. (C) Representative ECG traces of other atrial arrhythmias in KO mice: atrioventricular block (top), atrial premature beats (middle), and atrial flutter (bottom). Scale bar: 0.2 s. (D-I) ECG analysis at 8, 16, and 24 weeks (n=6 per group, biological replicates). (D-F) No significant differences were observed in P-wave duration, QRS interval, or QTc between groups (two-tailed unpaired Student's t-test). (G) P-wave amplitude was significantly lower in KO mice compared to WT and progressively declined with age (WT 8 weeks versus KO 8 weeks, Mann–Whitney U-test, other comparisons were performed using Student's t-test). (H) PR interval (the time from the onset of the P wave to the start of the QRS complex on the ECG) was significantly prolonged in KO mice and further increased with aging (Student's t-test). (I) RR interval (the time between two successive R waves on the ECG) showed an…

• 

  Fig. 3.. Atrial structure and histopathological changes in KO mice. (A) Echocardiography showed that the left atrial transverse diameter (LATD) in KO mice was significantly larger than that in WT mice at 8, 16, and 24 weeks of age, with progressive enlargement over time (n=6 per group; biological replicates, two-tailed unpaired Student's t-test). (B) The right atrial transverse diameter (RATD) increased with age in KO mice and was significantly greater than in WT mice at 24 weeks (n=6, Student's t-test). (C) Representative Masson's trichrome staining images of the left atrium from both groups of mice at 8, 16, and 24 weeks (n=6). Scale bar for the whole heart section: 1 mm, magnified view of the left atrium: 100 μm. (D) Quantitative analysis of left atrial fibrosis showed that the fibrosis burden in KO mice was significantly higher than that in the WT group at 8, 16, and 24 weeks, with fibrosis severity increasing with age in KO mice (n=6, Student's t-test). (E) H&E staining at 24 weeks revealed non-myocyte infiltration, disorganized cardiomyocyte arrangement, and inflammatory cell aggregation in KO mice (n=6). (F) TEM images of the left atrium at 24 weeks showed myofibrillar…

• 

  Fig. 4.. Ventricular structure and function in KO mice. (A) Echocardiographic analysis showed no significant difference in ejection fraction between WT and KO mice at 8, 16, and 24 weeks of age (n=6 per group; biological replicates, two-tailed unpaired Student's t-test). (B) Left ventricular end-diastolic volume (LVEDV) gradually increased with age in KO mice and was significantly higher than in WT mice at 16 weeks (n=6, WT 24 weeks versus KO 24 weeks, and KO 8 weeks versus KO 24 weeks, Mann–Whitney U test, other comparisons were performed using Student's t-test). (C) Left ventricular end-systolic volume (LVESV) also increased with age in KO mice, with a significant difference compared to WT mice at 16 weeks (n=6, Student's t-test). (D) Representative ventricular histology at 24 weeks (n=6). Top: Masson's trichrome staining revealed no significant difference in ventricular fibrosis between groups. Scale bars: 500 µm. Middle: H&E staining showed no apparent pathological abnormalities in ventricular cardiomyocytes. Scale bars: 50 µm. Bottom: TEM images showed no obvious ultrastructural changes. Scale bars: 2 μm. (E) Quantification of left ventricular fibrosis at 24 weeks showed no…

• 

  Fig. 5.. Gross cardiac morphology in KO mice. (A) Representative gross heart images from WT and KO mice at 8, 16, and 24 weeks of age. Each grid square on the scale equals 1 mm. (B) There was no significant difference in heart weight normalized to body weight between the two groups of mice (n=6 per group, biological replicates, two-tailed unpaired Student's t-test). Data are presented as mean±s.d. ns, not significant.

• 

  Fig. 6.. Molecular characteristics of AF onset and progression in KO mice: enhanced apoptosis, reduced gap junction protein expression, and elevated inflammation. (A) Representative IF images of left atrial tissue from 24-week-old WT and KO mice. Left: staining for cardiac troponin I (cTnI; red) and DAPI (blue), a fluorescent nuclear dye (4′,6-diamidino-2-phenylindole). Right: TUNEL staining highlighting apoptotic nuclei (green), indicating DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling). Arrows indicate TUNEL-positive apoptotic nuclei. Scale bars: 50 µm. (B) Quantification based on cTnI staining showed a significant reduction in the number of cardiomyocytes in KO mice (n=6 per group; biological replicates). (C) TUNEL staining revealed a marked increase in apoptotic nuclei and a significantly higher proportion of apoptotic cardiomyocytes in KO mice (n=6). (D) WB analysis of atrial tissue showing protein expression levels of Connexin 40 (Cx40, 34 kDa) and Connexin 43 (Cx43, 43 kDa), with Tubulin-α as the loading control. Full-length blots are provided in Fig. S1A. (E,F) Densitometric analysis showed that expression levels of Connexin 40 and Connexin 43 were…

• 

  Fig. 7.. DYNLT1 knockout may trigger AF in KO mice through TMCO1-mediated ER calcium overload in atrial myocytes. (A) iPSC-aCMs were subjected to Co-IP using an anti-DYNLT1 antibody. Immunoblotting (IB) detected TMCO1 (21 kDa) and DYNLT1 (14 kDa) in the precipitates, but not in the IgG isotype control. The input from iPSC-aCMs confirmed normal expression of DYNLT1 and TMCO1, demonstrating their presence in the cells before the Co-IP procedure. Representative of three independent experiments. Full-length blots are provided in Fig. S1B. (B) Representative IF co-localization staining shows that in the left atrium cardiomyocytes of WT mice, TMCO1 (green) highly co-localizes with the ER marker calnexin (red) (yellow); however, in KO mice, the degree of co-localization with calnexin is reduced. DAPI (blue) was used to label the cell nuclei, n=6, biological replicates. Scale bars: 10 μm. The enlarged image of the merged figure shows that, compared to WT mice, the co-localization of TMCO1 with the ER is reduced in KO mice. Scale bars: 2 μm. (C) WB analysis of TMCO1 (21 kDa) in ER extracted from mouse atrial tissue calnexin (90 kDa) was used as an internal control. Each sample was prepared by…