Journal: Biology Open

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

doi: 10.1242/bio.061895

Figure Lengend Snippet: 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 significantly decreased in KO mice compared to WT ( n =6). (G-I) Serum levels of interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-α (TNF-α) were significantly elevated in KO mice compared to WT ( n =6). All samples were collected from 24-week-old mice. Data are presented as mean±s.d. All statistical analyses were performed using two-tailed unpaired Student's t -tests. *** P <0.001, **** P <0.0001.

Article Snippet: DAPI staining reagent (G1012; 1 μg/ml), TUNEL detection kit (G1501), 4% paraformaldehyde fixative (G1101-500ML), 20× citric acid antigen retrieval solution (pH 6.0; G1202-250ML), and anti-fade mounting medium (G1401-5ML) were from Servicebio (Wuhan, China).

Techniques: Expressing, Staining, TUNEL Assay, Control, Two Tailed Test

Journal: Biology Open

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

doi: 10.1242/bio.061895

Figure Lengend Snippet: 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 pooling atrial tissues from two mice. The extracted ER fractions were characterized by minimal cytoplasmic or mitochondrial contamination. Full-length blots are provided in Fig. S1C . (D) Densitometric quantification of TMCO1, normalized to calnexin, confirmed a significant reduction in KO mice ( n =3; **** P <0.0001, two-tailed unpaired Student's t -test). (E) ER calcium fluorescence intensity, measured in relative fluorescence units (RFU), was significantly higher in KO mice compared to WT, suggesting ER calcium overload ( n =6; **** P <0.0001, Student's t -test). Data are presented as mean±s.d.

Article Snippet: DAPI staining reagent (G1012; 1 μg/ml), TUNEL detection kit (G1501), 4% paraformaldehyde fixative (G1101-500ML), 20× citric acid antigen retrieval solution (pH 6.0; G1202-250ML), and anti-fade mounting medium (G1401-5ML) were from Servicebio (Wuhan, China).

Techniques: Knock-Out, Co-Immunoprecipitation Assay, Western Blot, Control, Expressing, Staining, Marker, Two Tailed Test, Fluorescence