Fig 1: Molecular docking of ALDH6A1 with NAD+ and ultrastructural electron microscopic images of cardiomyocytes.a The binding conformation of ALDH6A1-NAD+ was simulated. The amino acids and hydrogen bonds in ALDH6A1 were changed by K113 propionylation, from 10 hydrogen bonds with 6 amino acids (S211, E212, R213, G261, S262 and M284) to 15 hydrogen bonds with 8 amino acids (F185, N186, S211, E212, R213, S262, R366 and E417). b Compared with the SR group, the ultrastructure of atrial cardiomyocytes in the AF group showed obvious mitochondrial swelling, vacuolization, and cristae damage. The yellow circles in the figure indicated mitochondrial swelling and vacuolization, and the red arrows indicated mitochondrial cristae damage (n = 3 per group).
Fig 2: Kpr expression of ALDH6A1-K113, ALDH6A1-K113Q, and ALDH6A1-K113R in HEK-293 cells and cell function verification.a Sequencing chromatograms of pCMV3-ALDH6A1-K113, pCMV3-ALDH6A1-K113Q and pCMV3-ALDH6A1-K113R point mutation sites. b Kpr expression levels of over-expressed pCMV3-Flag, ALDH6A1-K113, ALDH6A1-K113Q, and ALDH6A1-K113R in HEK-293 cells were verified by IP. IP was performed with anti-Flag antibody, and then detected with Pan-Kpr antibody (n = 3 per group). c Detection of NADH levels in HEK-293 cells transfected with pCMV3-Flag, ALDH6A1-K113, ALDH6A1-K113Q, and ALDH6A1-K113R. ALDH6A1 levels were detected by anti-Flag antibody for normalization (n = 5 per group). d Bright field and fluorescence images of HEK-293 cells transfected with pCMV3-Flag, ALDH6A1-K113, ALDH6A1-K113Q, and ALDH6A1-K113R and incubated with DCFH-DA. Scale bars, 100 μm. e Statistical analysis of relative ROS positive areas in HEK293 cells (n = 15 per group). All experimental data were expressed as Mean ± SD. ns no significance; ****P < 0.0001.
Fig 3: Up-regulated propionylated ALDH6A1 in right atrial appendage tissue of POAF.a Seven proteins with nine differential Kpr sites had significantly higher levels (fold change > 1.7, P < 0.05). b Mass spectrometry peak maps of the ALDH6A1 K264 and K113 propionylation. c Protein homology sequence analysis of the ALDH6A1 264 and 113 sites in different species. ALDH6A1 K264 and K113 were highlighted red. d Validation of ALDH6A1 propionylated expression in POSR and POAF right atrial appendage tissues by IP and WB (n = 6 per group). e ROC curve of Kpr-ALDH6A1 in the right atrial appendage tissues of patients with POSR and POAF (n = 6 per group). All experimental data were expressed as Mean ± SD. *P < 0.05.
Fig 4: Schematic mechanism of intraoperative Kpr protein in patients with POAF after CABG.After CABG, patients with POAF increased the Kpr level of ALDH6A1 in right atrial appendage tissue and enhanced its enzyme activity, resulting in increased intracellular production of NADH and accumulation of ROS. The increase of intracellular ROS may cause oxidative stress and atrial electrical remodeling, thus promoting arrhythmia, such as AF. POAF postoperative atrial fibrillation, CABG coronary artery bypass grafting, Kpr lysine propionylation, NADH nicotinamide adenine dinucleotide, ROS reactive oxygen species, Ikur ultra-rapid delayed rectifier K+ current, INa sodium current, ICa, L L-type calcium current. Some of the elements in this figure were created by Biorender.
Supplier Page from Sino Biological, Inc. for Human ALDH6A1 Gene ORF cDNA clone expression plasmid, N-Flag tag