A Common East Asian aldehyde dehydrogenase 2*2 variant promotes ventricular arrhythmia with chronic light-to-moderate alcohol use in mice

Figures

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  Fig. 1. QT prolongation phenotype in ALDH2 Vt alcohol users. ALDH2 Vt alcohol users showed a significantly longer QT interval (corrected to RR interval as QTc) compared to alcohol non-users (Alc−) and ALDH2 Wt alcohol users (430.1 vs 422.4 vs 415.6 ms in Alc−, Alc+ ALDH2 Wt and Alc+ ALDH2 Vt, respectively) (a). Box plot represents 25th and 75th percent quartiles, the line represents the median, and the whiskers are 10% (lower) and 90% (upper). * denotes p < 0.05 for Alc+ compared with Alc− within each genotype group (either ALDH Wt or ALDH Vt). Backward stepwise multi-variate regression models exploring the associations between clinical covariates and QTc interval in the current study (b). Higher daily alcohol intake amounts were associated with steeper QTc increments in subjects carrying ALDH2 Vt (pink) versus ALDH2 Wt (blue) (adjusted pinteraction: 0.017) (c). Bars in c represent the standard deviation for QTc and the standard error for alcohol dose, respectively. The shade areas represent 95% confidence interval area. *p < 0.05, **p < 0.001 for Alc+ versus Alc− within each genotype category (either ALDH Wt or ALDH Vt); #p < 0.05 for Alc+, ALDH Vt versus Alc+, ALDH Wt.

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  Fig. 2. Schematic of the establishment and generation of the EtOH-treated ALDH2 deficiency (ALDH2*2 KI) model. Four-month-old homozygous ALDH2*2 KI mice (C57BL/6, male) after 7 weeks of 4% Alc diet treatment (a). Pathology-related findings in representative gross morphology and cross-sections of mice hearts from different groups showing preserved yet slightly higher myocardial mass and LV wall thickness in EtOH-treated ALDH2*2 KI mice (b). Histological analysis (H&E) with Masson’s trichrome (MT) staining of heart sections (c). Heart weight indexed to body weight of mice revealed a slight but significant increase in 4% EtOH-treated mice groups (left), particularly in ALDH2*2 KI mice, compared to those of their respective normal diet control groups (d). Markedly extensive fibrosis (black arrows) within the myocardium (light blue staining) was also observed in EtOH-treated ALDH2*2 KI mice (right) (d). Illustrations of body surface electrocardiogram (ECG) in the mouse model. 4% EtOH-treated ALDH2*2 KI mice showed electrocardiographic phenotypes of more prolonged QRS duration, QT interval (e), and spontaneous onset of frequent VPCs (black arrow) (f) compared to other groups during 5 min of…

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  Fig. 3. Expression of Cx43 in cardiomyocytes by immuno-confocal microscopy. Immuno-confocal microscopy for myocardial double staining for WGA and Cx43 showed a significantly reduced total Cx43 expression area (with and without total myocardial area correction) in EtOH-treated ALDH2*2 KI mice compared to that of the EtOH-treated Wt and normal diet ALDH2*2 KI mice groups, with a featured increase in cytoplasmic Cx43 staining (white arrows) (a, b). A markedly increased ratio of Cx43 redistribution on lateral cardiomyocyte borders was observed in both EtOH-treated mice groups when compared to that of the other two normal diet control groups (a, c), with EtOH-treated ALDH2*2 KI mice exhibiting more pronounced increase of Cx43 lateralization than EtOH-treated Wt mice. For each group, 13–15 images were analyzed (3 mice in each group) (×40, 800 pixels). Error bars represent standard error. *p < 0.05, **p < 0.01, ***p < 0.001 for any 4% EtOH vs normal diet control within each genotype group (ALDH2*2 KI or Wt); #p < 0.05, ##p < 0.01, ###p < 0.001 for 4% EtOH ALDH2*2 KI vs 4% EtOH Wt.

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  Fig. 4. Western blotting analysis of heart tissue. Densitometric analysis of heart tissue TGF‐β1/collagen-1, Cx43 (total form), and a variety of ion channel proteins (including Nav1.5, Kv1.4, Kv4.2, Kv4.3, Cav1.2, and Cav1.3) of four groups of mice (a–q) via western blotting. Compared to normal diet Wt mice, EtOH-treated ALDH2*2 KI mice showed a 40.4% reduction in total Cx43, a 74.3% reduction in Kv1.4 and a 67.2% reduction in Kv4.2 (d, f, g), respectively. Cytoplasmic and membranous fractions of ion channel expression levels (k–q) were separately examined to determine the subcellular distribution of their alterations in distinctly ALDH2 functioning mice fed normal or 4% EtOH diet. Error bars represent standard error. *p < 0.05, **p < 0.01, ***p < 0.001 for any 4% EtOH vs normal diet control within each genotype group (ALDH2*2 KI or Wt); #p < 0.05, ##p < 0.01, ###p < 0.001 for 4% EtOH ALDH2*2 KI vs 4% EtOH Wt.

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  Fig. 5. Findings on programmed electrical stimulation induction at different pacing cycle lengths. a Optical action potential at 300 ms PCL. b APD at 70% repolarization. c An example of electrical propagation as isochrone maps in the ventricle at 300 ms PCL. d CV at different PCLs; n = 6 in each group; error bars are SD; p values were calculated using GraphPad Prism 8.0 with multiple t tests. APD action potential duration, CV conduction velocity, PCL pacing cycle length. e Simultaneous ECG recordings and ventricular optical mapping were performed during VA induction from an example of malignant VA in EtOH-treated ALDH2*2 KI mice. EtOH-treated Wt mice showed only one episode of VA with a slight wave break and failed to exhibit rotations during VA (Supplementary Fig. 2, Supplementary Movie 2). Wt and ALDH2*2 KI mice fed normal diet revealed smooth electrical propagation following PES induction (Supplementary Movies 3 and 4). f Dominant-frequency (DF) points and snapshots of the spiral wave corresponding to optical signal in time domain at a given point during VA with g multiple VA beats and initiation sites shown (white arrows). h, i Quantification of induced VA episodes and duration. (n =…

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  Fig. 6. Determination of action potential duration (APD) and maximal upstroke velocity of action potential (Vmax) in the cardiomyocytes. Comparisons of superimposed recordings of APD at 50% and 90% repolarization (a, b), Vmax (c), resting membrane potential (RMP), and action potential amplitude (APA) d among different mouse groups. Vmax was 97.6 ± 8.0 vs. 140.4 ± 13.9 mV/s for EtOH-treated and normal diet ALDH2*2 KI mice, respectively; and 125.9 ± 6.3 vs. 146.9 ± 9.6 9 mV/s for EtOH-treated and normal diet Wt mice, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 for any 4% EtOH vs normal diet control within each genotype group (ALDH2*2 KI or Wt); #p < 0.05, ##p < 0.01, ###p < 0.001 for 4% EtOH ALDH2*2 KI vs 4% EtOH Wt. n = 8 independent cells in each group. Error bars represent standard error.

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  Fig. 7. Single cardiomyocyte patch clamp recording of multiple ion current components. INa and ICa were elicited via a 50 (a) and 400 ms (e) depolarizing step, respectively, with representative traces (b, f for INa and ICa) and current-voltage relationships showing INa and ICa density (c, g) among different mouse groups. Markedly diminished INa density of cardiomyocytes in both EtOH-treated mouse groups at −45 mV (with the maximal current) compared to their respective normal diet controls irrespective of genotypes and EtOH-treated Wt mice (d). At 0 mV, ICa densities of cardiomyocytes in both EtOH-treated mouse groups were significantly lower, especially in ALDH2*2 KI mice, when compared to that in their respective normal diet control groups (h). Details about voltage-dependent INa/ICa inactivation curves and recovery of INa/ICa from inactivation among different mouse groups are further detailed in Supplementary Figs. 3 and 4. The elicited total outward potassium current (IK) (i), representative traces and current-voltage relationships showing transient outward potassium current (Ito) density among different mice groups. The Ito was defined as the difference between the peak (Ipeak) and…

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  Fig. 8. Schematic diagram illustrating the effect of ALDH2 knock-in (KI) on cardiac electrophysiological properties with and without alcohol treatment. Changes of cardiac electrophysiological properties in conditions of (1) Wild-type (Wt) without EtOH use (left panel), (2) Wt with EtOH use (middle panel), and (3) ALDH2 variant (polymorphism) with EtOH use (right panel).