Diabetic Aldehyde Dehydrogenase 2 Mutant (ALDH2*2) Mice Are More Susceptible to Cardiac Ischemic‐Reperfusion Injury Due to 4‐Hydroxy‐2‐Nonenal Induced Coronary Endothelial Cell Damage

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  Figure 1. ALDH2*2 mutant and wild type (WT) mice with type‐1 diabetes mellitus (DM) decreased cardiac function and increased cardiac fibrosis when compared with ALDH2*2 and WT control mice without DM. Fractional shortening (FS) % (A), ejection fraction (EF) % (B), heart rate (HR), (C), anterior wall thickness (AWT) (D), posterior wall thickness (PWT) (E), left ventricular end‐diastolic diameter (LVDd) (F), left ventricular end‐systolic diameter (LVDs) (G), microscopic images of cardiac fibrosis (H through K) and its quantification (L), ALDH2 activity (M), Western blots of 4‐hydroxy‐2‐nonenal (4HNE) adducts (N) and its quantification data (O) were shown from ALDH2*2 mutant diabetic mice and nondiabetic control ALDH2*2 mutant mice (Ctrl.) and respective WT mice. Data are presented as mean±SEM. *P<0.05, **P<0.01, and ***P<0.001. N=6. ALDH2*2 indicates aldehyde dehydrogenase 2 mutant allele; Ctrl., control.

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  Figure 2. Decrease in coronary endothelial cell (CEC)s, endothelial nitric oxide (eNOS) and eNOS phosphorylation in type‐1 diabetic hearts when compared between ALDH2*2 mutant and WT control mice. Representative micrographs of CD31+ immunostaining of CECs of cardiac sections (A through D) and the quantification of the number of CECs in cardiac sections (E) were shown from WT and ALDH2*2 control (Ctrl.) and diabetic mellitus (DM) mice. Quantification data of myocardial coronary perfusion pressure (F) were shown from WT and ALDH2*2 Ctrl. and DM mice. Data are presented as mean±SEM. Representative micrographs of double staining of CD31+ and endothelial nitric‐oxide synthase (eNOS) immunofluorescence staining in CECs of cardiac sections were shown from WT and ALDH2*2 Ctrl. and DM (G through R). The quantification data of CD31+ cells (S) and eNOS + cells (T) were shown. Representative Western blot images of eNOS, phospho eNOS (p‐eNOS), and porin levels (U) and eNOS quantification data (V), phospho eNOS S1177 (W), and the ratio of phospho eNOS S1177/eNOS (X) were shown. Data are presented as mean±SEM. *P<0.05, **P<0.01, and ***P<0.001. N=6. ALDH2*2 indicates aldehyde dehydrogenase 2 allele; and…

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  Figure 3. High glucose decreases cell viability and angiogenesis (tube formation) and increases mitochondrial membrane potential and oxidative stress (mitosox) in cultured coronary endothelial cell (CEC) lines. Representative Hoffman modulation‐contrast microscopic images of CECs were shown (A through D). The data quantification of viable cells was shown (E). Representative micrographs of tube formation of cultured CECs (F through I) and quantification of tube formation (J). Representative fluorescent images of JC‐1 staining of CECs (K through N) and quantification of the ratio of JC‐1 Red and JC‐1 Green (O) were shown. Data quantification of mitosox staining of cultured CECs (P). Data are presented as mean± SEM. *P<0.05, **P<0.01, and ***P<0.001. N=6. DSF indicates disulfiram; HG, high glucose; and WT, wild type.

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  Figure 4. Type 1 DM increases myocardial ischemia‐reperfusion injury in ALDH2*2 mutant diabetic mice as compared with WT control mice. Representative macroscopic cardiac sections with 2,3,5‐triphenyltetrazolium chloride (TTC) staining (A) and its quantification data (B), cardiac functional parameters heart rate (C), Left ventricular pressure (LVP) (D), rate of rise in left ventricular pressure (+dP/dt) (E) and rate of decline in left ventricular pressure (–dP/dt) (F), and quantification data of perfusion pressure (G) were shown in WT and ALDH2*2 diabetic mice with sham operation (Sham) or ischemia‐reperfusion injury (IRI). Representative micrographs of double staining of CD31 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunofluorescence staining in CECs of cardiac sections (H through S) and the quantification data of staining of CD31 (T), TUNEL (U) and double positive of CD31 and TUNEL (V) were shown in WT and ALDH2*2 diabetic mice with sham operation (Sham) or ischemia reperfusion injury (IRI). Data are presented as mean±SEM. *P<0.05, **P<0.01, and ***P<0.001. N=6. ALDH2*2 indicates aldehyde dehydrogenase 2 mutant allele; DM, diabetes mellitus; and WT, wild…

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  Figure 5. Alda‐1, an ALDH2 activator protects against ischemia‐reperfusion injury in ALDH2*2 diabetic mice. Representative macroscopic cardiac sections with 2,3,5‐triphenyltetrazolium chloride (TTC) staining (A) and its quantification data (B), cardiac functional parameters heart rate (C), left ventricular pressure (LVP) (D), rate of rise in left ventricular pressure (+dP/dt) (E) and rate of decline in left ventricular pressure (–dP/dt) (F), and quantification data of perfusion pressure (G) were shown in ALDH2*2 diabetic mice with 1‐day Vehicle (Vehicle) or Alda‐1 (Alda‐1) pretreatment, and hereafter sham operation (Sham) or ischemia reperfusion injury (IRI). Representative micrographs of double staining of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and CD31 immunofluorescence staining and the merging of both staining in CECs of cardiac sections (H through P) and the quantification data of staining of CD31 (Q) and TUNEL (R) in ALDH2*2 diabetic mice with Sham, or pretreated with Vehicle or Alda‐1 before IRI. Data are presented as mean±SEM. *P<0.05, **P<0.01, and ***P<0.001. N=6. ALDH2 indicates aldehyde dehydrogenase 2; ALDH2*2, ALDH2 mutant allele; CEC, coronary…

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  Figure 6. Pretreatment of mice with systemically delivered Alda‐1 protects coronary endothelial cell (CEC)s from 4‐hydroxy‐2‐nonenal (4HNE) induced damage in control ALDH2*2 mice. The quantification data of left ventricular pressure (LVP) (A), rate of rise in left ventricular pressure (+dP/dt) (B) rate of decline in left ventricular pressure (–dP/dt) (C) and perfusion pressure (D) were shown from 4HNE‐perfused hearts of nondiabetic ALDH2*2 mice with vehicle (Vehicle) or Alda‐1 pretreatment (Alda‐1). Representative micrographs of hematoxylin‐eosin (H‐E) stained cardiac sections were shown from 4HNE‐perfused hearts of nondiabetic ALDH2*2 mice with control (Ctrl or no 4HNE perfusion) (E), 4HNE perfusion (4HNE) (F) and Alda‐1 pretreatment before 4HNE perfusion (Alda‐1 & 4HNE) (G). The magnifications of the inset boxes were shown in (H through J) micrographs were in the interstitial region of the midventricle section. The cartoons of open and closed arterioles were shown. The quantification of data of open (K) and closed (L) coronary arterioles were shown from the interstitial region of the midventricle section. Representative micrographs of double staining of CD31+ and endothelial nitric‐oxide…