Cardiac-specific overexpression of aldehyde dehydrogenase 2 exacerbates cardiac remodeling in response to pressure overload

Fig. 1. Pressure overload-induced heart failure is associated with decreases in cardiac ALDH2 activity. Cardiac function and ALDH2 expression and activity in wild-type mice subjected to sham surgery or transverse aortic constriction (TAC) for 12 weeks: Shown are (A) ejection fraction measured by echocardiography. Additional echocardiographic parameters can be found in Table 1; (B) Gravimetric measurements of heart mass in Sham and TAC mice; (C) Representative Western blot of ALDH2; (D) Densitometric measurements from panel C; (E) ALDH2 activity measurements. n = 5–8 per group; *p ≤ 0.05, ***p < 0.001, ****p ≤ 0.0001; in panel E, Welch's correction was applied because equal variances cannot be assumed; (F) Representative Western blot of protein-4-hydroxynonenal (HNE) adducts from sham and TAC hearts. Heart homogenates incubated with reagent HNE served as a positive control; and (G) Densitometric measurements from panel F. n = 4 per group, *p < 0.05.

Fig. 2. Generation of a cardiac-specific ALDH2 overexpressing mouse. (A) Schematic of the α-MHC-hALDH2 overexpression construct. α-MHC, α-myosin heavy chain; hGH poly A, human growth hormone poly-adenylation signal. (B) Western blot analysis of ALDH2 expression in tissues from NTg and ALDH2 Tg mice. Amido Black served as a loading control. (C) ALDH2 activity measurements. n = 6 per group, *** *p < 0.0001. (D) Localization of ALDH2 in the cytosolic and mitochondrial fractions of the heart. Cytosolic marker, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mitochondrial marker, COX4I1, cytochrome c oxidase subunit 4 isoform 1. (E) Gravimetric measurements of heart size in naïve NTg and ALDH2 Tg mice. n = 10–11 per group, *p < 0.05.

Fig. 3. ALDH2 overexpression increases pressure overload-induced cardiac hypertrophy. Measurements of cardiac structure and gene expression in NTg and ALDH2 Tg mice subjected to 12 weeks of TAC: (A) Gravimetric measurements of heart size; (B) Representative images of WGA and DAPI-stained mid-ventricular histological sections from NTg and ALDH2 Tg TAC hearts; (C) Cardiomyocyte area; and (D) Expression of Nppa and Nppb measured by qRT-PCR. n = 12–15 per group, *p < 0.05.

Fig. 4. ALDH2 overexpression decreases protein-HNE adducts. Western blots of indicators of aldehyde burden and antioxidant enzymes in NTg and ALDH2 Tg mice subjected to 12 weeks of TAC: (A) Representative Western blot of protein-4-hydroxynonenal (HNE) adducts. Heart homogenates incubated with reagent HNE served as a positive control; (B) Densitometric measurements from panel A. n = 13–14 per group; (C) Representative Western blots of catalase and HO-1; (D) Densitometric measurement of catalase from panel C; (E) Densitometric measurement of HO-1 from panel C. E. n = 9 per group, *p < 0.05.

Fig. 5. ALDH2 overexpression diminishes capillary density in the context of pressure overload. Measurements of gene expression and cardiac structure in NTg and ALDH2 Tg mice subjected to 12 weeks of TAC: (A) Gene expression of fibrotic and vascular markers measured by qRT-PCR. n = 12–15 per group, *p < 0.05; (B) Representative images of fast green/sirius red-stained NTg and ALDH2 Tg TAC hearts. Red staining indicates fibrotic regions. Green-blue staining depicts non-fibrotic regions; (C) Quantification of perivascular fibrosis from panel B; (D) Quantification of interstitial fibrosis from panel B; (E) Representative images of NTg and ALDH2 Tg TAC hearts stained with isolectin B4; (F) Quantification of capillary density. n = 4–6 per group, *p < 0.05.

Supp Fig. 1. ALDH2 Tg overexpression does not alter the expression of genes indicative of heart failure following pressure overload. Measurements of gene expression in NTg and ALDH2 Tg mice subjected to 12 weeks of TAC. n = 12–15 per group.