Targeted Overexpression of Mitochondrial ALDH2 in Coronary Endothelial Cells Mitigates HFpEF in a Diabetic Mouse Model

Figure 1. Map of ALDH2 construct. Human ALDH2 was subcloned into the pCW3SL AAV vector. The ALHD2 HA P2a was PCR amplified from a previously generated construct, pCWB cTNT ALDH2 HA P2a eGFP using the following primers (SalI 5′-TCTAGACGCGTCGACCACCATGTTGCGCGCTGCCGCCCGCT, EcorI R 5′-AGGTTGATTATCTCGAGCGGAATTCCTAAGGACCGGGGTTTTCTTCCACGTCTC. The VE-cadherin promoter was amplified from mouse genomic DNA. Promoter and transgene were inserted into the BstAPI/EcoRI digested pCW3SL vector. Map shows single cutting restriction enzyme sites.

Figure 2. Overexpression of ALDH2 specifically in CVECs by the AAV-9 viral vector with a VEC-specific promoter by directly injecting into the db/db mouse hearts. Among the isolated myocardial cells, ALDH2 was overexpressed only in coronary ECs, not Mϕs, FBs, and CMs as shown in the immunoblots with their specific markers such as CD31, CD68, α-smooth muscle actin, and troponin T, respectively, and the loading control, GAPDH (A). Representative micrographs (B–G) from hearts of AAV-GFP– and AAV-ALDH2–treated mice demonstrate immunopositivity for CD31 (green fluorescence) and ALDH2 (red fluorescence). The micro graphical images of immunostaining show colocalization of endothelial marker CD31 with ALDH2 signal (D,G), supporting successful transgene expression in coronary endothelial cells.. Original western blots can be found at Supplementary Materials.

Figure 3. ALDH2 and 4HNE levels and ALDH2 activity in the myocardium with ALDH2 overexpression specifically in CVECs. Immunoblot images of ALDH2, 4HNE adducts, and β-actin (A). The quantification data of levels of ALDH2 (B) and 4HNE adducts (C), as well as ALDH2 activity (D), are shown. The data are the mean ± SEM. n > 3. * p < 0.05; ** p < 0.01; *** p < 0.001. Original western blots can be found at Supplementary Materials.

Figure 4. Changes in cardiac functional parameters. Echocardiographic evaluation of cardiac function in db/m, db/db, AL, and AF mice. The heart rate (HR) (A), percentage ejection fraction (%EF) (B), left ventricular end-systolic diameter (LVESD) (C), and left ventricular end-diastolic diameter (LVEDD) (D) showed no significant differences among groups. Left ventricular anterior wall thickness in diastole (LVAW;d) (E) and posterior wall thickness in diastole (LVPW;d) (F) were measured. The E/A ratio, defined as the ratio of early (E) to late (A) diastolic peak mitral inflow velocities, was evaluated (G). Isovolumic relaxation time (IVRT) (H) and the E/e′ ratio, calculated by dividing the peak early mitral inflow velocity (E) by the early diastolic mitral annular velocity (e′) (I) were evaluated. AAV9-GFP or AAV9-ALDH2 was administered to db/m and db/db mice as indicated. The data are the mean ± SEM. n = 6. * p < 0.05; ** p < 0.01; *** p < 0.001.