Cardiac-Specific Overexpression of a Dominant Negative (DN) p67phox Mutant Shows Reduction in Mouse Heart Size

The role of NADPH oxidase (NOX) was recently suggested in cardiac hypertrophy. Overexpression of a DN mutant of p67phox, a cytosolic subunit of NOX, DN-p67 with a V204A point mutation completely inhibited NOX enzymatic function of superoxide generation in vitro. We recently found that overexpression of DN-p67 in adult rat cardiomyocytes attenuated α1-adrenoceptor-induced hypertrophy in vitro, thus hypothesized that NOX plays a critical role in cardiac hypertrophy and remodeling. In order to investigate the role of cardiac NOX/p67phoxin vivo, we created a transgenic (Tg) mouse model with cardiac-specific overexpression of the above DN-p67 mutant along with a downstream independently-translated fluorescent tag gene EGFP led by an initiation sequence IRES. The DN-p67/IRES-EGFP sequence was then linked to a cardiac-specific α-MHC promoter generating a 9.1-Kb transgene containing α-MHC/DN-p67/IRES-EGFP, which was microinjected into the C57BL/6 mouse zygotes to generate Tg founder mice. Hearts from 1.5-, 3- and 6-month (M) old heterozygous Tg mice and their Non-Tg (NTg) littermates were harvested and compared. The heart weight /body weight (HW/BW) ratio in Tg group (n=23) was significantly (p<0.01) smaller (overall: 14%↓; 1.5-M: 10%↓; 3-M: 16%↓; 6-M: 12%↓) vs. NTg group (n=19). This result was confirmed by 2D echocardiography measuring the left ventricular myocardial area (LVMA) determined by subtracting endocardial area from epicardial area in short axis view at the end diastole. The LVMA (mm2) in 3-M and 6-M mice was significantly (p<0.05) smaller in Tg (0.44±0.02, n=21) vs. NTg (0.50±0.02, n=13) with equal BW. LV internal dimensions and systolic function were preserved without difference in Tg vs. NTg. The LV wall thickness also appeared to be smaller in LV cross-sections in Tg vs. NTg by hematoxylin/eosin staining, and no change in fibrosis was detected in both groups by trichrome staining (n=4). Interestingly, the expression of NOX catalytic subunit gp91phox in Tg hearts was significantly decreased (3-fold↓, n=5) vs. NTg by Western blot, whereas the cardiac expression of other NOX subunits p47phox and p22phox remained the same in both groups. In summary, we have generated a Tg mouse model with cardiac-specific overexpression of a DN p67phox, which shows significant reduction in myocardial mass/size and gp91phox expression with normal cardiac function and development. This study indicates that normal cardiac size and growth is dependent on the expression of a functional NOX/p67phox.