Cholesterol lowering attenuates pressure overload-induced heart failure in mice with mild hypercholesterolemia

Figure 1. AAV8-LDLr gene transfer normalizes lipoprotein cholesterol levels in C57BL/6 LDLr-/- mice. Bar graphs illustrating total cholesterol (A), non-HDL cholesterol (B), VLDL cholesterol (C), IDL cholesterol (D), LDL cholesterol (E), and HDL cholesterol (F) plasma levels in C57BL/6 LDLr-/- mice at day 10 after AAV8-null gene transfer (controls) or AAV8-LDLr gene transfer. Lipoproteins were isolated by ultracentrifugation. Data are expressed as means ± SEM (n=7).

Figure 2. Cholesterol lowering gene therapy improves survival, attenuates cardiac hypertrophy, and inhibits lung congestion after TAC. Kaplan-Meier survival curves during an 8 weeks follow-up period comparing survival in control TAC mice (black) and AAV8-LDLr TAC mice (red) (A). Bar graphs illustrating body weight (B), tibia length (C), heart weight (D) in control sham (n=10), AAV8-LDLr sham (n=10), control TAC (n=9), and AAV8-LDLr TAC (n=10) mice 8 weeks after operation. Left ventricular wall area (E), anterior wall thickness (F) cardiomyocyte-cross-sectional area (G), and capillary density (H) quantified by morphometric and histological analysis in control sham (n=14), AAV8-LDLr sham (n=11), control TAC (n=25), and AAV8-LDLr TAC (n=11) mice 8 weeks after operation. Bar graph illustrating wet lung weight (I) in control sham (n=10), AAV8-LDLr sham (n=10), control TAC (n=9), and AAV8-LDLr TAC (n=10) mice 8 weeks after operation. Data are expressed as means ± SEM. Insets show a 4x magnification of the boxed region.

Figure 3. Representative Sirius Red-stained cross-sections of sham hearts and TAC hearts at day 56 after operation. Scale bar represents 1 mm.

Figure 4. Immunohistochemical analysis of the myocardium of sham mice and TAC mice at day 56 after operation. Representative photomicrographs show laminin-stained cardiomyocytes and CD31-positive capillaries. Scale bar represents 50 μm.

Figure 5. Quantification of pro-hypertrophic myocardial proteins by western blot. Bar graphs illustrating Akt (A), p-Akt (B), mTOR (C), p-mTOR (D), p38 MAPK (E), and p-p38 MAPK (F) protein levels quantified by western blot in the myocardium of control sham (n=8), AAV8-LDLr sham (n=8), control TAC (n=8), and AAV8-LDLr TAC (n=8) mice 8 weeks after operation. All protein levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level. The GAPDH/ß-tubulin ratio is shown in panel g. Data are expressed as means ± SEM (n=8). Representative images of western blots are shown in panel h.

Figure 6. AAV8-LDLr gene transfer significantly reduces interstitial fibrosis, perivascular fibrosis, and apoptosis after TAC. Bar graphs illustrating the number of cleaved caspase-3 positive cells (A), the degree of interstitial fibrosis (B), and the degree of perivascular fibrosis (C) in control sham (n=14), AAV8-LDLr sham (n=11), control TAC (n=25), and AAV8-LDLr TAC (n=11) 8 weeks after operation. Representative photomicrographs showing Sirius Red-stained interstitial collagen viewed under polarized light (D). Scale bar represents 50 μm. Bar graphs illustrating the 25 kD isoform of TGF-ß1 (E), Smad2/3 (F), and Smad4 (G) myocardial protein levels quantified by western blot in the myocardium of control sham (n=8), AAV8-LDLr sham (n=8), control TAC (n=8), and AAV8-LDLr TAC (n=8) mice 8 weeks after operation. All protein levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level. Data are expressed as means ± SEM. Representative images of western blots are shown in panel h. Insets show a 4x magnification of the boxed region.

Figure 7. Cholesterol lowering gene therapy in TAC mice reduces pro-oxidative enzymes, increases anti-oxidant defence systems, and decreases nitro-oxidative stress in the myocardium. Bar graphs illustrating plasma TBARS expressed as plasma malondialdehyde equivalents (A), plasma xanthine oxidase activity (B), myocardial protein level of NADPH oxidase 2 (C), of NADPH oxidase 4 (D), and of superoxide dismutase (E), and plasma superoxide dismutase activity (F) (n=8 for each condition). Percentage of 3-nitrotyrosine-positive area in the myocardium in control sham (n=14), AAV8-LDLr sham (n=11), control TAC (n=25), and AAV8-LDLr TAC (n=11) mice 8 weeks after operation (G). Data are expressed as means ± SEM. Representative images of western blots are shown in panel h. Representative photomicrographs showing myocardial sections stained for 3-nitrotyrosine (I). Scale bar represents 100 μm. Insets show a 4x magnification of the boxed region.

Figure 8. AAV8-LDLr gene therapy counteracts metabolic remodelling. Capillary glucose (A) and plasma insulin levels (B) 8 weeks after operation (n=10 for each group). Quantification of glucose uptake in the myocardium determined by micro-PET as shown by the maximal standardized uptake (SUV) value (C) and total myocardial uptake (% of injected dose) (D) 8 weeks after sham operation or after TAC (n=10-13 in each group). Representative micro-PET images illustrating the uptake of [18F]-FDG in the myocardium of sham mice and TAC mice at day 56 after operation are shown in panel (E).

Figure 9. Representative images of western blots of metabolic proteins.