Fig. (5).
Myocardial fatty acid synthase, lipid accumulation and cell death. A. Immmunohistology detection of Fasn with anti-Fasn antibodies (Fasn) on frozen heart sections of three different heart failure models (upper panels), i.e. 10 month-old B6 mice with 6 months of AAC (B6 AAC-6mo), 6 month-old ApoE-/- mice with 2 months of AAC (ApoE-/- AAC), and 18 month-old ApoE-/- mice (ApoE-/--18 mo). Age-matched, 10 month-old sham-operated B6 mice (B6 sham), 6 month-old sham-operated ApoE-/- mice (ApoE-/- sham) and 18 month-old B6 mice (B6-18 mo) served as controls (lower panels; bar: 40 µm). B. Immunofluorescence co-localization of Fasn with α-sarcomeric actin on frozen heart sections of a 10 month-old B6 mouse with 6 months of AAC (B6 AAC-6mo). α-sarcomeric actin was detected with affinity-purified, mouse anti-actin antibodies followed by F(ab)2 fragments of Alexa Fluor 488-labeled (green) secondary antibodies (upper left/right panels). The actin-positive cardiomyocytes co-localized with Fasn, which was detected with affinity-purified, rabbit anti-Fasn antibodies followed by F(ab)2 fragments of Alexa Fluor 546-labeled (red) secondary antibodies (upper left/lower right panels). Cell nuclei were stained with DAPI (blue, left panels; bar: 20 µm). Immunohistology (A) and immunofluorescence (B) data are representative of at least 3 different mice/group. C. Cardiac triglyceride content (µmol/g wet weight) was determined with 5 month-old B6 mice after 1 month of AAC (AAC-1mo) and 10 month-old B6 mice after 6 months of AAC (AAC-6mo). Age-matched sham-operated B6 mice served as controls (Sham-1mo; Sham-6mo). Data represent mean ± S.D., n=4 mice/group (**, p<0.001; 6 months of AAC vs. 1 month of AAC). D. The number of TUNEL-positive cardiomyocytes (per 104 cells) was determined by an in situ cell death detection (TUNEL) assay on formalin-fixed, paraffin-embedded heart sections of 5 month-old B6 mice after 1 month of AAC (AAC-1mo) and 10 month-old B6 mice after 6 months of AAC (AAC-6mo). Agematched, sham-operated B6 mice served as controls (Sham-1mo; Sham-6mo). Data represent mean ± S. D., n=4 mice/group (**, p<0.001; 6 months of AAC vs. 1 month of AAC). E, F. Percentage (%) of TUNEL-positive cardiomyocytes was determined (E) with isolated neonatal cardiomyocytes incubated with 0.5 mM palmitic acid/BSA for 18 h (+Palmitate) relative to control cells (Cont.), and (F) with isolated cardiomyocytes over-expressing Fasn (+Fasn) relative to mock-transfected control cardiomyocytes (Cont.). Data represent mean ± S.D., n=3 different experiments performed in triplicates each (**, p<0.0001 and *, p<0.005). G. Over-expression of Fasn in transfected cardiomyocytes (+Fasn) was detected in immunoblot with Fasn-specific antibodies (IB: anti-Fasn) relative to mock-transfected cells (Cont.). The lower panel is a control immunoblot detecting actin (IB: anti-actin). H. Transfected cardiomyocytes expressing Fasn show increased lipogenic activity relative to mock-transfected controls. Lipogenic activity was determined by incorporation of 1,2-[14C]-acetate into lipids and is expressed as d.p.m. per well. Data represent mean ± S. D., n=3 different experiments performed in triplicates each (**, p<0.0005).