A critical role for PPARalpha-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: modulation by dietary fat content

Abstract

To explore the role of peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated derangements in myocardial metabolism in the pathogenesis of diabetic cardiomyopathy, insulinopenic mice with PPARalpha deficiency (PPARalpha(-/-)) or cardiac-restricted overexpression [myosin heavy chain (MHC)-PPAR] were characterized. Whereas PPARalpha(-/-) mice were protected from the development of diabetes-induced cardiac hypertrophy, the combination of diabetes and the MHC-PPAR genotype resulted in a more severe cardiomyopathic phenotype than either did alone. Cardiomyopathy in diabetic MHC-PPAR mice was accompanied by myocardial long-chain triglyceride accumulation. The cardiomyopathic phenotype was exacerbated in MHC-PPAR mice fed a diet enriched in triglyceride containing long-chain fatty acid, an effect that was reversed by discontinuing the high-fat diet and absent in mice given a medium-chain triglyceride-enriched diet. Reactive oxygen intermediates were identified as candidate mediators of cardiomyopathic effects in MHC-PPAR mice. These results link dysregulation of the PPARalpha gene regulatory pathway to cardiac dysfunction in the diabetic and provide a rationale for serum lipid-lowering strategies in the treatment of diabetic cardiomyopathy.

Figure 1

The diabetic cardiac phenotype is influenced by the activity of PPARα. (a) PPARα-null mice are resistant to development of diabetic ventricular hypertrophy. (Left) Bars represent mean (± SEM) BV/BW ratios (n ≥ 11) of PPARα^+/+ and PPARα^−/− mice 6 weeks after an injection of vehicle or STZ. (Right) Bars represent mean cellular capacitance (n ≥ 12) of isolated cardiomyocytes from PPARα^+/+ and PPARα^−/− mice 6 weeks after injection of vehicle or STZ. *, P < 0.05 vs. vehicle-injected PPARα^+/+ and all PPARα^−/− mice. (b) Ventricular hypertrophy and dysfunction are exacerbated in diabetic MHC-PPAR mice (Lower Left). Bars represent mean (± SEM) BV/BW ratios (n ≥ 7) of MHC-PPAR and NTG littermate mice after an injection of saline vehicle or STZ. (Lower Right) Bars represent mean percent LV fractional shortening (n ≥ 7 for each group) as assessed by echocardiographic analysis 6 weeks after an injection of vehicle or STZ. *, P < 0.05 vs. vehicle-injected NTG mice. **, P < 0.05 vs. NTG mice and vehicle-injected MHC-PPAR mice. (Upper) Representative two-dimensional guided M-mode images of the LV obtained from the parasternal view at the mid-ventricular level of control and diabetic NTG and MHC-PPAR mice.

Figure 2

Myocardial TAG accumulation in diabetic MHC-PPAR mice. (Upper) Photomicrographs depicting the histologic appearance of ventricular tissue from NTG and MHC-PPAR mice (404-3 line) after an injection of vehicle or STZ are shown. Red droplets indicate neutral lipid staining. (Lower) Representative spectra from ESIMS analyses for linoleic acid-containing TAGs. m/z ratios of 838, 862, and 888 denote TAGs containing acyl groups with chain lengths of 16:0/16:0/18:2, 16:0/18:2/18:2, and 18:1/18:2/18:2, respectively.

Figure 3

The cardiomyopathy of MHC-PPAR mice is worsened by consumption of a diet rich in LCFA. (a) Bars represent mean LV FS (Left) and LV internal diameter at diastole (LVIDd, Right) of NTG and MHC-PPAR (404-4 line) mice on HF chow rich in LCFA or calorie-matched control chow. (b) TAG and ceramide levels in hearts of NTG and MHC-PPAR mice after HF diet treatment, as determined by ESIMS. Bars represent mean levels of long-chain TAG (Left) and ceramide (Right). *, P < 0.05 vs. NTG mice. **, P < 0.05 vs. NTG mice and control chow-fed MHC-PPAR mice. (c) HF diet-induced ventricular dysfunction in MHC-PPAR mice is reversible. The graph displays mean FS of MHC-PPAR mice plotted as a function of time. Black squares represent transgenic mice fed the HF diet. Subgroups of mice returned to a standard rodent chow for 15 days at the 30th and 60th days of the trial are denoted by the white circles. *, P < 0.05 vs. corresponding HF-fed MHC-PPAR mice. (Upper) M-mode echocardiographic images of the LV of a representative MHC-PPAR mouse at baseline (Day 0), after 30 days on the HF diet (Day 30) and 15 days after being returned to the standard chow (Day 45).

Figure 4

Diet-induced cardiac lipotoxicity depends upon fatty acid chain length but does not require increased flux of fatty acids through mitochondrial pathways. (a) MCT diet rescues cardiac dysfunction in MHC-PPAR mice. Bars represent mean echocardiographic-determined LV fractional shortening of NTG and MHC-PPAR mice (404-3 line; n ≥ 5 for each group) 8 weeks after initiation of control, LCT, or MCT diet administration. *, P < 0.05 vs. NTG mice. **, P < 0.05 vs. NTG mice and MHC-PPAR mice on control or MCT chows. NS = nonsignificant. (b) LCT but not MCT HF diet increases myocardial TAG levels. Bars represent mean levels of TAG-associated fatty acids with chain length of 16:0, 18:1, and 18:2 in MHC-PPAR mouse ventricles after 8 weeks of control, LCT, or MCT diet administration. *, P < 0.05 vs. MHC-PPAR mice fed control or MCT chow. (c) Inhibition of mitochondrial fatty acid import exacerbates cardiac dysfunction in MHC-PPAR mice. Fractional shortening of MHC-PPAR mice (404-4 line; n ≥ 5 for each group) after 3 weeks of daily injections of etomoxir sodium (25 ng/kg/day) while receiving control or HF chow. *, P < 0.05 vs. MHC-PPAR mice on control chow. **, P < 0.05 vs. MHC-PPAR mice on control chow and saline-treated HF mice.

Figure 5

Activation of extra-mitochondrial lipid metabolic pathways correlates with the severity of the cardiomyopathic phenotype. (a) HF diet or STZ treatment activates the peroxisomal FAO pathway in a PPARα-dependent manner. Representative autoradiographs of Northern blot analyses performed with total RNA isolated from cardiac ventricle of NTG or MHC-PPAR mice after 4 weeks of HF or control (C) diet (Top), NTG or MHC-PPAR mice 6 weeks after STZ administration (Middle), PPARα^+/+ and PPARα^−/− mice 5 days after vehicle or STZ injection (Bottom) using cDNA probes denoted at left. (b) Bars represent mean hydrogen peroxide (H₂O₂) levels in cardiac extracts from NTG or MHC-PPAR mice after 4 weeks of HF diet. *, P < 0.05 vs. NTG mice. **, P < 0.05 vs. NTG mice and MHC-PPAR mice fed control chow. (c) PPARα is required for the generation of H₂O₂ in the diabetic heart. Bars represent mean H₂O₂ levels in cardiac extracts isolated from PPARα^+/+ and PPARα^−/− mice 5 days after an injection of vehicle or STZ. *, P < 0.05 vs. vehicle-injected wild-type and all PPARα-null mice. (d) Bars represent mean GSH/GSSG ratios (n ≥ 4) in cardiac extracts from MHC-PPAR mice after 4 weeks of control or HF diet. *, P < 0.05 vs. NTG mice given HF chow.