Rescue of cardiomyopathy in peroxisome proliferator-activated receptor-alpha transgenic mice by deletion of lipoprotein lipase identifies sources of cardiac lipids and peroxisome proliferator-activated receptor-alpha activators

Abstract

Background: Emerging evidence in obesity and diabetes mellitus demonstrates that excessive myocardial fatty acid uptake and oxidation contribute to cardiac dysfunction. Transgenic mice with cardiac-specific overexpression of the fatty acid-activated nuclear receptor peroxisome proliferator-activated receptor-alpha (myosin heavy chain [MHC]-PPARalpha mice) exhibit phenotypic features of the diabetic heart, which are rescued by deletion of CD36, a fatty acid transporter, despite persistent activation of PPARalpha gene targets involved in fatty acid oxidation.

Methods and results: To further define the source of fatty acid that leads to cardiomyopathy associated with lipid excess, we crossed MHC-PPARalpha mice with mice deficient for cardiac lipoprotein lipase (hsLpLko). MHC-PPARalpha/hsLpLko mice exhibit improved cardiac function and reduced myocardial triglyceride content compared with MHC-PPARalpha mice. Surprisingly, in contrast to MHC-PPARalpha/CD36ko mice, the activity of the cardiac PPARalpha gene regulatory pathway is normalized in MHC-PPARalpha/hsLpLko mice, suggesting that PPARalpha ligand activity exists in the lipoprotein particle. Indeed, LpL mediated hydrolysis of very-low-density lipoprotein activated PPARalpha in cardiac myocytes in culture. The rescue of cardiac function in both models was associated with improved mitochondrial ultrastructure and reactivation of transcriptional regulators of mitochondrial function.

Conclusions: MHC-PPARalpha mouse hearts acquire excess lipoprotein-derived lipids. LpL deficiency rescues myocyte triglyceride accumulation, mitochondrial gene regulatory derangements, and contractile function in MHC-PPARalpha mice. Finally, LpL serves as a source of activating ligand for PPARalpha in the cardiomyocyte.

Figure 1. LpL-deficiency rescues ventricular dysfunction in MHC-PPARα mice

A) Representative M-mode echocardiographic images of the LV from each genotype at baseline (one month) and after 4 weeks of HF diet. B) Bars represent mean (±SE) percent LV fractional shortening (FS), as determined by echocardiographic analyses. *p<0.05 vs WT and hsLpLko, †p<0.05 vs. MHC-PPARα mice on matched diet (n=6–8/group).

Figure 2. Reversal of myocardial lipid accumulation in MHC-PPARα/hsLpLko mice on HF diet

A) Representative photomicrograph depicting oil red O–stained ventricular tissue prepared from all genotypes following 4 weeks of HF diet. Red droplets indicate neutral lipid staining. B) Mean (±SE) myocardial TG levels for each genotype after HF diet (n=3–5/group). *p<0.05 vs WT and hsLpLko, †p<0.05 vs. MHC-PPARα.

Figure 3. LpL-deficiency normalizes fuel utilization of MHC-PPARα hearts

The oxidation rates of [U-¹⁴C]glucose (left) and [9,10-³H]palmitate (right) were assessed in IWHs (WT, n=8; hsLpLko, n=6; MHC-PPARα, n=9; MHC-PPARα/hsLpLko, n=6). Bars represent mean (±SE) oxidation rates expressed as nanomoles of substrate oxidized per minute per gram dry weight. *p<0.05 vs WT and hsLpLko, †p<0.05 vs. MHC-PPARα.

Figure 4. Mitochondrial gene expression and ultrastructure are improved in MHC-PPARα/hsLpLko hearts

A) Representative EMs from papillary muscle of WT, MHC-PPARα, and MHC-PPARα/hsLpLko hearts. White bars=2 microns. B) Q–rtPCR analysis of cardiac transcripts encoding PGC-1α in WT and MHC-PPARα hearts (n=9/genotype), C) PGC-1α in MHC-PPARα/hsLpLko and MHC-PPARα/CD36ko compared to WT and MHC-PPARα (n=6–7/group) and D) additional gene targets (abbreviations in text) in hearts from WT, MHC-PPARα, hsLpLko, MHC-PPARα and MHC-PPARα/hsLpLko (n=7–9/group). All bars represent mean (± SE) arbitrary unit (AU) normalized to the WT value (=1.0) in each case. *p<0.05 vs WT and hsLPLko, †p<0.05 vs. MHC-PPARα.

Figure 4. Mitochondrial gene expression and ultrastructure are improved in MHC-PPARα/hsLpLko hearts

A) Representative EMs from papillary muscle of WT, MHC-PPARα, and MHC-PPARα/hsLpLko hearts. White bars=2 microns. B) Q–rtPCR analysis of cardiac transcripts encoding PGC-1α in WT and MHC-PPARα hearts (n=9/genotype), C) PGC-1α in MHC-PPARα/hsLpLko and MHC-PPARα/CD36ko compared to WT and MHC-PPARα (n=6–7/group) and D) additional gene targets (abbreviations in text) in hearts from WT, MHC-PPARα, hsLpLko, MHC-PPARα and MHC-PPARα/hsLpLko (n=7–9/group). All bars represent mean (± SE) arbitrary unit (AU) normalized to the WT value (=1.0) in each case. *p<0.05 vs WT and hsLPLko, †p<0.05 vs. MHC-PPARα.

Figure 5. Cardiac metabolic PPARα target gene expression is normalized in LpL-deficient MHC-PPARα hearts

A) mRNA levels of FA metabolism gene targets and B) glucose metabolism gene targets (abbreviations in text) as determined by Q-rtPCR using RNA from hearts of 2 month-old mice after HF diet (n=7–9/genotype). Bars represent mean (± SE) AU normalized to the WT value (=1.0). *p<0.05 vs WT and hsLpLko, †p<0.05 vs. MHC-PPARα.

Figure 6. Heart uptake of VLDL-TG and -CE is altered in MHC-PPARα/hsLpLko hearts

Bars represent mean (±SE) cardiac lipid uptake as represented by radioactive count (dpm/gram of tissue), normalized to liver radioactive counts, for TG (¹⁴C-TG, left) and CE (³H-CE, right) in WT (n=6), MHC-PPARα (n=7), MHC-PPARα/hsLpLko (n=8), and MHC-PPARα/CD36ko (n=8) animals. *p<0.05 compared to all other groups.

Figure 7. Lipolysis of VLDL by LpL results in activation of PPARα

Transient co-transfections using the UAS₃tk.luc reporter and the Gal4-PPARα-LBD performed in NRVM. Cells were stimulated for the last 12–14 hours with A) BSA, 100mmol/L oleate, VLDL alone, LpL alone, or VLDL+LpL. B) BSA, or LpL+VLDL (original), re-isolated VLDL control (VLDLc) or TG-depleted VLDL (VLDLtg), or VLDL+LpL+THL. C) Same co-transfection conditions as in A, but with either siCtrl or siCD36. The bars represent mean (±SE) relative light units (RLU) for three or more experiments each done in triplicate, corrected for Renilla luciferase activity, and normalized to the activity of cells stimulated with BSA (=1.0). *p<0.05 compared to BSA treatment for each siRNA, †p≤0.05 compared to VLDL+LpL.