Effects of PPARs agonists on cardiac metabolism in littermate and cardiomyocyte-specific PPAR-γ-knockout (CM-PGKO) mice

Abstract

Understanding the molecular regulatory mechanisms controlling for myocardial lipid metabolism is of critical importance for the development of new therapeutic strategies for heart diseases. The role of PPARγ and thiazolidinediones in regulation of myocardial lipid metabolism is controversial. The aim of our study was to assess the role of PPARγ on myocardial lipid metabolism and function and differentiate local/from systemic actions of PPARs agonists using cardiomyocyte-specific PPARγ -knockout (CM-PGKO) mice. To this aim, the effect of PPARγ, PPARγ/PPARα and PPARα agonists on cardiac function, intra-myocyte lipid accumulation and myocardial expression profile of genes and proteins, affecting lipid oxidation, uptake, synthesis, and storage (CD36, CPT1MIIA, AOX, FAS, SREBP1-c and ADPR) was evaluated in cardiomyocyte-specific PPARγ-knockout (CM-PGKO) and littermate control mice undergoing standard and high fat diet (HFD). At baseline, protein levels and mRNA expression of genes involved in lipid uptake, oxidation, synthesis, and accumulation of CM-PGKO mice were not significantly different from those of their littermate controls. At baseline, no difference in myocardial lipid content was found between CM-PGKO and littermate controls. In standard condition, pioglitazone and rosiglitazone do not affect myocardial metabolism while, fenofibrate treatment significantly increased CD36 and CPT1MIIA gene expression. In both CM-PGKO and control mice submitted to HFD, six weeks of treatment with rosiglitazone, fenofibrate and pioglitazone lowered myocardial lipid accumulation shifting myocardial substrate utilization towards greater contribution of glucose. In conclusion, at baseline, PPARγ does not play a crucial role in regulating cardiac metabolism in mice, probably due to its low myocardial expression. PPARs agonists, indirectly protect myocardium from lipotoxic damage likely reducing fatty acids delivery to the heart through the actions on adipose tissue. Nevertheless a direct non-PPARγ mediated mechanism of PPARγ agonist could not be ruled out.

Figure 1. Effect of PPAR γ agonist on FFA plasma levels.

Effect of six weeks PPAR agonists treatments on plasma FFA levels of CM-PGKO mice (white bars) and littermate controls (black bars) fed with standard (SD) or high fat (HFD) diet. # p<0.05 HFD group vs SD group. * HFD +drugs vs HFD group. P = Pioglitazone; F = Fenofibrate; R = Rosiglitazone.

Figure 2. Effect of PPARγ agonist on PPARγ and PPARα mRNA expression and protein levels.

(A) Effect of six weeks PPAR agonists treatments on PPARγ and PPARα mRNA expression levels in CM-PGKO mice (white bars) and littermate controls (black bars) at baseline and after 24 weeks high fat (HFD) diet. P = Pioglitazone; F = Fenofibrate; R = Rosiglitazone. # p<0.05 HFD group vs baseline, * HFD +drugs vs HFD group (B) Representative immunoistochemical analysis of PPARγ and PPARα protein from ventricular biopsy specimens (×400).

Figure 3. Proteins expression involved in glucose uptake and lipid uptake, oxidation and accumulation.

Western blot showing the expression of proteins involved in glucose uptake and lipid uptake, oxidation and accumulation in CM-PGKO and littermate control fed with standard (SD) or high fat diet (HFD).

Figure 4. Effect of PPARγ agonist on mRNA expression of genes involved in glucose and lipid metabolism.

Effect of Rosiglitazone (R), Pioglitazone (P) and Fenofibrate (F) treatment on cardiomyocyte mRNA expression of genes involved in glucose and lipid metabolism of CM-PGKO mice (white bars) and littermate controls (black bars) fed with standard (SD) or high fat (HFD) diet for 24 weeks. # p<0.05 HFD group vs SD group. * HFD +drugs vs HFD group. +Piolitazone and Rosiglitazone group vs Fenofibrate.

Figure 5. Effect of PPAR agonists on Myocardial lipid content.

Effect of PPAR agonists on Myocardial lipid content in CM-PGKO mice (white bars) and littermate controls (black bars) fed with standard (SD) and high fat diet (HFD). A) Oil red O staining showed an abundance of neutral lipid droplets randomly scattered throughout the cytoplasm of cardiomyocytes (original magnification, ×400). (B) Oil red O staining was quantified using Molecular Analysis Software (n = 3 in each group). # p<0.05 HFD vs SD, * p<0.05 Drug vs only diet group, +p<0.05 CTLR vs CMPGKO group.