The Effects of PPAR Stimulation on Cardiac Metabolic Pathways in Barth Syndrome Mice

Abstract

Aim: Tafazzin knockdown (TazKD) in mice is widely used to create an experimental model of Barth syndrome (BTHS) that exhibits dilated cardiomyopathy and impaired exercise capacity. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that play essential roles as transcription factors in the regulation of carbohydrate, lipid, and protein metabolism. We hypothesized that the activation of PPAR signaling with PPAR agonist bezafibrate (BF) may ameliorate impaired cardiac and skeletal muscle function in TazKD mice. This study examined the effects of BF on cardiac function, exercise capacity, and metabolic status in the heart of TazKD mice. Additionally, we elucidated the impact of PPAR activation on molecular pathways in TazKD hearts. Methods: BF (0.05% w/w) was given to TazKD mice with rodent chow. Cardiac function in wild type-, TazKD-, and BF-treated TazKD mice was evaluated by echocardiography. Exercise capacity was evaluated by exercising mice on the treadmill until exhaustion. The impact of BF on metabolic pathways was evaluated by analyzing the total transcriptome of the heart by RNA sequencing. Results: The uptake of BF during a 4-month period at a clinically relevant dose effectively protected the cardiac left ventricular systolic function in TazKD mice. BF alone did not improve the exercise capacity however, in combination with everyday voluntary running on the running wheel BF significantly ameliorated the impaired exercise capacity in TazKD mice. Analysis of cardiac transcriptome revealed that BF upregulated PPAR downstream target genes involved in a wide spectrum of metabolic (energy and protein) pathways as well as chromatin modification and RNA processing. In addition, the Ostn gene, which encodes the metabolic hormone musclin, is highly induced in TazKD myocardium and human failing hearts, likely as a compensatory response to diminished bioenergetic homeostasis in cardiomyocytes. Conclusion: The PPAR agonist BF at a clinically relevant dose has the therapeutic potential to attenuate cardiac dysfunction, and possibly exercise intolerance in BTHS. The role of musclin in the failing heart should be further investigated.

Keywords: Barth syndrome; Ostn; PPAR agonist; bezafibrate; cardiomyopathy; musclin; tafazzin.

FIGURE 1

Bezafibrate (BF) ameliorates systolic function in TazKD mice. (A) Left ventricular ejection fraction. (B) Left ventricular fractional shortening. Asterisks denote significant differences between groups (^∗p < 0.05).

FIGURE 2

Exercise performance of mice on the treadmill. (A) Distance traveled on the treadmill until exhaustion of WT-, TazKD-, and BF-treated TazKD mice (TazKD-BF) that were housed with or without in-cage running wheels. Asterisks denote significant differences between groups (^∗p < 0.05). (B) Average daily running distance of WT-, untreated-, and BF-treated TazKD mice on running wheels. Asterisks denote significant differences between groups (p < 0.05).

FIGURE 3

Pathways that are affected by BF treatment in TazKD hearts. (A) Gene Ontology (GO) terms (FDR < 0.05) significantly enriched in regulated genes in hearts of TazKD- and BF-treated TazKD mice. (B) Subcellular localization of proteins, encoded by upregulated genes in BF-treated TazKD mice. (C) The heatmap of differentially expressed transcription factors in BF-treated TazKD hearts.

FIGURE 4

Disease network of regulated genes identified in (A) WT vs. TazKD and (B) TazKD vs. TazKD-BF datasets. Rectangles and circles correspond to disorders and disease genes, respectively. A link is placed between a disorder and a disease gene if mutations in that gene lead to a specific disorder. Network is generated with AltAnalyze software interrogating the Comparative Toxicogenomics Database (http://ctdbase.org/).

FIGURE 5

The clustered heatmap of differentially expressed genes in rows of untreated WT, untreated TazKD, and BF-treated TazKD groups. Log2 values of FCs of 262 differentially expressed genes are shown. Expression values intensities in rows are normalized to row means. Rows were clustered using the average Euclidian method. Zoomed fragment of the heatmap with the expression FC values of Asns, Ostn, Mthfd2, and Atf4 are magnified on the lower pane.

FIGURE 6

Musclin is upregulated in failing hearts. (A) Musclin mRNA in developing and adult heart (Y-axis is in the logarithmic scale). (B) DAB-staining of 3 months old left ventricles with anti-musclin antibodies. (C) Western blot of circulating musclin in plasma of TazKD mice. (D) Quantification of Ostn mRNA in non-failing and failing human hearts with Taqman assay.