Jean Bastin

Fatty Acid Oxidation Enzyme Gene Expression Is Downregulated in the Failing Heart

BACKGROUND During the development of heart failure (HF), the chief myocardial energy substrate switches from fatty acids to glucose. This metabolic switch, which recapitulates fetal cardiac energy substrate preferences, is thought to maintain aerobic energetic balance. The regulatory mechanisms involved in this metabolic response are unknown. METHODS AND RESULTS To characterize the expression of genes involved in mitochondrial fatty acid beta-oxidation (FAO) in the failing heart, levels of mRNA encoding enzymes that catalyze the first and third steps of the FAO cycle were delineated in the left ventricles (LVs) of human cardiac transplant recipients. FAO enzyme and mRNA levels were coordinately downregulated (> 40%) in failing human LVs compared with controls. The temporal pattern of this alteration in FAO enzyme gene expression was characterized in a rat model of progressive LV hypertrophy (LVH) and HF [SHHF/Mcc-facp (SHHF) rat]. FAO enzyme mRNA levels were coordinately downregulated (> 70%) during both the LVH and HF stages in the SHHF rats compared with controls. In contrast, the activity and steady-state levels of medium-chain acyl-CoA dehydrogenase, which catalyzes a rate-limiting step in FAO, were not significantly reduced until the HF stage, indicating additional control at the translational or post-translational levels in the hypertrophied but nonfailing ventricle. CONCLUSIONS These findings identify a gene regulatory pathway involved in the control of cardiac energy production during the development of HF.

A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator- activated receptor alpha- deficient mice.

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor implicated in the control of cellular lipid utilization. To test the hypothesis that PPARalpha is activated as a component of the cellular lipid homeostatic response, the expression of PPARalpha target genes was characterized in response to a perturbation in cellular lipid oxidative flux caused by pharmacologic inhibition of mitochondrial fatty acid import. Inhibition of fatty acid oxidative flux caused a feedback induction of PPARalpha target genes encoding fatty acid oxidation enzymes in liver and heart. In mice lacking PPARalpha (PPARalpha-/-), inhibition of cellular fatty acid flux caused massive hepatic and cardiac lipid accumulation, hypoglycemia, and death in 100% of male, but only 25% of female PPARalpha-/- mice. The metabolic phenotype of male PPARalpha-/- mice was rescued by a 2-wk pretreatment with beta-estradiol. These results demonstrate a pivotal role for PPARalpha in lipid and glucose homeostasis in vivo and implicate estrogen signaling pathways in the regulation of cardiac and hepatic lipid metabolism.

Activation of peroxisome proliferator activated receptor pathway stimulates the mitochondrial respiratory chain and can correct deficiencies in patients' cells lacking its components.

CONTEXT The mitochondrial respiratory chain (RC) disorders are the largest group of inborn errors of metabolism and still remain without treatment in most cases. OBJECTIVE We tested whether bezafibrate, a drug acting as a peroxisome proliferator-activated receptor (PPAR) agonist, could stimulate RC capacities. DESIGN Fibroblasts or myoblasts from controls or patients deficient in complex I (CI), complex III (CIII), or complex IV (CIV) were cultured with or without bezafibrate. MAIN OUTCOME MEASURES Enzyme activities, mRNA and protein expression, and respiration rates were measured. RESULTS In control cells, bezafibrate increased the CI, CIII, and CIV enzyme activities (+42 to +52%), as well as RC mRNAs (+40 to +120%) and RC protein levels (+50 to +150%). Nine of 14 patient cell lines tested exhibited a significant increase in the activity of the deficient RC complex after bezafibrate treatment (+46 to +133%), and full pharmacological correction could be achieved in seven cell lines. Similar effects were obtained using a PPARdelta agonist. These changes were related to a drug-induced increase in the mutated mRNAs and RC protein levels. Finally, the molecular mechanisms by which the PPAR pathway could induce the expression of genes encoding structural subunits or ancillary proteins of the RC apparatus, leading to stimulate the activity and protein levels of RC complex, likely involved the PPARgamma coactivator-1alpha. CONCLUSIONS This study suggests a rationale for a possible correction of moderate RC disorders due to mutations in nuclear genes, using existing drugs, and brings new insights into the role of PPAR in the regulation of the mitochondrial RC in human cells.

Bezafibrate for an Inborn Mitochondrial Beta-Oxidation Defect

To the Editor: Carnitine palmitoyltransferase II (CPT2) deficiency is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation. The most common form of this disorder is characteriz...

Current issues regarding treatment of mitochondrial fatty acid oxidation disorders.

Treatment recommendations in mitochondrial fatty acid oxidation (FAO) defects are diverse. With implementation of newborn screening and identification of asymptomatic patients, it is necessary to define whom to treat and how strictly. We here discuss critical questions that are currently under debate. For some asymptomatic long-chain defects, long-chain fat restriction plays a minor role, and a normal diet may be introduced. For patients presenting only with myopathic symptoms, e.g., during exercise, treatment may be adapted to energy demand. As a consequence, patients with exercise-induced myopathy may be able to return to normal activity when provided with medium-chain triglycerides (MCT) prior to exercise. There is no need to limit participation in sports. Progression of retinopathy in disorders of the mitochondrial trifunctional protein complex is closely associated with hydroxyacylcarnitine accumulation. A strict low-fat diet with MCT supplementation is recommended to slow or prevent progression of chorioretinopathy. Additional docosahexanoic acid does not prevent the decline in retinal function but does promote nonspecific improvement in visual acuity and is recommended. There is no evidence that L-carnitine supplementation is beneficial. Thus, supplementation with L-carnitine in a newborn identified by screening with either a medium-chain or long-chain defect is not supported. With respect to the use of the odd-chain medium-chain triglyceride triheptanoin in myopathic phenotypes, randomized trials are needed to establish whether triheptanoin is more effective than even-chain MCT. With increasing pathophysiological knowledge, new treatment options have been identified and are being clinically evaluated. These include the use of bezafibrates in myopathic long-chain defects.

Genetic Basis for Correction of Very-Long-Chain Acyl-Coenzyme A Dehydrogenase Deficiency by Bezafibrate in Patient Fibroblasts : Toward a Genotype-Based Therapy

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.

Correction of fatty acid oxidation in carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate.

Carnitine palmitoyltransferase 2 (CPTII) deficiency is among the most common inborn errors of mitochondrial fatty acid β-oxidation (FAO). Clinical phenotype varies in relation to the metabolic block, as assessed by studies of FAO in patient fibroblasts. Thus, fibroblasts from patients with mild manifestations have appreciable residual CPTII enzyme activity, in contrast to those from severely affected patients. In the present study, we hypothesized that the hypolipidemic drug bezafibrate, acting as an activator of the peroxisome proliferator-activated receptor α might stimulate FAO in CPTII-deficient cells. Data obtained show that bezafibrate treatment of mild-type CPTII-deficient cells resulted in a time- and dose- dependant increase in CPTII mRNA (from +47% to +66%) and residual enzyme activity (from +54% to 135%), and led to normalization of 3H-palmitate and 3H-myristate cellular oxidation rates. Bezafibrate did not correct FAO in fibroblasts from patients with severe phenotype. This study establishes for the first time that peroxisome proliferator-activated receptor activators, acting via stimulation of gene expression, can stimulate CPTII residual activity to a level sufficient to allow normal FAO flux in deficient human fibroblasts, and suggests that this approach should be tested in other inborn errors of mitochondrial β-oxidation.

Dietary lipids regulate β-oxidation enzyme gene expression in the developing rat kidney

This study examines the ability of dietary lipids to regulate gene expression of mitochondrial and peroxisomal fatty acid β-oxidation enzymes in the kidney cortex and medulla of 3-wk-old rats and evaluates the role of glucagon or of the α-isoform of peroxisome proliferator-activated receptor (PPARα) in mediating β-oxidation enzyme gene regulation in the immature kidney. The long-chain (LCAD) and medium-chain acyl-CoA dehydrogenases (MCAD) and acyl-CoA oxidase (ACO) mRNA levels were found coordinately upregulated in renal cortex, but not in medulla, of pups weaned on a high-fat diet from day 16 to 21. Further results establish that switching pups from a low- to a high-fat diet for only 1 day was sufficient to induce large increases in cortical LCAD, MCAD, and ACO mRNA levels, and gavage experiments show that this upregulation of β-oxidation gene expression is initiated within 6 h following lipid ingestion. Treatment of pups with clofibrate, a PPARα agonist, demonstrated that PPARα can mediate regulation of cortical β-oxidation enzyme gene expression, whereas glucagon was found ineffective. Thus dietary lipids physiologically regulate gene expression of mitochondrial and peroxisomal β-oxidation enzymes in the renal cortex of suckling pups, and this might involve PPARα-mediated mechanisms.

A potential link between peroxisome proliferator-activated receptor signalling and the pathogenesis of arrhythmogenic right ventricular cardiomyopathy

AIMS Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by major fibro-fatty replacement of the right ventricle (RV). We hypothesized that changes in peroxisome proliferator-activated receptor (PPAR) signalling contributed to myocardium fatty accumulation and contractile dysfunction in ARVC. METHODS AND RESULTS Real-time quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to assess cardiac expression of PPARalpha and gamma and two of their downstream target genes--medium-chain acyl-CoA dehydrogenase (MCAD) and phosphoenolpyruvate carboxykinase (PEPCK)--in both RV and left ventricle (LV) from five controls and five ARVC patients. In vitro motility assays were used to analyse functional properties of myosin. In the RV, sliding velocity was nearly two-fold lower in ARVC than in controls, whereas a 10% reduction in velocity values was noted between ARVC and non-failing myocardium in the LV. In controls, PPARalpha and MCAD mRNA and protein levels were higher in the RV compared with the LV. In ARVC, the expression of PPARalpha and MCAD mRNA and/or proteins was decreased in both RV and LV. RV from ARVC was also characterized by a dramatic activation of the PPARgamma pathway, as attested by the increase in PPARgamma mRNA and protein (500 and 270%, respectively, each P < 0.001) and by the induction of PEPCK gene. In contrast, the LV of ARVC heart exhibited no changes in the expression of the PPARgamma regulatory pathway compared with control. CONCLUSION ARVC is associated with major disturbances in the PPARalpha and PPARgamma signalling pathway in the RV that may contribute to intracellular lipid overload and severe myosin dysfunction.

Characterization of fatty acid oxidation in human muscle mitochondria and myoblasts

The mitochondrial oxidation of fatty acids (FAO) is the main energy-producing pathway in skeletal and cardiac muscle. Starting from standard muscle biopsies (100-200mg), we determined the optimal conditions of mitochondrial oxygen consumption by the FAO pathway, and in parallel we performed the isolation and primary culture of muscle cells to test their cellular FAO capacities. The determinations of maximal beta-oxidation rates in the presence of palmitoyl-CoA or palmitoyl-L-carnitine (mean+/-SEM: 32.5+/-2.0 and 34.1+/-1.3nmol O(2) min(-1) mg(-1) protein, n=16, respectively) provide a screening method of mitochondrial fatty acid transport system and intra-mitochondrial beta-oxidation. We also determined the conditions of tritiated palmitate oxidation by human myoblasts (mean+/-SEM: 6.6+/-0.1nmol 3H fatty acid h(-1) mg(-1) protein, n=8), and show that beta-oxidation defects can be detected in our experiments. Overall, we propose an original laboratory test to investigate FAO in human skeletal muscle and to screen for FAO disorders in myopathies and cardiomyopathies in human.