Olga Zhelyabovska

Peroxisome Proliferator-Activated Receptor δ Is an Essential Transcriptional Regulator for Mitochondrial Protection and Biogenesis in Adult Heart

Rationale: Peroxisome proliferator-activated receptors (PPARs) (&agr;, &ggr;, and &dgr;/&bgr;) are nuclear hormone receptors and ligand-activated transcription factors that serve as key determinants of myocardial fatty acid metabolism. Long-term cardiomyocyte-restricted PPAR&dgr; deficiency in mice leads to depressed myocardial fatty acid oxidation, bioenergetics, and premature death with lipotoxic cardiomyopathy. Objective: To explore the essential role of PPAR&dgr; in the adult heart. Methods and Results: We investigated the consequences of inducible short-term PPAR&dgr; knockout in the adult mouse heart. In addition to a substantial transcriptional downregulation of lipid metabolic proteins, short-term PPAR&dgr; knockout in the adult mouse heart attenuated cardiac expression of both Cu/Zn superoxide dismutase and manganese superoxide dismutase, leading to increased oxidative damage to the heart. Moreover, expression of key mitochondrial biogenesis determinants such as PPAR&ggr; coactivator-1 were substantially decreased in the short-term PPAR&dgr; deficient heart, concomitant with a decreased mitochondrial DNA copy number. Rates of palmitate and glucose oxidation were markedly depressed in cardiomyocytes of PPAR&dgr; knockout hearts. Consequently, PPAR&dgr; deficiency in the adult heart led to depressed cardiac performance and cardiac hypertrophy. Conclusions: PPAR&dgr; is an essential regulator of cardiac mitochondrial protection and biogenesis and PPAR&dgr; activation can be a potential therapeutic target for cardiac disorders.

Liver X receptors are negative regulators of cardiac hypertrophy via suppressing NF-κB signalling

AIMS Nuclear factor-kappaB (NF-kappaB) plays a critical role in cell growth and inflammation during the progression of cardiac hypertrophy and heart failure. Several members of nuclear receptor superfamily, including liver X receptors (LXRalpha and LXRbeta), have been shown to suppress inflammatory responses, but little is known about their effects in cardiomyocytes. METHODS AND RESULTS We investigated LXR expression patterns in pressure overload-induced hypertrophic hearts and the hypertrophic growth of the LXRalpha-deficient hearts from mice (C57/B6) in response to pressure overload. The underlying mechanisms were also explored using cultured myocytes. We found that cardiac expression of LXRalpha was upregulated in pressure overload-induced left ventricular hypertrophy in mice. Transverse aorta coarctation-induced left ventricular hypertrophy was exacerbated in LXRalpha-null mice relative to control mice. A synthetic LXR ligand, T1317, suppressed cardiomyocyte hypertrophy in response to angiotensin II and lipopolysaccharide treatments. In addition, LXR activation suppressed NF-kappaB signalling and the expression of associated inflammatory factors. Overexpression of constitutively active LXRalpha and beta in cultured myocytes suppressed NF-kappaB activity. CONCLUSION LXRs are negative regulators of cardiac growth and inflammation via suppressing NF-kappaB signalling in cardiomyocytes. This should provide new insights into novel therapeutic targets for treating cardiac hypertrophy and heart failure.

Peroxisome Proliferator-Activated Receptor β/δ Activation in Adult Hearts Facilitates Mitochondrial Function and Cardiac Performance Under Pressure-Overload Condition

Peroxisome proliferator-activated receptor &bgr;/&dgr; (PPAR&bgr;/&dgr;) is an essential transcription factor in myocardial metabolism. This study aims to investigate the effects of PPAR&bgr;/&dgr; activation in the adult heart on mitochondrial biology and oxidative metabolism under normal and pressure-overload conditions. We have investigated the effects of cardiac constitutively active PPAR&bgr;/&dgr; in adult mice using a tamoxifen-inducible transgenic approach with Cre-LoxP recombination. The expression of PPAR&bgr;/&dgr; mRNA and protein in cardiomyocytes of adult mice was substantially increased after short-term induction. In these mice, the cardiac expression of key factors involved in mitochondrial biogenesis, such as PPAR&ggr; coactivator-1, endogenous antioxidants Cu/Zn superoxide dismutase, and catalase, fatty acid, and glucose metabolism, such as carnitine palmitoyltransferase Ib, carnitine palmitoyltransferase II, and glucose transporter 4, were upregulated. Subsequently, myocardial oxidative metabolism was elevated concomitant with an increased mitochondrial DNA copy number and an enhanced cardiac performance. Moreover, activation of PPAR&bgr;/&dgr; in the adult heart improved cardiac function and resisted progression to pathological development in mechanical stress condition. We conclude that PPAR&bgr;/&dgr; activation in the adult heart will promote cardiac performance along with transcriptional upregulation of mitochondrial biogenesis and defense, as well as oxidative metabolism at basal and pressure-overload conditions.

Peroxisome Proliferator-activated Receptor δ Regulates Mitofusin 2 Expression in the Heart

Mitofusin 2 (Mfn2) has been proposed as an important mitochondrial protein in maintaining mitochondrial network and bioenergetics. Mfn2 is highly expressed in the heart, but is downregulated in response to hypertrophic stimuli. However, little is known about how Mfn2s expression is regulated in cardiomyocytes. Here, we have investigated how Mfn2 expression in the heart responds to fasting condition and determined if Mfn2 is one of those PPARdelta-selective target genes that are involved in myocardial energy metabolism. Fasting for 48 h in mice led to a robust increase of Mfn2 expression in the heart. On the other hand, cardiomyocyte-restricted PPARdelta deficiency in mice led to substantially diminished cardiac expression of Mfn2 transcript and protein compared to that of controls. Fasting induced cardiac expression of Mfn2 was blunted in cardiomyocyte-restricted PPARdelta deficient hearts. Moreover, PPARdelta-selective ligand treatment in cultured cardiomyocytes induced elevated Mfn2 expression. A functional PPRE consensus sequence located at -837 to -817 bp upstream of the mouse Mfn2 promoter was identified and confirmed by Electrophoretic Mobility Shift Assays and Luciferase Promoter Reporter Assays. We conclude that Mfn2 is a PPARdelta-selective target, which may play an important role in regulating myocardial energy homeostasis.

Generation of an inducible, cardiomyocyte-specific transgenic mouse model with PPAR β/δ overexpression.

Peroxisome proliferator-activated receptors (PPARs) consist of three subtypes, each displaying distinctive tissue distribution. In general, the three PPAR subtypes exert overlapping function in transcriptional regulation of lipid metabolism. However, each PPAR subtype possesses distinctive functions in different tissues dependent on their expression abundance, endogenous ligands, and the PPAR coregulators in a specific tissue. Transgenesis is an invaluable technique in defining the in vivo function of a particular gene and its protein. Cre/LoxP-mediated gene targeting has been extensively used to explore the tissue-specific function of PPARs. While this tissue-specific loss-of-function approach is extremely useful in determining the essential role of a PPAR, the tissue-specific gain-of-function approach is another important technique used to understand the effects of PPAR activation in a particular tissue. Transgenic overexpression of PPAR in a specific tissue has been used. However, this conventional technique requires generating the transgenic models individually for each target tissue. In this chapter, we describe the methodology for a more efficient generation of transgenic mouse models with a constitutively active form of PPARβ/δ in different tissues.

Transgenic cardiac-targeted overexpression of human thymidylate kinase

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the α-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In ‘2 by 2’ protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3′-azido-3′deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.