Malformations of the septum, outflow tract and aortic arch are the most common congenital cardiovascular defects and occur in mice lacking Cited2, a transcriptional coactivator of TFAP2. Here we show that Cited2 -/-mice also develop laterality defects, including right isomerism, abnormal cardiac looping and hyposplenia, which are suppressed on a mixed genetic background. Cited2 -/-mice lack expression of the Nodal target genes Pitx2c, Nodal and Ebaf in the left lateral plate mesoderm, where they are required for establishing laterality and cardiovascular development. CITED2 and TFAP2 were detected at the Pitx2c promoter in embryonic hearts, and they activate Pitx2c transcription in transient transfection assays. We propose that an abnormal Nodal-Pitx2c pathway represents a unifying mechanism for the cardiovascular malformations observed in Cited2 -/-mice, and that such malformations may be the sole manifestation of a laterality defect.Genetic, developmental and molecular studies over the past decade have identified a number of DNA-binding transcription factors that have key roles in cardiac morphogenesis and in the pathogenesis of common congenital heart defects 1 . The role of transcriptional coactivators, molecules that connect DNA-binding transcription factors to the core transcriptional machinery, in cardiac development has only recently become apparent. These coactivators are exemplified by the paralogous genes EP300 and CREBBP 2 . Mutations in CREBBP cause Rubinstein-Taybi Syndrome 3 and are frequently associated with cardiac malformations 4 . EP300 and CREBBP interact with high affinity with a ubiquitously expressed cytokine and hypoxia-inducible transcriptional coactivator called CITED2 (also called p35srj and Mrg1) [5][6][7][8] . Binding of CITED2 to EP300 competitively inhibits the binding of the transcription factor HIF1A to EP300, blocking hypoxia-activated gene transcription 5,8 . Cited2 is essential for normal development of the heart, adrenals and nervous system 9-13 and for fibroblast proliferation 14 . Mice lacking Cited2 die prenatally with diverse cardiovascular malformations, including atrial and ventricular septal defects, double-outlet right ventricle, common arterial trunk and aberrant aortic arches.In addition to functioning as a transcriptional repressor of HIF1A, CITED2 also physically interacts with and coactivates TFAP2 (transcription factor AP2, also called Tcfap2) and LIM-domain containing transcription factors by linking them to EP300 and CREBBP 9,15,16 . Mutations in Tcfap2a and TFAP2B (Char syndrome) result in cardiac and aortic arch malformations 17,18 , suggesting that coactivation of TFAP2 by CITED2, EP300 and CREBBP is necessary for the normal development of these structures 9 . An alternative explanation for the development of cardiac malformations in mice lacking Cited2 is dysregulation of hypoxia-activated gene transcription 12 .The cardiovascular malformations resulting from deficiency of Cited2 encompass a diverse and variable spectrum that is not explained by effects on...

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

Mutations in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer in affected individuals. FH-associated neoplasia is characterized by defective mitochondrial function and by upregulation of transcriptional pathways mediated by hypoxia-inducible factor (HIF), although whether and by what means these processes are linked has been disputed. We analysed the HIF pathway in Fh1-/- mouse embryonic fibroblasts (MEFs), in FH-defective neoplastic tissues and in Fh1-/- MEFs re-expressing either wild-type or an extra-mitochondrial restricted form of FH. These experiments demonstrated that upregulation of HIF-1alpha occurs as a direct consequence of FH inactivation. Fh1-/- cells accumulated intracellular fumarate and manifested severe impairment of HIF prolyl but not asparaginyl hydroxylation which was corrected by provision of exogenous 2-oxoglutarate (2-OG). Re-expression of the extra-mitochondrial form of FH in Fh1-/- cells was sufficient to reduce intracellular fumarate and to correct dysregulation of the HIF pathway completely, even in cells that remained profoundly defective in mitochondrial energy metabolism. The findings indicate that upregulation of HIF-1alpha arises from competitive inhibition of the 2-OG-dependent HIF hydroxylases by fumarate and not from disruption of mitochondrial energy metabolism.

Background— Chronic heart failure (CHF) is a major cause of morbidity and mortality that requires a novel approach to therapy. Perhexiline is an antianginal drug that augments glucose metabolism by blocking muscle mitochondrial free fatty acid uptake, thereby increasing metabolic efficiency. We assessed the effects of perhexiline treatment in CHF patients. Methods and Results— In a double-blind fashion, we randomly assigned patients with optimally medicated CHF to either perhexiline (n=28) or placebo (n=28). The primary end point was peak exercise oxygen consumption (V̇ o 2 max), an important prognostic marker. In addition, the effect of perhexiline on myocardial function and quality of life was assessed. Quantitative stress echocardiography with tissue Doppler measurements was used to assess regional myocardial function in patients with ischemic CHF. 31 P magnetic resonance spectroscopy was used to assess the effect of perhexiline on skeletal muscle energetics in patients with nonischemic CHF. Treatment with perhexiline led to significant improvements in V̇ o 2 max (16.1±0.6 to 18.8±1.1 mL · kg −1 · min −1 ; P <0.001), quality of life (Minnesota score reduction from 45±5 to 34±5; P =0.04), and left ventricular ejection fraction (24±1% to 34±2%; P <0.001). Perhexiline treatment also increased resting and peak dobutamine stress regional myocardial function (by 15% and 24%, respectively) and normalized skeletal muscle phosphocreatine recovery after exercise. There were no adverse effects during the treatment period. Conclusions— In patients with CHF, metabolic modulation with perhexiline improved V̇ o 2 max, left ventricular ejection fraction, symptoms, resting and peak stress myocardial function, and skeletal muscle energetics. Perhexiline may therefore represent a novel treatment for CHF with a good safety profile, provided that the dosage is adjusted according to plasma levels.