Hypoxic Regulation of <i>Hand1</i> Controls the Fetal-Neonatal Switch in Cardiac Metabolism

<div><p>Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia <i>in utero</i>. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several “fetal” genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.</p></div

<i>Hand1</i> levels fall in the heart immediately following birth, under control of hypoxia signaling.

<p>(A) RTPCR for <i>Hand1</i> RNA from whole hearts of perinatal mice at a range of stages around birth, showing a steep decline in expression from birth. Levels expressed as a multiple of average 6-wk-old adult levels (<i>n</i> = 4 each group). (B) Levels of cardiac <i>Hand2</i> RNA do not fall at birth. Levels of <i>Hand2</i> at p1.5 normalised to e18.5 levels (<i>n</i> = 6 each group). (C) Western blot of protein extract from e18 (prenatal) control, p0.5 <i>XMLC-Hand1</i>, and p0.5 control hearts showing reduction in Hand1 but not Hand2 protein levels after birth, and persistence of Hand1 expression in <i>XMLC2-Hand1</i> hearts. (D) RTPCR showing increased <i>Hand1</i> RNA levels in hearts of adult wild-type mice incubated at 12% oxygen for 2 wk (“hypoxia”) over controls at normoxia (20% O₂) (<i>n</i> = 4 each group) (<i>p</i> = 0.001, two-tailed <i>t</i> test). (E) RTPCR showing significantly increased <i>Hand1</i> RNA levels in the hearts of p0.5 neonatal <i>α-MHC-Cre::VHL^(fl/fl</i>⁾ mice compared with wild-type controls, <i>p</i> = 0.0002 two-tailed <i>t</i> test, <i>n</i> = 6 each group. (F) Western blot of protein extract from <i>VHL^(fl/fl)</i> and control hearts at p0.5, showing elevation of Hand1 and HIF1α in <i>VHL^(fl/fl)</i> hearts. (G) RTPCR of chromatin immunoprecipitation assay using anti-HIF1α antiserum and primers to the HIF motif-containing sequences in the <i>Hand1</i> promoter from e18 hearts, showing binding of HIF1α to two sites. Bars represent summation of three experiments, and results expressed as multiples of signal for nonamplified sequence. The <i>p</i> values are two-tailed <i>t</i> tests relative to nonamplified <i>γ-crystallin</i> primers.</p

Prevention of neonatal <i>Hand1</i> down-regulation in transgenic mouse hearts leads to cardiomyopathy and death.

<p>(A) Cardiac RNA levels of <i>Hand1</i> in e18.5 and p0.5 wild-type, and e18.5 and p0.5 transgenic (TG 18.5 and TG0.5, respectively) showing RNA levels in the transgenic heart around 2.5 times that of wild-type p0.5 heart. (B) Cardiac <i>Hand1</i> elevating pups appear grossly normal, but are cyanosed (c, control; oe, Hand1 overexpressing). (C, D) H and E stain of cryostat section through thorax of control and <i>Hand1</i> overexpressing hearts, showing thin ventricular wall of the Hand1 overexpressing heart, and ventricular rupture (arrowed) with blood in the pericardial space (rv, right ventricle; lv, left ventricle). (E, F) EFIC sectioning and reconstruction of control and Hand1 overexpressing hearts from 4-h-old fostered pups, showing small size but no gross structural defect. (G, H) Periodic acid-Schiff stain of control and Hand1 overexpressing heart, showing decreased glycogen levels in <i>Hand1</i> overexpressing heart (purple). Glycogen stain in intercostal muscle of transgenic pup arrowed in (H). (I) Quantification of glucose enzymatically released from glycogen in hearts of neonates 2 h after caesarian section. Levels of glycogen in XMLC-Hand1 hearts are 17.5% of XMLC controls (<i>p</i> value, two-tailed <i>t</i> test, <i>n</i> = 6 hearts each group).</p

Prolongation of neonatal cardiac <i>Hand1</i> expression prevents transcriptional up-regulation of lipid metabolizing genes.

<p>(A) Schematic showing myocardial lipid metabolism (adapted from Kodde et al. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666-Kodde1" target="_blank">[55]</a>). (B) RTPCR showing RNA expression in e16 control, p0.5 control, and p0.5 hand1 up-regulating hearts. Levels of <i>ACC</i>, <i>MCD</i>, <i>FABP4</i>, <i>ACSL</i>, <i>CPT1A CPT1B</i>, and <i>HSL</i> are significantly up-regulated around birth (<i>p</i><0.05 two-tailed <i>t</i> test, <i>n</i> = 4 each group). No postnatal rise in <i>ACC</i>, <i>MCD</i>, <i>FABP4</i>, <i>CPT1A</i>, and <i>HSL</i> is seen in Hand1 up-regulating hearts. Significantly increased RNA expression of <i>ACBP</i> and <i>ATGL</i> is seen in Hand1 up-regulating hearts. Genes whose expression is reduced in Hand1 overexpressing hearts are in red in (A). <i>ACC</i>, acetyl coA carboxylase; <i>MCD</i>, malonyl coA decarboxylase; <i>FABP</i>, fatty acid binding protein; <i>FATP</i>, fatty acid transport protein; <i>ACSL</i>, acyl coA synthase long chain 1; <i>HSL</i>, hormone sensitive lipase; <i>ATGL</i>, adipose triglyceride lipase; <i>ACBP</i>, acylcoA binding protein; <i>CPT</i>, Carnitine Palmitoyl Transferase. (C) RTPCR of mRNA from 2-mo-old adult XMLC2-Hand1 mice following doxycycline induction for 2 wk, showing changes in expression of RNA encoding fatty acid metabolising proteins relative to control non-up-regulating mice (*<i>p</i><0.05, **<i>p</i><0.005, two-tailed <i>t</i> test, <i>n</i> = 4 each group). (D) RTPCR of mRNA from e14.5 embryo hearts from αMHC-Cre::Hand1^(fl/fl) and control pups, showing up-regulation of genes encoding fatty acid metabolising enzymes(*<i>p</i><0.05, two-tailed <i>t</i> test, <i>n</i> = 4 each group). (E) RTPCR showing significant <i>PGC1-α</i> elevation in the heart around birth (<i>n</i> = 4 each group, <i>p</i> = 0.008, two-tailed <i>t</i> test), with no significant drop in <i>Hand1</i> up-regulating hearts (<i>p</i> = 0.26, <i>n</i> = 6 each group). (F) Western blot of protein extract from cultured HL1 cardiomyocytes nontransfected (“control”) and transfected with <i>PGC1-α</i> and <i>HIF1</i>, showing no elevation of Hand1 in <i>PGC1-α</i> elevated PGC1<i>α</i> and Hand1 but not Hand2 protein expression in HIF1 expressing cells. (G) PCR of nuclear genomic (<i>globin</i>) and mitochondrial (<i>COX2</i>) DNA showing unchanged ratio in <i>Hand1</i> elevating neonatal hearts and Hand1-transfected HL1 cells compared with controls, implying no change in mitochondrial number. Control HL1 cells are transfected with an empty vector. (H) Map of 5′ promoters of several putative <i>Hand1</i> transcriptional targets in the e18.5 heart. Numbers refer to fold enrichment over <i>γ crystallin</i> in chromatin immunoprecipitation assay using anti-Hand1 serum. For more detailed chromatin immunoprecipitation data, please see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666.s009" target="_blank">Text S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666.s002" target="_blank">Figure S2</a>. (I) Site-directed mutagenesis of the Hand1-binding canonical CANNTG e-box in the 5′ HSL luciferase promoter de-represses expression of luciferase in HL1 cells, both in untransfected cells and cells stably expressing Hand1 (transfections in triplicate, measurement in quadruplicate, <i>p</i> values, two-tailed <i>t</i> test).</p

Lipid metabolism is inhibited in neonatal <i>Hand1</i> overexpressing hearts.

<p>(A) LC-MS trace showing typical output for intact lipid extracted from control hearts (green trace) and Hand1 up-regulating hearts (red), showing significantly lower levels of triacylglycerides (TAG) compared to phospholipid (PL) in Hand1 up-regulating hearts. (B) Quantitative analysis of cardiac triacylglyceride levels showing significant reduction in Hand1 up-regulating hearts, expressed as the ratio of TAG to phospholipid (<i>n</i> = 6 each group, <i>p</i> = 0.006, two-tailed <i>t</i> test). (C) Quantitative analysis of cardiac malonyl coA levels showing significant reduction in Hand1 up-regulating hearts (<i>n</i> = 6 each group, <i>p</i> = 0.04, two-tailed <i>t</i> test). (D) Reduced levels of C6, C14, and C18 containing acylcarnitine species in Hand1 prolonging neonatal hearts compared with controls. For full dataset, please refer to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001666#pbio.1001666.s007" target="_blank">Table S3</a>. (E) Multivariate partial least squares discriminant analysis (PLS-DA) scores of acylcarnitine profiles showing a significant decrease in global levels of acylcarnitines in Hand1 up-regulated hearts relative to controls (R²X = 33%, R²Y = 62%, and Q² = 48%). (F) BODIPY-500/510C₁, C₁₂ uptake is significantly reduced in HL1 cells by transfection with Hand1. Graph shows quantification of fluorescence in Hand1 transfected and nontransfected controls, 10 high power fields each. (i) and (ii) show representative fluorescence micrographs of control and Hand1 transfected cells following labeled lipid incubation (<i>p</i> = 0.037, two-tailed <i>t</i> test).</p