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Cardiac myocyte-specific HIF-1 deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart

Department of Medicine, Yale University, New Haven, Connecticut, USA; and
^* Department of Biology, UC San Diego, San Diego, California, USA

¹Correspondence: BCMM 436C, 295 Congress Ave., New Haven, CT 06510, USA. E-mail: Frank.Giordano{at}yale.edu

SPECIFIC AIMS

The hypoxia-responsive factor HIF-1 is a central mediator of cellular transcriptional responses to oxygen tension. We created cardiac myocyte-specific HIF-1 null mice to determine the role of HIF-1 in defining basal cardiac vascularization, energy metabolism, contractile function, and gene expression.

PRINCIPAL FINDINGS

1. Cardiac myocyte-specific HIF-1 expression is essential for maintenance of normal cardiac contractile function
Using a Cre-lox approach, we crossed mice with lox-P sites flanking exon 2 of the HIF-1 gene with mice expressing Cre recombinase in left ventricular (LV) cardiac myocytes under direction of the myosin light chain 2v promoter. Cardiac HIF-1 null mice were viable with expected gene frequencies. Analysis of the effects of HIF-1 deletion on cardiac contractile performance in vivo with echocardiography and catheter-based hemodynamic assessment revealed reduced LV fractional shortening and reduced systolic and diastolic function in the absence of HIF-1 (Fig. 1 ). These data establish that HIF-1 is required for maintenance of cardiac contractility even during normoxia. To further investigate this finding, cell shortening and calcium reuptake kinetics were measured in isolated cardiac myocytes. These studies documented a reduction in cell shortening from 11.60 ± 1.42% of resting length in control cells to 4.95 ± 0.80% in HIF-1 null cardiomyocytes (P<0.001), and a lengthening of t1/2 [Ca²⁺]_in to 0.195 ± 0.001 s in HIF-1 null cells to 0.169 ± 0.001 s in controls (P<0.001), consistent with an intrinsic reduction in contractility at least partially attributable to altered calcium handling.

View larger version (18K): [in this window] [in a new window]   Figure 1. Expression of HIF-1 in cardiac myocytes is required to maintain normal heart function in vivo. Hemodynamic characteristics were compared in mice whose HIF-1 was deleted specifically from cardiac myocytes (open symbols; n=13) vs. littermate controls (filled squares; n=9). a, b) Maximum rates of left ventricular pressure development (+dP/dt) and pressure decline (–dP/dt) were significantly reduced in cardiac HIF-1 null mice under basal conditions and during adrenergic stimulation with increasing doses of dobutamine. c) As further evidence of diastolic dysfunction, isovolumic relaxation time (Tau) was significantly increased at low and moderate levels of dobutamine stimulation, indicating impaired relaxation. d) Heart rates were similar in both groups under all conditions. e, f) Peak developed pressures tended to be less in cardiac HIF-1 mice and left ventricular end-diastolic pressures tended to be mildly higher; these difference were not statistically significant for either parameter. g) Echocardiography correlated with the hemodynamic data, revealing reduced fractional shortening in the HIF-1 null hearts (n=25 HIF null, 28 littermate controls). *P < 0.01, **P < 0.001. Dobutamine doses are in µg/kg body wt/min.

2. Loss of cardiac myocyte-specific HIF-1 expression leads to diminished vascularization in conjunction with thicker myocardial walls and reduced LV chamber diameter
HIF-1 is a transcriptional activator of Vegf-A and PDGF-B expression, and therefore has proangiogenic properties. We investigated the vascularization of cardiac myocyte HIF-1 null hearts and found a relatively mild (15.6±6.18%; P<0.05) average reduction of vessel counts in the left ventricles (Fig. 2 ), suggesting that control of coronary vascularization is only partially HIF-1 dependent. Despite this reduction in vascularity, there was an increase in myocardial wall thickness concomitant with a reduction in LV chamber diameter. This geometric configuration of the LV is consistent with what has been reported during development in the embryonic lethal global HIF-1 null mice, and suggests that this configuration is adaptive to the loss of HIF-1, perhaps to alter wall tension and thus myocardial oxygen consumption.

View larger version (29K): [in this window] [in a new window]   Figure 2. Cardiac myocyte-specific HIF-1 deletion reduces cardiac vascularity without concomitant loss of LV mass. Blood vessels in left ventricle myocardium from control mice (a) and age/gender-matched cardiac HIF-1 null littermates (b) were fluorescently identified with an antibody against PECAM. Comparative vessel counts are expressed per 40x microscopic field and demonstrate a reduction in microvessels in HIF-1 null hearts (c). These data correlated with PECAM levels determined by Western blot using muscle-specific actin as a loading standard (d). Smooth muscle (SMC) -invested vessels were assessed with an anti-SMC actin antibody. Cardiac HIF-1 null hearts had similar numbers of SMC-invested vessels as controls (e), indicating that reduced vascularity in HIF-1 null hearts is at the microvessel level. Left ventricle (LV) to body weight (BW) ratios were increased in cardiac HIF-1 null mice (f) despite reduced vascularity, correlating with increased diastolic thickness of the intraventricular septum (IVSd) and the posterior wall (PWd) as determined by echocardiography (g, h). LV end-diastolic diameter (LVEDD) was significantly reduced in HIF-1 null hearts (i), indicating (in conjunction with the wall thickness data) that loss of HIF-1 leads to thicker hearts with reduced LV chamber size. HIF-1 null = gray bars; *P < 0.05; **P < 0.005.

3. Loss of cardiac myocyte-specific HIF-1 expression results in diminished cellular ATP, phosphocreatine, and lactate levels under normoxic conditions
HIF-1 transcriptionally coordinates expression of lactate dehydrogenase, most glycolytic enzymes, as well as the glucose transporter Glut-1. To determine whether HIF-1 helps define cardiac metabolism under normoxic conditions, we investigated the effects of cardiac HIF-1 deletion on lactate, ATP, and phosphocreatine content in the heart. Loss of HIF-1 resulted in a 23.8 ± 2.1% decrease in lactate levels (P0.05), 29.5 ± 5.6% reduction in ATP content (P0.05), and 15.2 ± 3.3% reduction in phosphocreatine levels (P0.05) without stress provocation, establishing a critical role of HIF-1 in cardiac myocyte energy metabolism under basal conditions.

4. HIF-1 coordinates gene expression in the heart under normoxia
HIF-1 protein levels are increased in the setting of hypoxia via a post-translational mechanism that involves prolyl hydroxylation, von Hippel-Lindau protein-mediated ubiquitylation, and destruction by the proteosome. Nonetheless, HIF-1 levels are detectable under normoxic conditions. We used quantitative RT-PCR analysis to investigate gene expression in normoxic cardiac HIF-1 null and control littermate hearts. Expression of representative metabolism-related genes Glut-1, LDH-A, and PGK was significantly reduced in cardiac HIF-1 null hearts, as were the angiogenesis-associated gene Vegf-A and the gene for the SERCA2 sarcoplasmic reticulum calcium pump. The angiogenic gene encoding platelet-derived growth factor (PDGF-B) was significantly up-regulated despite recent documentation that it is HIF-1 responsive. Endothelin-1 (ET-1), another HIF-1-responsive gene, was also up-regulated, suggesting either alternative regulatory pathways supercede HIF-1 effects or a paracrine source of ET-1. These mRNA reductions were corroborated for selected genes at the protein level (Glut-1, SERCA2) by Western blot.

CONCLUSIONS AND SIGNIFICANCE

Here we show that loss of the HIF-1 transcriptional control pathway in cardiac muscle alters gene expression and has deleterious effects on cardiac function, vascularity, energy availability, and calcium handling. These effects occur without the provocation of induced hypoxia or ischemia and establish that in cardiac muscle transcriptional control by HIF-1 is required during normoxia. In the heart, therefore, HIF-1 appears to act as an oxygen-sensing transcriptional modifier that coordinates gene expression at all oxygen levels, not just in response to hypoxia or ischemia.

The mechanism(s) by which loss of HIF-1 leads to cardiac contractile dysfunction are unclear but may involve the combined effects of hypovascularity, altered calcium handling, and altered energy metabolism with reduced high-energy phosphate content (Fig. 3 ). That we observed contractile abnormalities in isolated HIF-1 null cardiac myocytes establishes that at least some of the documented in vivo cardiac dysfunction is secondary to an intrinsic defect in myocyte contractility independent of altered coronary vascularity. This intrinsic decrease in contractility was accompanied by prolongation of calcium transients and a significant reduction in SERCA2 expression, indicating that altered calcium handling contributes to the phenotype. The importance of the observed alterations in high-energy phosphate content should not be underemphasized. Whether this reduction is directly related to decreased expression of GLUT-1 and the glycolytic enzymes or involves other metabolism-related genes not yet identified as HIF-1 responsive is unclear. What seems clear is that HIF-1 plays a critical role in defining cardiac energetics, even under normoxic conditions. More in-depth analysis of the alterations in glucose and fatty acid metabolism that occur in the absence of myocardial HIF-1 expression is needed to more fully define the role of HIF-1 as a transcriptional regulator of cardiac energy metabolism.

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic delineating the multiple effects of cardiac myocyte-specific deletion of HIF-1 and potential mechanisms. HIF-1 forms a heterodimer with the aryl hydrocarbon nuclear receptor nuclear translocator (ARNT) and binds a core hypoxia response element (HRE) in a growing repertoire of HIF-responsive genes. These include genes involved in angiogenesis, glucose metabolism, vasomotor control, and erythropoiesis, many of which are involved in delivery of oxygen and nutrients to cells or in controlling cellular utilization of these substrates. SERCA2 = sarcoplasmic reticulum calcium ATPase, ET-1 = endothelin 1, Glut-1 = glucose transporter 1, PDGF = platelet-derived growth factor, LV = left ventricle.

In summary, these studies establish that cardiac myocyte-specific HIF-1 expression is required for basal transcriptional activation of multiple genes in the heart during normoxia and that myocardial gene expression is oxygen sensitive at physiologic oxygen levels. Loss of this basal HIF-1-mediated transcriptional activation has deleterious effects on cardiac function, energetics, and vascularization, clear evidence that in the heart HIF-1 plays an important role under normal physiologic conditions. Although not addressed here, HIF-1 likely plays an even more important role in conditions of cardiac stress such as ischemia and pressure overload. Further elucidation of the role of HIF-1 in cardiomyocyte biology may give significant insight into cardiovascular disease states characterized by chronic or recurrent reductions in myocardial oxygen tension and perhaps identify new targets for therapeutic intervention.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1510fje; doi: 10.1096/fj.04-1510fje

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