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European Journal of Heart Failure 2006 8(4):347-354; doi:10.1016/j.ejheart.2005.10.009
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© 2006 European Society of Cardiology

Stabilization of hypoxia inducible factor rather than modulation of collagen metabolism improves cardiac function after acute myocardial infarction in rats

Sebastian Philippa,b,*, Jan Steffen Jürgensenc, Jens Fielitzb,d, Wanja M. Bernhardtc, Alexander Weidemannc, Astrid Schicheb, Bernhard Pilzb, Rainer Dietzb, Vera Regitz-Zagrosekd, Kai-Uwe Eckardtc and Roland Willenbrockb,e

a Department of Cardiology, West-German Heart Center Essen, University of Duisburg-Essen Essen, Germany
b Department of Cardiology, Charité Hospital und Helios Kliniken Berlin-Buch, Humboldt University Berlin Germany
c Division of Nephrology and Hypertension, Friedrich Alexander University Erlangen Nuernberg Erlangen, Germany
d Department of cardiovascular diseases in women, Charité, and German Heart Institute Berlin Germany
e St. Elisabeth Hospital Halle, Germany

* Corresponding author. Department of Cardiology, West-German Heart Center Essen, University of Duisburg-Essen Hufelandstraße 55, 45122 Essen, Germany. Tel.: +49 201 723 4857. E-mail address: sebastian.philipp{at}medizin.uni-essen.de


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Prolyl hydroxylase domain-containing enzymes (PHD) hydroxylate a proline residue that controls the degradation of hypoxia inducible factor (HIF). Hypoxia inhibits this hydroxylation thus increasing HIF levels. HIF is upregulated in ischemic tissues, growing tumors and in nonischemic, mechanically stressed myocardium. Pharmacological inhibition of prolyl 4-hydroxylase (P4-H) stabilizes HIF-protein in vitro and may modulate collagen turnover. The aims of this study were to investigate whether inhibition of P4-H protects myocardium against ischemia, and whether the observed effects are related to modulation of collagen metabolism or due to the stabilization of HIF.

Methods: Rats were treated with a specific P4-H inhibitor (P4-HI) or vehicle starting 2 days before induction of myocardial infarction (MI). Rats were investigated 7 or 30 days after MI. Induction of HIF-1{alpha} and -2{alpha} was visualized by immunohistochemistry. Expression of growth factors (connective tissue growth factor, Osteopontin) and mRNA expression and protein levels of Collagen I and III as well as HIF-2{alpha} were measured.

Results: P4-HI augments HIF in the myocardium as early as 24 h after treatment. P4-HI did not alter the MI-induced enhanced expression of growth factors and collagen. Treatment with P4-HI significantly reduced heart and lung weight, improved left ventricular contractility, prevented left ventricular enlargement and improved left ventricular ejection fraction without affecting infarct size after 30 days.

Conclusions: Specific inhibition of the P4-H improved cardiac function without affecting the infarct size after experimental myocardial infarction in rats. Stabilization of HIF rather than inhibition of collagen maturation by P4-HI may prevent cardiac remodeling after MI.

Key Words: Heart failure • Hypoxia inducible factor (HIF) • Infarction • Ischemia • Remodeling

Received February 19, 2005; Revised July 23, 2005; Accepted October 13, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Interruption of coronary blood flow leads to infarction of the left ventricle myocardium and induces complex remodeling processes with an array of adaptive reactions, which include changes in cell metabolism, new vessel formation, matrix production and myocyte replication [1,2]. Hypoxia inducible factor (HIF)-1 is a transcription factor that functions as a global regulator of oxygen homeostasis. The target genes regulated by the HIF-protein play an important role in structural and functional biology, including genes involved in energy metabolism, cytoprotection, vasomotor regulation, angiogenic growth, matrix metabolism, cell growth, and survival decisions [1,2]. HIF-1 activity is induced by hypoxia because oxygen-dependent prolyl hydroxylation targets HIF for ubiquitinylation and proteasomal degradation and plays critical roles in the responses of the cardiovascular and respiratory systems to hypoxia [3-6]. Comparatively few studies on HIF have been reported in cardiac myocytes [7,8]. Recently, HIF-1P protein was observed to be induced in the ischemic human myocardium. In biopsy specimen obtained from ischemic or infarcted myocardium, HIF-1P and VEGF proteins were detected by immunohistochemical staining, whereas they were undetected in specimen of the nonischemic myocardium [9]. HIF-1P is further induced in the nonischemic myocardium by a phosphatidylinositol 3-kinase-dependent induction in response to mechanical stress [10]. If HIF-1P is deleted cardiac contractile function is reduced, cellular ATP as well as phosphocreatinine and lactate levels are diminished even under normoxic conditions [11].

We showed previously that both HIF-subunits are induced in the heart after myocardial infarction, first in the infarct surrounding tissue, shortly thereafter also in the non-infarcted myocardium [12]. This illustrates that the early induction of HIF-1P mediates the transcription of the VEGF gene in the ischemic myocardium, which is one of the first adaptations of human myocardium to ischemia. Left ventricular (LV) remodeling after myocardial infarction (MI) contributes significantly to LV dilation and dysfunction, disability and death [13]. How the remodeling process is affected by hypoxia inducible factors is yet unknown.

LV remodeling due to myocardial infarction also involves activation of neuroendocrine systems such as renin-angiotensin-aldosterone system, enhanced growth factor expression and dysbalanced extracellular matrix turnover [13,14]. Posttranslational modification of proteins involved in collagen maturation are influenced by prolyl 4-hydroxylases (P4-H), which regulates their abundance, function and activity and are thereby involved in LV remodeling processes [15]. P4-H is a key enzyme in collagen synthesis and maturation catalysing hydroxylation of proline residues. Proline hydroxylation results in thermally stable triple helix collagen. Inhibition of P4-H has been shown to be effective in preventing LV remodeling following acute myocardial infarction [15].

In order to provide further understanding of the function of inhibition of the mechanism underlying the protection by inhibition of the P4-H we investigated the effect of a novel P4-HI on LV remodeling, collagen metabolism (Collagen I and III), growth factors (connective tissue growth factor (CTGF) and Osteopontin) and HIF following acute myocardial infarction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal experiments
To ligate the coronary arteries male Wistar rats (body weight 200-240 g) were anesthetized by chloral hydrate (400 µg/kg i.p.), intubated and artificially ventilated with a rodent ventilator with room air. Anterolateral thoracotomy was performed and the heart was rapidly exteriorized. A 7-0 silk suture was snared around the proximal left anterior descending (LAD) coronary artery and tightly ligated to occlude the vessel. The heart was then placed back and the chest was closed in 2 layers with 3-0 silk sutures. The animal was then allowed to recover. Sham-operated rats were treated in the same manner including snaring, but the coronary artery was not ligated.

Perioperative mortality was 45% in infarcted rats, which is usual in the rats species used in this study, and less than 5% in sham-operated rats. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the local committee. Study groups were as follow:

  1. This group was treated for 9 days starting 48 h prior to MI, echocardiography was performed 6 days after MI and hemodynamic measurements were made 7 days after MI (n=12 each for P4-HI and sham, n=14 for vehicle).
  2. This group was treated for 32 days starting 48 h prior to MI, echocardiography was performed 28 days after MI and hemodynamic measurements were made 30 days after MI (n=11 for P4-HI, n=11 for vehicle, n=15 for sham).
  3. To investigate whether inhibition of prolyl 4-hydroxylase by the specific inhibitor FG 2216 [16,17] will induce HIF in vivo, animals were treated for 24 h, 48 h and 7 days. Thereafter hearts, liver and kidneys were removed and HIF was measured in the tissue by immunohistochemistry. Vehicle-treated animals served as controls (n=3 for each group for immunohistochemistry).

At the termination of the study rats were anesthetized, organs were removed and weighed. The heart was dissected into left and right atrium, septum, left and right ventricle. All organs were immediately flash-frozen in liquid nitrogen and stored at –80 °C until further biochemical analysis. For analysis of HIF by immunohistochemistry the hearts were cut into 1.5 mm transverse slices and fixed by immersion in 3% paraformaldehyde and embedded in paraffin.

2.2. Treatment with P4-HI
The prolyl 4-hydroxylase inhibitor FG 2216 (30 mg/kg twice daily) was administered by oral gavage. The animals received either P4-HI or vehicle.

2.3. Hemodynamic measurements
Before sacrifice the rats were intubated and artificially ventilated under chloral hydrate anesthesia. A PE 50-catheter was inserted through the right jugular vein into the superior vena cava. Arterial blood pressure was measured directly via the left carotid artery with a Millar tip high fidelity catheter. Left ventricular hemodynamics were measured with a Millar catheter and registered with a Statham transducer (P23XL) and a Gould amplifier (AMP 4600). Left ventricular contractility (LV dP/dtmax) was obtained from the ventricular pressure curves. After completion of hemodynamic measurements the rats were sacrificed.

2.4. Echocardiography
A two-dimensional short-axis and long-axis view of the left ventricle was obtained with a 15-MHz transducer (Acuson Sequoia, Erlangen, Germany) in anesthetized rats. M-mode tracings were recorded and used to determine the diameter of the left ventricle at the end of the diastole (LVEDD) and systole (LVESD). Ejection fraction was calculated using short- and long-axis views. Measurements were done online by an observer blinded to treatment according to the American Society for Echocardiography leading-edge method.

2.5. Measurements of infarct size
Infarct size was determined using a technique described by Chien et al. [18]. Briefly, after sacrifice of the rats, the right ventricle (RV) and the left ventricle (LV), including the interventricular septum, were dissected, separated, and weighed. Incisions were made in the LV so that the LV tissue could be pressed flat. The LV circumference and region of infarction were outlined on a clear plastic sheet for both the endocardial and epicardial surfaces. The difference in size between the two marked areas on the sheet was used to determine the relative size of the MI using the NIH-Image software. The infarct size was expressed as a percentage of LV surface area.

2.6. Determination of HIF by immunohistochemistry
Immunohistochemistry was carried out on 4-µm sections of paraffin-embedded formaldehyde-fixed tissue as described previously [19]. HIF-1P was detected by a mouse monoclonal antibody, which reacts with human and rat HIF-1P from many species including the rat (P67, Novus Biologicals, Littleton) at a dilution of 1:10,000. HIF-2P was detected by polyclonal rabbit anti-mouse antibody (PM9, obtained from a rabbit immunized against a fusion-protein containing a 337-439 of mouse HIF-2P, a kind gift of Prof. Maxwell, London), at a dilution of 1:10,000. All incubations were performed in a humidified chamber. Between incubations, specimens were washed 2-4 times in TBST (50 mM Tris-HCl, 300 mM NaCl, 0.1% Tween 20, pH 7.6). Biotinylated secondary anti-mouse or anti-rabbit antibodies and a catalyzed signal amplification system (CSA-DAKO, Hamburg, Germany), based on a streptavidin-biotin-peroxidase reaction, were used. Controls included sham-operated animals, the omission of primary antibodies and the use of preimmune serum of animals immunized against HIF-2P. Signals were analyzed with a Leica DMRB microscope (Leica, Bensheim, Germany) in differential interference contrast. Photographs were digitally recorded by means of a Visitron system (Visitron, Puchheim, Germany).

2.7. HIF protein extraction and immunoblotting
Protein extraction and blotting from the left ventricles of rats after 30 days were performed essentially as described previously [20]. Sections of the frozen tissue were fractionated, weighed, and homogenized into 20-fold excess of extraction buffer (7 M urea, 10% glycerol, 10 mM Tris-HCl at pH 6.8, 1% SDS, 5 mM DTT (all chemicals from Sigma Chemical Co.-Aldrich, Taufkirchen, Germany) and Complete Mini Protease-Inhibitor Cocktail (Roche, Mannheim, Germany)) with an electric homogenizer (Ultra-Turrax; IKA, Staufen, Germany). Extracts were quantified with the Bio-Rad DC protein assay (Bio-Rad, Munich, Germany), and 300 µg of each extract were resolved on 8% polyacrylamide gels. Proteins were transferred onto Immobilon P membrane (Millipore, Bedford, MA) overnight and probed with anti-HIF-2P polyclonal rabbit anti-mouse antibody (PM9, see above) at a dilution of 1:400. As an additional standard, protein extracts of hypoxic rat PC12W cells were used. Signals were detected with horseradish peroxidase-conjugated antibodies (DAKO, Ely, United Kingdom) and enhanced chemiluminescence (SuperSignal West Dura Extended; Pierce, Rockford, IL). Blots were quantified using the luminescent image analyzer LAS 100 (Fujifilm) and AIDA image analyzer software (Raytest, Straubenhardt, Germany). After analysis, membranes were stained with Coomassie Blue (Sigma Chemical Co.) to verify equal protein loading and transfer.

2.8. Quantitation of mRNA by real time PCR
Total RNA preparation, deoxyribonuclease (DNase) digestion and reverse transcription were performed as described previously [21,22]. Briefly, RNA was extracted from LV samples using the RNAzol B2122; reagent (Lorei+Pasel, Germany). RNA was digested with RNase-free DNase I (Boehringer, Germany). From each sample, 250 ng DNase digested RNA was reverse transcribed with random hexamers and using SuperscriptTM RNaseH-Reverse Transcriptase according to the manufacturer's protocol (Gibco BRL, Germany). A "hot start" real time PCR procedure with SYBR Green that was validated in respect of reproducibility and linearity within the measuring range was performed in duplicates with the TaqManTM 7700 instrument (ABI). Primers for all target genes were designed using Dnasis version 2.1 and the PrimerExpress Software (PE Applied Biosystems). The primer sequences are shown in Table 1. All PCR reactions had efficiencies of about 1.9. A calibration curve containing 50, 25, 12.5, 6.25 and 3.125 ng cDNA pooled from all samples analyzed was used to estimate relative changes of mRNA expression within each sample and was run in each PCR reaction with the specific primers used. To correct for potential variances between samples in mRNA extraction or in RT-efficiency, the mRNA content of the target genes was normalized to the expression of the stably expressed reference gene GAPDH in the same sample by using the delta-delta-ct-method.


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  		Table 1 Primer sequence

 
2.9. Protein extraction and immunoblotting
LV myocardial samples were homogenized (30 s, 2000 rpm) in ice-cold extraction buffer (1:3 wt./vol) containing (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA 25% Glycerol, 0.5% SDS, 0.5% Nonident P-40, 0.1 mM DTT, 0.5 mM PMSF, 100 ng/ml Protease inhibitor cocktail) and then cleared by centrifugation (4 °C, 10 min, 14.000 rpm). The supernatant was assayed for protein concentration using Bio-Rad Protein Assay and flash-frozen in liquid nitrogen at –80 °C. Ten to twenty microgram of protein was separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes (Fa. Amersham). Membranes were incubated with specific monoclonal antibodies against pro-MMP-2 (Santa Cruz, SC10736, rabbit, 1:300) and Collagen I and III (DPC Biermann, R-1038, 1:2.000). As secondary antibody, specific HRP-conjugated antibodies were used and the signal was visualized with the ECL detection kit (Amersham Pharmacia Biotech). The immunoblots were digitized on a transluminate scanner and the specific protein band was analyzed with AlphaEaseFC (Software, version 3.1.2, Alpha Innotech Corporation). To normalize for different protein content, we stripped the membrane from the first antibody complex with buffer A (Buffer A: 200 mM Glycin pH 2.2, 0.1% SDS, 1% Tween 20) for 12 h at 4 °C and re-hybridized for GAPDH (primary antibody: Chemicon, MAB-374, 1:5.000; secondary antibody: Donkey anti-mouse, Dianova, 1:50.000) as described above.

2.10. Statistical methods
Values are given as mean±SEM except where indicated. Differences in echocardiographic and hemodynamic parameters as well as organ weights between MI-P4-HI, MI-control, and sham were analyzed by one-way ANOVA (Tukey-test), followed by a modified Student's t-test. RT-PCR statistics were calculated with the Excel 2000, Sigma Plot 8.0 and SPSS 11.0 software. The t-test was used to calculate differences between the examined groups and ANOVA corrections for multiple testing were used. A p-value≤0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Inhibition of prolyl 4-hydroxylase improves cardiac function 7 days after myocardial infarction
Rats were treated continuously with P4-HI for 9 days. Fig. 1 reveals that vehicle-treated animals with myocardial infarction showed an increased end-diastolic and end-systolic diameter over that in the sham-operated (no MI) animals. As expected, vehicle-treated rats with MI showed a reduced cardiac function compared to sham-operated rats. Treatment with P4-HI greatly blunted the left ventricular dilatation and the impairment of left ventricular function. There was no difference in heart (relative heart weight 4.0±0.1 mg/g for vehicle vs. 4.2±0.3 mg/g for FG 2216, p=ns) or lung weights (1168±21 mg for vehicle vs. 1182±25 mg for FG 2216, p=ns) among these groups. Hemodynamic parameters did not differ between the three groups.


Figure 1
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  		Fig. 1 Echocardiographic data 6 days after myocardial infarction. Echocardiographic variables of sham-operated rats, vehicle-treated rats and rats receiving P4-HI. Echocardiography was performed 6 days post surgery. Data are presented as mean value±standard error of the mean. LVEDD and LVESD = left ventricular end-diastolic and end-systolic diameter, respectively; FS = fractional shortening of left ventricle. *p<0.05 vs. sham, +p<0.05 vs. vehicle.

 
3.2. Inhibition of prolyl 4-hydroxylase reduces cardiac remodeling 28 days after myocardial infarction
Myocardial infarction increased heart and lung weight, reduced cardiac function and led to an enlargement of the left ventricle. Treatment with P4-HI prevented left ventricular end-diastolic dilatation, significantly reduced end-systolic dilatation and improved left ventricular function (Fig. 2). Heart weights and lung weights were significantly lower in the long-term treatment group compared to vehicle-treated rats (Table 2). Neither blood pressure nor left ventricular end-diastolic pressure (LVEDP) were different among the treated groups although there is a reduction of LVEDP (p=0.05) in the group receiving P4-HI (Table 2). Myocardial infarction reduced left ventricular contractility (dP/dtmax). Treatment with P4-HI significantly improved left ventricular contractility compared to vehicle-treated rats. The infarct size caused by ligation of the LAD was unaffected by P4-HI (32.2% with P4-HI vs. 33.8% with vehicle) indicating that the effect was not due to a reduced infarct size which could have resulted from drug-induced angiogenesis of new collaterals.


Figure 2
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  		Fig. 2 Echocardiographic data 28 days after myocardial infarction. Echocardiographic variables of sham-operated rats, vehicle-treated rats and rats receiving P4-HI. LVEDD = left ventricular end-diastolic diameter, LVESD = left ventricular end-systolic diameter, EF = left ventricular ejection fraction, measured in 4 chamber view. **p<0.01 vs. sham, +p<0.05, ++p<0.01 vs. vehicle. Data are expressed as the mean value±standard error of the mean.

 


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  		Table 2 Body weight, organ weight and hemodynamic parameters 30 days after myocardial infarction

 
3.3. Induction of HIF by inhibition of prolyl 4-hydroxylase in vivo
It has been shown for other compounds that hydroxylase inhibitors can be used to stabilize HIF-P and to enhance HIF activity and its downstream effects, including the induction of angiogenesis [23,24]. In this study we used a specific inhibitor of the prolyl 4-hydroxylase [16,17,25]. The inhibitor induced both HIF-1P and HIF-2P in the heart, kidney and liver after 24 h (Fig. 3). The regional distribution was ubiquitous and persisted in all organs over the treatment period. At the end of the study we were able to show a clear increase of HIF-2P protein in the left ventricle of the P4-HI-group compared to vehicle-treated and control rats (Fig. 4a and b). A statistical comparison between the groups (n=4) was not performed.


Figure 3
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  		Fig. 3 Expression of HIF after P4-HI. Immunohistochemistry of HIF-1P and HIF-2P after treatment with the prolyl 4-hydroxylase inhibitor (P4-HI). The nuclear accumulation of the transcript factor is detectable in cardiomyocytes, interstitial cells and endothelial cells.

 


Figure 4
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  		Fig. 4 Immunoblots for HIF-2P. (a) HIF-2P induction in the left ventricle: representative example of one out of four immunoblots for HIF-2 of tissue extracts from left ventricles at the time of sacrifice 30 days after myocardial infarction. (b) Induction of HIF-2P in left ventricles in relation to control after 30 days. Data are presented as mean value±SEM (n=4).

 
3.4. Effect of MI and P4-HI treatment on collagen expression
As expected, collagen I and III protein levels and mRNA were elevated in the nonischemic ventricle of vehicle-treated infarcted rats as compared to sham-operated rats. Treatment with P4-HI did not significantly affect the collagen I or III after 30 days of myocardial infarction (Fig. 5).


Figure 5
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  		Fig. 5 Collagen I and III mRNA and protein expression. Collagen I and III mRNA and protein expression are shown. mRNA or protein expression of each was normalized to GAPDH. Mean of sham rats was set as 100% and MI and MI+FG 2216 (P4-HI) are expressed in percentage of sham. Data are presented as mean value±SEM. *p<0.05 vs. sham.

 
3.5. Effect of MI and P4-HI treatment on growth factors
To further understand the mechanism involved in the cardioprotection by P4-HI we looked at the regulation of growth factors such as connective tissue growth factor (CTGF) and Osteopontin. CTGF mRNA was increased almost 6-fold, and Osteopontin mRNA was increased 9-fold in rats with MI (Fig. 6). P4-HI had no significant effect on Osteopontin or CTGF expression after 30 days of treatment (Fig. 6).


Figure 6
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  		Fig. 6 CTFG and OPN mRNA expression. mRNA expression of specific genes are normalized to GAPDH. Mean of sham rats was set as 100% and MI and MI+FG 2216 (P4-HI) are expressed in percentage of sham. Data are presented as mean value±SEM. *p<0.05 vs. sham.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We report for the first time that inhibition of prolyl 4-hydroxylase by an orally available isoquinoline based inhibitor prevents changes in cardiac function independent of a reduction of collagen maturation or altering growth factors. Inhibition of P4-H led to an improvement of cardiac function by increasing left ventricular contractility and prevention of left ventricular dilatation. Inhibition of prolyl 4-hydroxylase with the specific inhibitor FG 2216 increased myocardial HIF levels prior to induction of myocardial infarction and throughout the treatment period. This study shows that inhibition of prolyl 4-hydroxylase can modulate the myocardial response to infarction and identifies a new therapeutic approach to the preservation of function in the post-MI heart.

Ischemic myopathy is a major cause for morbidity and mortality in all industrialised countries, and effective molecular therapies are being intensively sought [26,27]. Since the HIF system is upregulated by inhibition of P4-H, this protective system might be involved in the beneficial effects observed in our study. The transcription factor HIF is a primary regulator of the hypoxic response, controlling genes involved in diverse processes that balance metabolic supply and demand within tissues [28,29]. This makes modulation of HIF activity an attractive approach for the treatment of ischemic disease. Stabilization of HIF-P polypeptides by pharmacological inhibitors of HIF-P4-Hs is believed to be therapeutically beneficial in diseases characterized by acute or chronic ischemia, such as myocardial infarction, stroke, peripheral vascular disease and diabetes [30-32]. Under normoxia HIF is continuously generated and then rapidly degraded by prolyl hydroxylase. Changes in mRNA expression are normally not seen. Neither hypoxia nor inhibition leads to an increase of HIF-RNA but was reported to alter the mRNA of the hydroxylase [33]. We demonstrated in this study that the P4-HI is capable of augmenting HIF in vivo thus demonstrating the potential of a new non-peptide protein hydroxylase inhibitor as a pharmacological activator of the HIF pathway and as a therapeutic tool in treating ischemic diseases. We clearly demonstrated that the P4-HI used in our study has no effect on extracellular collagen metabolism or growth factors such as CTGF and Osteopontin, although other effects, particularly on cellular metabolism was not looked at and therefore, cannot be ruled out. It has been described that the blockade of HIF degradation with a peptide inhibitor modulates cellular metabolism and angiogenesis by induction of VEGF, angiopoietin-2 and angiopoietin-4 [34]. Further it has recently been demonstrated by Warnecke et al., that inhibition of HIF degradation by P4-HI induces angiogenesis in a rat model [23].

We were able to show the cardioprotective effect of H4-PI as an early effect already after 7 days of MI as well as a chronic effect after 30 days of MI. Recently it has been demonstrated that intramyocardial injection of a DNA encoding HIF-1a/VP16 hybrid enhances angiogenesis in an acute myocardial infarction model in the rat [35]. Induction of HIF prior to MI could have stimulated collateral growth in the heart to the extent that a significant portion of the ischemic tissue might be spared from infarction. That could have improved cardiac function by reducing infarct size. However the infarct size was not affected by treatment with H4-PI, indicating that the beneficial effect must have occurred during the remodeling phase. Clearly inhibition of P4-H exerted its salutary effect not only at the time of infarction but in the days and weeks following, the crucial phase of cardiac remodeling.

The beneficial effect observed in this study is assumed to be the induction of HIF, which was elevated throughout the study. As systolic and diastolic blood pressures were unchanged, hemodynamic or afterload reducing effects are very unlikely to have contributed. In this setting a possible reduction of extracellular matrix collagen has to be discussed. At first sight, knowing that P4-H is an essential enzyme in collagen biosynthesis, it could be debated that the effects observed in this study are due to inhibition of collagen synthesis. While we could not observe any relevant effect of P4-HI on collagen mRNA nor protein levels, Nwogu et al. [15] suggested that the prevention of left ventricular enlargement might be due to effects on extracellular matrix collagen. This difference may be explained by the compounds used. Whereas Nwogu used a phenanthroline P4-HI (FG 0041) we used an isoquinoline based P4-HI (FG 2216). This compound has been shown to induce HIF and VEGF [17] and is currently involved in clinical trials focusing on anemia [25]. Apparently FG 2216 has no relevant effect on collagen metabolism in vivo. Different P4-HIs seem to target different subunits of the enzyme [30]. With the compound used by Nwogu, different effects, particularly on collagen synthesis were observed [15]. The compound we used in our study did not have any effect on collagen expression.

Although we cannot yet determine definitely the mechanism which confers the beneficial effects of prolyl 4-hydroxylase inhibition, we conclude that continuous treatment with the prolyl 4-hydroxylase inhibitor FG 2216 is able to prevent deleterious remodeling in this model of ischemic heart failure. It is unknown whether these protective effects are also present in nonischemic heart failure, and this issue warrants further investigation.


   Acknowledgements
 
The authors thank James M. Downey of the University of South Alabama for helping with the manuscript. Further the authors thank Jeannette Mothes, Jutta Meisel and Rita Günzel for excellent technical and secretarial assistance. This study has been in part financially supported by FibroGen Inc. Jens Fielitz received the Pfizer fellowship of the German Society of Cardiology. This study was in part supported by GIS, Munich.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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