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European Journal of Heart Failure 2007 9(4):336-342; doi:10.1016/j.ejheart.2006.10.006
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© 2007 European Society of Cardiology

Inhibition of prolyl 4-hydroxylase prevents left ventricular remodelling in rats with thoracic aortic banding

Jens Fielitzb,1, Sebastian Philippd,f,1, Lars Roman Herdaa, Evelyn Schucha, Bernhard Pilzd, Carola Schuberta, Volkmar Günzlere, Roland Willenbrockd,g and Vera Regitz-Zagroseka,c,*

a Center for Gender in Medicine (GiM), and Cardiovascular Research Center (CCR), Charité, Universitätsmedizin Berlin Germany
b Department of Cardiology, CVK, Charite, Universitätsmedizin Berlin Germany
c German Heart Center, DHZB, Berlin Germany
d Franz-Volhard-Clinic, HELIOS Clinics GmbH Charité CCB, Germany
e FibroGen Inc. San Francisco, CA, United States
f Department of Cardiology, West German Heart Centre, University Duisburg-Essen Essen, Germany
g St. Elisabeth Hospital Halle, Germany

* Corresponding author. Center for Gender in Medicine (GiM), and Cardiovascular Research Center (CCR), Charité, Universitätsmedizin Berlin, Germany, Hessische Street 3–4, 10115 Berlin, Germany. Tel.: +49 30 450 525 172; fax: +49 30 450 525 972. E-mail address: vera.regitz-zagrosek{at}charite.de


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Background: Pressure overload leads to myocardial remodelling with collagen accumulation, left ventricular hypertrophy (LVH), neurohormonal activation and myocardial dysfunction. Prolyl 4-hydroxylases (P4H) are involved in collagen maturation. Inhibition of P4H has been shown to prevent LV remodelling and improve survival post-myocardial infarction.

Aim: To evaluate the role of P4H in pressure overload-induced myocardial remodelling.

Methods: Male Wistar rats underwent thoracic aortic banding (AoB) and were treated with a P4H inhibitor (P4HI) or vehicle (control). Echocardiography and haemodynamic measurements were performed after 4 weeks. Collagens, matrix metalloproteinases (MMP), tissue inhibitors of MMPs (TIMP), growth factors and neurohormonal markers were quantitated in LV samples.

Results: AoB led to LVH, increased LV enddiastolic pressure (LVEDP) and decreased contractility compared to sham. P4HI reversed these effects. AoB increased collagen I and III expression, which was normalized by P4HI. AoB led to deregulation of matrix remodelling enzymes, enhanced expression of growth factors and activation of the endothelin system. P4HI partially prevented deregulation of the MMP/TIMP system, inhibited upregulation of growth factors and normalized AoB-induced ECE-1 and ETB expression.

Conclusions: P4HI leads to an improvement of AoB-associated LV dysfunction and reduces imbalance of extracellular matrix turnover and hypertrophy-associated gene expression. P4H inhibition could therefore be of value in treatment of myocardial remodelling accompanying pressure overload hypertrophy.

Key Words: Aortic banding • Hypertrophy • Remodelling • Growth factors • Prolyl 4-hydroxylase

Received June 2, 2006; Revised July 21, 2006; Accepted October 9, 2006


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Chronic pressure overload leads to left ventricular (LV) hypertrophy and fibrosis, which are independent cardiovascular risk factors, leading to an increase in morbidity and mortality of patients [1-4]. Hypertrophy-associated extracellular remodelling processes with collagen accumulation and fibrosis within the LV are early events in pressure overload such as human aortic stenosis [5] and lead to activation of neuroendocrine systems, enhanced growth factor expression and imbalanced extracellular matrix turnover. Ongoing stimulation of these profibrotic pathways causes LV dysfunction and heart failure [6]. In particular, an imbalance between matrix metalloproteinases (MMP) and their inhibitors (TIMP) disturbs myocardial collagen turnover and leads to collagen deposition in LVH [7-11] further promoting LV remodelling. Accumulation of myocardial collagen has been shown to be associated with an increase in LV enddiastolic pressure (LVEDP) [12] and reduction of LV systolic function in human aortic stenosis [5]. Therefore, we speculated that inhibition of myocardial collagen deposition and LV fibrosis may result in an improvement of cardiac function in chronic pressure overload.

Haemodynamic overload also induces myocardial growth factors like transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) which contribute to pathological LV remodelling [13]. In addition, pressure overload leads to activation of integrin β1 (ITGβ1) and osteopontin (OPN), which are thought to be stress mediators responsible for transformation of extracellular stress into intracellular profibrotic and prohypertrophic signals [14-17]. In addition to the disturbance in collagen synthesis and degradation, hypertrophic LV remodelling involves activation of neuroendocrine systems such as the endothelin- and renin-angiotensin-aldosterone systems [18]. In particular, cardiac endothelin 1 and the ETa receptor are upregulated by pressure overload [19] and congestive heart failure [20].

Post-translational modification of collagen proteins by prolyl 4-hydroxylase (P4H) is involved in LV remodelling processes [21]. P4H is a key enzyme in collagen maturation, catalysing hydroxylation of proline residues, resulting in thermally stable triple helix collagen. Inhibition of P4H is therefore expected to favour production of unstable collagen within the endoplasmatic reticulum, which then is rapidly degraded. The orally available P4H inhibitor (FG 0041), reduced the amount of collagen and collagen proline hydroxylation, prevented LV remodelling and improved survival when administered for 4 weeks, starting 48 h after induction of myocardial infarction in rats [21]. Our study extends this investigation. The aim of the present study was to determine whether administration of an orally available P4H inhibitor would also lead to beneficial myocardial effects in a rat model of aortic stenosis.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
2.1. Animal experiments
Four week-old male Wistar rats (80-100 g) (Moellegaard Animal Farms, Schoenwalde, Germany) were randomised to either sham operation (n=21) or banding of the thoracic aorta (AoB, n=42). Animals were housed under standard conditions and maintained on commercial rat chow and water ad libitum. Anaesthesia for surgery was induced by chloral hydrate (400 mg/kg, i.p.), followed by orotracheal intubation and ventilation. At the experimental end point, body and organ weights were recorded, the LV separated and immediately flash-frozen in liquid nitrogen and stored at –80 °C until further biochemical analysis. AoB was performed as previously described [22]. For induction of AoB, a tantalum clip with internal diameter of 0.51 mm (Ethicon, USA) was placed around the ascending aorta 0.5-1 mm above the aortic valve. Sham-operated rats were treated identically, except that no clip was placed. The mean pressure gradient over the clip was 15 mm Hg after 1 week and 35 mm Hg after 4 weeks following clip placement. The perioperative mortality rate was 10%. From days 2 to 30, AoB rats received P4HI (n=14; 25 mg/kg BW/d bid) or vehicle (n=15, carboxymethylcellulose (CMC)) by oral gavage. The P4HI (FG 0041, 3-Carboxy-4-oxo-3,4-dihydro-1,10-phenanthroline) was provided by Fibrogen, Inc, its efficacy in preventing LV remodelling following myocardial infarction in rats, its substrate specificity and chemical characterization has been published previously [23,24]. FG0041 inhibits collagen-P4H at low µM concentrations (Ki=2 µM) at which it does not inhibit HIF-P4H. Housing and experiments were performed in accordance with the guidelines of the Charité, Universitätsmedizin Berlin, and the recommendations of the Society for Laboratory Animal Science (GV-SOLAS) and the Federation of European Laboratory Animal Science Association (FELASA).

2.2. Echocardiographic measurements
Echocardiography was performed on treatment day 28. 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 anaesthetised rats. M-mode tracings were recorded and used to determine the thickness of the LV posterior wall (PW) and the interventricular septum (IVS) in diastole. Left ventricular ejection fraction (LVEF) was calculated using short and long-axis views. Transmitral and aortic outflow measurements could not be done due to artefacts caused by the tantalum clip. Measurements were done online by an observer blinded to treatment according to the American Society for Echocardiography leading-edge method [25].

2.3. Haemodynamic measurements
Thirty days after AoB, the animals were intubated and artificially ventilated under chloral hydrate anaesthesia. Haemodynamic data were obtained as previously described [26]. Briefly, 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. Left ventricular haemodynamics were measured after thoracotomy by inserting a PE 50-catheter with a 14G needle through the free left ventricular wall into the left ventricle as previously described [26]. Due to this we were not able to use a high fidelity catheter for left ventricular haemodynamics. For better comparison, all other parameters were obtained using a similar catheter system. Pressures were registered with a Statham transducer (P23XL) and a Gould amplifier (AMP 4600). Left ventricular contractility was obtained from the ventricular pressure curves.

2.4. Quantitation of mRNA by real time PCR
Total RNA preparation, deoxyribonuclease (DNase) digestion and reverse transcription were performed as described previously [12,27]. Briefly, RNA was extracted from LV samples using the RNAzol B– reagent (Lorei+Pasel, Germany). RNA was digested with RNase-free Dnase I (Boehringer, Germany) and reverse transcribed using Superscript– RNaseH-Reverse Transcriptase according to the manufacturers protocol (Gibco BRL, Germany). A "hot start" real time PCR procedure with SYBR Green was performed in duplicates with the TaqMan– 7700 instrument (ABI). Primers for all target genes were designed using Dnasis version 2.1 and the PrimerExpress Software (PE Applied Biosystems). Primer sequences are shown in supplementary electronic Table A. A calibration curve was used to estimate relative changes in mRNA expression and was run in each PCR reaction with the specific primers used. GAPDH was used as an internal standard.

2.5. Protein extraction, immunoblotting and zymography
LV myocardial samples were homogenized (30 s, 2000 rpm) in ice-cold extraction buffer (1:3 wt/vol) containing (10 mmol/L Tris HCL, pH 7.5, 140 mmol/L NaCl, 1 mmol/L EDTA 25% Glycerol, 0.5% SDS, 0.5% Nonidet P-40, 0.1 mmol/L DTT, 0.5 mmol/L PMSF, 100 ng/ml Protease inhibitor cocktail) and then cleared by centrifugation (4 °C, 10 min, 14.000 rpm (Roche Diagnostics GmbH, Germany)). The supernatant was assayed for protein concentration using Bio-Rad Protein Assay and flash frozen in liquid nitrogen. Ten to 20 µg of protein were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were incubated with an antibody against 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). To normalize for different protein content membranes were stripped with buffer A (Buffer A: 200 mmol/L Glycin pH 2.2, 0.1% SDS, 1% TWEEN 20) for 12 h at +4 °C and re-hybridised for GAPDH (primary antibody: Chemicon, MAB-374, 1:5.000; secondary antibody: Donkey anti-mouse, Dianova, 1:50.000) as described above. Zymography [28] and quantification of collagen cross linking by determination of hydroxyproline to proline ratio [21] were performed as described previously. The immunoblots and zymograms were digitised on a transluminate scanner and the specific band was analysed with AlphaEaseFC (Software, version 3.1.2, Alpha Innotech Corporation).

2.6. Statistical methods
Values are given as mean±SEM except where indicated. Normalized Gene/GAPDH or protein/GAPDH ratios were compared to the mean ratio of the respective sham-group which was set as 100%. Differences in morphologic and biochemical parameters between animal groups were analysed by Mann-Whitney U-Test, one way ANOVA comparison with Tukey post-hoc test, or two-sided student's t-test. Statistics were calculated with the Excel, Sigma Plot and SPSS software. A p-value<0.05 was considered to be statistically significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
3.1. Morphology and functional data
AoB significantly increased the relative heart and lung weight. P4HI significantly reduced the AoB-induced relative lung weight suggesting a reduction in pulmonary congestion (Fig. 1). A tendency towards reduction of relative heart weight by P4HI achieved only borderline statistical significance (p=0.07). AoB increased the thickness of the interventricular septum (IVS) and LV posterior wall (LVPW) (Table 1). P4HI did not have an effect on AoB induced increase of IVS or LVPW thickness. LV enddiastolic pressure (LVEDP) and LV systolic pressure (LVsys) (Table 1) were significantly increased by AoB. P4HI significantly reduced the elevated LVEDP but had no effect on LVsys. In addition, LV dp/dtmax was significantly reduced by AoB, which was reversed by P4HI (Table 1). The heart rate was unaffected in all groups.


Figure 01
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  		Fig. 1 Post-mortem morphologic measurements in sham, aortic banding and P4HI groups. Relative heart and lung weights are shown. Data are expressed as mean value±SEM. ** p<0.01 sham vs. AoB, § p<0.05 AoB vs. AoB+P4HI treatment.

 


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  		Table 1 Functional characterization of sham, aortic banding and P4HI groups

 
3.2. Effect of AoB and P4HI treatment on collagen expression and cross linking
Collagen type I and III mRNA expression were increased under AoB (p<0.01 for all). P4HI normalized AoB-induced collagen I and III mRNA expression (Fig. 2). Collagen type I and type III protein expression were in parallel with its respective mRNA, showing an increase under AoB (p<0.05 for all), and a decrease with P4HI.


Figure 02
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  		Fig. 2 Collagen I and III mRNA and protein expression in sham, aortic banding and P4HI groups. Collagen I and III mRNA and protein expression (a) and representative Western Blot for collagen I are shown (b). a) mRNA or protein expression of collagen I and III was normalized to GAPDH. Mean of sham rats was set as 100% and AoB and AoB+P4HI are expressed in % of sham. Data are presented as mean value±SEM. ** p<0.01 sham vs. AoB, §§ p<0.01 AoB vs. AoB+P4HI treatment. b) In Western Blot M denotes marker, PC, positive control.

 
To confirm efficacy of P4H inhibition we measured collagen cross-linking by determining the hydroxyproline to proline ratio (Hyp/Pro). A modest 6% increase in the Hyp/Pro ratio was observed in AoB LV relative to sham-operated rats. The P4HI significantly reduced the Hyp/Pro ratio by 31% compared to AoB operated and vehicle treated rats (Table 2) underscoring its effect on collagen P4H.


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  		Table 2 Effect of AoB and P4HI treatment on collagen cross-linking

 
3.3. Effect of AoB and P4HI treatment on the matrix remodelling MMP/TIMP system
MMP-2 mRNA (p<0.01) and MMP-9 mRNA (p<0.05) were induced by AoB, P4HI reduced elevated MMP-2 and MMP-9 mRNA levels (Fig. 3). MMP-2 and MMP-9 zymographic activity also showed an upregulation under AoB (p<0.05 for all). P4HI had a tendency to reduce AoB-induced MMP-2 and MMP-9 zymographic activity, but this did not reach statistical significance (Table 3a).


Figure 03
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  		Fig. 3 MMP-2,-9 and TIMP-1 and-2 mRNA expression in sham, aortic banding and P4HI groups. mRNA expression of specific genes was normalized to GAPDH. Mean of sham rats was set as 100% and AoB and AoB+P4HI are expressed in % of sham. Data are presented as mean value±SEM. ** p<0.01 sham vs. AoB, * p<0.05 sham vs. AoB, §§ p<0.01 AoB vs. AoB+P4HI, § p<0.05 AoB vs. AoB+P4HI.

 


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  		Table 3 MMP-2 and-9 zymographic activity and MT1-MMP, MMP-8, TIMP-3 and TIMP-4 mRNA expression in sham, aortic banding and P4HI groups

 
MT-1 MMP mRNA and MMP-8 mRNA were increased in AoB (p<0.05 for all) (Table 3b). This upregulation was not affected by P4HI treatment (Table 3b).

TIMP-1 and TIMP-2 mRNA were elevated following AoB (p<0.01 for all), respectively and this effect was decreased by P4HI (Fig. 3). TIMP-3 and TIMP-4 mRNA were unchanged under AoB and P4HI treatment (Table 3b).

3.4. Regulation of growth factors
CTGF, OPN, TGFβ1 and ITGβ1 mRNA were increased in AoB (p<0.01 for all) (Fig. 4). AoB-induced CTGF, TGFβ1 and ITGβ1 mRNA expression was significantly reduced by P4HI treatment and OPN showed a tendency towards downregulation (Fig. 4).


Figure 04
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  		Fig. 4 TGFβ1, CTGF, ITGβ1 and OPN mRNA expression in sham, aortic banding and P4HI groups. mRNA expression of TGFβ1, CTGF, ITGβ1 and OPN was normalized to GAPDH. Mean of sham rats was set as 100% and AoB and AoB+P4HI are expressed in % of sham. Data are presented as mean value±SEM. ** p<0.01 sham vs. AoB, §§ p<0.01 AoB vs. AoB+P4HI, § p<0.05 AoB vs. AoB+P4HI treatment.

 
3.5. Regulation of myocardial neuroendocrine systems
The ET-1, ETA, ETB and ECE-1 mRNA expression was increased in AoB (p<0.01 for all). P4HI led to a 40% reduction in ET-1 and 29% reduction in ETA which did not reach statistical significance and to a significant downregulation of AoB-induced ETB and ECE-1. The angiotensin II receptor type 1 (AT1) mRNA was increased under AoB (p<0.05) but was not modulated by P4HI (Fig. 5).


Figure 05
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  		Fig. 5 ET-1, ETA, ETB, ECE-1 and AT1 mRNA expression. mRNA expression of ET-1, ETA, ETB, ECE-1 and AT1 was normalized to GAPDH mRNA. Mean of sham rats was set as 100% and AoB and AoB+P4HI expressed in % of sham. Data are presented as mean value±SEM. * p<0.05 sham vs. AoB, § p<0.05 AoB vs. AoB+P4HI treatment.

 
3.6. Combined upregulation morphological markers of hypertrophy and growth factors
Relative heart weight was significantly correlated with the expression of TGFβ1 (r=0.722), CTGF (r=0.833), OPN (r=0.673), ET-1 (r=0.655), ECE-1 (r=0.550) and ETA (r=0.636; p<0.001 for all) mRNA. IVS thickness correlated with TGFβ1 (r=0.598), CTGF (r=0.69) and OPN (r=0.56; p<0.001 for all) mRNA expression.


   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 Acknowledgments
 References
 
We report for the first time that an orally available prolyl 4-hydroxylase (P4H) inhibitor counteracts changes in cardiac function and attenuates myocardial expression of some genes that are typically induced by AoB. Inhibition of collagen crosslinking by P4H inhibition led to a reduction of the pressure load-induced increase in LVEDP and relative lung weight and to an increase of the AoB-induced reduction of contractility. These findings suggest an improvement in LV function in the presence of persisting pressure overload by P4H inhibition. Functional improvement was accompanied by the reduction of load-induced collagen synthesis, reduction of load-induced alterations in the MMP/TIMP system, reduced myocardial growth factor induction and reduced activation of the cardiac endothelin system.

Collagen P4Hs are key enzymes in collagen synthesis, which catalyse the hydroxylation of proline residues leading to the formation of thermally stable triple helix collagen. Their inhibition is expected to favour production of unstable collagen within the endoplasmatic reticulum, which then is rapidly degraded. The orally available P4H inhibitor, FG 0041; which was used in the present study prevented collagen hydroxylation, LV remodelling and improved survival following acute myocardial infarction in rats [21].

In our study, FG 0041 reduced the AoB-induced increase in LVEDP, reduced the relative lung weight which was increased by AoB and increased the AoB-reduced dp/dtmax, suggesting a reduction in LV stiffness and an improvement of LV systolic function. Since FG 0041 reduced the AoB-induced increase in collagen I and III mRNA and protein, we hypothesize that the reduction of collagen content was responsible for the reduction in left ventricular stiffness. Previously, it has been shown that small molecule inhibitors of collagen P4Hs also inhibit the three known isoforms of HIF-P4Hs [23]. However, the Ki values for inhibition of HIF-P4H were 5-(HIF-P4H isoforms 2 and 3) to 15-fold (HIF-P4H isoform 1) higher compared to inhibition of collagen P4H (Ki 2 uM) [24] indicating that FG 0041 is a much more potent inhibitor of collagen P4Hs compared to HIF-P4Hs. We also showed that P4HI treatment reduces the hydroxylation of proline within the LV. This finding, which has been reported previously [21] demonstrates the activity of FG 0041 towards collagen P4H.

P4HI modified the AoB-induced collagen gene expression. The regulation of MMPs and TIMPs found in AoB in the present study is in agreement with previous studies [11,28]. The 8-fold increase in TIMP-1 may shift the equilibrium towards MMP inhibition and therefore collagen accumulation, as it is the case in human aortic stenosis [28]. The AoB-induced increase in TIMP-1 mRNA was reduced by P4HI. This may have contributed to a reduction of collagen protein accumulation.

The beneficial effects of P4HI on gene expression may partially be due to the reduction of left ventricular enddiastolic pressure and consequently enddiastolic wall stress. In addition, P4HI modifies collagen breakdown products that have direct effects on gene transcription. Regulation of gene expression by collagen fragments has been described for collagen IV fragments influencing MT1-MMP expression [29] and the C-terminal propeptide of type II procollagen regulating type II procollagen transcription [30].

The profibrotic and prohypertrophic growth factors TGFβ1, CTGF, ITGβ1 and OPN as well as the cardiac endothelin system were upregulated by AoB in close correlation with the increase in heart weight. P4HI led to a downregulation of AoB-induced expression of the growth factors and reduced the activation of the endothelin system. We speculate, that a reduced expression of the myocardial growth factors TGFβ1, CTGF and OPN following P4HI treatment is mainly related to the decreased LVEDP and therefore LV wall stress. In fact, we [12] and others [5] have shown that in human aortic stenosis increased expression of TGFβ1 and accumulation of myocardial collagen was strongly connected with elevated LVEDP and reduced cardiac function. These findings could mean that a reduction of LVEDP might facilitate a decrease of growth factor and collagen expression. However, neither LV wall thickness nor relative heart weight were normalized in AoB rats treated with P4HI during the 4 week experimental period and only a tendency towards downregulation of relative heart weight was observed. In this regard, we speculate that the duration of the study was too short to achieve a reduction of myocardial hypertrophy. In addition, persisting pressure overload probably led to increased systolic wall stress and to activation of the local renin angiotensin system as known from previous studies and indicated by AT1 upregulation in the present study [12,18]. This probably contributed to the persistence of hypertrophy.

4.1. Limitations
We investigated the effect of P4H inhibition on myocardial remodelling during the early stages of AoB-induced LVH. However, LVH in patients is a long lasting process and myocardial remodelling and fibrosis are already established at the time of clinical presentation. We cannot conclude from the present study that P4HI also has favourable effects on LV remodelling once fibrosis is present. Future studies need to address this issue.

In summary, inhibition of collagen prolyl 4-hydroxylase leads to favourable changes in cardiac function and hypertrophy-associated gene expression. P4H inhibition could be of potential value in treating the remodelling processes that accompany the development of left ventricular hypertrophy.


   Appendix A. Supplementary data
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
Acknowledgments
 References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejheart.2006.10.006.


   Acknowledgments
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
References
 
The work was supported by the DFG (Re 662/3-5), and GK 754 and the EU (EUGeneHeart project, contract 018833). We thank Ralph V. Shohet for carefully reading the manuscript and helpful discussions. Technical help by Jenny Thomas and Britta Hannack is highly appreciated. Jens Fielitz is a postdoctoral fellow of the Universitätsmedizin Charité Virchow Klinikum and received the Pfizer Fellowship of the German Society of Cardiology.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
1 Equal contribution. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 

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