Cellular Physiology and Biochemistry

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1 Zieseniss Accepted: et April al.: PHD3 09, 2015 and Myocardial Infarction /15/ $39.50/0 Original Paper This is an Open Access article licensed under the terms of the Creative Commons Attribution- NonCommercial 3.0 Unported license (CC BY-NC) ( applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Anke Zieseniss a,b Amke R. Hesse a,b Aline Jatho a Sabine Krull a Marion Hölscher a Sabine Vogel a Dörthe M. Katschinski a,b a Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, b DZHK (German Center for Cardiovascular Research), partner site Göttingen, Göttingen, Germany Aims: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far. Methods and Results: transgenic mice (cphd3tg). Resting cphd3tg mice did not show constitutive accumulation of followed up for 14 months in these mice and found to be unchanged. After challenging the cphd3tg of the cphd3tg compared to wild type mice. Conclusion: Whereas overexpression of PHD3 in Copyright Introduction Hypoxic transcriptional gene expression is mainly driven by the hypoxia-inducible hydroxylation and subsequent ubiquitination [1]. The prolyl-4-hydroxylase domain enzymes A. Zieseniss and A.R. Hesse contributed equally to this work Dörthe M. Katschinski Institute of Cardiovascular Physiology, University Medical Center Göttingen, Humboldtallee 23, Göttingen (Germany) Tel. +49-(0) , doerthe.katschinski@med.uni-goettingen.de

2 844 (PHD) 1-3 and the von Hippel Lindau protein are responsible for this regulation [2, 3]. Based on the fact that the K m values of the PHDs for oxygen are higher than the concentrations generally present in normoxic tissues in vivo biological oxygen sensors. [4] HIF-1 plays an important role in cardioprotection induced by ischemic preconditioning [5, 6]. Thus, induction of the HIF response via inhibition of the PHDs may provide a novel therapeutic strategy in the treatment of ischemic cardioprotection left anterior descending (LAD) artery than the control mice. [8] Cardiac tissue protection in normoxia, but highly inducible in hypoxia in various cell models and tissues including in vitro and in vivo important for balancing the hypoxic response based on its strong induction after oxygen deprivation [13, 14]. Despite these differential functions of PHD2 and PHD3 in regulating Besides in myeloid and neuronal cells, this has also been demonstrated in embryonic rat this can`t be deduced from the present correlative studies. To analyze if upregulated levels cphd3tg) and analyzed the consequences of the transgene under resting conditions and after LAD ligation. Mice cphd3tg) the pbsiisk- pbskiisk- Animal experimentation and echocardiography Phd3tg mice and the respective littermate

3 845 Echocardiography and measurement of left ventricular end systolic diameter (LVESD), left ventricular Measurement of infarct size Protein extraction and immunoblot analysis nitrocellulose membranes (Amersham Biosciences) by semi-dry blotting (PeqLab). Primary antibodies Cell Signaling, 2118), 2 O 2, qrt-pcr analysis CTG ACG CCC TTC ACC GCG AG AAA GGC ATC CCA GCC TCC GT ACA GCC GGA A CCT GAG GAG AGA ACT CAG CTG CCA TGA CTG CAC G Histological analysis

4 846 Fig. 1. Left ventricles of cphd3tg cphd3tg tricles of cphd3tg cphd2 and cphd2/3 least three independent experiments. Statistical analysis t p the Figure legends. cphd3tg and littermate increase of PHD2 (Fig. 1A and B). Histological assessment of the hearts revealed no gross abnormalities in the cphd3tg by echocardiography in cphd3tg wt unchanged in the hearts of cphd3tg mice (Fig. 1E). As a positive control for the successful

5 847 Fig. 2. resting cphd3tg extracted from left ventricles of cphd3tg mice and the respective group). Fig. 3. Overexpression of PHD3 in the heart does not alter cardiac function over time. cphd3tg mice (n analyzed for fractional shortening (FS), posterior PHD1, PHD2 and PHD3 in the cphd3tg mice. not affected by the transgenic overexpression responsible for the induction of genes that facilitate adaptation and survival of cells and expressed in the resting cphd3tg mice. To analyse, if over time PHD3 cphd3tg and wt mice over a cphd3tg mice (Fig. 3). cphd3tg mice

6 848 response and the functional outcome after myocardial infarction. Gain of PHD3 restrained detectable indicating that altered HIF 5E). Our data demonstrate that gain of PHD3 does not affect cardiac function in resting diminished adaptation of the heart to ischemia. even in older cphd3tg animals. Cardiac overexpression of other components of the cellular and PHD3 are resulting in the development of cardiomyopathies [24-26]. These mouse cphd3tg per se do not impair cardiac function. functions contrast to the resting cphd3tg cphd3tg group has previously demonstrated an augmented expression of cardiac PHD3 expression

7 849 Fig. 5. PHD3 overexpres- sponse and outcome after myocardial infarction. cphd3tg LAD ligation. 6 hours after ern blots (A) and quanti- protein loading control. (C) lyzed by Evans Blue perfusion and TTC stainings 6 hrs tive infarct size staining are cphd2tg mice under rest- sent the number of animals are representative of at least three independent experiments. of hyperglycemia-induced cardiomyopathy [32]. PHD inhibitors that activate HIF are an attractive therapeutic option for minimizing tissue damage from myocardial ischemia. Several PHD inhibitory drugs are in development of a PHD inhibitor protects the heart from an ischemic insult [34]. PHD3 is upregulated in out [25]. Therefore inhibition of the increased PHD3 activity in hypoxic tissue might be

8 850 PHD3 in the heart. From these data one could deduce that strong expression of PHD3 in the stimulus Cardiovasc 6 Hypoxia-inducible factor-1 is central to PHD3. 12 Age-dependent increase of prolyl-4-hydroxylase 13 of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. 14 Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing

9 Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress myosin heavy chain gene promoter in transgenic mice Pan-genomic binding of hypoxia-inducible transcription factors. Biol Chem 23 Hypoxia-inducible factor 1 reduces infarction and attenuates progression of cardiac dysfunction after myocardial infarction in the Loss of Hypoxia-Inducible Factor Prolyl Hydroxylase Activity in Cardiomyocytes Phenocopies Ischemic 26 inducible factor-dependent degeneration, failure, and malignant transformation of the heart in the absence Pre- and post-conditional inhibition of prolyl-4-hydroxylase domain enzymes protects the