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Transcriptome analysis of the response to chronic constant hypoxia in zebrafish hearts.

Marques IJ, Leito JT, Spaink HP, Testerink J, Jaspers RT, Witte F, van den Berg S, Bagowski CP - J. Comp. Physiol. B, Biochem. Syst. Environ. Physiol. (2007)

Bottom Line: We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus).We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels.We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Zoology, Institute of Biology, University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.

ABSTRACT
Insufficient blood supply during acute infarction and chronic ischemia leads to tissue hypoxia which can significantly alter gene expression patterns in the heart. In contrast to most mammals, some teleost fishes are able to adapt to extremely low oxygen levels. We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus). In order to identify the molecular basis for the adaptations to CCH, we profiled the gene expression changes in the hearts of adult zebrafish. We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels. Two notch receptors (notch-2 and notch-3) as well as regulatory genes linked to cell proliferation were transcriptionally upregulated in hypoxic hearts. We observed a simultaneous increase in expression of IGF-2 and IGFbp1 and upregulation of several genes important for the protection against reactive oxygen species (ROS). We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.

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Histological changes of zebrafish and cichlid hearts after exposure to chronic constant hypoxia. a shows zebrafish hearts that were dissected, sectioned and stained with a hematoxylin–eosin staining with A, B and E representing normoxic and C, D and F hypoxic conditions. Cell nuclei are seen in dark (dark blue in online version) and cell cytoplasm in light (pink in online version). Pictures A, C, E and F have the same magnification (10×). Images B and D represent a 20× magnification of cardiac muscle (D). Abbreviations used are: a atrium; v ventricle; vo ventricular outflow tract and ca conus arteriosus. b (A–F) corresponds to sections of cichlid hearts, which were treated the same way as the ones above from zebrafish and G and H show cichlid hearts which have been perfused prior to dissection and were stained with either hematoxylin–eosin (A–F) or Azan blue (G, H). In A, B, C and G pictures of normoxic conditions are shown and D, E, F and H represent the corresponding hypoxic conditions. Similar results for both the zebrafish and the cichlid hearts were observed in three independent experiments
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Fig2: Histological changes of zebrafish and cichlid hearts after exposure to chronic constant hypoxia. a shows zebrafish hearts that were dissected, sectioned and stained with a hematoxylin–eosin staining with A, B and E representing normoxic and C, D and F hypoxic conditions. Cell nuclei are seen in dark (dark blue in online version) and cell cytoplasm in light (pink in online version). Pictures A, C, E and F have the same magnification (10×). Images B and D represent a 20× magnification of cardiac muscle (D). Abbreviations used are: a atrium; v ventricle; vo ventricular outflow tract and ca conus arteriosus. b (A–F) corresponds to sections of cichlid hearts, which were treated the same way as the ones above from zebrafish and G and H show cichlid hearts which have been perfused prior to dissection and were stained with either hematoxylin–eosin (A–F) or Azan blue (G, H). In A, B, C and G pictures of normoxic conditions are shown and D, E, F and H represent the corresponding hypoxic conditions. Similar results for both the zebrafish and the cichlid hearts were observed in three independent experiments

Mentions: In comparison to the normoxic control groups, we observed a significantly smaller ventricular outflow tract and reduced lacunae within the central ventricular cavity and reduced lacunae around the trabeculae in midline sections of hearts of both zebrafish (Danio rerio) (Fig. 2a) and cichlids (Haplochromis piceatus) (Fig. 2b) exposed to CCH. In addition to the midline sections, none of the sections investigated from hypoxia treated fishes showed a ventricular outflow tract in comparable size to the normoxic controls (data not shown). In addition to the midline sections, none of the lateral sections (to both sides of the midline) investigated from hypoxia treated fishes showed a ventricular outflow tract as well as lacunae in a comparable size to the normoxic controls (data not shown). The larger cichlid hearts were also perfused and midline sectioned and showed similar results with a smaller ventricular outflow tract and reduced lacunae (Fig. 2b-H). This might represent ventricular hypertrophy or hyperplasia in both walls and trabeculae, which could lead to the observed cavity obliteration in these sections. We quantified the number of cardiac myocyte nuclei per area in the midline sections of both zebrafish and cichlids under normoxic, as well as hypoxic conditions. A significant difference in both species was observed and showed that hypoxia led to a 1.4- and 1.6-fold increase in the number of cardiac myocyte nuclei per area in zebrafish and cichlid hearts, respectively (Table 1). Cardiac myocyte nuclei in sections were clearly distinguishable from nuclei of other cells like erythrocytes and fibroblasts and only centralized nuclei in cardiac myocytes (which are more elongated than nuclei from erythrocytes) were counted. Furthermore, scanning electron microscopy (SEM) was used to confirm these findings in the smaller zebrafish hearts (Fig. 3). Future research is warranted in order to assess further how the cardiac myocytes adapt to CCH in the fish heart.Fig. 2


Transcriptome analysis of the response to chronic constant hypoxia in zebrafish hearts.

Marques IJ, Leito JT, Spaink HP, Testerink J, Jaspers RT, Witte F, van den Berg S, Bagowski CP - J. Comp. Physiol. B, Biochem. Syst. Environ. Physiol. (2007)

Histological changes of zebrafish and cichlid hearts after exposure to chronic constant hypoxia. a shows zebrafish hearts that were dissected, sectioned and stained with a hematoxylin–eosin staining with A, B and E representing normoxic and C, D and F hypoxic conditions. Cell nuclei are seen in dark (dark blue in online version) and cell cytoplasm in light (pink in online version). Pictures A, C, E and F have the same magnification (10×). Images B and D represent a 20× magnification of cardiac muscle (D). Abbreviations used are: a atrium; v ventricle; vo ventricular outflow tract and ca conus arteriosus. b (A–F) corresponds to sections of cichlid hearts, which were treated the same way as the ones above from zebrafish and G and H show cichlid hearts which have been perfused prior to dissection and were stained with either hematoxylin–eosin (A–F) or Azan blue (G, H). In A, B, C and G pictures of normoxic conditions are shown and D, E, F and H represent the corresponding hypoxic conditions. Similar results for both the zebrafish and the cichlid hearts were observed in three independent experiments
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Related In: Results  -  Collection

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Fig2: Histological changes of zebrafish and cichlid hearts after exposure to chronic constant hypoxia. a shows zebrafish hearts that were dissected, sectioned and stained with a hematoxylin–eosin staining with A, B and E representing normoxic and C, D and F hypoxic conditions. Cell nuclei are seen in dark (dark blue in online version) and cell cytoplasm in light (pink in online version). Pictures A, C, E and F have the same magnification (10×). Images B and D represent a 20× magnification of cardiac muscle (D). Abbreviations used are: a atrium; v ventricle; vo ventricular outflow tract and ca conus arteriosus. b (A–F) corresponds to sections of cichlid hearts, which were treated the same way as the ones above from zebrafish and G and H show cichlid hearts which have been perfused prior to dissection and were stained with either hematoxylin–eosin (A–F) or Azan blue (G, H). In A, B, C and G pictures of normoxic conditions are shown and D, E, F and H represent the corresponding hypoxic conditions. Similar results for both the zebrafish and the cichlid hearts were observed in three independent experiments
Mentions: In comparison to the normoxic control groups, we observed a significantly smaller ventricular outflow tract and reduced lacunae within the central ventricular cavity and reduced lacunae around the trabeculae in midline sections of hearts of both zebrafish (Danio rerio) (Fig. 2a) and cichlids (Haplochromis piceatus) (Fig. 2b) exposed to CCH. In addition to the midline sections, none of the sections investigated from hypoxia treated fishes showed a ventricular outflow tract in comparable size to the normoxic controls (data not shown). In addition to the midline sections, none of the lateral sections (to both sides of the midline) investigated from hypoxia treated fishes showed a ventricular outflow tract as well as lacunae in a comparable size to the normoxic controls (data not shown). The larger cichlid hearts were also perfused and midline sectioned and showed similar results with a smaller ventricular outflow tract and reduced lacunae (Fig. 2b-H). This might represent ventricular hypertrophy or hyperplasia in both walls and trabeculae, which could lead to the observed cavity obliteration in these sections. We quantified the number of cardiac myocyte nuclei per area in the midline sections of both zebrafish and cichlids under normoxic, as well as hypoxic conditions. A significant difference in both species was observed and showed that hypoxia led to a 1.4- and 1.6-fold increase in the number of cardiac myocyte nuclei per area in zebrafish and cichlid hearts, respectively (Table 1). Cardiac myocyte nuclei in sections were clearly distinguishable from nuclei of other cells like erythrocytes and fibroblasts and only centralized nuclei in cardiac myocytes (which are more elongated than nuclei from erythrocytes) were counted. Furthermore, scanning electron microscopy (SEM) was used to confirm these findings in the smaller zebrafish hearts (Fig. 3). Future research is warranted in order to assess further how the cardiac myocytes adapt to CCH in the fish heart.Fig. 2

Bottom Line: We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus).We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels.We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Zoology, Institute of Biology, University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.

ABSTRACT
Insufficient blood supply during acute infarction and chronic ischemia leads to tissue hypoxia which can significantly alter gene expression patterns in the heart. In contrast to most mammals, some teleost fishes are able to adapt to extremely low oxygen levels. We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus). In order to identify the molecular basis for the adaptations to CCH, we profiled the gene expression changes in the hearts of adult zebrafish. We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels. Two notch receptors (notch-2 and notch-3) as well as regulatory genes linked to cell proliferation were transcriptionally upregulated in hypoxic hearts. We observed a simultaneous increase in expression of IGF-2 and IGFbp1 and upregulation of several genes important for the protection against reactive oxygen species (ROS). We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.

Show MeSH
Related in: MedlinePlus