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Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism, intervention, and long-term consequences.

Tintu A, Rouwet E, Verlohren S, Brinkmann J, Ahmad S, Crispi F, van Bilsen M, Carmeliet P, Staff AC, Tjwa M, Cetin I, Gratacos E, Hernandez-Andrade E, Hofstra L, Jacobs M, Lamers WH, Morano I, Safak E, Ahmed A, le Noble F - PLoS ONE (2009)

Bottom Line: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis.Systemic administration of rhVEGF(165) to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass.This cardiomyopathy persists into adulthood.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Angiogenesis and Cardiovascular Pathology, Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT

Background: Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos.

Methods: Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine.

Principal findings: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF(165) to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype.

Conclusions/significance: Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

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Histology of E20 chick embryo hearts.A to D, LV dilatation in hypoxic hearts. Scale bars represent 1 mm. E, cardiac morphometry showing a larger LV lumen and a thinner LV wall in hypoxia. F and G, increased number of apoptotic cardiomyocytes (arrowheads) in hypoxic hearts. Scale bars represent 100 µm. H and I, HE staining showing increased intracellular spaces (arrows) in the hypoxic hearts. J to O, ultrastructural analysis showing myofibrillar disarray, disruption of sarcomeric architecture (L and M, red circles), endothelial gaps in capillaries (N, arrowheads; L = lumen) and infiltration of inflammatory cells (O, asterisk) in hypoxic hearts. P and Q, PAS staining demonstrating increased glycogen deposition (red) in hypoxic hearts. R, Q-PCR showing increased ANF expression in hypoxic hearts. Data are shown as mean±SE; * P<0.05 Hypoxia versus Normoxia.
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pone-0005155-g001: Histology of E20 chick embryo hearts.A to D, LV dilatation in hypoxic hearts. Scale bars represent 1 mm. E, cardiac morphometry showing a larger LV lumen and a thinner LV wall in hypoxia. F and G, increased number of apoptotic cardiomyocytes (arrowheads) in hypoxic hearts. Scale bars represent 100 µm. H and I, HE staining showing increased intracellular spaces (arrows) in the hypoxic hearts. J to O, ultrastructural analysis showing myofibrillar disarray, disruption of sarcomeric architecture (L and M, red circles), endothelial gaps in capillaries (N, arrowheads; L = lumen) and infiltration of inflammatory cells (O, asterisk) in hypoxic hearts. P and Q, PAS staining demonstrating increased glycogen deposition (red) in hypoxic hearts. R, Q-PCR showing increased ANF expression in hypoxic hearts. Data are shown as mean±SE; * P<0.05 Hypoxia versus Normoxia.

Mentions: Chick embryos that had been exposed to 15% O2 throughout embryonic development displayed lower arterial blood PO2 levels, elevated hematocrit, and lower total body, liver and heart weights as described previously [9], [10]. Hearts of hypoxic embryos displayed LV dilatation and loss of ventricular wall mass, without congenital cardiac defects (Figure 1A–E). Hearts of hypoxic chick embryos contained an increased number of apoptotic cardiomyocytes (Figure 1F–G) and histological analysis revealed disturbed arrangement of cardiomyocytes (Figure 1H–I). Cardiomyocyte degeneration, myofibrillar disarray with disruption of sarcomeric architecture, leaky blood vessels, and infiltration of inflammatory cells were observed at the ultrastructural level (Figure 1J–O). Hearts of hypoxic chick embryos also contained increased levels of glycogen (Figure 1P–Q), and expression levels of atrial natriuretic factor (ANF) were increased by a factor 2 (Figure 1R). Collagen content showed a 40% increase (Figure 2A–C), reflecting enhanced fibrosis. Levels of the sarcomeric proteins titin (Figure 2D–E) and myosin heavy chain (MHC, Table 1) were lowered in hypoxic embryonic chick hearts. The titin-to-MHC ratio was similar in normoxic and hypoxic hearts. Although the hypoxia-induced decrease in total titin content involved both the N2BA and the N2B isoform, the ratio between the isoforms changed significantly (Figure 2F, Table 1).


Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism, intervention, and long-term consequences.

Tintu A, Rouwet E, Verlohren S, Brinkmann J, Ahmad S, Crispi F, van Bilsen M, Carmeliet P, Staff AC, Tjwa M, Cetin I, Gratacos E, Hernandez-Andrade E, Hofstra L, Jacobs M, Lamers WH, Morano I, Safak E, Ahmed A, le Noble F - PLoS ONE (2009)

Histology of E20 chick embryo hearts.A to D, LV dilatation in hypoxic hearts. Scale bars represent 1 mm. E, cardiac morphometry showing a larger LV lumen and a thinner LV wall in hypoxia. F and G, increased number of apoptotic cardiomyocytes (arrowheads) in hypoxic hearts. Scale bars represent 100 µm. H and I, HE staining showing increased intracellular spaces (arrows) in the hypoxic hearts. J to O, ultrastructural analysis showing myofibrillar disarray, disruption of sarcomeric architecture (L and M, red circles), endothelial gaps in capillaries (N, arrowheads; L = lumen) and infiltration of inflammatory cells (O, asterisk) in hypoxic hearts. P and Q, PAS staining demonstrating increased glycogen deposition (red) in hypoxic hearts. R, Q-PCR showing increased ANF expression in hypoxic hearts. Data are shown as mean±SE; * P<0.05 Hypoxia versus Normoxia.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2663815&req=5

pone-0005155-g001: Histology of E20 chick embryo hearts.A to D, LV dilatation in hypoxic hearts. Scale bars represent 1 mm. E, cardiac morphometry showing a larger LV lumen and a thinner LV wall in hypoxia. F and G, increased number of apoptotic cardiomyocytes (arrowheads) in hypoxic hearts. Scale bars represent 100 µm. H and I, HE staining showing increased intracellular spaces (arrows) in the hypoxic hearts. J to O, ultrastructural analysis showing myofibrillar disarray, disruption of sarcomeric architecture (L and M, red circles), endothelial gaps in capillaries (N, arrowheads; L = lumen) and infiltration of inflammatory cells (O, asterisk) in hypoxic hearts. P and Q, PAS staining demonstrating increased glycogen deposition (red) in hypoxic hearts. R, Q-PCR showing increased ANF expression in hypoxic hearts. Data are shown as mean±SE; * P<0.05 Hypoxia versus Normoxia.
Mentions: Chick embryos that had been exposed to 15% O2 throughout embryonic development displayed lower arterial blood PO2 levels, elevated hematocrit, and lower total body, liver and heart weights as described previously [9], [10]. Hearts of hypoxic embryos displayed LV dilatation and loss of ventricular wall mass, without congenital cardiac defects (Figure 1A–E). Hearts of hypoxic chick embryos contained an increased number of apoptotic cardiomyocytes (Figure 1F–G) and histological analysis revealed disturbed arrangement of cardiomyocytes (Figure 1H–I). Cardiomyocyte degeneration, myofibrillar disarray with disruption of sarcomeric architecture, leaky blood vessels, and infiltration of inflammatory cells were observed at the ultrastructural level (Figure 1J–O). Hearts of hypoxic chick embryos also contained increased levels of glycogen (Figure 1P–Q), and expression levels of atrial natriuretic factor (ANF) were increased by a factor 2 (Figure 1R). Collagen content showed a 40% increase (Figure 2A–C), reflecting enhanced fibrosis. Levels of the sarcomeric proteins titin (Figure 2D–E) and myosin heavy chain (MHC, Table 1) were lowered in hypoxic embryonic chick hearts. The titin-to-MHC ratio was similar in normoxic and hypoxic hearts. Although the hypoxia-induced decrease in total titin content involved both the N2BA and the N2B isoform, the ratio between the isoforms changed significantly (Figure 2F, Table 1).

Bottom Line: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis.Systemic administration of rhVEGF(165) to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass.This cardiomyopathy persists into adulthood.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Angiogenesis and Cardiovascular Pathology, Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT

Background: Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos.

Methods: Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine.

Principal findings: Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF(165) to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype.

Conclusions/significance: Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

Show MeSH
Related in: MedlinePlus