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Reoxygenation after severe hypoxia induces cardiomyocyte hypertrophy in vitro: activation of CREB downstream of GSK3ß

Franz-Volhard Clinic, HELIOS Klinikum-Berlin, Charité Campus Buch, Medical Faculty of the Humboldt University Berlin and
^* Max Delbrück Center for Molecular Medicine, Berlin, Germany

¹Correspondence: Franz Volhard Clinic, Charité Campus Buch, Wiltbergstr. 50, 13125 Berlin, Germany. E-mail: M.Bergmann{at}mdc-berlin.de

SPECIFIC AIMS

Hypertrophy of viable cardiomyocytes contributes significantly to ventricular remodeling after ischemia/reperfusion in vivo. Our aim was to characterize signaling pathways activated in isolated cardiomyocytes subjected to hypoxia/reoxygenation in relation to protein synthesis focusing on reactive oxygen species (ROS), ß-adrenoreceptors (ß-AR), PI3-kinase, and downstream transcription factors.

PRINCIPAL FINDINGS

1. Hypoxia/reoxygenation induces cardiomyocyte hypertrophy in vitro
HIF1 protein was stabilized during hypoxia and rapidly degraded during reoxygenation in isolated neonatal rat cardiomyocytes (Fig. 1 A). ³H-Leucine incorporation was determined in cells cultured under hypoxia for 1, 3, or 6 h followed by reoxygenation to complete 48 h of culture. Protein synthesis was increased in correlation with prolonged culture under hypoxia. A maximum 1.7 ±0.15-fold increase was observed for 6 h hypoxia, followed by 42 h reoxygenation (Fig. 1B ). Immunohistochemical analysis showed that hypoxia/reoxygenation at these times induced an increase of cell size and -sarcomeric actin expression (Fig. 1C ). ANP mRNA levels were increased by hypoxia/reoxygenation (Fig. 1D ).

View larger version (42K): [in this window] [in a new window]   Figure 1. Hypoxia/reoxygenation induces hypertrophy. A) Hypoxia increased HIF1 protein levels rapidly down-regulated by reoxygenation. Cardiomyocytes were cultured under severe hypoxia (0% O2, 5% CO2, 95% N2) for 1, 3, or 6 h (H1, H3, H6) or under 6 h hypoxia followed by 5 min reoxygenation (H6R5'). B) Cardiomyocytes were cultured for various periods of hypoxia followed by reoxygenation to complete 48 h of culture (H1R47, H3R45, H6R42). Data indicate fold induction of protein synthesis vs. control cells and are mean ±SE of 3 to 6 independent experiments from as many independent cell preparations. *P <0.05; ***P <0.005. C) Immunofluorescence staining of -sarcomeric actin confirmed hypertrophy in cardiomyocytes after hypoxia 6 h and reoxygenation 42 h. D) Northern blot experiments with ANP probe demonstrated increased ANP levels. Top: ANP autoradiogram; bottom: ethidium bromide staining of the rRNA 18S and 28S.

2. Hypoxia/reoxygenation-induced hypertrophy involves reactive oxygen species, PI-3 kinase, MAPK ERK, and ß2-adrenergic receptor-dependent, pertussis toxin-sensitive pathways
The ROS scavenger N-acetyl cysteine (NAC) as well as Gi antagonist pertussis toxin completely blocked hypertrophy in this model system. The ß2-AR antagonist ICI 118,551 added at the beginning of reoxygenation inhibited hypertrophy (1.2±0.11-fold), whereas ß1-AR antagonist CGP20712A or the -receptor blocker prazosin had no significant effect (1.54±0.10). Hypoxia/reoxygenation increased phosphorylation of AKT and MAPK ERK phosphorylation. Protein synthesis was measured after hypoxia 6 h/reoxygenation 42 h in the presence of PI3K (LY294002), ERK (PD 98059), or PKA (H89) inhibitors. The hypoxia/reoxygenation-induced hypertrophy was sensitive to these inhibitors. Inhibition of PI3-kinase was most potent. These results suggest an important role for PI3-kinase/Akt as well as ERK and PKA signaling pathways possibly downstream of ß2-AR/Gi in hypoxia/reoxygenation-induced hypertrophy.

3. DNA binding activity of CREB, GATA and NF-B under hypoxia/reoxygenation
In addition to the ß-AR-responsive transcription factor CREB, GATA and NF-B were investigated since several studies have demonstrated their role in cardiomyocyte hypertrophy or reoxygenation-induced preconditioning. EMSA analysis showed that 6 h of hypoxia increased CREB, NF-B, and GATA DNA binding activity by 2- to 4-fold. By 30 min, reoxygenation had already decreased binding activities of NF-B and GATA. In contrast, CREB DNA binding increased even after 30 min of reoxygenation and was still enhanced after 2 h of reoxygenation.

Since phosphorylation of CREB at serine-133 is mediated by a variety of kinases, including PKA, AKT, and p90^RSK2 downstream of ERK, we tested the effect of H89, LY294002, and PD98059 on CREB phosphorylation. None of these inhibitors was able to inhibit CREB serine-133 phosphorylation in hypoxia/reoxygenation-treated cells, but these same inhibitors potently blocked CREB DNA binding.

We next tested the hypothesis that CREB DNA binding is regulated by phosphorylation of CREB serine-129 via GSK3ß. Hypoxia/reoxygenation induced GSK3ß serine-9 phosphorylation (2.5±0.4-fold), which was blocked by ROS, PI-3 kinase, PKA, and ERK inhibition (Fig. 2 B, C). CREB serine-129 was regulated in the opposite direction: corresponding to GSK3ß serine-9 phosphorylation resulting in kinase inactivation, serine-129 was phosphorylated only in the absence of stimulus. This effect was antagonized by ß2-AR and Gi inhibitor pertussis toxin (Fig. 2B ). The data demonstrate GSK3ß to integrate several signaling pathways toward CREB serine-129 phosphorylation.

View larger version (58K): [in this window] [in a new window]   Figure 2. Regulation of CREB by hypoxia/reoxygenation. Inhibition of PI3-kinase, MAPK ERK, and PKA has no effect on hypoxia/reoxygenation-induced CREB serine-133 phosphorylation but inhibits CREB DNA binding. Inhibition of DNA binding correlates inversely to CREB serine-129 phosphorylation and directly to GSK3ß serine-9 phosphorylation. Cardiomyocytes were subjected to hypoxia 6 h/reoxygenation 30 min with inhibitors present during reoxygenation (B). A) Up-regulation of CREB serine-133 phosphorylation occurs independent of PI3-kinase, ERK, PKA, or ß2-AR. B) CREB serine-129 phosphorylation is reduced by hypoxia 6 h/reoxygenation 30 min in correlation to AKT, ERK, and GSK3ß phosphorylation. ROS inhibitor NAC and ß2-AR inhibitor ICI antagonize this effect. The GSK3ß inhibitor II (GSK I) blocks CREB 129 phosphorylation after H/R and under baseline conditions. C) PI3-kinase, MAPK ERK, and PKA regulate phosphorylation of GSK3ß at serine-9. D) CREB DNA binding is blocked by inhibition of PI3-kinase, MAPK ERK, PKA, ROS, ß2-AR, and Gi. Data are representative of at least 3 independent experiments.

To further characterize CREB activation in hypoxia/reoxygenation-treatment, EMSA analysis was performed. Hypoxia/reoxygenation enhanced CREB DNA binding activity by 3-fold, significantly counteracted by inhibition of PI3-kinase, MAPK ERK, and PKA as well as upstream signaling pathways like Gi and ß2-AR (Fig. 2D ).

4. Inactivation of GSK3ß activity by hypoxia/reoxygenation correlates with enhanced CREB transcriptional activity
To evaluate the potential role of GSK3ß on CREB activity, reporter gene assays with a CRE-luciferase vector were performed. This CRE construct was not induced by forskolin in cardiomyocytes after liposomal transfection (efficiency: 5%), possibly relating to cell type-specific effects concerning this plasmid. Therefore, these experiments were performed in PC12 cells, which, similar to cardiomyocytes, activate the PI3K/Akt/GSK3ß pathway under hypoxia. We observed a 2.5 ±0.3-fold increase of CRE-dependent luciferase expression by hypoxia/reoxygenation. This effect was completely abolished in cells cotransfected with GSK3ßS9A, a mutant of GSK3ß that cannot be inactivated by phosphorylation.

5. Requirement of CREB for hypoxia/reoxygenation-induced hypertrophy
Next, we tested the hypothesis that CREB activation was essential for the development of hypertrophy in hypoxia/reoxygenation. Protein synthesis was determined in cells transfected with a dominant negative CREB mutant, K-CREB. K-CREB is able to dimerize with wild-type CREB but lacks the DNA binding domain, thereby preventing gene activation by titration of the endogenous CREB. Hypoxia 6 h/reoxygenation 42 h was able to increase protein synthesis in cells transfected with the control vector (1.51±0.1, P<0.05), whereas hypoxia/reoxygenation-induced hypertrophy was abrogated in K-CREB-transfected cells (0.9±0.2). These findings demonstrate that CREB is essential for hypoxia/reoxygenation-induced hypertrophy.

CONCLUSIONS AND SIGNIFICANCE

Our study demonstrates a direct effect of hypoxia 6 h followed by 42 h reoxygenation on cardiomyocyte hypertrophy. The hypertrophy depends on ROS, PI3-kinase, as well as ERK and PKA activation. The kinases appear to be downstream to signaling via ß2-adrenoreceptors linked to Gi. The transcription factor CREB, but not NF-B, was found to be essential for cardiomyocyte hypertrophy. Inactivation of GSK3ß was important for hypoxia/reoxygenation-induced CRE-dependent transcription. The data have significance with regard to current approaches to limit reperfusion injury and cardiac hypertrophy.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of signaling pathways involved in hypoxia/reoxygenation-induced cardiomyocyte hypertrophy. Reoxygenation after severe hypoxia (H/R) induces release of reactive oxygen species (ROS). Previous studies have shown that ROS target Gi proteins, which couple to ß2-AR in cardiomyocytes. ROS induce phosphorylation of transcription factor CREB at serine-133. CREB serine-133P is a target for hierarchical phosphorylation by GSK3ß, thereby inhibiting CREB-dependent gene activation. ROS relieve CREB inhibition by GSK3ß phosphorylation at serine-9. This phosphorylation inactivates GSK3ß kinase activity and leads to dephosphorylation of CREB serine-129. MAPK ERK and PKA are involved in regulation of GSK3ß activity. CREB-dependent gene transcription is necessary for cardiomyocyte hypertrophy induced by hypoxia/reoxygenation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1054fje; doi: 10.1096/fj.03-1054fje

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