Reoxygenation after severe hypoxia induces cardiomyocyte hypertrophy in vitro: activation of CREB downstream of GSK3β

ABSTRACT In vivo, left ventricular remodeling after myocardial infarction involves hypertrophy generally attributed to increased cardiac workload. We hypothesized that hypoxia/reoxygenation directly induces cardiomyocyte hypertrophy and studied several participating kinases and transcription factors in isolated cardiomyocytes. Hypoxia for 6 h followed by 42 h reoxygenation induced cardiomyocyte hypertrophy assessed by 3 H leucine incorporation and immunohistochemistry. Inhibition of reactive oxygen species (ROS), serine/threonine kinase AKT, and ERK abolished reoxygenation‐induced hypertrophy. In addition, a β2‐adrenergic receptor (β2‐AR) antagonist, as well as Gi inhibitor pertussis toxin, blocked reoxygenation‐induced hypertrophy. Hypoxia for 6 h increased transcription factors CREB, NF‐κ B, and GATA DNA binding activities. However, only CREB DNA‐binding was sustained during reoxygenation. Inhibition of PI3‐kinase, ERK, and PKA abrogated reoxygenation‐induced CREB DNA‐binding without affecting CREB serine‐133 phosphorylation. These same pathways were found to regulate hypoxia/reoxygenation‐induced GSK3β kinase activity and CREB serine‐129 de‐phosphorylation. GSK3β mutants resistant to phosphorylation blocked the stimulation of CRE‐dependent transcription induced by hypoxia/reoxygenation. Transfection of cardiomyocytes with a dominant‐negative mutant of CREB abrogated hypoxia/reoxygenation‐induced hypertrophy. We suggest that hypoxia/reoxygenation induces cardiomyocyte hypertrophy through CREB activation. Inactivation of GSK3β by hypoxia/reoxygenation, possibly integrating PI3‐kinase and ERK pathways downstream of β2‐AR and ROS, is a prerequisite for CRE‐dependent transcription. Transient hypoxia may contribute to cardiac hypertrophy in ischemic heart disease independent of cardiac workload.