ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation.
Extracellular signal-regulated kinase 1/2 (ERK1/2) is known to function in cell survival in response to various stresses; however, the mechanism of cell survival by ERK1/2 remains poorly elucidated in ischemic heart. Here we applied functional proteomics by two-dimensional electrophoresis to identify a cellular target of ERK1/2 in response to ischemic hypoxia. Approximately 1500 spots were detected by Coomassie Brilliant Blue staining of a sample from unstimulated cells. The staining intensities of at least 50 spots increased at 6-h reoxygenation after 2-h ischemic hypoxia. Of the 50 spots that increased, at least 4 spots were inhibited in the presence of PD98059, a MEK inhibitor. A protein with a molecular mass of 52 kDa that is strongly induced by ERK1/2 activation in response to ischemic hypoxia and reoxygenation was identified as alpha-enolase, a rate-limiting enzyme in the glycolytic pathway, by liquid chromatography-mass spectrometry and amino acid sequencing. The expressions of the alpha-enolase mRNA and protein are inhibited during reoxygenation after ischemic hypoxia in the cells containing a dominant negative mutant of MEK1 and treated with a MEK inhibitor, PD98059, leading to a decrease in ATP levels. alpha-Enolase expression is also observed in rat heart subjected to ischemia-reperfusion. The induction of alpha-enolase by ERK1/2 appears to be mediated by c-Myc. The introduction of the alpha-enolase protein into the cells restores ATP levels and prevents cell death during ischemic hypoxia and reoxygenation in these cells. These results show that alpha-enolase expression by ERK1/2 participates in the production of ATP during reoxygenation after ischemic hypoxia, and a decrease in ATP induces apoptotic cell death. Furthermore, alpha-enolase improves the contractility of cardiomyocytes impaired by ischemic hypoxia. Our results reveal that ERK1/2 plays a role in the contractility of cardiomyocytes and cell survival through alpha-enolase expression during ischemic hypoxia and reoxygenation.
Mizukami
Yoichi
Y
Center for Gene Research, Yamaguchi University, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. mizukami@yamaguchi-u.ac.jp
Iwamatsu
Akihiro
A
Aki
Toshihiko
T
Kimura
Masayasu
M
Nakamura
Kazuyuki
K
Nao
Tomoko
T
Okusa
Tomoko
T
Matsuzaki
Masunori
M
Yoshida
Ken-Ichi
K
Kobayashi
Sei
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D017202:Myocardial Ischemia / Q000378:metabolism*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D051381:Rats
D005419:Flavonoids; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020930:MAP Kinase Kinase Kinases; D010751:Phosphopyruvate Hydratase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one