活性氧族群及一氧化氮於心臟細胞中調控內皮素所誘發乙型肌凝重鍊蛋白基因所扮演的角色

OBJECTIVES: We dissected the molecular
regulatory mechanism of reactive oxygen
species (ROS) on endothelin-1-(
ET-1)-induced b-myosin heavy chain
(b-MyHC) gene expression and hypertrophic
signaling in neonatal rat cardiomyocytes.
BACKGROUND: ET-1 causes hypertrophy
in the cardiomyocytes. Expression of cardiac
b-MyHC gene is increased in response to
ET-1. Our previous study has demonstrated
that ET-1 increases intracellular reactive
oxygen species (ROS) in cardiomyocytes.
However, the intracellular regulatory
mechanism of ROS on ET-1-induced
b-MyHC gene expression and cardiac
hypertrophy still remains unclear.
METHODS: Cultured neonatal rat
cardiomyocytes were stimulated with ET-1,
3 H-leucine incorporation and the b-MyHC
gene promoter activities were examined. We
also examined the effects of antioxidant
pretreatment on ET-1-induced cardiac
hypertrophy and MAPK activties to elucidate
the redox-sensitive pathway in cardiomyocyte
hypertrophy and b-MyHC gene expression.
RESULTS: 3 H-Leucine incorporation and
b-MyHC promoter activities were increased
by ET-1. These ET-1 effects were blocked by
the specific ETA receptor antagonist BQ-485.
Cardiomyocytes treated with antioxidants
significantly reduced ET-1-induced
3 H-leucine incorporation and b-MyHC gene
promoter activities. ET-1 activated
mitogen-activated protein kinases (MAPKs;
extracellular signal-regulated kinase [ERK],
p38, and JNK), which were significantly
inhibited by antioxidants. Either PD98059 or SB203580 inhibited ET-1-increased 3 H-leucine incorporation, but only PD98059
decreased ET-1-induced b-MyHC promoter
activities. Co-transfection of dominant
negative mutant of Ras, Raf, and MEK1
decreased the ET-1-increased b-MyHC
promoter activities, suggesting that the
Ras-Raf-ERK pathway is required for ET-1
action. Deletion mapping revealed that the
deletion construct containing 273 base pairs
(bp) of b-MyHC 5’-flanking sequences
upstream from the transcription initiation site
is necessary and sufficient for ET-1-induced
increase of transcription, whereas the
construct containing only 188 bp of
5’-flanking region had no effect, indicating
the ET-1-responsive element(s) is located
between position -273 and –188 bp upstream
from the transcription start site. This minimal
construct of 273 bp upstream of transcription
initiation site in the b-MyHC promoter region
is also required for ROS-induced b-MyHC
expression.
CONCLUSIONS: These data demonstrate
that ROS involved in ET-1-induced
hypertrophic responses, b-MyHC expression
and mediated ET-1-induced activation of
MAPK pathways, which culminated in
hypertrophic responses and b-MyHC
expression. The ROS-MAPK
(ERK)-meditated pathway plays an essential
role in ET-1-induced hypertrophic responses
and b-MyHC expression in cardiomyocytes.