CARDIOVASCULAR JOURNAL OF AFRICA • Vol 21, No 1, January/February 2010
AFRICA
37
Cardiovascular Topics
The effect of hypoxia-inducible factor 1-alpha on
hypoxia-induced apoptosis in primary neonatal rat
ventricular myocytes
YAN-FANG ZHOU, XIAO-WEI ZHENG, GUO-HUI ZHANG, ZHI-HONG ZONG, GUO-XIAN QI
Summary
Aim:
To study the role of hypoxia-inducible factor 1-alpha
(HIF-1
α
) on hypoxia-induced apoptosis in primary neonatal
rat ventricular myocytes.
Methods:
Primary neonatal rat ventricular myocytes were
exposed to hypoxia for 24 hours. HIF-1
α
activity was
suppressed by treating the cells with 3-(5
′
-hydroxymethyl-2
′
-
furyl)-1-benzyl indazole (YC-1). The degree of cell apoptosis
was assessed by Hoechst 33258 DNA staining. The levels of
HIF-1
α
and the pro-apoptotic proteins Bnip3, Bax and Bad
were measured with western blotting.
Results:
On exposure to hypoxia, there was an increase in the
expression levels of HIF-1
α
, and the pro-apoptotic protein
Bnip3 was upregulated. Suppression of HIF-1
α
activity by
YC-1 treatment was followed by blockade of hypoxia-induced
apoptosis and Bnip3 expression; however, the changes in the
levels of Bax and Bad expression were unclear.
Conclusion:
Acute hypoxia enhanced primary neonatal rat
ventricular myocyte apoptosis through the activation of
HIF-1
α
and a mechanism that perhaps involved Bnip3.
Targeting HIF-1
α
may be a new strategy for reducing the
degree of hypoxia-induced apoptosis in ventricular myocytes.
Keywords:
HIF-1, primary neonatal rat ventricular myocytes,
hypoxia, apoptosis
Submitted 14/6/09, accepted 13/8/09
Cardiovasc J Afr
2010;
21
: 37–41
Currently, it is believed that cell death in ventricular myocytes
occurs via not only necrosis but also apoptosis. Necrosis is a
destructive and uncontrolled process, whereas apoptosis is a
highly regulated process of programmed cell suicide. The highly
organised nature of apoptotic signalling renders it amenable
to manipulation. However, the exact regulating mechanisms
of apoptosis in ventricular myocytes remain unclear. A better
understanding of the apoptotic pathways and their regulatory
mechanisms might help identify novel therapeutic targets for
alleviating myocardial damage and dysfunction.
HIF-1 is a transcription factor composed of a strictly regu-
lated
α
-subunit and a constitutive
β
-subunit.
1,2
HIF-1
α
is mainly
regulated by hypoxia; this protein undergoes rapid ubiquitination
and proteasomal degradation in the normal condition, but under
hypoxic conditions, the degradation of HIF-1
α
is suppressed and
it is translocated to the nucleus, where it dimerises with HIF-1
β
.
3
The heterodimeric HIF-1 molecule binds to hypoxia-responsive
elements (HRE) located in the promoter and enhancer regions of
hypoxia-regulated genes, causing their transactivation. HIF-1
α
is thought to be a crucial regulator of hypoxia-adaptational
responses.
4-6
Some genes that encode important proteins involved in the
apoptotic pathway, such as some of the Bcl-2 family proteins,
are regulated by HIF-1
α
. It was reported that the pro-apoptotic
proteins Bnip3, Bad and Bax
7-9
are regulated by HIF-1
α
. HIF-1
α
exerts both anti-apoptotic and pro-apoptotic effects, depending
on the cell type.
10
This differential effect is partly due to the
regulatory effect of HIF-1
α
on the pro-apoptotic proteins on the
Bcl-2 family.
To understand the function of HIF-1
α
in hypoxia-induced
apoptosis in primary neonatal rat ventricular myocytes and the
underlying molecular mechanisms, we induced apoptosis in
primary neonatal rat ventricular myocytes by subjecting them to
hypoxic conditions for 24 hours. HIF-1
α
activity was suppressed
by treating the cells with YC-1, which is an HIF-1
α
inhibitor
used widely in both
in vitro
and
in vivo
studies.
11
We observed
that hypoxia increased the expression levels of HIF-1
α
and pro-
apoptotic protein Bnip3 and the degree of apoptosis; however,
when HIF-1
α
was inhibited by YC-1, there was a corresponding
decrease in the level of Bnip3 protein expression and the degree
of apoptosis.
Methods
Cell culture, hypoxia, and inhibition of HIF-1
α
The hearts of one- to two-day-old rats were rapidly removed
from the chest cavity under anesthesia; the heart samples were
trimmed of atrial tissue, great vessels and pericardium. The
ventricles were washed in ice-cold phosphate-buffered saline
Department of Cardiology, First Affiliated Hospital of China
Medical University, Shenyang, China
YAN-FANG ZHOU, MD
XIAO-WEI ZHENG, MD
GUO-XIAN QI, MD,
Cardiovascular Disease Institute, First People’s Hospital,
Zhenjiang, China
YAN-FANG ZHOU, MD
GUO-HUI ZHANG, MD
Department of Biochemistry, China Medical University,
ShenYang, China
ZHI-HONG ZONG, MD
