Knockdown and transfection experiments in H9c2 cells using siRNA to
sequences in the open-reading frame of SDHa mRNA. A, RT-PCR for
SDHa normalized to 18 S rRNA. Lanes 1 and 2 shows SDHa mRNA
expression in control cells. Lanes 3, 4, lanes 5,
6, and lanes 7, 8 show effects of SDHa siRNA (80
μm) on mRNA expression at 24, 48, and 72 h, respectively. The
histogram represents SDHa induction by real time PT-PCR (n = 3;
*, p < 0.05 compared with control). B, Western
blotting for mitochondrial succinate-ubiquinol oxidoreductase (SDH) subunit A
and B proteins, the two functionally active subunits of Complex II. The blot
shows controls (lanes 1 and 2) and cells transfected with
siRNA to the SDHa subunit (lanes 3-8). Porin is
used as a loading comparison. Lanes 3-6 show SDHa siRNA
transiently inhibits SDHa but not SDHb protein expression. C, in-gel
quantification of Complex II enzyme activity in control and SDHa-silenced H9c2
cells at two concentrations of SDHa siRNA (40 and 80 μm). Native
gels were incubated in reaction buffer containing 10 mm succinate
and developed with nitroblue tetrazolium. D, nuclear HIF-1α and
NRF-1 in SDHa-silenced cells. SDHa silencing increases
HIF-1α but not NRF-1 nuclear protein accumulation. Cells were
transfected with either scRNA (controls) or siRNA to SDHa subunit.
After transfection, nuclear HIF1α and NRF-1 were analyzed by Western
blot. Tubulin is shown for a loading comparison. E, PHD activity in
SDHa-silenced cells. PHD activity was assessed by detection of
hydroxylated proline 564 of HIF-1α (Hyp-564) by
immunoprecipitation followed by Western blot analysis. Proteasomal degradation
was inhibited with 5 μm MG132 (2-h incubation). Aerobic H9c2
cells were incubated with 1 mm DMOG for 2 h to inhibit PHD. Cells
were transfected with either scRNA or siRNA for the SDHa subunit. The
last two lanes show the effect of siNRF-1. Total HIF-1α is
shown for comparison. F, SDHa transfection and NRF-1 knockdown in
primary rat cardiomyocytes. Left shows Western blot analysis of
nuclear HIF-1α and NRF-1 relative to tubulin. SDHa protein is shown
relative to β-actin. Lane 1, control cells. Lane 2,
SDHa overexpression and SDHa, NRF-1, and HIF-1α protein expression at 48
h. Lane 3, SDHa, NRF-1, and HIF-1α expression 48 h after
siNRF-1. Lane 4, co-transfection of SDHa-pCMV6-XL5 plasmid with
siNRF-1 (low NRF-1 and high SDHa expression) prevents nuclear HIF-1α
protein accumulation. The histogram (right) shows HIF-1α
nuclear protein relative to tubulin by densitometry. Data are means of
n = 4 (*, p < 0.05 compared with control).