1. • Approximately 600 and 400 spots were found in 2D gels run in the pH 4-7 and 6-11 ranges respectively, and stained with Colloidal
Coomassie Blue. Currently, a comparative analysis of the protein profile is underway and will later be statistically validated.
• Future work: identification of differentially expressed proteins present in samples and controls by mass spectrometry techniques;
optimization of the cardiomyocytes extraction process; validation of the cardiotoxicity biomarkers.
V.M. Costa, F. Carvalho, J.A. Duarte, M.D.L. Bastos, F. Remião, The heart as a target for
xenobiotic toxicity: the cardiac susceptibility to oxidative stress., Chem. Res. Toxicol. 26 (2013) 1285–311.
This work was supported by Fundação para a Ciência e Tecnologia (FCT) - project
[EXPL/DTP-FTO/0290/2012] – QREN’s iniciative funded by EU/FEDER through COMPETE - Operational
Programme for Competitiveness Factors.
HL-1
10 min
5000 rpm
4°C
Culture Incubation (MTX
1µM and 10µM, for
12h)
Harvest
(Scropping PBS + Ca2+ + Mg2+)
Cell Lysis
(7M Urea, 2M Thiourea,
4%CHAPS, 0.5% IPG Buffer)
Supernatant
Sonicate
15 x 2 s
bursts
Pellet
(Membrane proteins)
Supernatant
12 min
12000 rpm
4°C
Pellet
Supernatant
(Proteins)
Bradford
QuantificationIEFSDS-PAGE
Pellet
A
B
A
pH
MW
B
C D
Figure 2 – 2-D gel images of the control (A and B), 10 µM MTX
incubation for 12h (C and D), Proteins (150 µg) from cell extracts of
HL-1 cardiomyocytes were separated on a pH 4–7 strip (A and C)
and pH 6-11 strip (B and D), followed by SDS-PAGE on a 12.5%
acrylamide gel. Proteins were visualized by colloidal coomassie
blue staining. The image gels were analyzed by Image Master
Workstation software (Nonlinear).
Figure 1 – A: HL-1 cardiomyocytes control;
B: HL-1 cardiomyocytes with 10 µM MTX
incubation for 12h.
Cell cultures were assessed morphologically
by phase contrast microscopy at the selected
time-points (microphotographs were taken
with a Nikon Eclipse TS100 equipped with a
Nikon DS-Fi1 camera).
Mitoxantrone
(MTX)
,
a
synthe3c
anthracenedione,
has
been
used
as
an
an3carcinogen
drug
in
various
types
of
cancer
cancer
(prostate,
metastasized
breast
cancer,
lymphoma,
acute
myeloid
leukemia)
as
well
as
in
mul3ple
sclerosis.
This
molecule
has
the
ability
to
inhibit
type
II
topoisomerase
preven3ng
DNA
linkage
and
consequently
the
progression
of
the
cellular
cycle.
Studies
have
found
that
the
use
of
this
drug
has
cardiotoxic
effects
that
cause
heart
failurePrevious
studies
have
found
that
the
use
of
MTX
has
cardiotoxic
effects
causing
heart
failure
and
leading
to
an
increased
risk
of
death.
The
mechanisms
underlying
the
damage
caused
by
MTX
to
the
cardiac
muscle
are
being
studied
using
proteomic
tools
(2D
electrophoresis,
2DE).
This
approach
will
enable
the
iden3fica3on
of
novel
biomarkers
of
cardiac
toxicity.
The
HL-­‐1
cell
line
used
in
this
work
possesses
similar
characteris3cs
to
human
cardiomyocytes.
4 7 6 11
Control10µMMTX