•  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 Quantification IEF SDS-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

Co

ntro

l 10

µM

MTX