First published online July 14, 2008
Journal of Experimental Biology 211, 2524-2532 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018960
Differential roles of p38-MAPK and JNKs in mediating early protection or apoptosis in the hyperthermic perfused amphibian heart
Catherine Gaitanaki,
Michalis Mastri,
Ioanna-Katerina S. Aggeli and
Isidoros Beis*
Department of Animal and Human Physiology, School of Biology, University
of Athens, Panepistimioupolis, 157 84 Athens, Greece
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Fig. 1. (A) Representative records of the isolated perfused Rana ridibunda
heart electromechanical activity. Isolated hearts were perfused at either 25
or 42°C for up to 6 h with bicarbonate buffer pH 7.35, and the electrical
as well as mechanical activity were monitored throughout using an appropriate
setting as described in Materials and methods. (B) Time course of the
mechanical activity (%) of the isolated perfused heart at the two different
temperatures studied. *P<0.05 vs the
respective value at time zero.
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Fig. 2. Time profile of hyperthermia (42°C)-induced p38-MAPK (A) and Hsp27 (C)
phosphorylation in samples from isolated perfused Rana ridibunda
hearts. (A) Upper panel: phospho-p38-MAPK was detected in extracts (50 µg
of protein) from control hearts (0.5 h equilibration at 25°C followed by
0.5 h perfusion at 25°C) or hearts perfused after equilibration at
42°C for increasing periods of time as indicated. (C) Upper panel:
phospho-Hsp27 was detected in corresponding samples. As a control for equal
protein loading, an anti-actin antibody was used (A,C: bottom panels).
Densitometric analysis of phospho-p38 (B) and phospho-Hsp27 (D) bands was
performed by laser scanning. Western blots shown are representative of at
least three independent experiments while data are means ± s.e.m. from
at least three independent experiments. *P<0.05 and
**P<0.01 vs control (untreated) hearts.
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Fig. 3. Time course of phosphorylation of JNKs and c-Jun in samples from isolated
perfused Rana ridibunda hearts. (A) Upper panel: phospho-JNKs were
detected in extracts (50 µg of protein) from control hearts (0.5 h
equilibration at 25°C followed by 0.5 h perfusion at 25°C) or hearts
perfused after equilibration at 42°C for increasing periods of time as
indicated. (C) Upper panel: phospho-c-Jun was detected in corresponding
samples. The membranes were re-probed with an anti-actin antibody so as to
verify equal protein loading (A,C: bottom panels). Densitometric analysis of
phospho-JNKs (B) and phospho-c-Jun (D) bands was performed by laser scanning.
Western blots shown are representative of at least three independent
experiments while data are means ± s.e.m. for at least three
independent experiments. **P<0.01 and
 P<0.001 vs control (untreated)
hearts.
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Fig. 4. Profile of caspase 3 and poly(ADP-ribose) polymerase (PARP) cleavage in
samples from isolated Rana ridibunda hearts perfused after
equilibration at 42°C for increasing periods of time as indicated. (A)
Upper panel: pro-caspase 3 immunoreactivity was observed with no cleaved
active forms of caspase 3 detected. (B) Upper panel: proteolytic processing of
PARP was observed and quantification of its fragment was performed by laser
scanning densitometry (C). The membranes were re-probed with an anti-actin
antibody so as to verify equal protein loading (A,B: bottom panels). Western
blots shown are representative of at least three independent experiments while
data are means ± s.e.m. for at least three independent experiments.
*P<0.05 and P<0.001
vs control hearts (0.5 h equilibration followed by 1 h perfusion at
25°C).
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Fig. 5. Effect of HOE642 (HOE), ouabain (Ouab.), catalase (Cat.) and superoxide
dismutase (SOD) on hyperthermia-induced phosphorylation of p38-MAPK (A: upper
panel). The phosphorylated form of the kinase was detected in extracts (50
µg of protein) from control hearts (0.5 h equilibration followed by 1 h
perfusion at 25°C) or hearts perfused after equilibration for 1 h at
42°C in the presence or absence of HOE642 (5 µmol
l–1), ouab (100 µmol l–1), catalase (150
U ml–1) and SOD (30 U ml–1). The effects of
HOE642 and ouabain alone were also assessed. As a control for equal protein
loading an anti-actin antibody was used (A: bottom panel). Densitometric
analysis of phospho-p38-MAPK (B) was performed by laser scanning. (C)
Relationship of the net p38-MAPK phosphorylation levels induced by
hyperthermia in the presence of the agents assessed to the
hyperthermia-induced p38-MAPK phosphorylation levels. Western blots shown are
representative of at least three independent experiments while data are means
± s.e.m. for at least three independent experiments.
**P<0.001 vs control hearts and
P<0.001 vs hearts perfused at
42°C.
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Fig. 6. Effect of HOE642, ouabain, catalase and SOD on hyperthermia-induced
phosphorylation of JNKs (A: upper panel). Phosphorylated JNKs were detected in
extracts (50 µg of protein) from control hearts (0.5 h equilibration
followed by 1 h perfusion at 25°C) or hearts perfused after equilibration
for 1 h at 42°C in the presence or absence of HOE642 (5 µmol
l–1), ouabain (100 µmol l–1), catalase
(150 U ml–1) and SOD (30Uml–1). The effects
of HOE642 and ouabain alone were also assessed. As a control for equal protein
loading, an anti-actin antibody was used (A: bottom panel). Densitometric
analysis of phospho-JNKs (B) was performed by laser scanning. (C) Relationship
of the net JNK phosphorylation level (%) induced by hyperthermia in the
presence of the agents assessed to the hyperthermia-induced JNKs
phosphorylation level. Western blots shown are representative of at least
three independent experiments while data are means ± s.e.m. for at
least three independent experiments. P<0.001
vs control hearts and *P<0.01 vs
hearts perfused at 42°C.
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Fig. 7. Effect of HOE642, ouabain, catalase and SOD on hyperthermia-induced
phosphorylation of JNKs (A: upper panel). Phosphorylated JNKs were detected in
extracts (50 µg of protein) from control hearts (0.5 h equilibration
followed by 4 h perfusion at 25°C) or hearts perfused after equilibration
for 4 h at 42°C in the presence or absence of HOE642 (5 µmol
l–1), ouabain (100 µmol l–1), catalase
(150 U ml–1) and SOD (30Uml–1). The effects
of HOE642 and ouabain alone were also assessed. As a control for equal protein
loading, an anti-actin antibody was used (A: bottom panel). Densitometric
analysis of phospho-JNKs (B) was performed by laser scanning. (C) Relationship
of the net JNK phosphorylation level (%) induced by hyperthermia in the
presence of the agents assessed to the hyperthermia-induced JNK
phosphorylation level. Western blots shown are representative of at least
three independent experiments while data are means ± s.e.m. for at
least three independent experiments. **P<0.001
vs control hearts and P<0.001
vs hearts perfused at 42°C.
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Fig. 8. Effect of HOE642, ouabain, catalase, SOD, SP600125 (SP) and AS601245 (AS)
on hyperthermia-induced cleavage of PARP (A: upper panel). Proteolytical
processing of PARP was detected in extracts (50 µg of protein) from control
hearts (0.5 h equilibration followed by 4 h perfusion at 25°C) or hearts
perfused after equilibration for 4 h at 42°C in the presence or absence of
HOE642 (5 µmol l–1), ouab (100 µmol
l–1), catalase (150 U ml–1), SOD (30 U
ml–1), SP (10 µmol l–1) and AS (1 µmol
l–1). To confirm equal protein loading the membranes were
re-probed with an anti-actin antibody (A: bottom panel). Densitometric
analysis of the band corresponding to the fragment of PARP was performed by
laser scanning (B). Western blots shown are representative of at least three
independent experiments while data are means ± s.e.m. for at least
three independent experiments. P<0.001
vs control hearts and *P<0.001 vs
hearts perfused at 42°C.
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