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Epidermal Growth Factor Receptor Silencing Blunts the Slow Force Response to Myocardial Stretch

View Article: PubMed Central - PubMed

ABSTRACT

Background: Myocardial stretch increases force biphasically: the Frank‐Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]‐EGFR).

Methods and results: Silencing capability of p‐shEGFR was assessed in EGFR‐GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA‐EGFR–injected hearts showed ≈60% reduction of EGFR protein expression compared with shRNA‐SCR–injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was ≈130% of initial rapid phase in the shRNA‐SCR group, while it was blunted in shRNA‐EGFR–expressing muscles. Angiotensin II (Ang II)‐dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate‐free medium, demonstrating an increase in shRNA‐SCR–injected myocardium, an effect not observed in the silenced group. Ang II‐ or EGF‐triggered reactive oxygen species production was significantly reduced in shRNA‐EGFR–injected hearts compared with that in the shRNA‐SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox‐sensitive pathway, leading to p90RSK activation in shRNA‐SCR‐injected myocardium, an effect that was absent in the shRNA‐EGFR group.

Conclusions: Our results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox‐sensitive kinase activation pathway, leading to SFR development.

No MeSH data available.


Cardiac ERK1/2 and p90RSK activation by Ang II or EGF stimulation. Cardiac tissue strips were incubated during 15 minutes either with Ang II (1 nmol/L) or EGF (0.1 μg/mL) and then homogenized, electrophoresed, and immunoblotted with specific antibodies to detect ERK1/2 (A) and p90RSK (B) phosphorylation. In the shRNA‐SCR group, Ang II promoted a significant p90RSK phosphorylation/activation and a marginally significant increase in ERK1/2 (P=0.057), an effect that was not observed in the shRNA‐EGFR group, as it can be appreciated in the representative blots (top of each panel) as well as in the corresponding averaged results (bottom of each panel). (Mann‐Whitney test [A], t test [B], *P<0.05 vs nonstimulated control.) The number of independent experiments are included in the bars. Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; shRNA, small hairpin RNA.
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jah31787-fig-0006: Cardiac ERK1/2 and p90RSK activation by Ang II or EGF stimulation. Cardiac tissue strips were incubated during 15 minutes either with Ang II (1 nmol/L) or EGF (0.1 μg/mL) and then homogenized, electrophoresed, and immunoblotted with specific antibodies to detect ERK1/2 (A) and p90RSK (B) phosphorylation. In the shRNA‐SCR group, Ang II promoted a significant p90RSK phosphorylation/activation and a marginally significant increase in ERK1/2 (P=0.057), an effect that was not observed in the shRNA‐EGFR group, as it can be appreciated in the representative blots (top of each panel) as well as in the corresponding averaged results (bottom of each panel). (Mann‐Whitney test [A], t test [B], *P<0.05 vs nonstimulated control.) The number of independent experiments are included in the bars. Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; shRNA, small hairpin RNA.

Mentions: We have previously demonstrated that ROS‐sensitive kinases ERK1/2 and p90RSK are responsible for NHE1 activation during myocardial stretch.21 Randomly selected cardiac left ventricle muscle strips from each heart was stimulated with Ang II (1 nmol/L) or EGF (0.1 μg/mL) for 15 minutes and frozen immediately after. Immunoblot analysis of homogenized samples revealed an increase in ERK1/2 and p90RSK phosphorylation in the shRNA‐SCR group, an effect that was not observed in shRNA‐EGFR samples (Figure 6A and 6B). Samples from shRNA‐EGFR–injected hearts treated with Ang II produced a similar amount of ERK1/2 phosphorylation as the nonstimulated samples. Results from samples treated with EGF exposed a large mean and standard error, explained by the finding that one of the four samples analyzed presented an exceeded phosphorylation band compared with the rest of the samples. Duplicate analysis of this sample generated the same outcome. Overall analysis indicates that neither Ang II nor EGF stimulation of shRNA‐EGFR–injected heart samples induced a significant increase in ERK1/2 or p90RSK phosphorylation. Total kinases expression measured on immunoblots revealed no modified expression of the silenced versus nonsilenced group (Figure S2).


Epidermal Growth Factor Receptor Silencing Blunts the Slow Force Response to Myocardial Stretch
Cardiac ERK1/2 and p90RSK activation by Ang II or EGF stimulation. Cardiac tissue strips were incubated during 15 minutes either with Ang II (1 nmol/L) or EGF (0.1 μg/mL) and then homogenized, electrophoresed, and immunoblotted with specific antibodies to detect ERK1/2 (A) and p90RSK (B) phosphorylation. In the shRNA‐SCR group, Ang II promoted a significant p90RSK phosphorylation/activation and a marginally significant increase in ERK1/2 (P=0.057), an effect that was not observed in the shRNA‐EGFR group, as it can be appreciated in the representative blots (top of each panel) as well as in the corresponding averaged results (bottom of each panel). (Mann‐Whitney test [A], t test [B], *P<0.05 vs nonstimulated control.) The number of independent experiments are included in the bars. Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; shRNA, small hairpin RNA.
© Copyright Policy - creativeCommonsBy-nc-nd
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121502&req=5

jah31787-fig-0006: Cardiac ERK1/2 and p90RSK activation by Ang II or EGF stimulation. Cardiac tissue strips were incubated during 15 minutes either with Ang II (1 nmol/L) or EGF (0.1 μg/mL) and then homogenized, electrophoresed, and immunoblotted with specific antibodies to detect ERK1/2 (A) and p90RSK (B) phosphorylation. In the shRNA‐SCR group, Ang II promoted a significant p90RSK phosphorylation/activation and a marginally significant increase in ERK1/2 (P=0.057), an effect that was not observed in the shRNA‐EGFR group, as it can be appreciated in the representative blots (top of each panel) as well as in the corresponding averaged results (bottom of each panel). (Mann‐Whitney test [A], t test [B], *P<0.05 vs nonstimulated control.) The number of independent experiments are included in the bars. Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; shRNA, small hairpin RNA.
Mentions: We have previously demonstrated that ROS‐sensitive kinases ERK1/2 and p90RSK are responsible for NHE1 activation during myocardial stretch.21 Randomly selected cardiac left ventricle muscle strips from each heart was stimulated with Ang II (1 nmol/L) or EGF (0.1 μg/mL) for 15 minutes and frozen immediately after. Immunoblot analysis of homogenized samples revealed an increase in ERK1/2 and p90RSK phosphorylation in the shRNA‐SCR group, an effect that was not observed in shRNA‐EGFR samples (Figure 6A and 6B). Samples from shRNA‐EGFR–injected hearts treated with Ang II produced a similar amount of ERK1/2 phosphorylation as the nonstimulated samples. Results from samples treated with EGF exposed a large mean and standard error, explained by the finding that one of the four samples analyzed presented an exceeded phosphorylation band compared with the rest of the samples. Duplicate analysis of this sample generated the same outcome. Overall analysis indicates that neither Ang II nor EGF stimulation of shRNA‐EGFR–injected heart samples induced a significant increase in ERK1/2 or p90RSK phosphorylation. Total kinases expression measured on immunoblots revealed no modified expression of the silenced versus nonsilenced group (Figure S2).

View Article: PubMed Central - PubMed

ABSTRACT

Background: Myocardial stretch increases force biphasically: the Frank&#8208;Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]&#8208;EGFR).

Methods and results: Silencing capability of p&#8208;shEGFR was assessed in EGFR&#8208;GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA&#8208;EGFR&ndash;injected hearts showed &asymp;60% reduction of EGFR protein expression compared with shRNA&#8208;SCR&ndash;injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was &asymp;130% of initial rapid phase in the shRNA&#8208;SCR group, while it was blunted in shRNA&#8208;EGFR&ndash;expressing muscles. Angiotensin II (Ang II)&#8208;dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate&#8208;free medium, demonstrating an increase in shRNA&#8208;SCR&ndash;injected myocardium, an effect not observed in the silenced group. Ang II&#8208; or EGF&#8208;triggered reactive oxygen species production was significantly reduced in shRNA&#8208;EGFR&ndash;injected hearts compared with that in the shRNA&#8208;SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox&#8208;sensitive pathway, leading to p90RSK activation in shRNA&#8208;SCR&#8208;injected myocardium, an effect that was absent in the shRNA&#8208;EGFR group.

Conclusions: Our results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox&#8208;sensitive kinase activation pathway, leading to SFR development.

No MeSH data available.