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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.


Myocardial superoxide anion production induced by Ang II or EGF. Cardiac strips from shSCR‐ or shEGFR‐expressing hearts were stimulated with 1 nmol/L Ang II or 0.1 μg/mL EGF. While both significantly increased superoxide anion production in the scramble group, the effect was canceled in the EGFR‐silenced group (A, results expressed as percentage of nonstimulated control). Importantly, left ventricle basal oxidative stress (B, estimated by lipid peroxidation through the TBARS method) as well as basal NOX activity (C, estimated by superoxide production) was not statistically different between groups. Furthermore, NOX gp91 membrane subunit protein expression level was not affected by the experimental procedure (D, representative immunoblots and corresponding averaged results from band densitometry analysis). (ANOVA, *P<0.05 vs control.) The number of independent experiments are included in the bars). Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; NOX, NADPH oxidase; shRNA, small hairpin RNA; TBARS, thiobarbituric acid reactive substances.
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jah31787-fig-0005: Myocardial superoxide anion production induced by Ang II or EGF. Cardiac strips from shSCR‐ or shEGFR‐expressing hearts were stimulated with 1 nmol/L Ang II or 0.1 μg/mL EGF. While both significantly increased superoxide anion production in the scramble group, the effect was canceled in the EGFR‐silenced group (A, results expressed as percentage of nonstimulated control). Importantly, left ventricle basal oxidative stress (B, estimated by lipid peroxidation through the TBARS method) as well as basal NOX activity (C, estimated by superoxide production) was not statistically different between groups. Furthermore, NOX gp91 membrane subunit protein expression level was not affected by the experimental procedure (D, representative immunoblots and corresponding averaged results from band densitometry analysis). (ANOVA, *P<0.05 vs control.) The number of independent experiments are included in the bars). Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; NOX, NADPH oxidase; shRNA, small hairpin RNA; TBARS, thiobarbituric acid reactive substances.

Mentions: Increased NHE1 activity during stretch is dependent of reactive oxygen species (ROS) formation and activation of redox‐sensitive kinases.21 In the present study, cardiac left ventricle muscle strips were used to study myocardial O2˙ production in response to Ang II (1 nmol/L) or the specific EGFR agonist, EGF stimuli in both experimental groups. Figure 5A shows that Ang II significantly increased O2˙ production in cardiac samples of shRNA‐SCR–injected hearts, whereas this effect was not observed in those in the EGFR‐silenced group. Additionally, when an equipotent concentration of EGF (0.1 μg/mL) was used to directly stimulate the EGFR, a significant increase in O2˙ production was observed in the shRNA‐SCR group, an effect that was completely abrogated in the cardiac strips of the shRNA‐EGFR group (Figure 5A). Since EGFR activation increases ROS production, the chronic EGFR‐silencing effect on basal redox homeostasis was analyzed. Lipid peroxidation estimated by quantifying TBARS in shRNA‐SCR– or shRNA‐EGFR–injected hearts was of the same magnitude (Figure 5B), indicating that oxidative stress was similar in both experimental groups. On the other hand, NOX2, one of the most important sources of O−2 in cardiac tissue that plays an important role in Ang II‐induced cardiac hypertrophy,26 can change its expression in response to different stimuli.22 In the present study, total NOX activity (Figure 5C), as well as protein expression level of the NOX2 gp91 membrane subunit (Figure 5D), did not differ significantly between groups, indicating that it was not affected by chronic reduction of the EGFR expression.


Epidermal Growth Factor Receptor Silencing Blunts the Slow Force Response to Myocardial Stretch
Myocardial superoxide anion production induced by Ang II or EGF. Cardiac strips from shSCR‐ or shEGFR‐expressing hearts were stimulated with 1 nmol/L Ang II or 0.1 μg/mL EGF. While both significantly increased superoxide anion production in the scramble group, the effect was canceled in the EGFR‐silenced group (A, results expressed as percentage of nonstimulated control). Importantly, left ventricle basal oxidative stress (B, estimated by lipid peroxidation through the TBARS method) as well as basal NOX activity (C, estimated by superoxide production) was not statistically different between groups. Furthermore, NOX gp91 membrane subunit protein expression level was not affected by the experimental procedure (D, representative immunoblots and corresponding averaged results from band densitometry analysis). (ANOVA, *P<0.05 vs control.) The number of independent experiments are included in the bars). Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; NOX, NADPH oxidase; shRNA, small hairpin RNA; TBARS, thiobarbituric acid reactive substances.
© Copyright Policy - creativeCommonsBy-nc-nd
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121502&req=5

jah31787-fig-0005: Myocardial superoxide anion production induced by Ang II or EGF. Cardiac strips from shSCR‐ or shEGFR‐expressing hearts were stimulated with 1 nmol/L Ang II or 0.1 μg/mL EGF. While both significantly increased superoxide anion production in the scramble group, the effect was canceled in the EGFR‐silenced group (A, results expressed as percentage of nonstimulated control). Importantly, left ventricle basal oxidative stress (B, estimated by lipid peroxidation through the TBARS method) as well as basal NOX activity (C, estimated by superoxide production) was not statistically different between groups. Furthermore, NOX gp91 membrane subunit protein expression level was not affected by the experimental procedure (D, representative immunoblots and corresponding averaged results from band densitometry analysis). (ANOVA, *P<0.05 vs control.) The number of independent experiments are included in the bars). Ang II indicates angiotensin II; EGF, epidermal growth factor; EGFR, EGF receptor; NOX, NADPH oxidase; shRNA, small hairpin RNA; TBARS, thiobarbituric acid reactive substances.
Mentions: Increased NHE1 activity during stretch is dependent of reactive oxygen species (ROS) formation and activation of redox‐sensitive kinases.21 In the present study, cardiac left ventricle muscle strips were used to study myocardial O2˙ production in response to Ang II (1 nmol/L) or the specific EGFR agonist, EGF stimuli in both experimental groups. Figure 5A shows that Ang II significantly increased O2˙ production in cardiac samples of shRNA‐SCR–injected hearts, whereas this effect was not observed in those in the EGFR‐silenced group. Additionally, when an equipotent concentration of EGF (0.1 μg/mL) was used to directly stimulate the EGFR, a significant increase in O2˙ production was observed in the shRNA‐SCR group, an effect that was completely abrogated in the cardiac strips of the shRNA‐EGFR group (Figure 5A). Since EGFR activation increases ROS production, the chronic EGFR‐silencing effect on basal redox homeostasis was analyzed. Lipid peroxidation estimated by quantifying TBARS in shRNA‐SCR– or shRNA‐EGFR–injected hearts was of the same magnitude (Figure 5B), indicating that oxidative stress was similar in both experimental groups. On the other hand, NOX2, one of the most important sources of O−2 in cardiac tissue that plays an important role in Ang II‐induced cardiac hypertrophy,26 can change its expression in response to different stimuli.22 In the present study, total NOX activity (Figure 5C), as well as protein expression level of the NOX2 gp91 membrane subunit (Figure 5D), did not differ significantly between groups, indicating that it was not affected by chronic reduction of the EGFR expression.

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.