ErbB2/EGFR dimers are reduced in diabetes and by chronic treatment with AG1478 or AG825.

<p>Panel a) are represenatative Western Blots following immunoprecipitations (IP) with either total-EGFR or total-erbB2 antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG1478 (+AG1478) or AG825 (+AG825). N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).</p

Effect of combined treatment with Losartan and Epidermal Growth Factor (EGF) on post ischemic recovery in cadiac contractility and coronary flow.

<p>The data for Baseline was computed at 30 min perfusion period before ischemia and the data for Reperfusion (REP) was computed at 30 min reperfusion period, and expressed as mean±SEM (N=6); Pmax=Left ventricular developed pressure; LVEDP=Left ventricular end-diastolic pressure; +dp/dt=Positive derivative of pressure; −dp/dt=Negative derivative of pressure; %R=% recovery=(reperfusion/baseline)×100; R=drug given during Reperfusion after Ischemia;</p>*<p>Value significantly different from Diabetes, p<0.05;</p><p># Value significantly different from D+Losartan, p<0.05.</p

Diabetes is associated with reduced expression and phosphorylation of EGFR receptor at multiple tyrosine sites that can be further inhibited by chronic treatment with AG1478.

<p><b>a</b>) Representative Western blots showing levels of phosphorylated EGFR at Y992, Y1068, Y1086, and Y1148 as well as total EGFR (t-EGFR) and Actin as a control protein in non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG1478 (+AG1478). <b>b</b>) quantification of EGFR expression relative to actin and <b>c–f</b>) quantification of EGFR phosphorylation at the stated tyrosine site relative to total EGFR expression for all the groups studied by densitometry. N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).</p

Acute EGF treatment in diabetic hearts opposes the diabetes and/or ischemia-induced changes in phosphorylation of EGFR/erbB2 signaling cascade.

<p><b>a</b>) A representative Western blot of phosphorylation changes in key molecules following acute administration of EGF before ischemia (DEP) or after ischemia (DER) is compared to diabetic hearts subjected to 40 mins ischemia (DI); b–g) densitometry plots quantifying the relative intensity of bands for the stated molecule relative to actin. N=4; * significantly different to DI.</p

Schematic model summarising our findings on the role of EGFR/erbB2 signaling in diabetes-induced cardiac dysfunction.

<p>Diabetes and/or hyperglycemia via attenuation of the EGFR/erbB2 signaling and through subsequent modulation of downstream effectors such as ERK1/2, p38 MAPK or AKT/FOXO can lead to cardiac dysfunction. The effects of diabetes on EGFR/erbB2 pathway are exacerbated by blockade of this pathway by AG1478 or AG825 which leads to worsening cardiac recovery from I/R. However, the inhibitory effects of diabetes on EGFR/ErbB2 pathway may be opposed by administering EGF that also leads to improved cardiac function. The Angiotensin II (Ang II)/AT₁ receptor pathway can also activate EGFR/erbB2 pathway that can be blocked by Losartan. Co-administration of EGF with Losartan attenuates losartan-mediated EGFR blockade and improves cardiac function in diabetes beyond that attained by either drug alone.</p

Acute EGF administration prevented Losartan-mediated inhibition of EGFR phosphorylation in diabetic hearts.

<p><b>a</b>) A representative Western blot showing post-I/R levels of phosphorylated (p-EGFR) and total EGFR (T-EGFR) in diabetic hearts (D) and following acute administration during reperfusion of Losartan (LOS), EGF (EGF) or their combination (EGF+LOS) and b) densitometric histogram quantifying the relative intensity of p-EGFR/t-EGFR following normalization of each molecule to actin. N=4; * significantly different to D and ** significantly different to LOS.</p

Acute EGF treatment in non-diabetic control hearts opposes the ischemia-induced changes in phosphorylation of EGFR/erbB2 signaling cascade.

<p><b>a</b>) A representative Western blot of phosphorylation changes in key molecules following acute administration of EGF before ischemia (CEP) or after ischemia (CER) is compared to control hearts subjected to 40 mins ischemia (CI); b–g) densitometry plots quantifying the relative intensity of bands for the stated molecule relative to actin. N=4; * significantly different to CI.</p

The effect of erbB inhbitors on phosphorylation of ERK1/2, p38 MAP kinase and AKT signaling.

<p><b>a</b>) Representative Western blots and <b>b–d</b>) quantification of phosphorylation for the stated molecule relative to total actin expression for all the groups studied by densitometry. N=4; ** significantly different from diabetes (p<0.05).</p

Effect of chronic treatment with AG1478 or AG825 on post ischemic recovery in cardiac contractility and coronary flow.

<p>The data for Baseline was computed at 30 min perfusion period before ischemia and the data for Reperfusion (REP) was computed at 30 min reperfusion period, and expressed as mean±SEM (N=6). Pmax=Left ventricular developed pressure; LVEDP=Left ventricular end-diastolic pressure; +dp/dt=Positive derivative of pressure; −dp/dt=Negative derivative of pressure; %R=% recovery=(reperfusion/baseline)×100;</p>*<p>Value significantly different from Control, p<0.05;</p><p># Value significantly different from Diabetes, p<0.05.</p

Diabetes is associated with reduced expression and phosphorylation of erbB2 receptor at multiple tyrosine sites that can be further inhibited by chronic treatment with AG825.

<p><b>a</b>) Representative Western blots showing levels of phosphorylated erbB2 at Y877, Y1248, Y1248-a (which represents detection of Y1248 using an alternative antibody (p- erbB2-Antibody (Tyr1248)/EGFR (Tyr1173)) and Y12221/2 as well as total erbB2 (t-erbB2) and actin as a control protein in non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG825 (+AG825). <b>b</b>) quantification of erbB2 expression relative to actin and <b>c–f</b>) quantification of erbB2 phosphorylation at the stated tyrosine site relative to total erbB2 expression for all the groups studied by densitometry. N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).</p