The Anrep effect requires transactivation of the epidermal growth factor receptor

Abstract

Myocardial stretch elicits a biphasic contractile response: the Frank-Starling mechanism followed by the slow force response (SFR) or Anrep effect. In this study we hypothesized that the SFR depends on epidermal growth factor receptor (EGFR) transactivation after the myocardial stretch-induced angiotensin II (Ang II)/endothelin (ET) release. Experiments were performed in isolated cat papillary muscles stretched from 92 to 98% of the length at which maximal twitch force was developed (L(max)). The SFR was 123 +/- 1% of the immediate rapid phase (n = 6, P < 0.05) and was blunted by preventing EGFR transactivation with the Src-kinase inhibitor PP1 (99 +/- 2%, n = 4), matrix metalloproteinase inhibitor MMPI (108 +/- 4%, n = 11), the EGFR blocker AG1478 (98 +/- 2%, n = 6) or the mitochondrial transition pore blocker clyclosporine (99 +/- 3%, n = 6). Stretch increased ERK1/2 phosphorylation by 196 +/- 17% of control (n = 7, P < 0.05), an effect that was prevented by PP1 (124 +/- 22%, n = 7) and AG1478 (131 +/- 17%, n = 4). In myocardial slices, Ang II (which enhances ET mRNA) or endothelin-1 (ET-1)-induced increase in O(2)() production (146 +/- 14%, n = 9, and 191 +/- 17%, n = 13, of control, respectively, P < 0.05) was cancelled by AG1478 (94 +/- 5%, n = 12, and 98 +/- 15%, n = 8, respectively) or PP1 (100 +/- 4%, n = 6, and 99 +/- 8%, n = 3, respectively). EGF increased O(2)() production by 149 +/- 4% of control (n = 9, P < 0.05), an effect cancelled by inhibiting NADPH oxidase with apocynin (110 +/- 6% n = 7), mKATP channels with 5-hydroxydecanoic acid (5-HD; 105 +/- 5%, n = 8), the respiratory chain with rotenone (110 +/- 7%, n = 7) or the mitochondrial permeability transition pore with cyclosporine (111 +/- 10%, n = 6). EGF increased ERK1/2 phosphorylation (136 +/- 8% of control, n = 9, P < 0.05), which was blunted by 5-HD (97 +/- 5%, n = 4), suggesting that ERK1/2 activation is downstream of mitochondrial oxidative stress. Finally, stretch increased Ser703 Na(+)/H(+) exchanger-1 (NHE-1) phosphorylation by 172 +/- 24% of control (n = 4, P < 0.05), an effect that was cancelled by AG1478 (94 +/- 17%, n = 4). In conclusion, our data show for the first time that EGFR transactivation is crucial in the chain of events leading to the Anrep effect.

Figure 1. SFR and EGFR transactivation

A, a typical force record from a cat papillary muscle subjected to an increase in length from 92 to 98% of L_max; the biphasic response to stretch can be seen (vertical dotted lines indicate stretching interval). B–D, same as A but from muscles pretreated with matrix metalloproteinase inhibitor (MMPI, B), the Src kinase inhibitor PP1 (C) or the EGFR blocker AG1478 (D), interventions that cancel EGFR transactivation. As can be seen, all these pharmacological interventions prevented the development of the SFR to stretch. E, the averaged results obtained under the different experimental conditions expressed as a percentage of the initial rapid phase. *P < 0.05 control curve vs. others (2-way ANOVA). §P < 0.05 vs. initial rapid phase (for the sake of clarity, significance is indicated only for 15 min of stretch).

Figure 2. ERK1/2 phosphorylation after stretch

Five minutes of stretch significantly increased ERK1/2 phosphorylation, kinases upstream NHE-1, that we have previously shown to play a critical role in SFR development (Caldiz et al. 2007). This effect was blunted either by EGFR blockade with AG1478 (AG, A) or by Src kinase inhibition with PP1 (B), demonstrating that EGFR transactivation after stretch is necessary for ERK1/2 phosphorylation. It is important to highlight that either AG1478 or PP1 alone did not modify basal ERK1/2 phosphorylation (92 ± 4%, n= 4, and 107 ± 8%, n= 4, of control respectively). *P < 0.05 vs. non-stretched control.

Figure 3. O₂⁻ production after addition of Ang II or ET-1 in cardiac slices

The increase in O₂⁻ production induced either by 1 nmol l⁻¹ Ang II (A) or 1 nmol l⁻¹ ET-1 (B) was cancelled by AG1478 or PP1. These findings demonstrate that the well known increase in ROS production after Ang II/ET-1 treatment requires EGFR transactivation in the myocardium. *P < 0.05 vs. control.

Figure 4. Ang II–ET-1 crosstalk

Ang II (1 nmol l⁻¹) promoted a significant increase in preproET-1 mRNA expression detected by real-time RT-PCR, a result that confirms the Ang II–ET crosstalk in the cat myocardium. This increase in preproET-1 mRNA was, as expected, not only cancelled by AT1 receptor blockade with losartan (Los), but also by scavenging ROS with 2-mercaptopropionyl glycine (MPG) or by inhibiting NADPH oxidase with apocynin (Apo), indicating that NADPH oxidase-mediated ROS production is a key step to increase ET-1 expression. *P < 0.05 vs. control.

Figure 5. O₂⁻ production after addition of EGF in cardiac slices

Direct activation of the EGFR by EGF significantly enhanced O₂⁻ production in a dose-dependent manner (A). We selected the lower concentration of EGF (0.1 μg ml⁻¹) to perform subsequent experiments because it increased O₂⁻ production in a similar magnitude to 1 nmol l⁻¹ ET-1 (see Fig. 3). As expected, the 0.1 μg ml⁻¹ EGF effect was blunted by specific EGFR blockade with AG1478 (AG) but not by Src kinase inhibition with PP1 (B) since the latter is located upstream of EGFR. The EGF-induced increase in O₂⁻ production was also prevented by NADPH oxidase inhibition with apocynin (Apo), by two different mitochondrial ATP-sensitive K⁺ channel (mKATP) blockers (5-hydroxydecanoate (5-HD) and glybenclamide (Gly)), and also by the respiratory chain inhibitor rotenone (Rot). These data support the hypothesis that EGF is a necessary mediator in the stretch-induced mitochondrial ROS production. * and # indicate P < 0.05 vs. control and 0.1 μg ml⁻¹ EGF respectively.

Figure 6. ERK1/2 phosphorylation after EGF stimulation

EGF significantly increased ERK1/2 phosphorylation, an effect that was cancelled by 5-HD, glybenclamide (Gly), rotenone (Rot) or apocynin (Apo). These results further support the notion that mitochondrial ROS formation is a necessary upstream step to ERK 1/2 phosphorylation. *P < 0.05 vs. control.

Figure 7. Role of MPTP in the development of the SFR

MPTP inhibition with cyclosporine cancelled the SFR as shown in A (representative force record) and B (averaged results expressed as a percentage of the initial rapid phase). Interestingly, cyclosporine also cancelled the increase in O₂⁻ production induced by 0.1 ng ml⁻¹ EGF. These results suggest a role for MPTP in the development of the Anrep effect, probably by permitting the release of mitochondrial ROS. For the sake of comparison a control SFR already presented in Fig. 1E and a control EGF-promoted O₂⁻ production shown in Fig. 5B were also included in panels B and C. *P < 0.05 vs. control (panel B) or vs. EGF (panel C).

Figure 8. Stretch-induced NHE-1 phosphorylation

Five minutes of stretch significantly increased NHE-1 phosphorylation at Ser703 estimated by a phospho-Ser 14-3-3 binding motif antibody. This effect was cancelled when the EGFR was blocked by AG 1478 (AG). These results support a role of the EGFR transactivation in the chain of events leading to NHE-1 phosphorylation and SFR development. It is important to highlight that AG1478 alone did not modify basal NHE-1 phosphorylation (93 ± 4% of control, n= 4). *P < 0.05 vs. control.

Figure 9. Scheme of the proposed mechanism leading to the SFR

Stretch and/or angiotensin II (Ang II) through AT1 receptor stimulation triggers the release/formation of endothelin (ET). NADPH oxidase-generated reactive oxygen species (ROS) probably play a significant role in this step of the signalling cascade. ET activates the ET_A receptor and induces epidermal growth factor receptor (EGFR) transactivation. The communication between the ET_A receptor and the EGFR involves matrix metalloprotease (MMP) activation and possibly the ROS-sensitive Src kinase. EGFR activation triggers an intracellular signalling pathway that leads to mKATP channel opening increasing ROS production and release through the mitochondrial permeability transition pore (MPTP). This causes ERK1/2 activation and NHE-1 phosphorylation at Ser703, increasing NHE-1 activity and intracellular Na⁺ concentration that favours intracellular Ca²⁺ accumulation through the Na⁺/Ca²⁺ exchanger (NCX). The increase in ROS production can also contribute to the increase in intracellular Ca²⁺ through a direct stimulatory effect on the NCX (Eigel et al. 2004) and by increasing action potential duration (Zhou et al. 2006).