The insulin receptor family and protein kinase B (Akt) are activated in the heart by alkaline pH and α1-adrenergic receptors

Figures

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  Figure 1.. Alkaline pH signals to protein synthesis in perfused hearts via PI3K, activating PKB/Akt and MAPKs. Male rat hearts were perfused in the Langendorff mode (30 min) with Tris buffers at starting pHo 7.4 or 9.1 with/without 50 µM LY294002, Krebs Henseleit buffer (KHB) alone or in the presence of 50 mU/ml insulin or 50 nM A61603, or were subjected to ischaemia (20 min) with reperfusion in KHB (40 min). (A) The rate of protein synthesis was measured by incorporation of [14C]-Phe. (B–D) Protein samples were immunoblotted with antibodies to phosphorylated (Phospho-) or total kinases or Rps6 as indicated. Representative blots are on the left (positions of relative molecular mass markers are indicated). Densitometric analysis (ratio of phosphorylated/total protein) is on the right of each set of immunoblots, with individual data points shown and means ± SEM (n = 3–4 per group). Statistical analysis used one-way ANOVA with Holm–Sidak post-test.

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  Figure 2.. Alkaline pHo activates insulin receptor family members (InsRFs) in the heart. (A and B) Rat heart or kidney whole extracts (A) or heart fractions from differential centrifugation (B) were immunoblotted for the INSRR α subunit. WH, whole heart. (C and D) HEK293 cells expressing human FLAG-INSRRs were treated (1 min) with insulin (50 mU/ml) or pHo 9.1 buffer with/without the indicated concentrations of linsitinib. Samples were immunoblotted with antibodies to phosphorylated (Phospho-) InsRFs, FLAG or phospho- or total PKB/Akt. Blots are representative of three independent cell preparations. IC50 values for individual experiments were determined from densitometric data (D). Results are means ± SEM (n = 3). *P < 0.05 for IC50 for INSRR activated by alkaline pHo relative to IC50 for the insulin receptor (INSR) activated by insulin (unpaired two-tailed t-test). (E) Male rat hearts were perfused (15 min) with Tris buffer at starting pHo 9.1 or in Krebs Henseleit buffer (KHB) with/without 50 mU/ml insulin. Samples were immunoblotted with antibodies to phospho-InsRFs, or total PKB/Akt. Densitometric analysis is on the right as individual data points with means ± SEM (n = 4 per group).…

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  Figure 3.. α1-ARs signal to PKB/Akt and ERK1/2 via insulin receptor family members. (A) Male rat hearts were perfused (30 min) with Krebs Henseleit Buffer (KHB) alone or with 50 nM A61603, with/without 1 µM linsitinib. Samples were immunoblotted for phosphorylated (Phospho-) or total kinases. Representative blots are on the left with densitometric analysis on the right. (B and C) Neonatal rat cardiomyocytes were untreated (control), or exposed to 50 nM A61603 or 100 µM phenylephrine (PE) for 5 min with/without the indicated concentrations of linsitinib. Samples were immunoblotted for phospho- or total kinases. In B, representative blots are on the left with densitometric analysis on the right. Individual data points are shown with means ± SEM. In C, a representative experiment is shown. (D) Neonatal rat cardiomyocytes were untreated (control), or exposed to 50 nM A61603 (24 h) with/without 1 µM linsitinib. Cells were immunostained with antibodies to troponin T. Images from a representative experiment are on the left with assessment of cell area on the right. Individual data points are shown with the means ± SEM (n = 3–4 per group). Statistical analysis used one-way ANOVA with Holm–Sidak…

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  Figure 4.. Linsitinib inhibits acute (24 h) activation of ERK1/2 and cardiac hypertrophy induced by PE in vivo. (A) Schematic of the study. C57Bl/6J male mice were subjected to baseline echocardiography (Echo) then implanted with minipumps to deliver vehicle only (Control), or phenylephrine (PE; 40 mg/kg/d) with/without linsitinib (2 mg/kg/d). Echocardiograms were taken at 1 or 4 days and mice were killed. (B) Heart samples (25 µg) were immunoblotted for phosphorylated (Phospho-) or total kinases. Representative blots are on the left with densitometric analysis on the right. (C) RNA was prepared from the mouse hearts and mRNA expression of hypertrophic (Myh7, Nppa, Nppb) and collagen (Col1a1, Col3a1, Col4a1) genes was assessed by qPCR. (D) Representative M-mode images (left) are shown at baseline and 24 h post minipump implantation, with quantitative analysis (right) of ejection fraction and fractional shortening, plus systolic (s) and diastolic (d) cardiac dimensions at 24 h relative to baseline measurements for individual mice. LVPW, left ventricular posterior wall; LVID, LV internal diameter. Individual data points are shown with means ± SEM (n = 5–6 per group). Statistical analysis used…

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  Figure 5.. Linsitinib exacerbates cardiac hypertrophy induced by phenylephrine over 4 days. C57Bl/6J male mice were treated with vehicle alone (Control), or with phenylephrine (PE; 40 mg/kg/d) with/without linsitinib (2 mg/kg/d) using osmotic minipumps. Cardiac function/dimensions were assessed by echocardiography. (A) Representative M-mode images at baseline and 4 days post minipump implantation. Quantitative assessment of cardiac function (B) and left ventricular cardiac dimensions in diastole (C) and systole (D) at baseline (BL) and 4 days. WT:ID, ratio of wall thickness (WT = anterior wall + posterior wall) to internal diameter (ID). Individual data points are shown with means ± SEM (n = 7 per group). Statistical analysis used two-way ANOVA with Holm–Sidak's post-test.

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  Figure 6.. Linsitinib exacerbates cardiac fibrosis induced by phenylephrine in vivo. C57Bl/6J male mice were treated with vehicle alone (Control), or with phenylephrine (PE; 40 mg/kg/d) with/without linsitinib (2 mg/kg/d) for 4 days. (A) mRNA expression was measured by qPCR. Statistical analysis used two-way ANOVA with Holm–Sidak's post-test (1 day data are also shown in Figure 4 and are presented here for comparison). (B) Heart sections were stained for H&E (upper images) or Masson's Trichrome (lower images; yellow arrows indicate areas of fibrosis). Cardiomyocyte cross-sectional area and myocardial fibrosis were measured (right panels). Individual data points are shown with means ± SEM (n = 4–6 per group). Statistical analysis used one-way ANOVA with Holm–Sidak's post-test.

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  Figure 7.. Schematic model of receptor cross-talk system from α1-adrenergic receptors (α1-ARs) via insulin receptor family members to cardiomyocyte hypertrophy. (A) In cardiomyocytes, α1-ARs signal through Gαq and increase intracellular pHi. Normalisation of pHi increases extracellular pHo which may trigger INSRRs in T tubules. This may serve to prevent excessive intracellular alkalinisation. Alternatively, INSRRs/INSRs/IGF1Rs may become phosphorylated via an intracellular mechanism. On activation, INSRRs/INSRs/IGF1Rs become Tyr phosphorylated and activated protein kinase B (PKB; also known as Akt), signalling via phosphoinositide 3′ kinase (PI3K; green). The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade is also activated (yellow) partly via INSRRs/INSRs/IGF1Rs, but also through an alternative mechanism driven by Gαq. PKB/Akt and ERK1/2 signalling combine to promote protein synthesis, cardioprotection and adaptive hypertrophy. (B) Linsitinib inhibits INSRRs/INSRs/IGF1Rs, preventing PKB/Akt signalling and reducing ERK1/2 signalling, leading to increased fibrosis. This increases the workload of cardiomyocytes which hypertrophy, but the increased stress also causes cardiomyocyte…