Constitutive glycogen synthase kinase-3alpha/beta activity protects against chronic beta-adrenergic remodelling of the heart

Abstract

Aims: Glycogen synthase kinase 3 (GSK-3) signalling is implicated in the growth of the heart during development and in response to stress. However, its precise role remains unclear. We set out to characterize developmental growth and response to chronic isoproterenol (ISO) stress in knockin (KI) mice lacking the critical N-terminal serines, 21 of GSK-3alpha and 9 of GSK-3beta respectively, required for inactivation by upstream kinases.

Methods and results: Between 5 and 15 weeks, KI mice grew more rapidly, but normalized heart weight and contractile performance were similar to wild-type (WT) mice. Isolated hearts of both genotypes responded comparably to acute ISO infusion with increases in heart rate and contractility. In WT mice, chronic subcutaneous ISO infusion over 14 days resulted in cardiac hypertrophy, interstitial fibrosis, and impaired contractility, accompanied by foetal gene reactivation. These effects were all significantly attenuated in KI mice. Indeed, ISO-treated KI hearts demonstrated reversible physiological remodelling traits with increased stroke volume and a preserved contractile response to acute adrenergic stimulation. Furthermore, simultaneous pharmacological inhibition of GSK-3 in KI mice treated with chronic subcutaneous ISO recapitulated the adverse remodelling phenotype seen in WT hearts.

Conclusion: Expression of inactivation-resistant GSK-3alpha/beta does not affect eutrophic myocardial growth but protects against pathological hypertrophy induced by chronic adrenergic stimulation, maintaining cardiac function and attenuating interstitial fibrosis. Accordingly, strategies to prevent phosphorylation of Ser-21/9, and consequent inactivation of GSK-3alpha/beta, may enable a sustained cardiac response to chronic beta-agonist stimulation while preventing pathological remodelling.

Figure 1

Developmental growth and baseline characteristics of WT (GSK-3α/β^Ser-21/9) and KI (GSK-3α/β^Ala-21/9) mice. (A) Heart weight (HW), body weight (BW), and tibia length (TL) measurements in a cohort of male mice between 5 and 15 weeks of age. n = 25 (grey square, KI) and n = 20 (black circle, WT). (B) Representative western blots of isolated hearts perfused for 15 min with ISO (10⁻⁸ mol) or control (Krebs–Henseleit buffer). (C) GSK-3α/β phosphorylation profile in response to acute and chronic ISO exposure. Representative immunoblots are shown with quantitative analyses of repeat experiments expressed as the ratio of phosphorylated to total protein. Data expressed as mean ± SEM (n = 4), *P < 0.05 vs. control. (D) Concentration–response relationship for isolated hearts perfused with ISO; dP/dt_max, contractile performance; HR, heart rate; LVDP, left ventricular developed pressure. Black circle, WT; grey square, KI, n = 6/group, NS between genotypes in all categories.

Figure 2

Response to chronic exposure to ISO or vehicle control and/or BIO. (A) Heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) in mice exposed to 14-day ISO, vehicle control (CON), and/or intraperitoneal BIO; n ≥ 10/group, *P < 0.05. (B) Protocol for pharmacological GSK-3 inhibition; KI mice subjected to chronic 14-day exposure with ISO/CON additionally received daily intraperitoneal injections of BIO (300 µL of 50 nmol). (C) Representative western blots from hearts snap-frozen at 4 h after intraperitoneal BIO injection. (D) Morphological measurements of mean LV wall thickness recorded from 700 µm-thick sections at the mid-papillary level in hearts; n = 6/group, *P < 0.05. (E) Representative cross-sectional MR images of hearts at end-diastole at 14-day treatment. Images were acquired under isoflurane anaesthesia and are taken at the level of the mid-papillary muscle.

Figure 3

Myocyte cross-sectional area, capillary density, and TUNEL staining. Representative images (A) and quantitative analysis of (B) myocyte cross-sectional area (red; wheat germ agglutinin–rhodamine), (C) capillary density (green; isolectin B4), and (D) TUNEL-positive nuclei; n = 5/group, *P < 0.05.

Figure 4

Fibrosis and foetal gene reactivation. (A) Quantification of fibrosis and mRNA transcript abundance for procollagens IαI, IIIαI, and fibronectin; n = 5/group, *P < 0.05. Additional quantification of the foetal genes, ASA and ANF, was also preformed; n = 8/group, *P < 0.05. All transcript results are normalized to β-actin mRNA levels. (B) Representative examples of picrosirius red-stained LV sections (8 µm thick) viewed under circular polarized light demonstrating the effect of ISO on cardiac fibrosis.

Figure 5

Echocardiographic measures of hearts subjected to ISO or vehicle control (CON). Serial studies were performed under isoflurane inhalational anaesthesia at baseline (Day 0, black bar), after 2-week treatment (Day 14, grey bar), and after 2-week recovery (Day 28, white bar); n = 6/group, *P < 0.05 vs. within-group baseline measurements. (A) Representative long-axis parasternal echocardiographic images, IVS, interventricular septum; LVID, left ventricular internal dimension; LVPW, left ventricular posterior wall. (B) FS [= (LVIDd − LVIDs/LVIDd) × 100] and (C) Structural heart measurements taken at the mid-papillary level; LVIDd/s represent end-diastole and end-systole LVID measurements, respectively.

Figure 6

Chronic ISO treatment and recovery. (A) Experimental protocol: mice were treated for 2 weeks with subcutaneous ISO or vehicle and recovered for 2 weeks after pump extraction. Serial echocardiography was performed at baseline, 14, and 28 days. Parallel experiments were undertaken in chronic ISO-treated animals and hearts explanted for Langendorff-perfusion to test acute adrenergic sensitivity. (B) Dose–response profiles to acute ISO exposure in isolated Langendorff-perfused hearts. Baseline response is compared with that at 2-week ISO treatment (left panel) and 2-week recovery (right panel), respectively. Absolute values are plotted. LVDP, left ventricular developed pressure, +dP/dt, systolic contractile performance; HR, heart rate; n = 6/group, *P < 0.05 vs. WT baseline, ^#P < 0.05 vs. KI treated/recovery groups.