Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure

Abstract

Although increased external load initially induces cardiac hypertrophy with preserved contractility, sustained overload eventually leads to heart failure through poorly understood mechanisms. Here we describe a conditional transgenic system in mice characterized by the sequential development of adaptive cardiac hypertrophy with preserved contractility in the acute phase and dilated cardiomyopathy in the chronic phase following the induction of an activated Akt1 gene in the heart. Coronary angiogenesis was enhanced during the acute phase of adaptive cardiac growth but reduced as hearts underwent pathological remodeling. Enhanced angiogenesis in the acute phase was associated with mammalian target of rapamycin-dependent induction of myocardial VEGF and angiopoietin-2 expression. Inhibition of angiogenesis by a decoy VEGF receptor in the acute phase led to decreased capillary density, contractile dysfunction, and impaired cardiac growth. Thus, both heart size and cardiac function are angiogenesis dependent, and disruption of coordinated tissue growth and angiogenesis in the heart contributes to the progression from adaptive cardiac hypertrophy to heart failure.

Figure 1

Generation of cardiac-specific inducible Akt1–Tg mice. (A) Schematic illustration of binary Tg system. (B) DOX-dependent expression of Akt1 transgene. Top: Temporal profile of DOX treatment. Bottom: Western blot analysis of transgene expression. (C) Kinase assay in vitro. Left: Western blot analysis of Akt substrate. GSK3, glycogen synthase kinase 3. Right: Densitometric analysis of Akt activity.

Figure 2

Cardiac hypertrophy is induced by short-term Akt activation. (A) Top: Temporal profile of DOX treatment. Bottom: Representative gross morphology of the DTG hearts. (B) HW/BW ratio. *P < 0.01. (C) Time course of cardiac hypertrophy. Top: Time course of transgene expression. Bottom: Time course of HW/BW ratio. ^†P < 0.01 versus day 0; ^#P < 0.05 versus day 14. (D) Echocardiography. Top: Representative M-mode recordings. Bottom: Posterior wall thickness (PWT), LV end-diastolic dimension (LVDd), and percent fractional shortening (%FS). **P < 0.05. (E) Histological analysis. H&E, wheat germ agglutinin (WGA), and Masson’s trichrome (MT) staining of heart sections. Scale bars: 50 μm. (F) Fold induction of ANP and β-MHC expression after short-term Akt activation.

Figure 3

Extensive hypertrophy and contractile dysfunction induced by prolonged Akt activation. (A) Top: Temporal profile of DOX treatment. Bottom: Representative gross morphology. (B) HW/BW ratio. *P < 0.01. (C) Echocardiography. Top: Representative M-mode recordings. Bottom: posterior wall thickness, LV end-diastolic dimension, and percent fractional shortening. *P < 0.01; ^#P < 0.05. (D) Histology: H&E, wheat germ agglutinin, and Masson’s trichrome staining of heart sections. Scale bar: 50 μm. (E) Fold induction of ANP and β-MHC expression after prolonged Akt activation. **P < 0.01 versus control; ^†P < 0.05 versus control.

Figure 4

Prevention of cardiac growth and heart failure progression by rapamycin. (A) HW/BW ratio of mice in the acute (left) and chronic (right) phase after Akt transgene induction. *P < 0.01. (B) Western blot analysis of Akt transgene and S6K phosphorylation in the acute (left) and chronic (right) phase after Akt transgene induction. (C) Echocardiography. Top: Representative M-mode recordings. Bottom: posterior wall thickness, LV end-diastolic dimension, and percent fractional shortening 6 weeks after transgene induction. *P < 0.01. (D) Histological analysis. H&E, wheat germ agglutinin, and Masson’s trichrome staining of heart sections 6 weeks after transgene induction. Scale bar: 50 μm. (E) Fold induction of ANP and β-MHC expression. ^#P < 0.05 versus control. Cont, control; Rap, rapamycin treatment.

Figure 5

Coronary angiogenesis and angiogenic growth factor expression. (A) Left: Representative CD31 staining. Scale bar: 50 μm. Right: Capillary density. *P < 0.01. (B) Expressions of VEGF-A and Ang-2 in the heart. (C) Effects of rapamycin on capillary density and angiogenic growth factor expression in the acute phase. Left: Capillary density after 2 weeks of transgene induction. Right: Expression of VEGF-A and Ang-2 after 2 weeks of transgene induction. (D) Effects of rapamycin on capillary density and angiogenic growth factor expressions in the chronic phase. Left: Capillary density after 6 weeks of transgene induction. *P < 0.01. Right: Expression of VEGF-A and Ang-2 after 6 weeks of transgene induction.

Figure 6

Induction of VEGF-A and Ang-2 by Akt in cultured adult cardiac myocytes. (A) Top: Representative Western blot analysis of Ang-2, Akt, and S6K. Bottom: Densitometric analysis of Ang-2 expression levels. *P < 0.01; ^#P < 0.05. (B) VEGF concentration in the culture media as measured by ELISA. *P < 0.01.

Figure 7

Inhibition of coronary angiogenesis results in impaired cardiac growth and contractile dysfunction. (A) Echocardiography. Top: Schematic illustrations of adenoviruses and experimental protocol. KDR, kinase domain insert–containing receptor. Middle: Representative M-mode recordings. Bottom: Echocardiographic parameters. *P < 0.01; ^#P < 0.05. (B) HW/BW ratio of control or DTG hearts treated with a control vector (Ad-cont) or adenoviral vector encoding Flk1-Fc (Ad-Flk). *P < 0.01. (C) Representative Western blot of Akt, S6K, VEGF-A, and Ang-2. (D) Histology of control or DTG hearts treated with Ad-cont or Ad-Flk. Scale bars: 50 μm. (E) Capillary density of control or DTG hearts treated with Ad-cont or Ad-Flk. *P < 0.01. (F) Cross-talk between cardiac myocytes and coronary vasculature during cardiac growth. Secretion of multiple angiogenic growth factors including VEGF and Ang-2 from cardiomyocytes is thought to be responsible for enhanced coronary angiogenesis during adaptive cardiac growth. Coronary vasculature, on the other hand, is thought to contribute to cardiac growth and the maintenance of contractile function.