Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice

Abstract

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of beta-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat alpha-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dt(max)) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dt(min), was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, beta-adrenergic receptor (beta-AR) density, and beta-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-beta is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-beta/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting beta-AR signaling capacity.

Fig 1.

(A) Western blotting of myocardial cell extracts from three control (C1, C2, C3) and three transgenic (T1, T2, T3) animals. Upper lane, tissue extracts were blotted with an antibody recognizing both phosphorylated and unphosphorylated forms of Akt; lower lane, tissue extracts were blotted with an antibody against only the phosphorylated form of Akt. (B) Western blotting with an antibody against phospho-GSK3-β of myocardial cell extracts from Akt-transgenic and control mice. (Left) Responses are also shown in extracts of primary rat neonatal myocardial cells unstimulated and stimulated with ISO or infected with adenoviruses carrying a dominant-negative (Akt-dn) or a constitutively active (Akt-ca) form of Akt, respectively. (C) Immunohistochemistry using an anti-GATA4 antiserum in myocardial tissue from Akt transgenic or control mice. GATA4 is accumulated in the nucleus in the myocardium of Akt transgenic but not in control mice, where the coloration is more diffuse. (D) Western blotting with antibodies recognizing total MAPK p42/44 (upper lane) or phospho MAPK p42/44 (lower lane) in control (C1, C2, C3) vs. transgenic mice (T1, T2, T3).

Fig 2.

(A and B) Masson trichrome staining of longitudinal sections of the myocardium from wild-type (A) and transgenic mice (B). (C and D) Staining with FITC-labeled wheat germ hemagglutinin of crosssectional sections of myocardium from Akt control (C) and transgenic animals (D).

Fig 3.

Electron microscopy analysis of myocardial sections from control (A) or E40K transgenic mice (B). An increase in myofibrillar size is evident in transgenic mice.

Fig 4.

Hemodynamic analysis of cardiac function in Akt transgenic vs. control mice under resting conditions and with graded dobutamine infusion. (Top) Heart rate vs. dobutamine dosage in transgenic (•) vs. control (□) mice before vagotomy (control), after vagotomy (basal), and at different dobutamine dosages, as indicated in the figure. (Top Middle) Maximal LV pressure vs. dobutamine dosage in transgenic (•) vs. control (□) mice. (Bottom Middle) LV dP/dt_max in transgenic (•) vs. control mice (□). (Bottom) LV dP/dt_min in transgenic (•) vs. control mice (□). †, P< 0.05.

Fig 5.

(A) ISO-induced cAMP generation in control vs. transgenic mice. Base is represented in white bars, ISO in black bars. (B) β-AR density (fmol/mg of membranes) in control vs. transgenic myocardium. (C) β-AR affinity, expressed as % of high-affinity agonist binding. The transgenic overexpression of AKT in the heart did not alter β-AR signaling nor affect β-AR receptor density or the ability of the receptor to couple to Gs.