Akt2 deficiency promotes cardiac induction of Rab4a and myocardial β-adrenergic hypersensitivity

Abstract

Patients with diabetes mellitus can develop cardiac dysfunction in the absence of underlying coronary artery disease or hypertension; a condition defined as diabetic cardiomyopathy. Mice lacking the intracellular protein kinase Akt2 develop a syndrome that is similar to diabetes mellitus type 2. Expression profiling of akt2(-/-) myocardium revealed that Rab4a, a GTPase involved in glucose transporter 4 translocation and β-adrenergic receptor (βAR) recycling to the plasma membrane, was significantly induced. We therefore hypothesized that Akt2 deficiency increases myocardial β-adrenergic sensitivity. Confirmatory analysis revealed up-regulation of Rab4a mRNA and protein in akt2(-/-) myocardium. In cultured cardiomyocyte experiments, Rab4a was induced by pharmacological inhibition of Akt as well as by specific knockdown of Akt2 with siRNA. Isolated akt2(-/-) hearts were hypersensitive to isoproterenol (ISO) but exhibited normal sensitivity to forskolin. Prolonged ISO treatment led to increased cardiac hypertrophy in akt2(-/-) mice compared to wild type mice. In addition, spontaneous hypertrophy was noted in aged akt2(-/-) hearts that was inhibited by treatment with the βAR blocker propranolol. In agreement with previous results demonstrating increased fatty acid oxidation rates in akt2(-/-) myocardium, we found increased peroxisome proliferator-activated receptor α (PPARα) activity in the hearts of these animals. Interestingly, increased myocardial Rab4a expression was present in mice with cardiac-specific overexpression of PPARα and was also observed upon stimulation of PPARα activity in cultured cardiomyocytes. Accordingly, propranolol attenuated the development of cardiac hypertrophy in the PPARα transgenic mice as well. Our results indicate that reduced Akt2 leads to up-regulation of Rab4a expression in cardiomyocytes in a cell-autonomous fashion that may involve activation of PPARα. This maladaptive response is associated with hypersensitivity of akt2(-/-) myocardium to β-adrenergic stimulation.

Figure 1

Rab4a expression is increased in akt2^−/− myocardium. A. Quantitative real-time RT-PCR analysis of Rab4a mRNA levels in akt2^−/− and WT myocardium obtained from 2-month (n=3; *, p=0.0008 akt2^−/− vs. WT), 4-month (n=5; *, p=0.0002 akt2^−/− vs. WT) and 9-month (n=11; *, p=0.006 akt2^−/− vs. WT) old mice. Rab4a mRNA levels were normalized by GAPDH mRNA levels and are presented in arbitrary units. B. Rab4a protein levels in ventricular lysates obtained from 10-day-old mice and from 2-, 3-, 4-, 6- and 9-month-old mice ( WT and akt2^−/−). Results were normalized by actin protein levels (lower panel). C. Rab4a protein levels in skeletal muscle, brain and liver lysates obtained from 2–3 month-old WT and akt2^−/− mice. Rab4a bands from skeletal muscle were increased by 1.85±0.1 fold in akt2^−/− mice (lower panel, n=4–6; *, p=0.01). D. Rab4a and Rab5 protein levels in ventricular lysates obtained from 2-month-old WT (n=5) and akt2^−/− (n=4) mice.

Figure 2

Rab4a expression is induced in cultured rat neonatal ventricular cardiomyocytes by Akt inhibition. A. Analysis of phospho-Akt levels and phospho-GSK3β levels in cultured cardiomyocytes following 10 minutes of stimulation with 10 nM insulin under normal conditions (Cont) and in the presence of the Akt1/2 inhibitor A6730. Cardiomyocytes were exposed to the drug for 24 hours before the activation with insulin. B. Rab4a protein levels following exposure of cardiomyocytes to regular media (0) or to different concentrations of the Akt1/2 inhibitor A6730 (0.5–2.5 μM, n=4–6 wells for each concentration, lower panel *, p< 0.01 compared to control).C. Rab4a protein levels following incubation of cultured cardiomyocytes with 1μM of akt2 siRNA or 1μM of a non–targeting (cont) siRNA in an Accell medium. Rab4a and akt2 bands were normalized by actin protein levels (lower panel *, p< 0.0001 compared to control, n=5 wells in 3 independent experiments)

Figure 3

The sensitivity to acute stimulation with ISO is enhanced in akt2^−/− hearts. Isolated hearts obtained from 8-week-old akt2^−/− and WT mice were perfused with Tyrode's solution containing 100 nM ISO (n=8 and 10 for akt2^−/− and WT hearts, respectively) or 1 μM Forskolin (Forsk, n=6 in each group). Parameters at baseline and during the peak pressure response to ISO or Forsk were evaluated. A. Spontaneous heart rate. B. Developed LV pressure (*, p=0.005 akt2^−/− ( Iso) vs. WT (Iso)). C. Maximal rate of LV contraction (+dP/dT; *, p=0.0001 akt2^−/− ( Iso) vs. WT (Iso)). D. Maximal rate of LV relaxation (−dP/dT; *, p=0.0001 akt2^−/− ( Iso) vs. WT (Iso)). E. cAMP levels were measured in WT and akt2^−/− hearts at baseline (n=2 in each group) and following stimulation with Iso (100nM) + IBMX (0.25 mM) for 5 min (n=5 and 6 for WT and akt2^−/− hearts, respectively. *, p=0.04).

Figure 4

Increased β-adrenergic-mediated cardiac hypertrophy in akt2^−/− mice. 7-week-old akt2^−/− and WT mice were implanted with Alzet osmotic minipumps to deliver ISO (60 mg/kg/day, n=7–8) or PBS (n=5–6) for 14 days. Note increased ISO-mediated hypertrophy characterized by elevated heart weight-to-tibia length ratio (HW/T) in akt2^−/− mice compared with WT mice. *, p<0.001 for WT (iso) vs. WT (PBS) and akt2^−/− (iso) vs. akt2^−/− (PBS); †, p=0.007 akt2^−/− (iso) vs. WT (iso).

Figure 5

Spontaneous gradual development of cardiac hypertrophy in akt2^−/− mice. A. Heart weight to tibial length ratio (HW/T) was determined at various time points. The number of mice analyzed at each time point were: 2 months (3 WT,4 akt2^−/−), 3 months (3 WT, 5 akt2^−/−), 4 months (6 WT, 3 akt2^−/−), 6 months (5 WT, 5 akt2^−/−), and 9 months (9 WT, 8 akt2^−/−). B. Noninvasive blood pressure measurements obtained from 2 month old akt2^−/− and WT mice (n=4 in each group). C. Anatomic appearance of akt2^−/− and WT hearts obtained from 9-month-old animals. D. Histological sections from left and right verticals of akt2^−/− and WT mice stained with Masson’s trichrome-staining. Notice the absence of intra-myocardial fibrosis in akt2^−/− sections. E. Right: representative LV histological sections from 9-month-old akt2^−/− and WT mice stained with H&E. Left: Summary of the average cross-sectional area of cardiomyocytes. Results are normalized to WT.

Figure 6

Propranolol treatment of akt2^−/− mice inhibits cardiac hypertrophy. A. Reduced cardiac hypertrophy in akt2^−/− mice treated with propranolol for 6 weeks from the age of 7-weeks-old, compared to untreated akt2^−/− mice (n=4–6; *, p=0.008). Increased cardiac hypertrophy in untreated akt2^−/− mice compared to untreated WT mice (n=6; †, p=0.002). B. Decreased cardiac hypertrophy in 10-month-old akt2^−/− mice treated with propranolol for 6 weeks (n=3; *, p=0.004) compared to untreated akt2^−/− mice. Increased cardiac hypertrophy in untreated akt2^−/− mice compared to untreated WT (†, p=0.0004). C. Representative LV histological sections from 10-month old control akt2^−/− mice and akt2^−/− mice treated with propranolol. Summary of the average cross-sectional area of cardiomyocytes (Right panel)

Figure 7

Increase in PPARα and PPARα-target gene expression in akt2^−/− myocardium. LV tissue isolated from 9-month-old WT (n=5) and akt2^−/− (n=5) mice was used to purify RNA for quantitative RT-PCR analysis. Peroxisome proliferator-activated receptor α (PPARα ), carnitine palmitoyltranferase 1 (CPT-1) and medium-chain acyl-CoA dehydrogenase (MCAD) were normalized to the expression of GAPDH. The data is normalized to WT and is presented in arbitrary units (AU). PPARα (*, p=0.04), mCPT-1 (*, p=0.018) and mCAD (*, p=0.025).

Figure 8

Rab4a is induced in MHC-PPARα myocardium A. Increased Rab4a protein level in transgenic mice with cardiac-specific overexpression of PPARα . Rab4a protein levels in ventricular lysates obtained from 6 week-old MHC-PPARα mice and WT littermates, normalized by actin protein levels (lower panel, n=7–8; *, p= 0.002) B. Rab4a expression following incubation of cardiomyocytes with Ad-GFP-PPARα or Ad-GFP control (MOI of 100). Cell were evaluated with (n=8 wells) or without (n=5 wells) the PPARα agonist WY14643 (1μM). Bands were normalized by actin (lower panel *, p= 0.00005 compared to Ad-GFP+WY). C. Reduced cardiac hypertrophy in MHC-PPARα mice treated with propranolol for 6 weeks from the age of 2-weeks-old, compared to untreated MHC-PPARα (n=9 and 5; respectively *, p=0.0004). Increased cardiac hypertrophy in untreated MHC-PPARα mice compared to untreated WT (n=8; †, p=0.00001).