Catecholamine-induced cardiac hypertrophy in rats is associated with the activation of p70 kinase and c-Jun NH(2)-terminal kinase 2

Abstract

Objective: To assess the effect of intravenous infusion of adrenergic, hypertrophic agonists on the activity of the ribosomal S6 (p70(S6)) kinase and the c-Jun NH(2)-terminal kinase 2 (JNK2) in adult rat hearts.

Animals and methods: Noradrenaline (NA), isoproterenol (ISO), phenylephrine (PE), NA in combination with propranolol (NA+Prop) and NA in combination with prazosin (NA+Praz) were continuously intravenously infused in rat hearts for up to 72 h. The expression and phosphorylation status of JNK2 and p70(S6) kinase were investigated by western blotting and use of specific and phosphospecific antibodies. The activity of p70(S6) kinase was measured by incorporation of (32)P-labelled phosphate into the specific substrate S6 peptide.

Results: The ratio of left ventricular weight to body weight was increased by NA, and by alpha- (PE, NA+Prop) and beta-adrenergic (ISO, NA+Praz) stimulation. Right ventricular weight to body weight ratio was higher only after beta-adrenergic stimulation (ISO, NA+Praz). p70(S6) kinase activity was stimulated predominantly through beta-adrenoceptors in parallel with left and right ventricular hypertrophy. Activation of JNK2 was mainly dependent on alpha-adrenoceptor activation, which led to hypertrophy of only the left ventricle.

Conclusions: The activation of p70(S6) kinase and JNK2 may be implicated in the development of catecholamine-induced cardiac hypertrophy in vivo.

Keywords: Catecholamines; Hypertrophy; Rat; Ribosomal S6 kinase; c-Jun NH2-terminal kinase.

Figure 1)

Ratio of left (LVW/BW) and right (RVW/BW) ventricular weight to body weight. Female Sprague-Dawley rats weighing 210 to 260 g were intravenously infused with noradrenaline (NA), phenylephrine (PE), isoproterenol (ISO), NA plus propranolol (NA+Prop), and NA plus prazosin (NA+Praz), respectively, for 72 h. Controls consisted of NaCl-infused rat hearts. The number of experiments is given in parentheses. Data are mean ± SEM. *P<0.05 versus samples of NaCl-treated animals, ^†P<0.05 versus samples of NA-infused animals

Figure 2)

(A) Time course of ribosomal S6 (p70^S6) kinase activation in adult rat hearts during NaCl, noradrenaline (NA), phenylephrine (PE) and isoproterenol (ISO) infusion. The durations (t) of intravenous infusion are given. Samples from the left (LV) and right ventricle (RV), respectively, were examined by western blotting using an antibody specific for p70^S6 kinase. The active form of p70^S6 kinase was detected by the appearance of phosphorylation shifts. Three forms of phosphorylated p70^S6 kinase are indicated by arrows. Results are representative of data from five sets of experiments. (B) After 1.5 h, the activity of p70^S6 kinase was measured by immunoprecipitation followed by investigation of ³²P incorporation into an S6 peptide. The result of an individual representative test is shown. (C) Quantification of ³²P incorporation in the RV and LV. Data are mean ± SEM of five experiments. *P<0.05 versus control rats

Figure 3)

(A) Activity of ribosomal S6 (p70^S6) kinase in adult rat hearts after 1.5 h of intravenous infusion of NaCl, noradrenaline (NA), NA combined with prazosin (NA+Praz) and NA combined with propranolol (NA+Prop). Samples from the left (LV) and right ventricle (RV) were examined by western blotting using an antibody specific for p70^S6 kinase. The active form of p70^S6 kinase was detected by the appearance of phosphorylation shifts. Three forms of phosphorylated p70^S6 kinase are indicated by arrows. Results are representative of data from five sets of experiments. (B) The activity of p70^S6 kinase was measured by immunoprecipitation followed by measuring ³²P incorporation into an S6 peptide. The result of an individual representative test is shown. (C) Quantification of ³²P incorporation in the RV and LV. Data are mean ± SEM of five experiments after 1.5 h. *P<0.05 versus control rats; †P<0.05 versus samples of NA-infused animals

Figure 4)

(A) Time course of the expression and activation of p54 c-Jun NH₂-terminal kinase (JNK) in adult rat hearts during NaCl, nor-adrenaline (NA), phenylephrine (PE) and isoproterenol (ISO) infusion. The durations of infusion are given. Samples from the left (LV) and right ventricle (RV) were examined by western blotting using an antibody specific for phospho-JNK2 and total JNK2. Western blots representative of five sets of experiments are shown. (B) Bands from western blots on x-ray films at 1.5 h were quantified by laser scanning densitometry. The data are mean ± SEM of five experiments. *P<0.05 versus control rats

Figure 5)

(A) Expression and activation of p54 c-Jun NH2-terminal kinase (JNK) in adult rat hearts after 1.5 h intravenous infusion of NaCl, noradrenaline (NA), NA combined with prazosin (NA+Praz) and NA combined with propranolol (NA+Prop). Samples from the left (LV) and right ventricle (RV) were examined by western blotting using an antibody specific for phospho-JNK2 and total JNK2. Western blots representative of five sets of experiments are shown. (B) Bands from western blots on x-ray films were quantified by laser scanning densitometry. The data are mean ± SEM of five experiments. *P<0.05 versus control rats; ^†P<0.05 versus samples from NA-infused animals