Role of PI3-Kinase in Angiotensin II-Induced Cardiac Hypertrophy: Class I Versus Class III

Figures

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  FIGURE 1. Effect of blockade of PI3KC1 on Ang II-induced cardiac hypertrophy in rat hearts. Cardiomyocyte diameter and Akt phosphorylation were examined in SD rats that received subcutaneous infusion of Ang II or saline control with cardiac transduction of Lv-DNp85 or Lv-GFP. (A) Representative western blots of phosphorylated Akt (p-Akt) and total Akt in the heart of each group of rats. (B) Bar graphs summarizing the ratio of phosphorylated Akt vs. total Akt. Data are presented as means ± SE (n = 6 rats). * p < 0.05 as compared with the group of saline + Lv-GFP. #p < 0.05 as compared with the group of Ang II + Lv-GFP. (C) Micrographs showing representative heart sections stained with hematoxylin/eosin in each group of rats. (D) Bar graphs summarizing diameter of cardiac myocytes from transverse cardiac sections of each group of rats. 100 cells per rat were observed randomly and averaged. Scale bar: 50 μm. Data are means ± SE (n = 6 rats in each group). *p < 0.05 vs. rats that received saline + Lv-GFP. #p < 0.05 vs. rats that received Ang II + Lv-GFP.

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  FIGURE 2. Effect of Ang II on autophagy in cardiomyocytes. Autophagy was detected with MAP-LC3 antibody immunofluorescence by counting intracellular puncta as formation of autophagosomes. (A–F) Representative fluorescence micrographs of cultured cardiomyocytes stained with anti-LC3 antibodies after treatments with HBSS control (A), and Ang II at different concentrations (from 10−9 to 10−5 M, B-F). (G) Bar graphs showing the effect on autophagy under Ang II exposure at different concentrations. Data are mean ± SE, n = 10 in each group. *p < 0.05 vs. Control. **p < 0.01 vs. Control.

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  FIGURE 3. Effect of Ang II and blockade of PI3KC1 on autophagy in cardiomyocytes. Autophagy was examined in cardiomyocytes by immunostaining fluorescence with MAP-LC3 antibodies in cardiomyocytes treated with vehicle control, Ang II (10−6 M) with or without the PI3KC1 inhibitor, LY-294002 (10−6 M) for 24 h (A–D) Representative fluorescence micrographs of cultured cardiomyocytes stained with MAP-LC3 antibodies after treatments. (E) Bar graphs summarizing quantitative analysis of autophagic alterations in cardiomyocytes treated under the conditions described in the above. The scale in the images is 25 μm. Data are means ± SE, which were derived from three experiments and at least triplicate wells in each experiment.*p < 0.05 vs. cardiomyocytes that treated with vehicle control. **p < 0.01 vs. cardiomyocytes that treated with vehicle control. #p < 0.05 vs. cardiomyocytes that treated with Ang II.

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  FIGURE 4. Effect of Ang II and blockade of PI3KC1 on mitochondrial ROS production in cardiomyocytes. Mitochondrial ROS levels were determined using the oxidant-sensitive fluorogenic probe, MitoSOX, in cardiomyocytes treated with vehicle control, Ang II (10−6 M) with or without the PI3C1 inhibitor, LY294002 (10−6 M). (A–D) Representative fluorescence micrographs of cultured cardiomyocytes probed by MitoSOX after treatments. (E) Bar graphs summarizing the mitochondrial ROS levels in cardiomyocytes treated with the conditions described above. The scale in the images is 25 μm. Data are presented as means ± SE, which were derived from three experiments and at least triplicate wells in each experiment. *p < 0.01 vs. cardiomyocytes treated vehicle control. #p < 0.05 vs. cardiomyocytes treated with Ang II.

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  FIGURE 5. Effect of Ang II and blockade of PI3KC1 on cardiomyocyte hypertrophy. Morphology of cardiomyocytes were determined by immunocytochemistry using an α-sarcomeric actin antibody in cardiomyocytes after treated with vehicle control, Ang II (10−6 M) with or without the PI3KC1 inhibitor, LY294002 (10−6 M) for 24 h (A–D) Representative fluorescence micrographs of cultured cardiomyocytes stained with α-sarcomeric actin antibody after treatments. (E) Bar graph summarizing the size of cardiomyocytes after the indicated treatment. Data are means ± SE, derived from three experiments and at least triplicate wells in each experiment. *p < 0.05 vs. control; #p < 0.05 vs. Ang II treatment.

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  FIGURE 6. Effect of Ang II and blockade of PI3KC3 on cardiomyocyte hypertrophy. Cell morphology of cardiomyocytes was determined by immunocytochemistry using an α-sarcomeric actin antibody in cardiomyocytes after treatment with vehicle control, Ang II (10−6 M) with or without the PI3KC3 inhibitor, 3-methyladenine (3-MA, 10−6 M) for 24 h (A–D) Representative fluorescence micrographs of cultured cardiomyocytes stained with α-sarcomeric actin antibodies after treatments. (E) Bar graph summarizing the size of cardiomyocytes after the indicated treatment. Data are means ± SE, which were derived from three experiments and at least triplicate wells in each experiment. *p < 0.05 vs. control; #p < 0.05 vs. Ang II treatment.

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  FIGURE 7. Effect of Ang II and blockade of PI3KC3 on mitochondrial ROS production and autophagy in cardiomyocytes. Mitochondrial ROS levels were determined using the oxidant-sensitive fluorogenic probe, MitoSOX and autophagy was examined using MAP-LC3 antibodies in cardiomyocytes treated with vehicle control, Ang II with or without the PI3KC3 inhibitor, 3-methyladenine (3-MA). (A) Bar graphs summarizing the mitochondrial ROS levels in cardiomyocytes treated with vehicle control, Ang II (10−6 M), 3-MA (10−6 M), and Ang II + 3-MA. Data are presented as means ± SE, which were derived from three experiments and at least triplicate wells in each experiment. *p < 0.05 and **p < 0.01 vs. cardiomyocytes treated vehicle control. #p < 0.05 vs. cardiomyocytes treated with Ang II. (B) Bar graphs summarizing quantitative analysis of autophagic alterations in cardiomyocytes treated with vehicle control, Ang II (10−6 M), 3-MA (10−6 M), and Ang II + 3-MA. Data are means ± SE, which were derived from three experiments and at least triplicate wells in each experiment. *p < 0.05 and **p < 0.01 vs. cardiomyocytes that treated with vehicle control. #p < 0.05 vs. cardiomyocytes that treated with Ang II.

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  FIGURE 8. Proposed intracellular mechanisms involved in the action of PI3KC1 vs. PI3KC3 in the Ang II-induced cardiac hypertrophy. PI3KC1, PI3-kinase Class I; PI3KC3, PI3-kinase Class III; ROS, reactive oxygen species; MitoROS, mitochondrial ROS.