Fig 1.
Pik3ip1 is enriched in neonatal rat ventricular cardiomyocytes and interacts with p110α.
(A) mRNA levels of Pik3ip1 were measured in cardiomyocytes (CMs) and fibroblasts (FBs) using quantitative reverse transcription PCR (qRT-PCR) (n = 3, * p < 0.05, t test). (B) Western blot analysis was performed to compare CMs and FBs using anti-Pik3ip1, Vimentin, and α-actinin antibodies. (C, D) The interaction between Pik3ip1 and p110α was analyzed in adult mouse heart tissue (C) and NRCMs (D) using anti-p110α or anti-Pik3ip1 antibodies.
Fig 2.
Silencing of Pik3ip1 induces cardiomyocyte hypertrophy.
NRCMs were transfected with the indicated siRNA for 24 h and subsequently serum-starved for 24 h. (A) qRT-PCR analysis of the mRNA transcripts for Pik3ip1 in 50 nM siNegative or 50 nM siPik3ip1-transfected NRCMs. mRNA expression was normalized to 18S. (n = 3, ** p < 0.01, t test). (B) Representative immunoblot images (left) and quantified graphs (right) of Pik3ip1 protein in siNegative or siPik3ip1-transfected NRCMs. α-Tubulin served as an internal control. (n = 3, * p < 0.05, t test). (C) Representative images of NRCMs stained with anti-α-actinin antibody and DAPI in siNegative or siPik3ip1-transfected NRCMs. Scale bar: 100 μm. (D) Quantification of the relative cell surface areas. (n = 5, ** p < 0.01, t test). (E) Effect of Pik3ip1 knockdown on protein synthesis. Leucine incorporation was determined as described in the Materials and Methods. (n = 12, ** p < 0.01, t test).
Fig 3.
The effects of silencing Pik3ip1 on the PI3K/AKT/mTOR pathway.
NRCMs were transfected with the indicated siRNA for 24 h and subsequently serum-starved for 24 h. (A) PI3K activity (p110α) in the siNegative and siPik3ip1-transfected NRCMs. Western blot analyses of samples from siNegative and siPik3ip1-transfected NRCMs were performed after 48 h. (B–E) Representative immunoblot images of phosphorylated protein and total protein (upper), and the ratio of phosphorylated protein/total protein (bottom) for (B) AKT, (C) mTOR, (D) p70s6k, and (E) eEF2. (F) ERK 1/2 (n = 3, * p < 0.05 and ** p < 0.01, t test)
Fig 4.
Pik3ip1 silencing-induced cardiomyocyte hypertrophy is dependent on PI3K activity.
NRCMs were transfected with siNegative and siPik3ip1 for 24 h and subsequently treated with 0.05% DMSO or LY294002 for 24 h. (A) Representative images (left) of NRCMs stained with anti-α-actinin antibody in siNegative or siPik3ip1-transfected NRCMs with or without LY294002. Scale bar: 100 μm. Quantification of relative cell surface areas (right). (n = 5, * p < 0.05 compared with siNegative-transfected NRCMs treated with DMSO, and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (B) All siRNA-transfected NRCMs were incubated with DMSO or LY294002, after which protein synthesis was assessed using a leucine incorporation assay. (n = 12, ** p < 0.01 compared with siNegative-transfected NRCMs treated with DMSO and ## p < 0.01 compared with siPik3ip1-transfected NRCMs treated with DMSO, ANOVA). (C) The signaling molecules involved in the PI3K pathway in the 4 different types of samples (siNegative, siPik3ip1, siNegative plus LY294002, and siPik3ip1 plus LY294002) were verified by the indicated antibodies.
Fig 5.
Pik3ip1 attenuates IGF1-induced cardiomyocyte hypertrophy.
NRCMs were infected with the indicated adenovirus for 24 h and subsequently treated with or without 100 ng/ml IGF1. (A) Exogenous HA-tagged mouse Pik3ip1 expression was verified by RT-PCR and western blot analyses. Exogenous Pik3ip1 mRNAs were amplified using a mouse Pik3ip1-specific primer set (Table A in S1 File). The corresponding cell lysates from adenovirus-infected NRCMs were analyzed by immunoblot assay with anti-HA and anti-α-tubulin antibodies. (B-C) Adenovirus-infected NRCMs were incubated for 30 min with or without IGF1, after which p110α activity (B) and AKT phosphorylation (C) was assessed. (D) Representative images (left) of NRCMs stained with anti-α-actinin antibody in AdControl or AdPik3ip1-infected NRCMs for 24 h with or without IGF1. Scale bar: 100 μm. Quantification of relative cell surface area (right). (n = 5, ** p < 0.01 compared with AdControl-infected NRCMs and ## p < 0.01 compared with AdControl-infected NRCMs treated with IGF1, ANOVA). (E) Adenovirus-infected NRCMs were incubated for 24 h with or without IGF1, after which protein synthesis was assessed using a leucine incorporation assay (n = 5, ** p < 0.01 compared with AdControl-infected NRCMs and ## p < 0.01 compared with AdControl-infected NRCMs treated with IGF1, ANOVA). (F) Extracts from adenovirus-infected NRCMs treated for 30 min with or without IGF1 were verified by the indicated antibodies.
Fig 6.
Pik3ip1 expression was upregulated in 4-weeks exercise-induced hypertrophic hearts.
(A) qRT-PCR analysis of transcripts for Pik3ip1 in 1-week TAC, 2-weeks TAC, 2-weeks exercised and 4-weeks exercised mice hearts. mRNA expression was normalized to 18S. (n = 3, ** p < 0.01 compared with compared with the Sham or Sedentary mice, t test). (B) Representative immunoblot images p110α, AKT, Pik3ip1 and α-tubulin protein expression in 1-week TAC, 2-weeks TAC, 2-weeks exercised and 4-weeks exercised mice hearts. (C) Quantification of Pik3ip1 and p110α protein expression in 1-week TAC, 2-weeks TAC, 2-weeks exercised and 4-weeks exercised mice hearts.α-tubulin served as an internal control. (n = 3, * p < 0.05 compared with the Sham or Sedentary mice, t test) (D) Quantification of phosphorylated AKT in 1-week TAC, 2-weeks TAC, 2-weeks exercised and 4-weeks exercised mice hearts. Total AKT served as a control. (n = 3, * p < 0.05 compared with the Sham or Sedentary mice, t test).
Fig 7.
A working pathway model to show the effect of Pik3ip1 on exercise-induced cardiac hypertrophy.
A schematic model of Pik3ip1-deficiency-mediated cardiomyocyte hypertrophy. Pik3ip1 inhibits PI3K activity through interacting with p110. Knockdown of Pik3ip1 could increase AKT activity in basal conditions. Activated AKT induces hypertrophy by activation of the mTOR pathway. mTOR increases cell growth and protein synthesis through activation of p70s6k and eEF2. Pik3ip1-deficiency-mediated cardiomyocyte hypertrophy is attenuated by treatment with the PI3K inhibitor, LY294002. In addition, Pik3ip1 can attenuate IGF1-induced hypertrophy by inhibiting PI3K activity.