Pik3ip1 expression was upregulated in 4-weeks exercise-induced hypertrophic hearts.

<p><b>(A)</b> qRT-PCR analysis of transcripts for <i>Pik3ip1</i> 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>(B)</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. <b>(C)</b> 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) <b>(D)</b> 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).</p

AAV-mediated overexpression of Cytl1 induces CF.

<p>Control virus or AAV-Cytl1 (5 × 10¹⁰ viral genome) was injected into the tail vein of WT mice, and the phenotype of the heart was examined after 8 wks. (A) Picrosirius staining of heart cross-sections from WT mice injected with control virus or AAV-Cytl. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Cytokine-Like 1 Regulates Cardiac Fibrosis via Modulation of TGF-β Signaling

<div><p>Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in <i>cytl1</i> knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of <i>cytl1</i> alone led to the development of CF <i>in vivo</i>. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of <i>cytl1</i> was sufficient to induce these two critical CF-related processes <i>in vitro</i>, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-β receptor 1. Cytl1 induced the expression of TGF-β2 both <i>in vivo</i> and <i>in vitro</i>. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 <i>in vitro</i>. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-β-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies.</p></div

Pik3ip1 attenuates IGF1-induced cardiomyocyte hypertrophy.

<p>NRCMs were infected with the indicated adenovirus for 24 h and subsequently treated with or without 100 ng/ml IGF1. (<b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122251#pone.0122251.s001" target="_blank">S1 File</a>). The corresponding cell lysates from adenovirus-infected NRCMs were analyzed by immunoblot assay with anti-HA and anti-α-tubulin antibodies. (<b>B-C</b>) Adenovirus-infected NRCMs were incubated for 30 min with or without IGF1, after which p110α activity <b>(B)</b> and AKT phosphorylation <b>(C)</b> was assessed. (<b>D</b>) 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). (<b>E</b>) 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). <b>(F)</b> Extracts from adenovirus-infected NRCMs treated for 30 min with or without IGF1 were verified by the indicated antibodies.</p

A working pathway model to show the effect of Pik3ip1 on exercise-induced cardiac hypertrophy.

<p>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.</p

Cytl1 is upregulated under pathological conditions.

<p>Three groups of WT mice were subjected to TAC for 6 wks (TAC), ligation of coronary artery for 4 wks (MI), or ligation of coronary artery for 30 min followed by reperfusion for 24 hrs (I/R). Hearts were harvested and qRT-PCR was performed to determine the transcript levels of Cytl1. n = 3 for sham, n = 3 for TAC, n = 4 for MI, n = 4 for I/R. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

The effects of silencing Pik3ip1 on the PI3K/AKT/mTOR pathway.

<p>NRCMs were transfected with the indicated siRNA for 24 h and subsequently serum-starved for 24 h. <b>(A)</b> 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>B–E</b>) Representative immunoblot images of phosphorylated protein and total protein (upper), and the ratio of phosphorylated protein/total protein (bottom) for (<b>B</b>) AKT, (<b>C</b>) mTOR, (<b>D</b>) p70s6k, and (<b>E</b>) eEF2. <b>(F)</b> ERK 1/2 (n = 3, * p < 0.05 and ** p < 0.01, t test)</p

CF is attenuated in <i>cytl1</i> KO mice.

<p>WT and <i>cytl1</i> KO mice were subjected to TAC for 6 wks and the extent of fibrosis in the heart was analyzed. (A) Picrosirius staining of heart cross-sections from WT and <i>cytl1</i> KO mice subjected to TAC. Fibrotic areas in the interstitial and perivascular areas were quantified using MetaMorph software (right panels). (B) Quantification of the mRNA levels of several fibrotic markers (TGF-β2, collagen 1 and TNF-α) by qRT-PCR. (C) Activation of the TGF-β signaling pathway was investigated by western blotting. GAPDH served as the loading control. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Pik3ip1 is enriched in neonatal rat ventricular cardiomyocytes and interacts with p110α.

<p>(<b>A</b>) 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>B</b>) Western blot analysis was performed to compare CMs and FBs using anti-Pik3ip1, Vimentin, and α-actinin antibodies. (<b>C</b>, <b>D</b>) The interaction between Pik3ip1 and p110α was analyzed in adult mouse heart tissue (<b>C</b>) and NRCMs (<b>D</b>) using anti-p110α or anti-Pik3ip1 antibodies.</p

Cytl1 functions independently of CCR2.

<p>Adult primary cardiac FBs were treated with CCL2 (20 ng/ml) or Ad-Cytl1 (50 moi) for 48 h. In selected experiments, the cells were pretreated with the CCR2 antagonist CAS445679-97-0 (6 nM). (A) The cells were immunostained with an antibody against α-SMA. (B) Quantification of the mRNA levels of TGF-β2, Cytl1, collagen 1 and α-SMA by qRT-PCR. n = 3–5 for each experimental group. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p