LKB1IP promotes pathological cardiac hypertrophy by targeting PTEN/Akt signalling pathway

Abstract Pathological cardiac hypertrophy represents a leading cause of morbidity and mortality worldwide. Liver kinase B1 interacting protein 1 (LKB1IP) was identified as the binding protein of tumour suppressor LKB1. However, the role of LKB1IP in the development of pathological cardiac hypertrophy has not been explored. The aim of this study was to investigate the function of LKB1IP in cardiac hypertrophy in response to hypertrophic stimuli. We investigated the cardiac level of LKB1IP in samples from patients with heart failure and mice with cardiac hypertrophy induced by isoproterenol (ISO) or transverse aortic constriction (TAC). LKB1IP knockout mice were generated and challenged with ISO injection or TAC surgery. Cardiac function, hypertrophy and fibrosis were then examined. LKB1IP expression was significantly up‐regulated on hypertrophic stimuli in both human and mouse cardiac samples. LKB1IP knockout markedly protected mouse hearts against ISO‐ or TAC‐induced cardiac hypertrophy and fibrosis. LKB1IP overexpression aggravated ISO‐induced cardiomyocyte hypertrophy, and its inhibition attenuated hypertrophy in vitro. Mechanistically, LKB1IP activated Akt signalling by directly targeting PTEN and then inhibiting its phosphatase activity. In conclusion, LKB1IP may be a potential target for pathological cardiac hypertrophy.

predominantly mediate cardiac hypertrophy. 5 Therefore, Akt protein has a direct impact on the development of cardiac hypertrophy. Phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumour suppressor gene that dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and depresses the PI3K-Akt pathway. 6 Loss of PTEN in the myocardium induces cardiac hypertrophy, which suggests the important role of PTEN in the development of cardiac hypertrophy. 7 However, much less is known about the upstream regulatory mechanism of PTEN expression and activity.
Liver kinase B1 (LKB1) interacting protein 1 (LKB1IP; also known as LIP1 or STK11IP) consists of 25 exons; it maps to human chromosome 2q36 and encodes a leucine-rich repeat-containing protein of 121 kDa. LKB1IP appears to be a cytoplasmically located protein that may regulate LKB1 function by controlling its subcellular localization. 8 LKB1IP also binds to the common mediator Smad4 protein of the transforming growth factor β and bone morphogenetic protein pathways. 9 However, the role of LKB1IP in vivo, especially in regulating cardiac hypertrophy, remains unknown.
In this study, we used LKB1IP-knockout mice and found that LKB1IP, by targeting PTEN, acts as a novel positive regulator of pathological cardiac hypertrophy induced by isoproterenol (ISO) or pressure overload. We identified a new role for LKB1IP in the development of cardiac hypertrophy.

| Human heart samples
Samples of failing hearts were from patients with end-stage heart failure undergoing cardiac transplantation. Control tissues were taken from healthy heart organ donors, when the organ was not eligible for transplantation. The use of hearts samples from patients was approved by the Medical Institutional Ethics Committee of Qilu Hospital, Shandong University, China. The participants gave written informed consent prior to the inclusion in the study. The investigation conformed to the principles outlined in the 1964 Declaration of Helsinki and its later amendments.

| Cell culture
Primary neonatal rat cardiomyocytes (NRCMs)NRCMs were prepared from the hearts of 2-to 3-day-old SD rats according to the following protocol. 10 Briefly, PBS containing 0.03% trypsin and 0.04% collagenase type II was used to isolate cardiomyocytes, followed by fibroblast removal using a differential attachment technique. After removing fibroblasts, NRCMs were seeded at a density of 2 × 10 5 cells per well onto six-well culture plates coated with gelatin in plating medium consisting of DMEM/F12 medium supplemented with 20% foetal calf serum, BrdU (0.1 mmol/L, to inhibit the proliferation of fibroblasts) and penicillin/streptomycin at 37°C in 5% CO 2 . Transient transfection of siRNA targeting LKB1IP (siLKB1IP) in cardiomyocytes involved using Lipofectamine RNAiMAX (Invitrogen) to knock down LKB1IP according to the manufacturer's protocol. LKB1IP siRNA is a pool of 3 different adenoviruses were used to overexpress LKB1IP protein level.

| Establishment of cardiac hypertrophy
We used male mice 8 to 10 weeks old at 2 weeks after transverse aortic constriction (TAC) or sham surgery or those subcutaneously injected with β-adrenoceptor (β-AR) agonist ISO (10 mg/kg per day, 14 days) or saline for 14 days, as described previously. 11 Echocardiography and haemodynamic analyses were performed before mice were killed; then, the hearts were harvested to analyse the hypertrophic response.

| Echocardiography
Echocardiography measurements were performed with a high-resolution microimaging system equipped with a 30-MHz transducer (Vevo2100; VisualSonics Inc) at the indicated times to evaluate the cardiac function of mice. Mice were anaesthetized with 1.5% to 2% inhaled isoflurane inhalation, and heart rates were kept at 400-

| Histopathology
Heart tissues were arrested with a 10% potassium chloride solution at end diastole, then fixed in 4% paraformaldehyde and maintained at 4°C until use. The fixed tissues were dehydrated and processed for paraffin embedding, and 5-μm sections were stained with haematoxylin and eosin (H&E) or tetramethyl rhodamine isothiocyanate-conjugated wheat germ agglutinin (WGA) (L4895, Sigma) and nuclei with 4',6-diamidino-2-phenylindole (DAPI) (Ab104139, Abcam) to measure myocyte cross-sectional areas. The degree of collagen deposition was detected by using a picrosirius red staining kit (ab150681, Abcam). The LV collagen volume was measured as the area of the picrosirius red positive-stained cardiac field and was expressed as a percentage of the total area. Immunohistochemical staining was performed with LKB1IP antibody (C157105, LS bio, 1:200). Images were analysed by using Image-Pro Plus 6.0.

| Immunofluorescence
For immunocytochemical analysis, NRCMs were infected with adenoviruses for 24 hr or siRNA for 48 hr and then stimulated with ISO for 24 hr Cells were fixed with 4% formaldehyde in phosphate-buff- and analysed by using Image-Pro Plus 6.0. We observed 30 cells in randomly selected fields for each group to quantitatively analyse cell sizes.

| Immunoprecipitation
Cells were lysed in an ice-cold immunoprecipitation buffer (NP-40 lysis buffer) containing protease inhibitor cocktail tablets (539 131, Millipore) and centrifuged at 13,000 × g for 15 min. The obtained cell lysates were incubated with the indicated antibody on a rocking platform at 4 ℃ overnight. On the second day, cell lysates were incubated with protein A/G-agarose beads (SC2003; Santa Cruz Biotechnology) for 2 hr at 4℃. The immune complex was collected after washing with cold immunoprecipitation buffer and subjected to immunoblotting with the indicated primary antibodies and corresponding secondary antibodies.

| Western blot analysis
Protein extracts were prepared by lysing tissues or cells in RIPA lysis buffer, fractionated by SDS-PAGE and then transferred onto PVDF membranes. After blocking in 5% skim milk, blots were probed with specific primary antibodies. The membranes were immunoblotted with a primary antibody at 4℃ overnight. After 3 cycles of cleaning with TBST, membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies and observed by enhanced chemiluminescence (Pierce).

| Quantitative PCR (qPCR)
Total RNA was extracted from hearts or cells by using TRIzol reagent (Invitrogen, Carlsbad, CA). An amount of 1 μg RNA was reverse-transcribed into cDNA by using the PrimeScript

| Statistical analysis
All data are expressed as mean ± SEM. Analyses involved use of SPSS v23 (SPSS Inc, Chicago, IL) and data passed normality and equal variance tests. PASS 11 (NCSS Inc) was used to calculate the sample size.

Statistical comparisons between 2 groups involved Student's t test
and otherwise one-way ANOVA and Bonferroni post-tests. All statistical tests were two-tailed, and P < .05 was considered statistically significant.

| LKB1IP expression is up-regulated in the hypertrophic heart and cells
To explore the role of LKB1IP in the development of cardiac hypertrophy and heart failure, we first investigated LKB1IP abundance in human and mouse hypertrophic heart disease.
Immunohistochemistry revealed significantly greater expression of LKB1IP in the human failing heart than in normal controls (P < .05; Figure Figure 1J). Collectively, these data demonstrate elevated LKB1IP expression under pathological cardiac hypertrophy.

| Establishment and analysis of LKB1IP knockout mice
To evaluate the hypertrophic effects of LKB1IP in vivo, we first used a mouse model with global knockout of LKB1IP (LKB1IP -/-). Four guide RNAs (GAAAGGCTAGGATTTTTGCT, GTGGAATATCTCAAGTACAG, CCTGCCTTCATCACTCCCTA and AAGGAACATTCAGGTTAGCG) were designed to delete 479 bp in exon 3 of LKB1IP gene, and Cas9 nuclease was introduced into C57BL/6NJ-derived fertilized eggs with well-recognized pronuclei followed by transfer to pseudopregnant females ( Figure 2A). The absence of LKB1IP in the heart tissues was first confirmed by realtime PCR ( Figure 2B). LKB1IP expression in the main heart tissues was further detected by Western blot analysis, thus indicating effective LKB1IP deletion in LKB1IP -/mice ( Figure 2C). Wild-type (WT) and LKB1IP -/mice did not differ in bodyweight ( Figure 2D).
The blood pressure of WT and LKB1IP -/mice was measured. There were no significant differences between WT and LKB1IP -/mice (Supplementary Figure 4). To clarify the effect of LKB1IP knockout on LKB1 and its downstream AMP-activated protein kinase (AMPK), we assessed protein levels by Western blot analysis, which showed no significant differences in LKB1, phosphorylated AMPKα (T172) and total AMPKα levels between WT and LKB1IP -/mice ( Figure 2E).
However, ISO-induced cardiac hypertrophy was significantly attenuated in LKB1IP -/mice, as demonstrated by lower IVSd, IVSs, LVPWd Paraffin-embedded slides were stained with picrosirius red to determine the extent of fibrosis. Both interstitial and perivascular fibrosis were markedly increased in ISO-treated WT hearts but were greatly decreased in LKB1IP -/hearts ( Figure 3E and 3F).
The expression of cardiac hypertrophy-related genes such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and myosin heavy chain β (β-MHC) was significantly increased in ISO-treated WT mice ( Figure 3G). ANP and β-MHC levels in ISOinduced hypertrophic cardiac tissues were lower in LKB1IP -/than WT mice ( Figure 3G). However, BNP level did not significantly differ between WT and LKB1IP -/mice treated with ISO. Both capillary and arteriolar density were increased in mice treated with ISO compared with saline. However, there were no significantly differences between ISO-treated WT and LKB1IP -/mice (Supplementary Figure 1A-1B). These loss-of-function data indicate that LKB1IP deficiency alleviates the pathological cardiac remodelling induced by ISO.

| LKB1IP deficiency alleviates pressure overload-induced cardiac hypertrophy in mice
We further evaluated whether LKB1IP knockout could alleviate pres-  Table 3). On H&E and WGA staining, TAC surgery increased cardiomyocyte size and perivascular and interstitial fibrosis in WT mice ( Figure 4D-4F).
Remarkably, these hypertrophic features were alleviated in hearts of LKB1IP -/mice that underwent TAC ( Figure 4D-4F). Consistently, these hypertrophic pathological phenotypes were accompanied by up-regulated hypertrophic genes including ANP, BNP and β-MHC in WT mice with TAC, which was attenuated in LKB1IP -/mice ( Figure 4G). Besides, both capillary and arteriolar density were significantly increased in TAC compared with sham groups. However, there were no differences between WT and LKB1IP -/mice underwent TAC (Supplementary Figure1C-1D). Thus, LKB1IP knockout alleviated pressure overload-induced cardiac hypertrophy in mice.

| LKB1IP positively regulates ISO-induced cardiomyocyte hypertrophy in vitro
We investigated whether LKB1IP directly regulates cardiomyocyte ISO-elevated ANP, BNP and β-MHC mRNA levels ( Figure 5F). These data further demonstrate that LKB1IP positively regulated cardiomyocyte hypertrophy under hypertrophic stress.

| LKB1IP positively regulates Akt phosphorylation
To explore the molecular mechanism by which LKB1IP regulates cardiac hypertrophy, we detected hypertrophy-related signalling pathways. In the mouse models of cardiac hypertrophy, ISO or TAC significantly increased Akt phosphorylation at Thr 308 as compared with their controls in WT mice, which was attenuated in LKB1IP -/mice ( Figure 6A-6B). Consistently, ISO-induced Akt phosphorylation in NRCMs was enhanced with LKB1IP overexpression ( Figure 6C) or suppressed with LKB1IP knockdown ( Figure 6D). We performed

| LKB1IP promotes Akt phosphorylation via direct targeting PTEN
To investigate how LKB1IP regulates Akt phosphorylation, we detected whether LKB1IP could interact with PTEN, the negative regulator of PI3K/Akt signalling. NRCMs were infected with adenovirus expressing Flag-tagged LKB1IP and HA-tagged PTEN followed by immunoprecipitation, which showed that LKB1IP could bind to PTEN ( Figure 7A). To test whether LKB1IP directly interact with PTEN under stimulated condition, we performed coimmunoprecipitation assays in NRCMs. The interaction of LKB1IP and PTEN was increased after ISO treatment for 15 min ( Figure 7B).
Immunohistochemistry against PTEN and LKB1IP in the heart tissues from mouse pathological hypertrophy were performed, which further validated their interaction (Supplementary Figure 7).
Importantly, basal and ISO-induced phosphatase activity of PTEN was significantly reduced with LKB1IP overexpression ( Figure 7C).
However, LKB1IP overexpression did not affect PTEN protein level (Supplementary Figure 5). Thus, the phosphatase activity of PTEN but not protein level was regulated by LKB1IP. To further confirm that LKB1IP regulates Akt phosphorylation via PTEN, NRCMs were overexpressed with LKB1IP and/or PTEN followed by ISO treatment. LKB1IP overexpression enhanced Akt phosphorylation, which was suppressed by PTEN overexpression (Figure 7D), which suggests that LKB1IP promotes PTEN-mediated Akt phosphorylation.
Taken together, these data demonstrate that LKB1IP may positively activate Akt signalling by directly targeting PTEN and subsequently inhibit its phosphatase activity ( Figure 7E). LKB1 is a tumour suppressor and a serine/threonine kinase that phosphorylates AMPK family members. 12 Peutz-Jeghers syndrome is characterized by the accumulation of non-cancerous gastrointestinal polyps and increased risk of cancer; LKB1 germline mutations occur in patients with this disease. 13 In our previous study, we found that endothelial cell-specific LKB1 knockout led to hypertension and increased angiogenesis in mice. 14 inhibition. 9 However, much less is known about other functions of LKB1IP. In this study, we generated LKB1IP-knockout mice to explore its function. LKB1IP deletion did not affect the expression of LKB1 and AMPKα phosphorylation in the mouse heart, which suggests that LKB1IP may play a role independent of LKB1 and AMPK. Suppression of PTEN by miR-301a could promote cardiomyocytes proliferation. 30 Tripartite motif 10 (TRIM10) promoted ubiquitination of PTEN, consequently resulting in its proteasomal degradation and activation of hypertrophic signalling. 31 Besides, the 70 kDa isoform of PTEN, PTEN-L, interaction with MFN1 on the mitochondria, could cause cardiomyocyte apoptosis during ethanol toxicity in the heart. Inhibition of PTEN activity prevented mitochondrial PTEN-L-MFN1 interaction for organelle dysfunction, conferring resistance to alcohol-induced toxicity. 32 In this study, we demonstrated that LKB1IP could bind with PTEN and inhibit its phosphatase activity. Thus, LKB1IP may function as a new inhibitor of PTEN. This discovery uncovered a novel regulatory mechanism of PTEN activity in cardiac hypertrophy.

| D ISCUSS I ON
Cardiomyocytes proliferate rapidly during embryogenesis but lose their proliferative capacity soon after birth. 33,34 However, adult cardiomyocytes retain the ability to respond to a few common disease stimuli including myocardial infarction or ischaemia associated with coronary artery disease, chronic hypertension, valvular insufficiency and stenosis, myocarditis, congenital malformations, familial hypertrophic and dilated cardiomyopathies, and diabetic cardiomyopathy. [35][36][37] Over time, individuals with cardiac hypertrophy, which contributes to cardiac remodelling including hypertrophic growth and interstitial expansion, are vulnerable to heart failure, arrhythmia or sudden death. 34 Thus, the detailed mechanism in cardiac hypertrophy needs exploration.
The PI3K/Akt pathway is one of the most important regulatory signalling pathways in cardiac hypertrophy. Once phosphorylated by PIP3, Akt is activated and phosphorylates downstream targets, which results in cardiac hypertrophy. 5 In this study, In summary, this study provides both in vivo and in vitro evidence that LKB1IP functions as a novel positive regulator of pathological cardiac hypertrophy. These findings improve our understanding of the mechanism of pathological cardiac hypertrophy and suggest that LKB1IP may be a therapeutic target for ameliorating pathological cardiac hypertrophy.

ACK N OWLED G EM ENT
We sincerely appreciate all laboratory members.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.