PKM2 promotes angiotensin‐II‐induced cardiac remodelling by activating TGF‐β/Smad2/3 and Jak2/Stat3 pathways through oxidative stress

Abstract Hypertensive cardiac remodelling is a common cause of heart failure. However, the molecular mechanisms regulating cardiac remodelling remain unclear. Pyruvate kinase isozyme type M2 (PKM2) is a key regulator of the processes of glycolysis and oxidative phosphorylation, but the roles in cardiac remodelling remain unknown. In the present study, we found that PKM2 was enhanced in angiotensin II (Ang II)‐treated cardiac fibroblasts and hypertensive mouse hearts. Suppression of PKM2 by shikonin alleviated cardiomyocyte hypertrophy and fibrosis in Ang‐II‐induced cardiac remodelling in vivo. Furthermore, inhibition of PKM2 markedly attenuated the function of cardiac fibroblasts including proliferation, migration and collagen synthesis in vitro. Mechanistically, suppression of PKM2 inhibited cardiac remodelling by suppressing TGF‐β/Smad2/3, Jak2/Stat3 signalling pathways and oxidative stress. Together, this study suggests that PKM2 is an aggravator in Ang‐II‐mediated cardiac remodelling. The negative modulation of PKM2 may provide a promising therapeutic approach for hypertensive cardiac remodelling.


| INTRODUC TI ON
Hypertension is a key risk cause for cardiovascular diseases, remaining the predominant cause of mortality in the world and presenting a considerable economic burden. 1 Pressure overload induces cardiac remodelling that includes cardiomyocyte hypertrophy and fibrosis, and ultimately leads to the progression of heart failure. 2 As the predominant effector of the renin-angiotensin-aldosterone system (RAAS), angiotensin II (Ang II) promoted cardiac fibrosis by inducing fibroblasts proliferation and stimulating collagen synthesis and eventually caused cardiac remodelling and heart failure. 3 Multiple signalling pathways have been demonstrated to positively regulate cardiac fibrosis, including transforming growth factor β (TGFβ)/small mothers against decapentaplegic proteins 2 and 3 (Smad2/3), Janus kinase 2 (Jak2)/signal transducer and activator of transcription 3 (Stat3) signalling pathways. 4 TGFβ induced phosphorylation of Smad2 and Smad3 to promote extracellular matrix secretion which plays important roles in myocardial fibrosis. 5 Moreover, activation of Jak2/Stat3 signalling pathway enhances collagen-related gene transcription to accelerate fibrosis. 6 Hence, targeting these signalling pathways might provide new therapeutic strategies to reverse cardiac remodelling.
Oxidative stress, defined as disturbances in the pro-/anti-oxidant balance, is harmful to cells due to an excess production of reactive oxygen species (ROS). 7 Accumulating evidence indicates that oxidative stress plays a vital role in renal fibrosis, cardiovascular disease and other diseases. [8][9][10] It is reported that ROS mediates Ang-IIinduced fibrotic cardiomyopathy by activating transcription factors and fibrotic signalling kinases. 11 Besides, glutathione (GSH) and GSH peroxidase 4 (GPx4) act as negative regulators of oxidative stress by limiting ROS production. 12 Pyruvate kinase muscle isozyme 2 (PKM2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. The primary function of PKM2 is to mediate the conversion of phosphoenolpyruvate (PEP) to pyruvate as the last step of glycolysis to generate ATP. 13 PKM2 also has various biological functions, including proinflammatory cytokine production, cells proliferation and oxidative stress. [14][15][16] However, the potential role of PKM2 in Ang-II-induced cardiac remodelling remains unknown.
To explore the potential role of PKM2 in cardiac remodelling diseases, we analysed the expression of PKM2 in Ang-II-treated neonatal rat cardiac fibroblasts (NRCFs), neonatal rat cardiomyocytes (NRCMs) and mouse hearts. Furthermore, we checked the role of PKM2 in cardiac remodelling. Finally, we elucidated the potential underlying mechanism of PKM2 in the development of cardiac remodelling.

| Animal model
Male C57BL/6 mice were purchased from the Vital River Laboratory Animal Technology Company of Beijing in China. All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University. Hypertensive cardiac remodelling was induced in 8-to 12-week-old male mice by chronic subcutaneous infusion of Ang II (A9525, Sigma-Aldrich) at a dose of 1,500 ng/kg/min using the ALZET ® Osmotic Pumps (Model 1007D) for 7 days or 1,000 ng/kg/min using the pumps (Model 1002D) for 14 days. The control mice were infused with saline for 7 or 14 days.
For shikonin treatment, mice were randomized to be intraperitoneally injected with either vehicle or shikonin at a dose of 1.25 mg/ kg 3 times per week. All mice were analysed by echocardiography and haemodynamic measurements at 7 days or 14 days.

| Primary culture of NRCMs and NRCFs
Cardiomyocytes and fibroblasts were isolated from 1-day-old SD rat hearts using a conventional method as described. 17 Briefly, heart tissues were digested by 0.1% trypsin and 0.05% type II collagenase.
The dissociated cells in DMEM/F-12 medium with 15% FBS and 1% penicillin and streptomycin were plated to obtain adhered cardiac fibroblasts after 90 min in incubator (5% CO 2 ), and the cardiomyocytes in suspension were transferred to another dish with coating laminin. Both cardiomyocytes and fibroblasts were cultured in serum-free DMEM/F-12 for 12 h before experiments.
The sequence of negative control siRNA was as follows: 5′-UUCUCCGAACGUGUCACGU-3′. Transfections of the siRNAs were performed by using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, cells were seeded in 6-well plates to obtain approximately 30%-50% confluence. Twenty microlitres of mixture containing siRNA (20 pM for each) and transfection reagents (5 μl for each) were added to the cells for 6 h at 37℃ and then replaced with fresh culture medium. The efficiency of transfection was confirmed by Western blot analysis.

| Cell migration assay
Cell migration was examined by wound healing assay. NRCFs grown to 90% in 6-well plates were scratched via a 200 μl pipette tip to create the wound followed by washing detached cells with PBS.
Cultured cells were grown for 12 h and 24 h to allow wound healing.
Wound healing area was measured using Image J (National Institutes of Health, Bethesda, MD, USA), and % of wound area was analysed by GraphPad Prism 7.0 (GraphPad Software). % of wound area represents the ration of wound area at T 12h (or T 24h )/wound area at T 0h .

| Cell counting assay
The cell viability was detected by cell counting kit-8 (CCK-8) (YEASEN, Shanghai, China). Briefly, 10 μl of CCK-8 solution was added to each well and incubated at 37℃ for 2.5 h; then, the OD value for each well was read at wavelength 450 nm with a microplate reader (Multiskan, Thermo, USA). The assay was repeated 3 times.
The cell viability was calculated as follows:

| Cell oxidative stress assays
Reactive oxygen species levels were determined by Flow cytometry. Briefly, 5 × 10 5 NRCFs with pretreatment of 0.1 μM shikonin or DMSO for 2 h were treated with Ang II for 12 h, and then incu- Total glutathione (GSH+GSSG) and GSSG were measured according to the GSH and GSSG assay kit (Beyotime) using kinetic determination methods under a 412 nm wavelength, and the GSH/GSSG ratio was calculated.

| Pyruvate production assay
Pyruvate production was detected using PA assay kit (Solarbio, Beijing, China). Briefly, 5 × 10 5 NRCFs were treated with Ang II and then washed 3 times with PBS and were harvested with 1 ml extracting solution. After ultrasonic disruption, cells were freezing for 30 min and then centrifuged to collect the supernatant. Pyruvate levels in mouse serums and NRCFs were measured using the kit according to the manufacturer's instructions.

| Measurement of blood pressure
Mice were acclimatized for 7 days prior to experimentation. The blood pressure of all mice was measured before Ang II infusion and every day in 7-day model or every other day in 14-day model.

| Echocardiography
Mice were lightly anaesthetized with 1.5% isoflurane. Cardiac function was evaluated by the high-resolution Micro-Ultrasound system

| Histopathology analysis
For histological analysis, hearts were fixed, embedded and sectioned into 5μm-thick slices. Sections were used to stain wheat germ agglutinin (WGA) to assess cellular hypertrophy. Sections were stained with Masson's trichrome (collagen, blue; cytoplasm, red/pink) for collagen deposition analysis. Immunohistochemistry images were captured with Pannoramic SCAN II (3DHISTECH Ltd) and analysed by using Image J.

| Quantitative RT-PCR (qRT-PCR) analysis
Total RNA was extracted from the heart tissues and cells by using

| Western blot analysis
Cells or heart tissues were lysed with RIPA lysis buffer. Equal amounts of protein were loaded, separated by SDS-PAGE gels and transferred to a membrane (Millipore). The membrane was blocked and incubated with primary antibody at 4℃ overnight, and then incubated with secondary antibodies for 1 h at room temperature.
Western blot signals were detected with the enhanced chemiluminescence system (Millipore), and signal intensities were analysed with Image J.

| Statistical analysis
All data were expressed as mean ± standard error of the mean (SEM) and were analysed using GraphPad Prism 7.0 (GraphPad Software) or SPSS Version 21 software. Comparison between two groups was conducted using two-tailed, unpaired Student's t-test. In the result with more than two groups, analysis of variance (ANOVA) was applied to analyse the difference. Two-way ANOVA moderated by

| PKM2 expression is upregulated in hypertensive heart
To explore the role of PKM2 in cardiac remodelling, we examined the expression of PKM2 in Ang-II-induced hypertensive mouse hearts. PKM2 was boosted at both mRNA and protein levels after 7-and 14-day Ang II infusion ( Figure 1A, B). In vitro, PKM2 upregulated in NRCFs in a time-dependent manner after Ang II treatment ( Figure 1C). However, PKM2 expression was not altered in Ang-II-treated NRCMs ( Figure 1D). Consequently, accumulation of PKM2 led to increase pyruvate production in Ang-II-treated hypertensive mouse serums and NRCFs ( Figure S1A, B). In summary, these results suggest that PKM2 is boosted in response to fibrotic stimuli and may play a role in Ang-II-induced myocardial fibrosis. It is reported that mTOR as a key activator of PKM2 is known augmented by mTOR activation and reduced by mTOR suppression in cancer cells. 18 To determine whether mTOR involved in the regulation of PKM2, we used rapamycin, inhibitor of mTOR signalling pathway, in Ang-II-incubated NRCFs. We found that upregulation of PKM2 induced by Ang II was reduced by rapamycin. (Figure S1C). These data indicate that PKM2 is positively regulated by mTOR.

| Inhibition of PKM2 alleviates Ang-II-induced early cardiac remodelling
To determine whether enhanced PKM2 causes adverse cardiac remodelling in early-stage, wild-type (WT) mice were treated with PKM2 inhibitor, shikonin (1.25 mg/kg, i.p., 3 times per week) and Ang II (1,500 ng/min/kg) for 7 days (Figure 2A). PKM2 was downregulated after treatment of shikonin ( Figure S2A). Systolic blood pressure induced by Ang II infusion was not influenced by shikonin ( Figure 2B). In Ang-II-induced 7-day mouse cardiac remodelling, shikonin recovered cardiac pump function, which included left ventricular ejection fraction (EF %) and fractional shortening (FS %) ( Figure 2C). Concomitantly, shikonin decreased Ang-II-induced hypertrophic responses, including heart weight/body weight (HW/ BW) ratio, heart weight/tibia length (HW/TL) ratio, the crosssectional area of myocytes and the expression of hypertrophic markers atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) (Figure 2D-F). In addition, shikonin relieved Ang-II-induced an increase in the area of cardiac fibrosis and collagen I and III expression ( Figure 2G, H). Hence, inhibition of PKM2 significantly inhibits Ang-II-induced early cardiac remodelling and rescues cardiac function.

| PKM2 inhibition ameliorates Ang-II-induced late cardiac remodelling
Furthermore, we treated WT mice with shikonin and Ang II (1,000 ng/min/kg) for 14 days in late cardiac remodelling model ( Figure 3A). Consistently with 7-day model, PKM2 was downregulated after treatment of shikonin ( Figure S2B). Accordingly, systolic blood pressure increased by Ang II infusion was not altered by shikonin ( Figure 3B), and shikonin reduced cardiac hypertrophy/fibrosis and improved cardiac function with Ang II infusion ( Figure 3C-H).
Consistently, inhibition of PKM2 can also reverse late cardiac dysfunction and remodelling in Ang-II-infused mice.

| Inhibition of PKM2 suppresses Ang-IIinduced cardiac fibroblasts dysfunction
We next determined the effect of PKM2 inhibition on cardiac fibroblasts migration, proliferation and collagen synthesis in vitro.

| Inhibition of PKM2 improves Ang-II-induced oxidative stress
To further demonstrate the molecular mechanisms of pro-fibrotic effect of PKM2, we checked oxidative stress in these processes.
MDA, the product of oxidative stress showed a similar change to generation of ROS ( Figure 6B). We also evaluated anti-oxidants GSH/GSSG ratio and GPx4. As shown in Figure 6C, D, shikonin treatment significantly increases Ang-II-induced GSH/GSSG ratio and GPx4 in NRCFs downregulation. Furthermore, we detected oxidative stress in Ang-II-treated 7-and 14-day mouse hearts. Our data showed shikonin treatment significantly improved Ang-II-induced GPx4 and SOD2 downregulation ( Figure 6E, F). Hence, suppression of PKM2 significantly alleviates Ang-II-induced oxidative stress in cardiac remodelling.

| DISCUSS ION
In this study, we identified PKM2 as a detrimental factor in pathological cardiac remodelling. PKM2 was increased in both Ang-IItreated hearts and NRCFs, but not in NRCMs. Inhibition of PKM2 in mice alleviated Ang-II-induced cardiac remodelling and dysfunction.
Cardiac fibrosis is pivotal in cardiac remodelling and heart failure.
Persistent cardiac fibrosis causes an increase in cardiac muscle stiffness, eventually leads to cardiac dysfunction. 23 PKM2 is a key enzyme involved in the aerobic glycolysis of rapidly proliferation cells, which involves in fibroblast function and fibrosis. [24][25][26] Increasing evidence suggests that PKM2 contributes to pathological fibrosis process in different diseases. In chronic kidney diseases, overexpression of PKM2 remarkably induces myofibroblast activation and renal interstitial fibrosis, and inhibition of PKM2 by shikonin reverses these detrimental effects. 27 PKM2 is elevated in diabetic model mouse lungs and hearts, which regulates glucose-induced lung fibrogenesis. 28 The expression of PKM2 is markedly increased in TAC-induced hypertrophic hearts. 29 In our study, we also found that PKM2 expression was elevated in Ang-II-infused remodelling mouse hearts and cardiac fibroblasts. Inhibition of PKM2 by shikonin ameliorated cardiac fibrosis, hypertrophy and dysfunction in Ang-II-induced mouse cardiac remodelling. In vitro, suppression of PKM2 alleviated Ang-II-induced migration, proliferation and collagen synthesis.
Hence, PKM2 acts as a pro-fibrotic role in cardiac remodelling.
TGFβ/Smad2/3 and Jak2/Stat3 signalling are primary pathways in fibrogenesis and majorly contribute to reactive fibrosis in Ang-II-induced cardiac remodelling. 30 TGFβ, one of the best characterized fibrogenic growth factors, is markedly and consistently activated by Ang II in fibrotic animal hearts. 31 Overexpression of TGFβ stimulates the deposition of excessive collagen in cardiac interstitium and eventually induces ventricular fibrosis. 32 In addition, blockade of TGFβ prevents cardiac fibrosis in pressure overload model. 33 Smad2/3, reported as TGFβ transcriptional activators, promotes the activation of cardiac fibroblasts into myofibroblasts and aggravate pathological cardiac fibrosis. 34 We found that suppression of PKM2 restrained the activation of TGFβ/Smad2/3 signalling pathway. Numerous data indicate that the Jak2/Stat3 signalling pathway is involved in the development of cardiac fibrosis. 35 Selective inhibition of Stat3 phosphorylation ameliorates left-atrial fibrosis in mouse MI model through inhibiting collagen synthesis. 6 Our results showed that suppression of PKM2 inhibited phosphorylation of Jak2 and Stat3. These data indicate that PKM2-activated aggravates cardiac fibrosis by targeting TGFβ/Smad2/3 and Jak2/Stat3 signalling pathways.
Oxidative stress plays an important role in the pathophysiology of myocardial fibrosis. 36 In an in vivo, ROS promotes collagen synthesis and deposition in fructose-and high glucose-induced myocardial fibrosis. 37,38 In adult rat cardiac fibroblasts, the generation of ROS induced by Ang II also stimulates collagen production. 39 MDA, namely lipid peroxidation, is also a widely accepted biomarker of oxidative stress. 40 In our study, inhibition of PKM2 reduced Ang-II-induced ROS production and MDA levels. Diversespecific and -nonspecific anti-oxidant defence systems exist to scavenge ROS. GSH and GPx4, major anti-oxidants, protect cells against oxidative stress by decreasing the generation of ROS. 41,42 We found that Ang II decreased GSH/GSSG ratio and GPx4 levels, while inhibition of PKM2 opposed these detrimental effects.
In summary, we have discovered that PKM2 promotes angiotensin-II-induced cardiac remodelling by activating TGFβ/ Smad2/3 and Jak2/Stat3 pathways through oxidative stress. We propose negative modulation of PKM2 as a potential therapeutic strategy for the treatment of cardiac remodelling diseases.

ACK N OWLED G EM ENT
This study was supported by National Natural Science Foundation of China (81670380).

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.