Sialyltransferase7A promotes angiotensin II-induced cardiomyocyte hypertrophy via HIF-1α-TAK1 signalling pathway

Abstract

Aims

Sialylation is up-regulated during the development of cardiac hypertrophy. Sialyltransferase7A (Siat7A) mRNA is consistently over-expressed in the hypertrophic left ventricle of hypertensive rats independently of genetic background. The aims of this study were: (i) to detect the Siat7A protein levels and its roles in the pathological cardiomyocyte hypertrophy; (ii) to elucidate the effect of sialylation mediated by Siat7A on the transforming-growth-factor-β-activated kinase (TAK1) expression and activity in cardiomyocyte hypertrophy; and (iii) to clarify hypoxia-inducible factor 1 (HIF-1) expression was regulated by Siat7A and transactivated TAK1 expression in cardiomyocyte hypertrophy.

Methods and results

Siat7A protein level was increased in hypertrophic cardiomyocytes of human and rats subjected to chronic infusion of angiotensin II (ANG II). Delivery of adeno-associated viral (AAV9) bearing shRNA against rat Siat7A into the left ventricular wall inhibited ventricular hypertrophy. Cardiac-specific Siat7A overexpression via intravenous injection of an AAV9 vector encoding Siat7A under the cardiac troponin T (cTNT) promoter aggravated cardiac hypertrophy in ANG II-treated rats. In vitro, Siat7A knockdown inhibited the induction of Sialyl-Tn (sTn) antigen and cardiomyocyte hypertrophy stimulated by ANG II. Mechanistically, ANG II induced the activation of TAK1-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in parallel to up-regulation of Siat7A in hypertrophic cardiomyocytes. Siat7A knockdown inhibited activation of TAK1-NF-κB pathway. Interestingly, HIF-1α expression was increased in cardiomyocytes stimulated by ANG II but decreased after Siat7A knockdown. HIF-1α knockdown efficiently decreased TAK1 expression. ChIP and luciferase assays showed that HIF-1α transactivated the TAK1 promoter region (nt −1285 to −1274 bp) in the cardiomyocytes following ANG II stimulus.

Conclusion

Siat7A was up-regulated in hypertrophic myocardium and promoted cardiomyocyte hypertrophy via activation of the HIF-1α-TAK1-NF-κB pathway.

1. Introduction

Ventricular hypertrophy is a compensatory response of the heart to overload, particularly occurring under uncontrolled hypertension. As an early myocardial reaction to pathologic stresses, hypertrophic growth of cardiomyocytes may provide a short-term benefit. However, epidemiological research showed that ventricular hypertrophy is associated with the development of heart failure, one of the leading causes of death worldwide.^1–4 Specific features of pathological cardiomyocyte hypertrophy include activation of the foetal gene programme, reorganization of contractile proteins, and an increase in cell size, which are correlated with transcriptional and posttranslational events within the growing cardiomyocyte.^3,5 Angiotensin II (ANG II) protein, one of the major components of the renin–angiotensin system (RAS), is increased in hypertension and promotes the pathogenesis of cardiac hypertrophy.^3,6 The RAS-targeting therapy has been shown efficacious to ameliorate cardiac hypertrophy to some extent.^3,7 More recently, targeting molecules or crucial signalling pathways involved in maladaptive cardiac growth needs development as novel treatments.³

Glycosylation is a kind of post-translational modification of proteins, which is exerted by the glycosyltransferases and glycosidases.⁸ Aberrant glycosylation is involved in the pathogenesis of cardiac function, such as cardiac arrhythmias, cardiac hypertrophy, and heart failure.^9–12 The terminal glycosylation process is frequently modified with sialic acid, which is called sialylation. Different sialyltransferases conduct the transfer of cytidine 5′-monophosphono-N-acetylneuraminic acid (CMP-Neu5Ac) to specific oligosaccharide chains of glycoproteins or glycolipids.¹³ Sialylation regulates embryonic heart development and cardiac function in adults. Deficiency in sialic acid biosynthesis results in lethality in mice.^14,15 In the healthy heart, 169 synthesized sialylated proteins were identified. However, more than 200 sialylated proteins were detected in the hypertrophic heart.¹⁶

Sialyltransferase7A (Siat7A), which is also called sialyltransferase ST6GalNAc1, catalyzes the transfer of sialic acid with α-2, 6-linkage to GalNAc α-O-Serine/Threonine (Ser/Thr), synthesizing the Sialyl-Tn (sTn) antigen. The sTn antigen, carried by many kinds of glycoproteins, has been identified as an onco-foetal antigen and is involved in cancer development.^9,17,18 Siat7A and sTn are rarely observed in normal cardiac tissue. Nevertheless, it was reported that Siat7A is the only common gene consistently overexpressed at an early stage of left ventricular hypertrophy in three hypertensive rat models independent of genetic background.^17,19 We previously proved that Siat7A is highly expressed in ischaemic cardiomyocytes and promotes cardiomyocyte apoptosis.²⁰ However, the functions and the relevant mechanisms of Siat7A in the development of cardiac hypertrophy are still unknown.

TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. TAK1 activates several intracellular signalling pathways, including NF-κB and MAPK cascade.²¹ An elegant series of studies show that activation of TAK1-p38MAPK signalling plays an important role in the pathogenesis of cardiac hypertrophy.^22–29 The activation of TAK1-NF-κB pathway in cardiomyocytes in response to hypertrophic stress has not been reported yet. Moreover, the expression level of TAK1 is increased in cardiomyocytes stimulated by ANG II or after pressure overload in vitro or in vivo, respectively.^22,26 However, how TAK1 is transactivated in the process of cardiac hypertrophy is still unknown.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor, which is composed of two basic proteins; one is a highly inducible HIF-1α subunit and the other is a constitutively expressed HIF-1β subunit. Hypoxia stabilizes HIF-1α protein levels by inhibiting the rapid degradation of HIF-1α by the proteasome. Thereby, HIF-1 binds to a highly conserved core DNA motif (G/ACGTG) in hypoxia response elements to transactivate or transrepress the target genes adaption to low oxygen conditions.³⁰ Nevertheless, it was demonstrated that non-hypoxic stimuli such as ANG II, high glucose or pregnancy obviously elevate the HIF-1α subunit, allowing for the activation of HIF-1 transcription factor.^31–34 HIF-1α expression was increased in the early phase of pathological ventricular hypertrophy. Although HIF-1α has a short-term beneficial function to adapt to pressure overload or acute ischaemia, its chronic activation promotes profound cardiac decompensation.^35,36 Given that Siat7A is consistently increased at the early stage of left ventricular hypertrophy, we hypothesized that Siat7A might regulate HIF-1α expression.

In this study, we found that increased Siat7A, coinciding with the s-Tn antigen, promoted cardiomyocyte hypertrophy induced by ANG II. Siat7A overexpression aggravated the cardiac hypertrophy and increased the levels of HIF-1α and TAK1, whereas Siat7A knockdown significantly inhibited the activation of HIF-1α-TAK1 signalling in the myocardium in response to hypertrophic stress. Furthermore, as the transcription factor, HIF-1α transactivated TAK1-NF-κB pathway in hypertrophic cardiomyocytes.

2. Methods

2.1 Selection of human samples

We selected human heart tissue with cardiac hypertrophy (n = 5) or without any cardiac disease (n = 5). All the specimens were collected and paraffin embedded during autopsy. Four specimens were collected from fatal traffic accidents, four were from death caused by brain stem haemorrhage, and two were from haemorrhage of cerebral vascular malformation. Tissue specimens were stained for histological examination. Informed consent was obtained from the families of the subjects as approved by the Institutional Review Board protocol from Dalian Medical University. All of the procedures were complied with the principles outlined in the Declaration of Helsinki.

2.2 Animals and experimental protocols

Adult male Wistar rats, weighing about 180 g, were purchased from the Animal Medicine Center at Dalian Medical University. The housing type of facility was specific pathogen free. All procedures were approved by the Animal Research Committee at Dalian Medical University and in accordance with the Institutional Guidelines for Animal Research and complied with the Guide for the Care and Use of Laboratory Animals published by the US NIH (2011). Forty rats were randomly divided into the following three groups: Saline-infused rats receiving the delivery of scramble shRNA (shNC-saline, n = 12), ANG II-infused rats receiving the delivery of scramble shRNA (shNC-ANGII, n = 14), and ANG II-infused rats receiving the delivery of shSiat7A (shSiat7A-ANGII, n = 14). Experiments have shown that AAV9 vector is non-pathogenic and can be transfused into myocardium efficiently by direct injection in the cardiac myocardium.^37–40 In our experiment, the AAV9 vector expressing enhanced green fluorescence protein (GFP) for shRNA-mediated knockdown of Siat7A were purchased from Hanbio Biotechnology (Shanghai, China). The shRNA sequences were synthesized as follows: 5-CCACACCUCACCACACAAATT-3 and 5-CCAGUUUAUAGAGGACAAUTT-3, based on the efficient siRNA sequences targeting rat Siat7A gene used in our previous research.²⁰ The operation was performed as described previously (see details in the Supplementary material online).²⁰

On the other hand, 28 rats were randomly divided into the following four groups: AAV9-cTNT GFP-saline rats (n = 7), AAV9-cTNT GFP-ANGII rats (n = 7), AAV9-cTNT Siat7A-saline rats (n = 7), and AAV9-cTNT Siat7A-ANGII rats (n = 7). The AAV9 vectors carrying the cardiac troponin T (cTNT) promoter to drive the expression of GFP (AAV9-cTNT GFP) and rat Siat7A (AAV9-cTNT Siat7A) were purchased from Hanbio Biotechnology (Shanghai, China). Viral solution (1 × 10¹² vector genomes vg/mL, 200 µL/rat) was slowly injected via the jugular vein after the rats were anaesthetized by intraperitoneal administration of pentobarbital (50 mg/kg).

For infusion of ANGII, 2 weeks after injection of AAV9 vectors, osmotic minipumps (Alzet Model 2004; Durect Corporation, Cupertino, CA, USA) prefilled with ANG II (Aladdin, A107852) or saline were subcutaneously inserted as described previously (see details in the Supplementary material online).⁴¹

2.3 Echocardiography

Performed transthoracic echocardiography in the rats on Day 29 after ANG II stress (see details in the Supplementary material online). The data was analysed from three consecutive cardiac cycles for each measurement. The left ventricular internal dimension (LVID) at diastole and systole, left ventricular posterior wall thickness (LVPW) at diastole and systole, left ventricular anterior wall thickness (LVAW) at diastole and systole, left ventricular ejection fraction (EF%), and left ventricular fractional shortening (FS%) were calculated. Euthanasia of all the rats was performed by intraperitoneal administration of pentobarbital (150 mg/kg).

2.4 Bioluminescence imaging

Luciferase activity in rat hearts was assessed by ex vivo bioluminescence imaging of each isolated heart. All bioluminescence imaging was performed by CARESTREAM Image Station System (Micro Focus X-RAY Imaging Source, Carestream Health Inc., USA).

2.5 Histopathology

Haematoxylin and eosin (HE) and immunohistochemical staining were performed as described in the Supplementary material online.

2.6 Cell culture and treatment

The human cardiomyocyte-like cell line (AC16) was obtained from the American Type Culture Collection (Manassas, VA, USA). Manipulation was described in the Supplementary material online.

2.7 Transfection

TAK1 siRNA (5-AUUUGUAGAAGCAAUGUCCTT-3), HIF-1α siRNA (5-AAUGGGUUCACAAAUCAGCTT-3), and Scrambled siRNA (siNC, 5-ACGUGACACGUUCGGAGAATT-3) were purchased from Genepharma (Shanghai, China). Transfection was performed as described previously (see details in the Supplementary material online).²⁰

Particles for shRNA Siat7A and shRNA HIF-1α were purchased from GenePharma (Shanghai, China) for lentiviral transduction. The shRNA sequence targeting Siat7A was 5-GCTACACGATGAAGGGATAAT-3. The shRNA sequences targeting HIF-1α were 5-GCTGATTTGTGAACCCATT-3 (HIF-1α-1) and 5-GTGATGAAGAATTACCGAAT-3 (HIF-1α-2). The scrambled shRNA (shNC) sequences were 5-TTCTCCGAACGTGTCACGT-3. To establish the stable knockdown cell line, the puromycin was applied to the infected cells. The expression efficiency was evaluated by real-time RT-PCR and western blot analysis.

To generate HIF-1α-transfected AC16 cells, the full-length HIF-1α was cloned into the pCDH vector. The primers 5-GCTCTAGAATGGAGGGCGCCGGCGGCGC-3 and 5-GCGGATCCTCAGTTAACTTGATCCAAAG-3 were used to generate plasmids encoding full length HIF-1α. Generation of lentivirus expressing HIF-1α was performed as described previously.⁴²

2.8 Western blot analysis

Analysis was performed as described in the Supplementary material online.²⁰

2.9 RNA extraction and real-time RT-PCR

Total RNA was extracted by using Trizol (Ambion). cDNA was synthesized using the PrimeScript™ RT reagent kit (Takara) as described previously (see details in the Supplementary material online).²⁰ Real-time PCR primer sequences were described in Supplementary material online, Table S1.

2.10 Immunofluorescence and confocal microscopy

Immunofluorescence staining was performed as shown in the Supplementary material online.

2.11 Dual-luciferase reporter assay

The predicted sequences of TAK1 promoter (P1, P2, P3) and predicted binding sequences (BS1, BS2) were cloned into the pGL3-basic vector (Promega, Beijing, China). Simultaneously, mutant plasmids (BS1M, BS2M) were constructed with site-directed mutagenesis (see details in the Supplementary material online).

2.12 Chromatin immunoprecipitation (ChIP) assay

Detailed assay is shown in the Supplementary material online.

2.13 Statistical analysis

Results were presented as mean ± SEM. Statistical differences were evaluated by ANOVA followed by a post hoc Tukey test. P-values of <0.05 were considered statistically significant.

3. Results

3.1 Siat7A knockdown attenuated left ventricular hypertrophy

To evaluate the status of Siat7A expression under cardiac hypertrophy in humans, HE staining was performed to identify the left ventricular hypertrophy (Figure 1A-a–d). Immunohistochemical staining was applied simultaneously to analyse the expression of Siat7A (Figure 1A-e–h). Figure 1A-a and c show the morphology of the normal cardiomyocytes, whereas cardiac hypertrophy was shown in Figure 1A-b or d, which was indicated by the larger size of cardiomyocyte compared with that of the normal hearts. Siat7A expression was apparently stronger in the cytoplasm of the hypertrophic cardiomyocytes (Figure 1A-f and h), compared with that shown in the normal cardiomyocytes (Figure 1A-e and g).

To determine the effects of Siat7A knockdown on the cardiac geometry and contractile function under pathological stress in vivo, AAV9 vectors bearing scramble shRNA or shRNA Siat7A were delivered into the left ventricular wall, which was followed by continuous infusion of ANG II for 4 weeks. Ex vivo bioluminescence images of isolated hearts in the three group rats apparently documented GFP expression, which verified that the AAV9 vector effectively delivered the shRNA gene to the myocardium (Figure 1C). ANG II infusion apparently induced the elevation of mean arterial pressure (MAP) of either the shNC-ANGII group rats or the shSiat7A-ANGII group rats compared with that of the shNC-saline group rats from Day 10. MAP remained at a high level throughout the procedure (Figure 1B). As shown in Figure 1E–G, infusion of ANG II significantly increased Siat7A expression in the myocardium with the delivery of scramble shRNA, in comparison with infusion of saline. Siat7A knockdown in the myocardium efficiently inhibited Siat7A expression induced by infusion of ANG II and inhibited cardiac hypertrophic responses as shown by a lower heart weight-to-body weight ratio (HW/BW) and a smaller cardiomyocyte size compared with that of the shNC-ANG II group rats (Figure 1D, G,and H). Concordantly, cardiac hypertrophy markers including ANP, BNP and α-skeletal actin were significantly down-regulated in the shSiat7A-ANG II group rats (Figure 1I–K). To further verify the effect of Siat7A knockdown on the function of left ventricle in response to hypertrophic stress, the LVPWD, LVPWS, LVAWD, LVAWS, LVIDD, LVIDS, LV EF%, and LV FS% of all rats were calculated (Supplementary material online, Table S2). As shown in Figure 1L and M, the LVPWS, LV EF%, and FS% were significantly increased to compensate for the heart dysfunction in the shNC-ANGII group rats. Siat7A knockdown significantly reversed AGN II-induced dysfunction in cardiac hypertrophy. The LV EF% and FS% of shSiat7A-ANGII group rats were not significantly different from that of shNC-saline group rats.

3.2 Siat7A and TAK1 were increased in hypertrophic cadiomyocytes

We examined the effect of ANG II on Siat7A expression in vitro. The cultured human cardiomyocyte cell line, AC16, was used to perform Real-time PCR and immunoblotting as well as confocal microscopy analysis (Figure 2A–G). Figure 2A, B, C, F,and G show that Ang II stimulation of AC16 caused cardiomycyte hypertrophy indicated by significant increasing levels of ANP, BNP, β-MHC, α-skeletal actin and stronger expression of sarcomeric α-actinin. Expressions of Siat7A and TAK1 were significantly increased simultaneously in a concentration-dependent manner due to ANG II stimulus (Figure 2D–F). Moreover, the expression of s-Tn antigen which is the special substrate of Siat7A was stronger in the AC16 cells treated by ANG II, compared with the cells treated by saline (Figure 2G–c, d).

3.3 Siat7A knockdown inhibited cardiomyocyte hypertrophy via inactivation of TAK1-NF-κB

To mechanically verify the effects of Siat7A knockdown on cardiomyocyte hypertrophy, we transferred shRNA Siat7A into the cultured AC16 cells. As shown in Figure 3A, B, F, G, and H, compared with transfection of shRNA NC, transfection of shRNA Siat7A significantly down-regulated Siat7A expression and consistently inhibited the induction of s-Tn antigen as well as TAK1 expression at both mRNA and protein levels in the AC16 cells stimulated by ANG II. Furthermore, down-regulation of Siat7A before treatment with ANG II obviously restricted the expressions of ANP, BNP, β-MHC, α-skeletal actin and sarcomeric α-actinin in the AC16 cells, which indicated that cardiomyocyte hypertrophy was inhibited (Figure 3C–G).

To ascertain whether Siat7A exerted any effect on activation of TAK1 in the cardiomyocytes stimulated by ANG II, we detected the NF-κB activity which may be initiated by the activation of TAK1. As shown in Figure 3H, transfection of shRNA Siat7A simultaneously decreased TAK1 expression and phosphorylation of NF-κB as well as cardiac hypertrophic markers in the AC 16 cells stimulated by ANG II. Transfusion of TAK1 siRNA before the ANG II stimulus significantly decreased the NF-κB activity and cardiac hypertrophic markers, which was consistent with the significant down-regulation of TAK1 expression in the AC16 cells (Figure 3I–L). Therefore, it was proven that Siat7A expression increased TAK1 expression, which activated NF-κB and resulted in cardiomyocyte hypertrophy.

3.4 HIF-1α was regulated by Siat7A and mediated TAK1 in hypertrophic cardiomyocytes

To clarify the mechanisms for Siat7A-invovled regulation of TAK1 expression, we focused on HIF-1α. As shown in Figure 4A–C, HIF-1α expression was increased in the AC16 cells following ANG II stimulation. Transfection of shRNA Siat7A significantly inhibited the induction of HIF-1α at both mRNA and protein levels in the AC16 cells treated by ANG II. Although HIF-1β expression was detected in the AC16 cells, it was not affected by either ANGII stimulus or Siat7A knockdown (Supplementary material online, Figure S1). To identify the effect of HIF-1α expression on TAK1 expression, HIF-1α siRNA was transfected into the AC16 cells before the treatment of ANG II. As shown in Figure 4D–I, TAK1 expression and levels of cardiac hypertrophic markers were significantly decreased at both mRNA and protein levels due to significant HIF-1α knockdown in the AC16 cells.

3.5 HIF-1α transactivated TAK1 gene by binding to the TAK1 promoter region (nt −1285 to −1274) in cardiomyocytes treated by ANG II

Since HIF-1α was found to mediate TAK1 expression, we constructed three putative TAK1 promoter segments for HIF-1α binding sites using a transcription factor search website (TFSEARCH) (Figure 5A). A luciferase assay showed that the activity of TAK1 promoter in the nt −2000 to −1000 region was substantially increased, which was consistent with the increase in HIF-1α expression in the AC16 cells stimulated by ANG II (Figure 5A–C). Transfection of shRNA HIF-1α resulted in inhibition of HIF-1α in the AC16 cells treated by ANG II, simultaneously significantly decreasing TAK1 promoter activity (Figure 5D and E). However, transfection of HIF-1α gene up-regulated HIF-1α expression in the AC16 cells treated by ANG II, simultaneously significantly increasing TAK1 promoter activity (Figure 5F and G). To further clarify the TAK1 promoter sequence for HIF-1α binding sites within the nt −2000 to −1000 region, we constructed two segments which contained the highly conserved core DNA motif. As shown in Figure 5H–J, the activity of TAK1 promoter in the nt −1285 to −1274 region was increased, which was consistent with the induction of HIF-1α in the AC16 cells treated by ANG II. TAK1 promoter activity failed to be detected, while transfecting the mutagenic sequences or the segment in the nt −1250 to −1000 region in the AC16 cells before the treatment of ANG II. Moreover, the knockdown of HIF-1α significantly decreased the activity of the TAK1 promoter in the nt −1285 to −1274 region in the AC16 cells stimulated by ANG II, while the up-regulated HIF-1α obviously increased TAK1 promoter activity in the AC16 cells stimulated by ANG II (Figure 5K–N). To determine that HIF-1α binds to the identified HIF-1α element at the TAK1 promoter, we performed ChIP to detect the relevant DNA-protein interactions. As shown inFigure 5O and P, the identified sequence for the TAK1 promoter was detected using an anti-HIF-1α antibody. These results show that HIF-1α transactivated TAK1 gene expression by binding to the TAK1 promoter region (nt −1285 to −1274 bp).

3.6 Siat7A overexpression aggravated cardiac hypertrophy

To mimic the up-regulation of Siat7A observed during cardiac hypertrophy development, we used an AAV9 expressing Siat7A under the control of the cardiac-specific cTNT promoter. Two weeks after AAV9 injection, infusion of ANGII was followed for 4 weeks. GFP expression in rat hearts was strongly documented by ex vivo bioluminescence imaging (Figure 6D). As shown in Figure 6A, ANGII apparently induced the elevation of MAP of either the AAV9-cTNT GFP-ANGII group or AAV9-cTNT Siat7A-ANGII group rats from Day 10. Continuous infusion of ANGII resulted in cardiac hypertrophic responses indicated by significant increases in HW/BW, cardiomyocyte size and reactivations of myocardial foetal genes such as ANP, BNP and α-skeletal actin. In addition, LVIDs was significantly decreased, whereas LV EF% and FS% were significantly increased following ANGII stimulus. Interestingly, Siat7A overexpression by intravenous injection of AAV9-cTNT Siat7A viral solution led to more severe cardiac hypertrophic responses compared with AAV9-cTNT GFP-ANGII rats (Figure 6B, C, E, F, G, K, L,and M).

Next, we studied the effect of Siat7A expression on the HIF-1α-TAK1 signalling pathway in vivo. Siat7A overexpression significantly induced the increase in HIF-1α at both mRNA and protein levels (Figure 6I and N). Similarly, the level of TAK1 was obviously up-regulated in parallel to the increased Siat7A and HIF-1α in either AAV9-cTNT GFP-ANGII or AAV9-cTNT Siat7A-ANGII group rats (Figure 6H, J and N). Furthermore, Siat7A knockdown in cardiac myocardium significantly inhibited the levels of HIF-1α and TAK1 in rats subjected to ANGII stimulus (Figure 6O and Supplementary material online, Figure S2). These in vivo results show that overexpression of Siat7A aggravated cardiac hypertrophy, simultaneously up-regulated the levels of HIF-1α and TAK1 in cardiac myocardium following ANGII stimulation.

4. Discussion

In this study, we demonstrated that Siat7A was increased in the hypertrophic cardiomyocytes. Siat7A overexpression aggravated cardiac hypertrophy following hypertension. Siat7A knockdown depressed cardiomyocyte hypertrophy stimulated by ANG II both in vivo and in vitro. Increased Siat7A expression promoted cardiomyocyte hypertrophy by up-regulated HIF-1α expression. HIF-1α, as a transcription factor, directly bound to the TAK1 promoter in the nt −1285 to −1274 region and up-regulated TAK1 expression, thereby initiating the activation of TAK1-NF-κB signalling in the hypertrophic cardiomyocyte. Siat7A knockdown suppressed the activity of HIF-1α-TAK1-NF-κB in the cardiomyocytes stimulated by ANG II and inhibited hypertrophy. Our findings shown in the schematic representation (Figure 7), provide the target molecules for therapeutic strategy of inhibiting cardiomyocyte hypertrophy through the Siat7A-HIF-1α-TAK1 pathway.

Metabolic glycan staining reveals that sialylation is up-regulated during cardiac hypertrophy. Especially newly synthesized sialylated glycans were significantly increased on the surface of cardiomyocyte.¹⁶ Moreover, Siat7A mRNA is found consistently over-expressed in the left cardiac myocardium of hypertensive rat models.¹⁹ In this study, we set out to identify Siat7A protein level and its effect on cardiomyocyte hypertrophy. We found that Siat7A protein was apparently over-expressed in cardiomyocytes of the hypertrophic left ventricle of humans. To detect the correlation between increased Siat7A with hypertrophic cardiomyocytes under hypertension, we used ANG II-induced hypertensive rat model because AGN II plays an important role in cardiac hypertrophy.^3,7 In this study, cardiac hypertrophy at early stage was indicated by the significant increases in HW/BW, LVPWs, LV EF%, FS% cardiomyocyte size, and reactivations of cardiac hypertrophic markers. Down-regulation of Siat7A significantly inhibited development of cardiac hypertrophy induced by ANG II in vivo, as shown in our results. Although cardiac hypertrophy with stimulation of ANG II sustained unchanged or even increased contractile function, this actually was a compensatory response by the heart to haemodynamic overload and provided only short-term benefit. A relative reduction of capillary density due to pathologic cardiomyocyte hypertrophy may lead to increased cardiomyocyte hypoxia and apoptosis.^3,43,44 We previously found that Siat7A in cardiomyocytes is elevated and accelerates cardiomyocyte apoptosis under the shortage in oxygen supply to heart muscle.²⁰ Therefore, together with our present data, we concluded that Siat7A was increased at early stage of cardiomyocyte hypertrophy following ANG II stimulus, which was implicated in progressive cardiomyocyte remodelling. Fortunately, Siat7A knockdown effectively suppressed cardiomyocyte remodelling at an early stage. Next, we will explain which pathway was regulated by increased Siat7A expression in the hypertrophic cardiomyocyte.

TAK1 protein is highly expressed in embryonic or neonatal cardiacmyocytes, but significantly decreased in adult cardiomyocytes. However, TAK1 expression and the TAK1-p38MAPK signalling are implicated in chronic pressure overload induced-cardiomyocyte hypertrophy.^22–29 In this study, we found that the transcriptional and translational up-regulation of TAK1 paralleled the over-expression of Siat7A in the hypertrophic cardiomyocyts stimulated by ANGII both in vivo and in vitro. sTn antigen synthesized by Siat7A was also increased. Siat7A knockdown effectively decreased production of sTn and TAK1 expression as well as cardiac hypertrophic response in the cardiomyocytes treated by ANG II. We further detected the phosphorylation of NF-κB which is one of canonical downstream target molecules of TAK1.²¹ Moreover, NF-κB is implicated in the regulation of cardiomyocyte, and sustained activation of NF-κB appears to be detrimental to cardiac cell fate.⁴⁵ Our results showed that NF-κB was obviously phosphorylated in parallel to the increased TAK1 expression in the cardiomyocytes treated by ANG II, and down-regulation of either Siat7A or TAK1 effectively inhibited phosphorylation of NF-κB. Thus, we concluded that ANGII-induced Siat7A activated TAK1-NF-κB signalling in the cardiomyocytes treated by ANG II, thereby promoting cardiomyocyte hypertrophy. So how did Siat7A transactivate TAK1 expression during cardiomyocyte hypertrophy?

HIF-1α functions as a transcription activator to regulate oxygen homeostasis.³⁰ It was demonstrated that HIF-1α expression is significantly increased in vascular smooth muscle cells stimulated by ANG II. Two separate pathways are responsible for the non-hypoxic induction of HIF-1α.^31,32 In this study, we detected that HIF-1α, but not HIF-1β, was apparently increased in cardiomyocytes treated by ANG II. Interestingly, we found that Siat7A knockdown significantly inhibited HIF-1α, but not HIF-1β, in the cardiomyocyte stimulated by ANG II. These results showed that HIF-1α was regulated by Siat7A in cardiomyocytes stimulated by ANG II. In the future, further clarification is needed regarding the relevant mechanisms for the regulation of HIF-1α by Siat7A. In the present study, when HIF-1α was effectively down-regulated, TAK1 expression at both mRNA and protein levels was significantly suppressed in the cardiomyocyte stimulated by ANG II. We further confirmed whether or not TAK1 was transactivated by HIF-1α. Luciferase assay clarified the sequences within TAK1 promoter range (nt −1285 to −1274) that was bound and transactivated by HIF-1α. Additional results from CHIP tests suggest that TAK1 was directly bound by HIF-1α. Therefore, these results identified that HIF-1α functioned as a transcription factor of TAK1 gene in the cardiomyocyte.

In conclusion, our study provides a novel route contributing to cardiomyocyte hypertrophy following hypertension. Siat7A expression and s-Tn, which is synthesized by Siat7A, were increased in cardiomyocytes and promoted cardiomyocyte hypertrophy through activation of HIF-1α-TAK1- NF-κB pathway following ANG II stimulation. ANGII-induced Siat7A increased HIF-1α expression which then directly transactivated TAK1 expression via binding the promoter of TAK1. Cardiac hypertrophy was markedly attenuated by down-regulation of Siat7A. These findings may provide a crucial therapeutic target for inhibiting cardiomyocyte hypertrophy after hypertension.

Time for primary review: 24 days

Acknowledgements

We kindly thank Professor Yuanshan Fu in the Anatomy Department of Dalian Medical University to provide us sincere help.

Conflict of interest: none declared.

Funding

This work was supported by the National Nature Science Foundation of China [81670383 to D.Z.; 81874136 to C.H.]; and the Liaoning Provincial Natural Science Foundation of China [2015020683 to D.Z.].

References

Author notes