
==== Front
J Adv Res
J Adv Res
Journal of Advanced Research
2090-1232
2090-1224
Elsevier

S2090-1232(23)00299-0
10.1016/j.jare.2023.10.007
Medicine
Leucine zipper protein 1 attenuates pressure overload-induced cardiac hypertrophy through inhibiting Stat3 signaling
Fan Di ab1
Jiang Wan-li c1
Jin Zhi-li ab
Cao Jian-lei ab
Li Yi ab
He Tao ab
Zhang Wei ab
Peng Li ab
Liu Hui-xia ab
Wu Xiao-yan ab
Chen Ming ab
Fan Yong-zhen ab
He Bo ab
Yu Wen-xi ab
Wang Hai-rong ab
Hu Xiao-rong huxrzn@whu.edu.cn
ab⁎
Lu Zhi-bing luzhibing222@a63.com
ab⁎
a Department of Cardiology, Zhongnan Hospital of Wuhan University, Wuhan 430062, China
b Institute of Myocardial Injury and Repair, Wuhan University, Wuhan 430062, China
c Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China
⁎ Corresponding authors at: Department of Cardiology, Zhongnan Hospital of Wuhan University, Institute of Myocardial Injury and Repair, Wuhan University at Donghu Road a69, Wuhan 430062, China. huxrzn@whu.edu.cnluzhibing222@a63.com
1 Co-first authors.

06 10 2023
9 2024
06 10 2023
63 117128
11 4 2023
30 9 2023
6 10 2023
© 2024 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

LUZP1 expression was increased in pressure overload-induced cardiac hypertrophy in postnatal mice. By constructing cardiac-specific Luzp1 knockout and transgenic mice, we demonstrated that cardiac-specific LUZP1 deficiency aggravated, while cardiac-specific LUZP1 overexpression attenuated pressure overload-induced cardiac hypertrophy and dysfunction. Mechanistically, LUZP1 elevated SHP1 expression to inactivate Stat3 pathway, thereby preventing pathological cardiac hypertrophy. Accordingly, SHP1 silence blocked the anti-hypertrophic effects of LUZP1 in vivo and in vitro.

Highlights

• LUZP1 expression was progressively increased in hearts after TAC surgery.

• Cardiac-specific LUZP1 deficiency aggravated pressure overload-induced cardiac hypertrophy.

• Cardiac-specific LUZP1 overexpression attenuated pressure overload-induced cardiac hypertrophy.

• Stat3 pathway was a downstream target of LUZP1 in regulating pathological cardiac hypertrophy.

• LUZP1 elevated SHP1 expression to inactivate Stat3 pathway.

Introduction

Cardiac hypertrophy is an important contributor of heart failure, and the mechanisms remain unclear. Leucine zipper protein 1 (LUZP1) is essential for the development and function of cardiovascular system; however, its role in cardiac hypertrophy is elusive.

Objectives

This study aims to investigate the molecular basis of LUZP1 in cardiac hypertrophy and to provide a rational therapeutic approach.

Methods

Cardiac-specific Luzp1 knockout (cKO) and transgenic mice were established, and transverse aortic constriction (TAC) was used to induce pressure overload-induced cardiac hypertrophy. The possible molecular basis of LUZP1 in regulating cardiac hypertrophy was determined by transcriptome analysis. Neonatal rat cardiomyocytes were cultured to elucidate the role and mechanism of LUZP1 in vitro.

Results

LUZP1 expression was progressively increased in hypertrophic hearts after TAC surgery. Gain- and loss-of-function methods revealed that cardiac-specific LUZP1 deficiency aggravated, while cardiac-specific LUZP1 overexpression attenuated pressure overload-elicited hypertrophic growth and cardiac dysfunction in vivo and in vitro. Mechanistically, the transcriptome data identified Stat3 pathway as a key downstream target of LUZP1 in regulating pathological cardiac hypertrophy. Cardiac-specific Stat3 deletion abolished the pro-hypertrophic role in LUZP1 cKO mice after TAC surgery. Further findings suggested that LUZP1 elevated the expression of Src homology region 2 domain-containing phosphatase 1 (SHP1) to inactivate Stat3 pathway, and SHP1 silence blocked the anti-hypertrophic effects of LUZP1 in vivo and in vitro.

Conclusion

We demonstrate that LUZP1 attenuates pressure overload-induced cardiac hypertrophy through inhibiting Stat3 signaling, and targeting LUZP1 may develop novel approaches to treat pathological cardiac hypertrophy.

Keywords

Cardiac hypertrophy
Fibrosis
LUZP1
SHP1
Stat3
Abbreviations

Stat3 Signal transducer and activator of transcription 3

TAC Transverse aortic constriction

LUZP1 Leucine zipper protein 1

PE Phenylephrine

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

SHP1 Src homology region 2 domain-containing phosphatase 1

NRCMs Neonatal rat cardiomyocytes

MOI Multiplicity of infection

PTP1B SHP2 or protein tyrosine phosphatase, non-receptor type 1

LVIDd Left ventricular internal dimension at the diastole

LVIDs Left ventricular internal dimension at the systole

FS Fractional shortening

HE Hematoxylin and Eosin

PSR Picric Sirius Red

DEGs Differentially expressed genes

KEGG Kyoto Encyclopedia of Genes and Genomes

HCM Hypertrophic cardiomyopathy
==== Body
pmcIntroduction

Cardiac hypertrophy, characterized as cardiomyocyte enlargement, fibrotic remodeling and the re-activation of fetal genes, is a predominant predisposing factor for congestive heart failure [1]. Various signaling pathways contribute to the pathogenesis of cardiac hypertrophy, including mitogen-activated protein kinases, protein kinase B pathways [2]. Signal transducer and activator of transcription 3 (Stat3) is involved in regulating various cardiovascular diseases, such as ischemic heart injury, cardiac fibrosis and doxorubicin-induced cardiotoxicity, etc. A previous study found that Stat3 played an indispensable role in the proliferation of endothelial cells and fibroblasts, thereby maintaining the postnatal capillary vasculature and interstitial matrix deposition balance in ischemic hearts [3]. Therapeutic activation of Stat3 dramatically inhibited cardiac fibrosis after myocardial infarction, while cardiac-specific Stat3 deletion facilitated cardiac remodeling in infarcted hearts [4], [5]. Consistently, our recent findings revealed that activating Stat3 pathway ameliorated oxidative damage, cardiomyocyte apoptosis and cardiac dysfunction in doxorubicin-treated mice [6]. In addition, Stat3 pathway is also implicated in the initiation and progression of cardiac hypertrophy. Kunisada et al. demonstrated that cardiac-specific Stat3 overexpression resulted in spontaneous concentric hypertrophic growth in mice [7]. Zhuang et al. recently identified that Stat3 phosphorylation and nuclear accumulation significantly impaired mitochondrial bioenergetics, thereby driving hypertrophic growth and cardiac dysfunction in transverse aortic constriction (TAC)-operated mice [8]. In contrast, inhibiting Stat3 activity dramatically prevented pressure overload-elicited cardiac hypertrophy and dysfunction [9]. Moreover, we recently also determined that Stat3 inhibition could prevent pressure overload-induced hypertrophic growth and heart failure [10]. These researches have defined Stat3 as a promising therapeutic target to manage cardiac hypertrophy.

Leucine zipper protein 1 (LUZP1) is mainly localized at the actin cytoskeleton and centrioles, and primarily regulates actin filament bundling and ciliogenesis [11], [12], [13]. Accordingly, Bozal-Basterra et al. found that LUZP1 dysregulation correlated with abnormal formation of cilia in human fibroblasts, eventually leading to Townes-Brocks Syndrome [14]. Interestingly, Hsu and colleagues found that Luzp1 ablation resulted in complex cardiovascular defects in mice, thereby causing perinatal death [15]. Yet, the role and molecular basis of LUZP1 in pressure overload-induced cardiac hypertrophy in postnatal mice remain unclear. The present study establishes cardiac-specific Luzp1 knockout (cKO) and transgenic (cTG) mice to elucidate its role and underlying mechanisms in TAC-induced cardiac hypertrophy.

Materials and methods

Reagents

Phenylephrine (PE, #P6126) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Y-27632 dihydrochloride (#HY-10583) and Y-33075 dihydrochloride (#HY-10069), two independent inhibitors of Rho associated coiled-coil containing protein kinase (ROCK), were obtained from MedChemExpress LLC (Princeton, NJ, USA). Anti-LUZP1 (#17483–1-AP) was acquired from Proteintech (Rosemont, IL, USA). Anti-α-actinin (#69758), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, #2118), anti-phospho-Stat3 (p-Stat3, #9145), anti-total Stat3 (t-Stat3, #9139S) and anti-Src homology region 2 domain-containing phosphatase 1 (SHP1, #26516) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Ethics statement

All experiments involving animals were approved by the Animal Care and Use Committee of our hospital (approval no. 20200708), and were also complied with the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health.

Animals and experimental protocols

All animals were housed in a SPF environment with a constant temperature (20 ± 2 °C) and humidity (50 ± 5 %) on regular 12/12 h light/dark cycles. These mice were allowed free access to food and water, and kept for at least 1 week before study commenced. CRISPR/Cas9 technology was applied to introduce loxP inserts flanking exons 3 and 4 of the Luzp1 gene to generate Luzp1 floxed (Flox) mice in C57BL/6J background, and the genotype was validated by two pairs of flox-specific primers as following: 5′arm F1 5′-GTTGCTGTTTCCAGCACTATTCTGT-3′, R1 5′-GACAAAGCATCTGTGAGGCAAGT-3′; 3′arm F1 5′-TCTGAGGCGGAAAGAACCAG-3′, R1 5′-CATGGCAGGACTATGCTGAATTG-3′. To create Luzp1 cKO mice, Luzp1 floxed mice and α-Mhc-MerCreMer transgenic mice (C57BL/6J background, stock no. 005650, the Jackson Laboratory) were crossed as previously described [10]. For the induction of Luzp1 deletion, Luzp1 cKO mice were intraperitoneally injected with tamoxifen (25 mg/kg/day) for 5 consecutive days. To establish Luzp1 cTG mice, mouse Luzp1 cDNA (#MC223433; OriGene Technologies, Inc., Rockville, MD, USA) was cloned under the α-Mhc promoter, and the plasmid was injected into fertilized mouse embryos as previously described [16]. To generate pressure overload-induced cardiac hypertrophy, 8–10-week-old male mice were exposed to TAC operation [10]. For the induction of anesthesia, mice were intraperitoneally injected with sodium pentobarbital (50 mg/kg), which then received a thoracotomy with the thoracic aorta isolated. Next, the aortic arch was identified and ligated with a 27 gauge needle, and the adequacy of ligation was validated using Doppler echocardiogram after removing the needle. For postoperative analgesia, mice were subcutaneously injected with temgesic (0.1 mg/kg/day) for 7 consecutive days. Mice in sham groups received the same operation without constricting the aorta. All mice were sacrificed 4 weeks post-TAC operation with hearts collected for further examination. To establish Stat3 cKO mice, Stat3 floxed mice and α-Mhc-MerCreMer transgenic mice were crossed as we recently described [10]. Luzp1 cKO mice were crossed with Stat3 cKO mice to generate cardiac-specific Luzp1 and Stat3 double knockout (DKO) mice. To silence SHP1 specifically in the heart, mice were injected with AAV9 vectors (1 × 1011 viral genome particles per mouse) carrying shShp1 (#TR502526; OriGene Technologies, Inc.) under a cTnT promoter from tail vein 4 weeks before TAC surgery [17], [18].

Cell culture and treatments

Neonatal rat cardiomyocytes (NRCMs) were isolated as we previously described, and 0.1 mmol/L 5-bromo-3′-deoxyuridine was added to block the proliferation of cardiac fibroblasts [10], [19]. To knock down or overexpress LUZP1 in vitro, NRCMs were infected with adenovirus carrying shRNA against LUZP1 (shLuzp1) at the multiplicity of infection (MOI) of 100 or rat full-length LUZP1 (AdLuzp1, MOI = 30) for 4 h, which were then cultured in DMEM/F12 containing 15 % fetal bovine serum (FBS) for an additional 24 h before further stimulation [20]. To elicit hypertrophic growth, these NRCMs were then stimulated with 50 μmol/L PE for 24 h as we previously described [10], [19]. To block the remodeling of actin cytoskeleton, Y-27632 (10 μmol/L) and Y-33075 (10 μmol/L) were used to inhibit ROCK [21], [22]. For Stat3 silence in vitro, NRCMs were transfected with 50 nmol/L siStat3 (#sc-29494; Santa Cruz, Dallas, Texas, USA) for 6 h, and subsequently cultured in fresh medium for 24 h before PE stimulation [10]. For Stat3 inhibition, NRCMs were also treated with 5 μmol/L Stattic when stimulated with PE [23]. To silence SHP2 or protein tyrosine phosphatase, non-receptor type 1 (PTP1B), siShp2 (#sc-36489, Santa Cruz) or siPtp1b (#sc-36329, Santa Cruz) were used.

Echocardiography

To evaluate cardiac function, echocardiographic analysis was carried out via referring to our previous studies [24], [25]. First, mice were inhaled with 1.5 % isoflurane for anesthetization, and then two-dimension mode echocardiography was applied to identify the long-axis as well as short-axis structures of the heart. Next, M−mode tracings were used to measure the structural and functional parameters, such as left ventricular internal dimension at the diastole (LVIDd), LVID at the systole (LVIDs), fractional shortening (FS) and heart rate. All data were calculated from 5 or more cardiac cycles.

Hematoxylin and Eosin (HE) and Picric Sirius Red (PSR) staining

After being harvested, arrested and fixed in 10 % neutral formalin, hearts underwent paraffin embedding and subsequently cut to 5-μm-thick slices. To determine cell area, cardiac slices were exposed to HE staining according to standard protocols, with more than 100 cells per group calculated blindly [26]. To evaluate cardiac fibrosis, cardiac slices were stained at room temperature with PSR solution for 2 h and then incubated with 0.2 % phosphomolybdenum acid solution for an additional 2 min [27], [28], [29]. More than 60 fields per group were counted to evaluate collagen deposition.

Immunofluorescence staining

For paraffin-embedded heart samples, the slices underwent dewaxing, rehydration and antigen retrieval using 1 × citric acid solution, with the non-specific binding epitope blocked by 10 % goat serum. For cell coverslips, cells were fixed for 15 min with 4 % paraformaldehyde, permeabilized for 5 min with 1 % Triton X-100 and blocked for an additional 1 h with 10 % goat serum at room temperature. Next, anti-LUZP1 (1:50 dilution) and anti-α-actinin (1:100 dilution) were added to the slices or cell coverslips, and allowed for incubation overnight at 4 °C, which were then stained for an additional 1 h with Alexa Fluor 488 goat anti-rabbit or 568 goat anti-mouse secondary antibodies (1:200 dilution) at 37 °C. Cell nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI), and the images were recorded with an OLYMPUS DX51 fluorescence microscope (Tokyo, Japan) [10], [30].

Western blot

RIPA lysis buffer and a BCA Protein Assay Kit (#23227; Invitrogen, Carlsbad, CA, USA) were used for protein extraction and quantification. Next, SDS-PAGE was performed to separate these proteins, which were then transferred onto PVDF membranes [31], [32], [33]. After being blocked with 5 % non-fat milk, the membranes were incubated with the primary antibodies at 4 °C overnight, and then the secondary antibody at 37 °C for 1 h. The protein signals were identified with the ECL system on a Bio-Rad ChemiDoc™ XRS + machine. Protein levels were normalized to GAPDH or matched total proteins using the Bio-Rad Image Lab software (version 6.0).

Quantitative real-time PCR

Total RNA was prepared and converted to cDNA as we previously described, with a LightCycler® 480 detection system (Roche; Basel, Switzerland) employed to quantify mRNA levels [10], [19]. The primer sequences were as following: rat Anp, forward 5′-AAAGCAAACTGAGGGCTCTGCTCG-3′, reverse 5′-TTCGGTACCGGAAGCTGTTGCA-3′; rat Myh6, forward 5′-GCTCCAGGGGTGATGGACAA-3′, reverse 5′-GATTCGATACCTCTGCCGGA-3′; rat Myh7, forward 5′-TCTGGACAGCTCCCCATTCT-3′, reverse 5′-CAAGGCTAACCTGGAGAAGATG-3′; rat Gapdh, forward 5′-GACATGCCGCCTGGAGAAAC-3′, reverse 5′-AGCCCAGGATGCCCTTTAGT-3′; mouse Anp, forward 5′-ACCTGCTAGACCACCTGGAG-3′, reverse 5′-CCTTGGCTGTTATCTTCGGTACCGG-3′; mouse Myh6, forward 5′-GTCCAAGTTCCGCAAGGT-3′, reverse 5′-AGGGTCTGCTGGAGAGGTTA-3′; mouse Myh7, forward 5′-CCGAGTCCCAGGTCAACAA-3′, reverse 5′-CTTCACGGGCACCCTTGGA-3′; mouse Col-1, forward 5′-AGGCTTCAGTGGTTTGGATG-3′, reverse 5′-CACCAACAGCACCATCGTTA-3′; mouse Col-3, forward 5′-CCCAACCCAGAGATCCCATT-3′, reverse 5′-GAAGCACAGGAGCAGGTGTAGA-3′; mouse Gapdh, forward 5′-ACTCCACTCACGGCAAATTC-3′, reverse 5′-TCTCCATGGTGGTGAAGACA-3′.

Transcriptome analysis

Total RNA was extracted from the whole murine hearts, and RNA integrity was assessed by the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies). RNA-sequencing was performed on an Illumina HiSeq 2500 platform, and differential expression analysis was performed using the DESeq2 R package. Differentially expressed genes (DEGs) were defined as those with an adjusted p value < 0.05 and |fold change| ≥ 2. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was implemented using the clusterProfiler R package. Of note, the hearts contain a lot of different types of cells, not only cardiomyocytes; however, they are the major cell type.

Human heart samples

Hypertrophic cardiomyopathy (HCM) samples were harvested from the left ventricles of HCM patients, while the control samples were harvested from the left ventricles of the donors whose hearts were unsuitable for transplantation for non-cardiac reasons [16], [34]. Informed consent was obtained from each patient or legal guardian. All experimental procedures were approved by the Review Board of our hospital and also conformed to the principles in the Declaration of Helsinki.

Statistical analysis

Data were expressed as the mean ± standard deviation (SD) and analyzed using the SPSS software (version 22.0). Student′s t-test was performed to compare differences between two groups, and one-way analysis of variance (ANOVA) was conducted to compare differences among multiple groups. Statistical significance was set at p < 0.05.

Results

LUZP1 expression is elevated in hypertrophic hearts

To clarify the role of LUZP1 in cardiac hypertrophy, we first determined LUZP1 expression in the myocardium from sham- or TAC-operated mice. Using the Human Protein Atlas database, we recently found that LUZP1 was mainly expressed by human hearts and localized to the cardiomyocytes [35]. As illustrated in Fig. 1A, the protein levels of LUZP1 were progressively increased in the myocardium after TAC surgery. In line with the results from the public database, immunofluorescence staining also showed that LUZP1 was mainly localized to cardiomyocytes, and was upregulated in the myocardium 4 weeks post-TAC surgery (Fig. 1B). In addition, the protein level of LUZP1 was also dramatically elevated in human HCM hearts, compared with those from donors (Fig. 1C). To further validate LUZP1 upregulation in cardiomyocytes, we isolated NRCMs and stimulated its hypertrophic growth with PE in vitro. As shown in Fig. 1D, LUZP1 protein expression was elevated in PE-stimulated cardiomyocytes. Previous studies have shown that LUZP1 acted as a centrosomal and actin cytoskeleton-localizing protein, and played critical roles in the remodeling of actin cytoskeleton [12], [13], [14]. Therefore, we speculated the upregulation of LUZP1 in hypertrophic hearts might be associated with the remodeling of actin cytoskeleton during cardiac hypertrophy. ROCK pathway is essential for the remodeling of actin cytoskeleton, and therefore, we used two independent ROCK inhibitors to block actin cytoskeleton remodeling in vitro. As shown in Fig. 1E, we found that both of the two ROCK inhibitors dramatically decreased the protein levels of LUZP1 in PE-stimulated NRCMs. Collectively, we demonstrate that LUZP1 expression is elevated in hypertrophic hearts.Fig. 1 LUZP1 expression is elevated in hypertrophic hearts. (A) LUZP1 protein level in mouse hearts with or without TAC surgery (n = 6). (B) Immunofluorescence staining of LUZP1 and α-actinin in sham- or TAC-operated hearts (n = 6). (C) LUZP1 protein level in HCM or donor hearts (n = 6). (D) LUZP1 protein level in NRCMs stimulated with or without PE (n = 6). (E) LUZP1 protein level in PE-stimulated NRCMs with or without the treatment of ROCK inhibitors (n = 6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

LUZP1 inhibits PE-induced hypertrophic growth of cardiomyocytes in vitro

Next, we performed gain- and loss-of-function studies to investigate the role of LUZP1 in cardiac hypertrophy in vitro. NRCMs were firstly infected with shLuzp1 to silence LUZP1 in vitro (Fig. 2A). As illustrated in Fig. 2B, LUZP1 silence dramatically increased the mRNA levels of hypertrophic markers, including Anp and Myh7. In contrast, Myh6 mRNA level in PE-stimulated NRCMs was further decreased by LUZP1 silence (Fig. 2C). As expected, PE-induced enlargement of cell area was further amplified in Luzp1-silenced NRCMs (Fig. 2D). Meanwhile, we also overexpressed LUZP1 in NRCMs (Fig. 2E). As shown in Fig. 2F-G, LUZP1 overexpression dramatically reduced the mRNA levels of Anp, Myh7, but elevated Myh6 mRNA level in PE-treated NRCMs. Consistently, PE-induced hypertrophic growth of NRCMs was also suppressed by LUZP1 overexpression (Fig. 2H). Collectively, we demonstrate that LUZP1 inhibits PE-induced hypertrophic growth of cardiomyocytes in vitro.Fig. 2 LUZP1 inhibits PE-induced hypertrophic growth of cardiomyocytes in vitro. (A) LUZP1 protein in NRCMs with shRNA or shLuzp1 infection (n = 6). (B-C) Relative Anp, Myh6 and Myh7 mRNA levels (n = 6). (D) Cell area as determined by immunofluorescence staining of α-actinin (n = 6). (E) LUZP1 protein in NRCMs with AdRNA or AdLuzp1 infection (n = 6). (F-F) Relative Anp, Myh6 and Myh7 mRNA levels (n = 6). (H) Cell area as determined by immunofluorescence staining of α-actinin (n = 6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

LUZP1 deletion aggravates TAC-induced cardiac hypertrophy and dysfunction in mice

To further clarify the protective effects of LUZP1 against cardiac hypertrophy, we established a LUZP1 cKO mice (Fig. 3A). Notably, LUZP1 cKO mice displayed no evident abnormities in the heart at baseline (data not shown). Yet, TAC-induced elevation of cardiac mass was further increased in LUZP1 cKO mice, as determined by the increased heart weight/tibial length (HW/TL) (Fig. 3B). Meanwhile, pulmonary congestion, as determined by the increased lung weight (LW)/TL, was also aggravated by LUZP1 deletion in response to TAC surgery (Fig. 3C). Accordingly, histological analysis revealed a significantly increased cardiomyocyte size in LUZP1 cKO mice in response to TAC operation (Fig. 3D). Consistent with the phenotypic alterations, the mRNA level of Myh6 was reduced, while the mRNA levels of Anp and Myh7 were elevated by LUZP1 ablation in hypertrophic hearts (Fig. 3E-F). Compared with LUZP1 floxed mice, blood pressure (BP) and heart rate were not affected in LUZP1 cKO mice (Fig. 3G-H). Yet, TAC-induced cardiac dilation and dysfunction were dramatically exacerbated by LUZP1 deficiency, as evidenced by increased LVIDd, LVIDs and decreased FS (Fig. 3I-J). Fibrotic remodeling is the other key characteristic during hypertrophic growth, and accelerates heart failure [28], [29]. Interestingly, fibrotic area in TAC-operated mice was aggravated by LUZP1 deficiency, accompanied with increased mRNA levels of fibrotic markers, including Col-1 and Col-3 (Fig. 3K-L). Collectively, we demonstrate that LUZP1 deletion aggravates TAC-induced cardiac hypertrophy and dysfunction in mice.Fig. 3 LUZP1 deletion aggravates TAC-induced cardiac hypertrophy and dysfunction in mice. (A) LUZP1 protein level in the myocardium from LUZP1 cKO or Flox mice (n = 6). (B-C) Quantifications of HW/TL and LW/TL (n = 6). (D) Cell area as determined by HE staining (n = 6). (E-F) Relative Anp, Myh6 and Myh7 mRNA levels (n = 6). (G-H) BP and heart rate (n-6). (I-J) Echocardiographic parameters as determined by LVIDd, LVIDs and FS (n-6). (K) Fibrotic area as determined by PSR staining (n = 6). (L) Relative Col-1 and Col-3 mRNA levels (n = 6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

LUZP1 overexpression attenuates TAC-induced cardiac hypertrophy and dysfunction in mice

We then explored whether LUZP1 overexpression could prevent TAC-induced hypertrophic remodeling using LUZP1 cTG mice (Fig. 4A). As illustrated in Fig. 4B-C, TAC-induced elevations of HW/TL and LW/TL were reduced in LUZP1 cTG mice. HE staining revealed that TAC-induced cardiomyocyte hypertrophy was also inhibited by LUZP1 overexpression (Fig. 4D). Accordingly, Myh6 mRNA was increased, while Anp and Myh7 mRNA were decreased in LUZP1 cTG hearts after TAC surgery (Fig. 4E-F). No alterations of BP and heart rate were found between LUZP1 cTG and NTG mice pre- or post-TAC operation (Fig. 4G-H). Consistent with the molecular and morphological results, functional studies revealed that LUZP1 overexpression dramatically ameliorated TAC-induced cardiac dysfunction (Fig. 4I-J). Meanwhile, TAC-induced elevations of Col-1 and Col-3 mRNA were also prevented in LUZP1 cTG mice (Fig. 4K-L). Collectively, we demonstrate that LUZP1 overexpression attenuates TAC-induced cardiac hypertrophy and dysfunction in mice.Fig. 4 LUZP1 overexpression attenuates TAC-induced cardiac hypertrophy and dysfunction in mice. (A) LUZP1 protein level in the myocardium from LUZP1 cTG or NTG mice (n = 6). (B-C) Quantifications of HW/TL and LW/TL (n = 6). (D) Cell area as determined by HE staining (n = 6). (E-F) Relative Anp, Myh6 and Myh7 mRNA levels (n = 6). (G-H) BP and heart rate (n-6). (I-J) Echocardiographic parameters as determined by LVIDd, LVIDs and FS (n-6). (K) Fibrotic area as determined by PSR staining (n = 6). (L) Relative Col-1 and Col-3 mRNA levels (n = 6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

LUZP1 suppresses Stat3 pathway to prevent TAC-induced cardiac hypertrophy in mice

We then performed transcriptome analysis using TAC-operated LUZP1 cKO or Flox hearts to notify the underlying molecular basis. The Heatmap demonstrated that LUZP1 deletion dramatically elevated the expression of hypertrophic and fibrotic genes (Fig. 5A). KEGG analysis revealed that Jak-Stat pathway was the most significantly altered pathways in LUZP1 cKO hearts (Fig. 5B). Consistent with the transcriptome results, we found that LUZP1 ablation elevated, while LUZP1 overexpression reduced Stat3 phosphorylation in TAC-operated hearts (Fig. 5C-D). To determine the necessity of Stat3 pathway in LUZP1 deficiency-mediated hypertrophy-promoting effects in vivo, we generated Luzp1 and Stat3 DKO mice (Fig. 5E). As shown in Fig. 5F-G, Stat3 deficiency decreased TAC-induced elevations of HW/TL and LW/TL in LUZP1 cKO mice. Meanwhile, the increased cardiac hypertrophy and fibrosis in LUZP1 cKO mice after TAC surgery were negated by Stat3 ablation, as evidenced by the decreased cell area, fibrotic area and mRNA levels of fibrotic markers (Fig. 5H-J). As expected, LUZP1 deficiency failed to impair cardiac function in Stat3 cKO mice after TAC operation (Fig. 5K-L). Collectively, we demonstrate that LUZP1 suppresses Stat3 pathway to prevent TAC-induced cardiac hypertrophy in mice.Fig. 5 LUZP1 suppresses Stat3 pathway to prevent TAC-induced cardiac hypertrophy in mice. (A) Heatmaps showing the significantly altered genes related to cardiac hypertrophy (n = 3). (B) The top ten altered KEGG pathways by LUZP1 (n = 3). (C-D) Protein levels of p-Stat3 and t-Stat3 in LUZP1 cKO, LUZP1 cTG or matched control hearts after TAC surgery (n = 6). (E) Protein levels of LUZP1 and t-Stat3 in the myocardium from mice with different genotypes (n = 6). (F-G) Quantifications of HW/TL and LW/TL (n = 6). (H) Cell area as determined by HE staining (n = 6). (I) Fibrotic area as determined by PSR staining (n = 6). (J) Relative Col-1 and Col-3 mRNA levels (n = 6). (K-L) Echocardiographic parameters as determined by LVIDd, LVIDs and FS (n-6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

LUZP1 deficiency facilitates PE-induced hypertrophic growth of cardiomyocytes through activating Stat3

To validate the involvement of Stat3 in vitro, endogenous Stat3 expression was silenced using siStat3 before shLuzp1 infection as we recently described [10]. As shown in Fig. 6A, LUZP1 silence further increased cell area in PE-stimulated NRCMs, which was blocked in those with siStat3 transfection. Meanwhile, the decreased Myh6, and the increased Anp and Myh7 mRNA levels in Luzp1-deficient cells were reversed by Stat3 silence (Fig. 6B-C). We also inhibited Stat3 activity using Stattic to further assess whether the role of Stat3 depends on its expression or the activity [23]. As shown in Fig. 6D-F, Luzp1 deficiency-mediated hypertrophy-promoting effects were attenuated by Stat3 inhibition. Collectively, we demonstrate that LUZP1 deficiency facilitates PE-induced hypertrophic growth of cardiomyocytes through activating Stat3.Fig. 6 LUZP1 deficiency facilitates PE-induced hypertrophic growth of cardiomyocytes through activating Stat3. (A) Cell area as determined by immunofluorescence staining of α-actinin (n = 6). (B-C) Relative Anp, Myh6 and Myh7 mRNA levels (n = 6). (D) Cell area as determined by immunofluorescence staining of α-actinin (n = 6). (E-F) Relative Anp, Myh6 and Myh7 mRNA levels (n = 6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

LUZP1 blocks Stat3 pathway through upregulating SHP1 in vivo and in vitro

Finally, we investigated the precise molecular basis through which LUZP1 inhibited Stat3 pathway. Stat3 is inactivated by various protein tyrosine phosphatases, such as SHP1, SHP2 and PTP1B [36]. As shown in Fig. 7A, SHP1 silence dramatically blocked the inhibitory effects of LUZP1 on Stat3 phosphorylation in PE-stimulated NRCMs. Yet, neither SHP2 knockdown nor PTP1B silence prevented LUZP1 overexpression-mediated Stat3 de-phosphorylation in PE-stimulated NRCMs (Fig. 7B). Meanwhile, we found that SHP1 transcripts were downregulated in LUZP1 cKO hearts after TAC operation using transcriptome data; however, no alterations of SHP2 and PTP1B were observed (Fig. 7C). Consistently, SHP1 protein level was also reduced in LUZP1 cKO hearts, but elevated in LUZP1 cTG hearts in response to TAC operation (Fig. 7D). These findings implied that SHP1 might be required for Stat3 inhibition by LUZP1. To further clarify the necessity of SHP1, NRCMs were pre-transfected with siShp1 to silence endogenous SHP1 before LUZP1 overexpression. As shown in Fig. 7E-H, LUZP1 overexpression-mediated anti-hypertrophic effects were blocked by SHP1 silence, as evidenced by the elevated cell area, Anp, Myh7 mRNA levels and decreased Myh6 mRNA level. In accordance with the in vitro results, we found that the decreased HW/TL and LW/TL in LUZP1 cTG mice after TAC operation were increased by SHP1 silence (Fig. 8A-B). LUZP1-mediated anti-hypertrophic and anti-fibrotic effects were also blocked by shShp1 injection (Fig. 8C-E). As expected, LUZP1 overexpression failed to preserve cardiac function in Shp1-silenced mice after TAC operation (Fig. 8F-H). Collectively, we demonstrate that LUZP1 blocks Stat3 pathway through upregulating SHP1 in vivo and in vitro.Fig. 7 LUZP1 blocks Stat3 pathway through upregulating SHP1 in vitro. (A) Protein levels of p-Stat3 and t-Stat3 in AdLuzp1-infected NRCMs with or without siShp1 transfection (n = 6). (B) Protein levels of p-Stat3 and t-Stat3 in AdLuzp1-infected NRCMs with siShp2 or siPtp1b transfection (n = 6). (C) Heatmaps showing SHP1, SHP2 and PTP1B gene expression (n = 3). (D) SHP1 protein level in LUZP1 cKO, LUZP1 cTG or matched control hearts after TAC surgery (n = 6). (E) Cell area as determined by immunofluorescence staining of α-actinin (n = 6). (F-G) Relative Anp, Myh6 and Myh7 mRNA levels (n = 6). (H) Relative Shp1 mRNA level in NRCMs with siRNA or siShp1 transfection (n = 6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

Fig. 8 LUZP1 blocks Stat3 pathway through upregulating SHP1 in vivo. (A-B) Quantifications of HW/TL and LW/TL (n = 6). (C) Cell area as determined by HE staining (n = 6). (D) Relative Col-1 and Col-3 mRNA levels (n = 6). (E) Fibrotic area as determined by PSR staining (n = 6). (F-G) Echocardiographic parameters as determined by LVIDd, LVIDs and FS (n-6). (H) Relative Shp1 mRNA level in the myocardium with shRNA or shShp1 infection (n = 6). All results were presented as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

Discussion

Cardiac hypertrophy is an indispensable contributor of heart failure, and the underlying mechanisms remain unclear. The present study determines a previously unrecognized role of LUZP1 in pressure overload-induced cardiac hypertrophy and dysfunction. First, during the progression of cardiac hypertrophy, LUZP1 level is progressively increased in hypertrophic hearts. Second, using gain- and loss-of-function approaches, we reveal that cardiac-specific LUZP1 deficiency aggravates, while cardiac-specific LUZP1 overexpression attenuates pressure overload-induced cardiac hypertrophy and dysfunction in vivo and in vitro. Third, the transcriptome data identify Stat3 pathway as a key downstream target of LUZP1 in regulating pathological cardiac hypertrophy. Cardiac-specific Stat3 knockout abolishes the pro-hypertrophic effects in LUZP1 cKO mice after TAC surgery. Finally, LUZP1 elevates SHP1 expression to inactivate Stat3 pathway, and SHP1 silence blocks the anti-hypertrophic effects of LUZP1 in mice and NRCMs. Overall, we demonstrate that LUZP1 attenuates pressure overload-induced cardiac hypertrophy through inhibiting Stat3 signaling.

Stat3 is a key molecular node of the complex signaling network that transmits extracellular stimuli and cytosolic signals to the nucleus upon different stimuli. Emerging studies have found that Stat3 is constitutively active in hypertrophic hearts and predicts poor outcomes in clinical patients [37], [38]. Under physiological conditions, Stat3 is sequestered in the cytoplasm as inactive monomers. Canonical Stat3 activation involves the phosphorylation at Tyr705 by receptor-associated Janus kinases in response to stimulations with pro-inflammatory cytokines and growth factors, which allows Stat3 homodimerization or heterodimerization, translocates to the nucleus and directly regulates gene expression as a transcription factor [39]. Zhuang et al. recently found that Stat3 activation and nuclear translocation impair mitochondrial bioenergetics, facilitate expressions of fibrotic genes and drive cardiac hypertrophy [8]. In addition, phosphorylated Stat3 also interacts with Smad3, and subsequently synergistically provokes fibrotic remodeling of the heart [40]. Consistently, we recently found that Stat3 activation promoted, while Stat3 inactivation prevented hypertrophic growth and cardiac dysfunction in TAC-operated mice [10]. To overcome the constitutive activation of Stat3, multiple protein tyrosine phosphatases (SHP1, SHP2 and PTP1B, etc) are elevated to balance the homeostasis of Stat3 pathway. Zhang et al. previously demonstrated that Stat3 activation was dramatically suppressed in Noonan syndrome patients bearing SHP2-activating mutations [41]. Deletion of SHP2 led to dilated cardiomyopathy, heart failure and premature mortality [42]. In addition, PTP1B deficiency also caused Stat3 over-activation, thereby preventing hyperinsulinemia and endotoxemia in high fat diet and lipopolysaccharide-treated mice [43]. Interestingly, we found that neither SHP2 knockdown nor PTP1B silence prevented Stat3 de-phosphorylation in AdLuzp1-infected NRCMs after PE stimulation in our study. In contrast, SHP1 silence dramatically blocked the inhibitory effects of LUZP1 on Stat3 phosphorylation in PE-stimulated NRCMs. Consistently, transcriptome data also implied that SHP1 mRNA, but not SHP2 or PTP1B, was decreased in LUZP1 cKO hearts after TAC surgery. These findings suggest that LUZP1 elevates SHP1 to inactivate Stat3 pathway, thereby preventing pressure overload-induced cardiac hypertrophy and dysfunction. However, the specific mechanism mediating LUZP1 upregulation in hypertrophic hearts remains unclear. In our study, we found that the protein level of LUZP1 was dramatically elevated in hypertrophic hearts, and LUZP1 overexpression attenuated pressure overload-induced cardiac hypertrophy. Based on the results, we reasonably concluded that LUZP1 was upregulated to exert the compensatory cardioprotection during hypertrophic stimuli. Accordingly, we recently also detected an upregulated LUZP1 expression in doxorubicin-treated hearts, and defined it as a cardioprotectant [35]. Previous studies have shown that LUZP1 acted as a centrosomal and actin cytoskeleton-localizing protein, and played critical roles in the remodeling of actin cytoskeleton [12], [13], [14]. Therefore, we speculated the upregulation of LUZP1 in hypertrophic hearts might be associated with the remodeling of actin cytoskeleton during cardiac hypertrophy. As expected, our findings revealed that blocking actin cytoskeleton remodeling with two ROCK inhibitors dramatically reduced the elevation of LUZP1 protein in PE-stimulated NRCMs.

Overall, we demonstrate that LUZP1 attenuates pressure overload-induced cardiac hypertrophy through inhibiting Stat3 signaling, and targeting LUZP1 may develop novel approaches to treat pathological cardiac hypertrophy.

Compliance with Ethics Requirements

All experiments involving animals were approved by the Animal Care and Use Committee of our hospital (approval no. 20200708), and were also complied with the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health.

Availability of data and materials

All data that support the findings in this study are available from the corresponding author upon reasonable request.

Funding

This study was supported by a grant from the 10.13039/501100001809 National Natural Science Foundation of China (No. 82272233 , 82070425 , 82272185 , 82370290, 82100368 ), 10.13039/501100003819 Natural Science Foundation of Hubei Province of China (No. 2021CFA011 ), Program of Excellent Doctoral (Postdoctoral) Study of Zhongnan Hospital of Wuhan University (No. ZNYB2022014 ), Youth Interdisciplinary Special Fund of Zhongnan Hospital of Wuhan University (No. ZNQNJC2022010 ), Science and Technology Innovation Cultivation Fund of Zhongnan Hospital (No. CXPY2022095 ), Medical Science and Technology Innovation Platform Support Project of Zhongnan Hospital of Wuhan University (No. PTXM2023016 ).

CRediT authorship contribution statement

Di Fan: Conceptualization, Data curation, Methodology, Writing – original draft, Writing – review & editing, Funding acquisition. Wan-li Jiang: Data curation, Methodology, Writing – original draft, Writing – review & editing. Zhi-li Jin: Data curation, Methodology. Jian-lei Cao: Data curation, Methodology. Yi Li: . Tao He: Data curation, Methodology. Wei Zhang: Data curation, Methodology. Li Peng: Data curation, Methodology. Hui-xia Liu: Data curation, Methodology. Xiao-yan Wu: . Ming Chen: Formal analysis, Investigation, Software. Yong-zhen Fan: Formal analysis, Investigation, Software. Bo He: Data curation, Methodology, Formal analysis, Investigation, Software. Wen-xi Yu: Data curation, Methodology. Hai-rong Wang: Writing – original draft. Xiao-rong Hu: Conceptualization, Writing – original draft, Writing – review & editing. Zhi-bing Lu: Conceptualization, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

None.
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