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KLK11 Promotes the Activation of Mtor and Protein Synthesis To Wang et al. BMC Cardiovasc Disord (2021) 21:266 https://doi.org/10.1186/s12872-021-02053-y RESEARCH ARTICLE Open Access KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy Yi Wang1, Hongjuan Liao2, Yueheng Wang1* , Jinlin Zhou1, Feng Wang1, Yingxin Xie1, Kun Zhao3 and Weinian Gao2 Abstract Background: Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods: Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quan- titative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiog- raphy. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results: The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hyper- trophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the efects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regula- tion of cardiomyocyte hypertrophy. Conclusions: Our fndings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis. Keywords: KLK11, Cardiac hypertrophy, Protein synthesis, MTOR, Akt Background During the last decades, cardiovascular diseases have *Correspondence: [email protected]; [email protected] become the leading cause of death worldwide [1]. Te 1 Department of Ultrasound, The Second Hospital of Hebei Medical morbidity and mortality of cardiac diseases and heart University, Shijiazhuang, China Full list of author information is available at the end of the article failure keep growing [2]. Cardiac hypertrophy is one of © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea- tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. BMC Cardiovasc Disord (2021) 21:266 Page 2 of 12 the primary mechanisms that participate in the devel- including cancer biology. For instance, KLK11 suppresses opment of cardiac dysfunction and heart failure [3]. esophageal squamous cell carcinoma by inhibiting cel- Cardiomyocytes within the heart tissues are terminally lular proliferation via inhibition of the Wnt/β-catenin diferentiated cells unable to divide to support the car- signaling pathway [12]. Knockdown of KLK11 reversed diac injury. Tese cardiomyocytes undergo hypertrophic oxaliplatin resistance by inhibiting proliferation and acti- growth to support the increase in demand in response to vating apoptosis [13]. KLK11 also participated in colo- cardiac injury [4]. As thus, cardiac hypertrophy has been rectal adenocarcinoma [14, 15]. However, the roles of considered a promising target for the treatment of heart KLK11 in cardiovascular diseases, such as cardiac hyper- failure [3]. However, the mechanisms underlying cardiac trophy, remain to be explored. In the present work, we hypertrophy remain not fully understood. aimed to investigate the potential functions of KLK11 in Protein synthesis is one of the critical features of car- cardiac hypertrophy and the underlying mechanisms. diomyocyte hypertrophy [5]. In cardiomyocytes, pro- tein synthesis is controlled by the mammalian target of Methods rapamycin (mTOR) signaling pathway [6]. mTOR phos- Human samples phorylated the ribosomal protein p70 S6K1 and 4EBP1 Five control heart samples and fve hypertrophic heart to facilitate de novo protein synthesis [7]. Within cardio- samples were collected at Te Second Hospital of Hebei myocytes, mTOR can be regulated by various pathways. Medical University from May 2016 to Jan 2019. Te left For instance, AMP-activated protein kinase (AMPK) ventricle heart tissues were frozen in liquid nitrogen and represses mTOR and protein synthesis via regulating stored at − 80 °C within two hours of tissue harvesting. the phosphorylation of the TSC1/TSC2 complex, the Each of the patients or control donors signed the writ- upstream inhibitor of mTOR [8]. Besides, our current ten form of consent. Te study was approved by the Eth- work demonstrated that Carboxypeptidase A4 promotes ics Committee of Clinical Research of Hebei Medical mTOR signaling during cardiomyocyte hypertrophy University. Te information of the patients is shown in through activating PI3K-AKT signaling [9]. Furthermore, Table 1. we demonstrated that 5-hydroxytryptamine receptor 2A (HTR2A) fosters the development of cardiac hypertro- Mouse model of cardiac hypertrophy phy by activating PI3K-PDK1-AKT-mTOR signaling [10]. C57BL/6 mice were purchased from Charles River Labo- Repression of mTOR with rapamycin can inhibit protein ratories (Beijing). 8–12 weeks old male C57BL/6 mice synthesis and repress cardiac hypertrophy induced by were subjected to sham or transverse aortic constriction diferent pathological stress [7]. (TAC) surgery. Four weeks later, cardiac function (frac- Kallikreins (KLK) are a subgroup of serine proteases tion shortening and ejection fraction) was analyzed by that have many physiological functions. Accumulating echocardiography based on a protocol described previ- evidence suggests that many kallikreins are implicated in ously [16]. Double-bind was applied for the echocardi- carcinogenesis, and some have potential as novel markers ography analysis. Mice were sacrifced by an overdose of for cancer and other diseases [11]. KLK11 is a member isofurane (5% isofurane with 2 l min−1 oxygen) anesthe- of the human kallikrein gene family. KLK11 has signif- sia until lack of respiration for 5 min to ensure euthana- cant roles in physiological and pathological processes, sia [17, 18], followed by heart dislocation. Heart weight, Table 1 Patient information in this study Patient Age Gender Diagnosis β-adrenergic blocker ACE inhibitor Control-1 54 Male Ventricular septal defect − − Control-2 60 Female Mitral valve defect − − Control-3 47 Female Ventricular septal defect − − Control-4 61 Male Ventricular septal defect − − Control-5 49 Male Mitral valve defect − − Hypertrophy-1 58 Female Hypertrophic obstructive cardiomyopathy + − Hypertrophy-2 62 Male Hypertrophic obstructive cardiomyopathy + + Hypertrophy-3 65 Male Hypertrophic obstructive cardiomyopathy − + Hypertrophy-4 54 Female Hypertrophic obstructive cardiomyopathy − + Hypertrophy-5 56 Male Hypertrophic obstructive cardiomyopathy + + Wang et al. BMC Cardiovasc Disord (2021) 21:266 Page 3 of 12 cardiomyocyte size, and gene expression were also ana- Te knockdown efciency was confrmed by quantitative lyzed. Te animal study was approved by the Ethics Com- real-time PCR. mittee of Animal Research of Hebei Medical University. Histological analysis Isolation and culture of neonatal mouse cardiomyocytes For histological analysis, the heart tissues were fxed in Mouse hearts were subjected to cardiomyocyte isolation 4% paraformaldehyde (Solarbio) and then embedded in based on a protocol described previously [19, 20]. Te parafn overnight. Te heart tissues were cut into 5-μm mouse cardiomyocytes were cultured in DMEM (Gibco, slices, and hematoxylin & eosin (H&E) staining was per- #C11965500BT) supplemented with 10% FBS (Gibco, formed to analyze the histological change with the Hema- #A3840001). BrdU (Sigma, #B5002) was used to repress toxylin–Eosin/H&E
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