Elsevier

Matrix Biology

Volume 110, June 2022, Pages 40-59
Matrix Biology

Asporin, an extracellular matrix protein, is a beneficial regulator of cardiac remodeling

https://doi.org/10.1016/j.matbio.2022.04.005Get rights and content

Highlights

  • ASPN is the top differentially expressed gene in ischemic cardiomyopathy.

  • Aspn expression is induced in response to cardiac pressure overload or ischemia-reperfusion.

  • Genetic deletion of ASPN led to enhanced fibrosis and decreased cardiac function in mice after pressure overload or ischemia-reperfusion.

  • ASPN regulates mitochondrial bioenergetics and protects cardiomyocytes from hypoxia-reoxygenation mediated cell death.

  • ASPN-derived peptide prevented pressure-overload induced fibrosis and preserved heart function.

  • The peptide also reduced infarct size after Ischemia-reperfusion injury in mice.

Abstract

Heart failure is accompanied by adverse cardiac remodeling involving extracellular matrix (ECM). Cardiac ECM acts as a major reservoir for many proteins including growth factors, cytokines, collagens, and proteoglycans. Activated fibroblasts during cardiac injury can alter the composition and activity of these ECM proteins. Through unbiased analysis of a microarray dataset of human heart tissue comparing normal hearts (n = 135) to hearts with ischemic cardiomyopathy (n = 94), we identified Asporin (ASPN) as the top differentially regulated gene (DEG) in ischemic cardiomyopathy; its gene-ontology terms relate closely to fibrosis and cell death. ASPN is a Class I small leucine repeat protein member implicated in cancer, osteoarthritis, and periodontal ligament mineralization. However, its role in cardiac remodeling is still unknown. Here, we initially confirmed our big dataset analysis through cells, mice, and clinical atrial biopsy samples to demonstrate increased Aspn expression after pressure overload or cardiac ischemia/reperfusion injury. We tested the hypothesis that Aspn, being a TGFβ1 inhibitor, can attenuate fibrosis in mouse models of cardiac injury. We found that Aspn is released by cardiac fibroblasts and attenuates TGFβ signaling. Moreover, Aspn−/− mice displayed increased fibrosis and decreased cardiac function after pressure overload by transverse aortic constriction (TAC) in mice. In addition, Aspn protected cardiomyocytes from hypoxia/reoxygenation-induced cell death and regulated mitochondrial bioenergetics in cardiomyocytes. Increased infarct size after ischemia/reperfusion injury in Aspn−/− mice confirmed Aspn's contribution to cardiomyocyte viability. Echocardiography revealed greater reduction in left ventricular systolic function post-I/R in the Aspn−/− animals compared to wild type. Furthermore, we developed an ASPN-mimic peptide using molecular modeling and docking which when administered to mice prevented TAC-induced fibrosis and preserved heart function. The peptide also reduced infarct size after I/R in mice, demonstrating the translational potential of ASPN-based therapy. Thus, we establish the role of ASPN as a critical ECM molecule that regulates cardiac remodeling to preserve heart function.

Graphical abstract: model representing the protective effects of Asporin in heart

Introduction

Heart Failure (HF) is the one of the leading causes of morbidity and mortality worldwide, generating significant health and economic burdens. Significant advances to limit ischemic injury by thrombolytic therapy or primary percutaneous coronary intervention (PCI) have decreased mortality from myocardial infarction, but long-term consequences of reperfusion injury and pathological cardiac remodeling contribute significantly to mortality [1]. Dynamic changes in extracellular matrix (ECM) regulate cellular responses mediating cardiac remodeling [2]. Due to increased activity of transforming growth factor (TGF)β1 during pathological remodeling, activated fibroblasts deposit fibrillar collagens and other ECM proteins [3]. Changes in the composition of ECM play crucial roles in providing structural integrity to heart and altering signaling pathways in numerous cell types including cardiomyocytes and fibroblasts [4]. However, adverse cardiac remodeling due to excessive fibrosis increases ventricular stiffness leading to impaired cardiac function and increased risk of mortality.

Despite great advances in the acute management of myocardial I/R injury, strategies to inhibit or reverse adverse cardiac remodeling remains elusive. ECM components are integral to the remodeling process and can play protective and deleterious effects [5]. Among these, small leucine rich proteoglycan (SLRP) decorin, lumican, biglycan, and fibromodulin play crucial roles in heart valve development and cardiac remodeling [6], [7], [8], [9], [10]. Waehre et al. [11] showed that decreased presence of SLRPs in heart results in loosely packed ECM, leading to left ventricular dilation and increased mortality after pressure overload. In the current study, we found Asporin (ASPN), an ECM protein, is the top differentially expressed genes (DEGs) in ischemic cardiomyopathy compared to normal controls. ASPN is a member of SLRP class I family, which acts as natural TGFβ inhibitor by regulating the latter's interaction with its receptor [12]. ASPN, also known as periodontal ligament associated protein-1 (PLAP-1), was first identified in human cartilage where its overexpression was found to be associated with osteoarthritis [13]. ASPN directly binds to type I collagen through its LRR domain [13] and can play a crucial part in collagen fibrillogenesis [13]. Inhibition of ASPN stimulates TGFβ-induced smad2/3 signaling [12]. ASPN contains a pro-peptide sequence, D-repeat region (varies from 9 to 20 aa), 10 tandem leucine-rich repeats (LRR), and cysteine residues on both its N- and C-termini [14]. ASPN has been implicated as an oncoprotein in prostate cancer [15], pancreatic cancer [16] and gastric cancer [17, 18], but as a tumor suppressor in breast cancer [19, 20]. It is also involved in metastatic progression by regulating mesenchymal stromal cell differentiation [21]. However, the role ASPN in cardiac remodeling has not been studied yet.

In the present study, we explored the requirement of Aspn in pathological cardiac remodeling in different preclinical models. TGFβ1 treatment activated fibroblasts and increased the expression and release of Aspn into ECM space, which in turn, can inhibit TGFβ-induced SMAD2/3 signaling. Using paired clinical atrial biopsies obtained before and after cardiopulmonary bypass (CPB), we found increased ASPN expression in human heart tissue as well after surgery; the role of which is further determined using different animal models of cardiac injury. Genetic deficiency of Aspn in mice resulted in exacerbated fibrosis in pressure overload model and cardiomyocyte cell death in ischemia-reperfusion injury model leading to decline in diastolic and systolic function, respectively. Importantly, addition of exogenous ASPN to cultured H9C2 cardiomyocytes reduced cell death induced by hypoxia/reoxygenation. Further, using molecular modeling and docking studies, we designed an ASPN-mimic peptide, which we documented to reduce cardiac fibrosis and cardiomyocyte cell death in vivo. This, in turn, attenuated the decline in heart function after cardiac insults. Thus, our study reveals the important role of ASPN in pathological remodeling and myocardial preservation and may be an attractive candidate for treatment of heart failure.

Section snippets

ASPN is one of the top differentially regulated genes in ischemic cardiomyopathy

In an unbiased approach, we first sought to explore the differential gene expression from ischemic cardiomyopathy clinical samples and compared to control donor hearts. We utilized publicly available microarray dataset of Magnet consortiums obtained from Gene Expression Omnibus (GSE57345). Analysis using R language script pointed to ASPN as one of the top upregulated genes (log2FC=1.77; p = 7.43E-30) in ischemic cardiomyopathy samples (n = 94) compared to non-failing donor heart controls (n

Discussion

In the last 2 decades or so, many advances have been made to better understand the regulation of adverse cardiac remodeling in HF. Controlled scarring is a normal repair process of cardiac remodeling; however, excessive fibrosis is deleterious [26]. Stress signals can also induce compensatory mechanisms to inhibit adverse cardiac signaling [27]. Here, we report the comprehensive role of an ECM protein, Aspn, in cardiac remodeling for the first time as an important compensatory signal. We

Cell culture

H9c2 and 3T3 cells were obtained from ATCC and maintained in growth media (DMEM: 10 mM glucose, 10% FBS, antibiotic and antimycotic, pH 7.4). For H9c2 cells, differentiation was initiated by switching to differentiation media (DMEM: 10 mM glucose, 1% FBS, antibiotic and antimycotic, 1 nM retinoic acid, pH 7.4) in the manner described previously [48]. Differentiation was sustained for 5 days before starting experiments.

Animal ethics

All animal procedures followed the National Institutes of Health standards

Funding

This work was supported by the NHLBI HL144509 (RAG), NHLBI HL155553 (HP) and institutional funding (RAG).

Acknowledgments

We would like to acknowledge the Cedars-Sinai Biobank and Translational research core for histology services. We thank Ian Williamson for help in getting Aspn knock out mice and excellent lab management. We also would like to acknowledge Dr. Tanuj Sharma (Yonsei University, South Korea) for in-depth discussion of molecular docking models.

Author contributions

Conceptualization: HP, RAG, AS; Supervision: HP, RAG, AS, RM; Data Collection and Analysis: HP, HC, AS, RT, JG, YS, SS, JS, AMA, DS, MK; Data Interpretation: HP, HC, AS, RT, JG, RMM Jr, RAG, MK; Writing: HP, RAG, AS, RMM Jr; Funding Acquisition: RAG, HP. All authors intellectually contributed and provided approval for publication.

References (76)

  • X. Jiang et al.

    Knockdown of asporin affects transforming growth factor-beta1-induced matrix synthesis in human intervertebral annulus cells

    J. Orthop. Translat.

    (2016)
  • S.M. Weis et al.

    A role for decorin in the remodeling of myocardial infarction

    Matrix Biol.

    (2005)
  • S. Deckx et al.

    Osteoglycin prevents the development of age-related diastolic dysfunction during pressure overload by reducing cardiac fibrosis and inflammation

    Matrix Biol.

    (2018)
  • N. Beetz et al.

    Ablation of biglycan attenuates cardiac hypertrophy and fibrosis after left ventricular pressure overload

    J. Mol. Cell. Cardiol.

    (2016)
  • B. Jaishy et al.

    Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity

    J. Lipid Res.

    (2015)
  • Y. Liu et al.

    RNA-Seq identifies novel myocardial gene expression signatures of heart failure

    Genomics

    (2015)
  • N.M. Dhutia et al.

    A new automated system to identify a consistent sampling position to make tissue Doppler and transmitral Doppler measurements of E, E' and E/E'

    Int. J. Cardiol.

    (2012)
  • P.G. Scott et al.

    Crystal structure of the biglycan dimer and evidence that dimerization is essential for folding and stability of class I small leucine-rich repeat proteoglycans

    J. Biol. Chem.

    (2006)
  • S.F. Altschul et al.

    Basic local alignment search tool

    J. Mol. Biol.

    (1990)
  • C. Ramakrishnan et al.

    Stereochemical criteria for polypeptide and protein chain conformations. II. Allowed conformations for a pair of peptide units

    Biophys. J.

    (1965)
  • D.J. Hausenloy et al.

    Myocardial ischemia-reperfusion injury: a neglected therapeutic target

    J. Clin. Invest.

    (2013)
  • N.G. Frangogiannis

    The extracellular matrix in myocardial injury, repair, and remodeling

    J. Clin. Invest.

    (2017)
  • D. Fan et al.

    Matrix as an interstitial transport system

    Circ. Res.

    (2014)
  • L.E. Dupuis et al.

    Small leucine-rich proteoglycans exhibit unique spatiotemporal expression profiles during cardiac valve development

    Dev. Dyn.

    (2014)
  • K. Andenaes et al.

    The extracellular matrix proteoglycan fibromodulin is upregulated in clinical and experimental heart failure and affects cardiac remodeling

    PLoS ONE

    (2018)
  • D. Westermann et al.

    Biglycan is required for adaptive remodeling after myocardial infarction

    Circulation

    (2008)
  • A. Waehre et al.

    Lack of chemokine signaling through CXCR5 causes increased mortality, ventricular dilatation and deranged matrix during cardiac pressure overload

    PLoS ONE

    (2011)
  • A. Rochette et al.

    Asporin is a stromally expressed marker associated with prostate cancer progression

    Br. J. Cancer

    (2017)
  • Q. Ding et al.

    Asporin participates in gastric cancer cell growth and migration by influencing EGF receptor signaling

    Oncol. Rep.

    (2015)
  • R. Satoyoshi et al.

    Asporin activates coordinated invasion of scirrhous gastric cancer and cancer-associated fibroblasts

    Oncogene

    (2015)
  • B. Castellana et al.

    ASPN and GJB2 Are Implicated in the Mechanisms of Invasion of Ductal Breast Carcinomas

    J. Cancer

    (2012)
  • P. Maris et al.

    Asporin Is a Fibroblast-Derived TGF-beta1 Inhibitor and a Tumor Suppressor Associated with Good Prognosis in Breast Cancer

    PLoS Med.

    (2015)
  • R.M. Hughes et al.

    Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression

    Cancer Res.

    (2019)
  • A.V. Kuznetsov et al.

    The Role of Mitochondria in the Mechanisms of Cardiac Ischemia-Reperfusion Injury

    Antioxidants (Basel)

    (2019)
  • I. Kou et al.

    Binding characteristics of the osteoarthritis-associated protein asporin

    J. Bone Miner. Metab.

    (2010)
  • C. Dominguez et al.

    HADDOCK: a protein-protein docking approach based on biochemical or biophysical information

    J. Am. Chem. Soc.

    (2003)
  • Y. Liu et al.

    Editorial: cardiac Hypertrophy: from Compensation to Decompensation and Pharmacological Interventions

    Front. Pharmacol.

    (2021)
  • H.B. Wang et al.

    Identification of differentially expressed genes and preliminary validations in cardiac pathological remodeling induced by transverse aortic constriction

    Int. J. Mol. Med.

    (2019)
  • Cited by (16)

    • ASPORIN: A root of the matter in tumors and their host environment

      2024, Biochimica et Biophysica Acta - Reviews on Cancer
    • Fibroblasts and immune cells: at the crossroad of organ inflammation and fibrosis

      2024, American Journal of Physiology - Heart and Circulatory Physiology
    View all citing articles on Scopus
    1

    Equal 1st author.

    2

    Equal 2nd author.

    View full text