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