Original article
Absence of thrombospondin-2 increases cardiomyocyte damage and matrix disruption in doxorubicin-induced cardiomyopathy

https://doi.org/10.1016/j.yjmcc.2011.05.010Get rights and content

Abstract

Clinical use of the antineoplastic agent doxorubicin (DOX) is limited by its cardiomyocyte toxicity. Attempts to decrease cardiomyocyte injury showed promising results in vitro, but failed to reduce the adverse effects of DOX in vivo, suggesting that other mechanisms contribute to its cardiotoxicity as well. Evidence that DOX also induces cardiac injury by compromising extracellular matrix integrity is lacking. The matricellular protein thrombospondin-2 (TSP-2) is known for its matrix-preserving function, and for modulating cellular function. Here, we investigated whether TSP-2 modulates the process of doxorubicin-induced cardiomyopathy (DOX-CMP). TSP-2-knockout (TSP-2-KO) and wild-type (WT) mice were treated with DOX (2 mg/kg/week) for 12 weeks to induce DOX-CMP. Mortality was significantly increased in TSP-2-KO compared to WT mice. Surviving DOX-treated TSP-2-KO mice had depressed cardiac function compared to WT animals, accompanied by increased cardiomyocyte apoptosis and matrix damage. Enhanced myocyte damage in the absence of TSP-2 was associated with impaired activation of the Akt signaling pathway in TSP-2-KO compared to WT. The absence of TSP-2, in vivo and in vitro, reduced Akt activation both under non-treated conditions and after DOX. Importantly, inhibition of Akt phosphorylation in cardiomyocytes significantly reduced TSP-2 expression, unveiling a unique feedback loop between Akt and TSP-2. Finally, enhanced matrix disruption in DOX-treated TSP-2-KO hearts went along with increased matrix metalloproteinase-2 levels. Taken together, this study is the first to provide evidence for the implication of the matrix element TSP-2 in protecting against DOX-induced cardiac injury and dysfunction.

Research highlights

► Deterioration of the extracellular matrix contributes to DOX-cardiotoxicity. ► A matrix element mediates cardiomyocyte injury and matrix disruption after DOX. ► Reduced Akt activation in TSP-2-KO mice increases cardiomyocyte injury in DOX-CMP. ► Reduced Akt signaling blunts TSP-2 expression, forming a feedback loop in DOX-CMP. ► Enhanced MMP-2 activity in TSP-2-KO mice increases matrix disruption after DOX.

Introduction

Doxorubicin (DOX) is an anticancer drug commonly used for the treatment of hematologic malignancies and solid tumors. Its clinical application is limited by a dose-related cardiotoxicity, which is characterized by progressive cardiac dilatation, contractile dysfunction and ultimately heart failure [1]. Formation of reactive oxygen species (ROS) and resulting cardiomyocyte damage is generally believed to be the primary cause of its cardiotoxicity [2]. Interestingly, interventions that aim to decrease oxidative stress by treatment with exogenous antioxidants or overexpression of endogenous antioxidant enzymes showed promising results in vitro and in vivo [3], [4], [5], [6], but failed to reduce the incidence of cardiotoxicity in patients treated with DOX. This suggests that mechanisms other than anti-oxidants contribute to the response to DOX as well.

DOX-induced cardiomyopathy (DOX-CMP) has been associated with loss of myocardial interstitial collagen and enhanced expression of non-structural glycoproteins in the matrix [7], [8], [9], [10], [11], but clear evidence on the implication of specific matrix components in DOX-cardiotoxicity is completely lacking. By recognizing the possible duality of DOX-cardiotoxicity, we hypothesize that matrix components mediate the development of DOX-CMP. Here, we address whether the matricellular protein thrombospondin-2 (TSP-2) affects DOX-induced cardiotoxic remodeling by mediating matrix alterations or cardiomyocyte toxicity.

Thrombospondins (TSPs) are secreted glycoproteins within the ECM that belong to the group of matricellular proteins, and act as key players in the cross-talk between the matrix and surrounding cells. TSP-2 is known to control matrix metalloproteinase (MMP) activity, whereas its binding to cell surface receptors regulates cellular function during wound healing and tumor formation by linking the matrix to intracellular pathways [12], [13], [14]. In the heart, TSP-2 was initially considered to be essential for strengthening the cardiac matrix in hypertensive heart disease [15]. Recent evidence supports an additional role for TSP-2 as it was shown to have a pro-survival effect on cardiomyocytes during physiological aging of the heart [16]. Here, we show that the pro-survival and matrix-preserving actions of TSP-2 protect against cardiomyocyte loss and ECM weakening that contribute to DOX-induced heart failure.

Section snippets

Methods

An expanded Methods section is available in the Online Supplement.

Increased susceptibility to DOX-CMP in TSP-2-KO mice

No mortality was observed in all sham-treated groups, except for the TSP-2-KO males where two mice died (18%, 2 out of 11) (Fig. 1A). Chronic DOX-exposure significantly induced mortality in male WT mice (29%, 5 out of 17), but not in WT females (7%, 1 out of 15) compared to sham-treated mice (Fig. 1A). DOX-associated mortality further increased in the absence of TSP-2 and was significantly higher in male (67%, 16 out of 24) and female (38%, 10 out of 26) TSP-2-KO mice compared to gender-matched

Discussion

DOX is extremely effective in the treatment of many cancers, however its cardiotoxicity severely limits its clinical utilization as an antineoplastic agent. Although it is well established that DOX cardiotoxicity involves cardiac injury and remodeling, most studies solely addressed the cardiomyocyte effects, leaving the role of the ECM relatively unexplored. The present study is the first to provide evidence for the implication of a matrix element in cardiac injury and remodeling following

Funding

This study was supported by a Marie Curie Exellence Progam to S.H. and M.S. and Y.M.P., an Ingenious Hypercare NoE from the European Union (EST 2005-020706-2), research grants from the Netherlands Heart Foundation (2007B036, 2008B011 and 2009) and a VIDI grant of the Dutch Scientific Organisation (NWO) to S.H.

Conflict of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Hans Duimel (Maastricht University Medical Center, Department of Pathology, Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands) for excellent technical support and Paul Veulemans (Department of Cardiovascular diseases, K.U. Leuven, Leuven, Belgium) for echocardiographic analysis.

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