Thrombospondin-4 Is Required for Stretch-Mediated Contractility Augmentation in Cardiac Muscle Oscar H. Cingolani, Jonathan A. Kirk, Kinya Seo, Norimichi Koitabashi, Dong-ik Lee, Genaro Ramirez-Correa, Djahida Bedja, Andreas S. Barth, An L. Moens and David A. Kass Circ Res. 2011;109:1410-1414; originally published online October 27, 2011; doi: 10.1161/CIRCRESAHA.111.256743 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2011 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/109/12/1410 Data Supplement (unedited) at: http://circres.ahajournals.org/content/suppl/2011/10/27/CIRCRESAHA.111.256743.DC1.html Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/ Downloaded from http://circres.ahajournals.org/ at SWETS SUBSCRIPTION SERVICE on November 25, 2013 Integrative Physiology Thrombospondin-4 Is Required for Stretch-Mediated Contractility Augmentation in Cardiac Muscle Short Communication Oscar H. Cingolani, Jonathan A. Kirk, Kinya Seo, Norimichi Koitabashi, Dong-ik Lee, Genaro Ramirez-Correa, Djahida Bedja, Andreas S. Barth, An L. Moens, David A. Kass Rationale: One of the physiological mechanisms by which the heart adapts to a rise in blood pressure is by augmenting myocyte stretch-mediated intracellular calcium, with a subsequent increase in contractility. This slow force response was first described over a century ago and has long been considered compensatory, but its underlying mechanisms and link to chronic adaptations remain uncertain. Because levels of the matricellular protein thrombospondin-4 (TSP4) rapidly rise in hypertension and are elevated in cardiac stress overload and heart failure, we hypothesized that TSP4 is involved in this adaptive mechanism. Objective: To determine the mechano-transductive role that TSP4 plays in cardiac regulation to stress. Methods and results: In mice lacking TSP4 (tsp4؊/؊), hearts failed to acutely augment contractility or activate stretch-response pathways (ERK1/2 and Akt) on exposure to acute pressure overload. Sustained pressure overload rapidly led to greater chamber dilation, reduced function, and increased heart mass. Unlike controls, tsp4؊/؊ cardiac trabeculae failed to enhance contractility and cellular calcium after a stretch. However, the contractility response was restored in tsp4؊/؊ muscle incubated with recombinant TSP4. Isolated tsp4؊/؊ myocytes responded normally to stretch, identifying a key role of matrix-myocyte interaction for TSP4 contractile modulation. Conclusion: These results identify TSP4 as myocyte-interstitial mechano-signaling molecule central to adaptive cardiac contractile responses to acute stress, which appears to play a crucial role in the transition to chronic cardiac dilatation and failure. (Circ Res. 2011;109:1410-1414.) Key Words: cardiac mechanics Ⅲ mechano-transduction Ⅲ extracellular matrix Ⅲ ventricular function Ⅲ Anrep pproximately one-third of the world’s population has transduction pathways involving matrix structural and signal- Aincreased blood pressure, a leading cause of cardio- ing proteins.5 Which of these proteins mediate the Anrep vascular morbidity and mortality. In such individuals, the effect is unknown. left ventricle is subjected to higher subacute and sustained On the basis of our own and published data in stressed left wall stress due to high pressures and muscle stretch. The ventricles, we hypothesized that thrombospondin 4 (TSP-4),6 heart normally responds to such increased loading by posed an intriguing candidate. TSP-4 is a matricellular protein augmenting its contractility within minutes, thereby main- whose gene expression is among the most profoundly upregu- taining cardiac output. This response was first attributed to lated in hearts with pathological remodeling in humans7 and Anrep in 1912,1 yet nearly 100 years later, its underlying experimental animals.8,9 Each of the 5-member thrombospondin mechanisms still remain unclear. Most existing data fo- family contains epidermal growth factor-like type II domains cuses on the myocyte signal, showing stretch-induced (EGF-like repeats), calcium-binding type III repeats, and a activation of angiotensin type 1 and endothelin-1 recep- highly conserved C-terminal domain. TSP4 is a pentamer that tors, leading to enhanced intracellular calcium through the has been linked to the regulation and cross-linking of matrix sodium-hydrogen exchanger (NHE-1)2 and/or transient re- proteins.10 Human TSP4 is predominantly expressed in cardiac ceptor potential (TRP) channels.3 Extracellular signal-related and skeletal muscle; however, its role in regulating mechanical kinase (ERK1/2), which stimulates NHE-1, may also be responses is unknown. We studied mice with a global knockout Ϫ Ϫ important.4 Yet, myocytes are not directly stressed in intact of tsp4 (tsp4 / ). The mice have no demonstrable cardiovascu- muscle but must receive the signal through mechano- lar phenotype at baseline but display an abnormal adaptation to Original received September 14, 2011; revision received October 12, 2011; accepted October 19, 2011. In September 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 16 days. From the Division of Cardiology, Department of Medicine, Department of Biomedical Engineering, The Johns Hopkins University Medical Institutions, Baltimore, MD. Correspondence to David A. Kass, MD, Ross 858, Johns Hopkins Medical Institutions, Ross Bldg, 858, 720 Rutland Ave, Baltimore, MD 21205. E-mail [email protected] © 2011 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.111.256743 Downloaded from http://circres.ahajournals.org/ at SWETS1410 SUBSCRIPTION SERVICE on November 25, 2013 Cingolani et al Thrombospondin-4 and Cardiac Function 1411 subacute loading stress, revealing TSP4 to be central for matrix- Non-standard Abbreviations and Acronyms myocyte mechano-transduction. EF ejection fraction Methods EGF(R) epidermal growth factor (receptor) Ϯ Tsp4 mice were obtained from The Jackson Laboratory and bred in ERK1/2 extracellular signal-regulated kinase 1⁄2 Ϫ/Ϫ ϩ/ϩ our facility to derive tsp4 and littermate controls (tsp4 ), FS Frank-Starling backcrossed onto a C57BL/6 background for 12 generations. An expanded Methods section is available in the Data Supplement at NHE-1 sodium hydrogen exchanger http://circres.ahajournals.org. TAC transaortic constriction TRP transient receptor potential Results TSP4 thrombospondin 4 TSP4 Is Upregulated by Acute and Chronic Stress Rat cardiomyocytes subjected to 60 minutes of 10% cyclic stretch at 1 Hz displayed a 2-fold rise in gene expression (Figure 1A), whereas intact hearts subjected to 3 weeks of by a decline in function (Figure 1C, right). Summary data transaortic constriction (TAC) displayed a near 40-fold in- confirmed this disparity in chamber response (Figure 1D). Acute Ϫ Ϫ crease (Figure 1B). Tsp4 / mice served as negative controls. TAC stimulated stretch-response kinases ERK1/2 and Akt11 in controls but not in tsp4Ϫ/Ϫ hearts (Figure 1E). The increase in ؊/؊ TSP4 Hearts Appear Normal But Fail to pressure load was similar in both genotypes (Online Figure III) Respond to Acute or Chronic Stress and therefore could not explain the different responses. Ϫ/Ϫ Tsp4 mice were born in normal Mendelian ratios with The disparity in short-term afterload response translated to normal appearance, behavior, and lifespan. Whole myocardi- worsened adaptations to chronic overload (Figure 2). Within um microarray revealed few differentially expressed genes in 48 hours after TAC, tsp4Ϫ/Ϫ hearts showed greater dilation/ Ϫ/Ϫ tsp4 versus controls, primarily some reduced extracellular dysfunction, and this increased further in the ensuing 3 matrix genes (Online Table I). Baseline cardiac structure and weeks. Controls showed full compensation (no dilation, function out to at least 1 year (Online Figure I and Online preserved ejection fraction) at 1 week and less dysfunction Table II), and isolated myocyte function and calcium tran- after that. Thus, lack of TSP4 limited the heart’s ability to sients both at rest and with isoproterenol stimulation were mount an early compensation to TAC. Interstitial fibrosis similar to littermate controls (Online Figure II). assessed with a qualitative scale (0–3, eg, none to severe) Ϫ/Ϫ In contrast, tsp4 hearts failed to respond normally to an rose similarly in both groups (0.9Ϯ0.13–1.7Ϯ0.2 for WT, acute rise in left ventricular afterload. Acute TAC in normal 0.9Ϯ0.14–1.8Ϯ0.1 for tsp4Ϫ/Ϫ). hearts immediately reduced ejection fraction,