Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Circ Res. Author manuscript; available in PMC 2012 April 11.
Published in final edited form as:
PMCID: PMC3324097

Thrombospondin-4 Is Required for Stretch-Mediated Contractility Augmentation in Cardiac Muscle



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.


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.


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.

Keywords: cardiac mechanics, mechano-transduction, extracellular matrix, ventricular function, Anrep

Approximately one-third of the world’s population has increased blood pressure, a leading cause of cardiovascular morbidity and mortality. In such individuals, the left ventricle is subjected to higher subacute and sustained wall stress due to high pressures and muscle stretch. The heart normally responds to such increased loading by augmenting its contractility within minutes, thereby maintaining cardiac output. This response was first attributed to Anrep in 1912,1 yet nearly 100 years later, its underlying mechanisms still remain unclear. Most existing data focuses on the myocyte signal, showing stretch-induced activation of angiotensin type 1 and endothelin-1 receptors, leading to enhanced intracellular calcium through the sodium-hydrogen exchanger (NHE-1)2 and/or transient receptor potential (TRP) channels.3 Extracellular signal-related kinase (ERK1/2), which stimulates NHE-1, may also be important.4 Yet, myocytes are not directly stressed in intact muscle but must receive the signal through mechano-transduction pathways involving matrix structural and signaling proteins.5 Which of these proteins mediate the Anrep effect is unknown.

On the basis of our own and published data in stressed left ventricles, we hypothesized that thrombospondin 4 (TSP-4),6 posed an intriguing candidate. TSP-4 is a matricellular protein whose gene expression is among the most profoundly upregulated in hearts with pathological remodeling in humans7 and experimental animals.8,9 Each of the 5-member thrombospondin family contains epidermal growth factor-like type II domains (EGF-like repeats), calcium-binding type III repeats, and a highly conserved C-terminal domain. TSP4 is a pentamer that has been linked to the regulation and cross-linking of matrix proteins.10 Human TSP4 is predominantly expressed in cardiac and skeletal muscle; however, its role in regulating mechanical responses is unknown. We studied mice with a global knockout of tsp4 (tsp4−/−). The mice have no demonstrable cardiovascular phenotype at baseline but display an abnormal adaptation to subacute loading stress, revealing TSP4 to be central for matrixmyocyte mechano-transduction.


Tsp4± mice were obtained from The Jackson Laboratory and bred in our facility to derive tsp4−/− and littermate controls (tsp4+/+), backcrossed onto a C57BL/6 background for 12 generations. An expanded Methods section is available in the Data Supplement at


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 transaortic constriction (TAC) displayed a near 40-fold increase (Figure 1B). Tsp4−/− mice served as negative controls.

Figure 1
Impact of loading stress on TSP4 expression, and of TSP4 gene silencing on intact heart acute pressure-overload response

TSP4−/− Hearts Appear Normal But Fail to Respond to Acute or Chronic Stress

Tsp4−/− mice were born in normal Mendelian ratios with normal appearance, behavior, and lifespan. Whole myocardium microarray revealed few differentially expressed genes in tsp4−/− versus controls, primarily some reduced extracellular matrix genes (Online Table I). Baseline cardiac structure and function out to at least 1 year (Online Figure I and Online Table II), and isolated myocyte function and calcium transients both at rest and with isoproterenol stimulation were similar to littermate controls (Online Figure II).

In contrast, tsp4−/− hearts failed to respond normally to an acute rise in left ventricular afterload. Acute TAC in normal hearts immediately reduced ejection fraction, yet over the ensuing 30 minutes, contractility increased as denoted by a leftward shift of the pressure-volume loop and restoration of stroke volume (ie, Anrep effect) (Figure 1C, left). In tsp4−/− hearts, the immediate response to higher afterload was similar, but the subsequent rise in contractility was absent and replaced by a decline in function (Figure 1C, right). Summary data confirmed this disparity in chamber response (Figure 1D). Acute TAC stimulated stretch-response kinases ERK1/2 and Akt11 in controls but not in tsp4−/− hearts (Figure 1E). The increase in pressure load was similar in both genotypes (Online Figure III) and therefore could not explain the different responses.

The disparity in short-term afterload response translated to worsened adaptations to chronic overload (Figure 2). Within 48 hours after TAC, tsp4−/− hearts showed greater dilation/ dysfunction, and this increased further in the ensuing 3 weeks. Controls showed full compensation (no dilation, preserved ejection fraction) at 1 week and less dysfunction after that. Thus, lack of TSP4 limited the heart’s ability to mount an early compensation to TAC. Interstitial fibrosis assessed with a qualitative scale (0–3, eg, none to severe) rose similarly in both groups (0.9±0.13–1.7±0.2 for WT, 0.9±0.14 –1.8±0.1 for tsp4−/−).

Figure 2
Tsp4−/− hearts display greater chamber dilation and hypertrophy to sustained pressure-overload

Abnormal Stretch Activation in TSP4−/− Muscle

In isolated muscle contracting at constant length, acute stretch augments developed force without a change in intracellular calcium (Frank-Starling [FS] mechanism12) and then exhibits a second and slower rise in force and Ca2+ (Anrep effect).

Both were observed in control cardiac trabeculae (Figure 3A); however, tsp4−/− muscle only displayed the acute FS component; thereafter, force declined (Figure 3B and 3C). Ca2+ rose gradually in control but not tsp4−/− muscle after stretch (Figure 3D and 3E). This defect was restored by preincubation with recombinant TSP4 monomer for 15 minutes (Figure 3F). This efficacy from such a short period of incubation favors a signaling action of TSP4 rather than its modifying fundamental matrix structure.

Figure 3
Impact of TSP4 on acute and slow force and calcium response following stretch in isolated muscle

Slow Force Response Is Normal in TSP4−/− Myocytes

TSP4 is expressed in both interstitial cells and myocytes. To test its role to mechanical responses in the myocyte itself, we studied isolated adult cells using a carbon fiber force-length Servo control system.13 Cells were set to contract auxotonically, and data were analyzed by net developed force and by force-sarcomere length relations. Unlike intact heart and muscle, myocytes from both control and tsp4−/− hearts displayed similar acute and slow force responses (Figure 4A). The ability of the stretch activated channel blocker GsMtx-4 to suppress the myocyte SFR was tested as a positive control (Figure 4B).

Figure 4
Effect of TSP4 on isolated myocyte response to auxotonic stress


This study reveals TSP4 as an essential protein linking the imposition of subacute myocardial stress to the heart’s capacity to adapt by augmenting intracellular myocyte calcium and improving contractility. TSP4 is known to function as a matrix-modulating protein, and our myocardial microarray data support this. Yet, its absence had no impact on the immediate contractile response to stretch (FS) in whole heart, muscle, or isolated myocytes. By contrast, the slow force response (Ca2+-coupled) was absent, but this was only in whole heart and muscle, not in myocytes. One interpretation of these data is structural, wherein TSP4-collagen interactions are required to sustain an imposed matrix stress to the myocyte (eg, minutes versus 1 beat). However, the fact that exposure to TSP4 monomer rapidly restored delayed force augmentation in tsp4−/− muscle favors an alternative signaling role. With this model, signaling is required only if matrix is present, because the myocyte response was normal with or without TSP4 and it also implies a negative influence of sustained and stressed matrix on myocyte function that is normally countered by TSP4. Upregulation of TSP4 in stress-induced cardiac disease would be viewed as adaptive, offsetting such negative signaling.

One limitation of our study is that tsp4−/− mice were a global knockout, and it is possible that effects beyond the heart were affected, particularly in the chronic TAC experiment. For example, TSP4 has been linked with vascular inflammation and atherogenesis14 and could be activated by vascular distension. However, the load-response behavior that we observed in 10 minutes is probably not related to such changes nor to enhanced protein expression, and the recapitulation of the behavior in isolated muscle reduces the likelihood that this reflects vascular modulation.

The precise interactome coupling TSP4 matrix-to-myocyte remains to be determined, though one possibility is through its EGF-like motifs, as transactivation of EGFR is an upstream component of the Anrep effect,4 and TSP4 EGF-like repeats can activate EGFR in other tissues.15 The fact that tsp4−/− hearts failed to activate ERK1/2 after acute loading the mechanics and Ca2+ data, as the kinase maybe important to the Anrep effect,4 whereas Akt activation has been coupled to integrin-mediated mechano-transduction.16 Further studies will be required to dissect these mechanisms, and these will likely require a preparation with both myocyte and matrix as their interaction appears central to TSP4 regulation. Our results, coming nearly 100 years after Anrep’s description, shed new light on the mechanism coupling subacute myocardial stress and contractility and identify TSP4 as a potential therapeutic target to further enhance adaptive cardiac stress responses.

Supplementary Material



Sources of Funding

This work was funded by National Health Service Grants HL089297, HL059408, and HL-077180, T32HL-0772, Fondation Leducq, and Belfer Laboratory Foundation (D.A.K.), KO8-HL-109074-01 (OHC) postdoctoral fellowship awards T32HL-0227 (O.H.C. and J.A.K.), and American Heart Association postpoctoral fellowship awards (N.K., D.L., K.S., and A.L.M.).

Non-standard Abbreviations and Acronyms

ejection fraction
epidermal growth factor (receptor)
extracellular signal-regulated kinase ½
sodium hydrogen exchanger
transaortic constriction
transient receptor potential
thrombospondin 4





1. von Anrep G. On the part played by the suprarenals in the normal vascular reactions of the body. J Physiol. 1912;45:307–317. [PubMed]
2. Cingolani HE, Perez NG, Aiello EA, Ennis IL, Garciarena CD, Villa-Abrille MC, Dulce RA, Caldiz CI, Yeves AM, Correa MV, Nolly MB, Chiappe de CG. Early signals after stretch leading to cardiac hypertrophy. Key role of nhe-1. Front Biosci. 2008;13:7096–7114. [PubMed]
3. Ward ML, Williams IA, Chu Y, Cooper PJ, Ju YK, Allen DG. Stretch-activated channels in the heart: contributions to length-dependence and to cardiomyopathy. Prog Biophys Mol Biol. 2008;97:232–249. [PubMed]
4. Villa-Abrille MC, Caldiz CI, Ennis IL, Nolly MB, Casarini MJ, Chiappe de Cingolani GE, Cingolani HE, Perez NG. The Anrep effect requires transactivation of the epidermal growth factor receptor. J Physiol. 2010;588:1579–1590. [PubMed]
5. Kakkar R, Lee RT. Intramyocardial fibroblast myocyte communication. Circ Res. 2010;106:47–57. [PMC free article] [PubMed]
6. Lawler J, Duquette M, Whittaker CA, Adams JC, McHenry K, DeSimone DW. Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family. J Cell Biol. 1993;120:1059–1067. [PMC free article] [PubMed]
7. Tan FL, Moravec CS, Li J, Apperson-Hansen C, McCarthy PM, Young JB, Bond M. The gene expression fingerprint of human heart failure. Proc Natl Acad Sci U S A. 2002;99:11387–11392. [PubMed]
8. Rysa J, Leskinen H, Ilves M, Ruskoaho H. Distinct upregulation of extracellular matrix genes in transition from hypertrophy to hypertensive heart failure. Hypertension. 2005;45:927–933. [PubMed]
9. Mustonen E, Aro J, Puhakka J, Ilves M, Soini Y, Leskinen H, Ruskoaho H, Rysa J. Thrombospondin-4 expression is rapidly upregulated by cardiac overload. Biochem Biophys Res Commun. 2008;373:186–191. [PubMed]
10. Narouz-Ott L, Maurer P, Nitsche DP, Smyth N, Paulsson M. Thrombospondin-4 binds specifically to both collagenous and non-collagenous extracellular matrix proteins via its c-terminal domains. J Biol Chem. 2000;275:37110–37117. [PubMed]
11. Honsho S, Nishikawa S, Amano K, Zen K, Adachi Y, Kishita E, Matsui A, Katsume A, Yamaguchi S, Nishikawa K, Isoda K, Riches DW, Matoba S, Okigaki M, Matsubara H. Pressure-mediated hypertrophy and mechanical stretch induces il-1 release and subsequent igf-1 generation to maintain compensative hypertrophy by affecting akt and jnk pathways. Circ Res. 2009;105:1149–1158. [PubMed]
12. de Tombe PP, Mateja RD, Tachampa K, Mou YA, Farman GP, Irving TC. Myofilament length dependent activation. J Mol Cell Cardiol. 2010;48:851–858. [PubMed]
13. Sugiura S, Nishimura S, Yasuda S, Hosoya Y, Katoh K. Carbon fiber technique for the investigation of single-cell mechanics in intact cardiac myocytes. Nat Protoc. 2006;1:1453–1457. [PubMed]
14. Frolova EG, Pluskota E, Krukovets I, Burke T, Drumm C, Smith JD, Blech L, Febbraio M, Bornstein P, Plow EF, Stenina OI. Thrombospondin-4 regulates vascular inflammation and atherogenesis. Circ Res. 2010;107:1313–1325. [PMC free article] [PubMed]
15. Liu A, Garg P, Yang S, Gong P, Pallero MA, Annis DS, Liu Y, Passaniti A, Mann D, Mosher DF, Murphy-Ullrich JE, Goldblum SE. Epidermal growth factor-like repeats of thrombospondins activate phospholipase cgamma and increase epithelial cell migration through indirect epidermal growth factor receptor activation. J Biol Chem. 2009;284:6389–6402. [PMC free article] [PubMed]
16. De Acetis M, Notte A, Accornero F, Selvetella G, Brancaccio M, Vecchione C, Sbroggio M, Collino F, Pacchioni B, Lanfranchi G, Aretini A, Ferretti R, Maffei A, Altruda F, Silengo L, Tarone G, Lembo G. Cardiac overexpression of melusin protects from dilated cardiomyopathy due to long-standing pressure overload. Circ Res. 2005;96:1087–1094. [PubMed]