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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.
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.
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 http://circres.ahajournals.org.
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.
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−/−).
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.
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).
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.
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.).