Absence of Thrombospondin-2 Causes Age-Related Dilated Cardiomyopathy
Background— The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart.
Methods and Results— TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas <10% of the wild-type mice died. In the absence of TSP-2, older mice displayed a severe dilated cardiomyopathy with impaired systolic function, increased cardiac dilatation, and fibrosis. Ultrastructural analysis revealed progressive myocyte stress and death, accompanied by an inflammatory response and replacement fibrosis, in aging TSP-2 KO animals, whereas capillary or coronary morphology or density was not affected. Importantly, adeno-associated virus-9 gene–mediated transfer of TSP-2 in 7-week-old TSP-2 KO mice normalized their survival and prevented dilated cardiomyopathy. In TSP-2 KO animals, age-related cardiomyopathy was accompanied by increased matrix metalloproteinase-2 and decreased tissue transglutaminase-2 activity, together with impaired collagen cross-linking. At the cardiomyocyte level, TSP-2 deficiency in vivo and its knockdown in vitro decreased the activation of the Akt survival pathway in cardiomyocytes.
Conclusion— TSP-2 expression in the heart protects against age-dependent dilated cardiomyopathy.
Received March 13, 2009; accepted August 17, 2009.
Whether aging of the heart and related functional changes should be regarded as a physiological or pathological process is a puzzling question. In the absence of other diseases such as hypertension and diabetes mellitus or detrimental environmental factors such as smoking, the heart is perfectly able to “survive” for a human lifespan. Hence, disease processes such as lamin A/C mutations that result in accelerated aging of the heart and dilated cardiomyopathy point toward essential protective mechanisms that are mandatory for normal physiological aging of the heart.1,2
Clinical Perspective on p 1597
The extracellular matrix is a crucial system that hinders functional and structural deterioration of the heart with aging. Matrix elements not only provide structural support, as collagen does, but also are implicated in maintaining cellular homeostasis and regulating intracellular signaling during normal cardiac physiology.2 Thrombospondin-2 (TSP-2) belongs to a family of nonstructural matricellular proteins implicated in regulating cell-matrix interactions. Its expression is low in the normal postnatal heart but reappears at high levels during cardiac pathology3,4 and helps to preserve cardiac integrity during hypertension.5 However, its function in normal physiological aging of the heart remains unknown. We therefore investigated whether TSP-2, by affecting survival pathways in cardiomyocytes, may provide necessary molecular support for the heart with aging and thus increase lifespan. We provide data showing that hearts lacking TSP-2 progress toward dilated cardiomyopathy with advanced age but have normal morphology and function at a young age. Importantly, postnatal adeno-associated viral vector (AAV9) –mediated transfer of TSP-2 in young TSP-2 knockout (KO) mice completely normalized their survival and prevented the development of cardiac failure. With aging, lack of TSP-2 resulted in decreased activation of the Src/Akt survival pathway, increased matrix metalloproteinase 2 (MMP-2) activity, and decreased collagen cross-linking, leading to progressive cardiomyocyte dropout and overall cardiac failure and dilatation.
Materials and Methods
See the online-only Data Supplement for additional details.
Transgenic Mice and Experimental Procedures
This study was approved by the Institutional Animal Research committees, and all experiments were performed according to official rules formulated under Dutch and Belgian law on the care and use of experimental animals. Eight- to 60-week-old TSP-2 KO mice and their wild-type (WT) littermates on a C57Bl6/129SvJ/EMS+Ter genetic background were used.6 At a young age, male and female mice were divided into 3 age groups for further analysis: young mice, 8 to 12 weeks old (n=20 for TSP-2 WT mice, n=20 for TSP-2 KO mice); intermediate-age mice, 25 to 30 weeks old (n=20 for TSP-2 WT mice, n=20 for TSP-2 KO mice); and older mice, 50 to 60 weeks old (n=100 for TSP-2 WT mice, n=100 for TSP-2 KO mice). Consequently, only mortality rates within the third group were included in the survival curve.
Next, to rescue cardiac TSP-2 expression, AAV9 gene transfer of TSP-2 was performed in TSP-2 KO mice. Additional details on the construction and production of the AAV9–TSP-2 and AAV9–green fluorescent protein (GFP) vectors, with both TSP-2 and GFP driven under the cytomegalovirus promoter, are provided in the online-only Data Supplement. We injected 100 μL containing 1×1011 viral genomic copies of AAV9–TSP-2 or the control AAV9-GFP intravenously into the tail vein of adult (7-week-old) male (n=10 for AAV9–TSP-2, n=25 for AAV9-GFP) and female (n=19 for AAV9–TSP-2, n=10 for AAV9-GFP) TSP-2 KO mice and monitored them until 60 weeks of age. All analyses were performed following standard operating procedures and confirmed by independent observers blinded to genotype or treatment group.
Cardiac Function, Histopathological, and Molecular Analyses
After the study period, all mice were anesthetized, followed by transthoracic echocardiographic examination. Subsequently, hearts were taken out and prepared for further histological and molecular analysis, including immunohistochemical and electron microscopic analysis, determination of zymographic MMP and tissue transglutaminase (tTG) activity with aging, RNA isolation and real-time polymerase chain reaction, immunoblotting, integrin-linked kinase (ILK) activity and active transforming growth factor-β (TGF-β) assays, in vitro experimental approaches, force measurements in single permeabilized cardiomyocytes, and phosphorylation status of myofilament proteins. Experimental materials and methods are described more extensively in the online-only Data Supplement.
All data are expressed as mean±SEM. Mann–Whitney U test, unpaired t test, or 2-way ANOVA was used as appropriate to assess statistical significance between groups. Survival curves were obtained by the Kaplan–Meier method and compared by the log-rank test. A 2-sided value of P<0.05 was considered statistically significant.
TSP-2 KO Mice Develop Accelerated Aging-Induced Cardiomyopathy
To investigate the effect of TSP-2 on aging, a prospective study for mortality was conducted (Figure 1A). Whereas no significant mortality was noted until 20 weeks of age, the survival rate of TSP-2 KO mice progressively declined thereafter compared with WT mice (Figure 1A). By 60 weeks of age, 55% of the TSP-2 KO mice died (55 of 100), whereas 90% of the WT mice (90 of 100) remained alive (P<0.01). Interestingly, mortality was significantly higher in male (84%, 42 of 50) compared with female (26%, 13 of 50) TSP-2 KO mice (P<0.01; Figure 1A).
TSP-2 transcript and protein levels were significantly increased (3.4- and 3.6-fold, respectively) in older compared with young TSP-2 WT hearts (Table 1 and Figure 1B). Moreover, immunoblotting revealed that TSP-2 protein levels were more elevated in older female compared with older male WT hearts (Figure 1B). Immunohistochemical analysis confirmed that TSP-2 protein expression was low in young WT hearts (Figure 1C) but abundantly present and located primarily in the extracellular matrix surrounding the cardiomyocytes in both older female and male TSP-2 WT hearts (Figure 1C).
To investigate whether the absence of TSP-2 resulted in compensatory changes in expression of other TSPs before the onset of heart failure, transcript and/or protein levels of TSP-1, -3, and -4 were determined in the hearts of young and older TSP-2 KO and WT mice (Table 1). Compared with WT hearts, a clear trend toward increased TSP-4 transcript levels was observed in TSP-2 KO hearts (P=0.08 at young age, P=0.07 at older age; Table 1). However, no significant differences in TSP-1, -3, and -4 transcript levels were noted between WT and KO hearts at both young and older age (Table 1). Concordantly, TSP-1 and -3 immunoblotting did not reveal any significant differences between the TSP-2 WT and KO hearts (Table 1).
Further analysis of the aged hearts revealed that the increased mortality was due to progressive and severe dilated cardiomyopathy, with increasing cardiac fibrosis and dilatation with aging in TSP-2 KO mice (Figure 1D through 1K and Tables 2 and 3⇓). In concordance, echocardiographic analysis showed depressed systolic function and increased cardiac dilatation in surviving TSP-2 KO compared with older WT mice (Table 2). Cardiac fibrosis, dysfunction, and dilatation were more pronounced in the older male compared with the older female TSP-2 KO hearts (Figure 1D through 1K and Tables 2 and 3⇓). Increased cardiac failure in TSP-2 KO mice was further validated by an increased ratio of lung to body weight compared with WT mice at 60 weeks (Table 2). At young age, cardiac function and dimensions did not differ in TSP-2 KO compared with age-matched WT animals (Table 2).
Structural Features of Accelerated Aging of the Heart in TSP-2 KO Mice
To study the underlying mechanisms of the age-related cardiomyopathy, the morphological features of cardiomyocytes, interstitial matrix, and vessels, including markers of apoptosis and cell stress, were examined within the surviving male and female TSP2 WT and KO hearts at young (8 to 12 weeks), intermediate (25 to 30 weeks), and older (50 to 60 weeks) age.
Histopathological analysis revealed a progressive decrease in cardiomyocyte density and increase in scar-forming fibrosis with advanced age in the male and female TSP-2 KO hearts but not in aged WT hearts (Figure 1D through 1K, Table 3, and Figure I of the online-only Data Supplement). No differences were seen at young age. Cardiomyocyte dropout was further indicated by a significantly reduced ratio of left ventricular weight to body weight in older TSP-2 KO mice, whereas this ratio did not differ between young WT and TSP-2 KO mice (Figure 1D through 1K and Table 3). Myocyte stress in older TSP-2 KO hearts was confirmed by desmin staining,7 revealing a clear desmin disorganization (Figure 2A). Ubiquitin, a marker of cell stress, and p16, an indicator of aging,8 were significantly increased in older TSP-2 KO hearts (Figure 2B and 2C and Table 3).
Electron microscopy substantiated cardiomyocyte stress and a disorganization of the extracellular matrix in older TSP-2 KO compared with WT hearts, whereas myocytes and matrix were normal in younger TSP-2 KO and WT hearts (Figure 2E and 2F). Cardiomyocytes of old TSP-2 KO hearts showed mitochondrial enlargement and lysis of myofilaments (Figure 2E and 2F).
Decreased cardiac mass was not due to significant changes in myocyte cross-sectional area of older TSP-2 KO hearts compared with age-matched WT hearts (Table 3). Myocyte death and fibrosis were not caused by differences in vascularity (Table 3).
AAV9–TSP-2 Treatment of TSP-2 KO Mice Prevents Accelerated Aging-Induced Cardiomyopathy
To determine whether the age-related cardiomyopathy in TSP-2 KO mice could be rescued by postnatal gene transfer of TSP-2, we treated young male and female TSP-2 KO mice with AAV9–TSP-2 compared with control AAV9-GFP (Figure 3A through 3F). At 60 weeks of age, both immunoblotting and immunostaining revealed that AAV9-mediated transfer of TSP-2 resulted in significant and widespread cardiac TSP-2 protein expression, whereas TSP-2 staining was absent in the control AAV9-GFP–treated mice (Figure 3B through 3F).
Importantly, AAV9–TSP-2 blunted the mortality observed in the older control AAV9-GFP–treated TSP-2 KO mice (68%, 24 of 35: 3 of 10 females and 21 of 25 males; Figure 3A). AAV9–TSP-2 also prevented cardiomyocyte dropout, fibrosis, and cardiac dilatation and dysfunction, still present in the aged control-treated TSP-2 KO mice (Figure 3A through 3F and Table 4). Together, these data confirm that postnatal TSP-2 expression is essential for maintaining myocardial architecture and function with age.
Increased Inflammation in Older TSP-2 KO Hearts
Next, we investigated whether a prolonged low-grade inflammation in response to aging-associated free radical generation could underlie the age-related cardiomyopathy observed in TSP-2-KO mice.9 Cardiac aging and progressive myocyte stress in the absence of TSP-2 were accompanied by increased inflammation. The number of CD45-positive inflammatory cells increased progressively with age, reaching significance in TSP-2 KO compared with WT hearts at older age (Figure 2D and Table 3), but no significant differences were noticed at young age (Figure 2D and Table 3). Inflammation was more pronounced in older male compared with female TSP-2 KO mice (Figure 2D and Table 3). In concordance, cardiac transcript levels of TGF-β1 and interleukin (IL) -1β, IL-6, and IL-12 significantly increased with age and in older TSP-2 KO compared with WT hearts (Table 1). The activation of TGF-β1 did not differ (Table 1). In addition, transcript levels of enzymes influencing the oxidation status of the heart, including superoxide dismutase-2 and glutathione peroxidase-1, did not significantly differ between TSP-2 WT and KO hearts (Table 1), whereas catalase-110 was significantly higher in older WT compared with KO hearts (Table 1). In conclusion, age-related cardiomyopathy in the absence of TSP-2 is due to progressive cardiomyocyte stress and dropout, accompanied by increased inflammation and scar-forming reparative fibrosis.
Higher MMP-2 and Lower tTG-2 Activity in TSP-2 KO Mice
Previous studies have shown that a lack of TSP-2 results in an increase in MMP-2 activity, which in turn inactivates tTG-2. The latter enzyme is involved in collagen cross-linking and may protect against cardiac dilatation and dysfunction.11,12 To investigate whether the increased cardiac dilatation in the absence of TSP-2 may relate to increased MMP-2 and consequently decreased tTG activity, their activity levels were measured in young and older TSP-2 KO and WT hearts.
Whereas no significant differences were noted in TSP-2 KO hearts, MMP-2 zymographic activity decreased significantly in TSP-2 WT hearts with progressing age (Figure 4A). MMP-2 activity levels were significantly lower in young KO compared with WT hearts but significantly higher in older KO compared with older WT hearts. MMP-9 zymographic activity did not differ significantly in TSP-2 KO compared with WT hearts at both young or old age (Figure 4B).
Decreased MMP-2 activity with progressing age in TSP-2 WT hearts was paralleled by increased tTG activity (Figure 4C). Concordantly, increased MMP-2 activity in older TSP-2 KO compared with older WT hearts was paralleled by reduced activity of tTG-2 and a decrease in ε-lysyl γ-glutaminyl cross-links (Figure 4D and 4E). A significant decrease in tTG-2 activity in TSP-2 KO compared with WT hearts was also observed at younger age (Figure 4A and 4C).
Sirius red–polarization microscopy revealed mainly well-aligned, thick, and tightly packed orange-red collagen fibers in young and older WT hearts. Loosely assembled yellow-green collagen fibers predominated the older TSP-2 KO hearts (Figure 4F and 4G and Table 3). Thus, increased MMP-2, decreased tTG-2 activity, and impaired collagen maturation in the absence of TSP-2 contributed to increased cardiac dilatation with aging.
Loss of TSP-2 Results in Impaired Activation of Akt In Vivo and In Vitro
The Akt pathway is centrally involved in promoting myocyte survival, and its activation protects against cardiac injury, fibrosis, and failure.13,14 TSP-2 contains 2 conserved domains that are able to activate the Akt pathway, namely the CD47-binding site within its C-terminal domain and the N-terminal β1-integrin recognition site that is proximal to the CD36-binding domain.15 Therefore, we investigated whether these interactions might contribute to the cardiomyocyte survival pathway.
First, significantly reduced activation of Src and Akt was demonstrated in TSP-2 KO compared with WT hearts (Figure 5A and 5B). Immunoblotting for ILK and in vitro kinase activity assays were performed to determine the relative level and activity of ILK in WT and TSP-2 KO hearts. However no differences in ILK activity could be detected in the hearts of TSP-2 WT and KO mice (Figure II of the online-only Data Supplement). Next, an interaction of TSP-2 with the Src/Akt pathway was confirmed in cardiac myocytes in vitro. The use of a lentiviral short-hairpin RNA produced an 80% knockdown of TSP-2 and resulted in significantly reduced phosphorylation of Src and Akt (Figure 5C through 5G), whereas a control, unrelated short-hairpin RNA lentiviral vector did not alter their phosphorylation status.
Finally, monoclonal blocking antibodies against either the CD47- (α-TSPCD47) or the CD36- (α-TSPCD36) binding sites of TSP were administered to neonatal rat cardiomyocytes in vitro (Figure 6A through 6C). Blocking of the TSP/CD47 interaction resulted in reduced phosphorylation of Src and Akt (Figure 6E through 6G) and caused obvious myocyte stress compared with control treatment. Here, myocyte stress was indicated by changes in the cytoskeletal organization as revealed by altered phalloidin staining, a significantly decreased cardiomyocyte density, and increased protein expression of activated caspase-3 (Figure 6A through 6D and 6H). Blocking of the CD36-binding site of TSP, however, did not significantly alter Src or Akt phosphorylation, nor did it change cardiomyocyte appearance or expression of activated caspase-3 (Figure 6A through 6H).
Absence of TSP-2 Affects Cardiomyocyte Function With Aging
To investigate the effect of age-related cardiomyopathy on the function of surviving cardiomyocytes, active and passive forces and Ca2+ sensitivity were determined through the use of individual permeabilized cardiomyocytes isolated from young and older TSP-2 KO and WT left ventricles. Passive and active forces and Ca2+ sensitivity did not differ in young TSP-2 KO compared with WT myocytes (n=10 cardiomyocytes from 5 hearts per group; P=NS; Figure 7A and 7B). Forces increased with advanced age in both TSP-2 KO and WT cardiomyocytes, but active forces did not differ between TSP-2 KO and WT cardiomyocytes (Figure 7B). Passive forces were significantly higher in older TSP-2 KO compared with WT cardiomyocytes, indicating increased cardiomyocyte stiffness. Normalized force–Pca curves showed similar myofilament Ca2+ sensitivity at young and older ages in TSP-2 WT and KO mice (data not shown). Impaired diastolic function of isolated cardiomyocytes was not due to an alteration in myofilament phosphorylation because treatment with the catalytic subunit protein kinase-A (PKA) provided similar effects in TSP-2 KO and WT cardiomyocytes (Figure 7C). PKA treatment resulted in nonsignificant reduced passive forces in young TSP-2 KO mice (passive force before PKA: WT, 1.4±0.2; KO, 2.3±0.5: passive force after PKA: WT, 1.1±0.1; KO, 1.8±0.4). Similarly, passive forces in the older mice did not change significantly. Phosphorylation levels of myosin binding protein c, myosin light chain 2, and troponin-T and -I did not significantly differ in TSP-2 KO compared with WT hearts at both young and older ages (Figure 7D).
With advanced medical assistance and better control of cardiovascular risk factors, the general population is getting older, and many people may live to be >90 years of age without experiencing cardiac dysfunction. Still, little is known about the physiological survival mechanisms that help to “adapt” the heart and prevent myocyte death, fibrosis, and dysfunction in response to the cumulative hemodynamic stress related to aging. An understanding of these processes may lead to new therapeutic approaches that promote the longevity of the heart, even in the absence of hypertension, diabetes mellitus, smoking, or coronary disease.
We provide evidence that cardiac expression of the matricellular protein TSP-2 protects against aging-related functional decline of the heart. Loss of TSP-2 resulted in progressive cardiomyocyte death, accompanied by inflammation and reparative scar-forming fibrosis, all of which contributed to the development of progressive cardiac dilatation and dysfunction. Importantly, postnatal AAV9-mediated transfer of TSP-2 in young TSP-2 KO mice completely rescued the progressive structural and functional declines with aging.
In view of its matrix-cell–regulating properties, a protective role for TSP-2 against age-related heart disease may be mediated by regulating outward-inward cell signaling and function, thereby affecting cardiac remodeling. First, TSP-2 seems to protect against age-related myocyte death, at least in part, by promoting the Akt survival pathway in cardiomyocytes.15 The absence of TSP-2 in vivo or its knockdown in vitro resulted in reduced activation of Src and Akt, which are both involved in promoting myocyte survival.13 Moreover, our data indicate that the conserved C-terminal CD47 binding of TSP-2 is implicated in activating the Src/Akt survival pathway in cardiomyocytes. A blocking antibody against the CD47-binding domain (α-TSPCD47), but not against the CD36-binding domain (α-TSPCD36), significantly reduced activation of Src/Akt and increased myocyte stress and apoptosis in vitro. To exclude that other upstream regulators are responsible for activating the effector protein Akt, the interaction with ILK was investigated in vivo. ILK functions downstream and independently of phosphatidylinositol 3-kinase to phosphorylate Akt. Immunoblotting for ILK, together with ILK activity measurements, did not show differences in KO and WT hearts. Together, our data indicate that the effect of TSP-2 on Akt phosphorylation is Src dependent. Previous reports have shown that the Akt pathway is regulated through several receptor and receptor-independent pathways (ie, mechanical stretch).16,17 Concordantly, we cannot exclude that TSP-2 might also regulate myocyte survival by influencing other survival pathways, including ERK1-2 and p38-mitogen-activated protein kinases,18 focal adhesion kinases,19 or STAT3 signaling pathways.20
A protective role for TSP-2–mediated Akt activation in physiological aging, however, fits with a crucial role for Akt1 and Akt2 in physiological growth of cardiomyocytes during exercise; Akt1- or Akt2-deficient mice are not able to develop physiological hypertrophy during exercise but develop heart failure in response to pressure overload.13,21 Moreover, the implication of outside-inside signaling and, more specifically, Akt activation in promoting (cardio)myocyte survival has also been demonstrated for other matricellular proteins. Periostin-22 and CCN-1– (Cyr-61)23 deficient mice have impaired myocyte survival resulting from reduced activation of the Akt survival pathway.
Beside its role in outside-inside signaling, TSP-2 also regulates extracellular matrix remodeling. It forms a complex with pro–MMP-2 and tissue inhibitor of metalloproteinase-2, which is then internalized by the low-density lipoprotein–related scavenger receptor LRP1 and thereby downregulates total MMP-2 activity.24 MMP-2 in turn decreases total tTG activity11 and may diminish the binding of TSP to CD47, thereby decreasing its downstream activity.25 Aging in mice lacking TSP-2 results in increased MMP-2 activity, decreased tTG activity, and reduced collagen cross-linking, all contributing to increased cardiac dilatation and dysfunction.2 In concordance, Agah et al26 previously reported increased dermal TSP-2 expression and decreased clearance of MMP-2 from the pericellular environment as a function of age in TSP-2 WT mice.
TSP-2 was also shown to regulate angiogenesis and inflammation.9,26,27 Here, we show that myocyte death and fibrosis with progressing age were not caused by differences in vascularity. However, inflammation was increased in the absence of TSP-2 in the older, but not in the younger, hearts. Both progressive cardiomyocyte death and the loss of the CD47-mediated antiinflammatory effect of TSP-29 might have contributed to this enhanced inflammatory influx. Whether the increased inflammatory response in aged TSP-2 KO mice acts mainly to remove the necrotic cardiomyocytes or whether the increased inflammation represents a primary contributing factor that aggravates cardiomyocyte injury, fibrosis, and age-related cardiomyopathy remains to be resolved.
The morphological and functional features of this age-related cardiomyopathy and mortality occurred mainly in male KO mice. Several previous studies have shown that female mice display a lower mortality and less severe cardiac pathology compared with their male counterparts.28 Factors such as hormonal status, higher blood pressure, and increased physical activity may predispose male KO mice to severe myocardial damage, dilatation, and dysfunction and mortality compared with female mice with increasing age.28–30
Intriguingly, AAV9-mediated expression of TSP-2 in 7-week-old TSP-2 KO mice rescued this lethal phenotype, confirming that postnatal expression of TSP-2 is crucial for the normal physiological aging of the heart. These results also make it less probable that prenatal or early postnatal morphological changes in the heart of TSP-2 KO mice could underlie this progressive phenotype. Because of its cardiotrophic properties, AAV9-mediated gene transfer rescues a severe cardiac phenotype. AAV9-mediated gene transfer deserves to be further explored as a novel therapeutic tool for the clinical application of gene therapy in human cardiac diseases.31
To study whether aging of cardiomyocytes in the absence of TSP-2 may affect their function per se, isometric force measurements were performed in isolated, permeabilized cardiomyocytes. Active forces did not differ in aged TSP-2 KO compared with WT cardiomyocytes, whereas passive forces indicative of diastolic dysfunction were increased in the surviving TSP-2 KO cardiomyocytes. These alterations were independent of exogenous PKA activity, excluding myofilament phosphorylation as the underlying cause of cardiac dysfunction of isolated cardiomyocytes. Together, these data indicate that the surviving individual cardiomyocytes in the aged TSP-2 KO heart are characterized by increased stiffness, whereas active forces are not affected. Thus, progressive myocyte death, but not reduced contractility of individual cardiomyocytes, is responsible for the progressive cardiac dysfunction in the absence of TSP-2.
Our present study reveals a novel and pivotal role for TSP-2 in the protection against age-related cardiomyopathy. Decreased activation of the Src/Akt survival pathway in the absence of TSP-2, together with increased inflammation, MMP-2 activity, and decreased collagen cross-linking, all contributed to impaired cardiomyocyte survival and increased cardiac dilatation and dysfunction with advanced age.
TSP-2 seems to represent a novel and crucial survival mechanism that protects against senescence of the heart.
We thank Rick van Leeuwen, Wouter Verhesen, Kevin Custers, and Hans Duimel for their technical support.
Sources of Funding
This study was supported by the Marie Curie Excellence Program to Dr Heymans, M. Swinnen, and Dr Pinto; a research grant from the Research Fund K.U. Leuven (PDMK/08/175) to Dr Vanhoutte; long-term structural funding–Methusalem funding by the Flemish government to Dr Carmeliet; a grant for SFB-TR 19 (project A2) to Dr Westermann from the Deutsche Forschungsgemeinschaft; a research grant from the Research Foundation Flanders (G.0601.09 and WOG) to Drs VandenDriessche and Chuah; a research grant from the Research Foundation Flanders (G.0740.09.N10) to Dr Van de Werf; grants to Dr Sage from the Gilbertson Foundation and National Institute of Health (GM40711) and Ingenious Hypercare NoE from the European Union (EST 2005–020706–2); and research grants from the Netherlands Heart Foundation (2007B036, 2008B011) and a VIDI grant from the Netherlands Organization for Scientific Research to Dr Heymans. Dr Pinto is an established investigator of the Netherlands Heart Foundation.
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The incidence of heart failure increases with advanced aging. Still, some people get old (>80 years of age) without having their heart affected. With a healthy lifestyle and in the absence of hypertension, diabetes mellitus, or smoking, the heart is perfectly able to “endure” for a human lifetime. Identification of the protective mechanisms that allow the heart to age “for a long time” could lead to new therapeutic targets to delay its senescence and to treat heart failure in general. The present study proposes thrombospondin-2 as such a protective mechanism. The absence of this matrix protein in mice resulted in progressive dilated cardiomyopathy beginning in their middle age as a result of progressive cardiomyocyte death and matrix disruption. Importantly, overexpression of thrombospondin-2 via adeno-associated virus-9 gene transfer, a promising technique for gene therapy in human diseases, completely prevented this age-related cardiomyopathy. Thus, the postnatal presence of thrombospondin-2 is essential for “healthy” aging of the heart. Recent findings that modification of the extracellular environment by aging cells protects against age-related pathology should encourage the use of thrombospondin-2 and other matrix components as a novel therapeutic strategy for the prevention of heart failure in the aging individual.
↵*The first 2 authors contributed equally to this work.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.863266/DC1.