Versican accumulation drives Nos2 induction and aortic disease in Marfan syndrome via Akt activation.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening condition associated with Marfan syndrome (MFS), a disease caused by fibrillin-1 gene mutations. While various conditions causing TAAD exhibit aortic accumulation of the proteoglycans versican (Vcan) and aggrecan (Acan), it is unclear whether these ECM proteins are involved in aortic disease. Here, we find that Vcan, but not Acan, accumulated in Fbn1C1041G/+ aortas, a mouse model of MFS. Vcan haploinsufficiency protected MFS mice against aortic dilation, and its silencing reverted aortic disease by reducing Nos2 protein expression. Our results suggest that Acan is not an essential contributor to MFS aortopathy. We further demonstrate that Vcan triggers Akt activation and that pharmacological Akt pathway inhibition rapidly regresses aortic dilation and Nos2 expression in MFS mice. Analysis of aortic tissue from MFS human patients revealed accumulation of VCAN and elevated pAKT-S473 staining. Together, these findings reveal that Vcan plays a causative role in MFS aortic disease in vivo by inducing Nos2 via Akt activation and identify Akt signaling pathway components as candidate therapeutic targets.

Increased Proteoglycanases in Pulmonary Valves after Birth Correlate with Extracellular Matrix Maturation and Valve Sculpting.

Increased mechanical forces on developing cardiac valves drive formation of the highly organized extracellular matrix (ECM) providing tissue integrity and promoting cell behavior and signaling. However, the ability to investigate the response of cardiac valve cells to increased mechanical forces is challenging and remains poorly understood. The developmental window from birth (P0) to postnatal day 7 (P7) when biomechanical forces on the pulmonary valve (PV) are altered due to the initiation of blood flow to the lungs was evaluated in this study. Grossly enlarged PV, in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from postnatal day 0 (P0) to P7; the Adamts5-/- aortic valves (AV) did not exhibit a phenotypic correction. We hypothesized that blood flow, initiated to the lungs at birth, alters mechanical load on the PV and promotes ECM maturation. In the Adamts5-/- PV, there was an increase in localization of the proteoglycan proteases ADAMTS1, MMP2, and MMP9 that correlated with reduced Versican (VCAN). At birth, Decorin (DCN), a Collagen I binding, small leucine-rich proteoglycan, exhibited complementary stratified localization to VCAN in the wild type at P0 but colocalized with VCAN in Adamts5-/- PV; concomitant with the phenotypic rescue at P7, the PVs in Adamts5-/- mice exhibited stratification of VCAN and DCN similar to wild type. This study indicates that increased mechanical forces on the PV at birth may activate ECM proteases to organize specialized ECM layers during cardiac valve maturation.

Excess Provisional Extracellular Matrix: A Common Factor in Bicuspid Aortic Valve Formation.

A bicuspid aortic valve (BAV) is the most common cardiac malformation, found in 0.5% to 2% of the population. BAVs are present in approximately 50% of patients with severe aortic stenosis and are an independent risk factor for aortic aneurysms. Currently, there are no therapeutics to treat BAV, and the human mutations identified to date represent a relatively small number of BAV patients. However, the discovery of BAV in an increasing number of genetically modified mice is advancing our understanding of molecular pathways that contribute to BAV formation. In this study, we utilized the comparison of BAV phenotypic characteristics between murine models as a tool to advance our understanding of BAV formation. The collation of murine BAV data indicated that excess versican within the provisional extracellular matrix (P-ECM) is a common factor in BAV development. While the percentage of BAVs is low in many of the murine BAV models, the remaining mutant mice exhibit larger and more amorphous tricuspid AoVs, also with excess P-ECM compared to littermates. The identification of common molecular characteristics among murine BAV models may lead to BAV therapeutic targets and biomarkers of disease progression for this highly prevalent and heterogeneous cardiovascular malformation.

Adamts5-/- Mice Exhibit Altered Aggrecan Proteolytic Profiles That Correlate With Ascending Aortic Anomalies.

OBJECTIVE: Investigate the requirement of Aggrecan (Acan) cleavage during aortic wall development in a murine model with ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin-type motifs) 5 deficiency and bicuspid aortic valves. APPROACH: Mice with altered extracellular matrix remodeling of proteoglycans will be examined for anomalies in ascending aortic wall development. Neo-epitope antibodies that recognize ADAMTS cleaved Acan fragments will be used to investigate the mechanistic requirement of Acan turnover, in aortic wall development. RESULTS: Adamts5-/-;Smad2+/- mice exhibited a high penetrance of aortic anomalies (n=17/17); Adamts5-/-;Smad2+/- mice with bicuspid aortic valves (7/17) showed a higher number of anomalies than Adamts5-/-;Smad2+/- mice with tricuspid aortic valves. Single mutant Adamts5-/- mice also displayed a high penetrance of aortic anomalies (n=19/19) compared with wild type (n=1/11). Aortic anomalies correlated with Acan accumulation that was apparent at the onset of elastogenesis in Adamts5-/- mice. Neo-epitope antibodies that recognize the initial amino acids in the Acan cleaved fragments neo-FREEE, neo-GLGS, and neo-SSELE were increased in the Adamts5-/- aortas compared with WT. Conversely, neo-TEGE, which recognizes highly digested Acan core fragments, was reduced in Adamts5-/- mice. However, mice containing a mutation in the TEGE373↓374ALGSV site, rendering it noncleavable, had low penetrance of aortic anomalies (n=2/4). Acan neo-DIPEN and neo-FFGVG fragments were observed in the aortic adventitia; Acan neo-FFGVG was increased abnormally in the medial layer and overlapped with smooth muscle cell loss in Adamts5-/- aortas. CONCLUSIONS: Disruption of ADAMTS5 Acan cleavage during development correlates with ascending aortic anomalies. These data indicate that the mechanism of ADAMTS5 Acan cleavage may be critical for normal aortic wall development.

Intercalated cushion cells within the cardiac outflow tract are derived from the myocardial troponin T type 2 (Tnnt2) Cre lineage.

BACKGROUND: The origin of the intercalated cushions that develop into the anterior cusp of the pulmonary valve (PV) and the noncoronary cusp of the aortic valve (AV) is not well understood. RESULTS: Cre transgenes in combination with the Rosa TdTomato-EGFP reporter were used to generate three-dimensional lineage mapping of AV and PV cusps during intercalated cushion development. Tie2-Cre;EGFP was used to mark endothelial-derived mesenchymal cells, Wnt1-Cre;EGFP for cardiac neural crest and cardiac Troponin T (Tnnt2)Cre;EGFP, for myocardial lineage. The highest percentage of intercalated cushion cells at embryonic day (E) 12.5 was Tnnt2-Cre; EGFP positive; 68.0% for the PV and 50.0% AV. Neither Tnnt2 mRNA nor Tnnt2-Cre protein was expressed in the intercalated cushions; and the Tnnt2-Cre lineage intercalated cushion cells were also positive for the mesenchymal markers Sox9 and versican. Tnnt2-Cre lineage was present within the forming intercalated cushions from E11.5 and was present in the intercalated cushion derived PV and AV cusps and localized to the fibrosa layer at postnatal day 0. CONCLUSIONS: Intercalated cushions of the developing outflow tract are populated with Tnnt2-Cre derived cells, a Cre reporter previously used for tracing and excision of myocardial cells and not previously associated with mesenchymal cells. Developmental Dynamics 247:1005-1017, 2018. © 2018 Wiley Periodicals, Inc.

Development of myotendinous-like junctions that anchor cardiac valves requires fibromodulin and lumican.

BACKGROUND: There are many patients that exhibit connective tissue related cardiac malformations but do not have mutations in collagen genes. The Small Leucine Rich Proteoglycans (SLRP) fibromodulin (FMOD) and lumican (LUM) bind collagen and regulate fibril assembly in other biological contexts. RESULTS: FMOD deficient mice and double deficient FMOD; LUM mice exhibited anomalies in regions where cardiac valve tissue interdigitates with adjacent muscle for support. Ectopic connective and/or myocardial tissue(s) was associated with the more severe cardiac valve anomalies in FMOD; LUM deficient mice. At postnatal day 0 (P0) there was an increase in the mesenchymal cell number in the regions where valve cusps anchor in FMOD; LUM deficient mice compared to WT. The cardiac valve anomalies correlated with the highest levels of FMOD expression in the heart and also where myotendinous junctions (MTJ) components biglycan, collagen type I alpha 1, and collagen type VI, are also localized. CONCLUSIONS: The postnatal assembly of the collagen-rich ECM in regions where cardiac valves anchor, that we have designated 'myotendinous-like junctions' (MTLJ) requires the SLRPs FMOD and LUM. Moreover, FMOD and LUM may facilitate mesenchymal cell differentiation in late stages of cardiac valve development. Developmental Dynamics 245:1029-1042, 2016. © 2016 Wiley Periodicals, Inc.

Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development.

Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly.

The ability of the heart to adapt to increased stress is dependent on the modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest that LUM deficient mice (lum(-/-)) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum(-/-) hearts have not been evaluated. These studies show that LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum(-/-) neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum(-/-) hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum(-/-) hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum(-/-) hearts. There was also a reduction in the ß and γ forms of collagenα1(I) in lum(-/-) hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum(-/-) hearts, indicating that LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart. Further investigation into the role of LUM may yield novel therapeutic targets and/or biomarkers for patients with cardiovascular disease.

Inhibition of class I histone deacetylase activity represses matrix metalloproteinase-2 and -9 expression and preserves LV function postmyocardial infarction.

Left ventricular (LV) remodeling, after myocardial infarction (MI), can result in LV dilation and LV pump dysfunction. Post-MI induction of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated as causing deleterious effects on LV and extracellular matrix remodeling in the MI region and within the initially unaffected remote zone. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We assessed the efficacy of both class I/IIb- and class I-selective HDAC inhibitors on MMP-2 and MMP-9 abundance and determined if treatment resulted in the attenuation of adverse LV and extracellular matrix remodeling and improved LV pump function post-MI. MI was surgically induced in MMP-9 promoter reporter mice and randomized for treatment with a class I/IIb HDAC inhibitor for 7 days post-MI. After MI, LV dilation, LV pump dysfunction, and activation of the MMP-9 gene promoter were significantly attenuated in mice treated with either the class I/IIb HDAC inhibitor tichostatin A or suberanilohydroxamic acid (voronistat) compared with MI-only mice. Immunohistological staining and zymographic levels of MMP-2 and MMP-9 were reduced with either tichostatin A or suberanilohydroxamic acid treatment. Class I HDAC activity was dramatically increased post-MI. Treatment with the selective class I HDAC inhibitor PD-106 reduced post-MI levels of both MMP-2 and MMP-9 and attenuated LV dilation and LV pump dysfunction post-MI, similar to class I/IIb HDAC inhibition. Taken together, these unique findings demonstrate that selective inhibition of class I HDACs may provide a novel therapeutic means to attenuate adverse LV remodeling post-MI.

Fibulin-1 suppresses endothelial to mesenchymal transition in the proximal outflow tract.

Endothelial to mesenchymal transition (EMT) that occurs during cardiac outflow tract (OFT) development is critical for formation of the semilunar valves. Fibulin-1 (Fbln1) is an extracellular matrix protein that is present at several sites of EMT, including the OFT (i.e., E9.5-10.5). The aim of this study was to determine the role of Fbln1 in EMT during the earliest events of OFT development. Examination of proximal OFT cushions in Fbln1 null embryos detected hypercellularity at both E9.5 (93% increase; p = 0.002) and E10.5 (43% increase; p = 0.01) as compared to wild type, suggesting that Fbln1 normally suppresses OFT endocardial cushion EMT. This was supported by studies of proximal OFT cushion explants, which showed that explants from Fbln1 null embryos displayed a 58% increase in cells migrating from the explants as compared to wild type (p = 0.005). We next evaluated the effects of Fbln1 deficiency on the expression of factors that regulate proximal OFT EMT. At E9.5, Fbln1 null proximal OFT endocardium and EMT-derived mesenchyme showed increased TGFß2 (58% increase; p = 0.01) and increased Snail1-positive nuclei (27% increase; p = 0.0003). Histological examination of OFT cushions in Fbln1 null embryos (E9.5) also detected cells present in the cushion that were determined to be erythrocytes based on round morphology, autofluorescence, and positive staining for hemoglobin. Erythrocytes were also detected in Fbln1 null OFT cushions at E10.5. Together, the findings indicate that Fbln1 normally suppresses proximal OFT EMT preventing proximal cushion hypercellularity and blood cell accumulation.

Imbalanced expression of Vcan mRNA splice form proteins alters heart morphology and cellular protein profiles.

The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan ((tm1Zim)), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan ((tm1Zim)) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.

Small leucine-rich proteoglycans exhibit unique spatiotemporal expression profiles during cardiac valve development.

BACKGROUND: Small Leucine Rich Proteoglycans (SLRPs) play a role in collagen fiber formation and also function as signaling molecules. Given the importance of collagen synthesis to the cardiovascular extracellular matrix (ECM), we examined the spatiotemporal expression of SLRPs, not previously investigated in the murine heart. RESULTS: Cardiac expression using antibodies specific for biglycan (BGN), decorin (DCN), fibromodulin (FMOD), and lumican (LUM) revealed distinct patterns among the SLRPs in mesenchymal-derived tissues. DCN showed the most intense localization within the developing valve cusps, while LUM was evident primarily in the hinge region of postnatal cardiac valves. BGN, DCN, and FMOD were immunolocalized to regions where cardiac valves anchor into adjacent tissues. Medial (BGN) and adventitial (BGN, DCN, FMOD and LUM) layers of the pulmonary and aortic arteries also showed intense staining of SLRPs but this spatiotemporal expression varied with developmental age. CONCLUSIONS: The unique expression patterns of SLRPs suggest they have adapted to specialized roles in the cardiovascular ECM. SLRP expression patterns overlap with areas where TGFß signaling is critical to the developing heart. Therefore, we speculate that SLRPs may not only be required to facilitate collagen fiber formation but may also regulate TGFß signaling in the murine heart.

Insufficient versican cleavage and Smad2 phosphorylation results in bicuspid aortic and pulmonary valves.

Bicuspid or bifoliate aortic valve (BAV) results in two rather than three cusps and occurs in 1-2% of the population placing them at higher risk of developing progressive aortic valve disease. Only NOTCH-1 has been linked to human BAV, and genetically modified mouse models of BAV are limited by low penetrance and additional malformations. Here we report that in the Adamts5(-/-) valves, collagen I, collagen III, and elastin were disrupted in the malformed hinge region that anchors the mature semilunar cusps and where the ADAMTS5 proteoglycan substrate versican, accumulates. ADAMTS5 deficient prevalvular mesenchyme also exhibited a reduction of α-smooth muscle actin and filamin A suggesting versican cleavage may be involved in TGFß signaling. Subsequent evaluation showed a significant decrease of pSmad2 in regions of prevalvular mesenchyme in Adamts5(-/-) valves. To test the hypothesis that ADAMTS5 versican cleavage is required, in part, to elicit Smad2 phosphorylation we further reduced Smad2 in Adamts5(-/-) mice through intergenetic cross. The Adamts5(-/-);Smad2(+/-) mice had highly penetrant BAV and bicuspid pulmonary valve (BPV) malformations as well as increased cusp and hinge size compared to the Adamts5(-/-) and control littermates. These studies demonstrate that semilunar cusp malformations (BAV and BPV) can arise from a failure to remodel the proteoglycan-rich provisional ECM. Specifically, faulty versican clearance due to ADAMTS5 deficiency blocks the initiation of pSmad2 signaling, which is required for excavation of endocardial cushions during aortic and pulmonary valve development. Further studies using the Adamts5(-/-); Smad2(+/-) mice with highly penetrant and isolated BAV, may lead to new pharmacological treatments for valve disease.

Maladaptive matrix remodeling and regional biomechanical dysfunction in a mouse model of aortic valve disease.

Aortic valve disease (AVD) occurs in 2.5% of the general population and often requires surgical intervention. Aortic valve malformation (AVM) underlies the majority of cases, suggesting a developmental etiology. Elastin haploinsufficiency results in complex cardiovascular problems, and 20-45% of patients have AVM and/or AVD. Elastin insufficient (Eln+/-) mice demonstrate AVM and latent AVD due to abnormalities in the valve annulus region. The objective of this study was to examine extracellular matrix (ECM) remodeling and biomechanical properties in regional aortic valve tissue and determine the impact of early AVM on late AVD in the Eln+/- mouse model. Aortic valve ECM composition and remodeling from juvenile, adult, and aged stages were evaluated in Eln+/- mice using histology, ELISA, immunohistochemistry and gelatin zymography. Aortic valve tissue biomechanical properties were determined using micropipette aspiration. Cartilage-like nodules were demonstrated within the valve annulus region at all stages identifying a developmental abnormality preceding AVD. Interestingly, maladaptive ECM remodeling was observed in early AVM without AVD and worsened with late AVD, as evidenced by increased MMP-2 and MMP-9 expression and activity, as well as abnormalities in ADAMTS-mediated versican processing. Cleaved versican was increased in the valve annulus region of aged Eln+/- mice, and this abnormality correlated temporally with adverse alterations in valve tissue biomechanical properties and the manifestation of AVD. These findings identify maladaptive ECM remodeling in functional AVM as an early disease process with a progressive natural history, similar to that seen in human AVD, emphasizing the importance of the annulus region in pathogenesis. Combining molecular and engineering approaches provides complementary mechanistic insights that may be informative in the search for new therapeutic targets and durable valve bioprostheses.

Fibulin-1 is required during cardiac ventricular morphogenesis for versican cleavage, suppression of ErbB2 and Erk1/2 activation, and to attenuate trabecular cardiomyocyte proliferation.

BACKGROUND: Trabeculation is an integral component of cardiac ventricular morphogenesis and is dependent on the matrix metalloproteinase, ADAMTS1. A substrate of ADAMTS1 is the proteoglycan versican which is expressed in the developing ventricle and which has been implicated in trabeculation. Fibulin-1 is a versican and ADAMTS1-binding extracellular matrix protein required for ventricular morphogenesis. Here we investigated the involvement of fibulin-1 in ADAMTS1-mediated cleavage of versican in vitro, and the involvement of fibulin-1 in versican cleavage in ventricular morphogenesis. RESULTS: We show that fibulin-1 is a cofactor for ADAMTS1-dependent in vitro cleavage of versican V1, yielding a 70-kDa amino-terminal fragment. Furthermore, fibulin-1-deficiency in mice was found to cause a significant reduction (>90%) in ventricular levels of the 70-kDa versican V1 cleavage product and a 2-fold increase in trabecular cardiomyocyte proliferation. Decreased versican V1 cleavage and augmented trabecular cardiomyocyte proliferation in fibulin-1 null hearts is accompanied by increased ventricular activation of ErbB2 and Erk1/2. By contrast, versican deficiency was found to lead to decreased cardiomyocyte proliferation and reduced ventricular trabeculation. CONCLUSION: We conclude that fibulin-1 regulates versican-dependent events in ventricular morphogenesis by promoting ADAMTS1 cleavage of versican leading to suppression of trabecular cardiomyocyte proliferation mediated by the ErbB2-Map kinase pathway.

Altered versican cleavage in ADAMTS5 deficient mice; a novel etiology of myxomatous valve disease.

In fetal valve maturation the mechanisms by which the relatively homogeneous proteoglycan-rich extracellular matrix (ECM) of endocardial cushions is replaced by a specialized and stratified ECM found in mature valves are not understood. Therefore, we reasoned that uncovering proteases critical for 'remodeling' the proteoglycan rich (extracellular matrix) ECM may elucidate novel mechanisms of valve development. We have determined that mice deficient in ADAMTS5, (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) which we demonstrated is expressed predominantly by valvular endocardium during cardiac valve maturation, exhibited enlarged valves. ADAMTS5 deficient valves displayed a reduction in cleavage of its substrate versican, a critical cardiac proteoglycan. In vivo reduction of versican, in Adamts5(-/-) mice, achieved through Vcan heterozygosity, substantially rescued the valve anomalies. An increase in BMP2 immunolocalization, Sox9 expression and mesenchymal cell proliferation were observed in Adamts5(-/-) valve mesenchyme and correlated with expansion of the spongiosa (proteoglycan-rich) region in Adamts5(-/-) valve cusps. Furthermore, these data suggest that ECM remodeling via ADAMTS5 is required for endocardial to mesenchymal signaling in late fetal valve development. Although adult Adamts5(-/-) mice are viable they do not recover from developmental valve anomalies and have myxomatous cardiac valves with 100% penetrance. Since the accumulation of proteoglycans is a hallmark of myxomatous valve disease, based on these data we hypothesize that a lack of versican cleavage during fetal valve development may be a potential etiology of adult myxomatous valve disease.

Long-term localized high-frequency electric stimulation within the myocardial infarct: effects on matrix metalloproteinases and regional remodeling.

BACKGROUND: Disruption of the balance between matrix metalloproteinases (MMP) and MMP inhibitors (TIMPs) within a myocardial infarct (MI) contributes to left ventricular wall thinning and changes in regional stiffness at the MI region. This study tested the hypothesis that a targeted regional approach through localized high-frequency stimulation (LHFS) using low-amplitude electric pulses instituted within a formed MI scar would alter MMP/TIMP levels and prevent MI thinning. METHODS AND RESULTS: At 3 weeks after MI, pigs were randomized for LHFS (n=7; 240 bpm, 0.8 V, 0.05-ms pulses) or were left unstimulated (UNSTIM; n=10). At 4 weeks after MI, left ventricular wall thickness (echocardiography; 0.89+/-0.07 versus 0.67+/-0.08 cm; P<0.05) and regional stiffness (piezoelectric crystals; 14.70+/-2.08 versus 9.11+/-1.24; P<0.05) were higher with LHFS than in UNSTIM. In vivo interstitial MMP activity (fluorescent substrate cleavage; 943+/-59 versus 1210+/-72 U; P<0.05) in the MI region was lower with LHFS than in UNSTIM. In the MI region, MMP-2 levels were lower and TIMP-1 and collagen levels were higher with LHFS than in UNSTIM (all P<0.05). Transforming growth factor-beta receptor 1 and phosphorylated SMAD-2/3 levels within the MI region were higher with LHFS than in UNSTIM. Electric stimulation (4 Hz) of isolated fibroblasts resulted in reduced MMP-2 and MT1-MMP levels but increased TIMP-1 levels compared with unstimulated fibroblasts. CONCLUSIONS: These unique findings demonstrate that LHFS of the MI region altered left ventricular wall thickness and material properties, likely as a result of reduced regional MMP activity. Thus, LHFS may provide a novel means to favorably modify left ventricular remodeling after MI.

Reduced versican cleavage due to Adamts9 haploinsufficiency is associated with cardiac and aortic anomalies.

Here, we demonstrate that ADAMTS9, a highly conserved versican-degrading protease, is required for correct cardiovascular development and adult homeostasis. Analysis of Adamts9(+/LacZ) adult mice revealed anomalies in the aortic wall, valvulosinus and valve leaflets. Abnormal myocardial projections and 'spongy' myocardium consistent with non-compaction of the left ventricle were also found in Adamts9(+/LacZ) mice. During development, Adamts9 was expressed in derivatives of the Secondary Heart Field, vascular smooth muscle cells in the arterial wall, mesenchymal cells of the valves, and non-myocardial cells of the ventricles, but expression also continued in the adult heart and ascending aorta. Thus, the adult cardiovascular anomalies found in Adamts9(+/LacZ) hearts could result from subtle developmental alterations in extracellular matrix remodeling or defects in adult homeostasis. The valvular and aortic anomalies of Adamts9(+/LacZ) hearts were associated with accumulation of versican and a decrease in cleaved versican relative to WT littermates. These data suggest a potentially important role for ADAMTS9 cleavage of versican, or other, as yet undefined substrates in development and allostasis of cardiovascular extracellular matrix. In addition, these studies identify ADAMTS9 as a potential candidate gene for congenital cardiac anomalies. Mouse models of ADAMTS9 deficiency may be useful to study myxomatous valve degeneration.

beta-Adrenergic receptor stimulated Ncx1 upregulation is mediated via a CaMKII/AP-1 signaling pathway in adult cardiomyocytes.

The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart.