Biljana Skrbic

Syndecan-4 Is Essential for Development of Concentric Myocardial Hypertrophy via Stretch-Induced Activation of the Calcineurin-NFAT Pathway

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4−/− mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4−/−-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling.

Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5.

Pressure overload-induced TGF-β signaling activates cardiac fibroblasts (CFB) and leads to increased extracellular matrix (ECM) protein synthesis including fibrosis. Excessive ECM accumulation may in turn affect cardiac function contributing to development of heart failure. The aim of this study was to examine the effects of SM16, an orally active small molecular inhibitor of ALK5, on pressure overload-induced cardiac fibrosis. One week after aortic banding (AB), C57Bl/6J mice were randomized to standard chow or chow with SM16. Sham operated animals served as controls. Following 4 weeks AB, mice were characterized by echocardiography and cardiovascular magnetic resonance before sacrifice. SM16 abolished phosphorylation of SMAD2 induced by AB in vivo and by TGF-β in CFB in vitro. Interestingly, Masson Trichrome and Picrosirius Red stained myocardial left ventricular tissue revealed reduced development of fibrosis and collagen cross-linking following AB in the SM16 treated group, which was confirmed by reduced hydroxyproline incorporation. Furthermore, treatment with SM16 attenuated mRNA expression following induction of AB in vivo and stimulation with TGF-β in CFB in vitro of Col1a2, the cross-linking enzyme LOX, and the pro-fibrotic glycoproteins SPARC and osteopontin. Reduced ECM synthesis by CFB and a reduction in myocardial stiffness due to attenuated development of fibrosis and collagen cross-linking might have contributed to the improved diastolic function and cardiac output seen in vivo, in combination with reduced lung weight and ANP expression by treatment with SM16. Despite these beneficial effects on cardiac function and development of heart failure, mice treated with SM16 exhibited increased mortality, increased LV dilatation and inflammatory heart valve lesions that may limit the use of SM16 and possibly also other small molecular inhibitors of ALK5, as future therapeutic drugs.

Lumican is increased in experimental and clinical heart failure, and its production by cardiac fibroblasts is induced by mechanical and proinflammatory stimuli

During progression to heart failure (HF), myocardial extracellular matrix (ECM) alterations and tissue inflammation are central. Lumican is an ECM‐localized proteoglycan associated with inflammatory conditions and known to bind collagens. We hypothesized that lumican plays a role in the dynamic alterations in cardiac ECM during development of HF. Thus, we examined left ventricular cardiac lumican in a mouse model of pressure overload and in HF patients, and investigated expression, regulation and effects of increased lumican in cardiac fibroblasts. After 4 weeks of aortic banding, mice were divided into groups of hypertrophy (AB) and HF (ABHF) based on lung weight and left atrial diameter. Sham‐operated mice were used as controls. Accordingly, cardiac lumican mRNA and protein levels were increased in mice with ABHF. Similarly, cardiac biopsies from patients with end‐stage HF revealed increased lumican mRNA and protein levels compared with control hearts. In vitro, mechanical stretch and the proinflammatory cytokine interleukin‐1β increased lumican mRNA as well as secreted lumican protein from cardiac fibroblasts. Stimulation with recombinant glycosylated lumican increased collagen type I alpha 2, lysyl oxidase and transforming growth factor‐β1 mRNA, which was attenuated by costimulation with an inhibitor of the proinflammatory transcription factor NFκB. Furthermore, lumican increased the levels of the dimeric form of collagen type I, decreased the activity of the collagen‐degrading enzyme matrix metalloproteinase‐9 and increased the phosphorylation of fibrosis‐inducing SMAD3. In conclusion, cardiac lumican is increased in experimental and clinical HF. Inflammation and mechanical stimuli induce lumican production by cardiac fibroblasts and increased lumican altered molecules important for cardiac remodeling and fibrosis in cardiac fibroblasts, indicating a role in HF development.

Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart.

Sustained pressure overload induces heart failure, the main cause of mortality in the Western world. Increased understanding of the underlying molecular mechanisms is essential to improve heart failure treatment. Despite important functions in other tissues, cardiac proteoglycans have received little attention. Syndecan‐4, a transmembrane heparan sulfate proteoglycan, is essential for pathological remodeling, and we here investigated its expression and shedding during heart failure. Pressure overload induced by aortic banding for 24 h and 1 week in mice increased syndecan‐4 mRNA, which correlated with mRNA of inflammatory cytokines. In cardiac myocytes and fibroblasts, tumor necrosis factor‐α, interleukin‐1β and lipopolysaccharide through the toll‐like receptor‐4, induced syndecan‐4 mRNA. Bioinformatical and mutational analyses in HEK293 cells identified a functional site for the proinflammatory nuclear factor‐κB transcription factor in the syndecan‐4 promoter, and nuclear factor‐κB regulated syndecan‐4 mRNA in cardiac cells. Interestingly, tumor necrosis factor‐α, interleukin‐1β and lipopolysaccharide induced nuclear factor‐κB‐dependent shedding of the syndecan‐4 ectodomain from cardiac cells. Overexpression of syndecan‐4 with mutated enzyme‐interacting domains suggested enzyme‐dependent heparan sulfate chains to regulate shedding. In cardiac fibroblasts, lipopolysaccharide reduced focal adhesion assembly, shown by immunohistochemistry, suggesting that inflammation‐induced shedding affects function. After aortic banding, a time‐dependent cardiac recruitment of T lymphocytes was observed by measuring CD3, CD4 and CD8 mRNA, which was reduced in syndecan‐4 knockout hearts. Finally, syndecan‐4 mRNA and shedding were upregulated in failing human hearts. Conclusively, our data suggest that syndecan‐4 plays an important role in the immune response of the heart to increased pressure, influencing cardiac remodeling and failure progression.

Slow Ca2 + sparks de-synchronize Ca2 + release in failing cardiomyocytes: Evidence for altered configuration of Ca2 + release units?

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca(2+) transient which contributes to the impaired contractility observed in this condition. We investigated whether alterations in ryanodine receptor function promote slowing of Ca(2+) release in a murine model of congestive heart failure (CHF). Myocardial infarction was induced by left coronary artery ligation. When chronic CHF had developed (10 weeks post-infarction), cardiomyocytes were isolated from viable regions of the septum. Septal myocytes from SHAM-operated mice served as controls. Ca(2+) transients rose markedly slower in CHF than SHAM myocytes with longer time to peak (CHF=152 ± 12% of SHAM, P<0.05). The rise time of Ca(2+) sparks was also increased in CHF (SHAM=9.6 ± 0.6 ms, CHF=13.2 ± 0.7 ms, P<0.05), due to a sub-population of sparks (≈20%) with markedly slowed kinetics. Regions of the cell associated with these slow spontaneous sparks also exhibited slowed Ca(2+) release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca(2+) release across the cell. Dyssynchronous Ca(2+) transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms, but slow sparks were not associated with orphaned ryanodine receptors. Rather, mathematical modeling suggested that slow sparks could result from an altered composition of Ca(2+) release units, including a reduction in ryanodine receptor density and/or distribution of ryanodine receptors into sub-clusters. In conclusion, our findings indicate that slowed, dyssynchronous Ca(2+) transients in CHF result from alterations in Ca(2+) sparks, consistent with rearrangement of ryanodine receptors within Ca(2+) release units.

Inhibition of SMAD2 phosphorylation preserves cardiac function during pressure overload

AIMS Left ventricular (LV) pressure overload leads to myocardial remodelling and reduced cardiac function. Both cardioprotective and deleterious effects have been attributed to SMAD2/3 (SMAD, small mothers against decapentaplegic) signalling, but the role of these important molecules in pressure overload remains unclear. The aim of this study was to examine the effects of SMAD2 inhibition on cardiac function and remodelling in mice subjected to aortic banding (AB), using a small molecule inhibitor (SM16) of SMAD2 signalling. METHODS AND RESULTS C57BL/6 mice were subjected to 1 week of AB, which led to a three-fold increased phosphorylation of SMAD2 that was reduced by SM16 (P≤ 0.05), as measured by western blotting. Cardiac function was evaluated by echocardiography and was preserved by SM16, as fractional shortening was increased by 38% (P≤ 0.05) and mitral flow deceleration reduced by 28% compared with AB mice not receiving SM16 (P≤ 0.05). In accordance with this, SM16 abolished the 21% increase in lung weight in AB mice (P≤ 0.05). Cardiomyocyte hypertrophy and foetal gene expression, as measured by qPCR, were also reduced. Myocardial collagen protein was unaltered 1 week after AB. LV sarcoplasmic reticulum Ca(2+)ATPase (SERCA2) reduction in AB mice and in transforming growth factor-β1-stimulated rat cardiomyocytes was diminished by SM16. Ca(2+) transient decay kinetics were improved in cardiomyocytes isolated from AB mice receiving SM16. CONCLUSION In pressure overload, pharmacological inhibition of SMAD2 signalling attenuated cardiomyocyte hypertrophy and preserved cardiac function. SM16 prevented SMAD2-mediated downregulation of SERCA2 in vivo and in cardiomyocytes, suggesting improved cardiomyocyte Ca(2+) handling as a possible cardioprotective mechanism.

Syndecan-4 is a key determinant of collagen cross-linking and passive myocardial stiffness in the pressure-overloaded heart

AIMS Diastolic dysfunction is central to the development of heart failure. To date, there is no effective treatment and only limited understanding of its molecular basis. Recently, we showed that the transmembrane proteoglycan syndecan-4 increases in the left ventricle after pressure overload in mice and man, and that syndecan-4 via calcineurin/nuclear factor of activated T-cells (NFAT) promotes myofibroblast differentiation and collagen production upon mechanical stress. The aim of this study was to investigate whether syndecan-4 affects collagen cross-linking and myocardial stiffening in the pressure-overloaded heart. METHODS AND RESULTS Aortic banding (AB) caused concentric hypertrophy and increased passive tension of left ventricular muscle strips, responses that were blunted in syndecan-4(-/-) mice. Disruption of titin anchoring by salt extraction of actin and myosin filaments revealed that the effect of syndecan-4 on passive tension was due to extracellular matrix remodelling. Expression and activity of the cross-linking enzyme lysyl oxidase (LOX) increased with mechanical stress and was lower in left ventricles and cardiac fibroblasts from syndecan-4(-/-) mice, which exhibited less collagen cross-linking after AB. Expression of osteopontin (OPN), a matricellular protein able to induce LOX in cardiac fibroblasts, was up-regulated in hearts after AB, in mechanically stressed fibroblasts and in fibroblasts overexpressing syndecan-4, calcineurin, or NFAT, but down-regulated in fibroblasts lacking syndecan-4 or after NFAT inhibition. Interestingly, the extracellular domain of syndecan-4 facilitated LOX-mediated collagen cross-linking. CONCLUSIONS Syndecan-4 exerts a dual role in collagen cross-linking, one involving its cytosolic domain and NFAT signalling leading to collagen, OPN, and LOX induction in cardiac fibroblasts; the other involving the extracellular domain promoting LOX-dependent cross-linking.

A mouse model of reverse cardiac remodelling following banding‐debanding of the ascending aorta

Aim:  Myocardial remodelling during pressure overload might contribute to development of heart failure. Reverse remodelling normally occurs following aortic valve replacement for aortic stenosis; however, the details and regulatory mechanisms of reverse remodelling remain unknown. Thus, an experimental model of reverse remodelling would allow for studies of this process. Although models of aortic banding are widely used, only few reports of debanding models exist. The aim of this study was to establish a banding–debanding model in the mouse with repetitive careful haemodynamic evaluation by high‐resolution echocardiography.

Decorin, lumican, and their GAG chain-synthesizing enzymes are regulated in myocardial remodeling and reverse remodeling in the mouse

On the basis of the role of small, leucine-rich proteoglycans (SLRPs) in fibrogenesis and inflammation, we hypothesized that they could be involved in cardiac remodeling and reverse remodeling as occurs during aortic stenosis and after aortic valve replacement. Thus, in a well-characterized aortic banding-debanding mouse model, we examined the SLRPs decorin and lumican and enzymes responsible for synthesis of their glycosaminoglycan (GAG) chains. Four weeks after banding of the ascending aorta, mice were subjected to a debanding operation (DB) and were subsequently followed for 3 or 14 days. Sham-operated mice served as controls. Western blotting revealed a 2.5-fold increase in the protein levels of glycosylated decorin in mice with left ventricular pressure overload after aortic banding (AB) with a gradual decrease after DB. Interestingly, protein levels of three key enzymes responsible for decorin GAG chain synthesis were also increased after AB, two of them gradually declining after DB. The inflammatory chemokine (C-X-C motif) ligand 16 (CXCL16) was increased after AB but was not significantly altered following DB. In cardiac fibroblasts CXCL16 increased the expression of the GAG-synthesizing enzyme chondroitin polymerizing factor (CHPF). The protein levels of lumican core protein with N-linked oligosaccharides increased by sevenfold after AB and decreased again 14 days after DB. Lumican with keratan sulfate chains was not regulated. In conclusion, this study shows alterations in glycosylated decorin and lumican core protein that might be implicated in myocardial remodeling and reverse remodeling, with a potential important role for CS/DS GAG chain-synthesizing enzymes.

Lack of collagen VIII reduces fibrosis and promotes early mortality and cardiac dilatation in pressure overload in mice

AIMS In pressure overload, left ventricular (LV) dilatation is a key step in transition to heart failure (HF). We recently found that collagen VIII (colVIII), a non-fibrillar collagen and extracellular matrix constituent, was reduced in hearts of mice with HF and correlated to degree of dilatation. A reduction in colVIII might be involved in LV dilatation, and we here examined the role of reduced colVIII in pressure overload-induced remodelling using colVIII knock-out (col8KO) mice. METHODS AND RESULTS Col8KO mice exhibited increased mortality 3-9 days after aortic banding (AB) and increased LV dilatation from day one after AB, compared with wild type (WT). LV dilatation remained increased over 56 days. Forty-eight hours after AB, LV expression of main structural collagens (I and III) was three-fold increased in WT mice, but these collagens were unaltered in the LV of col8KO mice together with reduced expression of the pro-fibrotic cytokine TGF-β, SMAD2 signalling, and the myofibroblast markers Pxn, α-SMA, and SM22. Six weeks after AB, LV collagen mRNA expression and protein were increased in col8KO mice, although less pronounced than in WT. In vitro, neonatal cardiac fibroblasts from col8KO mice showed lower expression of TGF-β, Pxn, α-SMA, and SM22 and reduced migratory ability possibly due to increased RhoA activity and reduced MMP2 expression. Stimulation with recombinant colVIIIα1 increased TGF-β expression and fibroblast migration. CONCLUSION Lack of colVIII reduces myofibroblast differentiation and fibrosis and promotes early mortality and LV dilatation in response to pressure overload in mice.