Adipokine profile in long-term juvenile dermatomyositis, and associations with adipose tissue distribution and cardiac function: a cross-sectional study.

OBJECTIVES: In long-term juvenile dermatomyositis (JDM), altered adipose tissue distribution and subclinical cardiac dysfunction have been described. Our aims were to compare adipokine levels in patients with JDM after long-term disease with controls, and explore associations between adipokines and (1) adipose tissue distribution and (2) cardiac function. METHODS: The study cohort included 59 patients with JDM (60% female, mean age 25.2 years, mean disease duration 16.9 years), and 59 age/sex-matched controls. Updated Pediatric Rheumatology International Trials Organization criteria for clinically inactive JDM were used to stratify patients into active (JDM-active) or inactive (JDM-inactive) disease groups. Lipodystrophy was clinically assessed in all patients. In all study participants, we measured adipose tissue distribution by dual-energy X-ray absorptiometry and cardiac function by echocardiography. Serum adipokines (adiponectin, apelin-12, lipocalin-2, leptin, visfatin and resistin) were analysed using ELISA. RESULTS: Patients with JDM had higher leptin levels compared with controls (p≤0.01). In JDM-active, apelin-12 and visfatin were higher compared with JDM-inactive (p≤0.05). In JDM-total and JDM-active, lower adiponectin correlated with lipodystrophy and total fat mass. Also, systolic dysfunction correlated with: lower adiponectin in JDM-total, JDM-inactive and JDM-active, and with lower apelin-12 in JDM-total and JDM-active and resistin in JDM-active (all p≤0.05). Lower adiponectin correlated with diastolic dysfunction in JDM-total and JDM-active. CONCLUSION: After long-term disease, leptin levels were unfavourably regulated in patients with JDM compared with controls, and apelin-12 and visfatin in JDM-active versus JDM-inactive. We found associations between adipokines and both adipose tissue distribution and cardiac systolic function in all patients with JDM, which was most prominent in patients with active disease.

Adipose tissue distribution is associated with cardio-metabolic alterations in adult patients with juvenile-onset dermatomyositis.

OBJECTIVES: Primary aims were to compare adipose tissue distribution in adult patients with juvenile-onset DM (JDM), with matched controls. Secondary aims were to explore how adipose tissue distribution is associated with cardio-metabolic status (cardiac dysfunction and metabolic syndrome) in patients. METHODS: Thirty-nine JDM patients (all aged ≥18 y, mean age 31.7 y and 51% female) were examined mean 22.7 y (s.d. 8.9 y) after disease onset and compared with 39 age/sex-matched controls. In patients, disease activity and lipodystrophy were assessed by validated tools and use of prednisolone noted. In all participants, dual-energy X-ray absorptiometry (DXA) and echocardiography were used to measure visceral adipose tissue (VAT)(g) and cardiac function, respectively. Risk factors for metabolic syndrome were measured and associations with adipose tissue distribution explored. For primary and secondary aims, respectively, P-values ≤0.05 and ≤0.01 were considered significant. RESULTS: Patients exhibited a 2.4-fold increase in VAT, and reduced HDL-cholesterol values compared with controls (P-values ≤ 0.05). Metabolic syndrome was found in 25.7% of the patients and none of the controls. Cardiac dysfunction (systolic and/or diastolic) was found in 23.7% of patients and 8.1% of controls (P = 0.07). In patients, VAT levels were correlated with age, disease duration and occurrence of metabolic syndrome and cardiac dysfunction. Occurrence of lipodystrophy (P = 0.02) and male sex (P = 0.04) tended to be independently associated with cardiac dysfunction. CONCLUSION: Adults with JDM showed more central adiposity and cardio-metabolic alterations than controls. Further, VAT was found increased with disease duration, which was associated with development of cardio-metabolic syndrome.

Bone mineral density and explanatory factors in children and adults with juvenile dermatomyositis at long term follow-up; a cross sectional study.

BACKGROUND: Juvenile dermatomyositis (JDM) is the most common idiopathic inflammatory myopathy in children and adolescents. Both the disease and its treatment with glucocorticoids may negatively impact bone formation. In this study we compare BMD in patients (children/adolescence and adults) with long-standing JDM with matched controls; and in patients, explore how general/disease characteristics and bone turnover markers are associated with BMD. METHODS: JDM patients (n = 59) were examined median 16.8y (range 6.6-27.0y) after disease onset and compared with 59 age/sex-matched controls. Dual-energy X-ray absorptiometry (DXA) was used to measure BMD of the whole body and lumbar spine (spine) in all participants, and of ultra-distal radius, forearm and total hip in participants ≥20y only. Markers of bone turnover were analysed, and associations with outcomes explored. RESULTS: Reduced BMD Z-scores (<-1SD) were found in 19 and 29% of patients and 7 and 9% of controls in whole body and spine, respectively (p-values < 0.05). BMD and BMD Z-scores for whole body and spine were lower in all patients and for < 20y compared with their respective controls. In participants ≥20y, only BMD and BMD Z-score of forearm were lower in the patients versus controls. In patients, BMD Z-scores for whole body and/or spine were found to correlate negatively with prednisolone use at follow-up (yes/no) (age < 20y), inflammatory markers (age ≥ 20y) and levels of interferon gamma-induced protein 10 (IP-10) (both age groups). In all patients, prednisolone use at follow-up (yes/no) and age ≥ 20y were independent correlates of lower BMD Z-scores for whole body and spine, respectively. CONCLUSION: In long-term JDM, children have more impairment of BMD than adults in spine and whole-body. Associations with BMD were found for both prednisolone and inflammatory markers, and a novel association was discovered with the biomarker of JDM activity, IP-10.

Novel associations between cytokines and pulmonary involvement in juvenile dermatomyositis - a cross-sectional study of long-term disease.

OBJECTIVES: To examine associations between cytokines and pulmonary involvement in patients with medium- to long-term JDM. METHODS: In a cross-sectional study, 58 patients examined median (range) 16.8 (6.6-27.0) years after symptom onset were stratified in inactive (JDM-inactive) and active (JDM-active) disease (updated PRINTO criteria); 56 age/sex matched controls were included. Twenty-nine cytokines (in serum) were analysed (Luminex technology/ELISA). Pulmonary function test included forced vital capacity, total lung capacity (TLC) and diffusing capacity for carbon monoxide reported as % of predicted and low forced vital capacity/TLC/diffusing capacity for carbon monoxide. In patients, the presence of clinical pulmonary damage was assessed and high resolution computed tomography scans were scored for interstitial lung disease, chest wall calcinosis and airways disease. RESULTS: Median age of patients was 21 (7-55) years, 59% were female and 36% inactive. In JDM-active and all patients, higher MCP-1, IP-10 and eotaxin correlated with high-resolution computed tomography findings (rs 0.34-0.61; P < 0.05). MCP-1 and eotaxin correlated with pulmonary damage in JDM-active and all patients (rs 0.41-0.49; P < 0.01). Higher TGF-ß1 and PDGF (growth factors) were associated with lower lung volumes (forced vital capacity/TLC measures) in all patients; PDGF in JDM-active and TGF-ß1 in JDM-inactive patients. IP-10 correlated with TLC% in JDM-active patients. No associations between cytokines and pulmonary function test were found in controls. CONCLUSIONS: In JDM, we found a novel association (not previously described in myositis) between eotaxin and pulmonary involvement; we have previously shown an association between eotaxin and cardiac dysfunction. The associations between IP-10/growth factors/MCP-1 and pulmonary involvement are novel in JDM and were mostly seen in JDM-active patients.

Functional and Structural Adaptations of Skeletal Muscle in Long-Term Juvenile Dermatomyositis: A Controlled Cross-Sectional Study.

OBJECTIVE: To compare muscle strength and endurance of the knee extensors between patients with long-term juvenile dermatomyositis (DM) and controls and between patients with active disease and those with inactive disease, and to explore associations between strength/endurance and 1) clinical parameters, 2) physical activity, and 3) humoral/structural adaptation in the skeletal muscle of patients. METHODS: In a cross-sectional study (44 patients and 44 age- and sex-matched controls), we tested isometric muscle strength (peak torque, in Nm) and dynamic muscle endurance (total work, in Joules) of the knee extensors, physical activity (measured by accelerometer), and serum myokine levels (by enzyme-linked immunosorbent assay). Patients were examined with validated tools (clinical muscle tests and measures of disease activity/damage and inactive disease) and using magnetic resonance imaging of the thigh muscles, which included evaluation of the quadriceps cross-sectional area (CSA). Needle biopsy samples of the vastus lateralis muscle (obtained from 12 patients ages ≥18 years) were assessed by histochemistry. RESULTS: After a mean ± SD disease duration of 21.8 ± 11.8 years, peak torque was lower in patients with juvenile DM compared to controls (mean difference 29 Nm, 95% confidence interval 13-46; P = 0.001). Similarly, total work of the knee extensors was lower in patients compared to controls (median 738J [interquartile range 565-1,155] versus 1,249J [interquartile range 815-1,665]; P < 0.001). Both peak torque and total work were lower in patients with active juvenile DM compared to those with inactive disease (both P < 0.019); in analyses controlled for quadriceps CSA, only total work remained lower in patients with active disease. Moreover, peak torque and total work correlated with findings from clinical muscle tests in patients with active disease (r = 0.57-0.84). Muscle biopsy results indicated that the fiber type composition was different, but capillary density was similar, between patients with active disease and those with inactive disease. CONCLUSION: In patients with long-term juvenile DM, both muscle strength and endurance of the knee extensors were lower when compared to matched controls, and also lower in patients with active disease compared to those with inactive disease. Our results indicate a need for more sensitive muscle tests in this clinical setting. We hypothesize that impaired muscle endurance in patients with active juvenile DM may be influenced by structural/functional adaptations of muscle tissue independent of muscle size.

The extracellular matrix proteoglycan lumican improves survival and counteracts cardiac dilatation and failure in mice subjected to pressure overload.

Left ventricular (LV) dilatation is a key step in transition to heart failure (HF) in response to pressure overload. Cardiac extracellular matrix (ECM) contains fibrillar collagens and proteoglycans, important for maintaining tissue integrity. Alterations in collagen production and cross-linking are associated with cardiac LV dilatation and HF. Lumican (LUM) is a collagen binding proteoglycan with increased expression in hearts of patients and mice with HF, however, its role in cardiac function remains poorly understood. To examine the role of LUM in pressure overload induced cardiac remodeling, we subjected LUM knock-out (LUMKO) mice to aortic banding (AB) and treated cultured cardiac fibroblasts (CFB) with LUM. LUMKO mice exhibited increased mortality 1-14 days post-AB. Echocardiography revealed increased LV dilatation, altered hypertrophic remodeling and exacerbated contractile dysfunction in surviving LUMKO 1-10w post-AB. LUMKO hearts showed reduced collagen expression and cross-linking post-AB. Transcriptional profiling of LUMKO hearts by RNA sequencing revealed 714 differentially expressed transcripts, with enrichment of cardiotoxicity, ECM and inflammatory pathways. CFB treated with LUM showed increased mRNAs for markers of myofibroblast differentiation, proliferation and expression of ECM molecules important for fibrosis, including collagens and collagen cross-linking enzyme lysyl oxidase. In conclusion, we report the novel finding that lack of LUM attenuates collagen cross-linking in the pressure-overloaded heart, leading to increased mortality, dilatation and contractile dysfunction in mice.

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-ß, 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-ß, 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-ß 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.

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

Pressure overload-induced TGF-ß 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-ß 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-ß 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-1ß 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-ß1 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.

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.

Differential regulation of extracellular matrix constituents in myocardial remodeling with and without heart failure following pressure overload.

Patients with aortic stenosis develop various degrees of myocardial hypertrophy and heart failure (HF) despite comparable transvalvular gradients. An important element in the transition from compensated hypertrophy to HF is dilatation of the left ventricle (LV). The molecular pathology associated with LV dilatation and development of HF is not known. Thus, we examined potential differences in the regulation of myocardial extracellular matrix (ECM) constituents in mice with hypertrophy only (ABnonHF) and with HF (ABHF) as response to comparable pressure overload. The ascending aorta was banded, or left loose in sham-operated mice. Increased lung weight and left atrial diameter indicating pulmonary congestion were used to identify ABHF mice. Cardiac function and geometry were evaluated by echocardiography. Despite comparable pressure gradients and cardiac output, ABHF had reduced fractional shortening (23%), reduced systolic (28%) and diastolic (32%) tissue velocity and increased LV internal dimension in diastole (10%) and systole (17%) (LVIDd/s) compared to ABnonHF (p≤0.05). Microarray analyses identified 120 differently regulated genes related to ECM in ABHF compared to ABnonHF (p≤0.05). Interestingly, in ABHF, we found a 24% (p≤0.05) reduction of the LV collagen VIII protein levels despite increased levels of LV total collagen by 23% (p≤0.05). LV collagen VIII correlated negatively with LVIDd (R=0.55, p=0.03) and LVIDs (R=0.72, p=0.002). As this protein may function as a "sealant" binding collagen fibrils together, reduction of collagen VIII could potentially contribute to LV dilatation and development of HF.

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-ß1-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.