Maurice Hayot

Julie Rodriguez

Barbara Vernus

Gilles Carnac

Elise Jean

David Allen

Lucie Goret

Philippe Obert

Robin Candau

Dr Anne Bonnieu
email: bonnieu@supagro.inra.fr

Myostatin up-regulation is associated with the skeletal muscle response to hypoxic stimuli

Keywords: hypoxic stimulus, myostatin, skeletal muscle atrophy, COPD, myotubes

Myostatin and hypoxia signalling pathways are able to induce skeletal muscle atrophy, but whether a relationship between these two pathways exists is currently unknown. Here, we tested the hypothesis that a potential mechanism for hypoxia effect on skeletal muscle may be through regulation of myostatin. We reported an induction of myostatin expression in muscles of rats exposed to chronic hypoxia. Interestingly, we also demonstrated increased skeletal muscle myostatin protein expression in skeletal muscle of hypoxemic patients with severe chronic obstructive pulmonary disease (COPD). Parallel studies in human skeletal muscle cell cultures showed that induction of myostatin expression in myotubes treated with hypoxiamimicking agent such as cobalt chloride (CoCl 2 ) is associated with myotube atrophy. Furthermore, we demonstrated that inhibition of myostatin by means of genetic deletion of myostatin or treatment with blocking antimyostatin antibodies inhibits the CoCl 2 -induced atrophy in muscle cells. Finally, addition of recombinant myostatin restored the CoCl 2 induced atrophy in myostatin deficient myotubes. These results strongly suggest that myostatin can play an essential role in the adaptation of skeletal muscle to hypoxic environment.

Myostatin, a member of the TGF superfamily of ligands, has been shown to be a negative regulator of skeletal muscle mass during embryogenesis and early postnatal muscle growth [START_REF] Jean | Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle[END_REF][START_REF] Mcpherron | Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[END_REF]. In mice, inactivation of myostatin leads to robust muscle growth resulting from a combination of muscle cell hyperplasia and hypertrophy. Subsequently, naturally inactivating mutations in the myostatin gene have been demonstrated to cause increased muscling in cattle, sheep, dogs and humans suggesting that the function of myostatin is conserved -across species [START_REF] Clop | A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep[END_REF][START_REF] Grobet | A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[END_REF][START_REF] Jean | Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle[END_REF][START_REF] Mcpherron | Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member[END_REF][START_REF] Mosher | A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs[END_REF][START_REF] Schuelke | Myostatin mutation associated with gross muscle hypertrophy in a child[END_REF].

More recently, data have demonstrated that myostatin negatively regulates muscle mass not only during early embryogenesis but later during adult muscle growth [START_REF] Grobet | Modulating skeletal muscle mass by postnatal, muscle-specific inactivation of the myostatin gene[END_REF][START_REF] Whittemore | Inhibition of myostatin in adult mice increases skeletal muscle mass and strength[END_REF]. In line with these findings, is the discovery that myostatin can inhibit the activation and self renewal of satellite cells [START_REF] Mccroskery | Myostatin negatively regulates satellite cell activation and self-renewal[END_REF] which are adult stem cells resident in skeletal muscle, responsible for postnatal muscle cell growth and new muscle protein production [START_REF] Moss | Satellite cells as the source of nuclei in muscles of growing rats[END_REF].

Several studies indicate that myostatin is an important mediator of muscle atrophy. For example, administration of myostatin in vivo to adult mice induces profound muscle loss analogous to that seen in human cachexia syndromes [START_REF] Zimmers | Induction of cachexia in mice by systemically administered myostatin[END_REF]. A decrease in muscle mass has been reported in transgenic mice overexpressing myostatin in skeletal muscle [START_REF] Reisz-Porszasz | Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin[END_REF]. Similarly, ectopic expression of myostatin through gene electrotransfer of a myostatin expression vector induces atrophy of skeletal adult muscle associated with decreased muscle gene expression [START_REF] Durieux | Ectopic expression of myostatin induces atrophy of adult skeletal muscle by decreasing muscle gene expression[END_REF]. Myostatin expression also appears to be sensitive to atrophic and/or hypertrophic signalling. For example, the anabolic agent growth hormone (GH) decreased myostatin expression in muscles of GH-A c c e p t e d M a n u s c r i p t 4 deficient adults [START_REF] Liu | Myostatin is a skeletal muscle target of growth hormone anabolic action[END_REF] and androgens negatively regulate myostatin expression in an androgen-dependent skeletal muscle [START_REF] Mendler | Androgens negatively regulate myostatin expression in an androgen-dependent skeletal muscle[END_REF]. In contrast, increased myostatin expression has been observed in several atrophic states, including prolonged bed rest in young men [START_REF] Zachwieja | Plasma myostatin-immunoreactive protein is increased after prolonged bed rest with low-dose T3 administration[END_REF], chronic disuse atrophy in older patients [START_REF] Reardon | Myostatin, insulin-like growth factor-1, and leukemia inhibitory factor mRNAs are upregulated in chronic human disuse muscle atrophy[END_REF] and age-related muscle wasting (sarcopenia) [START_REF] Yarasheski | Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men with muscle wasting[END_REF]. Despite this progress in our understanding of the role of myostatin in muscle atrophy, it is not currently known how myostatin is connected with atrophic signalling pathways.

Evidence has strongly suggested that hypoxia can also induce muscle atrophy. Hypoxia can be caused by a generalized reduction in oxygen delivery, such as altitude and pulmonary diseases, or by disruption in the local blood supply, such as an ischemic disorder. It is known that chronic exposure to high-altitude hypoxia [START_REF] Hoppeler | Morphological adaptations of human skeletal muscle to chronic hypoxia[END_REF][START_REF] Howald | Performing at extreme altitude: muscle cellular and subcellular adaptations[END_REF][START_REF] Macdougall | Operation Everest II: structural adaptations in skeletal muscle in response to extreme simulated altitude[END_REF] can cause a reduction of muscle fibre size and loss of skeletal muscle mass in mountaineers. Similar findings have been reported in healthy people who live at high altitude for generations and in rats chronically exposed to a simulated hypobaric altitude [START_REF] Sillau | Effects of hypoxia on capillary density and fiber composition in rat skeletal muscle[END_REF]. In addition, the decrease in muscle mass at high altitude is associated with an anorexic effect. However, even when the anorexic effect is controlled using pair feed experimental design the decrease in muscle mass is observed indicating that part of hypoxia-induced skeletal muscle atrophy is independent of undernutrition associated with hypoxia [START_REF] Bigard | Changes in dietary protein intake fail to prevent decrease in muscle growth induced by severe hypoxia in rats[END_REF][START_REF] Favier | Downregulation of Akt/mammalian target of rapamycin pathway in skeletal muscle is associated with increased REDD1 expression in response to chronic hypoxia[END_REF][START_REF] Pison | In vivo hypoxic exposure impairs metabolic adaptations to a 48 hour fast in rats[END_REF]. Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation. During the past ten years, COPD has emerged as a multi-organ-system disease [START_REF] Wouters | Management of severe COPD[END_REF]. Besides primary effects in the lung, COPD as a chronic disease has secondary effects on other systems including skeletal muscle. Specifically, COPD is characterized by skeletal muscle dysfunction and atrophy [START_REF] Agusti | Systemic effects of chronic obstructive pulmonary disease[END_REF][START_REF] Wouters | Local and systemic inflammation in chronic obstructive pulmonary disease[END_REF]. Chronic hypoxaemia, a common physiopathological consequence of COPD, has been proposed as a potential contributor, A c c e p t e d M a n u s c r i p t 5 among others, of peripheral muscle dysfunction in this disease [START_REF] Couillard | From muscle disuse to myopathy in COPD: potential contribution of oxidative stress[END_REF][START_REF] Gosker | Skeletal muscle fibre-type shifting and metabolic profile in patients with chronic obstructive pulmonary disease[END_REF]. However, the exact mechanisms underlying skeletal muscle dysfunction in COPD remain unclear. Understanding the mechanisms underlying the muscle response to hypoxia may provide insights into how muscle mass is regulated both in physiological and pathological situations.

Taking into consideration the importance of hypoxia and myostatin in skeletal muscle atrophy, we hypothesized that the muscle hypoxia effects might involve an up-regulation of myostatin gene expression. The aims of this study were first to characterize myostatin levels in skeletal muscles of rats exposed to hypobaric hypoxia and in skeletal muscles of hypoxemic COPD patients. We then investigated whether myostatin is required for the action of cobalt chloride (a hypoxic mimetic agent) in skeletal muscle cells.

Materials and Methods

Animals

Ten-week-old Wistar male rats (Harlan laboratories, France; n=30) were randomly assigned into 3 groups. Hypoxic animals (H rats, n=10) lived continuously for 5 weeks in hypobaric hypoxia (barometric pressure  475 mm Hg ; PIO 2 ≈90 mm Hg, altitude ≈ 4500m).

The two other groups of rats were maintained in normoxia (barometric pressure  760 mm Hg; PIO 2 ≈159 mm Hg, altitude ≈ 80m, N rats). The corresponding environments were created in steel chambers with a vacuum pump as previously described [START_REF] Goret | Training does not affect the alteration in pulmonary artery vasoreactivity in pulmonary hypertensive rats[END_REF]. Hypoxic rats (H) and normoxic rats (N) were fed ad libitum. Because of hypoxic impact on food intake and consequently on animal growth, a pair feed model was applied to normoxic rats (Npf) as previously described [START_REF] Bigard | Changes in dietary protein intake fail to prevent decrease in muscle growth induced by severe hypoxia in rats[END_REF]. Thus, the pair fed animals (Npf) were restricted to have the same food intake than the H rats.

Study population

COPD patients and Healthy control subjects

The study population consisted of 10 patients with clinically diagnosed very severe COPD as defined by the American Thoracic Society (ATS) and European Respiratory Society (ERS) [START_REF] Nici | American Thoracic Society/European Respiratory Society statement on pulmonary rehabilitation[END_REF] and by the "Global Initiative for Chronic Obstructive Lung Disease (GOLD) and a group of healthy sedentary age-matched controls. All patients suffered from chronic airflow limitation, defined as a measured Forced expiratory volume in 1 second (FEV1)/Forced vital capacity (FVC) < 60% of predicted values. They were under long term oxygen therapy for at least the last 6 months before the study. This group of patients showed low hypoxemia at rest with a mean value of PaO 2 = 64 ± 2 mm Hg. At the time of the study, patients were in stable clinical state, i.e. no clinical exacerbation during the last 4 weeks. All had sedentary way of life as confirmed by the Voorrips et al. questionnaire score <9 [START_REF] Voorrips | A physical activity questionnaire for the elderly[END_REF] and had not been enrolled in a pulmonary rehabilitation in the last year. Five healthy sedentary control subjects, volunteered to participate in the study, were non smokers and had no history or physical examination of lung or heart disease. They also had a low level of physical activity (Voorrips score <9). Exclusion criteria were no other potentially confounding factors such as neuromuscular disease, chronic heart failure, renal diseases, and liver diseases. No patient was on anticoagulant medication. Informed written consent was obtained from all patients, and the research protocol was approved by the institutional ethics committee of Montpellier Hospital and conducted in accordance with the Helsinki declaration and the European guidelines for "good clinical practice".

Localized muscle function tests:

The maximal voluntary contraction (MVC) and endurance of the quadriceps were assessed on an exercise bench according to the technique previously described by our group [START_REF] Delample | Implication of xanthine oxidase in muscle oxidative stress in COPD patients[END_REF].

Muscle biopsy of the vastus lateralis

Muscle biopsy procedures: A percutaneous muscle biopsy of the vastus lateralis muscle of the dominant leg was performed in each subject, at mid-thigh (15 cm above the patella) as described [START_REF] Bergstrom | Percutaneous needle biopsy of skeletal muscle in physiological and clinical research[END_REF] and often used by our group [START_REF] Hayot | Skeletal muscle microbiopsy: a validation study of a minimally invasive technique[END_REF]. The specimens were immediately frozen in isopentane cooled to freezing point with liquid nitrogen and stored at -80°C until analysis.

Assessment of body composition

Fat mass and fat-free mass (FFM) were determined by bioelectrical impedance analysis using a body composition analyser (model TBF-300; TANITA France, Neuilly-sur-Seine, France). Body mass index (Weight/Height²) and fat-free mass index (FFMI = FFM/Height²) were then calculated.

Cell culture products

Dulbecco's modified Eagle's medium (DMEM), nutrient mixture F-12 (Ham), nutrient mixture F-10 (Ham), horse serum were purchased from Sigma, fetal calf serum (FCS) from

Primary human muscle cell culture

Quadriceps muscle biopsie of a human adult (male, age 38 years) was obtained from the "AFM-BTR Banque de tissus pour la recherche" (Hôpital de la Pitié-Salpêtrière, Paris, France). Human myoblasts were initially prepared, purified and cultured as previously described [START_REF] Jean | Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle[END_REF]. Cells were plated at a density of 3×10 4 cells/cm 2 on Collagencoated Petri dishes (BD Biosciences), in growth medium DMEM supplemented with 20% serum and 2% ultroser (Gibco BRL). They were maintained at 37°C in a water-saturated atmosphere containing 5% CO2 in air. When human myoblasts reach confluency, differentiation was induced by serum depletion (2% FCS). Cells were kept in differentiation medium for 4 days.

Primary mouse muscle cell culture

Myostatin-/-mice, harboring a constitutive deletion of the third myostatin exon, have been described previously [START_REF] Grobet | Modulating skeletal muscle mass by postnatal, muscle-specific inactivation of the myostatin gene[END_REF] and were generously provided by L. Grobet (Faculty of Veterinary Medicine, University of Liège, Belgium). Animals were maintained on a 12h:12h light/dark cycle and were allowed free access to food. Mice were genotyped by polymerase chain reaction analysis of tail DNA. In vitro studies were performed using male mice between 4 and 6 weeks of age. Briefly, murine myoblasts were isolated from the whole mice muscles of the paw after enzymatic digestion by pronase [START_REF] Descamps | Inhibition of myoblast differentiation by Sfrp1 and Sfrp2[END_REF][START_REF] Levin | SFRP2 expression in rabbit myogenic progenitor cells and in adult skeletal muscles[END_REF]. Cells were plated at a density of 2x10 4 cells/cm 2 on Matrigel®-coated Petri dishes (BD Biosciences, France), in 80% Ham"s F10 supplemented with 20% horse serum (HS) (Sigma). They were maintained at 37°C in a water-saturated atmosphere containing 5%

A c c e p t e d M a n u s c r i p t 9 CO 2 in air. After 2 days, cells were washed with Ham"s F10 and placed in complete medium supplemented with 5 ng/ml basic fibroblast growth factor (bFGF). After two days, to achieve differentiation, satellite cells were switched to differentiation medium consisting of Ham"s F10, 20% HS without bFGF. Cells were kept in differentiation medium for 3 days.

Western blot

Muscle tissues or cells were lysed in hypotonic buffer (20mM Tris pH 7.5, 10mM NaCl) and protein concentrations were determined using the BioRad DC kit (Biorad). 50g of total proteins diluted in 1x Laemmli buffer (100mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 200mM DTT, bromophenol blue) were transferred to nitrocellulose membranes (Schleicher and Schull Bioscience, Dassel, Germany). Western blots were performed as previously described [START_REF] Froeschle | Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis[END_REF]. The following primary antibodies were used: monoclonal anti-MHC fast (Sigma) diluted 1/1000, which recognizes all three adult fast MHC isoforms; monoclonal anti-tubulin  (Sigma) diluted 1/5000; polyclonal anti-myostatin (Abcam) diluted 1/500, monoclonal anti-Hif1  (Novus/Interchim) diluted 1/200, monoclonal anti-actin sarcomeric (Sigma) diluted 1/1000. After washing, primary antibodies were detected with peroxidase-conjugated secondary antibodies (Amersham, Les Ulis, France) at a dilution of 1/5000 and an ECL kit.

To detect myostatin in serum, human serum samples were removed of albumin and IgG using Albumin and IgG removal kit (Amersham-Biosciences). This procedure allows an increase in the protein load of the less abundant proteins. Protein concentrations from human serum samples were determined using the BioRad DC kit (Biorad). Human serum samples (50 µg per lane) supplemented with laemmli loading buffer were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and Western blot analysis was performed with

RNA analysis

Total RNA was prepared using the guanidium thiocyanate procedure and analyzed by Northern blot as previously described [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF]. For Northern blot analysis, total RNA (40µg) were run on a 2M formaldehyde-containing 1% agarose gel, transferred and bound to nylon membranes (Hybond, Amersham) as described by supplier.

Filters were hybridized using atrogin-1/MAFbx cDNA (from S. Leibovitch, INRA Montpellier) and GAPDH cDNA [START_REF] Fort | Regulation of c-fos gene expression in hamster fibroblasts: initiation and elongation of transcription and mRNA degradation[END_REF] probes which were labeled by random priming. Radioactivity on the nylon membranes was determined on a PhosphoImager analyser (Molecular Dynamics). Expression of atrogin-1/MAFbx was normalized to GAPDH mRNA signal.

For semi-quantitative RT-PCR, 1µg of total RNA was reverse transcribed into cDNA with Reverse transcription System (Promega) following the manufacturer"s instructions. One-tenth of the cDNA was used in each polymerase chain reaction using Promega GoTaq polymerase.

Ethidium bromide intensity of agarose electrophoretic bands was quantified with ImageJ software in the linear amplification range determined from 20 to 35 PCR cycle amplifications. Primers for the rat myostatin [START_REF] Mendler | Myostatin levels in regenerating rat muscles and in myogenic cell cultures[END_REF] and rat GAPDH [START_REF] Zador | Changes in mRNA levels of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase isoforms in the rat soleus muscle regenerating from notexin-induced necrosis[END_REF] genes were used for amplification as described previously.

Immunofluorescence and myotube analysis

Human myotubes or murine myotubes were fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences) and treated with PBS 0.5% triton X-100.

Expression of myosin heavy chain was analysed using mouse monoclonal antibody against

A c c e p t e d M a n u s c r i p t 11 monoclonal anti-fast myosin heavy chain (MHC) diluted 1/100 (Sigma). Primary antibody diluted in PBS/BSA was incubated for 1 h at 37°C, then washed in PBS, followed by a 30-min incubation with fluorescein-conjugated anti-mouse (1/5000; Amersham). DNA was stained with Hoechst (0.1 g/ml; Sigma). The diameter of at least 300 myotubes was measured using PerfectImage software. The average diameter per myotube was calculated as the mean of 5 measurements taken along the length of the myotube.

Antibodies and recombinant myostatin treatments

To analyse the effects of antimyostatin antibodies, human myoblasts were seeded at a density of 3x10 4 cells/cm 2 in 35 mm plates in growth medium consisting of DMEM supplemented with 20% serum and 2% ultroser (Gibco BRL). Three days later, culture medium was changed to DMEM-2% FCS supplemented daily with rabbit antimyostatin antibodies (directed against the COOH-terminal region of human myostatin; diluted 1/50) or with a preimmune serum as control. CoCl 2 treatment was performed three days after the shift of medium for additional 24h, then the cultures were harvested for immunofluorescence analysis.

Prior addition to cell medium, antibodies were dialysed against DMEM.

To analyse the effects of recombinant myostatin, murine myoblasts plated at a density of 2×10 4 cells/cm 2 on Matrigel-coated Petri dishes (BD Biosciences), were grown successively in growth medium Ham"s F10 supplemented with 20% horse serum (HS) during 48h, then for additional 48h in growth medium supplemented with 5 ng/ml basic fibroblast growth factor.

Then to achieve differentiation, myoblasts were switched to differentiation medium consisting of Ham"s F10, 20% HS without bFGF. Two days after the shift of medium, myotubes were treated for 24h with either or both of CoCl 2 (200µM) or recombinant myostatin (1µg/ml), and then harvested for immunofluorescence analysis. Primary murine myoblasts were plated at a density of 1x10 4 cells/cm 2 and of respectively (in 24 multiwell plates) in growth medium. After 3 days myoblasts were transfected using a dreamfect gold reagent as described by the supplier (OZBiosciences) with 1 µg of total DNA composed of 100 ng of the luciferase reporter construct (pGL3-(CAGA)12-MLP-Luc) and 100 ng of RL-tk-luc (Promega, Charbonnieres, France). The total amount of DNA used for each plate was normalized with the empty expression vector pcDNA3. To evaluate luciferase activity, 24h after transfection cells were refed with differentiation medium supplemented or not with recombinant myostatin (1µg/ml). 48h after transfection, transfected cells were harvested for luciferase assay. RL-tk-luc plasmid was included in transfections as internal control for transfection efficiency. Activities of firefly and renilla luciferases were measured sequentially using the luciferase assay kit reagent as described by the supplier (Promega, Charbonnières, France). Luciferase activity (reporter activity) was measured and corrected with respect to pRL-tk activity and protein content of the extracts.

Statistical analysis

The results are expressed as mean ± SEM. Experimental conditions were compared to control conditions by Student"s tests after checking the standard assumptions of parametric tests. When the standard assumptions were violated, a Mann-Whitney test was performed.

When experimental designs included one factor and more than two experimental conditions, one-way ANOVA was conducted after testing standard assumptions. When a main effect was identified, Dunnet post -hoc test was used to localise the differences. A two-way ANOVA was conducted to analyse the data presented in figure 6. The experimental design included two factors: CoCl 2 treatment (four doses) and myostatin expression (+/+ and -/-myostatin A c c e p t e d M a n u s c r i p t 13 muscles). Significant interaction between these two factors were checked (Prism 3.0, GraphPad, San Diego, CA). Statistical significance was set at P-values < 0.05 (*)

Results

Myostatin gene expression is induced in muscles of rats chronically exposed to hypoxia

To explore the potential relationship between hypoxia and myostatin, we used a model of long-term exposure of rats (5 weeks) to chronic hypoxia (mimicking an altitude of 4 500 m) associated with pair fed experimental design. As shown in Table 1, after 5 weeks the final body weight was significantly reduced in chronic hypoxic rats (H) and in normoxic pairfed rats (Npf) in comparison with corresponding normoxic control animals (N). Similarly, Gastrocnemius (GAS) muscle weights were lower for H and Npf groups compared to N group (Table 1). Soleus (SOL) mass was significantly decreased in H animals versus N animals while it was only modestly and non-significantly lower in the Npf group than in the N group.

Moreover we observed a significant decreased of total protein content in the GAS between H and N group but not in the SOL. Comparison between H group vs Npf group showed a significant effect of hypoxia on the decrease of relative GAS muscle weight and of absolute SOL muscle weight. It should be noted that except for the values of the total protein content in SOL, all the values tended to be lower in H rats than in Npf rats. Since in multiple models of skeletal muscle atrophy, the muscle-specific F-box protein Atrogin-1/MAFbx is upregulated [START_REF] Gomes | Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy[END_REF], we then compared by Northern blot analysis atrogin-1/MAFbx mRNA expression in hypoxic (H) versus pair-fed normoxic (Npf) and normoxic control muscles (N). Muscles showed an increase in atrogin-1/MAFbx mRNA expression in H vs N (P<0.001) and vs Npf (P<0.05) (Fig. 1A andB). Note that normoxic pair-fed animals exhibited an increased atrogin-1/MAFbx expression both in the SOL (Npf vs N, P<0.01) and the GAS muscle (Npf vs N, P<0.05). Such observation indicates that part of hypoxia-induced In this model, we then examined the expression at the protein level of the myostatin gene in Soleus (SOL) and Gastrocnemius (GAS) muscles. We performed western blotting analysis using polyclonal antimyostatin antibody. In accordance with previous data [START_REF] Mendler | Androgens negatively regulate myostatin expression in an androgen-dependent skeletal muscle[END_REF], we detected a single protein band (at about 28 kDa) in total protein extracts of rat muscles corresponding to active myostatin. We found that SOL and GAS muscles chronically exposed to hypoxia displayed increased levels of myostatin protein as compared with normoxic pair fed SOL and GAS muscles (approximately a 1.7-fold increase in SOL and a 1.3 -fold increase in GAS) (Fig. 2A andB). Myostatin mRNA levels relative to GAPDH significantly increased upon hypoxia (Fig. 2C). Levels of GAPDH mRNA were not significantly affected by hypoxia. Thus these results indicate that in hypoxic muscles the changes in the pool of myostatin protein appear to parallel the fluctuations in myostatin mRNA level (Fig. 2C). Note that there is no variation of myostatin expression between normoxic and normoxic pair fed animals.

Myostatin levels are increased in COPD patients

To extend these findings, we investigated the effect of chronic hypoxaemia on myostatin in COPD patients. We hypothesized that an increase in myostatin might occur in patients with COPD. To test this hypothesis, we explored myostatin protein level in muscle biopsies obtained from COPD patients showing hypoxaemia. These patients were compared with sedentary control subjects of a similar age (in a attempt to diminish the influence of aging and physical activity). The main characteristics of the studied subjects are given (Table 2). As listed, no significant differences in weight, BMI were found between patients with A c c e p t e d M a n u s c r i p t 15 COPD and sedentary age-matched healthy subjects. Similarly the mean fibre cross sectional area did not significantly differ between controls and patients with COPD (our unpublished observation). Pulmonary function testing confirmed the presence of very severe disease among the patient group. On average, COPD patients also had severe airflow limitation (FEV 1 , and FEV 1 /FVC were significantly lower in patients compared with controls), and low values of PaO 2 . In addition, both peripheral muscle function as assessed by quadriceps force and endurance, and exercise tolerance, as assessed by 6-MWD, were significantly lower in patients compared with controls. Therefore these results attest muscle dysfunction in our patient group without signs of muscle atrophy. A representative western blot for myostatin is provided in Fig. 3A showing that myostatin protein level is increased in the vastus lateralis quadriceps of patients with COPD compared with control subjects. The mean myostatin protein level is 2.5-fold higher in the COPD group than in the control group (P<0.05) (Fig. 3B). These results indicate that levels of myostatin are elevated in skeletal muscle of patients with COPD. Together our results suggest a potential regulation of myostatin by hypoxia.

Several experiments have shown that myostatin circulates in serum as part of a latent complex. Myostatin is detected by western blot in serum samples from mice and human [START_REF] Gonzalez-Cadavid | Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting[END_REF][START_REF] Heineke | Genetic deletion of myostatin from the heart prevents skeletal muscle atrophy in heart failure[END_REF][START_REF] Schuelke | Myostatin mutation associated with gross muscle hypertrophy in a child[END_REF][START_REF] Zimmers | Induction of cachexia in mice by systemically administered myostatin[END_REF]. This prompted us to measure myostatin levels in some of our patient"s serum samples.

A representative Western blot for myostatin is provided and revealed a band at approximately 15 kD, consistent with the secreted form of myostatin [START_REF] Hill | The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum[END_REF][START_REF] Zimmers | Induction of cachexia in mice by systemically administered myostatin[END_REF]. We found that myostatin protein levels tended to be increased in the serum of COPD group compared with that of the group of healthy controls (Fig. 3C andD). function [START_REF] Bruick | A conserved family of prolyl-4hydroxylases that modify HIF[END_REF][START_REF] Wang | Hypoxiainducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension[END_REF]. Briefly, in differentiating medium (2% FCS) confluent human myoblasts exit the cell cycle and spontaneously differentiate into multinucleated cells (myotubes) that express muscle-specific structural proteins. Human myoblasts were differentiated in low serum medium for 3 days, then myotubes were treated with CoCl 2 (200µM) for an additional 24h and examined for myotube size by immunofluorescence analysis using anti-myosin heavy chain antibody (Fig. 4A). Four days after differentiation, the control myotubes were of constant size and myoblast fusion no longer occurred [START_REF] Rommel | Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways[END_REF]. Myotubes exposed to CoCl 2 for 24h display a significant decreased size with a 24 % reduction (P<0.01) in mean myotube diameter compared with non treated myotubes (Fig. 4B). We next wanted to investigate whether expression of myostatin was altered by CoCl 2 treatment in human cells. Western blot analysis revealed that addition of CoCl 2 elicited an increase in myostatin protein level in human myotubes (about 2-fold) (Fig. 4C). As expected, Western blot analysis revealed that addition of CoCl 2 elicited an increase in HIF1- protein after 24h of treatment, attesting that CoCl 2 mimics hypoxia in these cells (Fig. 4C). Furthermore, in the same conditions, levels of -the muscle structural protein MHC were decreased in human myotubes treated with CoCl 2 . These experiments clearly demonstrate that CoCl 2 induces atrophy of myotubes. They also indicate an association between the level of myostatin expression and the CoCl 2 -induced atrophy.

CoCl 2 treatment induces myostatin expression and causes atrophy of myotubes

Myostatin is necessary to mediate the effects of CoCl 2

To confirm the role of myostatin in CoCl 2 -induced muscle atrophy, we employed a battery of approaches. First, we investigated whether targeted inhibition of myostatin, through addition of blocking antimyostatin antibodies, could inhibit CoCl 2 mediated atrophy of human A c c e p t e d M a n u s c r i p t 17 myotubes. We have previously validated such strategy in human tumoral muscle cells [START_REF] Ricaud | Response of human rhabdomyosarcoma cell lines to retinoic acid: relationship with induction of differentiation and retinoic acid sensitivity[END_REF]. Human muscle cells were induced to differentiate in low serum medium daily supplemented with antimyostatin antibodies (Ab) or a preimmune serum as control (Ctl) for 3 days, then they were treated for an additional 24h with either or both of CoCl 2 (200µM) or Ab (20 µg/ml), and examined for myotube size by immunofluorescence analysis using antimyosin heavy chain antibody (Fig. 5A). We observed that culture in presence of antimyostatin antibodies significantly induced the formation of larger myotubes as compared with culture cell incubated with preimmune serum (Fig. 5B). As expected, CoCl 2 treatment induced myotube atrophy in preimmune treated cells. In contrast, myotubes treated with antimyostatin antibodies were refractory to atrophic CoCl 2 effect. Second, we asked whether myostatin deletion was sufficient to impair the atrophic response of myotubes to CoCl 2 . To this end we established primary cultures of myoblasts from wild-type and myostatin -/-mice muscles. We differentiated myoblasts to multinucleated myotubes by mitogen removal. Two days after differentiation, differentiated myotubes are predominant and are treated with increasing concentrations of CoCl 2 for one day and examined for changes in myotube diameter. It is of note that the mean diameter of myostatin -/-myotubes was significantly 12% higher than that of the wild-type (Fig. 6A andB). The decrease in wild-type myotube diameter by CoCl 2 was detectable at a concentration of 25µM with a 20% decrease and reaching a maximum (30% decrease) at 50µM (Fig. 6B).

Conversely, the effect of CoCl 2 was significantly blunted in myostatin -/-cells, the maximal effect (10%) being detectable at 50µM. Thus myotubes lacking myostatin have an impaired atrophic response to CoCl 2 . We next examined the effect of recombinant myostatin on the CoCl 2 mediated atrophy in this system model. We first tested the activity of recombinant myostatin following transient transfection into murine cells with the use of the pGL3-(CAGA)12 luciferase reporter assay which reflects myostatin activity [START_REF] Thies | GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding[END_REF] A c c e p t e d M a n u s c r i p t 18 [START_REF] Zimmers | Induction of cachexia in mice by systemically administered myostatin[END_REF]. We found that CAGA reporter was activated by myostatin (Fig. 7A).

Next we asked whether addition of recombinant myostatin was sufficient to restore the atrophic response of myostatin deficient myotubes to CoCl 2 . Two days after differentiation, wild type and myostatin -/-cells were treated for 24h with either or both of CoCl 2 (200µM) or recombinant myostatin (1µg/ml). We then examined myotube size by immunofluorescence analysis using anti-myosin heavy chain antibody (Fig. 7B and7C). In wild-type cells, myostatin did not modify the atrophic response of myotubes to CoCl 2 . Conversely, in myostatin -/-cells, myostatin was sufficient to restore the atrophic response to CoCl 2 . Note that at this low dose and during this short period of treatment myostatin by itself did not significantly induce myotube atrophy.

Discussion

In this study, we report upregulation of myostatin expression in muscles of rats chronically exposed to severe hypoxia as well as in muscles of patients with chronically hypoxemic COPD. Furthermore, our data show that CoCl 2 , an hypoxia-mimicking agent, increases the expression of myostatin and causes atrophy of myotubes in human and mouse cell cultures. Finally, we provided compelling evidence that the CoCl 2 atrophic effect is linked to myostatin expression. Indeed, this atrophic effect is not observed in muscle cells where myostatin is inactivated either through genetic deletion or by using blocking antimyostatin antibodies. Inversely, the presence of myostatin restores the CoCl2 induced atrophy in myostatin deficient myotubes.

In an attempt to identify proper effects of hypoxia on myostatin in muscle we took advantage of a model of rats chronically exposed to severe hypoxia with a pair fed experimental design which excludes the anorexic impact of hypoxia on skeletal muscle mass.

A c c e p t e d M a n u s c r i p t 19 Accordingly, our results show a trend towards a higher diminution of skeletal muscle mass in hypoxic rats than in normoxic pair fed rats. In addition, at the molecular level this effect is associated with a significant induction of the MAFbx, suggesting an activation of proteasomal degradation which could contribute to a reduction in skeletal muscle mass [START_REF] Gomes | Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy[END_REF]. Thus these data are in agreement with previous studies suggesting a direct effect of hypoxia per se in the induction of skeletal muscle atrophy independent of energy intake status [START_REF] Bigard | Changes in dietary protein intake fail to prevent decrease in muscle growth induced by severe hypoxia in rats[END_REF][START_REF] Favier | Downregulation of Akt/mammalian target of rapamycin pathway in skeletal muscle is associated with increased REDD1 expression in response to chronic hypoxia[END_REF][START_REF] Pison | In vivo hypoxic exposure impairs metabolic adaptations to a 48 hour fast in rats[END_REF]. Interestingly, comparison between H vs Npf groups showed a significant decrease in muscle weight in the SOL but not the GAS muscle (Table 1). This muscle specific effect of hypoxia may be explained by the difference in the metabolism of slow and fast muscles. Indeed, compared to GAS muscle SOL muscle exhibits a more oxidative metabolism and is more dependent on the availability of O 2 . Thus, SOL muscle could show a greater sensitivity to hypoxia. However the mechanisms producing the differences in response remain to be elucidated.

An interesting feature of the present study was the induction of myostatin by hypoxia.

Our data clearly show that myostatin expression is induced in hypoxic muscles compared to normoxic pair-fed muscles whereas the decreased food intake failed to increase myostatin expression. However, it should be noted that myostatin expression is induced in a model of muscle atrophy induced by a more severe food deprivation [START_REF] Allen | Myostatin Expression is Increased by Food Deprivation in a Muscle-Specific Manner and Contributes to Muscle Atrophy during Prolonged Food Deprivation in Mice[END_REF]. This discrepancy may be related to differences in both severity of the food deprivation stress and length of exposure. Therefore, our observation indicates that hypoxia exerts its inducer effect on myostatin expression independently of nutritional status.

Skeletal muscle dysfunction is a prominent feature of COPD, and hypoxia, among other factors, is a proposed contributor to this process while the underlying molecular mechanisms remain to be identified. There is evidence that COPD patients are prone to develop muscle atrophy which is now recognized as a predictor of mortality in this disease A c c e p t e d M a n u s c r i p t 20 [START_REF] Doucet | Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Marquis | Midthigh muscle cross-sectional area is a better predictor of mortality than body mass index in patients with chronic obstructive pulmonary disease[END_REF]. Several recent reports suggested that upregulation of myostatin is involved in the development of muscle atrophy associated with COPD [START_REF] Plant | Cellular Markers of Muscle Atrophy in Chronic Obstructive Pulmonary Disease (COPD)[END_REF][START_REF] Testelmans | Atrophy and hypertrophy signalling in the diaphragm of patients with COPD[END_REF]. In the present study, our results showed an increased skeletal muscle myostatin expression in hypoxemic COPD patients (PaO 2 = 64 ± 2 mm Hg) compared with that in healthy controls. Consistent with these data, we also found that myostatin protein level tend to increase in the serum of COPD group compared with that of the group of healthy controls. However, it is of note that we obtained this result with a limited number of subjects (n=3-4 /group). Thus to definitely conclude a greater number of subjects should be analysed.

It is thus tempting to suggest that the increase in myostatin is causally related to muscle atrophy in this pathological context. However, no characteristics of global muscle atrophy, such as fat-free mass index, BMI and mean fibre cross sectional area were found in our patients compared to age-matched sedentary controls, although COPD patients showed reduced quadriceps force. It remains possible that an increase in myostatin expression could precede the induction of muscle atrophy. Collectively our data tend to implicate the hypoxic regulation of myostatin in patients with COPD and suggest that myostatin induction could be related to the presence of COPD itself rather to muscle atrophy. However, it should be noted that, in addition to hypoxaemia, several factors including inflammation, oxidative stress have been implicated in muscle limb dysfunction in COPD [START_REF] Couillard | From muscle disuse to myopathy in COPD: potential contribution of oxidative stress[END_REF]. It is thus conceivable that inflammation and oxidative stress may also contribute alone or in concert with hypoxaemia to the higher myostatin expression levels. Further studies should be designed to address this point.

In agreement with our in vivo observations, our results showed that addition of the hypoxia mimic cobalt chloride CoCl 2 elicits a significant increase in myostatin expression and causes atrophy of human myotubes. We further demonstrated that CoCl 2 requires the presence A c c e p t e d M a n u s c r i p t 21 of myostatin to elicit its action on muscle cells since: i) the atrophic CoCl 2 effect is not observed both in human myotubes treated with antimyostatin antibodies and in murine myostatin deficient myotubes while ii) myostatin deficient myotubes treated with recombinant myostatin displayed an atrophic response to CoCl 2 . Altogether, these data strongly argue for a critical role for myostatin in mediating hypoxia effects in skeletal muscle cells. Can we link the hypoxia-induced atrophy to the up-regulation of myostatin expression? Several studies have established the catabolic effect of myostatin on skeletal muscle. In vitro, myostatin not only inhibits myoblast proliferation and differentiation but also induces atrophy of resulting myotubes [START_REF] Mccroskery | Myostatin negatively regulates satellite cell activation and self-renewal[END_REF][START_REF] Rios | Myostatin is an inhibitor of myogenic differentiation[END_REF][START_REF] Taylor | Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells[END_REF][START_REF] Thomas | Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation[END_REF]. In vivo, systemic administration of myostatin to adult mice or overexpression of myostatin in skeletal muscle also results in muscle atrophy [START_REF] Durieux | Ectopic expression of myostatin induces atrophy of adult skeletal muscle by decreasing muscle gene expression[END_REF][START_REF] Reisz-Porszasz | Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin[END_REF][START_REF] Zimmers | Induction of cachexia in mice by systemically administered myostatin[END_REF]. Inversely, myostatin gene deletion prevents glucocorticoid-induced muscle atrophy [START_REF] Gilson | Myostatin gene deletion prevents glucocorticoid-induced muscle atrophy[END_REF] and the effect of glutamine on prevention of glucocorticoid-induced skeletal muscle atrophy is associated with myostatin suppression [START_REF] Salehian | The effect of glutamine on prevention of glucocorticoid-induced skeletal muscle atrophy is associated with myostatin suppression[END_REF]. Supporting its atrophic action, it has been demonstrated that myostatin decreases phosphorylation and nuclear export of the FoxO transcription factors by Akt (McFarlane et al., 2006), impairs protein synthesis in vitro [START_REF] Taylor | Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells[END_REF] and negatively regulates Akt/mTOR signalling pathway, which controls protein synthesis (Amirouche et al., 2009). Recently, Favier et al. demonstrated that Akt/mTOR pathway is down-regulated in rat skeletal muscle in response to chronic hypoxia indicating that hypoxia negatively regulates protein synthesis in skeletal muscle [START_REF] Favier | Downregulation of Akt/mammalian target of rapamycin pathway in skeletal muscle is associated with increased REDD1 expression in response to chronic hypoxia[END_REF]. Therefore, the parallel actions of myostatin and hypoxia on protein synthesis suggest that induction of myostatin by hypoxia could represent a potential mechanism for hypoxia-induced atrophy.

The present data suggest the contribution of myostatin, a member of TGF family, in the skeletal muscle response to hypoxic stimuli. Interestingly, earlier findings on other tissues A c c e p t e d M a n u s c r i p t 22 have shown that various effects of hypoxia appear to be mediated by TGF signalling. TGF expression is up-regulated in lung of hypoxia-adapted animals [START_REF] Bartram | The role of transforming growth factor beta in lung development and disease[END_REF]. In human peritoneal fibroblasts, hypoxia increases TGF 1 and TGF- type I and II receptors mRNA levels [START_REF] Saed | Transforming growth factors beta1, beta2 and beta3 and their receptors are differentially expressed in human peritoneal fibroblasts in response to hypoxia[END_REF]. Chronic hypoxia-induced pulmonary hypertension and vascular remodeling are attenuated in absence of TGF signalling [START_REF] Chen | Dominant negative mutation of the TGF-beta receptor blocks hypoxia-induced pulmonary vascular remodeling[END_REF].

Similarly, TGF signalling is required for the inhibition of adipocyte differentiation by hypoxia. These previous results, together with our findings, support the hypothesis that TGF- signalling could mediate the response to hypoxic stress in several tissues. To our knowledge, a link between myostatin and hypoxia would represent a novel facet of the hypoxic response in muscle. However, additional in vivo experiments are further needed to detail the myostatin action in the skeletal muscle response to hypoxia.

In conclusion, the data presented here demonstrate a link between hypoxia and myostatin and provide insights into how hypoxia contributes to muscle dysfunction. We believe that they could have important biologic and therapeutic implications, since they offer important clues to understand the mechanisms of hypoxia action in muscle and may open possibilities for the development of new treatments for skeletal muscle disease.

ACKNOWLEDGMENTS:

This study was supported by funds from the Institut National de la Recherche

  performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996) and with the approval of the French Ministry of Agriculture.

  test (6-MWT): A 6-MWT was conducted according to a standardized protocol(2002).

  2 (Aldrich) was diluted in DMEM. Recombinant myostatin was purchased from R&D Systems.

  the rabbit polyclonal anti-myostatin (Abcam) raised against the COOH-terminal of human myostatin.

  due to caloric restriction associated with hypoxia. Collectively, our results suggest that hypoxic rats show a tendency to lose more muscle mass than normoxic pair fed rats.

  effects of hypoxia in vitro on myostatin expression we treated human myotubes with the cobalt chloride (CoCl 2 ) which mimics hypoxia by inducing HIF-1

  Agronomique (INRA), the "fédération ANTADIR (Association Nationale pour le Traitement à Domicile, l"Innovation et la Recherche)", and the Centre Hospitalier Universitaire de Montpellier. The authors would like to thank: Drs Guillaume Py, Christelle Koechlin and Arnaud Chatonnet for their useful discussions and Adrien Gibault for technical assistance. We thank Miss B. Bonafos, and Miss V. Rana for taking care of animals used in the present study

  s c r i p t and their genotyping, respectively. J.R. is the recipient of a doctoral fellowship cofinanced by INRA and Région Languedoc-Roussillon.

  FIGURE LEGENDS

Fig. 2 .

 2 Fig. 2. Myostatin expression is induced in soleus and gastronemius muscles of rats

Fig. 3 .

 3 Fig. 3. Increased levels of myostatin in patients with COPD

Fig. 5 .

 5 Fig. 5. Inhibition of myostatin blocks CoCl 2 -mediated atrophy in human muscle cells

Fig. 6 .

 6 Fig. 6. Myostatin deficient myotubes are resistant to CoCl 2 -mediated atrophy

  were obtained from at least five mice of the indicated genotype. The two-way ANOVA revealed a significant CoCl 2 effect (P<0.001). The second main effect was a significant myostatin expression effect (P<0.001); the myostatin -/-muscles being significantly less sensitive to CoCl 2 treatment than myostatin +/+ muscles. No significant interaction effect could be detected between CoCl 2 and myostatin factors (P>0.05) indicating that the difference in treatment response between -/-and +/+ myostatin was not different among the CoCl 2 doses. ‡, P<0.05 versus untreated wild type; *, P<0.05 versus untreated wild type. Scale bar, 20 µm. At least 300 myotubes were analysed.

Fig. 7 .

 7 Fig. 7. Myostatin restored CoCl 2 -induced atrophy

Figure 7

 7 Figure 1 Hayot et al

TABLE LEGEND Table 1 : Body weights and muscle weights in normoxic (N), normoxic pair fed (Npf) and hypoxic (H) rats

 LEGEND1

	SOL, soleus muscle; GAS, gastrocnemius muscle. Values are means ± SEM. Significant
	difference * P< 0.05; ** P<0.01 compared to N group, and ‡ P<0.05 compared to Npf group
	is given (n=10 per group)

Table 2 : Subject characteristics

 2


Yan Borné
email: yan.borne@med.lu.se

Gunnar Engström

Birgitta Essén

Jan Sundquist

Bo Hedblad

Country of birth and risk of hospitalization due to heart failure: a Swedish population-based cohort study

Introduction

Heart failure (HF) is one of the leading causes for morbidity and mortality, particularly in elderly [START_REF] Cowie | Incidence and aetiology of heart failure; a population-based study[END_REF][START_REF] Bleumink | Quantifying the heart failure epidemic: prevalence, incidence rate, lifetime risk and prognosis of heart failure The Rotterdam Study[END_REF]. In Europe, it is estimated that at least 15 million patients have HF [START_REF] Dickstein | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM)[END_REF]. In Sweden, HF is the most common hospitalization diagnosis in subjects aged 65 and over, and the total annual costs for HF management are approximately 2% of the Swedish health care budget [START_REF] Cline | The management of heart failure in Sweden[END_REF]. HF can be the result of various cardiac diseases; however, most cases of HF are caused by hypertension or coronary heart disease (CHD) [START_REF] Cowie | Incidence and aetiology of heart failure; a population-based study[END_REF][START_REF] Mckee | The natural history of congestive heart failure: the Framingham study[END_REF][START_REF] Fox | New perspectives on heart failure due to myocardial ischaemia[END_REF][START_REF] Nieminen | EuroHeart Failure Survey II (EHFS II): a survey on hospitalized acute heart failure patients: description of population[END_REF].

Immigration to Sweden has increased markedly during the last decades, especially of refugees during the Balkan wars in the 1990s, from the Middle East in the first decade of this millennium and of labour immigrants from the European Community. Previously the labour immigration has been very restricted with exception from the Nordic countries, which have been without barriers, dominated by the Finns. The proportion of foreign-born people in Sweden increased from 9.2% in 1990 to 13.8% in 2008 [START_REF]Malmö residents with foreign background 2008[END_REF]. The corresponding figures in Malmö, the third largest city in Sweden, were 16% to 28% [START_REF]Malmö residents with foreign background 2008[END_REF]. It has been reported that foreign-born compared to Swedish-born people have higher risk for and higher mortality rate from cardiovascular disease (CVD) [START_REF] Gadd | Are there differences in all-cause and coronary heart disease mortality between immigrants in Sweden and in their country of birth? A follow-up study of total populations[END_REF][START_REF] Gadd | Morbidity in cardiovascular diseases in immigrants in Sweden[END_REF][START_REF] Hedlund | Acute myocardial infarction incidence in immigrants to Sweden. Country of birth, time since immigration, and time trends over 20 years[END_REF]. Incidence of HF among immigrants in Sweden is however largely unknown.

Previous studies from Malmö have shown that there are substantial differences in risk of CVD between residential areas with high and low proportion of immigrants [START_REF] Engstrom | Geographic distribution of stroke incidence within an urban population: relations to socioeconomic circumstances and prevalence of cardiovascular risk factors[END_REF][START_REF] Tyden | Intra-urban variations in incidence and mortality in myocardial infarction. A study from the myocardial infarction register in the city of Malmo, Sweden[END_REF][START_REF] Tyden | Myocardial infarction in an urban population: worse long term prognosis for patients from less affluent residential areas[END_REF][START_REF] Engstrom | Trends in long-term survival after myocardial infarction: less favourable patterns for patients from deprived areas[END_REF]. The purpose of the present population-based study is to explore the relationship between country of birth and risk of HF hospitalizations after taking age, sex and socioeconomic indicators into account.

Methods

Study population and follow up

November 1 st , 1990, a total of 118,134 subjects, aged 40 to 89 years, were registered as residents in the city of Malmö. After excluding subjects with history of myocardial infarction (MI) (n=2,133) or those who were hospitalized due to HF (n=1,714) before November 1 st 1990, 114,917 (50,981 men and 63,936 women) subjects remained. All subjects were followed from November 1 st , 1990 until first hospitalization attributable to HF, MI, death, emigration from Sweden, or December 31 st , 2007, whichever came first. Since HF is very uncommon in young subjects, the study was restricted to individuals aged 40 to 89 years [START_REF] Fox | New perspectives on heart failure due to myocardial ischaemia[END_REF].

Population Registers

Information about the background characteristics was retrieved from the National Swedish Census Data ("Folk-och bostadsräkning, FoB") in 1990 [START_REF]Population and Housing Census 1990[END_REF]. This survey is a total register of the Swedish population November 1 st , 1990, and includes information on marital status, rented and self-owned homes, annual income and country of birth. This database consists of information from a mailed self-administered questionnaire and data from other population registers. Failure to complete and return the questionnaire was associated with a financial penalty. Information on annual income was retrieved from the Swedish income register, i.e. assessment of taxes in 1991 (i.e. for the income year 1990). Information on country of birth and marital status were retrieved from the Swedish total population register (TPR).

Information on education, occupation and housing condition were based on self-reported questionnaires. The response rate of the questionnaire was 97.5%. Information about migration and deaths during the follow-up period was retrieved from the Swedish TPR and the Swedish Cause of Death Register. HF cases were retrieved by record linkage with the Swedish Hospital Discharge Register (SHDR) [START_REF] Ingelsson | The validity of a diagnosis of heart failure in a hospital discharge register[END_REF]. Subjects with a hospital discharge diagnosis of HF (International Classification of Diseases, ICD, 8th code 427.00, 427.10, 428.99; ICD, 9th code 428; and ICD, 10th code I50, I11.0, respectively) as the primary diagnosis were considered to have HF. As previously reported from this study population, cases of acute MI was retrieved by record linkage with registers of hospital discharges and deaths in accordance to diagnosis criteria used in the National MI register [START_REF] Rosvall | Income and short-term case fatality after myocardial infarction in the whole middle-aged population of Malmö, Sweden[END_REF]. A validation study, at the National Board of Health and Welfare in Sweden, has shown that the diagnosis of MI in the SHDR is valid in 90-95% of the cases [START_REF]The validity of the diagnosis acute myocardial infarction in the patient registry 1987 and 1995[END_REF].

Subjects with non-fatal MI were followed until the day of infarction and censored thereafter.

In addition, risk of HF was explored for all HF cases, including those with MI during the follow-up. Non-fatal MI was defined as 410 (ICD-9) or I21, I22 (ICD-10). The data from 1983-1990 prior to November 1 st , 1990 were used to exclude subjects with previous hospitalization due to HF or MI. In a study that reviewed hospital records of patients with HF according to the Swedish hospital discharge register, it was shown that the validity was 95% if HF was the primary diagnosis, irrespective of clinical type [START_REF] Ingelsson | The validity of a diagnosis of heart failure in a hospital discharge register[END_REF]. In that study a review board examined the validity of the diagnosis according to European Society of Cardiology definition of HF [START_REF]The Task Force on Heart Failure of the European Society of Cardiology. Guidelines for the diagnoses of heart failure[END_REF]. No major difference in validity was found whether patients had been treated at an internal medicine or cardiology clinic (86% and 91%, respectively [START_REF] Ingelsson | The validity of a diagnosis of heart failure in a hospital discharge register[END_REF].

Country of birth

Information about country of birth was available for 99.96% of the population (51 missing).

Immigrants from countries (i.e. USA, Iraq, Ghana, etc) for which the total follow-up time was less than 2,500 person years were excluded. As in a previous study from this cohort, the Peoples Republic of China and Vietnam were grouped into one category [START_REF] Khan | Incidence of stroke and stroke subtypes in Malmo, Sweden, 1990-2000: marked differences between groups defined by birth country[END_REF]. Thus, selected countries in the analysis contained 98.1% of the cohort.

In an additional analysis, country of birth was also categorized into high income countries (i.e. <= $610), middle income countries (i.e. $611-$7,620) and low income countries (i.e. > $7,620) based on 1990 GNI (Gross national income) per capita in US$ [START_REF]World Bank Analytical Classifications (presented in World Development Indicators) GNI per capita in US$ (Atlas methodology)[END_REF], Table 1.

Socioeconomic status

Socioeconomic indicators used for the analysis were marital status, total annual income and housing status as previous studies have shown their association with HF [START_REF] Chung | Marital status as an independent predictor of event-free survival of patients with heart failure[END_REF][START_REF] Vollman | Coping and depressive symptoms in adults living with heart failure[END_REF][START_REF] Rohrbaugh | Effect of marital quality on eight-year survival of patients with heart failure[END_REF][START_REF] Ingelsson | Socioeconomic factors as predictors of incident heart failure[END_REF][START_REF] Luttik | Marital status, quality of life, and clinical outcome in patients with heart failure[END_REF][START_REF] Struthers | Social deprivation increases cardiac hospitalisations in chronic heart failure independent of disease severity and diuretic non-adherence[END_REF][START_REF] Philbin | Socioeconomic status as an independent risk factor for hospital readmission for heart failure[END_REF].

Marital status was categorized into married, single, divorced, or widowed [START_REF] Khan | Incidence of stroke and stroke subtypes in Malmo, Sweden, 1990-2000: marked differences between groups defined by birth country[END_REF]. The total annual income 1990 (in Swedish Kronor (SEK)) was categorized into 6 categories: 0 to 50,000; 50,100 to 100,000; 100,100 to 150,000; 150,100 to 200,000; 200,100 to 250,000; and >250,000 SEK per year. In November 1 st , 1990, $1 corresponded to 5.62 SEK.

Annual income might not fully reflect the socioeconomic differences among people, i.e. retired people or women with low income in the high income household. Therefore, the results were also adjusted for housing status. Housing status was grouped into self-owned home (house or apartment) or rented home. Among the population, 55.6% owned their house or apartment, and 40.5% rented their apartment first or second hand. Subjects with missing data on housing status (3.9%) were in the analysis coded in a separate category.

Statistics

Hazard ratios (HR), with 95% confidence interval (CI) were calculated using Cox proportional hazards regression models. In terms of country of birth, Sweden was used as the reference group in all analysis. HRs were first adjusted for baseline age and sex, then additionally adjusted for marital status (4 categories), annual income (6 categories) and housing status (3 categories). Possible interaction between age and income, and between country of birth and sex, respectively, on risk of HF was explored by introducing interaction terms in the multivariate models. Analysis involved the computer software SPSS (15.0). This study was approved by the Lund University Ethics review Committee (LU 78-02 and 2009/46).

Results

Baseline characteristics of study cohort

Baseline characteristics of Swedish natives and selected immigrant groups are presented in Table 1. Overall, mean (SD) age of participants at entry was 60.8 ±13.2 years and 55.6% (n=63,936) were women. A total of 99,976 subjects were born in Sweden and 15.9% outside Sweden, of whom 25% in other Nordic countries.

Incidence of MI in relation to immigration to Sweden

During the 16 year follow-up period 12,712 subjects (6,924 men and 5,788 women) had an acute MI. The incidence was 103 in men and 67 in women, respectively, per 10,000 personyears. Foreign born subjects had a higher risk for MI as compared to subjects born in Sweden (HR: 1.08; 95% CI: 1.02-1.14, p=0.008), taking age, sex and socio-economic indicators into account.

Risk of hospitalization due to HF in relation to country of birth

During a mean follow-up of 13.5±5.3 years, a total of 7,640 individuals (3,624 men and 4,016 women) were discharged from hospital with first time HF as primary diagnosis. The incidence of hospitalized HF per 10,000 person-years was 53 in men and 46 in women, respectively.

Incident HF increased significantly and linearly by age, in 5-year age groups from 3 per 10,000 person-years aged 40-44 years to 267 per 10,000 person-years aged 85-89 years (p<0.001). Of the 7,640 cases with HF, 1,243 (677 men and 566 women) had an MI before or concurrent with the hospitalization of HF. The latter group was censored at the time of the infarction in the first analysis.

The overall analysis showed substantial differences in risk in terms of hospitalization due to HF between countries of birth (p<0.001, 19df), Table 2. Adjusted for age and sex, the risk of HF was significantly higher among immigrants from Finland, Denmark, Former Yugoslavia and Hungary compared to Swedish natives. This risk increase remained among immigrants from Finland (HR: 1.40; 95% CI, 1.10-1.81), Former Yugoslavia (HR: 1.45; 1.23-1.72) and Hungary (HR: 1.48; 1.16-1.89), after taking marital status, annual income and housing conditions into account, Table 2.

The results were only marginally changed when HF cases with incident MI before or concurrent with hospitalization for HF (n=1,243) were included in the analysis, i.e. the risk increase for HF remained among immigrants from Finland (HR: 1.42; 1.13-1.78), Former Yugoslavia (HR: 1.34; 1.14-1.57) and Hungary (HR: 1.41; 1.12-1.77), Table 2.

In addition, 960 subjects died outside hospital with HF as the underlying death cause. These potential HF cases had no prior hospitalization due to HF, however including them as cases in the multivariate analysis did only marginally affect the results and immigrants from Finland, Hungary and Former Yugoslavia has significantly increased risk for HF (data not shown).

Risk of hospitalization due to HF in relation to age, sex and socioeconomic indicators

Age and male sex were, independently of marital status, housing condition and annual income, related to an increased risk of HF hospitalization, Table 3. Single, divorced and widowed compared to married subjects were also independently associated with increased risk for hospitalization due to HF. In addition, renting house and low annual income were associated with higher risk for HF. Excluding subjects with missing information on housing conditions (i.e. 3.9%) did only marginally change the results, i.e. the significantly risk increase for HF remained among immigrants from Finland, Former Yugoslavia and Hungary.

There was a significant interaction (p<0.001) between annual income and age on risk of HF hospitalizations. When stratifying the cohort under and equal or above 65 years the effect of income on risk for HF was higher for younger than elderly subjects (data not shown). No significant interaction (p=0.368) between country of birth and sex on risk of HF hospitalization was observed.

Additional analysis

Additional analyses were performed by categorizing country of birth for immigrants into high, middle and low income countries. When the analysis was restricted to risk of HF hospitalization without previous or concurrent MI, a significant higher risk, compared to Swedish natives, was observed among immigrants from high-income countries (HR: 1.12;

1.01-1.25) and middle-income (HR: 1.14; 1.02-1.26). This risk increase was independent of age, sex, marital status and socioeconomic indicators. Including HF cases with MI (n=1,243) before or concurrent with HF during the follow-up in the analysis did only marginally change the results, Table 2, Figure 1.

Discussion

Our analysis of prospective associations between country of birth and risk of hospitalization due to HF has yielded some novel and potentially important findings. First, we have demonstrated that immigrants from Finland, Former Yugoslavia, and Hungary, compared to Swedish-born subjects had significantly higher risk for hospitalization due to HF. These differences persisted when taking socioeconomic factors into account. Secondly, in an additional analysis we also found that the risk for HF among immigrants from high-and middle-income countries was significantly higher than their counter parts. These findings are in line with previous studies showing substantial differences in immigration, country of birth and incidence of CVD in other countries and in Sweden [START_REF] Bleumink | Quantifying the heart failure epidemic: prevalence, incidence rate, lifetime risk and prognosis of heart failure The Rotterdam Study[END_REF][START_REF] Gadd | Are there differences in all-cause and coronary heart disease mortality between immigrants in Sweden and in their country of birth? A follow-up study of total populations[END_REF][START_REF] Gadd | Morbidity in cardiovascular diseases in immigrants in Sweden[END_REF][START_REF] Hedlund | Acute myocardial infarction incidence in immigrants to Sweden. Country of birth, time since immigration, and time trends over 20 years[END_REF][START_REF] Larson | Lifetime risk for developing congestive heart failure: the Framingham Heart Study[END_REF][START_REF] Loehr | Heart failure incidence and survival (from the Atherosclerosis Risk in Communities study)[END_REF][START_REF] Ng | Trends and ethnic differences in hospital admissions and mortality for congestive heart failure in the elderly in Singapore, 1991 to 1998[END_REF]. In Malmö, Sweden and in other cities, residential areas with high proportion of immigrants are often characterized by high incidence of CVD [START_REF] Engstrom | Geographic distribution of stroke incidence within an urban population: relations to socioeconomic circumstances and prevalence of cardiovascular risk factors[END_REF][START_REF] Tyden | Intra-urban variations in incidence and mortality in myocardial infarction. A study from the myocardial infarction register in the city of Malmo, Sweden[END_REF][START_REF] Tyden | Myocardial infarction in an urban population: worse long term prognosis for patients from less affluent residential areas[END_REF][START_REF] Engstrom | Trends in long-term survival after myocardial infarction: less favourable patterns for patients from deprived areas[END_REF]. The increased cardiovascular risk among immigrants in this population is consistent with what have been reported previously from other Swedish studies [START_REF] Gadd | Are there differences in all-cause and coronary heart disease mortality between immigrants in Sweden and in their country of birth? A follow-up study of total populations[END_REF][START_REF] Gadd | Morbidity in cardiovascular diseases in immigrants in Sweden[END_REF][START_REF] Hedlund | Acute myocardial infarction incidence in immigrants to Sweden. Country of birth, time since immigration, and time trends over 20 years[END_REF].

There are several possible explanations of the findings in the present study. First, the observed increased risk of HF hospitalizations among immigrants from Finland, Former Yugoslavia and Hungary might reflect influences from their countries of origin. Most cases of HF are caused by hypertension or CHD (1, 5-7). There are substantial differences in CVD mortality between countries and regions, Eastern European countries generally have high rates of CVD or CHD mortality [START_REF] Sans | The burden of cardiovascular diseases mortality in Europe. Task Force of the European Society of Cardiology on Cardiovascular Mortality and Morbidity Statistics in Europe[END_REF]. In Hungary an increasing CVD mortality have been reported from the early 1970s, with the highest mortality rate in 1985. [START_REF] Kesteloot | Nutrition and health[END_REF]. Elevated body mass index, high intake of saturated fat and salt have been suggested as possible risk factors explaining the high risk for CVD among Hungarians [START_REF] Biro | Nutrition survey of the Hungarian population in a randomized trial between 1992-1994[END_REF]. High incidence of CVD has also been reported in Finland and former Yugoslavia [START_REF] Sans | The burden of cardiovascular diseases mortality in Europe. Task Force of the European Society of Cardiology on Cardiovascular Mortality and Morbidity Statistics in Europe[END_REF]. Hence, we can not exclude the possibility that high CVD risk of their country of origin might explain, at least partly, the increased risk of hospitalization due to HF among immigrants from Finland, Former Yugoslavia, and Hungary in our study.

Secondly, another possible explanation could be different circumstances and reasons to migrate to Sweden. During 1965-1973 former Yugoslavians were often recruited by Swedish companies as labour immigrants with low skilled jobs, and from 1973 to 1990s, when the labour immigration was stopped, they often immigrated to Sweden for family reunion and as asylum. After the politic instability in Hungary 1956, many Hungarian immigrants came to Sweden as refugees. Finnish immigrants came to Sweden as labour migrants from 1945 to 1970s. This suggests that immigrants from the above mentioned countries to Sweden could be regarded as selected groups. To what extend this matter might influence risk of HF is unclear.

In addition, before the baseline examination in 1990 the majority of all these three immigration groups have lived in Sweden at least 20 years or more. One could assume that the high risk for hospitalization due to HF in the present study might also be the result of stress from cultural and psychosocial change to acculturate and integrate into the Swedish society. However, another Swedish study has shown that Finnish labour migrants had higher risks for MI compared to Swedish natives, and that this increased risk still remained after 20 years in Sweden [START_REF] Alfredson | Incidence of myocardial infarction among male Finnish immigrants in relation to length of stay in Sweden[END_REF].

Third, another explanation could be associated with low socioeconomic status of immigrants compared to Swedish natives. It has been shown that Former Yugoslavians had disadvantage in labour markets compared to native Swedes [START_REF] Kogan | Ex-Yugoslavs in the Austrian and Swedish labour markets: the significance of the period of migration and the effect of citizenship acquisition[END_REF]. In addition it also matters where you live, as substantial socioeconomic differences between residential areas in cities have been demonstrated. In the city of Malmö, Sweden, residential areas with low socioeconomic circumstances and high proportion of immigrants have been characterized by high incidence of CVD [START_REF] Engstrom | Geographic distribution of stroke incidence within an urban population: relations to socioeconomic circumstances and prevalence of cardiovascular risk factors[END_REF][START_REF] Tyden | Intra-urban variations in incidence and mortality in myocardial infarction. A study from the myocardial infarction register in the city of Malmo, Sweden[END_REF][START_REF] Tyden | Myocardial infarction in an urban population: worse long term prognosis for patients from less affluent residential areas[END_REF][START_REF] Engstrom | Trends in long-term survival after myocardial infarction: less favourable patterns for patients from deprived areas[END_REF]. International and national studies have reported that prevalence of cardiovascular risk factors is associated with socioeconomic status [START_REF] Engstrom | Geographic distribution of stroke incidence within an urban population: relations to socioeconomic circumstances and prevalence of cardiovascular risk factors[END_REF][START_REF] Engstrom | Marital dissolution is followed by an increased incidence of stroke[END_REF][START_REF] Li | Stroke incidence, recurrence, and case-fatality in relation to socioeconomic position: a population-based study of middle-aged Swedish men and women[END_REF][START_REF] Sundquist | The influence of country of birth on mortality from all causes and cardiovascular disease in Sweden 1979-1993[END_REF]. In a Swedish study rented home was associated with cardiovascular mortality [START_REF] Sundquist | The influence of country of birth on mortality from all causes and cardiovascular disease in Sweden 1979-1993[END_REF]. In the present study, risk of HF was related to socioeconomic status. However, when several socioeconomic indicators were adjusted for in the analysis, the observed difference in risk of HF among immigrants from Finland, former Yugoslavia and Hungary still remained. This suggests that the increased risk of HF in these groups is not completely mediated by the socioeconomic indicators in this study. On the other hand, the significantly increased risk of HF for Danish subjects was reduced and non-significant when adjusted for SES, which suggest that socioeconomic circumstances still play a role.

Strength and limitation

It is important to be aware of strength but also limitations in the study design, measurement procedures, and methodological issues that might influence the interpretation of the results.

Rates of first hospitalizations due to HF, as presented in Table 2, were consistent with what have been reported by others [START_REF] Cowie | The epidemiology of heart failure[END_REF]. In that study the crude incidence for different populations ranged from 1.0-5.0 per 1000 person-years in the general population [START_REF] Cowie | The epidemiology of heart failure[END_REF]. Some methodological issues should also be considered. Sweden has a health care system which is financed by the community, and no insurance is needed. According to recently published Swedish studies, the Swedish health care system has achieved equality in the care and treatment of patients with HF [START_REF] Hedemalm | Symptom recognition and health care seeking among immigrants and native Swedish patients with heart failure[END_REF][START_REF] Hedemalm | Equality in the care and treatment of immigrants and native Swedes--a comparative study of patients hospitalised for heart failure[END_REF]. In these studies symptom recognition and health care seeking were rather similar among immigrants and natives Swedes who were hospitalised due to HF [START_REF] Hedemalm | Symptom recognition and health care seeking among immigrants and native Swedish patients with heart failure[END_REF][START_REF] Hedemalm | Equality in the care and treatment of immigrants and native Swedes--a comparative study of patients hospitalised for heart failure[END_REF]. In the city of Malmö there is only one hospital and patients from all residential areas can reach the hospital within 15 minutes, so the availability of the hospital could be regarded to be similar for all patients.

Emigration, vital status and hospitalizations at the end of the follow-up were up-dated on all individuals by data linkage with regional and national registers. The completeness and validity of these registers have been documented in several other studies from the city [START_REF] Engstrom | Geographic distribution of stroke incidence within an urban population: relations to socioeconomic circumstances and prevalence of cardiovascular risk factors[END_REF][START_REF] Khan | Incidence of stroke and stroke subtypes in Malmo, Sweden, 1990-2000: marked differences between groups defined by birth country[END_REF].

We were unable to include cases of HF which are diagnosed and treated as out of hospital patients. Thus, we cannot draw any conclusions about less severe cases. Since all cases were treated in-hospital with a primary diagnosis of HF, we can assume that the diagnosis was valid and that the HF was quite severe in most cases [START_REF] Kumler | Accuracy of a heart failure diagnosis in administrative registers[END_REF]. Furthermore, a validation study of cases retrieved from the Swedish hospital discharge register has shown that the validity of the diagnosis was 95%, irrespective of clinic type, if HF was the primary diagnosis [START_REF] Ingelsson | The validity of a diagnosis of heart failure in a hospital discharge register[END_REF]. We also included those 960 subjects who died outside hospital with HF as the underlying death cause as potential HF cases, however this did only marginally affect the results. In addition, the large number of individuals and events, and the complete and reliable data about the background population are considerable study strengths. The main limitation of the present study is that we lack information on cardiovascular risk factors, e.g. blood pressure, dietary habits, smoking habits and physical activity patterns. However, to collect these data from a complete urban population is not possible.

We did not observe any risk increase for hospitalization due to HF among immigrants from low-income countries. However, only 16 events was observed in this group (n=600) limiting the statistical power due to few events. Information on country of birth was available for almost all study subjects. Those who during the follow-up emigrated were censored at the time of emigration. However, re-migration to Sweden without reporting to the authorities could possibly exaggerate the population at risk and cause low risk estimates for some immigrant groups. A report from Sweden Statistics estimated that ~ 25000 to 50000 foreign-born individuals who officially live in Sweden have left the country [START_REF] Qvist | Täckningsproblem i Registret över totalbefolkningen RTB[END_REF]. A previous study from the present cohort explored whether exclusion of individuals without income, according to the assessments of taxes, had any impact on the relationships between country of birth and incidence of disease [START_REF] Khan | Incidence of stroke and stroke subtypes in Malmo, Sweden, 1990-2000: marked differences between groups defined by birth country[END_REF]. No major influence on hazards ratios for incident stroke was found in that study. Therefore, we believe that a remigrant effect is a minor problem in our study and should not explain the observed increased risk for HF related to country of birth. Finally, it is also possible that the chosen SES indicators (i.e. housing conditions, annual income and marital status) in the present study do not fully eliminate the effect of SES on hospitalization due to HF. Information on annual income and marital status in the present study was based on data from the TPR; however, housing conditions was questionnaire-based and self-reported. Annual income might not fully reflect the socioeconomic differences for all people, e.g. retired people or women with low income in households with high income.

However, we lacked information for example on household income and psychosocial stressors. The latter is known to vary by social position [START_REF] Brunner | Toward a new social biology[END_REF]. It could also be discussed whether socio-economic circumstances are mediators, rather than confounders, in the relationship between country of birth and HF.

In conclusion, there are substantial differences in risk of hospitalization due to HF among immigrants from different countries that can not be explained by socioeconomic factors.

Immigrants from Finland, Former Yugoslavia and Hungary had compared to Swedish natives significantly higher risk. To what extent these differences could be explained by biological risk factors remains to be explored.

Legend to figure 1. Age-and sex-adjusted risk for hospitalization due to HF in relation to country of birth of immigrants and annual income based on 1990 GNI (Gross National Income).

Figure1.

  Figure1. Age-and sex-adjusted risk for hospitalization due to HF in relation to country of birth of immigrants and annual income based on 1990 GNI (Gross National Income).

Table 1 .

 1 Baseline characteristics of 40-89 years old Swedish natives and selected immigrant groups by country of birth, living in Malmö,Sweden, 1990.

		N	Men	Mean	Married	Self-	Mean	Mean
	Country of Birth		(%)	Age	(%)	owned	Income	Years in
				(years)		Home	(1000	Sweden
						(%)	SEK)	(years)
	Sweden	96674	44	62±13	54	58	136	-
	Denmark (1)	2639	46	60±11	48	46	118	20
	Finland (1)	1392	40	55±10	42	52	121	21
	Norway (1)	513	44	60±12	43	43	113	20
	Germany (1)	1484	41	61±12	52	57	125	22
	Austria (1)	221	48	59±12	57	57	123	21
	Italy (1)	238	70	55±10	63	52	120	21
	Former Yugoslavia (2)	3372	52	53±9	63	34	111	19
	Poland (2)	2221	36	55±13	45	41	94	14
	Portugal (2)	236	51	53±8	72	46	116	20
	Romania (2)	392	50	55±11	58	25	78	7
	Hungary(2)	1128	55	56±11	52	48	115	19
	Former Soviet Union (2)	475	40	62±13	47	45	107	18
	Czechoslovakia (2)	447	52	55±11	46	45	107	17
	Turkey (2)	213	55	53±11	59	10	93	16
	Greece (2)	284	56	53±9	67	19	97	17
	Iran (2)	237	57	50±10	58	10	31	3
	Chile (2)	337	46	51±10	38	22	87	9
	China, Vietnam (3)	284	46	57±13	69	12	51	8
	Countries with p-y < 2500	2130	58	53±11	51	32	93	13
	Total	114917	44	61±13	53	56	132	17
	Country according to income
	Sweden	96674	44	62±13	54	58	136	-
	High-income countries (1)	7439	45	59±12	48	49	118	20
	Middle-income countries (2)	10153	49	54±11	55	35	100	16
	Low-income countries (3)	600	61	52±12	59	17	70	10
	Not applicable #	51	61	61±13	43	29	84	12
	Total	114917	44	61±13	53	56	132	17
	Figures are shown as mean (SD) or as proportions
	SEK, Swedish Kronor
	# Missing data on country of birth
	Number attached to each specific country belonged to country classification according to GNI (Gross National Income)

Table 2 .

 2 Incidence and risk of hospitalization due to heart failure in relation to country of birth. HF, heart failure; MI, myocardial infarction; HR, hazard ratio; CI, confidence interval; p-y; person-years * HR adjusted for age and sex † HR adjusted for age, sex, marital status, annual income and self-owned home

			HF excluding previous MI	HF including previous MI
		n	HR*	HR †	n	HR*	HR †
	Country of Birth	(per 10,000	(95% Cl)	(95% Cl)	(per 10,000	(95% Cl)	(95% Cl)
		p-y)			p-y)
	Sweden	5589 (44)	Reference	Reference	6677 (51)	Reference	Reference
	Denmark	149 (43)	1.25 (1.07-1.48) 1.16 (0.99-1.37)	177 (51)	1.23 (1.06-1.43) 1.13 (0.98-1.32)
	Finland	64 (32)	1.52 (1.19-1.94) 1.40 (1.10-1.81)	78 (39)	1.52 (1.22-1.91) 1.42 (1.13-1.78)
	Norway	24 (35)	0.95 (0.64-1.42) 0.90 (0.61-1.36)	28 (41)	0.92 (0.63-1.33) 0.88 (0.61-1.28)
	Germany	90 (45)	1.16 (0.94-1.43) 1.13 (0.92-1.39)	109 (55)	1.17 (0.97-1.42) 1.14 (0.94-1.38)
	Austria	7 (23)	0.69 (0.33-1.45) 0.66 (0.31-1.38)	7 (25)	0.57 (0.27-1.20) 0.55 (0.26-1.15)
	Italy	8 (23)	0.94 (0.47-1.87) 0.85 (0.42-1.69)	11 (31)	1.04 (0.58-1.88) 0.94 (0.52-1.69)
	Former Yugoslavia	144 (29)	1.72 (1.45-2.03) 1.45 (1.23-1.72)	164 (33)	1.58 (1.35-1.85) 1.34 (1.14-1.57)
	Poland	100 (31)	1.20 (0.99-1.46) 1.05 (0.86-1.28)	119 (37)	1.18 (0.98-1.41) 1.03 (0.86-1.24)
	Romania	17 (30)	1.36 (0.84-2.19) 1.12 (0.69-1.80)	22 (38)	1.41 (0.93-2.15) 1.17 (0.77-1.78)
	Hungary	65 (41)	1.65 (1.29-2.10) 1.48 (1.16-1.89)	76 (48)	1.57 (1.25-1.97) 1.41 (1.12-1.77)
	Former Soviet Union	31 (47)	1.11 (0.78-1.57) 1.04 (0.73-1.48)	35 (56)	1.04 (0.75-1.46) 0.98 (0.70-1.37)
	Czechoslovakia	14 (21)	0.85 (0.51-1.44) 0.76 (0.45-1.29)	20 (30)	1.01 (0.65-1.55) 0.90 (0.58-1.40)
	Turkey	6 (19)	0.86 (0.39-1.92) 0.70 (0.31-1.55)	8 (25)	0.94 (0.47-1883) 0.76 (0.38-1.53)
	Greece	3 (8)	0.47 (0.15-1.45) 0.39 (0.13-1.20)	7 (17)	0.89 (0.42-1.86) 0.73 (0.35-1.54)
	Iran	6 (16)	1.05 (0.47-2.34) 0.78 (0.35-1.74)	9 (24)	1.27 (0.66-2.45) 0.96 (0.50-1.85)
	Chile	8 (16)	1.01 (0.50-2.01) 0.79 (0.39-1.58)	9 (18)	0.92 (0.48-1.78) 0.73 (0.38-1.40)
	China, Vietnam	9 (22)	0.77 (0.40-1.48) 0.62 (0.32-1.20)	10 (24)	0.71 (0.38-1.32) 0.58 (0.31-1.07)
	p value (df=19)		p<0.001	p<0.001		p<0.001	p<0.001
	Country according to
	income
	Sweden	5589 (44)	Reference	reference	6677 (51)	reference	reference
	High-income countries	377 (37)	1.19 (1.07-1.32) 1.12 (1.01-1.25)	451(44)	1.18 (1.07-1.30) 1.11 (1.01-1.22)
	Middle-income countries	415 (28)	1.31 (1.19-1.45) 1.14 (1.02-1.26)	493 (33)	1.28 (1.16-1.40) 1.10 (1.01-1.21)
	Low-income countries	16 (18)	0.91 (0.56-1.49) 0.74 (0.45-1.22)	19 (21)	0.89 (0.57-1.39) 0.73 (0.46-1.14)
	p value (df=4)		p<0.001	p= 0.023		p<0.001	p=0.035

Table 3 .

 3 Risk of hospitalization due to heart failure in relation to age, sex and socioeconomic status at baseline HF, heart failure; MI, myocardial infarction; HR, hazard ratio; Cl, confidence interval † HR adjusted for age, sex, and when appropriate for marital status, annual income and self-owned home

		HF excluding	HF including
		previous MI	previous MI
		HR † (95% Cl)	HR † (95% Cl)
	Age
	Per year	1.11 (1.10-1.12)	1.11 (1.10-1.11)
	p value	p<0.001	p<0.001
	Sex
	Woman	reference	reference
	Man	1.99 (1.88-2.11)	2.08 (1.98-2.20)
	p value	p<0.001	p<0.001
	Marital Status
	Married	reference	reference
	Single	1.16 (1.07-1.26)	1.10 (1.02-1.18)
	Divorced	1.18 (1.09-1.28)	1.19 (1.10-1.27)
	Widowed	1.20 (1.12-1.28)	1.19 (1.12-1.27)
	p value	p<0.001	p<0.001
	Housing Condition
	Self-owned	reference	reference
	Rented	1.16 (1.10-1.22)	1.16 (1.11-1.21)
	p value	p<0.001	p<0.001
	Annual Income (SEK)
	0 to 49,000	reference	reference
	50,000 -100,000	0.99 (0.93-1.07)	1.01 (0.94-1.07)
	100,100-150,000	0.85 (0.78-0.92)	0.86 (0.80-0.93)
	150,100-200,000	0.66 (0.60-0.74)	0.67 (0.61-0.74)
	200,100 -250,000	0.53 (0.45-0.61)	0.52 (0.46-0.60)
	>250,000	0.56 (0.48-0.66)	0.55 (0.48-0.63)
	p value	p<0.001	p<0.001

Acknowledgements

This work was supported by the Swedish Heart and Lung Foundation and by funds from the Medical Faculty of Uppsala University and Lund University.

Conflict of interest: none declared.