Nicolas Vignier

Maria Chatzifrangkeskou

Blanca Morales Rodriguez

Mathias Mericskay

Nathalie Mougenot

Karim Wahbi

Gisèle Bonne

Antoine Muchir
email: a.muchir@institut-myologie.org

Rescue of biosynthesis of nicotinamide adenine dinucleotide protects the heart in cardiomyopathy caused by lamin A/C gene mutation

Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscle and electrical dysfunction of the heart, often leading to heart failurerelated disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. Recent studies suggested that nicotinamide adenine dinucleotide (NAD + ) cellular content could be a critical determinant for heart function. Biosynthesis of NAD + from vitamin B3 (known as salvage pathways) is the primary source of NAD + . We showed here that NAD + salvage pathway was altered in the heart of mouse and human carrying LMNA mutation, leading to an alteration of one of NAD + co-substrate enzymes, PARP-1. Oral administration of nicotinamide riboside, a natural NAD + precursor and a pyridine-nucleoside form of vitamin B3, leads to a marked improvement of the NAD + cellular content, an increase of PARylation of cardiac proteins and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our results provide mechanistic and therapeutic insights into dilated cardiomyopathy caused by LMNA mutations.

Introduction

Nicotinamide adenine dinucleotide (NAD) is a metabolic cofactor found in cells either in its oxidized (NAD + ) or reduced (NADH) form. NAD + can be synthesized from various precursors. Biosynthesis from nicotinic acid or nicotinamide (NAM)-both present in our diet as vitamin B3-is the primary source of NAD + [START_REF] Bogan | Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition[END_REF][START_REF] Chi | Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection[END_REF][START_REF] Houtkooper | The secret life of NAD+: an old metabolite controlling new metabolic signaling pathways[END_REF]. This pathway, also known as the salvage, is important for NAD + homeostasis. There is another NAD + precursornicotinamide riboside (NR)-that enhances NAD + levels through the salvage pathway when catalyzed by nicotinamide riboside kinase (Nmrk) [START_REF] Bieganowski | Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans[END_REF][START_REF] Ratajczak | NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells[END_REF]. In mammals there are two Nmrk enzymes, Nmrk1 and Nmrk2, the latter being highly restricted to muscle. In the salvage pathway of NAD + biosynthesis, nicotinamide phosphoribosyltransferase (Nampt) was identified as a rate-limiting enzyme that converts NAM to nicotinamide mononucleotide (NMN) [START_REF] Garten | Physiological and pathophysiological roles of NAMPT and NAD metabolism[END_REF]. NAD + is then synthesized from NMN by NMN adenylyltransferase (Nmnat) [START_REF] Stein | The dynamic regulation of NAD metabolism in mitochondria[END_REF].

Studies have indicated that NAD + content is a critical determinant for heart function and structure [START_REF] Hsu | Nicotinamide phosphoribosyltransferase regulates cell survival through NAD+ synthesis in cardiac myocytes[END_REF][START_REF] Pillai | Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway[END_REF] and that Nampt may play an important role in the development of dilated cardiomyopathy (DCM) [START_REF] Dou | Association of nicotinamide phosphoribosyltransferase (NAMPT) gene polymorphisms and of serum NAMPT levels with dilated cardiomyopathy in a Chinese population[END_REF]. In models of DCM, modulation of the different actors of the NAD salvage improves cardiac function [START_REF] Diguet | Nicotinamide riboside preserves cardiac function in a mouse model of dilated cardiomyopathy[END_REF][START_REF] Martin | Nicotinamide mononucleotide requires SIRT3 to improve cardiac function and bioenergetics in a Friedreich's ataxia cardiomyopathy model[END_REF][START_REF] Kuno | Resveratrol improves cardiomyopathy in dystrophin-deficient mice through SIRT1 protein-mediated modulation of p300 protein[END_REF]. DCM is characterized by dilatation and systolic dysfunction of the left or both ventricles [START_REF] Elliott | Classification of the cardiomyopathies: a position statement from the european society of cardiology working group on myocardial and pericardial diseases[END_REF]. The ventricular walls become thin and stretched, compromising cardiac contractility and resulting in altered left ventricular function. Despite current strategies to aggressively manage DCM, the disorder remains a common cause of heart failure and a prevalent diagnosis in individuals referred for cardiac transplantation. Mutations in LMNA encoding nuclear lamin A/C [START_REF] Lin | Structural organization of the human gene encoding nuclear lamin A and nuclear lamin C[END_REF] were reported to cause DCM (hereafter referred to LMNA cardiomyopathy) [START_REF] Fatkin | Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease[END_REF][START_REF] Bonne | Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy[END_REF]. Despite our gaps in understanding many of their fundamental functions, much of the current research on the lamin A/C is focused on how mutations within these proteins cause DCM. We asked here whether NAD + salvage pathway is altered and could play a role in the development of LMNA cardiomyopathy.

Results

Alteration of NAD + salvage pathway in LMNA cardiomyopathy

To explore the role of NAD + salvage pathway in the development of DCM, we studied a mouse model that displays an Lmna mutation changing the histidine in position 222 into a proline [START_REF] Arimura | Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies[END_REF]. The male mice develop a progressive contractile dysfunction (Supplementary Material, Table 1), cardiac remodeling and die by 32-34 weeks of age [START_REF] Arimura | Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies[END_REF]. Lmna p.H222P corresponds to a human disease-causing mutation associated with DCM [START_REF] Bonne | Clinical and molecular genetic spectrum of autosomal dominant Emery-Dreifuss muscular dystrophy due to mutations of the lamin A/C gene[END_REF]. Given that a consequence of altered NAD + salvage pathways would lead to an aberrant cellular NAD + content (Fig. 1A), we first assessed the steady-state cardiac NAD + level in both male wild-type (WT) and Lmna H222P/H222P mice. We showed that the NAD + content was significantly lowered in hearts from Lmna H222P/H222P mice compared with WT mice, when the left ventricular function was altered at 26 weeks (symptomatic) (Fig. 1B andC). There was a correlation between altered cardiac function and cardiac NAD + content, as there was no detectable decreased NAD + level in the heart of Lmna H222P/H222P mice at 14 weeks (pre-symptomatic) (Fig. 1B andC; Supplementary Material, Table 1).

We next assessed the expression level of enzymes from the salvage pathway of NAD + biosynthesis by real-time Polymerase Chain Reaction (PCR) using RNA extracted from Lmna H222P/H222P mouse hearts at 14 and 26 weeks of age. There was a significant decreased Nampt mRNA expression and a significant increased Nmrk2 mRNA expression in the hearts of Lmna H222P/H222P mice compared with WT mice (Fig. 1D). In addition, Nmnat1 mRNA expression (the nucleus isoform) was decreased at 14 weeks of age, and both cardiac mRNA expression levels of Nmnat1 and Nmnat3 (the mitochondrial isoform) were significantly decreased in Lmna H222P/H222P mice at 26 weeks of age, compared with WT mice (Fig. 1D). We then evaluated the expression of Nampt, Nmrk2 and Nmnat1 at the protein level. The deregulation of the protein expression level was in concordance with mRNA expression level in hearts from Lmna H222P/H222P mice compared with WT mice at 26 weeks of age (Fig. 1E). While these results strongly suggest that abnormal NAD + salvage pathway contributes to the development of cardiomyopathy in Lmna H222P/H222P mice, its pathogenic role in affected skeletal muscles is unknown. Based on our findings in heart, we hypothesize that abnormal NAD + salvage pathway is similarly involved in the pathogenesis of skeletal muscular dystrophy in the Lmna H222P/H222P mouse model. Furthermore, we showed a significant drop of NAD + content (Fig. S1A), a significant decreased Nampt and Nmnat1 and increased Nmrk2 mRNAs expression (Fig. S1B) in soleus from Lmna H222P/H222P mice at 26 weeks of age, compared with WT mice. We showed a similar decreased Nampt mRNA expression and increased Nmrk2 mRNA expression in the hearts of another mouse model of LMNA cardiomyopathy, Lmna delK32/+ mice [START_REF] Cattin | Heterozygous LmnadelK32 mice develop dilated cardiomyopathy through a combined pathomechanism of haploinsufficiency and peptide toxicity[END_REF], at 26 weeks of age compared with WT mice, when the left ventricular function was severely altered (Fig. S2A andS2B). The same expression pattern of these key genes was also found in other mouse models of inherited DCM (GEO accession number: GSE54838 and GSE17478) (Fig. S1C) [START_REF] Akbay | D-2-hydroxyglutarate produced by mutant IDH2 causes cardiomyopathy and neurodegeneration in mice[END_REF][START_REF] Wang | Particulate matter induces cardiac arrhythmias via dysregulation of carotid body sensitivity and cardiac sodium channels[END_REF]. We obtained samples of heart tissue from two human subjects with LMNA cardiomyopathy obtained after cardiac transplantation. Control heart samples were obtained from age-matched human subjects without LMNA mutation. We showed decreased expression of Nampt and increased expression of Nmrk2, in heart tissue of the patients with LMNA mutations compared with control subjects (Fig. 1F). Taken together these results showed that the metabolism of NAD + is altered in LMNA cardiomyopathy.

Rescue of cardiac NAD + content by NR improves cardiac function

Given that the expression of Nmrk2 was increased in heart from Lmna H222P/H222P mice, we tested whether NR, the substrate for Nmrk2 [START_REF] Trammell | Nicotinamide riboside is uniquely and orally bioavailable in mice and humans[END_REF], could increase NAD + content in the heart and therefore, blunt the development of left ventricular dysfunction. Lmna H222P/H222P mice were fed with NR-supplemented diet (400 mg/kg/day), starting at 17 weeks of age for 9 weeks duration (Fig. 2A). We first determined the NAD + content of NR-fed Lmna H222P/H222P mice compared with chow diet-fed Lmna H222P/H222P mice. We similarly treated WT mice and assessed the NAD + content. Lmna H222P/H222P mice fed with NR showed an increase of their NAD + content in both liver and heart (Fig. 2B). Feeding WT mice with the same concentration of NR leads similarly to increased NAD + content in both tissues (Fig. 2B). While around 25% of chow diet-fed Lmna H222P/H222P mice died during the duration of the protocol, we did not observed any death in the group of Lmna H222P/H222P mice fed with NR (data not shown). No significant difference in mortality was observed between NR-fed and chow diet-fed WT mice (data not shown). After NR treatment, heart size and left ventricular dilatation were reduced in Lmna H222P/H222P mice compared with chow diet-fed Lmna H222P/H222P mice (Fig. 2C). Cardiac structure and function were assessed by echocardiography after 4 weeks of treatment and at the end of the treatment in the four different groups of mice. Compared with chow diet-fed Lmna H222P/H222P mice, NR-fed Lmna H222P/H222P mice had significantly decreased left ventricular end-systolic and left ventricular end-diastolic diameters (Table 1). NR-fed Lmna H222P/H222P mice also have improved cardiac contractility, indicated by increased left ventricular fractional shortening, starting as early as 4 weeks of treatment (Fig. 2D, Table 1). Hence, feeding with NR for 9 weeks delayed the development of left ventricular dilatation and cardiac contractile dysfunction in Lmna H222P/H222P mice. Compared with WT mice, Lmna H222P/H222P mice had significantly increased expression of Myh7, which encodes myosin heavy chain, Nppa, which encodes the atrial natriuretic peptide type A and Col1a2, which encodes collagen (Fig. 2E). Feeding Lmna H222P/H222P mice with NR led to a significant lowering of the expression of these markers of cardiac remodeling compared with chow diet-fed animals (Fig. 2E). NR-fed Lmna H222P/H222P mice exhibited a decreased mRNA expression of Nmrk2 but Nampt mRNA expression was unchanged, compared with chow diet-fed Lmna H222P/H222P (Fig. S3A). In a pilot study, we assessed the effect of NR treatment on survival. The mean survival was significantly prolonged in NR-fed Lmna H222P/H222P mice compared with chow diet-fed mice (Fig. 2F). Taken together, these results showed that NR supplemented diet is efficient to partially restore the left ventricular function and increase the survival in a mouse model of LMNA cardiomyopathy.

A critical question relevant to potential treatment of patients with NR regards their effectiveness after the onset of cardiac dysfunction. To help answer this question, we assessed the benefit of NR after dilatation and decreased cardiac fractional shortening have already occurred. Lmna H222P/H222P mice were treated with NR (daily oral dose, 400 mg/kg per day), starting when left ventricular fractional shortening was significantly lowered (30%) compared with WT mice. NR increased NAD + content in both liver and heart compared with placebo (Fig. 2H.a). We used M-mode echocardiograph to image in vivo and assess left ventricular diameters. It showed that left ventricular dimensions and fractional shortening remained stable during the course of the treatment in Lmna H222P/H222P mice while this parameters were decreased in placebo-treated mice (Fig. 2H.b; Supplementary Material, Table S2)

Nicotinamide supplement is not beneficial for the cardiac function in LMNA cardiomyopathy

We next assessed the benefit of NAM (the substrate for Nampt) on the left ventricular function. If our hypothesis posits that NAD + salvage signaling plays a role in the pathogenesis of LMNA cardiomyopathy, and given that Nampt cardiac expression level is decreased in Lmna H222P/H222P mice, providing NAM should neither restore the cardiac NAD + content nor improve the leftventricular dysfunction. We treated Lmna H222P/H222P mice with NAM (500 mg/kg every other day) starting at 17-weeks of age, for 9 weeks (Fig. 3A). Lmna H222P/H222P mice treated with NAM did not exhibit changes of their NAD + content in both liver and heart compared with placebo-treated animals (Fig. 3B). Left ventricular structure and function were assessed by echocardiography after 4 weeks of treatment and at the end of the NAM treatment. Compared with placebo-treated Lmna H222P/H222P mice, NAM-treated Lmna H222P/H222P mice had no significant changes in left ventricular end-systolic and left ventricular end-diastolic diameters as well as fractional shortening (Fig. 3C andD, Table 2). NAM-treated Lmna H222P/H222P mice had no changed expression of cardiac Myh7, Nppa and Col1a2 (Fig. 3E). Thus, our data showed that treating Lmna H222P/H222P mice with NAM is not efficient to restore the cardiac NAD + content and the cardiac left ventricular function.

Decreased cardiac NAD + content alters global PARylation

We then asked the consequences of decreased cardiac NAD + on sirtuin 1 (SIRT1) and Poly(ADP-ribose) polymerase 1 (PARP-1) activities. The sirtuins are protein deacetylases, which play roles on mitochondria metabolism [START_REF] Stein | The dynamic regulation of NAD metabolism in mitochondria[END_REF][START_REF] Verdin | NAD+ in aging, metabolism, and neurodegeneration[END_REF]. The NAD + level has been shown to activate SIRT1, which regulates mitochondria biogenesis through the deacetylation of target proteins. We showed that the level of SIRT1 mRNA (Fig. 4A) and protein (Fig. 4B) expressions were not altered in hearts from Lmna H222P/H222P mice compared with WT mice. The levels of acetylated FOXO1 (Fig. 4C), P65 (Fig. 4D) and p53 (Fig. 4E), known targets of SIRT1 (25-27), were unchanged in hearts from Lmna H222P/H222P mice compared with WT mice. Thus, our data suggested that the decreased cardiac NAD + content is not altering SIRT1 activity in LMNA cardiomyopathy. PARP enzymes bind and cleave NAD + to produce NAM and ADP-ribose, and couple one or more ADP-ribose units to promote PARylation of acceptor proteins [START_REF] Bai | Biology of poly(ADP-Ribose) polymerases: the factotums of cell maintenance[END_REF]. The level of PARylation detected by poly(ADP-ribose) (PAR) monoclonal antibody was decreased in hearts from Lmna H222P/H222P mice compared with WT mice (Fig. 5A). Feeding Lmna H222P/H222P mice with NR led to a marked increase of cardiac PARylation (Fig. 5A). We next showed that mRNA (Fig. 5B) and protein (Fig. 5C) levels of cardiac PARP-1, the most abundant isoform of the PARP enzyme family, were lowered in Lmna H222P/H222P mice compared with WT mice and increased after NR feeding. We performed immunoprecipitation using extracts of hearts from Lmna H222P/H222P mice and WT mice and antibody against poly(ADP-ribose). We found that the PARylation of cardiac PARP-1 was lowered in Lmna H222P/H222P mice compared with WT mice (Fig. 5D). Therefore, these results showed that the drop in NAD + cardiac content in Lmna H222P/H222P mice was altering PARP-1 activity.

Discussion

Altered NAD + metabolism through the salvage pathway has been described in muscular dystrophies [START_REF] Ryu | NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation[END_REF][START_REF] Khan | Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3[END_REF][START_REF] Hori | Resveratrol ameliorates muscular pathology in the dystrophic mdx mouse, a model for duchenne muscular dystrophy[END_REF][START_REF] Goody | NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy[END_REF] and aging process [START_REF] Balan | Life span extension and neuronal cell protection by Drosophila nicotinamidase[END_REF][START_REF] Belenky | Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+[END_REF][START_REF] Massudi | Age-associated changes in oxidative stress and NAD+ metabolism in human tissue[END_REF][START_REF] Mouchiroud | The NAD(+)/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling[END_REF][START_REF] Zhang | NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice[END_REF][START_REF] Zhu | In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences[END_REF]. We expand the current knowledge by showing that altered NAD + salvage causes heart failure and inherited cardiomyopathy. We demonstrate that oral supplementation with NR, a vitamin B3 form and NAD + precursor, efficiently prevented development and progression of LMNA cardiomyopathy in mice. We show that NR supplementation significantly delayed disease progression even in mice that already manifested the disease, which is the time frame when medicated treatment would typically be started for patients. These results are remarkable, as they underline the utmost importance of specific vitamin co-factors, as modifiers of metabolism in cardiac disease, and emphasize the role of nutritional signaling in the pathogenesis of LMNA cardiomyopathy. The results presented in this study are in line with previous work showing that exogenous NAD + content blocks cardiac hypertrophy, [START_REF] Pillai | Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway[END_REF][START_REF] Diguet | Nicotinamide riboside preserves cardiac function in a mouse model of dilated cardiomyopathy[END_REF]. A recent study demonstrated that NR treatment transiently rescued the cardiomyopathy in iron deficiency mouse model [START_REF] Xu | Lethal cardiomyopathy in mice lacking transferrin receptor in the heart[END_REF]. Similarly, increasing the NAD + content using precursors of NAD + has been proven efficient to rescue hallmark of mitochondrial myopathy both in mouse [START_REF] Khan | Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3[END_REF] and human [START_REF] Weijer | Evidence for a direct effect of the NAD+ precursor acipimox on muscle mitochondrial function in humans[END_REF], as well as in aging [START_REF] Zhang | NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice[END_REF][START_REF] Rajman | Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence[END_REF]. How decreased NAD + results in cardiac tissue injury in LMNA cardiomyopathy is yet to be fully elucidated and this would need further investigation. We showed that PARylation is altered in LMNA cardiomyopathy, ensuing decreased cardiac NAD + . PARP enzymes bind and cleave NAD + to nicotinamide and charged PAR polymers on a large set of target proteins [START_REF] Alvarez-Gonzalez | Dissection of ADP-ribose polymer synthesis into individual steps of initiation, elongation, and branching[END_REF]. Given its DNA-binding zinc fingers [START_REF] Alvarez-Gonzalez | Dissection of ADP-ribose polymer synthesis into individual steps of initiation, elongation, and branching[END_REF][START_REF] Langelier | Structural basis for DNA-dependent poly(ADP-ribosyl)ation by human PARP-1[END_REF] PARP-1 can be activated through DNA breaks [START_REF] Benjamin | Poly(ADP-ribose) synthesis in vitro programmed by damaged DNA. A comparison of DNA molecules containing different types of strand breaks[END_REF] and irregular DNA structures [START_REF] Kun | Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone H1 trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I)[END_REF], and thus cleaves NAD + generating NAM and ADP-ribose. In the attempt to repair DNA lesions, PARP-1 activation takes place by adding ADP-ribose units to form chains of PAR covalently attached to acceptor proteins, among which PARP-1, to facilitate the DNA repair machinery [START_REF] Javle | The role of PARP in DNA repair and its therapeutic exploitation[END_REF][START_REF] De Vos | The diverse roles and clinical relevance of PARPs in DNA damage repair: current state of the art[END_REF]. It would be of interest to further investigate that the inhibition of PARP-1, and its PARylation, by the decreased cardiac NAD + content in Lmna H222P/H222P mice, could fail to trigger adaptive mechanisms, such as DNA repair, in the development of LMNA cardiomyopathy.

In conclusion, our experiments demonstrate a novel contributory metabolic mechanism for LMNA cardiomyopathy triggered by altered NAD + salvage pathway. Moreover, we have shown that modulation of cardiac NAD + content using NR is a straightforward therapeutic strategy. Given that vitamin B3 supplement is potentially translatable into therapy, our work supports more studies on human to begin to evaluate the therapeutic benefits of increasing NAD + and assess the benefit of such therapy on LMNA cardiomyopathy. Accordingly, recent work described that normalization of NAD + content could be used as a pharmacologic strategy in failing heart [START_REF] Diguet | Nicotinamide riboside preserves cardiac function in a mouse model of dilated cardiomyopathy[END_REF][START_REF] Lee | Normalization of NAD+ redox balance as a therapy for heart failure[END_REF] and a clinical trial to assess the benefit of NR in systolic heart failure is on-going (NCT03423342). This novel therapy would need to be associated with reduced risks, particularly as the treatment may be needed for years. The translation from bench-to-bedside could be seen in years to come since precursors of NAD + [e.g. Acipimox [START_REF] Fuccella | Inhibition of lipolysis by nicotinic acid and by acipimox[END_REF][START_REF] Musatti | Bioavailability and pharmacokinetics in man of acipimox, a new antilipolytic and hypolipemic agent[END_REF], Nicobion, Nicorandil] have already been used in therapeutics [START_REF] Ball | Acipimox in the treatment of patients with hyperlipidaemia: a double blind trial[END_REF][START_REF] Chen | A phase 3 randomized trial of nicotinamide for skin-cancer chemoprevention[END_REF][START_REF] Ishii | Impact of a single intravenous administration of nicorandil before reperfusion in patients with ST-segment-elevation myocardial infarction[END_REF][START_REF] Study | Effect of nicorandil on coronary events in patients with stable angina: the impact of nico-randil in angina (IONA) randomised trial[END_REF]. Recently, it has been shown that a single dose of NR resulted in significant increases in NAD + in healthy human volunteers [START_REF] Trammell | Nicotinamide riboside is uniquely and orally bioavailable in mice and humans[END_REF]. Overall, our work provides adequate grounds for a therapeutic approach based on NR for patients with LMNA cardiomyopathy.

Experimental Procedures

Human heart tissue

Explanted hearts from human subjects with LMNA cardiomyopathy were obtained from Myobank-AFM from the lnstitut de Myologie (Paris, France). The subjects were a 23-year-old man

Animals

All animal experiments were approved by the French Ministry of Health at the Center for Research in Myology for the Care and Use of Experimental Animals (approval number #6455). The animal experiments were performed according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Lmna H222P/H222P mice were fed chow and housed in a disease-free barrier facility with 12 h light/12 h dark cycles. Chow supplemented with 0.25% NR (400 mg/kg) (Chromadex), as previously reported [START_REF] Vaziri | hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase[END_REF], was formulated by Scientific Animal Food and Engineering. For postsymptomatic treatment, NR was delivered to Lmna H222P/H222P mice at a dose of 400/kg per day orally by gavage. NR was formulated in water and water was used as placebo. NAM was delivered to a concentration of 500 mg/kg every other day by intraperitoneal injection using a 27 G 5/8 syringe. NAM was formulated in physiologic serum and physiologic serum was used as placebo. For survival analysis, Lmna H222P/H222P mice were fed chow supplemented with 0.25% NR when they were 17 weeks of age and continued until they suffered from significant distress or died.

Echocardiography

Lmna H222P/H222P and WT mice were anesthetized with 0.75% isoflurane in O 2 and placed on a heating pad (25 • C). Echocardiography was performed using an ACUSON 128XP/10 ultrasound with an 11 MHz transducer applied to the chest wall. Cardiac ventricular dimensions and fractional shortening were measured in 2D mode and M-mode 3 times for the number of animals indicated. A 'blinded' echocardiographer, unaware of the genotype and the treatment, performed the examinations.

qPCR analysis

Total RNA was extracted using the RNeasy Mini kit (Qiagen). cDNA was synthesized using the SuperScript III synthesis system according to the manufacturer's instructions (Invitrogen). Real-time qPCR reactions were performed with SYBR Green I Master mix (Roche) using with the LightCycler 480 (Roche). Relative levels of mRNA expression calculated using the C T method were normalized by comparison to housekeeping mRNA [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. The listing of primers can be found in Supplementary Material Table S3.

Immunoblotting

Mouse heart tissues were homogenized in sample extraction buffer (Cell Signaling) completed with deacetylation inhibitors cocktail (Santa Cruz Biotechnology) then separated by 10% SDSpolyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted with primary antibodies (Supplementary Material, Table S4). Secondary antibodies were horseradish peroxidate-conjugate (Jackson ImmunoResearch). Recognized proteins were visualized by enhanced chemiluminescence.

NAD + concentration quantification.

Fresh mouse tissues were homogenized and processed according to previous report [START_REF] Sander | Quantitative analysis of pyridine nucleotides in red blood cells: a single-step extraction procedure[END_REF]. The concentration of cardiac and liver NAD + was determined using a spectrophotometric assay as described previously [START_REF] Bernofsky | An improved cycling assay for nicotinamide adenine dinucleotide[END_REF]. Absorption measurements were carried out in 96-well plates by spectrophotometry (Tecan).

Statistics.

Values for real-time q-PCR, immunoblots and NAD + content were compared using an unpaired Student's t-test. Comparisons of echocardiographic parameters between groups of Lmna H222P/H222P mice (treated and untreated) were performed using a Welch t-test; to validate these results, a non-parametric test (Mann-Whitney) was performed and concordance checked. Statistical analyses were performed using Prism 6.0 software (Graphpad).

Figure 1 .

 1 Figure 1. NAD + salvage pathway is altered in LMNA cardiomyopathy (A) Schematic representation of the NAD + salvage pathway. (B) Quantification of cardiac NAD + content (pmol/mg tissue) in WT (n = 6) and Lmna H222P/H222P (n = 9) mice at 14 weeks and in WT (n = 6) and Lmna H222P/H222P (n = 7) mice at 26 weeks. Bars indicate mean ± standard error of mean. **P-value ≤ 0.001. (C) Box-and-whisker plots showing median fractional shortening (FS) in 14-weeks old and 26-weeks old WT and Lmna H222P/H222P mice. Values are shown as 25 th to 75 th percentiles of the ranked set of data values. The line in the middle is plotted as the median. Whiskers (Tukey method) extend down to the minimum value and up to the maximum value. ***P-value ≤ 0.0001.(D) Expression of Nampt, Nmrk2, Nmnat1, Nmnat2 and Nmnat3 mRNAs obtained by qPCR from WT (n = 5) and Lmna H222P/H222P (n = 5) mice at 14 weeks and from WT (n = 5) and Lmna H222P/H222P (n = 5) mice at 26 weeks of age. *P-value ≤ 0.01, **P-value ≤ 0.001 and ***P-value ≤ 0.0001. (E) Immunoblots showing Nampt, Nmrk2 and Nmnat1 protein levels in hearts from 26-weeks-old WT (n = 4) and Lmna H222P/H222P (n = 4) mice. Representative of three independent experiments each performed in triplicates. The bar graphs represent protein relative expression (mean ± standard error of means) of Nampt and Nmrk2 (relative to ERK1/2). *P-value ≤ 0.01. (F) Immunoblots showing Nampt and Nmrk2 protein levels in hearts from controls (n = 2) and LMNA patients (n = 2). The bar graphs represent protein relative expression (mean ± standard error of mean) of Nampt (relative to ERK1/2).

Figure 2 .

 2 Figure 2. NR supplementation leads to cardiac improvement in Lmna H222P/H222P mice. (A) Schematic representation of the treatment protocol of WT and Lmna H222P/H222P mice with NR. (B) Quantification of NAD + content (pmol/mg tissue) in heart and liver from chow diet-fed WT (n = 7) and Lmna H222P/H222P (n = 8) mice and in NR fed WT (n = 7) and Lmna H222P/H222P (n = 26) mice at 26 weeks. Bars indicate means ± standard errors of means. ***P-value ≤ 0.0001. (C) Gross histological examination of heart from chow diet-fed or NR-fed WT and Lmna H222P/H222P mice at 26 weeks of age. (D) Box-and-whisker plots showing median fractional shortening (FS) in 17-, 21and 26-weeks-old chow diet-fed or NR-fed WT and Lmna H222P/H222P mice. Values are shown as 25 th to 75 th percentiles of the ranked set of data values. The line in the middle is plotted as the median. Whiskers (Tukey method) extend down to the minimum value and up to the maximum value. *P-value ≤ 0.01, **P-value ≤ 0.001, ***P-value ≤ 0.0001. (E) Expression of Myh7, Nppa and Col1a2 genes by qPCR from cDNAs of heart from chow diet-fed or NR-fed WT (n = 5) and Lmna H222P/H222P (n = 5) mice. ***P-value ≤ 0.0001. (F) Kaplan-Meier survival curves for chow diet-fed WT (n = 5) and Lmna H222P/H222P (n = 9) mice in NR-fed WT (n = 5) and Lmna H222P/H222P (n = 4) mice. ***P-value ≤ 0.0001. (G) Schematic representation of the treatment protocol of post symptomatic Lmna H222P/H222P mice with NR. (H.a) Quantification of NAD + content (pmol/mg tissue) in heart and liver from placebo-treated Lmna H222P/H222P (n = 5) mice and NR-treated Lmna H222P/H222P (n = 5) mice at 26 weeks. Bars indicate means ± standard errors of means. *P-value ≤ 0.01. (H.b) Box-and-whisker plots showing median fractional shortening (FS) in 17-, 21-and 26-weeks-old placebo-treated or NR-treated Lmna H222P/H222P mice. Values are shown as 25 th to 75 th percentiles of the ranked set of data values. The line in the middle is plotted as the median. Whiskers (Tukey method) extend down to the minimum value and up to the maximum value. ***P-value ≤ 0.0001, NS: non significant.

Figure 3 .

 3 Figure 3. NAM supplementation does not improve cardiac function in Lmna H222P/H222P mice (A) Schematic representation of the treatment protocol of Lmna H222P/H222P mice with NAM. (B) Quantification of NAD + content (pmol/mg tissue) in heart and liver from placebo-treated Lmna H222P/H222P (n = 6) mice and NAM-treated Lmna H222P/H222P (n = 7) mice at 26 weeks. Bars indicate means ± standard errors of means. (C) Gross histological examination of heart from placebo-treated and NAM-treated Lmna H222P/H222P (n = 7) mice at 26 weeks of age. (D) Box-and-whisker plots showing median fractional shortening (FS) in 17-, 21-and 26-weeks-old placebo-treated or NAM-treated Lmna H222P/H222P mice. Values are shown as 25 th to 75 th percentiles of the ranked set of data values. The line in the middle is plotted as the median. Whiskers (Tukey method) extend down to the minimum value and up to the maximum value. (E) Expression of Myh7, Nppa and Col1a2 genes by qPCR from cDNAs of heart from placebo or NAM-treated Lmna H222P/H222P (n = 5) mice.

Figure 4 .

 4 Figure 4. NAD + depletion does not alter SIRT1-dependent lysine acetylation in LMNA cardiomyopathy. (A) Expression of Sirt1 mRNA in hearts from a 26-week-old male WT (n = 4) and Lmna H222P/H222P (n = 4) mice. Data are represented as means ± standard errors of means. (B) Immunoblots showing Sirt1 protein level expression in a 26-week-old male WT and Lmna H222P/H222P mice. (C) Immunoblots showing acetylated-FOXO1 and total FOXO1 protein level expression in a 26-week-old male WT and Lmna H222P/H222P mice. (D) Immunoblots showing acetylated-P65 and total P65 protein level expression in a 26-week-old male WT and Lmna H222P/H222P mice. (E) Immunoblots showing acetylated-P53 and total P53 protein level expression in a 26-week-old male WT and Lmna H222P/H222P mice.

  carrying the LMNA c.781-783delAAG, p.Lys261del mutation and 47-year-old woman carrying the LMNA c.178C > G, p.Arg60Gly mutation. Control human heart samples were obtained from the National Disease Research Interchange (Philadelphia, PA, USA) from a 15-year-old girl who died of overdose and a 57-yearold man who died of cerebrovascular accident. Tissue samples received from either autopsy or transplanted consent donors were not obtained specifically for this study and provided by Myobank-AFM or the National Disease Research Interchange without patient identifiers.

Figure 5 .

 5 Figure 5. NAD + depletion causes decrease in global PARylation. (A) Immunoblots showing total PARylated protein level expression (PAR antibody) in hearts from a 26-week-old WT (n = 3) and from a 26-week-old chow diet-fed Lmna H222P/H222P (H222P) (n = 6) mice and in NR-fed Lmna H222P/H222P (n = 3) mice. ERK1/2 was shown as loading control. The bar graphs represent PARylated protein relative expression (mean ± standard error of means) (relative to ponceau). (B) Expression of Parp1 gene by qPCR from cDNAs of heart from WT (n = 5), chow diet-fed (n = 5) or NR-fed Lmna H222P/H222P (n = 5). *P-value ≤ 0.01, **P-value ≤ 0.001. (C) Immunoblots showing PARP-1 protein level in hearts from a 26-week-old WT (n = 3) and from a 26-week-old chow diet-fed Lmna H222P/H222P (H222P) (n = 6) mice and in NR-fed Lmna H222P/H222P (n = 3) mice. The bar graphs represent protein relative expression (means ± standard errors of means) of PARP-1 (relative to ERK1/2). *P-value ≤ 0.01. (D) Representative immunoprecipitation showing PARylated PARP-1 level in hearts from a 26-week-old WT, Lmna H222P/H222P and NR fed Lmna H222P/H222P mice. Immunoprecipitation showing PARP-1 level in hearts from a 26-week-old WT Lmna H222P/H222P and NR-fed Lmna H222P/H222P mice was shown as control.

Table 1 .

 1 Echocardiographic parameters for WT and Lmna H222P/H222P mice fed with either NR or chow diet

	Genotype	WT	WT	Lmna H222P/H222P	Lmna H222P/H222P
	Treatment	Chow-diet	NR-diet	Chow-diet	NR-diet
	n	1 3	1 2	1 1	3 1
	Age, weeks	17	17	17	17
	Heart rate, bpm	534.8 ± 35.5	553.9 ± 17.8	531.1 ± 34.8	549.2 ± 31,6
	IVSd, mm	0.62 ± 0.01	0.63 ± 0.02	0.59 ± 0.01	0.62 ± 0.01
	LVPWd, mm	0.58 ± 0.03	0.60 ± 0.02	0.62 ± 0.02	0.62 ± 0.01
	LVDd, mm	3.73 ± 0.11	3.68 ± 0.06	4.07 ± 0.11 #	3.80 ± 0.06
	IVSs, mm	0.98 ± 0.03	1.02 ± 0.02	0.93 ± 0.02	1.00 ± 0.02 *
	LVPWs, mm	0.96 ± 0.04	1.00 ± 0.03	0.95 ± 0.03	0.98 ± 0.02
	LVDs, mm	2.25 ± 0.11	2.12 ± 0.04	2.80 ± 0.14 #	2.42 ± 0.06 **
	FS, %	39.99 ± 1.26	42.91 ± 0.44	31.65 ± 1.82 ##	36.57 ± 0.94 *
	Genotype	WT	WT	Lmna H222P/H222P	Lmna H222P/H222P
	Treatment	Chow-diet	NR-diet	Chow-diet	NR-diet
	n	1 3	1 2	1 1	2 4
	Age, weeks	21	21	21	21
	Heart rate, bpm	560.1 ± 26.3	570.3 ± 42.13	551.1 ± 34.7	564.2 ± 36.2
	IVSd, mm	0.67 ± 0.01	0.68 ± 0.01	0.54 ± 0.02 ###	0.63 ± 0.01 ***
	LVPWd, mm	0.69 ± 0.02	0.72 ± 0.02	0.52 ± 0.03 ###	0.61 ± 0.02 **
	LVDd, mm	3.76 ± 0.07	3.73 ± 0.10	4.37 ± 0.15 ###	3.96 ± 0.05 **
	IVSs, mm	1.15 ± 0.02	1.13 ± 0.03	0.77 ± 0.04 ###	1.03 ± 0.02 ***
	LVPWs, mm	1.11 ± 0.03	1.13 ± 0.01	0.75 ± 0.03 ###	0.97 ± 0.03 ***
	LVDs, mm	2.10 ± 0.04	2.06 ± 0.08	3.42 ± 0.22 ###	2.53 ± 0.07 ***
	FS, %	44.42 ± 0.46	45.01 ± 0.87	26.14 ± 2.41 ###	36.20 ± 1.26 **
	Genotype	WT	WT	Lmna H222P/H222P	Lmna H222P/H222P
	Treatment	Chow-diet	NR-diet	Chow-diet	NR-diet
	n	1 3	1 2	9	3 1
	Age, weeks	26	26	26	26
	Heart rate, bpm	537.6 ± 28.9	558.5 ± 26.8	536.2 ± 37.8	555.1 ± 32.7
	IVSd, mm	0.64 ± 0.01	0.66 ± 0.01	0.57 ± 0.02 #	0.63 ± 0.01 *
	LVPWd, mm	0.73 ± 0.02	0.70 ± 0.03	0.48 ± 0.04 ###	0.58 ± 0.02 *
	LVDd, mm	3.97 ± 0.13	3.78 ± 0.08	4.32 ± 0.19	4.18 ± 0.06
	IVSs, mm	1.05 ± 0.05	1.05 ± 0.03	0.71 ± 0.04 ###	0.97 ± 0.03 ***
	LVPWs, mm	1.06 ± 0.03	1.11 ± 0.04	0.71 ± 0.04 ###	0.94 ± 0.03 ***
	LVDs, mm	2.52 ± 0.18	2.23 ± 0.10	3.47 ± 0.23 #	2.90 ± 0.10 *
	FS, %	37.62 ± 2.49	41.20 ± 1.48	20.63 ± 2.17 ###	30.89 ± 1.57 **

IVS, inter ventricular septum; LVPW, left ventricular posterior wall; LVD, left ventricular diameter; FS, fractional shortening; s, systole; d, diastole. Values are means ± standard errors of means. # P-value ≤ 0.01, ## P-value ≤ 0.001 and ### P-value ≤ 0.0001 between chow diet-fed WT mice and chow diet-fed Lmna H222P/H222P mice. *P-value ≤ 0.01, **P-value ≤ 0.001 and ***P-value ≤ 0.0001 between NR die-fed Lmna H222P/H222P mice and chow diet-fed Lmna H222P/H222P mice.

Table 2 .

 2 Echocardiographic parameters for male Lmna H222P/H222P mice treated or not with NAM at 17, 21 and 26 weeks of age

	Genotype	Lmna H222P/H222P	Lmna H222P/H222P	Lmna H222P/H222P	Lmna H222P/H222P	Lmna H222P/H222P	Lmna H222P/H222P
	Treatment	Placebo	NAM	Placebo	NAM	Placebo	NAM
	n	6	7	5	6	5	5
	A g e , w e e k s	1 7	1 7	2 1	2 1	2 6	2 6
	Heart rate, bpm	534.0 ± 35.52	553.9 ± 17.82	531.1 ± 34.84	549.2 ± 31.65	560.1 ± 26.31	570.3 ± 42.13
	IVSd, mm	0.65 ± 0.03	0.71 ± 0.03	0.64 ± 0.02	0.68 ± 0.02	0.56 ± 0.02	0.64 ± 0.02
	LVPWd, mm	0.60 ± 0.03	0.63 ± 0.02	0.56 ± 0.05	0.60 ± 0.03	0.58 ± 0.04	0.46 ± 0.02
	LVDd, mm	4.10 ± 0.21	3.94 ± 0.23	4.42 ± 0.15	4.45 ± 0.27	4.76 ± 0.13	4.68 ± 0.22
	IVSs, mm	1.10 ± 0.05	1.09 ± 0.05	0.92 ± 0.04	0.95 ± 0.04	0.86 ± 0.07	0.92 ± 0.07
	LVPWs, mm	1.07 ± 0.03	1.07 ± 0.04	0.88 ± 0.05	0.78 ± 0.04	0.70 ± 0.05	0.72 ± 0.05
	LVDs, mm	2.60 ± 0.27	2.50 ± 0.24	3.22 ± 0.18	3.33 ± 0.36	3.92 ± 0.19	3.64 ± 0.28
	FS, %	37.08 ± 3.30	37.40 ± 2.55	27.49 ± 1.97	26.18 ± 3.55	18.34 ± 2.33	21.55 ± 2.76

IVS, inter ventricular septum; LVPW, left ventricular posterior wall; LVD, left ventricular diameter; FS, fractional shortening; s: systole; d: diastole. Values are means ± standard errors of means.

Acknowledgements

The authors gratefully acknowledge ChromaDex for providing NR.

Funding

Association Française contre les Myopathies, Penn Medicine Orphan Disease Center (MDBR-17-110-CMD); and Agence Nationale de la Recherche (ANR-17-CE17-0015-03).

Supplementary Material

Supplementary Material is available at HMG online.


Quentin Boehler
email: q.boehler@unistra.fr

Salih Abdelaziz

Marc Vedrines

Philippe Poignet

Pierre Renaud

Towards the control of tensegrity mechanisms for variable stiffness applications: a case study

Keywords: Tensegrity mechanism, cable-driven mechanism, tensegrity modeling and control, tension distribution algorithm, variable stiffness

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

A tensegrity mechanim is a self-stressed system comprising a set of compressed bars in a set of tensioned elements [START_REF] Motro | Tensegrity: Structural Systems for the Future[END_REF] that can be either cables or springs. Actuation of the mechanism allows for device reconfiguration and such kind of mechanism has been demonstrated to be of interest in applications ranging from mobile robotics [START_REF] Koizumi | Rolling tensegrity driven by pneumatic soft actuators[END_REF] to manipulators [START_REF] Aldrich | Control synthesis for a class of light and agile robotic tensegrity structures[END_REF]. Besides, so-called tensegrity systems have been considered for variable stiffness applications [START_REF] Azadi | A variable spring using a tensegrity prism[END_REF], as the stiffness along specific directions can be dramatically modified through the level of prestress within its components. In this case, elastic cables or springs are preferred in order to bring compliance to the system around a fixed configuration. Introducing tensegrity mechanisms in which both the configuration and the stiffness can be controlled seems of great interest for robotic applications where variable stiffness is desired. This paper is thus focused on the control of such tensegrity mechanisms, an aspect which has not been yet investigated in the literature.

The modeling of tensegrity mechanisms is complex, as the tensions in the cables or springs have to remain positive, and static conditions must be taken into account as shown in [START_REF] Boehler | Definition and computation of tensegrity mechanism workspace[END_REF]. Cable-actuated tensegrity mechanisms therefore interestingly share

1 θ q 2 A B C D L q 1 Actuators Springs α 1 α 2 R Γ
Fig. 1 Cable-driven planar tensegrity mechanism.

properties with cable-driven robots. A cable-driven robot is composed of a platform connected to the base by cables which lengths are modified in a coordinated way to control the platform pose. As a cable can only be tensioned, unidirectional constraints exist, contrary to classical parallel manipulators in which the kinematic redundancy can be managed through bidirectional constraints [START_REF] Ebert-Uphoff | On the connections between cable-driven robots, parallel manipulators and grasping[END_REF]. In this paper, we propose to take advantage of recent developments in the control of cable-driven robots to develop a control strategy of tensegrity mechanism for variable stiffness applications.

The analysis we perform is divided into two steps. First, the position control of cable-actuated tensegrity mechanisms is investigated. An original controller is proposed based on so-called tension distribution algorithms [START_REF] Gouttefarde | A versatile tension distribution algorithm for n-DOF parallel robots driven by n+2 cables[END_REF]. Second, a stiffness modulation strategy through cable prestress is proposed. This strategy is implemented on an original planar cable-driven tensegrity mechanism. Thanks to the architecture of the mechanism, meaningful simulations results related to the configuration and stiffness control are obtained.

In section 2, the considered system is presented together with its modeling. In Section 3, a description of the control strategy is provided before introducing control behavior through simulation in section 4. Conclusions and future works are developed in section 5.

2 System description and modeling

Description

The considered system is depicted in Fig. 1. It is composed of one static and three moving bars of length L, articulated in four pin joints A, B, C and D. The resulting parallelogram linkage is actuated by two cables attached in C and D and passing through A and B. The system is indeed a tensegrity mechanism as the three bars are compressed by two tensioned actuated cables. As a parallelogram linkage, this system exhibits one degree of freedom. The mechanism configuration is represented by the angle θ .

Two linear springs of stiffness k are integrated along each cable to bring compliance to the system. The resulting angular stiffness of the end-effector BC is denoted K θ . The distances AC and BD are respectively denoted l 1 and l 2 and can be computed as

l 1 = 2L cos(θ /2) l 2 = 2L sin(θ /2) (1)
The lengths of the cables, denoted q 1 and q 2 (see Fig. 1), are controlled by two actuators of angular positions α 1 and α 2 through pulleys of radius R. In the follow- ing q = [q 1 , q 2 ] T , α α α = [α 1 , α 2 ] T and therefore q = -Rα α α.

Kinematic and static modeling

As a tensegrity composed of deformable springs, this system is submitted to selfequilibrium constraints that have to be considered together with the kinematic ones [START_REF] Boehler | Definition and computation of tensegrity mechanism workspace[END_REF]. Using the same energetic approach than in [START_REF] Arsenault | Kinematic, static, and dynamic analysis of a planar one-degreeof-freedom tensegrity mechanism[END_REF], the equilibrium equation is expressed as

∂U ∂ θ = 0 (2)
with U the potential energy assumed to be fully stored in the springs. The angular stiffness K θ is computed using the second derivative of U with respect to θ so that

K θ = ∂ 2 U ∂ θ 2 (3)
The two cables are used to control the configuration of the parallelogram, so we consider q as the articular variables. The system is kinematically redundant, i.e. each configuration can be reached with different levels of prestress. It also means that Eq. ( 2) can be satisfied for different values of K θ . Two variables are required to fully represent the system state, namely its configuration θ and its angular stiffness K θ . In the following, we thus designate the vector X = [θ , K θ ] T as the operational variables, as they are the variables to be simultaneously modified for a given task. Eq. ( 2) and (3) lead to the so-called Forward Static Model, i.e. the expression of X with respect to q: The first equation in (4) can be retrieved using the virtual work principle that gives a relationship between the cable tension set F = [F 1 , F 2 ] T and the torque Γ applied on the output bar BC (see Fig. 1):

       θ = 2 arctan(q 2 /q 1 ) K θ = kL 2 q 2 1 + q 2 2 q 2 1 + q 2 2 ( 4
) Tensegrity mechanism + - + - θ * θ F Tension distribution Γ v F * α α α Position controller Tension controller
L sin(θ /2)F 1 -L cos(θ /2)F 2 = Γ (5)
that can be noted

WF = Γ (6) with W = [L sin(θ /2), -L cos(θ /2)].
The equilibrium is reached when Γ = 0, and then the first equation in ( 4) can be retrieved from ( 6) knowing that F i = k(l iq i ) for the i-th spring. The reader should notice that two forces are used to generate a single torque. This redundancy, specific to cable-driven systems, means that a given torque can be generated for several couple of forces i.e. for several levels of prestress within the system. This specificity in the mechanism modeling is the starting point of the strategy we propose in order to reconfigure the system while controlling its prestress and thus its stiffness.

3 Control strategy

Tension distribution algorithm

The system position has to be controlled while ensuring that the tension set F remains in the user-defined positive interval bounded by F min and F max . In this paper, we propose the control strategy depicted in Fig. 2 adapted from [START_REF] Khosravi | Robust PID control of fully-constrained cable driven parallel robots[END_REF]. An external loop controls the reference configuration θ * using a position controller that generates a virtual torque Γ v to be applied to the system. This torque is used as an input to the tension distribution block that solves

WF * = Γ v , subjected to F min ≤ F * ≤ F max (7)
The inner loop then uses a tension controller to feed the actuators with the velocities α α α. It can be proved from [START_REF] Gouttefarde | A versatile tension distribution algorithm for n-DOF parallel robots driven by n+2 cables[END_REF] that the solution to Eq. ( 7) is of the form

F * = W + Γ v + N W λ (8)
where W + denotes the Moore-Penrose pseudoinverse of W, N W a basis of W nullspace, and λ a scalar to be chosen so that the inequality in Eq. ( 7) is satisfied, namely that

F min -W + Γ v ≤ N W λ ≤ F max -W + Γ v ( 9
)
which is equivalent to

λ min ≤ λ ≤ λ max ( 10
)
The existence of a solution to Eq. ( 7) depends on the value of the torque Γ v to be applied. The feasibility of Γ v is verified and its value is saturated accordingly using the approach of [START_REF] Bouchard | On the ability of a cable-driven robot to generate a prescribed set of wrenches[END_REF] to ensure the existence of a solution set. Then using the approach described in [START_REF] Gouttefarde | A versatile tension distribution algorithm for n-DOF parallel robots driven by n+2 cables[END_REF], the set Λ = {λ |λ min ≤ λ ≤ λ max } can be computed.

The second term of ( 8) corresponds to the level of prestress within the system. This latter does not modify the current equilibrium configuration of the system as it is chosen within W nullspace, so that it generates no torque Γ on the end-effector. The modulation of λ within Λ has thus a direct impact on the stiffness of the system and no influence on the system configuration. This property is at the center of the stiffness modulation strategy.

Stiffness modulation through prestress

In existing applications such as described in [START_REF] Gouttefarde | A versatile tension distribution algorithm for n-DOF parallel robots driven by n+2 cables[END_REF], the tension distribution algorithm is only used to provide a single feasible solution to Eq. ( 7) among the infinite number of solutions in Λ . The scalar λ can be chosen as the center of gravity of Λ , which has been proved to be continuous along continuous trajectories [START_REF] Gouttefarde | A versatile tension distribution algorithm for n-DOF parallel robots driven by n+2 cables[END_REF]. In that case, it means that λ = (λ max + λ min )/2, so that the level of prestress is chosen as far away as possible from the upper and lower bounds of F * .

In this paper, our idea is to modulate the choice of λ in Λ to modify the level of prestress and thus the stiffness. Around an equilibrium configuration (i.e. Γ v = 0), the first term of Eq. ( 8) vanishes and it becomes

F * = N W λ ( 11
)
where N W = [-L cos(θ /2), -L sin(θ /2)] T is a basis of W nullspace. As F i = k(l iq i ) for the i-th spring, Eq. ( 11) can be combined with Eq. ( 4) and (1) to give

K θ = L 2 2 (λ + 2k) ( 12
)
The stiffness K θ can thus be explicitly controlled through λ using the tension distribution algorithm. This constitutes an original use of the algorithm in order to modify the stiffness of the mechanism at a fixed configuration through a prestress modulation.

Simulations

In the following, the behavior obtained with the proposed approach is analyzed through a trajectory tracking simulation. In addition, the stiffness modulation is assessed to provide a first insight of the system ability to be controlled in position and stiffness simultaneously. We choose L = 70 mm, k = 0.27 N/mm and R = 8.4 mm as consistent values and to ensure future experimental validations. Thanks to the particular arrangement of the elements within a tensegrity mechanism, important loads can be supported using only slender and lightweight components. The weights and inertia within the system are therefore neglected. The control scheme of Fig. 2 is implemented in Matlab Simulink (The Mathworks, Inc.) in which the tensegrity mechanism is represented by its static model. The dynamics of the system as well as those of the actuators are neglected. These assumptions are justified by the trajectories to be tracked in the simulation, which exhibits low accelerations.

Trajectory tracking

The simulated trajectory aims at demonstrating the reconfiguration capacity of the system, as well as the impact of the configuration on Λ . For didactic purposes, we also show the impact of the tensions bounds on the system behavior. The trajectory is chosen accordingly as commented in the following.

The simulation results are illustrated in Fig. 3. During the first 20 s, the mechanism is driven from θ * = 90 • to θ * = 45 • with constant bounds on F * of 4 N and 10 N. For the next 5 s, the upper bound is linearly increased to 20 N for θ * = 45 • . During the last 5 s, both bounds on F * and θ * are fixed, so that neither the configuration nor the tensions are modified by the algorithm.

The cable tensions are equal at θ * = 90 • thanks to the symmetrical arrangement of the cables, and they are equal to (F max + F min )/2, the center of gravity of the feasible tensions. Between 0 and 20 s, Λ shrinks as the cables tensions get closer to their bounds. The limit of the tension distribution for these bounds is met at 45 • where F 1 = F max and F 2 = F min , and Λ is then reduced to a single value. Finally, when F max is increased from 10 to 20 N, the tensions continuously adapt to the new bounds thus keeping the same configuration for different tension sets.

These results show the ability of the system to track a trajectory in θ , but also to reach the same configuration for different tension sets.

Stiffness modulation

In order to demonstrate the stiffness modulation ability of the system, the angle is set to a constant value: θ * = 90 • for the first test and then θ * = 75 • . The scalar λ is imposed so that K θ lineary goes from 0.5 to 1 Nm/rad during 5 s (see Eq. ( 12)). The results are depicted in Fig. 4.

As already noticed in the previous simulation, the cable tensions are equal all along the modulation for θ = 90 • . This is no more the case for θ = 75 • . In both cases, λ is kept in Λ so that the cable tensions remain between the preset bounds (here between 4 and 20 N). It should be noticed that for this particular mechanism, the results may be counter-intuitive as the stiffness is increased when the prestress decreases in the cables. These simulation results show the original use of the tension distribution algorithm coupled with Eq. ( 12) to provide a stiffness modulation through a variation of the prestress within the mechanism.

Conclusions and perspectives

In this paper, a novel control strategy for cable-actuated tensegrity mechanisms is proposed. Existing works on cable-driven robots are exploited to design an adapted control strategy. This strategy is implemented on a 1-DOF tensegrity mechanism which state is defined as its configuration together with its stiffness about its DOF. Using an original adaptation of the tension distribution algorithm, both the configuration and the stiffness can be modified. Simulations notably show encouraging results on the stiffness variation capacity of the system. These results clearly emphasize the potential of this mechanism and the control strategy for variable stiffness robotic devices.

Further works will be carried out on the experimental validation of these results as well as on the development of alternate control strategies for further applications.

Fig. 2

 2 Fig. 2 Control strategy.

Fig. 3 Fig. 4

 34 Fig. 3 Simulation on a trajectory tracking between θ * = 90 • to θ * = 45 • . (a) Reference and current angle ; (b) Evolution of λ and Λ ; (c) Evolution of the cable tensions and their bounds.

Acknowledgements This work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme (Robotex ANR-10-EQPX-44, Labex CAMI -ANR-11-LABX-0004) and by the Région Alsace and Aviesan France Life Imaging infrastructure.


Cristian Rodrigo
email: cristian.rodrigo@unab.cl

Jenny M Blamey
email: jblamey@bioscience.cl

Oliver Huhn
email: ohuhn@uni-bremen.de

Christine Provost
email: christine.provost@locean.upmc.fr

Is there an active hydrothermal flux from the Orca seamount in the Bransfield Strait, Antarctica?

Keywords: Hydrothermal, Helium isotope, Thermophiles, Seamount, Volcanism, Rift system, Antarctica

The rifting zone of Bransfield Strait, Antarctica, is tectonically and geologically unique. It is a back-arc basin that was opened by extensional forces associated to roll-back subduction after cessation of spreading activity of the Phoenix Ridge, and the transtension of the westward ending of Scotia-Antarctica Plate boundary. The Bransfield Rift/ Ridge is still active generating volcanism or magma rise to force hydrothermal activity. During the ANT-XXV/4 cruise onboard R/V "Polarstern", standard CTD and beam transmission measurements were done to determine temperature anomaly and turbidity. Water sampling was performed to determine δ 3 He and to find thermophilic microorganisms to examine the Orca seamount hydrothermal activity. A temperature anomaly of ~0.08 °C, a pick of turbidity, and high value of δ 3 He (>10%) were found inside Orca seamount. Results are consistent with a hydrothermal flux coming from the seamount. The report of the first observation of thermophilic and hyperthermophilic microorganisms in cold deep Antarctic waters is part of this study. Inside Orca seamount these microorganisms were found at three different depth levels close to the bottom. We suggest that the fluid migration from the volcano resulted from recent magmatic activity and provided the required elemental nutrients for microbial growth. Besides some thermophiles were found outside the seamount in a small quantity close to the seafloor. These would probably be related to subsidiary structures of the Orca seamount, or were transported by currents from other active volcanic sites as Deception Island. The finding of these thermophilic and hyperthermophilic microorganisms raise questions about the dispersal and their resistance in these extreme environments.

centers and intra-plate volcanoes (e.g., [START_REF] German | Hydrothermal plumes above the East Scotia Ridge: an isolated highlatitude back-arc spreading centre[END_REF]. Every mentioned site has its own associated tectonics, geodynamics and geological characteristics that produce distinctive geophysical and geochemical signatures, which are reflected in the variability in composition, size or volume, spatial distribution, frequency and transience of the deep-sea hydrothermal vents [START_REF] Lupton | Tracking the Evolution of a Hydrothermal Event Plume with a RAFOS Neutrally Buoyant Drifter[END_REF][START_REF] Baker | Hydrothermal plumes along segments of contrasting magmatic influence, 15°20'-18°30'N, East Pacific Rise: Influence of axial faulting[END_REF][START_REF] Hey | Tectonic/volcanic segmentation and controls on hydrothermal venting along Earth's fastest seafloor spreading system[END_REF][START_REF] Hannington | Seaf loor tectonics and submarine hydrothermal systems[END_REF].

The rifting zone of the Bransfield Strait, located between the South Shetland Islands and the Antarctic Peninsula (Fig. 1) is tectonically and geologically unique (Lawyer et al., 1996). Subduction and extension processes occur simultaneously and there is currently no clarity in their geological evolution frías profundas de la Antártica. Dentro del cráter del monte submarino Orca, estos microorganismos se localizaron en tres niveles de profundidad diferentes cerca del fondo. Nosotros sugerimos que la circulación de fluidos hidrotermales, producto de la actividad magmática reciente, proporcionaron los nutrientes y el ambiente propicio para el crecimiento de estos microorganismos. Además, se encontraron fuera del volcán, cerca del lecho marino, microorganismos termófilos en una pequeña cantidad. Estos estarían relacionados, probablemente, con estructuras subsidiarias del monte submarino Orca o fueron transportadas por corrientes desde otros centros volcánicos activos como la isla Decepción. El descubrimiento de estos microorganismos termófilos e hipertermófilos genera nuevas preguntas sobre su dispersión y resistencia en estos ambientes extremos.

Introduction

The seaf loor hydrothermal activity has a great impact on the chemistry of the oceans and is responsible for extensive alteration of the oceanic crust [START_REF] Herzig | Polymetallic massive sulfides at the modern seafloor-a review[END_REF]. The water from vents also plays an important role in the heat budget and circulation patterns of the oceans [START_REF] Baker | Hydrothermal plume measurements: a regional perspective[END_REF]. Typical submarine hydrothermal vents support unusual ecosystem communities where primary production, which is the basis for the local food chain, depends on chemosynthetic microorganisms [START_REF] Baross | Growth of "black smokers" bacteria at temperatures of at least 250°C[END_REF]). This phenomenon is not unique to mid-ocean ridges since it also occurs in volcanic arcs, back-arc spreading FIG. 1. Regional tectonic setting of the area studied based on [START_REF] Barker | Rift propagation, detachment faulting, and associated magmatism in Bransfield Strait, Antarctic Peninsula[END_REF]. The location of former Phoenix Plate, other plates and tectonic structures are indicated. Zoom of the Bransfield Strait area with its tectonic structures, volcanic edifices are showed (modified after [START_REF] Pedrera | The fracture system and the melt emplacement beneath the Deception Island active volcano, South Shetland Islands, Antarctica[END_REF], and also seismicity from Robertson [START_REF] Robertson Maurice | Seismicity and tectonics of the South Shetland Islands and Bransfield Strait from a regional broadband seismograph deployment[END_REF] (epicenters in black dots). [START_REF] Barker | The Cenozoic subduction history of the Pacific margin of the Antarctic Peninsula: ridge crest-trench interactions[END_REF]Austin, 1994, 1998;[START_REF] Fretzdorff | Magmatism in the Bransfield Basin: Rifting of the South Shetland Arc?[END_REF][START_REF] Solari | Geotectonic evolution of the Bransfield Basin, Antarctic Peninsula: insights from analogue models[END_REF][START_REF] Poblete | Paleomagnetism and tectonics of the South Shetland Islands and the northern Antarctic Peninsula[END_REF]. The former Phoenix Plate, located between the Hero and Shackleton Fracture Zones (Fig. 1), was formed by seafloor spreading on the Antarctic-Phoenix Ridge (APR) since the Jurassic [START_REF] Barker | The Cenozoic subduction history of the Pacific margin of the Antarctic Peninsula: ridge crest-trench interactions[END_REF].

Oblique subduction under the Antarctic Plate ceased progressively from SW to NE during the Cenozoic as a result of successive ridge crest-trench collisions [START_REF] Herron | Sea-floor magnetic patterns and basement structure in the southeastern Pacific[END_REF][START_REF] Barker | The Cenozoic subduction history of the Pacific margin of the Antarctic Peninsula: ridge crest-trench interactions[END_REF]. Since the Pliocene the APR spreading stopped, and subduction slowed and roll-back started at the South Shetland Trench (Fig. 1) [START_REF] Livermore | Autopsy on a dead spreading center: The Phoenix Ridge, Drake Passage, Antarctica[END_REF][START_REF] Jabaloy | The transition from an active to a passive margin (SW end of the South Shetland Trench, Antarctic Peninsula)[END_REF][START_REF] Galindo-Zaldívar | Tectonic development of the Bransfield Basin and its prolongation to the South Scotia Ridge, northern Antarctic Peninsula[END_REF]. These mechanisms and the transtension associated with the westward ending of Scotia-Antarctica Plate boundary, enable the opening of the Bransfield Basin and associated Quaternary volcanism [START_REF] Galindo-Zaldívar | Tectonic development of the Bransfield Basin and its prolongation to the South Scotia Ridge, northern Antarctic Peninsula[END_REF][START_REF] Solari | Geotectonic evolution of the Bransfield Basin, Antarctic Peninsula: insights from analogue models[END_REF][START_REF] Pedrera | The fracture system and the melt emplacement beneath the Deception Island active volcano, South Shetland Islands, Antarctica[END_REF].

In this way, the Bransfield Rift/Ridge is still active producing magma rise and hydrothermalism [START_REF] Barker | The Cenozoic subduction history of the Pacific margin of the Antarctic Peninsula: ridge crest-trench interactions[END_REF]Austin, 1994, 1998;[START_REF] Rey | Tectonic, Volcanic, And Hydrothermal Event Sequence On Deception Island (Antarctica)[END_REF][START_REF] Somoza | Evidence for hydrothermal venting and sediment volcanism discharged after recent shortlived volcanic eruptions at Deception Island, Bransfield Strait, Antarctica[END_REF]. The Bransfield Strait area is a solitary Antarctic zone with its peculiar hydrothermal activity associated to submarine volcanic edifices [START_REF] Klinkhammer | Results of a search for hydrothermal activity in the Bransfield Strait, Antarctica[END_REF][START_REF] Bohrmann | Hydrothermal activity at Hook Ridge in the Central Bransfield Basin, Antarctica[END_REF]Dählman et al., 2001;[START_REF] Klinkhammer | Discovery of new hydrothermal vent sites in Bransfield Strait, Antarctica[END_REF][START_REF] Petersen | Hydrothermal precipitates associated with bimodal volcanism in the Central Bransfield Strait[END_REF]. As part of the most important volcanic edifices of the Bransfield Strait, is the Deception Island (Fig. 2). It has showed recent volcanic events occurred during the last decades producing subaerial fumaroles and hot springs, but also submarine hydrothermal vents inside the submerged caldera of the island [START_REF] Rey | Tectonic, Volcanic, And Hydrothermal Event Sequence On Deception Island (Antarctica)[END_REF][START_REF] Somoza | Evidence for hydrothermal venting and sediment volcanism discharged after recent shortlived volcanic eruptions at Deception Island, Bransfield Strait, Antarctica[END_REF]. Hydrothermal activity may persist up to decades after volcanic eruptions helped by geological structures under the seaf loor [START_REF] Somoza | Evidence for hydrothermal venting and sediment volcanism discharged after recent shortlived volcanic eruptions at Deception Island, Bransfield Strait, Antarctica[END_REF].

Another prominent volcanic structure on the Bransfield Ridge is the Orca seamount, an apparently inactive volcano, about 500 m of elevation above the seabed, with 3 km wide crater. (Fig. 2) [START_REF] Hatzky | The Orca Seamount Region, Antarctica[END_REF] [START_REF] Schreider | The stages of the development of the basin of the Bransfield Strait[END_REF]. Oceanographic measurements were performed within the Orca seamount to check the hydrothermal activity, however, no evidences were found [START_REF] Lawver | Distributed, active extension in Bransfield Basin, Antarctic Peninsula: Evidence from multibeam bathymetry[END_REF]. The seismic activity observed in the vicinity of Orca seamount has been interpreted as associated with magmatic activity (Fig. 1) [START_REF] Kaminuma | A Possibility of Earthquake Swarms around ORCA Sea Mount in the Bransfield Strait, the Antarctic[END_REF][START_REF] Robertson Maurice | Seismicity and tectonics of the South Shetland Islands and Bransfield Strait from a regional broadband seismograph deployment[END_REF][START_REF] Dziak | Tectonomagmatic activity and ice dynamics in the Bransfield Strait back-arc basin, Antarctica[END_REF][START_REF] Kanao | Seismicity in the Antarctic Continent and Surrounding ocean[END_REF], led us to begin an exploration of the volcano to check for its hydrothermal activity. This study was made considering that hydrothermal activity may persist long-time after magmatic discharges. The use of a multidisciplinary approach (oceanography, geochemistry and microbiology) would serve as background for future studies on the interaction of submarine volcanic hydrothermal f lux on very cold water.

Methodology

Oceanographic cruise

During the ANT-XXV/4 cruise on board R/V "Polarstern" in April 2009 in the Drake Passage and the Bransfield Strait, Antarctica, a hydrographic station was performed inside the crater of Orca seamount (Station A), and another station was setup immediately to the north of Orca seamount (Station B) (Fig. 2A and2B), to examine its potential hydrothermal activity. Seawater was collected using 12 l Niskin bottles mounted on a Sea-Bird SBE 32 Rosette equipped with a CTD (Conductivity, Temperature and Depth) Sea-Bird Electronics SBE911 plus. Deeper samples were taken 10 m from the seaf loor (nk1 samples in table 1). The bathymetric profile in figure 2C, shows the shape and size of the seamount, the interior of the crater, and the location of the stations and depths (levels) of bottle sampling (nk). The CTD was supplemented by an oxygen sensor SBE 43, a transmissiometer (WetLabs C-Star, 660 nm wavelength), and a chlorophyll-sensitive fluorimeter (Chelsea Aquatracka). Details of the cruise methods and data analysis can be consulted in Provost (2010).

Oceanographic and gas analyses

Temperature, salinity, oxygen and light beam transmission data were taken by the CTD and converted to workable oceanographic variables using standard procedures [START_REF] Provost | Two repeat crossings of Drake Passage in austral summer 2006: short term variations and evidence for considerable ventilation of intermediate and deep waters[END_REF]. The processing of the data for plotting consisted in manual spike removal and the application of a low pass filter using a time of 0.5 s and, for beam transmission, 2 s. The potential temperature anomaly (Δθ) calculation was performed graphically according to [START_REF] Baker | Hydrothermal venting along Earth's fastest spreading center[END_REF] using a first order fit of the potential temperature as function of the potential density.

Helium-3 is an isotope that has been used as tracer of hydrothermal activity in mid-ocean ridges (e.g., [START_REF] Baker | Hydrothermal venting along Earth's fastest spreading center[END_REF][START_REF] Jean-Baptiste | Arctic Ocean (communication arising): Hydrothermal activity on Gakkel Ridge[END_REF], and it has a solely magmatic origin. Thus its presence provides unequivocal evidence of magmatic activity [START_REF] Lupton | Mantle helium in hydrothermal plumes in the Galapagos Rift[END_REF]. Ocean water samples for noble gas analysis were stored from the Niskin bottles into gas tight copper tubes (50 ml). The noble gas samples were analysed in the Institute of Environmental Physics (IUP, Bremen) and in the noble gas mass spectrometry laboratory [START_REF] Sültenfuß | The Bremen mass spectrometric facility for the measurement of helium isotopes, neon, and tritium in water[END_REF]. They were processed in a first step with a UHV (Ultra High Vacuum) gas extraction system. Sample gases were transferred via water vapour into a glass ampoule were kept at liquid nitrogen temperature. Quality checks of this sample preparation line were done on a routine basis. This includes special vacuum checks with the Quadrupole Mass Spectrometer (QMS) and preparation of accurately defined samples for internal control measurement. For analysis of the noble gas isotopes the glass ampoule is connected to a fully automated UHV mass spectrometric system equipped with a two stage cryo system. Every 2 to 4 samples the system is calibrated with standard atmospheric air measurements. Measurements of line blanks and linearity were also performed. Calibration of the data has to be done to obtain consistent data sets.

For δ 3 He the error was 0.2% accuracy and 0.1% precision from replicates.

Microbiological analysis

For the microbiological analysis, at both stations, samples were taken at three levels from the bottom (Table 1 and Fig. 2C) using Niskin bottles and were stored at 4 °C in sterile containers. Microorganisms were grown using techniques similar to those used by [START_REF] Summit | Thermophilic subseafloor microorganism from the 1996 North Gorda Ridge eruption[END_REF], emulating the conditions of mid-ocean ridge environments. The first step was isolation and culture of samples. They were incubated in various rich media (see [START_REF] Muñoz | Thermophilic bacteria present in a sample from Fumarole Bay, Deception Island[END_REF]. The microorganisms in the enrichment cultures were grown at temperatures from 70-97 ºC. Solid media were prepared at 1% solid agar. Colonies formed on the solid media were taken individually and incubated at 70 °C in liquid media to observe the generation of pure cultures. Growing conditions were improved with the determination of the optimal conditions of pH, temperature, NaCl, sources of carbon and oxygen. Gram stain reaction was determined using a Merck gram stain kit according to the manufacturers recommended protocol. In order to identifiy the microorganosms isolated we performed DNA isolation and Polymerase Chain Reaction (PCR) amplification. Genomic DNA was extracted from cultures grown for 48 h at 80 °C and 85 °C using two different cellular disruption methods followed by Phenol:Chloroform:Isoamyl alcohol precipitation (PCI). Gene Clean Purification kit (Bio 101) was used according to manufacturer's instructions to clean the DNA samples. The PCR amplification of 16S rDNA gene was performed using Taq DNA polymerase, universal primer 1492R and the domain Bacteria-specific primer 27F. Archaeal 16S rDNA was amplified with the universal primer 1492R and the domain Archaea-specific primer 21F. A ~1,500 kb fragment of the 16S rDNA gene was expected to be amplified. Verification of DNA extraction and PCR amplification was carried out by running the samples on a 1% agarose gel. Fluorescent microscopy was performed, 0.1 ml of sample of each cell suspension was removed from the main culture and immediately stained with the commercial kit LIVE/DEAD BacLightTM (invitrogen). Samples were incubated in a dark room at the temperatures for at least 15 minutes, before analysis. Samples also were visualised using an electronic microscope JEOL JSM-T300.

Results and discussion

Oceanographic and noble gas tracers

In general, the profiles of temperature (T) and salinity (S) for stations A (Orca) and B show similar patterns and represent the typical hydrographic characteristics of the area (Fig. 3A) (e.g., [START_REF] Gordon | The basin waters of the Bransfield Strait[END_REF][START_REF] Wilson | Hydrography within the Central and East Basins of the Bransfield Strait, Antarctica[END_REF][START_REF] García | Water masses and distribution of physico-chemical properties in the western Bransfield Strait and Gerlache Strait during Austral summer 1995/96[END_REF][START_REF] Sangrà | The Bransfield current system[END_REF]. Both stations show a flow of warmer and saltier water between 200 and 450 m below the sea level (b.s.l.) that corresponds to the Modified Circumpolar Deep Water (CDW), which centres around σ θ =27.75 (Fig. 3B). This flow has its maximum in T and S at ~400 m b.s.l., which coincides with the oxygen minimum, and it is more marked at the Orca Station profiles, indicating weaker influence of the Southern Ocean waters near the slope of the King George Island. The profiles also show a colder, fresher and more oxygenated layer at ~200 m depth. Deeper waters for Station B show uniform salinity below 1,000 m b.s.l., and they are colder and fresher relative to water at the same depth outside the strait in the Weddell Sea [START_REF] Wilson | Hydrography within the Central and East Basins of the Bransfield Strait, Antarctica[END_REF], reflecting the local formation of bottom water in this basin [START_REF] Gordon | The basin waters of the Bransfield Strait[END_REF][START_REF] Wilson | Hydrography within the Central and East Basins of the Bransfield Strait, Antarctica[END_REF].

Below the seamount summit level (~600 m) oceanographic variables for A Station (Orca) are uniform with depth, which is considered normal inside a seamount (Figs. 3A andB). In this station, close to the bottom, the water temperature is under -1 °C, that is very cold compared with mid-ocean ridge average temperature at ~2,000 m depth (~2 °C) [START_REF] Baker | Hydrothermal venting along Earth's fastest spreading center[END_REF]. There is a difference of 0.25 °C comparing the temperatures between stations A and B at 1,033 m b.s.l. Both stations show a significant temperature increase at mid-depths due to CDW influence (right side of the D-T graphs, Fig. 3C) which coincides with a decrease in turbidity (transmission) (right side of the D-Tr graphs, Fig. 3C). To avoid confusion due to the influence of the warm CDW waters, the temperature anomaly (Δθ) was determined on the part of the curves that corresponds to the vicinity of the seabed, matching with turbidity picks (Fig. 3C). In this way, inside the crater of the volcano the temperature anomaly is ~0.08 °C, and for station B close to the bottom is ~0.03 °C. The Orca station value is higher than temperature anomalies observed by [START_REF] Klinkhammer | Results of a search for hydrothermal activity in the Bransfield Strait, Antarctica[END_REF] along the Hook and Middle Ridges, and Three Sisters (0.010-0.025 °C), meanwhile the anomaly for station B is similar. On the other hand, the determined values are lower than typical temperature anomalies for the East Pacific Rise (0.260 °C) [START_REF] Baker | Hydrothermal venting along Earth's fastest spreading center[END_REF].

The transmission is taken as a relative value, and there is a higher peak inside the Orca crater (Fig. 3C). This is consistent with the idea that there is a hydrothermal venting present, demonstrating that the turbid environment perhaps is due to the dispersion and particle re-suspension due to hydrothermal activity. There is also a higher peak value close to the bottom of station B. Maybe there is a hydrothermal influence outside the volcano, or there is a particle flow due to reasons above explained.

The high values of δ 3 He data also confirm the Orca hydrothermal activity (Fig. 3D). δ 3 He is mainly the ratio between 3 He and 4 He (normalized to atmospheric values) in %. In equilibrium with the atmosphere and typical polar surface temperatures δ 3 He is in the order of -1.8%. Only sources for 3 He (and thus for enhanced δ 3 He ratios) are primordial helium (typically released by hydrothermal activities) or tritium decay (50 years after hydrogen bomb test negligible) [START_REF] Huhn | Evidence of deep-and bottom-water formation in the western Weddell Sea[END_REF]. The Orca station shows below 500 m b.s.l. a value of δ 3 He >10% (Fig. 3D). Previous measurements in the Bransfield Strait show a δ 3 He maximum of ~7%, interpreted as a local injection of a 3 He-rich helium component into deep waters of the strait from the rift [START_REF] Schlosser | 3He in the Bransfield Strait waters: indication for local injection from back-arc rifting[END_REF]. Tritiogenic 3 He and excess 3 He from mixing with CDW were excluded as possible sources.

If it would be a CDW admixture, it could only come from the Weddell Sea (Transitional Zonal Water with Weddell Sea influence, TWW) via the northeastern entry of the Bransfield Strait. In the Weddell Sea the highest δ 3 He values are in the order of <9.7%, and there the δ 3 He maximum layer is on ~300-400 m depth. On a short section at the north eastern entry of Bransfield Strait the δ 3 He values are even far smaller (<2%) [START_REF] Huhn | Evidence of deep-and bottom-water formation in the western Weddell Sea[END_REF], and the entry via Joinville Island and the Antarctic Peninsula is too shallow being the values mentioned lower than 9.7% inside the Weddell Basin. On the other hand, the TWW goes to the SW close to the Antarctic Peninsula and limited to the North by the Peninsula Front (PF) [START_REF] Sangrà | The Bransfield current system[END_REF], so there is not influence in shallow and mid waters. The influence of the TWW in depth reaches the Bransfield Front (BF) under the CDW, but also the influence should be minimum due to the predominance of local cold deep water formation on the Bransfield Basins [START_REF] Wilson | Hydrography within the Central and East Basins of the Bransfield Strait, Antarctica[END_REF]. Therefore, the maximum value of δ 3 He inside the crater of Orca seamount (δ 3 He=13.94%) is high and it is interpreted due to a source from the crater bottom. The position of the helium Reference Station is shown in figure 2A ("RS" point), whose data were obtained during the cruise ANT-XIII/4 on board R/V "Polarstern" in 1996, using same methodology of sampling and analysis. The RS station δ 3 He profile shows similar structure and values compared with those of [START_REF] Schlosser | 3He in the Bransfield Strait waters: indication for local injection from back-arc rifting[END_REF], and also with Orca values up to ~500 m depth (Fig. 3D). The characteristics of the general vertical distribution of helium does not seem to have changed much over time in the Bransfield area, so, the hydrothermal influence at depths greater than 1,000 m coming from the rift is also maintained. On the other hand, in 2013 the helium measurements were repeated inside the Orca seamount (ANT-XXIX/3 cruise on board R/V "Polarstern", [START_REF] Dorschel | Environmental information for a marine ecosystem research approach for the northern Antarctic Peninsula (RV Polarstern Expedition PS81, ANT XXIX/3)[END_REF], and the values inside the volcano were very high (>27%), confirming the hydrothermal activity.

Microbiological tracers

The morphology and Gram stain of microorganisms isolated for samples taken in stations A and B are shown in table 2, meanwhile the characteristics of each type of microorganisms are shown in table 3. The information regarding the type of media used for culturing, oxygen, temperature, pH and general phylogeny for the different cultures is also indicated in the tables 2 and 3. Thermophilic and hyperthermophilic microorganisms were found in both stations. Inside the crater of the Orca volcano the amount of microorganisms found was significantly higher for all levels from where samples were taken, specifically when anaerobic medium was used (Table 3). Fluorescent microscopy images (Fig. 4) clearly show the variations in the relative amounts of microorganisms and their morphology present in different samples. At station A the majority of microorganisms present in the sample are Gram negative with cocci and rod morphology (Fig. 5). Most of the microorganisms present in the sample grew between 70° and 80 ºC, but some of them were able to grow at 90 °C under identical experimental conditions (sample volume and time of growth). Information regarding use of substrates for cultivation, oxygen requirements, temperature, pH, NaCl requirements in conjunction with DNA analysis revealed that the microorganisms present in the samples belong to both Archaea and Bacteria Domains. Even more, hyperthermophiles and thermophiles (Table 3) as well as halophilic microorganisms could be identified in the samples analyzed. Most of these microbial strains grew in a wide range of temperatures (65-90 °C). Optimum growth temperatures are between 80 ºC and 90 °C and doubling times are about an hour. None of the strains grew at mesophilic temperatures or below.

The finding of high-temperature microorganisms at the bottom of station B, suggests a hydrothermal influence from the Orca area and its surroundings. Other possibility could be that some high temperature microorganisms were transported from the Central Rift or other active volcanic area, such as Deception Island [START_REF] Somoza | Evidence for hydrothermal venting and sediment volcanism discharged after recent shortlived volcanic eruptions at Deception Island, Bransfield Strait, Antarctica[END_REF] (Fig. 6) or even transported by sediment re-suspension. In the case of Deception Island the transport of themophilic microoganisms could be helped by the Bransfield Current (BC) that goes to the NE close to South Shetland Islands coast (Fig. 6).

It is known that microorganisms thrive particularly under the surface of active oceanic ridges, where the circulation derived from volcanic activity and chemical energy sources have the ability to maintain a robust ecosystem, which could be potentially selfcontained [START_REF] Baross | Growth of "black smokers" bacteria at temperatures of at least 250°C[END_REF]. The presence of microorganisms, thermophiles and hyperthermophiles, in an area which generally maintains low temperature flows, indicates a stable warm, anoxic habitat below the seafloor, where these microorganisms can multiply and proliferate. Although high temperature microorganisms are not the only components of subsurface microbial community, the presence of thermophiles and hyperthermophiles provides a biological indicator of submarine groundwater conditions, as hyperthermophiles do not proliferate in cold water grounds [START_REF] Summit | Thermophilic subseafloor microorganism from the 1996 North Gorda Ridge eruption[END_REF].

Conclusions

The value of the temperature anomaly found (~0.08 °C), the distribution pattern and values of δ 3 He (>13.9%), the presence of a turbidity (transmission) peak correlating with the other measurements and the finding of thermophilic and hyperthermophilic microorganisms inside the crater of the Orca Seamount growing in cultures at temperatures >70 °C and in a medium that emulated mid-ocean ridge environments, can confirm the existence of an active hydrothermal flux from the bottom of the seamount crater. Although the water inside the volcanic edifice is very cold (<-1°C), all measurements suggest a hydrothermal migration from the bottom. We suggest that this flux could result from past eruptions, in a similar way to submarine hydrothermal venting in Deception Island, or triggered by recent magmatic activity that facilitated the upward water f low through fractures or faults. Additionally, there is a hydrothermal influence outside of the volcano, perhaps related to subsidiary structures of the Orca seamount, or due to the dispersal of hydrothermal plumes from other volcanic structures of the Bransfield Rift and/or active volcanoes of the region (e.g., Deception Island). The observations, inside and outside of Orca seamount, raise questions about the dispersal of microorganisms and their resistance in this cold environment. The biological results represent the first observations of thermophilic and hyperthermophilic microorganisms in deep cold Antarctic waters.

FIG. 2 .

 2 FIG. 2. Topography model of the studied area using multibeam bathymetry data available from Marine Geoscience Data System (hosted by LDEO-Columbia University). A. Location of the Orca Seamount in the Bransfield Strait and the "Reference Station" for δ 3 He data (RS); B. 3D submarine topography model of Orca seamount and the location of Stations A (red line) and B (blue line); C. Bathymetric profile that cross stations A and B, showing depth levels of Niskin bottle sampling.

  FIG. 3. Oceanographic profiles for Stations A (Orca) and B. A. Salinity (S) and Potential Temperature (T) profiles versus depth. The vertical position of the Modified Circumpolar Deep Water (CDW) is indicated; B. Oxygen (O) and Potential Density (D) versus depth; C. Potential Temperature (T) and Beam Transmission (Tr) versus Potential Density. Also is included temperature anomaly (Δθ) determination (curve fits: segmented lines); D. δ 3 He versus depth. Reference Station for helium is indicated (RS).

FIG. 4 .

 4 FIG. 4. Fluorescent (left side) and electronic microscopy (right side) of thermophilic and hyperthermophilic microorganisms for bottle samples of Station A (Orca seamount). Scale bars indicate 10 μm. A. nk 3: at 998 m depth; B. nk 2: at 1,033 m depth; C. nk 1: at 1,082 m depth.

TABLE 1 . SAMPLED LEVELS FOR THE ANALYSIS OF MICROORGANISMS AND ASSOCIATED OCEANOGRAPHIC DATA FOR STATIONS A (ORCA) AND B.

 1

	Station Bottle	Latitude (S)	Longitude (W)	Station depth (m)	Bottle depth (m)	Temp. (°C)	Salinity (psu)	Oxygen (μmol/kg)	pH	δ 3 He (%)
	A	nk1	62°25.378' 58°24.352'	1,094	1,082	-1.14	34.53	304.8	7.69	11.52
	A	nk2	62°25.378' 58°24.352'	1,094	1,033	-1.05	34.53	301.7	-	13.94
	A	nk3	62°25.378' 58°24.352'	1,094	988	-1.04	34.54	301.7	7.71	13.91
	B	nk1	62°22.153' 58°23.6'	1,444	1,435	-1.50	34.53	314.3	7.70	-
	B	nk2	62°22.153' 58°23.6'	1,444	1,186	-1.40	34.53	312.3	-	-
	B	nk3	62°22.153' 58°23.6'	1,444	1,036	-1.30	34.53	309.7	7.61	-

TABLE 2 . MORPHOLOGY AND GRAM STAIN OF MICROORGANISMS ISOLATED AND TEMPERATURE OF GROWING.

 2

	Station	Bottle	Morphology and Gram stain	Temperature (°C)
	A	nk1	Cocci, Gram negative	70
	A	nk1	Cocci, Gram negative	80
	A	nk1	Cocci, Gram negative	90
	A	nk2	Cocci and Rods, Gram negative	70
	A	nk3	Cocci and Rods, Gram negative	70
			Rods, Gram positive
	B	nk1	Rods, Gram positive	70
	B	nk2	Cocci, Gram positive	70
	B	nk3	Cocci and Rods, Gram negative
			Rods, Gram positive

TABLE 3 . CULTURING OF THERMOPHILIC AND HYPTHERMOPHILIC MICROORGANISMS.

 3

	Station	Bottle	MMM medium	TL medium	MMM Anaerobic medium	Temperature (°C)	pH	Domain
	A	nk1	+	+	+++	70	5.8	Archaea/Bacteria
	A	nk2	+	+	+++	70	5.8	Archaea/Bacteria
	A	nk3	+	+	++	70	5.8	Not identified
	A	nk1	+	+	+++	80	6.5	Archaea/Bacteria
	A	nk2	+	+	+++	80	6.5	Not identified
	A	nk3	+	+	+++	80	6.5	Archaea
	B	nk1	+	+	++	70	5.8	Not identified
	B	nk2	+	+	+	70	5.8	Not identified
	B	nk3	+	+	+	70	5.8	Bacteria
	B	nk1	+	+	+	80	6.5	Not identified
	B	nk2	+	+	+	80	6.5	Bacteria
	B	nk3	+	+	+	80	6.5	Not identified

Positive signs indicate positive growing of microorganisms at the different media. MMM: master medium that emulates seabed environment with trace elements; TL: specific medium to grow Thermococcales).

Palabras clave: Hidrotermal, Isótopo de helio, Termófilos, Monte submarino, Volcanismo, Sistema de rift, Antártica.

Acknowledgements

We greatly appreciate the support of the captain and crew of the R/V Polarsten, technical and scientific party of the ANT XXV-4/Drake Cruise. Special thanks to the Argentine and French scientific group on board. Many thanks to C. Balestrini for the pH data, and to E. Baker for his commentaries. Helium measurements and analysis were granted by the German Science Foundation DFG/SPP1159 (Grant RH25/32 and and HU 1544/4). Also we thank DIFROL, SHOA and AWI for managing the embarking to the ship, and to the Instituto Antártico Chileno (INACH). We would also like to thank the Editor W. Vivallo and the reviewer L. Somoza for their constructive comments; and S. Stipetic, UNAB geology student, for helping in the review of the manuscript.


Catherine Fillonneau

Angélique Villière

Carole Prost

Potentialities of the InnOscent chromatographic system for wine aroma analysis

A new chromatographic device was developed to go further in aroma analysis. Its innovative configuration not only permits to realize classical analysis by gas chromatography coupled with multiple detectors such as mass spectrometry, flame ionization detector and single or dual olfactometric port but also incorporates preparative fraction collection possibilities to recover bespoke aromatic samples. Accordingly, it becomes possible to evaluate the contribution of any compound to the overall aroma of a product. These experiments enable to point out 33 odorant zones among which a majority were associated to identified compounds. Despite their modest olfactometric detection, the contribution of IPMP and IBMP on the typical pyrazic-green odor of wines made of cabernet franc vineyard was evidenced.

Collection of designed extracts

InnOscent device in an innovative selective recovering configuration

Capillary column

These results exemplify the suitability of the approach to improve knowledge of mixture of odorants in a wine aroma context.

Flexible

  potentialities of the InnOscent system is presented through the aromatic analysis of a wine made of Cabernet franc vineyard after extraction of volatile compounds by a Car/PDMS SPME fiber. A particular focus was given to the contribution of pyrazines to the wine aroma.