Evaluation of losartan plus hydrochlorothiazide combination therapy against amlodipine monotherapy in patients of hypertension

Background: Microalbuminuria has been shown to predict cardiovascular disease (CVD) in patients with hypertension. Recently the FDC of losartan and hydrochlorothiazide (HCTZ) has been reported to be effective for achieving a target BP level and also improvement in cardiovascular prognosis. The present study was conducted to compare effect of losartan plus hydrochlorothiazide combination therapy and high dose amlodipine monotherapy on blood pressure and microalbuminuria.Methods: Total 184 patients with hypertension were randomly allocated to two groups. The patients in group 1 received Amlodipine 5 mg orally for first 4 weeks. The  patients  from  group  2  received  losartan  50  mg  orally  for  first  4  weeks. Patients  in  group  1  were  titrated  to  amlodipine  10  mg  orally  for next 4 weeks. The patients in group 2 were titrated to FDC of losartan (50 mg) plus HCTZ (12.5 mg) for next 4 weeks. Follow–up visits were scheduled at 4 weeks and 8 weeks. Pulse rate, sSBP and sDBP were estimated at each follow–up. Microalbuminuria was estimated at 8 weeks.Results: There was no significant difference in mean change in sSBP, sDBP and pulse rate between two treatment groups (p>0.05). There was greater reduction in microalbuminuria in group 2 patients (p<0.0001). The adverse effects such as flushing and lower extremity oedema were significantly more in amlodipine group (p<0.05).Conclusions: Losartan plus HCTZ has similar effect on BP, better safety profile and superior effect on microalbuminuria level  reduction

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy

: SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired

Selenoprotein N as a novel regulator of the muscle progenitor’s cell fate decision process : balancing differentiation and self-renewal

Les mutations du gène codant la sélénoprotéine N (SEPN1) provoquent une myopathie congénitale nommée SEPN1-related myopathy (SEPN1-RM), caractérisée par une faiblesse et une amyotrophie majeures des muscles du cou et du tronc, une scoliose et une insuffisance respiratoire potentiellement létale. SEPN1-RM a été associée avec un stress oxydant, une diminution de la population de cellules souches musculaires (cellules satellites) et des défauts de la régénération musculaire. Avec l’objectif de rechercher les mécanismes impliqués dans ces défauts, et en particulier un rôle potentiel de SEPN1 dans la régulation de l’équilibre entre le renouvellement et la différentiation du pool de cellules satellites, j’ai étudié des cellules satellites primaires de souris knocked-out pour Sepn1et la lignée musculaire murine C2C12 knocked-down pour Sepn1à différents stades de différentiation (cellules quiescentes, myoblastes etmyotubes). Utilisant un système de suspension pour générer une quiescence synchronisée des C2C12, j’ai trouvé que l’absence de SEPN1 dans les cellules en G0 n’est pas incompatible avec la sortie et le retour dans le cycle cellulaire, mais entraîne une moindre sous-régulation de l’expression de deux facteurs clé de la différentiation myogénique (augmentation des transcrits de MYOD1etMYOG par rapport aux contrôles) et une augmentation des niveaux de Cycline D1 (mRNAdeCCND1) en conditions de quiescence. Des études de microarrayet deqRT-PCR ont montré que la déplétion de SEPN1 dans des C2C12 prolifératives est associée à une augmentation significative de l’expression des facteurs de transcription myogéniques MYOG and MYOD1. En parallèle, des études d’immunoblot ont confirmé un niveau augmenté des protéines régulatrices du cycle cellulaire p21 and Cyclin D3 en conditions de prolifération. De plus, des cellules satellites primaires isolées à partir des muscles gastrocnemiusetplantarisde souris KO Sepn1ont montré une fusion accélérée des myoblastes au cours de la différentiation myogénique initiale. Par la suite, j’ai explore les voies mécanistiques impliquées dans ce phénotype cellulaire par western blot et/ou qRT-PCR utilisant des cellulesC2C12 knocked-down pour Sepn1. J’ai pu montrer l’absence de changements nets des voies de l’AMPK et p38, ainsi que du taux d’expression des marqueurs de stress du réticulum endoplasmique GRP78 oucalnexine. Par contre, nos données suggèrent que les voies HDAC5 etmTOR pourraient être impliquées dans le phénotype de différentiation musculaire accélérée. En conclusion, la déplétion de SEPN1 entraîne une quiescence incomplète et une différentiation myogénique accélérée. Par conséquent, ce travail identifie SEPN1 comme un nouveau régulateur du processus de décision du destin cellulaire des progéniteurs musculaires, l’absence de SEPN1 favorisant la différentiation au détriment du renouvellement cellulaire. Ces résultats peuvent contribuer à expliquer la déplétion de la population de cellules satellites et les défauts de régénération observés dans les modèles de SEPN1-RM, et aider à identifier de nouveaux biomarqueurs cellulaires qui seront utiles à l’avenir pour évaluer des approches thérapeutiques.Mutations of Selenoprotein N (SEPN1) cause a congenital myopathy, SEPN1-related myopathy (SEPN1-RM), characterized by severe weakness and wasting of neck and trunk muscles, scoliosis and lethal respiratory failure. SEPN1-RM has been associated with oxidative stress, reduced satellite cell population and defective muscle regeneration. To investigate the underlying mechanisms, particularly a potential role of SEPN1 in regulating the balance between self-renewal and differentiation of the satellite cell pool, I used Sepn1 KO mice primary satellite cells and C2C12 cells knocked down for Sepn1, at different stages of differentiation (quiescent cells, myoblasts and myotubes). Using a suspension system to generate synchronized quiescence on C2C12, I found that Sepn1 absence in G0 cells does not prevent cell cycle exiting and re-entering but prevents normal downregulation of two key myogenic factors (MYOD1 and MYOG mRNAs) and leads to higher Cyclin D1 levels (CCND1 mRNA) in quiescence conditions. Microarray and qRT-PCR studies showed that Sepn1 depletion in proliferative C2C12 cells leads to significant increase in the levels of the transcription factors MYOG and MYOD1. In parallel, immunoblot analysis showed an increased expression of the cell cycle regulator proteins p21 and Cyclin D3. Moreover, primary murine satellite cells isolated from gastrocnemius and plantaris muscles from the Sepn1 KO mice showed increased myoblast fusion during early myogenic differentiation. Next, I explored the mechanistic pathways leading to this cell phenotype by western blots and/or qRT-PCR using Sepn1 knockdown C2C12 cells. I found no clear-cut abnormalities of the AMPK or the p38 mediated pathways, and no consistent changes in the expression of the ER stress markers GRP78 or calnexin. In contrast, our data suggest that HDAC5 and mTOR could be involved in the accelerated differentiation phenotype. Other mechanistic studies are in the progress. In conclusion, lack of SEPN1 leads to incomplete quiescence and accelerated myogenic differentiation. Thus, we identify SEPN1 as a novel regulator of the muscle progenitor’s cell fate decision process and SEPN1 depletion favors differentiation over self-renewal. These results potentially explain the depletion of the satellite cell population and the regeneration defect in SEPN1-RM models, and identify novel biomarkers useful to assess potential therapeutic interventions

Development of versatile allele-specific siRNAs able to silence all the dominant dynamin 2 mutations

International audienceDominant centronuclear myopathy (CNM) is a rare form of congenital myopathy associated with a wide clinical spectrum, from severe neonatal to milder adult forms. There is no available treatment for this disease due to heterozygous mutations in the DNM2 gene encoding Dynamin 2 (DNM2). Dominant DNM2 mutations also cause rare forms of Charcot-Marie-Tooth disease and hereditary spastic paraplegia, and deleterious DNM2 overexpression was noticed in several diseases. The proof of concept for therapy by allele-specific RNA interference devoted to silence the mutated mRNA without affecting the normal allele was previously achieved in a mouse model and patient-derived cells, both expressing the most frequent DNM2 mutation in CNM. In order to have versatile small interfering RNAs (siRNAs) usable regardless of the mutation, we have developed allele-specific siRNAs against two non-pathogenic single-nucleotide polymorphisms (SNPs) frequently heterozygous in the population. In addition, allele-specific siRNAs against the p.S619L DNM2 mutation, a mutation frequently associated with severe neonatal cases, were developed. The beneficial effects of these new siRNAs are reported for a panel of defects occurring in patient-derived cell lines. The development of these new molecules allows targeting the large majority of the patients harboring DNM2 mutations or overexpression by only a few siRNAs