BIO101 stimulates myoblast differentiation and improves muscle function in adult and old mice.

BACKGROUND: Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse. Age-related imbalance of protein synthesis and degradation, mainly regulated by AKT/mTOR pathway, leads to progressive loss of muscle mass. Maintenance of anabolic and regenerative capacities of skeletal muscles may be regarded as a therapeutic option for sarcopenia and other muscle wasting diseases. Our previous studies have demonstrated that BIO101, a pharmaceutical grade 20-hydroxyecdysone, increases protein synthesis through the activation of MAS receptor involved in the protective arm of renin-angiotensin-aldosterone system. The purpose of the present study was to assess the anabolic and pro-differentiating properties of BIO101 on C2C12 muscle cells in vitro and to investigate its effects on adult and old mice models in vivo. METHODS: The effects of BIO101 on C2C12 differentiation were assessed using myogenic transcription factors and protein expression of major kinases of AKT/mTOR pathway by Western blot. The in vivo effects of BIO101 have been investigated in BIO101 orally-treated (50 mg/kg/day) adult mice (3 months) for 28 days. To demonstrate potential beneficial effect of BIO101 treatment in a sarcopenic mouse model, we use orally treated 22-month-old C57Bl6/J mice, for 14 weeks with vehicle or BIO101. Mice body and muscle weight were recorded. Physical performances were assessed using running capacity and muscle contractility tests. RESULTS: Anabolic properties of BIO101 were confirmed by the rapid activation of AKT/mTOR, leading to an increase of C2C12 myotubes diameters (+26%, P < 0.001). Pro-differentiating effects of BIO101 on C2C12 myoblasts were revealed by increased expression of muscle-specific differentiation transcription factors (MyoD, myogenin), resulting in increased fusion index and number of nuclei per myotube (+39% and +53%, respectively, at day 6). These effects of BIO101 were like those of angiotensin (1-7) and were abolished with the use of A779, a MAS receptor specific antagonist. Chronic BIO101 oral treatment induced AKT/mTOR activation and anabolic effects accompanied with improved physical performances in adult and old animals (maximal running distance and maximal running velocity). CONCLUSIONS: Our data suggest beneficial anabolic and pro-differentiating effects of BIO101 rendering BIO101 a potent drug candidate for treating sarcopenia and possibly other muscle wasting disorders.

QGC606: A Best-in-Class Orally Active Centrally Acting Aminopeptidase A Inhibitor Prodrug for Treating Heart Failure Following Myocardial Infarction.

BACKGROUND: Blockade of brain renin-angiotensin system (RAS) overactivity by firibastat, the first centrally acting aminopeptidase A (APA) inhibitor prodrug, has already demonstrated its effectiveness in improving cardiac function after myocardial infarction (MI). We developed QGC606, a more potent and more selective APA inhibitor prodrug and studied its effects after long-term oral administration in mice post-MI. METHODS: Two days after MI induced by the left anterior descending artery ligation, adult male mice were randomized into 4 groups to receive oral treatment during 4 weeks with vehicle; QGC606; firibastat; or the angiotensin-I converting enzyme inhibitor ramipril, used as positive control. RESULTS: Four weeks post-MI, brain APA was overactivated in vehicle-treated MI mice. QGC606 treatment normalized brain APA hyperactivity to control values measured in sham-operated mice. Four weeks post-MI, QGC606-treated mice had higher left ventricular (LV) ejection fractions, significantly smaller LV end-systolic diameter and volume, significantly lower HF biomarkers mRNA expression (Myh7 and Anf) and plasma N-terminal pro B-type natriuretic peptide (NT-pro-BNP) and noradrenaline levels than saline-treated mice. QGC606 treatment significantly improved the dP/dt max and min, LV end-diastolic pressure without affecting blood pressure (BP), whereas we observed a decrease in BP in ramipril-treated mice. We observed also a reduction of cardiac fibrosis, highlighted by lower connective tissue growth factor mRNA levels and a reduction of both the fibrotic area and MI size in QGC606-treated mice. CONCLUSIONS: Chronic oral QGC606 administration in post-MI mice showed beneficial effects in improving cardiac function and reducing cardiac remodeling and fibrosis but, unlike ramipril, without lowering BP.

NMRK2 Gene Is Upregulated in Dilated Cardiomyopathy and Required for Cardiac Function and NAD Levels during Aging.

Dilated cardiomyopathy (DCM) is a disease of multifactorial etiologies, the risk of which is increased by male sex and age. There are few therapeutic options for patients with DCM who would benefit from identification of common targetable pathways. We used bioinformatics to identify the Nmrk2 gene involved in nicotinamide adenine dinucleotde (NAD) coenzyme biosynthesis as activated in different mouse models and in hearts of human patients with DCM while the Nampt gene controlling a parallel pathway is repressed. A short NMRK2 protein isoform is also known as muscle integrin binding protein (MIBP) binding the α7ß1 integrin complex. We investigated the cardiac phenotype of Nmrk2-KO mice to establish its role in cardiac remodeling and function. Young Nmrk2-KO mice developed an eccentric type of cardiac hypertrophy in response to pressure overload rather than the concentric hypertrophy observed in controls. Nmrk2-KO mice developed a progressive DCM-like phenotype with aging, associating eccentric remodeling of the left ventricle and a decline in ejection fraction and showed a reduction in myocardial NAD levels at 24 months. In agreement with involvement of NMRK2 in integrin signaling, we observed a defect in laminin deposition in the basal lamina of cardiomyocytes leading to increased fibrosis at middle age. The α7 integrin was repressed at both transcript and protein level at 24 months. Nmrk2 gene is required to preserve cardiac structure and function, and becomes an important component of the NAD biosynthetic pathways during aging. Molecular characterization of compounds modulating this pathway may have therapeutic potential.

Efficacy of epicardial implantation of acellular chitosan hydrogels in ischemic and nonischemic heart failure: impact of the acetylation degree of chitosan.

This work explores the epicardial implantation of acellular chitosan hydrogels in two murine models of cardiomyopathy, focusing on their potential to restore the functional capacity of the heart. Different chitosan hydrogels were generated using polymers of four degrees of acetylation, ranging from 2.5% to 38%, because the degree of acetylation affects their degradation and biological activity. The hydrogels were adjusted to a 3% final polymer concentration. After complete macromolecular characterization of the chitosans and study of the mechanical properties of the resulting hydrogels, they were sutured onto the surface of the myocardium, first in rat after four-weeks of coronary ligation (n=58) then in mice with cardiomyopathy induced by a cardiac-specific invalidation of serum response factor (n=20). The implantation of the hydrogels was associated with a reversion of cardiac function loss with maximal effects for the acetylation degree of 24%. The extent of fibrosis, the cardiomyocyte length-to-width ratio, as well as the genes involved in fibrosis and stress were repressed after implantation. Our study demonstrated the beneficial effects of chitosan hydrogels, particularly with polymers of high degrees of acetylation, on cardiac remodeling in two cardiomyopathy models. Our findings indicate they have great potential as a reliable therapeutic approach to heart failure.

Aged Nicotinamide Riboside Kinase 2 Deficient Mice Present an Altered Response to Endurance Exercise Training.

Background: Skeletal muscle aging is marked by the development of a sarcopenic phenotype, a global decline of muscle energetic capacities, and an intolerance to exercise. Among the metabolic disorders involved in this syndrome, NAD metabolism was shown to be altered in skeletalmuscle, with an important role for the NAMPT enzyme recycling the nicotinamide precursor. An alternative pathway for NAD biosynthesis has been described for the nicotinamide riboside vitamin B3 precursor used by the NMRK kinases, including the striated muscle-specific NMRK2. Aim: With this study, our goal is to explore the ability of 16-month-old Nmrk2 -/- mice to perform endurance exercise and study the consequences on muscle adaptation to exercise. Methods: 10 control and 6 Nmrk2 -/- mice were used and randomly assigned to sedentary and treadmill endurance training groups. After 9 weeks of training, heart and skeletal muscle samples were harvested and used for gene expression analysis, NAD levels measurements and immunohistochemistry staining. Results: Endurance training triggered a reduction in the expression of Cpt1b and AcadL genes involved in fatty acid catabolism in the heart of Nmrk2 -/- mice, not in control mice. NAD levels were not altered in heart or skeletal muscle, nor at baseline neither after exercise training in any group. Myh7 gene encoding for the slow MHC-I was more strongly induced by exercise in Nmrk2 -/- mice than in controls. Moreover, IL-15 expression levels is higher in Nmrk2 -/- mice skeletal muscle at baseline compared to controls. No fiber type switch was observed in plantaris after exercise, but fast fibers diameter was reduced in aged control mice, not in Nmrk2 -/- mice. No fiber type switch or diameter modification was observed in soleus muscle. Conclusion: In this study, we demonstrated for the first time a phenotype in old Nmrk2 -/- mice in response to endurance exercise training. Although NMRK2 seems to be predominantly dispensable to maintain global NAD levels in heart and skeletal muscle, we demonstrated a maladaptive metabolic response to exercise in cardiac and skeletal muscle, showing that NMRK2 has a specific and restricted role in NAD signaling compared to the NAMPT pathway.

Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy.

BACKGROUND: Myocardial metabolic impairment is a major feature in chronic heart failure. As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress and oxidative stress response, nicotinamide adenine dinucleotide (NAD+) is emerging as a metabolic target in a number of diseases including heart failure. Little is known on the mechanisms regulating homeostasis of NAD+ in the failing heart. METHODS: To explore possible alterations of NAD+ homeostasis in the failing heart, we quantified the expression of NAD+ biosynthetic enzymes in the human failing heart and in the heart of a mouse model of dilated cardiomyopathy (DCM) triggered by Serum Response Factor transcription factor depletion in the heart (SRFHKO) or of cardiac hypertrophy triggered by transverse aorta constriction. We studied the impact of NAD+ precursor supplementation on cardiac function in both mouse models. RESULTS: We observed a 30% loss in levels of NAD+ in the murine failing heart of both DCM and transverse aorta constriction mice that was accompanied by a decrease in expression of the nicotinamide phosphoribosyltransferase enzyme that recycles the nicotinamide precursor, whereas the nicotinamide riboside kinase 2 (NMRK2) that phosphorylates the nicotinamide riboside precursor is increased, to a higher level in the DCM (40-fold) than in transverse aorta constriction (4-fold). This shift was also observed in human failing heart biopsies in comparison with nonfailing controls. We show that the Nmrk2 gene is an AMP-activated protein kinase and peroxisome proliferator-activated receptor α responsive gene that is activated by energy stress and NAD+ depletion in isolated rat cardiomyocytes. Nicotinamide riboside efficiently rescues NAD+ synthesis in response to FK866-mediated inhibition of nicotinamide phosphoribosyltransferase and stimulates glycolysis in cardiomyocytes. Accordingly, we show that nicotinamide riboside supplementation in food attenuates the development of heart failure in mice, more robustly in DCM, and partially after transverse aorta constriction, by stabilizing myocardial NAD+ levels in the failing heart. Nicotinamide riboside treatment also robustly increases the myocardial levels of 3 metabolites, nicotinic acid adenine dinucleotide, methylnicotinamide, and N1-methyl-4-pyridone-5-carboxamide, that can be used as validation biomarkers for the treatment. CONCLUSIONS: The data show that nicotinamide riboside, the most energy-efficient among NAD precursors, could be useful for treatment of heart failure, notably in the context of DCM, a disease with few therapeutic options.

Voluntary Exercise Improves Cardiac Function and Prevents Cardiac Remodeling in a Mouse Model of Dilated Cardiomyopathy.

Objective: Despite the indubitable beneficial effect of exercise to prevent of cardiovascular diseases, there is still a lack of studies investigating the impact of exercise in non-ischemic dilated cardiomyopathy. Here, we investigated the impact of voluntary exercise on cardiac function in a mouse model of non-ischemic dilated cardiomyopathy (αMHC-MerCreMer:Sf/Sf), induced by cardiac-specific inactivation of the Serum Response Factor. Materials and Methods: Seven days after tamoxifen injection, 20 αMHC-MerCreMer:Sf/Sf mice were assigned to sedentary (n = 8) and exercise (n = 12) groups. Seven additional αMHC-MerCreMer:Sf/Sf mice without tamoxifen injection were used as control. The exercise group performed 4 weeks of voluntary running on wheel (1.8 ± 0.12 km/day). Cardiac function, myocardial fibrosis, and mitochondrial energetic pathways were then blindly assessed. Results: Exercised mice exhibited a smaller decrease of left ventricular (LV) fractional shortening and ejection fraction compared to control mice. This was associated with a lower degree of LV remodeling in exercised mice, as shown by a lower LV end-systolic intrerventricular septal and posterior wall thickness decrease from baseline values compared to sedentary mice. Moreover, exercised mice displayed a reduced gene expression of atrial and brain natriuretic factors. These benefits were associated by a reduced level of myocardial fibrosis. In addition, exercised mice exhibited a higher mitochondrial aconitase, voltage-dependent anion-selective channel 1 and PPAR gamma coactivators-1 alpha proteins levels suggesting that the increase of mitochondrial biogenesis and/or metabolism slowed the progression of dilated cardiomyopathy in exercised animals. Conclusions: In conclusion, our results support the role of voluntary exercise to improve outcomes in non-ischemic dilated heart failure (HF) and also support its potential for a routine clinical use in the future.