Short-term nicotinamide riboside treatment improves muscle quality and function in mice and increases cellular energetics and differentiating capacity of myogenic progenitors.

OBJECTIVES: Nicotinamide adenine dinucleotide (NAD+), an essential cofactor for mitochondrial function, declines with aging, which may lead to impaired physical performance. Nicotinamide riboside (NR), a NAD+ precursor, restores cellular NAD+ levels. The aim of this study was to examine the effects of short-term NR supplementation on physical performance in middle-aged mice and the effects on mouse and human muscle stem cells. METHODS: We treated 15-mo-old male C57BL/6J mice with NR at 300 mg·kg·d-1 (NR3), 600 mg·kg·d-1 (NR6), or placebo (PLB), n = 8 per group, and assessed changes in physical performance, muscle histology, and NAD+ content after 4 wk of treatment. RESULTS: NR increased total NAD+ in muscle tissue (NR3 P = 0.01; NR6 P = 0.004, both versus PLB), enhanced treadmill endurance and open-field activity, and prevented decline in grip strength. Histologic analysis revealed NR-treated mice exhibited enlarged slow-twitch fibers (NR6 versus PLB P = 0.014; NR3 P = 0.16) and a trend toward more slow fibers (NR3 P = 0.14; NR6 P = 0.22). We next carried out experiments to characterize NR effects on mitochondrial activity and cellular energetics in vitro. We observed that NR boosted basal and maximal cellular aerobic and anaerobic respiration in both mouse and human myoblasts and human myotubes. Additionally, NR treatment improved the differentiating capacity of myoblasts and increased myotube size and fusion index upon stimulation of these progenitors to form multinucleated myotubes. CONCLUSION: These findings support a role for NR in improving cellular energetics and functional capacity in mice, which support the translation of this work into clinical settings as a strategy for improving and/or maintaining health span during aging.

Vitamin D Insufficiency Reduces Grip Strength, Grip Endurance and Increases Frailty in Aged C57Bl/6J Mice.

Low 25-OH serum vitamin D (VitD) is pervasive in older adults and linked to functional decline and progression of frailty. We have previously shown that chronic VitD insufficiency in "middle-aged" mice results in impaired anaerobic exercise capacity, decreased lean mass, and increased adiposity. Here, we examine if VitD insufficiency results in similar deficits and greater frailty progression in old-aged (24 to 28 months of age) mice. Similar to what we report in younger mice, older mice exhibit a rapid and sustained response in serum 25-OH VitD levels to differential supplementation, including insufficient (125 IU/kg chow), sufficient (1000 IU/kg chow), and hypersufficient (8000 IU/kg chow) groups. During the 4-month time course, mice were assessed for body composition (DEXA), physical performance, and frailty using a Fried physical phenotype-based assessment tool. The 125 IU mice exhibited worse grip strength (p = 0.002) and inverted grip hang time (p = 0.003) at endpoint and the 8000 IU mice transiently displayed greater rotarod performance after 3 months (p = 0.012), yet other aspects including treadmill performance and gait speed were unaffected. However, 125 and 1000 IU mice exhibited greater frailty compared to baseline (p = 0.001 and p = 0.038, respectively), whereas 8000 IU mice did not (p = 0.341). These data indicate targeting higher serum 25-OH vitamin D levels may attenuate frailty progression during aging.

Absence of complement factor H reduces physical performance in C57BL6 mice.

Complement (C) system is a double edge sword acting as the first line of defense on the one hand and causing aggravation of disease on the other. C activation when unregulated affects different organs including muscle regeneration. However, the effect of factor H (FH), a critical regulator of the alternative C pathway in muscle remains to be studied. FH deficiency results in excessive C activation and generates proinflammatory fragments C5a and C3a as byproducts. C3a and C5a signal through their respective receptors, C5aR and C3aR. In this study, we investigated the role of FH and downstream C5a/C5aR signaling in muscle architecture and function. Using the FH knockout (fh-/-) and fh-/-/C5aR-/double knockout mice we explored the role of C, specifically the alternative C pathway in muscle dysfunction. Substantial C3 and C9 deposits occur along the walls of the fh-/- muscle fibers indicative of unrestricted C activation. Physical performance assessments of the fh-/- mice show reduced grip endurance (76 %), grip strength (14 %) and rotarod balance (36 %) compared to controls. Histological analysis revealed a shift in muscle fiber populations indicated by an increase in glycolytic MHC IIB fibers and reduction in oxidative MHC IIA fibers. Consistent with this finding, mitochondrial DNA (mtDNA) and citrate synthase (CS) expression were both reduced indicating possible reduction in mitochondrial biomass. In addition, our results showed a significant increase in TGFß expression and altered TGFß localization in this setting. The architecture of cytoskeletal proteins actin and vimentin in the fh-/- muscle was changed that could lead to contractile weakness and loss of skeletal muscle elasticity. The muscle pathology in fh-/- mice was reduced in fh-/-/C5aR-/- double knockout (DKO) mice, highlighting partial C5aR dependence. Our results for the first time demonstrate an important role of FH in physical performance and skeletal muscle health.

High intensity interval training improves physical performance in aged female mice: A comparison of mouse frailty assessment tools.

Frailty syndrome increases the risk for disability and mortality, and is a major health concern amidst the geriatric shift in the population. High intensity interval training (HIIT), which couples bursts of vigorous activity interspersed with active recovery intervals, shows promise for the treatment of frailty. Here we compare and contrast five Fried physical phenotype and one deficit accumulation based mouse frailty assessment tools for identifying the impacts of HIIT on frailty and predicting functional capacity, underlying pathology, and survival in aged female mice. Our data reveal a 10-minute HIIT regimen administered 3-days-a-week for 8-weeks increased treadmill endurance, gait speed and maintained grip strength. One frailty tool identified a benefit of HIIT for frailty, but many were trending suggesting HIIT was beneficial for physical performance in these mice, but the 8-week timeframe may have been insufficient to induce frailty benefits. Finally, most frailty tools distinguished between surviving or non-surviving mice, whereas half correlated with functional capacity measured by nest building ability, and none correlated with underlying pathology. In summary, this study supports the ongoing development of mouse assessment tools as useful instruments for frailty research.

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

C5a induces caspase-dependent apoptosis in brain vascular endothelial cells in experimental lupus.

Blood-brain barrier (BBB) dysfunction complicates central nervous system lupus, an important aspect of systemic lupus erythematosus. To gain insight into the underlying mechanism, vascular corrosion casts of brain were generated from the lupus mouse model, MRL/lpr mice and the MRL/MpJ congenic controls. Scanning electron microscopy of the casts showed loss of vascular endothelial cells in lupus mice compared with controls. Immunostaining revealed a significant increase in caspase 3 expression in the brain vascular endothelial cells, which suggests that apoptosis could be an important mechanism causing cell loss, and thereby loss of BBB integrity. Complement activation occurs in lupus resulting in increased generation of circulating C5a, which caused the endothelial layer to become 'leaky'. In this study, we show that C5a and lupus serum induced apoptosis in cultured human brain microvascular endothelial cells (HBMVECs), whereas selective C5a receptor 1 (C5aR1) antagonist reduced apoptosis in these cells, demonstrating C5a/C5aR1-dependence. Gene expression of initiator caspases, caspase 1 and caspase 8, and pro-apoptotic proteins death-associated protein kinase 1, Fas-associated protein (FADD), cell death-inducing DNA fragmentation factor 45 000 MW subunit A-like effector B (CIDEB) and BCL2-associated X protein were increased in HBMVECs treated with lupus serum or C5a, indicating that both the intrinsic and extrinsic apoptotic pathways could be critical mediators of brain endothelial cell apoptosis in this setting. Overall, our findings suggest that C5a/C5aR1 signalling induces apoptosis through activation of FADD, caspase 8/3 and CIDEB in brain endothelial cells in lupus. Further elucidation of the underlying apoptotic mechanisms mediating the reduced endothelial cell number is important in establishing the potential therapeutic effectiveness of C5aR1 inhibition that could prevent and/or reduce BBB alterations and preserve the physiological function of BBB in central nervous system lupus.