Dietary phosphate restriction prevents the appearance of sarcopenia signs in old mice.

BACKGROUND: Sarcopenia is defined by the progressive and generalized loss of muscle mass and function associated with aging. We have previously proposed that aging-related hyperphosphataemia is linked with the appearance of sarcopenia signs. Because there are not effective treatments to prevent sarcopenia, except for resistance exercise, we propose here to analyse whether the dietary restriction of phosphate could be a useful strategy to improve muscle function and structure in an animal model of aging. METHODS: Five-month-old (young), 24-month-old (old) and 28-month-old (geriatric) male C57BL6 mice were used. Old and geriatric mice were divided into two groups, one fed with a standard diet (0.6% phosphate) and the other fed with a low-phosphate (low-P) diet (0.2% phosphate) for 3 or 7 months, respectively. A phosphate binder, Velphoro®, was also supplemented in a group of old mice, mixed with a standard milled diet for 3 months. Muscle mass was measured by the weight of gastrocnemius and tibial muscles, and quality by nuclear magnetic resonance imaging (NMRI) and histological staining assays. Muscle strength was measured by grip test and contractile properties of the tibialis muscle by electrical stimulation of the common peroneal nerve. Gait parameters were analysed during the spontaneous locomotion of the mice with footprinting. Orientation and motor coordination were evaluated using a static rod test. RESULTS: Old mice fed with low-P diet showed reduced serum phosphate concentration (16.46 ± 0.77 mg/dL young; 21.24 ± 0.95 mg/dL old; 17.46 ± 0.82 mg/dL low-P diet). Old mice fed with low-P diet displayed 44% more mass in gastrocnemius muscles with respect to old mice (P = 0.004). NMRI revealed a significant reduction in T2 relaxation time (P = 0.014) and increased magnetization transfer (P = 0.045) and mean diffusivity (P = 0.045) in low-P diet-treated mice compared with their coetaneous. The hypophosphataemic diet increased the fibre size and reduced the fibrotic area by 52% in gastrocnemius muscle with respect to old mice (P = 0.002). Twitch force and tetanic force were significantly increased in old mice fed with the hypophosphataemic diet (P = 0.004 and P = 0.014, respectively). Physical performance was also improved, increasing gait speed by 30% (P = 0.032) and reducing transition time in the static rod by 55% (P = 0.012). Similar results were found when diet was supplemented with Velphoro®. CONCLUSIONS: The dietary restriction of phosphate in old mice improves muscle quantity and quality, muscle strength and physical performance. Similar results were found using the phosphate binder Velphoro®, supporting the role of phosphate in the impairment of muscle structure and function that occurs during aging.

Hyperphosphatemia-Induced Oxidant/Antioxidant Imbalance Impairs Vascular Relaxation and Induces Inflammation and Fibrosis in Old Mice.

Aging impairs vascular function, but the mechanisms involved are unknown. The aim of this study was to analyze whether aging-related hyperphosphatemia is implied in this effect by elucidating the role of oxidative stress. C57BL6 mice that were aged 5 months (young) and 24 months (old), receiving a standard (0.6%) or low-phosphate (0.2%) diet, were used. Isolated mesenteric arteries from old mice showed diminished endothelium-dependent vascular relaxation by the down-regulation of NOS3 expression, increased inflammation and increased fibrosis in isolated aortas, compared to those isolated from young mice. In parallel, increased Nox4 expression and reduced Nrf2, Sod2-Mn and Gpx1 were found in the aortas from old mice, resulting in oxidant/antioxidant imbalance. The low-phosphate diet improved vascular function and oxidant/antioxidant balance in old mice. Mechanisms were analyzed in endothelial (EC) and vascular smooth muscle cells (SMCs) treated with the phosphate donor ß-glycerophosphate (BGP). In EC, BGP increased Nox4 expression and ROS production, which reduced NOS3 expression via NFκB. BGP also increased inflammation in EC. In SMC, BGP increased Collagen I and fibronectin expression by priming ROS production and NFκB activity. In conclusion, hyperphosphatemia reduced endothelium-dependent vascular relaxation and increased inflammation and vascular fibrosis through an impairment of oxidant/antioxidant balance in old mice. A low-phosphate diet achieved improvements in the vascular function in old mice.

Aging-related hyperphosphatemia impairs myogenic differentiation and enhances fibrosis in skeletal muscle.

BACKGROUND: Hyperphosphatemia has been related to the development of sarcopenia in aging mice. We describe the intracellular mechanisms involved in the impairment of the myogenic differentiation promoted by hyperphosphatemia and analyse these mechanisms in the muscle from older mice. METHODS: C2 C12 cells were grown in 2% horse serum in order to promote myogenic differentiation, in the presence or absence of 10 mM beta-glycerophosphate (BGP) for 7 days. Troponin T, paired box 7 (Pax-7), myogenic factor 5 (Myf5), myogenic differentiation 1 (MyoD), myogenin (MyoG), myocyte enhancer factor 2 (MEF2C), P300/CBP-associated factor (PCAF), histone deacetylase 1 (HDAC1), fibronectin, vimentin, and collagen I were analysed at 48, 72, and 168 h, by western blotting or by immunofluorescence staining visualized by confocal microscopy. Studies in mice were performed in 5- and 24-month-old C57BL6 mice. Three months before sacrifice, 21-month-old mice were fed with a standard diet or a low phosphate diet, containing 0.6% or 0.2% phosphate, respectively. Serum phosphate concentration was assessed by a colorimetric method and forelimb strength by a grip test. Fibrosis was observed in the tibialis anterior muscle by Sirius Red staining. In gastrocnemius muscle, MyoG, MEF2C, and fibronectin expressions were analysed by western blotting. RESULTS: Cells differentiated in the presence of BGP showed near five times less expression of troponin T and kept higher levels of Pax-7 than control cells indicating a reduced myogenic differentiation. BGP reduced Myf5 about 50% and diminished MyoD transcriptional activity by increasing the expression of HDAC1 and reducing the expression of PCAF. Consequently, BGP reduced to 50% the expression of MyoG and MEF2C. A significant increase in the expression of fibrosis markers as collagen I, vimentin, and fibronectin was found in cells treated with BGP. In mice, serum phosphate (17.24 ± 0.77 mg/dL young; 23.23 ± 0.81 mg/dL old; 19.09 ± 0.75 mg/dL old with low phosphate diet) correlates negatively (r = -0.515, P = 0.001) with the muscular strength (3.13 ± 0.07 gf/g young; 1.70 ± 0.12 gf/g old; 2.10 ± 0.09 gf/g old with low phosphate diet) and with the expression of MyoG (r = -0.535, P = 0.007) and positively with the expression of fibronectin (r = 0.503, P = 0.001) in gastrocnemius muscle. The tibialis anterior muscle from old mice showed muscular fibrosis. Older mice fed with a low phosphate diet showed improved muscular parameters relative to control mice of similar age. CONCLUSIONS: Hyperphosphatemia impairs myogenic differentiation, by inhibiting the transcriptional activity of MyoD, and enhances the expression of fibrotic genes in cultured myoblasts. Experiments carried out in older mice demonstrate a close relationship between age-related hyperphosphatemia and the decrease in the expression of myogenic factors and the increase in factors related to muscle fibrosis.

Uraemic toxins impair skeletal muscle regeneration by inhibiting myoblast proliferation, reducing myogenic differentiation, and promoting muscular fibrosis.

Uraemic toxins increase in serum parallel to a decline in the glomerular filtration rate and the development of sarcopenia in patients with chronic kidney disease (CKD). This study analyses the role of uraemic toxins in sarcopenia at different stages of CKD, evaluating changes in the muscular regeneration process. Cultured C2C12 cells were incubated with a combination of indoxyl sulphate and p-cresol at high doses (100 µg/mL) or low doses (25 µg/mL and 10 µg/mL) resembling late or early CKD stages, respectively. Cell proliferation (analysed by scratch assays and flow cytometry) was inhibited only by high doses of uraemic toxins, which inactivated the cdc2-cyclin B complex, inhibiting mitosis and inducing apoptosis (analysed by annexin V staining). By contrast, low doses of uraemic toxins did not affect proliferation, but reduced myogenic differentiation, primed with 2% horse serum, by inhibiting myogenin expression and promoting fibro-adipogenic differentiation. Finally, to assess the in vivo relevance of these results, studies were performed in gastrocnemii from uraemic rats, which showed higher collagen expression and lower myosin heavy chain expression than those from healthy rats. In conclusion, uraemic toxins impair the skeletal muscular regeneration process, even at low concentrations, suggesting that sarcopenia can progress from the early stages of CKD.

Endothelin-1 induces cellular senescence and fibrosis in cultured myoblasts. A potential mechanism of aging-related sarcopenia.

Endothelial dysfunction, with increased endothelin-1 (ET-1) synthesis, and sarcopenia, characterized by the loss of muscular mass and strength, are two aging-related conditions. However, a relationship between them has not been already established. The aim of this study was to determine whether ET-1 induces senescence and fibrosis in cultured murine myoblasts, which could be involved in the development of sarcopenia related to aging. For this purpose, myoblasts were incubated with ET-1 to assess cellular senescence, analyzed by senescence associated ß-galactosidase activity and p16 expression; and fibrosis, assessed by fibronectin expression. ET-1 induced myoblast senescence and fibrosis through ETA receptor. The use of antioxidants and several antagonists revealed that ET-1 effect on senescence and fibrosis depended on ROS production and activation of PI3K-AKT-GSK pathway. To stress the in vivo relevance of these results, circulating ET-1, muscular strength, muscular fibrosis and p16 expression were measured in male C57Bl6 mice from 5-18-24-months-old. Old mice shown high levels of ET-1 correlated with muscular fibrosis, muscular p16 expression and loss of muscle strength. In conclusion, ET-1 promotes fibrosis and senescence in cultured myoblasts, similar results were found in old mice, suggesting a potential role for ET-1 in the development of sarcopenia related to aging.