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

14 p.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 C2C12 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.Instituto de Salud Carlos IIIPrincipado de AsturiasComunidad de Madri

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 NF kappa B. BGP also increased inflammation in EC. In SMC, BGP increased Collagen I and fibronectin expression by priming ROS production and NF kappa B 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

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

17 p.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 mu g/mL) or low doses (25 mu g/mL and 10 mu g/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.Instituto de Salud Carlos II

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 beta-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

Hyperphosphatemia Promotes Senescence of Myoblasts by Impairing Autophagy Through Ilk Overexpression, A Possible Mechanism Involved in Sarcopenia

In mammalians, advancing age is associated with sarcopenia, the progressive and involuntary loss of muscle mass and strength. Hyperphosphatemia is an aging-related condition involved in several pathologies. The aim of this work was to assess whether hyperphosphatemia plays a role in the age-related loss of mass muscle and strength by inducing cellular senescence in murine myoblasts and to explore the intracellular mechanism involved in this effect. Cultured mouse C2C12 cells were treated with 10 mM beta-glycerophosphate (BGP] at different periods of time to induce hyperphosphatemia. BGP promoted cellular senescence after 24 h of treatment, assessed by the increased expression of p53, acetylated-p53 and p21 and senescence associated beta-galactosidase activity. In parallel, BGP increased ILK expression and activity, followed by mTOR activation and autophagy reduction. Knocking-down ILK expression increased autophagy and protected cells from senescence induced by hyperphosphatemia. BGP also reduced the proliferative capacity of cultured myoblasts. Old mice (24months-old] presented higher serum phosphate concentration, lower forelimb strength, higher expression of p53 and ILK and less autophagy in vastus muscle than young mice (5-months-old]. In conclusion, we propose that hyperphosphatemia induces senescence in cultured myoblasts through ILK overexpression, reducing their proliferative capacity, which could be a mechanism involved in the development of sarcopenia, since old mice showed loss of muscular strength correlated with high serum phosphate concentration and increased levels of ILK and p53

Análisis molecular de las relaciones entre hiperlipidemia y fibrosis vascular

La hiperlipidemia es uno de los desórdenes metabólicos más implicados en enfermedades cardiovasculares como la aterosclerosis que cursa con un aumento de LDL oxidadas (OxLDL) en sangre. El objetivo del estudio fue establecer la relación entre hiperlipidemia y fibrosis vascular, evaluando los mecanismos implicados. Para ello, se incubaron células endoteliales con OxLDL encontrando un aumento tanto en la expresión de fibronectina como en la producción de radicales libres. Las OxLDL revelaron la activación del factor de transcripción NFkB vía producción de radicales libres. Además, experimentos con EMSA confirman que el factor NFkB está implicado directamente en el aumento de la expresión de fibronectina estimulado por las OxLDL en células endoteliales. Por otro lado, las OxLDL son capaces de inducir la expresión del enzima conversor de endotelina-1 y la producción de endotelina-1 (ET-1). La ET-1 sintetizada en células endoteliales se libera y llega a otros tipos celulares. Por tanto, se analizó el efecto directo de la ET-1 en células musculares lisas (CML). La ET-1 también estimula fibrosis en CML medida como expresión de la proteína pro-fibrótica CTGF y las proteínas de MEC, fibronectina y colágeno I. La ET-1 también favorece la producción de radicales libres. El efecto de la ET-1 sobre la fibrosis fue específico de su unión al receptor ETA presente en CML, porque el antagonista específico BQ123 bloqueó completamente el efecto de la ET-1. En conclusión, durante la aterosclerosis se favorecerían los procesos de fibrosis tanto a nivel endotelial por las OxLDL como a nivel muscular por la ET-1 generada por ellas, que podría ser la causa de la disfunción vascular presente en la aterosclerosi