DNA methylation of skeletal muscle function-related secretary factors identifies FGF2 as a potential biomarker for sarcopenia.

BACKGROUND: Sarcopenia is characterized by progressive loss of muscle mass and function due to aging. DNA methylation has been identified to play important roles in the dysfunction of skeletal muscle. The aim of our present study was to explore the whole blood sample-based methylation changes of skeletal muscle function-related factors in patients with sarcopenia. METHODS: The overall DNA methylation levels were analysed by using MethlTarget™ DNA Methylation Analysis platform in a discovery set consistent of 50 sarcopenic older adults (aged ≥65 years) and 50 age- and sex-matched non-sarcopenic individuals. The candidate differentially methylated regions (DMRs) were further validated by Methylation-specific PCR (MSP) in another two independent larger sets and confirmed by pyrosequencing. Receiver operating characteristic (ROC) curve analysis was used to determine the optimum cut-off levels of fibroblast growth factor 2 (FGF2)_30 methylation best predicting sarcopenia and area under the ROC curve (AUC) was measured. The correlation between candidate DMRs and the risk of sarcopenia was investigated by univariate analysis and multivariate logistic regression analysis. RESULTS: Among 1149 cytosine-phosphate-guanine (CpG) sites of 27 skeletal muscle function-related secretary factors, 17 differentially methylated CpG sites and 7 differentially methylated regions (DMRs) were detected between patients with sarcopenia and control subjects in the discovery set. Further methylation-specific PCR identified that methylation of fibroblast growth factor 2 (FGF2)_30 was lower in patients with sarcopenia and the level was decreased as the severity of sarcopenia increased, which was confirmed by pyrosequencing. Correlation analysis demonstrated that the methylation level of FGF2_30 was positively correlated to ASMI (r = 0.372, P < 0.001), grip strength (r = 0.334, P < 0.001), and gait speed (r = 0.411, P < 0.001). ROC curve analysis indicated that the optimal cut-off value of FGF2_30 methylation level that predicted sarcopenia was 0.15 with a sensitivity of 84.6% and a specificity of 70.1% (AUC = 0.807, 95% CI = 0.756-0.858, P < 0.001). Multivariate logistic regression analyses showed that lower FGF2_30 methylation level (<0.15) was significantly associated with increased risk of sarcopenia even after adjustment for potential confounders including age, sex, and BMI (adjusted OR = 9.223, 95% CI: 6.614-12.861, P < 0.001). CONCLUSIONS: Our results suggest that lower FGF2_30 methylation is correlated with the risk and severity of sarcopenia in the older adults, indicating that FGF2 methylation serve as a surrogate biomarker for the screening and evaluation of sarcopenia.

Association of AST/ALT (De Ritis) ratio with sarcopenia in a Chinese population of community-dwelling elderly.

Background: The aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio, also known as De Ritis ratio, has been reportedly associated with malnutrition which plays a crucial role in sarcopenia. The aim of this study was to examine the relationship between AST/ALT ratio and sarcopenia in the Chinese community-dwelling elderly. Methods: A cross-sectional study with 2751 participants (1343 men and 1408 women) aged ≥60 years was performed. Appendicular skeletal muscle mass index (ASMI), grip strength, and gait speed were measured to diagnose sarcopenia according to the latest Asian Working Group for Sarcopenia (AWGS) consensus. The association of AST/ALT ratio with sarcopenia was examined using logistic regression analysis. Results: The prevalence of sarcopenia in the present study was 4.4%. AST/ALT ratio was higher in the sarcopenia group than in the non-sarcopenia group (1.30 ± 0.33 vs. 1.16 ± 0.62, P = 0.010). AST/ALT ratio was negatively correlated with the components of sarcopenia, including ASMI, grip strength, and gait speed. Logistic regression analysis indicated that high AST/ALT ratio (>1.20) was associated with increased risk of sarcopenia even after adjustment for potential confounders (adjusted OR = 2.33, 95%CI = 1.48-3.68, P < 0.001). Stratification analyses indicated that the association of high AST/ALT ratio with high risk of sarcopenia was more significant in males and the elderly with ≥70 years. Conclusions: Our findings demonstrate that high AST/ALT ratio is associated with increased risk of sarcopenia in a Chinese population of community-dwelling elderly.

Serum Retinol Binding Protein 4 as a Potential Biomarker for Sarcopenia in Older Adults.

Sarcopenia is characterized by progressive loss of muscle mass and function due to aging. Retinol-binding protein 4 (RBP4) is an adipokine with pro-inflammatory effects. However, the change of RBP4 concentration and its role in sarcopenia remains unclear. The aim of this study was to evaluate the association of serum RBP4 level with sarcopenia in the older adults. A total of 816 community-dwelling older adults aged ≥60 years were enrolled. Serum RBP4 was measured by enzyme-linked immunosorbent assay. Appendicular skeletal muscle mass index (ASMI), grip strength, and gait speed were measured. We found that serum RBP4 levels were higher in patients with sarcopenia when compared with those without sarcopenias (44.3 [33.9-57.7] vs 38.0 [28.0-48.4] µg/mL). Receiver operating characteristic curve analysis indicated that the optimal cutoff value of serum RBP4 level that predicted sarcopenia was 38.79 µg/mL with a sensitivity of 67.8% and a specificity of 53.3%. Multivariate logistic regression analysis showed that the subjects with a higher level of RBP4 had a higher risk of sarcopenia (adjusted odds ratio [OR] = 2.036, 95% CI = 1.449-2.861). Serum RBP4 concentration was negatively correlated with grip strength (r = -.098), gait speed (r = -.186), and AMSI (r = -.096). Moreover, serum RBP4 levels were higher in patients with severe sarcopenia when compared with those with moderate sarcopenia (49.0 [37.3-61.2] vs 40.4 [31.3-51.2] µg/mL). Taken together, our results demonstrate that serum RBP4 level is correlated with the risk and severity of sarcopenia in the older adults, indicating that RBP4 might serve as a surrogate biomarker for the screening and evaluation of sarcopenia.

Retinol-Binding Protein 4 Promotes Cardiac Injury After Myocardial Infarction Via Inducing Cardiomyocyte Pyroptosis Through an Interaction With NLRP3.

Background Acute myocardial infarction (AMI) is one of the leading causes of cardiovascular morbidity and mortality worldwide. Pyroptosis is a form of inflammatory cell death that plays a major role in the development and progression of cardiac injury in AMI. However, the underlying mechanisms for the activation of pyroptosis during AMI are not fully elucidated. Methods and Results Here we show that RBP4 (retinol-binding protein 4), a previous identified proinflammatory adipokine, was increased both in the myocardium of left anterior descending artery ligation-induced AMI mouse model and in ischemia-hypoxia‒induced cardiomyocyte injury model. The upregulated RBP4 may contribute to the activation of cardiomyocyte pyroptosis in AMI because overexpression of RBP4 activated NLRP3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome, promoted the precursor cleavage of Caspase-1, and subsequently induced GSDMD (gasdermin-D)-dependent pyroptosis. In contrast, knockdown of RBP4 alleviated ischemia-hypoxia‒induced activation of NLRP3 inflammasome signaling and pyroptosis in cardiomyocytes. Mechanistically, coimmunoprecipitation assay showed that RBP4 interacted directly with NLRP3 in cardiomyocyte, while genetic knockdown or pharmacological inhibition of NLRP3 attenuated RBP4-induced pyroptosis in cardiomyocytes. Finally, knockdown of RBP4 in heart decreased infarct size and protected against AMI-induced pyroptosis and cardiac dysfunction in mice. Conclusions Taken together, these findings reveal RBP4 as a novel modulator promoting cardiomyocyte pyroptosis via interaction with NLRP3 in AMI. Therefore, targeting cardiac RBP4 might represent a viable strategy for the prevention of cardiac injury in patients with AMI.

Trimetazidine attenuates dexamethasone-induced muscle atrophy via inhibiting NLRP3/GSDMD pathway-mediated pyroptosis.

Skeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can improve skeletal muscle performance both in humans and mice. We here showed that dexamethasone-induced atrophy, as evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression, and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and gasdermin-D (GSDMD). Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine treatment ameliorated dexamethasone-induced muscle pyroptosis and atrophy both in vivo and in vitro. Activation of NLRP3 using LPS and ATP not only increased the cleavage and activation of Caspase-1 and GSDMD, but also increased the expression levels of atrophy markers MuRF1 and Atrogin-1 in trimetazidine-treated C2C12 myotubes. Mechanically, dexamethasone inhibited the phosphorylation of PI3K/AKT/FoxO3a, which could be attenuated by trimetazidine. Conversely, co-treatment with a PI3K/AKT inhibitor, picropodophyllin, remarkably increased the expression of NLRP3 and reversed the protective effects of trimetazidine against dexamethasone-induced C2C12 myotube pyroptosis and atrophy. Taken together, our study suggests that NLRP3/GSDMD-mediated pyroptosis might be a novel mechanism for dexamethasone-induced skeletal muscle atrophy. Trimetazidine might be developed as a potential therapeutic agent for the treatment of dexamethasone-induced muscle atrophy.

Serum retinol-binding protein 4 as a predictor of cardiovascular events in elderly patients with chronic heart failure.

AIMS: RBP4 is an adipokine with adverse effects on cardiovascular system. Increased circulating retinol-binding protein 4 (RBP4) has been linked to chronic heart failure (CHF). However, whether elevated RBP4 is correlated with a poor prognosis in elderly patients with CHF remains unclear. The aim of this study was to evaluate the prognostic value of serum RBP4 in elderly patients with CHF. METHODS AND RESULTS: We enrolled 934 consecutive elderly patients (aged 60 years and older) with CHF and 138 age-matched and sex-matched control subjects in a prospective cohort study and explored the association of serum RBP4 levels with the clinical outcomes using multivariate Cox regression analyses. Serum RBP4 levels were elevated in CHF patients when compared with controls (46.66 ± 12.38 µg/mL vs. 40.71 ± 7.2 µg/mL, P < 0.001). Patients with the highest RBP4 concentrations had higher N terminal pro brain natriuretic peptide (NT-proBNP) levels but lower left ventricular eject fraction (LVEF) and estimated glomerular filtration rate (P < 0.001). Serum RBP4 levels were increased as the New York Heart Association functional class increased and LVEF decreased (P < 0.001) and were negatively correlated with LVEF (r = -0.154, P < 0.001) but positively correlated with NT-proBNP levels (r = 0.074, P = 0.023). Multivariate Cox regression analysis suggested that log RBP4 was an independent predictor for major adverse cardiac event(s) [hazard ratio (HR) = 2.61, 95% confidence interval (CI) = 1.19-5.70], together with age, male, LVEF, log NT-proBNP, and estimated glomerular filtration rate. Moreover, log RBP4 was also an independent predictor for cardiovascular mortality (HR = 2.24, 95% CI = 1.35-5.39) and CHF rehospitalization (HR = 2.54, 95% CI = 1.09-5.60) even after adjustment for the established adverse prognostic factors for CHF. The Kaplan-Meier survival curves showed that high concentration of RBP4 was a prognostic indicator of major adverse cardiac event(s) in patients with CHF. CONCLUSIONS: Our findings demonstrate for the first time that elevated serum RBP4 is correlated with worse outcome in elderly patients with CHF.

Serum retinol-binding protein 4 is associated with the presence and severity of coronary artery disease in patients with subclinical hypothyroidism.

Subclinical hypothyroidism (SCH) plays a crucial role in the development and progression of coronary heart disease (CAD). Retinol-binding protein 4 (RBP4) is an adipokine correlated with cardiovascular diseases. Recent studies found that RBP4 levels are increased in patients with SCH. However, the relationship of RBP4 with CAD in SCH patients remains unclear. A total of 199 SCH patients (148 with CAD and 51 without CAD) and 102 healthy controls were enrolled in this study. Serum RBP4 was increased in SCH patients than controls. Moreover, serum RBP4 was higher in SCH patients with CAD. Although there was no significant difference of metabolic parameters between SCH patients with and without CAD, serum RBP4 was positively correlated with body mass index, total cholesterol, and low-density lipoprotein cholesterol, as well as thyroid stimulating hormone. Multivariable logistic regression analyses revealed elevated RBP4 was correlated with increased risk for CAD in SCH patients. Serum RBP4 levels were also increased as the number of stenosed vessels increased. Furthermore, increased RBP4 was positively correlated with the severity of CAD quantified by the Gensini score. Our findings demonstrate that serum RBP4 is associated with the presence and severity of CAD in patients with SCH.

MiR-191 inhibit angiogenesis after acute ischemic stroke targeting VEZF1.

Acute ischemic stroke (AIS) is a major public health problem in China. Impaired angiogenesis plays crucial roles in the development of ischemic cerebral injury. Recent studies have identified that microRNAs (miRNAs) are important regulators of angiogenesis, but little is known the exact effects of angiogenesis-associated miRNAs in AIS. In the present study, we detected the expression levels of angiogenesis-associated miRNAs in AIS patients, middle cerebral artery occlusion (MCAO) rats, and oxygen-glucose deprivation/reoxygenation (OGD/R) human umbilical vein endothelial cells (HUVECs). MiR-191 was increased in the plasma of AIS patients, OGD/R HUVECs, and the plasma and brain of MCAO rats. Over-expression of miR-191 promoted apoptosis, but reduced the proliferation, migration, tube-forming and spheroid sprouting activity in HUVECs OGD/R model. Mechanically, vascular endothelial zinc finger 1 (VEZF1) was identified as the direct target of miR-191, and could be regulated by miR-191 at post-translational level. In vivo studies applying miR-191 antagomir demonstrated that inhibition of miR-191 reduced infarction volume in MCAO rats. In conclusion, our data reveal a novel role of miR-191 in promoting ischemic brain injury through inhibiting angiogenesis via targeting VEZF1. Therefore, miR-191 may serve as a biomarker or a therapeutic target for AIS.