A genome-wide association study identifies SLC8A3 as a susceptibility locus for ACPA-positive rheumatoid arthritis.

OBJECTIVE: RA patients with serum ACPA have a strong and specific genetic background. The objective of the study was to identify new susceptibility genes for ACPA-positive RA using a genome-wide association approach. METHODS: A total of 924 ACPA-positive RA patients with joint damage in hands and/or feet, and 1524 healthy controls were genotyped in 582 591 single-nucleotide polymorphisms (SNPs) in the discovery phase. In the validation phase, the most significant SNPs in the genome-wide association study representing new candidate loci for RA were tested in an independent cohort of 863 ACPA-positive patients with joint damage and 1152 healthy controls. All individuals from the discovery and validation cohorts were Caucasian and of Southern European ancestry. RESULTS: In the discovery phase, 60 loci not previously associated with RA risk showed evidence for association at P < 5×10(-4) and were tested for replication in the validation cohort. A total of 12 loci were replicated at the nominal level (P < 0.05, same direction of effect as in the discovery phase). When combining the discovery and validation cohorts, an intronic SNP in the Solute Carrier family 8 gene (SLC8A3) was found to be associated with ACPA-positive RA at a genome-wide level of significance RA [odds ratio (95% CI): 1.42 (1.25, 1.6), Pcombined = 3.19×10(-8)]. CONCLUSIONS: SLC8A3 was identified as a new risk locus for ACPA-positive RA. This study demonstrates the advantage of analysing relevant subsets of RA patients to identify new genetic risk variants.

Sequence of cellular events in pancreatic islets leading to impaired insulin secretion in chronic kidney disease.

OBJECTIVES: In chronic renal failure (CRF), a multitude of metabolic derangements occur in the pancreatic islets, resulting in impaired glucose-induced insulin secretion. These abnormalities include a rise in the basal level of cytosolic calcium ([Ca(2+)]i) in the islets, a decrease in their basal and stimulated adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content, a reduction in the V(max) of Ca(2+) ATPase and Na(+)-K(+) ATPase, and an impaired glucose-induced calcium signal. The sequence of events that leads to these derangements and to the impairment in insulin secretion during the evolution of CRF has not been defined. This study examined this particular issue by measuring the metabolic profiles of pancreatic islets weekly during the evolution of CRF over a period of 6 weeks. RESULTS: The results showed that serum levels of parathyroid hormone (PTH) begin to rise during the first week of CRF. The V(max) of Ca(2+) ATPase and Na(+)-K(+) ATPase increased during weeks 1 to 3 of CRF but decreased to low levels thereafter. At week 3 of CRF, the basal level of [Ca(2+)]i began to rise, whereas basal and stimulated ATP and ADP content started to fall. Glucose-induced calcium signal, Δ[Ca(2+)]i, and insulin secretion became abnormally low between weeks 3 and 6 of CRF. CONCLUSION: The data obtained allow for the inference of the following formulation: as serum levels of PTH begin to rise, calcium entry into islets is augmented, which in turn will stimulate the activity of Ca(2+) ATPase and the Na(+)-Ca(2+) exchanger, and therefore, calcium extrusion out of the islets is increased. Thus, [Ca(2+)]i remains normal during the first 2 weeks of CRF. Activation of the Na(+)-Ca(2+) exchanger may result in accumulation of sodium in the islets, an event that would activate the Na(+)-K(+) ATPase. Because calcium entry is further augmented by the progressive rise in serum PTH levels, mitochondrial oxidation and ATP production would be reduced, resulting in lower ATP content. This fall in ATP causes a reduction in the V(max) of Ca(2+) ATPase and Na(+)-K(+) ATPase, and therefore calcium extrusion out of the islets is reduced; consequently, [Ca(2+)]i rises. With the decrease in ATP content and the rise in [Ca(2+)]i, glucose-induced insulin secretion is impaired because of alterations in the closure of ATP-dependent potassium channels and reduction in the glucose-induced calcium signal (Δ[Ca(2+)])i.

Alterations in calcium regulatory protein expression in patients with preserved left ventricle systolic function and mitral valve stenosis.

BACKGROUND: In heart failure, alterations in the expression of proteins relevant to calcium homeostasis are involved in depressed contractility and diminished relaxation. However the regulation of genes expression is only partially known. The aim was to assess expression of calcium regulatory proteins in left ventricle (LV) myocardium characterised by a preserved global function in mitral valve stenosis (MVS) model but increased neurohumoral/cytokine (N/C) activation. METHODS AND RESULTS: Plasma N/C activation was evaluated in MVS-patients (n = 27), where expression of calcium regulatory proteins (L-type channel, sarcoplasmic reticulum Ca2+-ATPase type2 - SERCA2, Na+/Ca2+ exchanger -NCX, calsequestrin, phospholamban) in LV myocardium was assessed (Western Blot) in comparison with non-failing hearts (NFH). Out of all proteins assessed in MVS, only SERCA2 and NCX expression revealed highly variable changes between subjects, with significant reduction of SERCA2 (15%) level compared to NFH. Moreover, SERCA2 was negatively correlated with BNP (univariate/regression analysis r = -0.63, P = 0.005/r2 = 0.74, P <0.001, respectively), whereas NCX was positively correlated only with noradrenaline (univariate/stepwise analysis r = 0.59 P = 0.002/r2 = 0.59; P = 0.003). CONCLUSIONS: In MVS-patients LV becomes remodelled, although its global function is preserved. It seems that apart from alterations in LV load and wall stress, also such neurohumoral factors as BNP/noradrenaline may influence the Ca2+ handling proteins expression.

The cardiac sodium-calcium exchanger gene (NCX-1) is a potential canine cardiac biomarker of chronic mitral valvular insufficiency.

BACKGROUND: The cardiac sodium-calcium exchanger gene (NCX-1) is upregulated in humans and mice with congestive heart failure (CHF). HYPOTHESIS: NCX-1 expression is upregulated in dogs with heart failure from chronic mitral valvular insufficiency (CMVI). ANIMALS: Client-owned 14 healthy control dogs and 30 dogs with CMVI. METHODS: Prospective, controlled, observational study. We investigated the levels of NCX-1 expression in dogs at different stages of CMVI with real-time reverse transcription polymerase chain reaction. RESULTS: The mRNA expression levels of NCX-1 were determined in peripheral blood samples obtained from the animals used in this study. Dogs were graded by the severity of disease. The fold differences in the levels of mRNA expression compared with controls were 1.39 +/- 0.88 (group I), 1.32 +/- 0.65 (group II), 4.86 +/- 1.25 (group III), and 5.96 +/- 1.69 (group IV). NCX-1 expression was significantly higher in groups III and IV (P < .05) compared with the healthy controls, whereas groups I and II were not. CONCLUSIONS AND CLINICAL IMPORTANCE: The level of NCX-1 expression was significantly higher in groups of dogs with moderate to severe CMVI (groups III and IV) compared with the controls. Our findings indicate that NCX-1 can be a biomarker for chronic valvular disease in dogs and is a potential biomarker for severity of heart disease.

Altered platelet calsequestrin abundance, Na⁺/Ca²âº exchange and Ca²âº signaling responses with the progression of diabetes mellitus.

INTRODUCTION: Downregulation of calsequestrin (CSQ), a major Ca(2+) storage protein, may contribute significantly to the hyperactivity of internal Ca(2+) ([Ca(2+)]i) in diabetic platelets. Here, we investigated changes in CSQ-1 abundance, Ca(2+) signaling and aggregation responses to stimulation with the progression of diabetes, especially the mechanism(s) underlying the exaggerated Ca(2+) influx in diabetic platelets. MATERIALS AND METHODS: Type 1 diabetes was induced by streptozotocin in rats. Platelet [Ca(2+)]i and aggregation responses upon ADP stimulation were assessed by fluorescence spectrophotometry and aggregometry, respectively. CSQ-1 expression was evaluated using western blotting. RESULTS: During the 12-week course of diabetes, the abundance of CSQ-1, basal [Ca(2+)]i and ADP-induced Ca(2+) release were progressively altered in diabetic platelets, while the elevated Ca(2+) influx and platelet aggregation were not correlated with diabetes development. 2-Aminoethoxydiphenyl borate, the store-operated Ca(2+) channel blocker, almost completely abolished ADP-induced Ca(2+) influx in normal and diabetic platelets, whereas nifedipine, an inhibitor of the nicotinic acid adenine dinucleotide phosphate receptor, showed no effect. Additionally, inhibition of Na(+)/Ca(2+) exchange induced much slower Ca(2+) extrusion and more Ca(2+) influx in normal platelets than in diabetic platelets. Furthermore, under the condition of Ca(2+)-ATPase inhibition, ionomycin caused greater Ca(2+) mobilization and Ca(2+) influx in diabetic platelets than in normal platelets. CONCLUSIONS: These data demonstrate that platelet hyperactivity in diabetes is caused by several integrated factors. Besides the downregulation of CSQ-1 that mainly disrupts basal Ca(2+) homeostasis, insufficient Na(+)/Ca(2+) exchange also contributes, at least in part, to the hyperactive Ca(2+) response to stimulation in diabetic platelets.

Evidence for mechanistic alterations of Ca2+ homeostasis in Type 2 diabetes mellitus.

Altered cytosolic Ca2+ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca2+ regulation. Using human lymphocytes, we studied cytosolic calcium (Cai) and various Ca2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca2+-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca2+ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), was used to study Ca2+-store dependent Ca2+ fluxes. Significant (P<0.05) elevation of basal Ca, levels was observed in lymphocytes from diabetic subjects. Cai levels were positively correlated with fasting plasma glucose and HbA1c. There was also a significant (P<0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca2+ influx (as measured both by Fluo-3 fluorescence and 45Ca assays) as a consequence of thapsigargin-mediated Ca2+ store depletion. Upon addition of Mn2+ (a surrogate of Ca2+), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P<0.05) difference in the Na+/Ca2+ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca2+ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca2+ entry. We conclude that cellular Ca2+ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na+/Ca2+ exchange and (3) increased Ca2+ influx across the plasma membrane.

Platelet hyperactivity and abnormal Ca(2+) homeostasis in diabetes mellitus.

We sought to determine the mechanisms for hyperactivity and abnormal platelet Ca(2+) homeostasis in diabetes. The glycosylated Hb (HbA(1c)) level was used as an index of glycemic control. Human platelets were loaded with Ca- green-fura red, and cytosolic Ca(2+) ([Ca(2+)](i)) and aggregation were simultaneously measured. In the first series of experiments, the platelets from diabetic and normal subjects were compared for the ability to release Ca(2+) or to promote Ca(2+) influx. A potent and relatively specific inhibitor of Na(+)/Ca(2+) exchange, 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), increased the second phase of thrombin-induced Ca(2+) response, suggesting that the Na(+)/Ca(2+) exchanger works in the forward mode to mediate Ca(2+) efflux. In contrast, in the platelets from diabetics, CB-DMB decreased the Ca(2+) response, indicating that the Na(+)/Ca(2+) exchanger works in the reverse mode to mediate Ca(2+) influx. In the second series of experiments we evaluated the direct effect of hyperglycemia on platelets in vitro. We found that thrombin- and collagen-induced increases in [Ca(2+)](i) and aggregation were not acutely affected by high glucose concentrations of 45 mM. However, when the platelet-rich plasma was incubated with a high glucose concentration at 37 degrees C for 24 h, the second phase after thrombin activation was inhibited by CB-DMB. In addition, collagen-stimulated [Ca(2+)](i) response and aggregation were also increased. Thus in diabetes the direction and activity of the Na(+)/Ca(2+) exchanger is changed, which may be one of the mechanisms for the increased platelet [Ca(2+)](i) and hyperactivity. Prolonged hyperglycemia in vitro can induce similar changes, suggesting hyperglycemia per se may be the factor responsible for the platelet hyperactivity in diabetes.