SHP2-mediated signaling cascade through gp130 is essential for LIF-dependent I CaL, [Ca2+]i transient, and APD increase in cardiomyocytes

Leukemia inhibitory factor (LIF), a cardiac hypertrophic cytokine, increases L-type Ca(2+) current (I(CaL)) via ERK-dependent and PKA-independent phosphorylation of serine 1829 in the Cav(1.2) subunit. The signaling cascade through gp130 is involved in this augmentation. However, there are two major cascades downstream of gp130, i.e. JAK/STAT3 and SHP2/ERK. In this study, we attempted to clarify which of these two cascades plays a more important role. Knock-in mouse line, in which the SHP2 signal was disrupted (gp130(F759/F759) group), and wild-type mice (WT group) were used. A whole-cell patch clamp experiment was performed, and intracellular Ca(2+) concentration ([Ca(2+)](i) transient) was monitored. The I(CaL) density and [Ca(2+)](i) transient were measured from the untreated cells and the cells treated with LIF or IL-6 and soluble IL-6 receptor (IL-6+sIL-6r). Action potential duration (APD) was also recorded from the ventricle of each mouse, with or without LIF. Both LIF and IL-6+sIL-6r increased I(CaL) density significantly in WT (+27.0%, n=16 p<0.05, and +32.2%, n=15, p<0.05, respectively), but not in gp130(F759/F759) (+9.4%, n=16, NS, and -6.1%, n=13, NS, respectively). Administration of LIF and IL-6+sIL-6r increased [Ca(2+)](i) transient significantly in WT (+18.8%, n=13, p<0.05, and +32.0%, n=21, p<0.05, respectively), but not in gp130(F759/F759) (-3.8%, n=7, NS, and -6.4%, n=10, NS, respectively). LIF prolonged APD(80) significantly in WT (10.5+/-4.3%, n=12, p<0.05), but not in gp130(F759/F759) (-2.1+/-11.2%, n=7, NS). SHP2-mediated signaling cascade is essential for the LIF and IL-6+sIL-6r-dependent increase in I(CaL), [Ca(2+)](i) transient and APD.     

Ca2 + Sparks and Ca2 + waves are the subcellular events underlying Ca2 + overload during ischemia and reperfusion in perfused intact hearts

Abnormal intracellular Ca²⁺ cycling plays a key role in cardiac dysfunction, particularly during the setting of ischemia/reperfusion (I/R). During ischemia, there is an increase in cytosolic and sarcoplasmic reticulum (SR) Ca²⁺. At the onset of reperfusion, there is a transient and abrupt increase in cytosolic Ca^2+ +, which occurs timely associated with reperfusion arrhythmias. However, little is known about the subcellular dynamics of Ca²⁺ increase during I/R, and a possible role of the SR as a mechanism underlying this increase has been previously overlooked. The aim of the present work is to test two main hypotheses:  (1) An increase diastolic Ca²⁺ sparks frequency (cspf) constitutes a mayor substrate for the ischemia-induced diastolic Ca²⁺ increase; (2) an increase in cytosolic Ca²⁺ pro-arrhythmogenic events (Ca²⁺ waves), mediates the abrupt diastolic Ca²⁺ rise at the onset of reperfusion. We used confocal microscopy on mouse intact hearts loaded with Fluo-4. Hearts were submitted to global I/R (12/30 min) to assess epicardial Ca²⁺ sparks in the whole heart. Intact heart sparks were faster than in isolated myocytes whereas cspf was not different. During ischemia, cspf significantly increased relative to preischemia (2.07 ± 0.33 vs. 1.13 ± 0.20 sp/s/100 μm, n = 29/34, 7 hearts). Reperfusion significantly changed Ca²⁺ sparks kinetics, by prolonging Ca²⁺ sparks rise time and decreased cspf. However, it significantly increased Ca²⁺ wave frequency relative to ischemia (0.71 ± 0.14 vs. 0.38 ± 0.06 w/s/100 μm, n = 32/33, 7 hearts). The results show for the first time the assessment of intact perfused heart Ca^2 + sparks and provides direct evidence of increased Ca²⁺ sparks in ischemia that transform into Ca²⁺ waves during reperfusion. These waves may constitute a main trigger for reperfusion arrhythmias.     

Calcium release near l-type calcium channels promotes beat-to-beat variability in ventricular myocytes from the chronic AV block dog

Beat-to-beat variability of ventricular repolarization (BVR) has been proposed as a strong predictor of Torsades de Pointes (TdP). BVR is also observed at the myocyte level, and a number of studies have shown the importance of calcium handling in influencing this parameter. The chronic AV block (CAVB) dog is a model of TdP arrhythmia in cardiac hypertrophy, and myocytes from these animals show extensive remodeling, including of Ca^2 + handling. This remodeling process also leads to increased BVR. We aimed to determine the role that (local) Ca^2 + handling plays in BVR.In isolated LV myocytes an exponential relationship was observed between BVR magnitude and action potential duration (APD) at baseline. Inhibition of Ca^2 + release from sarcoplasmic reticulum (SR) with thapsigargin resulted in a reduction of [Ca^2 +]_i, and of both BVR and APD. Increasing I_CaL in the presence of thapsigargin restored APD but BVR remained low. In contrast, increasing I_CaL with preserved Ca^2 + release increased both APD and BVR. Inhibition of Ca^2 + release with caffeine, as with thapsigargin, reduced BVR despite maintained APD. Simultaneous inhibition of Na⁺/Ca^2 + exchange and I_CaL decreased APD and BVR to similar degrees, whilst increasing diastolic Ca^2 +. Buffering of Ca^2 + transients with BAPTA reduced BVR for a given APD to a greater extent than buffering with EGTA, suggesting subsarcolemmal Ca^2 + transients modulated BVR to a larger extent than the cytosolic Ca^2 + transient.In conclusion, BVR in hypertrophied dog myocytes, at any APD, is strongly dependent on SR Ca^2 + release, which may act through modulation of the l-type Ca^2 + current in a subsarcolemmal microdomain.     

Glycoprotein 130 polymorphism predicts soluble glycoprotein 130 levels

ObjectiveInterleukin-6 (IL-6) is a key cytokine in inflammatory diseases. It exerts its biological function via binding to a homodimer of its signal transducer glycoprotein 130 (gp130). Soluble gp130 (sgp130) is the natural inhibitor of IL-6 trans-signalling. The aim of this study was to test a possible influence of the gp130 genotype on sgp130 serum levels.Material and methodsIn two separate populations, subjects were genotyped for the gp130 polymorphism G148C. Sgp130, IL-6 and soluble interleukin-6 receptor (sIL-6R) levels were measured. The OSLO population consisted of 546 male subjects at high risk for CAD. The VIENNA population consisted of 299 male subjects with angiographically proven CAD.ResultsIn the OSLO population, 124 (22.7%) subjects were hetero- or homozygote for the rare C allele. Individuals carrying the polymorphism had significantly higher levels of sgp130. In a multivariate linear regression model this association remained significant (adjusted p = 0.001). In the VIENNA population, 48 (16.1%) subjects were hetero- or homozygote for the rare C allele. Consistent with the former study, sgp130 levels were significantly higher in carriers of the polymorphism compared to wildtype carriers (adjusted p = 0.038). In the VIENNA population, sgp130 levels were significantly higher in diabetic patients. In the OSLO population, sgp130 was higher in patients with increased body mass index and in smokers (p < 0.05).ConclusionsSgp130 serum levels are significantly higher in subjects carrying the gp130 polymorphism G148C compared to wildtype carriers. This finding proposes a possible genetical influence on sgp130 levels which may alter individual coping mechanisms in inflammatory diseases.     

Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2

AimsIn cardiac muscle, Ca^2 + release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca^2 + release refractoriness. We recently reported that the SR luminal Ca^2 + binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refractoriness.Methods and resultsGene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca^2 + release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca^2 +-dependent inactivation of L-type Ca^2 + current (I_Ca), which is responsible for triggering SR Ca^2 + release, significantly contributed to loss of Ca^2 + release refractoriness in Casq2 KO myocytes. Recovery from Ca^2 +-dependent inactivation of I_Ca was significantly accelerated in Casq2 KO compared to wild-type (WT) myocytes. In contrast, voltage-dependent inactivation measured by using Ba^2 + as charge carrier was not significantly different between WT and Casq2 KO myocytes. Ca^2 +-dependent inactivation of I_Ca was normalized by intracellular dialysis of excess apo-CaM (20 μM), which also partially restored physiological Ca^2 + release refractoriness in Casq2 KO myocytes.ConclusionsOur findings reveal that the intra-SR protein Casq2 is largely responsible for the phenomenon of SR Ca^2 + release refractoriness in murine ventricular myocytes. We also report a novel mechanism of impaired Ca^2 +-CaM-dependent inactivation of Ca_v1.2, which contributes to the loss of SR Ca^2 + release refractoriness in the Casq2 KO mouse model and, therefore, may further increase risk for ventricular arrhythmia in vivo.     

Toll-like receptor 4 activation promotes cardiac arrhythmias by decreasing the transient outward potassium current (Ito) through an IRF3-dependent and MyD88-independent pathway

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca^2 + transients, as well as the L-type Ca^2 + current (I_CaL) and the transient outward K⁺ current (I_to), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, I_to amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The I_to decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca^2 + transients and I_CaL were not affected by LPS; however, Na⁺/Ca^2 + exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased I_to, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca^2 + efflux via NCX, could explain the presence of arrhythmias in the LPS group.     

QTLs influencing IGF-1 levels in a LOU/CxFischer 344 F2 rat population. Tracks towards the metabolic theory of Ageing

ObjectiveSince a reduction of the insulin/IGF-1 signaling cascade extends life span in many species and IGF-1 signaling might partly mediate the effects of caloric restriction (CR), an experimental intervention for increasing longevity, the purpose of the present study was to use quantitative trait loci (QTL) analysis, an unbiased genetic approach, to identify particular regions of the genome influencing plasma IGF-1 levels in an F2 intercross between F344 and LOU/C rats; the latter being an inbred strain of Wistar origin, considered as a model of healthy aging since it resists to age (and diet)-induced obesity.DesignF1 hybrids were obtained by crossbreeding LOU/C with F344 rats, and then F1 were bred inter se to obtain the F2 population, of which 93 males and 94 females were studied. Total plasma IGF-1 levels were determined by radioimmunoassay. A genome scan of the F2 population was made with 100 microsatellite markers) selected for their polymorphism between LOU/C and F344 strains (and by covering evenly the whole genome.ResultsBy simple interval mapping sex-dependent QTLs were found on chromosome 17 in males and on chromosome 18 in females. By multiple interval mapping, additional QTLs were found on chromosomes 1, 4, 5, 6, 12, 15 and 19 in males and on chromosomes 3, 5, 6, 12 and 17 in females. Only the markers D1Rat196 and D12Mgh5 were found in both males and females. The majority of QTLs corresponded to metabolic syndrome (cardiac function: n = 45 (30%), obesity/diabetes: n = 22 (15%), inflammation: n = 19 (13%) and only a limited number to body weight: n = 13 (9%), proliferation (n = 10 (7%) or ossification: n = 7 (5%). Ninety-six candidate genes were located on the different QTLs. A significant proportion of these genes are connected to IGF-1 production and receptor pathways (n = 18) or metabolic syndrome (n = 11).ConclusionsSubsequent studies are necessary to determine whether the genetic networks underscored are also involved in age-associated obesity, diabetes and inflammation as well as cardiovascular impairments.     

Relationship between renal capacity to reabsorb glucose and renal status in patients with diabetes

AimsInterindividual variability in capacity to reabsorb glucose at the proximal renal tubule could contribute to risk of diabetic kidney disease. Our present study investigated, in patients with diabetes, the association between fractional reabsorption of glucose (FR_GLU) and degree of renal disease as assessed by urinary albumin excretion (UAE) and estimated glomerular filtration rate (eGFR).MethodsFR_GLU [1-(glucose clearance/creatinine clearance)] was assessed in 637 diabetes patients attending our tertiary referral centre, looking for correlations between FR_GLU and UAE (normo-, micro-, macro-albuminuria) and Kidney Disease: Improving Global Outcomes (KDIGO) eGFR categories: >90 (G1); 90–60 (G2); 59–30 (G3); and < 30–16 (G4) mL/min/1.73 m². Patients were stratified by admission fasting plasma glucose (FPG) into three groups: low (<6 mmol/L); intermediate (6–11 mmol/L); and high (>11 mmol/L).ResultsMedian (interquartile range, IQR) FR_GLU levels were blood glucose-dependent: 99.90% (0.05) for low (n = 106); 99.90% (0.41) for intermediate (n = 288); and 96.36% (12.57) for high (n = 243) blood glucose categories (P < 0.0001). Also, FR_GLU increased with renal disease severity in patients in the high FPG group: normoalbuminuria, 93.50% (17.74) (n = 135); microalbuminuria, 96.56% (5.94) (n = 77); macroalbuminuria, 99.12% (5.44) (n = 31; P < 0.001); eGFR G1, 94.13% (16.24) (n = 111); G2, 96.35% (11.94) (n = 72); G3 98.88% (7.59) (n = 46); and G4, 99.11% (2.20) (n = 14; P < 0.01). On multiple regression analyses, FR_GLU remained significantly and independently associated with UAE and eGFR in patients in the high blood glucose group.ConclusionHigh glucose reabsorption capacity in renal proximal tubules is associated with high UAE and low eGFR in patients with diabetes and blood glucose levels > 11 mmol/L.     

One-year metabolic outcomes in patients with type 2 diabetes treated with exenatide in routine practice

AimThe study objective was to analyze, in everyday practice, the long-term metabolic effects of exenatide (for 9 and 12 months) in patients with type 2 diabetes not responding to treatments with metformin and sulphonylurea at maximum dosages.MethodsA total of 299 type 2 diabetics were recruited from 14 centres specializing in diabetes care across Belgium. Main study endpoints were changes in HbA_1c, weight and waist circumference, and tolerability and compliance. Two patient cohorts were analyzed for effectiveness, with data available at 9 (n = 90) and 12 (n = 94) months of follow-up.ResultsSignificant decreases in HbA_1c of −1.3% and −1.6% were observed in the 9- and 12-month cohorts, respectively (P < 0.001). The decrease in HbA_1c was greater in patients with higher baseline levels (P < 0.001), and the response was independent of baseline weight, body mass index (BMI), age, gender and diabetes duration. A progressive reduction of weight (4.9 kg) was also observed in the two cohorts at 9 and 12 months (P < 0.001), with greater weight loss in patients with higher baseline BMI (P = 0.046) and in female subjects (P = 0.025). Waist circumference also decreased from baseline to endpoints. A correlation was observed between reduction in HbA_1c and weight loss (P = 0.019). Side effects, mainly of gastrointestinal origin, were reported in 33% (93/284 patients in the safety cohort). The rate of hypoglycaemia was 3.5%. Treatment was discontinued in 27% of patients (n = 77) mainly due to drug inefficacy (53%, n = 41) or adverse events (26%, n = 20), or both (8%, n = 6).ConclusionExenatide leads to long-term improvement of glycaemic control as well as weight loss in a majority of patients not responding to combined oral drug therapy in real-world clinical practice. However, no baseline factors predictive of response could be identified. Exenatide can be considered an effective treatment option in such patients, including those with high baseline HbA_1c and long duration of diabetes.     

Effect of isoprenaline chronic stimulation on APD restitution and ventricular arrhythmogenesis

BackgroundIsoprenaline (ISO) acts through β-adrenergic receptors to increase the intracellular Ca²⁺, which has effects on action potential duration (APD) restitution and arrhythmogenesis. Thus, we investigated the effect of chronic stimulation with isoprenaline on APD restitution and ventricular tachyarrhythmias (VA) in the rabbit heart.Methods and resultsRabbits were randomly selected to receive an injection of isoprenaline (ISO group) or an equal volume of 0.9% saline (CTL group). The S₁–S₂ protocol (n = 15) and S₁ dynamic pacing (n = 15) were performed to construct APD restitution and to induce APD alternans or arrhythmia in 10 sites of Langendorff-perfused hearts. Compared with the same sites in the control group, long-term ISO administration (7 days) shortened the APD₉₀ and the effective refractory period (ERP), and greatly increased the spatial dispersion of APD and ERP (p < 0.01). Compared to CTL group, the APD restitution curves were significantly changed (p < 0.01) and showed increased spacial dispersion of maximal slope (S_max) among each site in the ISO group (p < 0.05). In induction of VA and APD alternans, the threshold of VA and alternans was both decreased in each site of the ISO group.ConclusionChronic stimulation with ISO facilitated VA, possibly through the increased spatial dispersion of APD restitution.     

Efficacy and Safety of Oral Conivaptan,a Vasopressin-Receptor Antagonist,Evaluated in a Randomized,Controlled Trial in Patients With Euvolemic or Hypervolemic Hyponatremia

BackgroundIn most cases of hyponatremia, arginine vasopressin secretion is inappropriately high. This placebo-controlled, randomized, double-blind multicenter study evaluated the efficacy and safety of oral conivaptan, a V_1A/V₂-receptor antagonist, in patients with euvolemic or hypervolemic hyponatremia.MethodsEighty-three patients with serum [Na⁺] less than 130 mEq/L were stratified by volume status and randomly assigned to placebo or conivaptan 40 or 80 mg/d for 5 days.ResultsConivaptan increased the baseline-adjusted area under the serum [Na⁺]-time curve significantly more than placebo (P = 0.0001). Patients given either dose of conivaptan demonstrated a serum [Na⁺] of 4 mEq/L or greater above baseline significantly faster than those given placebo (P < 0.001) and maintained that increase for a greater total time (P = 0.0001). The least squares mean change in serum [Na⁺] from baseline to end of treatment was also significantly greater with conivaptan 40 and 80 mg/d (6.8 and 8.8 mEq/L, respectively) (P = 0.0001) than that with placebo (1.2 mEq/L). The percentage of patients who obtained an increase from baseline in serum [Na⁺] of 6 mEq/L or greater or normal serum [Na⁺] was significantly higher among patients given conivaptan 40 and 80 mg/d (67% and 88%, respectively) than among those given placebo (20%; P < 0.001). Conivaptan was well tolerated; the most frequent adverse events were urinary tract infection, anemia, pyrexia, cardiac failure, hypotension, and hypokalemia.ConclusionOral conivaptan was effective in increasing serum [Na⁺] in patients with euvolemic or hypervolemic hyponatremia and had a favorable safety profile.     

Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP,contractility, and relaxation

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca²⁺-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca²⁺ fluorescence were monitored by video microscopy. At 0.5 Hz stimulation, the extent of shortening was increased ~ 40% and maximal rate of shortening was increased ~ 80% in cardiomyocytes overexpressing Rrm1 + Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~ 150% with Rrm1 + Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1 + Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca²⁺], indicating increased contractility is primarily due to increased myofilament activity without altering Ca²⁺ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1 + Rrm2 overexpression as stimulation frequency was increased (1 Hz and 2 Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~ 1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca²⁺ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin–myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca²⁺. This article is part of a Special issue entitled "Possible Editorial".     

Aldosterone stimulates the cardiac sodium/bicarbonate cotransporter via activation of the g protein-coupled receptor gpr30

Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO₃⁻/CO₂ solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJ_HCO3⁻; control: 100 ± 5.82%, n = 7 vs Ald: 151.88 ± 11.02%, n = 5; P < 0.05), which was prevented by G15 (GPR30 blocker, 90.53 ± 7.81%, n = 7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13 ± 18.28%, n = 6; P < 0.05) and this effect was abrogated by G15 (124.19 ± 10.96%, n = 5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.     

Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and disease activity in ulcerative colitis

Background and aimsPotassium channels, K_V1.3 and K_Ca3.1, have been suggested to control T-cell activation, proliferation, and cytokine production and may thus constitute targets for anti-inflammatory therapy. Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by excessive T-cell infiltration and cytokine production. It is unknown if K_V1.3 and K_Ca3.1 in the inflamed mucosa are markers of active UC. We hypothesized that K_V1.3 and K_Ca3.1 correlate with disease activity and cytokine production in patients with UC.MethodsMucosal biopsies were collected from patients with active UC (n = 33) and controls (n = 15). Protein and mRNA expression of K_V1.3 and K_Ca3.1, immune cell markers, and pro-inflammatory cytokines were determined by quantitative-real-time-polymerase-chain-reaction (qPCR) and immunofluorescence, and correlated with clinical parameters of inflammation. In-vitro cytokine production was measured in human CD3⁺ T-cells after pharmacological blockade of K_V1.3 and K_Ca3.1.ResultsActive UC K_V1.3 mRNA expression was increased 5-fold compared to controls. Immunofluorescence analyses revealed that K_V1.3 protein was present in inflamed mucosa in 57% of CD4⁺ and 23% of CD8⁺ T-cells. K_V1.3 was virtually absent on infiltrating macrophages. K_V1.3 mRNA expression correlated significantly with mRNA expression of pro-inflammatory cytokines TNF-α (R² = 0.61) and IL-17A (R² = 0.51), the mayo endoscopic subscore (R² = 0.13), and histological inflammation (R² = 0.23). In-vitro blockade of T-cell K_V1.3 and K_Ca3.1 decreased production of IFN-γ, TNF-α, and IL-17A.ConclusionsHigh levels of K_V1.3 in CD4 and CD8 positive T-cells infiltrates are associated with production of pro-inflammatory IL-17A and TNF-α in active UC. K_V1.3 may serve as a marker of disease activity and pharmacological blockade might constitute a novel immunosuppressive strategy.     

A novel phosphorylation site,Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca^2 +] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca^2 +-sensitivity (pCa₅₀) at 2.2 μm was significantly higher in 199D (pCa₅₀ = 5.79 ± 0.01) compared to 199A (pCa₅₀ = 5.65 ± 0.01) and Wt (pCa₅₀ = 5.66 ± 0.02) at ~ 63% cTn exchange. Myofilament Ca^2 +-sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~ 6%) increase myofilament Ca^2 +-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI.     

Circulating level of microparticles and their correlation with arterial elasticity and endothelium-dependent dilation in patients with type 2 diabetes mellitus

ObjectiveThis study was designed to measure the circulating level of microparticles (MP) in patients with type 2 diabetes mellitus versus healthy volunteers and to evaluate their correlation with arterial elasticity and endothelium-dependent dilation.MethodsFlow cytometry was used to measure the circulating levels of MP, including annexinV+MP, platelet-derived microparticles (PMP), leukocyte-derived microparticles (LMP) and endothelial microparticles (EMP), in 63 patients with type 2 diabetes mellitus and 29 healthy volunteers. Brachial ankle pulse wave velocity (baPWV) and endothelium-dependent flow-mediated dilation (FMD) of the brachial artery were also measured.ResultsThe levels of annexinV+MP, PMP, LMP, CD31+/CD42?EMP and CD51+EMP increased significantly in diabetic patients compared with healthy controls (P < 0.001). Correlation analysis showed that HbA_1c was positively correlated to CD31+/CD42?EMP (r_s = 0.337, P = 0.008) and CD51+EMP (r_s = 0.266, P = 0.038). FMD in diabetic patients was significantly lower than that in healthy individuals (P = 0.007). FMD was negatively correlated to CD31+/CD42?EMP (r_s = ?0.441, P = 0.008) and CD51+EMP (r_s = ?0.405, P = 0.016). baPWV level in diabetic patients was significantly higher than that in healthy individuals (P < 0.001). baPWV was positively correlated to CD31+/CD42?EMP (r_s = 0.497, P < 0.001) and CD51+EMP (r_s = 0.428, P = 0.001). Multiple regression analysis indicated that EMP was an independent risk factor of FMD and baPWV.ConclusionsThe circulating level of microparticles increases in patients with type 2 diabetes. The level of endothelial microparticles is closely associated with vascular dysfunction.     

Prediction of peak oxygen pulse (O2Ppeak) without exercise testing in older adults

Peak oxygen pulse has been considered a surrogate of cardiovascular function and an independent predictor of all cause mortality. However, O₂P_peak depends on maximal volitional effort which may limit its utility in older subjects. The aim of this study was to develop a model to estimate O₂P_peak without exercise in an elderly sample. This cross-sectional study enrolled 67 community-dwelling older adults (69.4 ± 7.1 years; 41 men) for the non-exercise model development and 30 community-dwelling older adults (67.7 ± 6.4 years; n = 30; 17 men) for cross-validation. The non-exercise model was derived through hierarchical regression model and cross-validated by means of PRESS statistics and comparison against an independent sample. Classification accuracy of the model for tertiles of estimated and actual O₂P_peak was tested by gamma (γ) nonparametric correlation. The following prediction equation was generated: −3.416 + 0.137 × weight (kg) + 1.226 × Veterans Specific Activity Questionnaire (VSAQ) (metabolic equivalents, METs) + 1.987 × gender (0 = women, 1 = men) − 2.045 × β-Blockers use (0 = no, 1 = yes) − 0.044 × resting heart rate (HR) (R² = 0.83; standard error of estimate (SEE) = 1.68 mL beat⁻¹). Correlation in cross-validation group was 0.80 (P < 0.001). A high probability was observed for the model to rank the values in the same tertile in validation and cross-validation groups (γ = 0.98; γ = 0.92, respectively, P < 0.05). In conclusion, O₂P_peak can be estimated with reasonable precision without exercise testing, providing an alternative for elder subjects not capable to perform maximal effort.     

Slow Ca2 + sparks de-synchronize Ca2 + release in failing cardiomyocytes: Evidence for altered configuration of Ca2 + release units?

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca^2 + transient which contributes to the impaired contractility observed in this condition. We investigated whether alterations in ryanodine receptor function promote slowing of Ca^2 + release in a murine model of congestive heart failure (CHF). Myocardial infarction was induced by left coronary artery ligation. When chronic CHF had developed (10 weeks post-infarction), cardiomyocytes were isolated from viable regions of the septum. Septal myocytes from SHAM-operated mice served as controls. Ca^2 + transients rose markedly slower in CHF than SHAM myocytes with longer time to peak (CHF = 152 ± 12% of SHAM, P < 0.05). The rise time of Ca^2 + sparks was also increased in CHF (SHAM = 9.6 ± 0.6 ms, CHF = 13.2 ± 0.7 ms, P < 0.05), due to a sub-population of sparks (≈ 20%) with markedly slowed kinetics. Regions of the cell associated with these slow spontaneous sparks also exhibited slowed Ca^2 + release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca^2 + release across the cell. Dyssynchronous Ca^2 + transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms, but slow sparks were not associated with orphaned ryanodine receptors. Rather, mathematical modeling suggested that slow sparks could result from an altered composition of Ca^2 + release units, including a reduction in ryanodine receptor density and/or distribution of ryanodine receptors into sub-clusters. In conclusion, our findings indicate that slowed, dyssynchronous Ca^2 + transients in CHF result from alterations in Ca^2 + sparks, consistent with rearrangement of ryanodine receptors within Ca^2 + release units. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.     

The immediate effect of HCM causing actin mutants E99K and A230V on actin–Tm–myosin interaction in thin-filament reconstituted myocardium

Human cardiac actin mutants E99K and A230V were expressed with baculovirus/insect cells and used to reconstitute the thin-filament of bovine cardiac (BVC) muscle fibers, together with tropomyosin (Tm) and troponin (Tn) purified from bovine ventricles. Effects of [Ca^2 +], [ATP], and [phosphate] on tension and its transients were studied at 25 °C. In the absence of Tm/Tn, both mutants significantly decreased the tension of actin filament reconstituted fibers (WT: 0.75 ± 0.06 T₀, E99K: 0.58 ± 0.04 T₀, A230V: 0.58 ± 0.03 T₀), where T₀ is active tension of native fibers (T₀ = 26.9 ± 1.1 kPa, N = 41), indicating diminished actin–myosin interactions. However, in the presence of Tm and Tn, WT, E99K, and A230V recovered tension (0.85 ± 0.06 T₀, 0.89 ± 0.06 T₀, and 0.85 ± 0.05 T₀, respectively), demonstrating the compensatory effect of Tm/Tn. Ca^2 + sensitivity (pCa₅₀) increased (5.59 ± 0.02, 5.80 ± 0.03, 5.77 ± 0.03, respectively) and cooperativity (nH) decreased (2.6 ± 0.3, 1.87 ± 0.21, 1.60 ± 0.11, respectively). The kinetic constants of the cross-bridge cycle were deduced using sinusoidal analysis. E99K did not show any significant changes in any of the kinetic constants compared to those of WT. A230V caused a decrease in K₁ (ATP association constant), k₂ and k_− 2 (rate constants of the cross-bridge detachment step). The cross-bridge distribution was similar among WT, E99K, and A230V. In conclusion, our experiments demonstrate that the first step of HCM pathogenesis with E99K is increased pCa₅₀ and decreased n_H, which result in larger tension during partial activation to cause a diastolic problem. The effect on n_H is more severe with A230V. In addition, A230V has a problem of decreased cross-bridge kinetics, which affects the normal functions of the cross-bridge cycle and may contribute to the first step of the HCM pathogenesis.