Meloxicam-loaded nanocapsules as an alternative to improve memory decline in an Alzheimer’s disease model in mice: involvement of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase

The objective of this study was to investigate the effect of meloxicam-loaded nanocapsules (M-NC) on the treatment of the memory impairment induced by amyloid β-peptide (aβ) in mice. The involvement of Na⁺, K⁺-ATPase and cyclooxygenase-2 (COX-2) activities in the hippocampus and cerebral cortex was also evaluated. Mice received aβ (3 nmol/ 3 μl/ per site, intracerebroventricular) or vehicle (3 μl/ per site, i.c.v.). The next day, the animals were treated with blank nanocapsules (17 mL/kg) or M-NC (5 mg/kg) or free meloxicam (M-F) (5 mg/kg). Treatments were performed every other day, until the twelfth day. Animals were submitted to the behavioral tasks (open-field, object recognition, Y-maze and step-down inhibitory avoidance tasks) from the twelfth day. Na⁺, K⁺-ATPase and COX-2 activities were performed in hippocampus and cerebral cortex. aβ caused a memory deficit, an inhibition of the hippocampal Na⁺, K⁺-ATPase activity and an increase in the hippocampal COX-2 activity. M-NC were effective against all behavioral and biochemical alterations, while M-F restored only the COX-2 activity. In conclusion, M-NC were able to reverse the memory impairment induced by aβ, and Na⁺, K⁺-ATPase is involved in the effect of M-NC.     

Synaptic Plasma Membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity is Significantly Reduced by the α-Keto Acids Accumulating in Maple Syrup Urine Disease in Rat Cerebral Cortex

The objective of the present study was to investigate the in vitro effects of the branched-chain α-keto acids accumulating in maple syrup urine disease, namely L-2-ketoisocaproic acid, L-2-keto-3-methylvaleric acid and L-2-ketoisovaleric acid on Na⁺, K⁺-ATPase activity in synaptic plasma membranes from cerebral cortex of 35-day-old rats. All keto acids significantly inhibited Na⁺, K⁺-ATPase activity at concentrations similar (1 mM) or even lower (0.5 mM) than those found in blood and cerebrospinal fluid of maple syrup urine disease patients. We also tested the effects of alanine on this enzyme activity. Alanine per se did not alter Na⁺, K⁺-ATPase activity, but totally prevented the branched-chain α-keto acids-induced Na⁺, K⁺-ATPase inhibition, indicating that alanine and the keto acids may possibly bind to the same site on the enzyme. We also observed that the branched-chain amino acids leucine, isoleucine and valine also inhibited Na⁺ K⁺-ATPase activity to a similar degree as that of the branched-chain α-keto acids and that alanine was able to fully prevent these effects. Considering that Na⁺, K⁺-ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings may be involved in the pathophysiology of the neurological dysfunction of maple syrup urine disease.     

HERG K<Superscript>+</Superscript> channel expression in CD34<Superscript>+</Superscript>/CD38<Superscript>−</Superscript>/CD123<Superscript>high</Superscript> cells and primary leukemia cells and analysis of its regulation in leukemia cells

The human ether-a-go-go-related (herg) gene encoding K⁺ channels (HERG) belongs to an evolutionarily conserved multigene family of voltage activated K⁺ channels. The functional properties of HERG K⁺ channels are complex and their contribution to the repolarization of the cardiac action potential are well understood. Recent studies revealed that HERG K⁺ channels are preferentially expressed in different histogenesis of tumor cells. Leukemia is a cancer that originates in the bone marrow hematopoietic stem cells (HSCs). Leukemia stem cells (LSCs) are critical in the perpetuation of the disease. A better understanding of LSCs and molecular biology will allow the design of more effective therapies. We report in this study that herg was expressed in CD34⁺/CD38⁻/CD123^high LSCs but not expressed in normal bone marrow CD34⁺/CD38⁻ HSCs. In addtion, herg is also expressed in leukemia cell lines K562 and HL-60 and almost all the primary leukemia cells whereas not in the normal bone marrow cells. In addition, the expression of herg mRNA was not associated with the clinical and cytogenetic feature of leukemia. Moreover, HERG K⁺ channels can regulate leukemia cells proliferation and cell cycle. These data provide evidence for the oncogenic potential of HERG K⁺ channels and it may be a novel, potential pharmacological target for leukemia therapy in the future.     

Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

Dietary supplementation with N-acetyl cysteine, α-tocopherol and α-lipoic acid reduces the extent of oxidative stress and proinflammatory state in aged rat brain

The present study has attempted to understand how oxidative stress contributes to the development of proinflammatory state in the brain during aging. Three groups of rats have been used in this study: young (4–6?months, Group I), aged (22–24?months, Group II) and aged with dietary antioxidant supplementation (Group III). The antioxidants were given daily from 18?months onwards in the form of a combination of N-acetyl cysteine (50?mg/100?g body weight), α-lipoic acid (3?mg/100?g body weight), and α-tocopherol (1.5?mg/100?g body weight) till the animals were used for the experiments between 22 and 24?months. Several measurements have been made to evaluate the ROS (reactive oxygen species) production rate, the levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and the activation status of NF-κβ (p65 subunit) in brain of the three groups of rats under the study. Our results reveal that brain aging is accompanied with a significant increase in NADPH oxidase activity and mitochondrial ROS production, a distinct elevation of IL-1β, IL-6 and TNF-α levels along with increased nuclear translocation of NF-κβ (p65 subunit) and all these phenomena are partially but significantly prevented by the long-term dietary antioxidant treatment. The results imply that chronic dietary antioxidants by preventing oxidative stress and proinflammatory state may produce beneficial effects against multiple age-related deficits of the brain.     

Enhanced activity of the myocardial Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger contributes to left ventricular hypertrophy in the Goto–Kakizaki rat model of type 2 diabetes: critical role of Akt

Aims/hypothesis Diabetes mellitus is a strong risk factor for the development of heart failure, and left ventricular (LV) hypertrophy has been detected in a significant proportion of diabetic patients. Because several studies have suggested that the Na⁺/H⁺ exchanger (NHE1) plays a part in the molecular mechanisms involved in cardiac hypertrophy, we investigated its activity and its role in LV myocytes from the Goto–Kakizaki (GK) rat model of type 2 diabetes. Materials and methods Fluorometric measurements were used to assess sarcolemmal NHE1 activity in isolated myocytes. NHE1 levels and the possible molecular pathways driving and/or related to NHE1 activity were investigated in relation to the diabetic LV phenotype. Results Enhanced NHE1 activity was associated with LV myocyte hypertrophy. This occurred in the absence of any change in NHE1 protein levels; however, activation of several molecular pathways related to NHE1 activity was demonstrated. Thus, phosphorylation of the extracellular signal-regulated protein kinase (Erk), of the protein kinase Akt (also known as protein kinase B) and of the Ca²⁺/calmodulin-dependent kinase II was increased in GK LV myocytes. Intracellular Ca²⁺ levels were also increased. Chronic treatment (10–12 weeks) with the NHE1 inhibitor cariporide normalised NHE1 activity, decreased levels and reduced LV myocyte hypertrophy. Moreover, among the various activated pathways, cariporide treatment markedly reduced Akt activity only. Conclusions/interpretation These findings indicate that activation of the Akt pathway represents a likely mechanism mediating the hypertrophic effect of increased NHE1 activity in the GK model of type 2 diabetes.     

Molecular characterization of a novel 27.6-kb deletion causing α<Superscript>+</Superscript> thalassemia in a Chinese family

Over 80% of the α-thalassemia cases in southern China are caused by large deletions involving the α-globin gene cluster on chromosome 16p13.3. Here, we characterized a novel 27.6-kb deletion on the α-globin gene cluster in a Chinese family. Its breakpoints were detected to lie between coordinates 9079 and 36718 of the α-globin gene cluster (NG_000006.1), with a total of 27,640 nucleotides deleted. It was designated as −α ^27.6 deletion. The proband is a compound heterozygote of −−^SEA and −α ^27.6 and he displayed very mild hemoglobin H disease phenotype with Hb 7.9–9.3 g/dl. Phenotypic analysis on heterozygote of this deletion revealed it as α⁺ mutation. It leads to a very mild phenotype as adult heterozygotes have normal hematological parameters with the values at the lower border of the normal range. RT-PCR analysis showed that the α-globin mRNA level of the heterozygotes was decreased when compared with that of normal people.     

Diastolic dysfunction and arrhythmias caused by overexpression of CaMKIIδ<Subscript>C</Subscript> can be reversed by inhibition of late Na<Superscript>+</Superscript> current

Transgenic (TG) Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) δ_C mice develop systolic heart failure (HF). CaMKII regulates intracellular Ca²⁺ handling proteins as well as sarcolemmal Na⁺ channels. We hypothesized that CaMKII also contributes to diastolic dysfunction and arrhythmias via augmentation of the late Na⁺ current (late I _Na) in early HF (8-week-old TG mice). Echocardiography revealed severe diastolic dysfunction in addition to decreased systolic ejection fraction. Premature arrhythmogenic contractions (PACs) in isolated isometrically twitching papillary muscles only occurred in TG preparations (5 vs. 0, P < 0.05) which could be completely terminated when treated with the late I _Na inhibitor ranolazine (Ran, 5 μmol/L). Force–frequency relationships revealed significantly reduced twitch force amplitudes in TG papillary muscles. Most importantly, diastolic tension increased with raising frequencies to a greater extent in TG papillary muscles compared to WT specimen (at 10 Hz: 3.7 ± 0.4 vs. 2.5 ± 0.3 mN/mm²; P < 0.05). Addition of Ran improved diastolic dysfunction to 2.1 ± 0.2 mN/mm² (at 10 Hz; P < 0.05) without negative inotropic effects. Mechanistically, the late I _Na was markedly elevated in myocytes isolated from TG mice and could be completely reversed by Ran. In conclusion, our results show for the first time that TG CaMKIIδ_C overexpression induces diastolic dysfunction and arrhythmogenic triggers possibly via an enhanced late I _Na. Inhibition of elevated late I _Na had beneficial effects on arrhythmias as well as diastolic function in papillary muscles from CaMKIIδ_C TG mice. Thus, late I _Na inhibition appears to be a promising option for diastolic dysfunction and arrhythmias in HF where CaMKII is found to be increased.     

HbE Level and Red Cell Parameters in Heterozygous HbE With and Without α<Superscript>0</Superscript>-Thalassemia Trait

We compared hemoglobin (Hb) E levels and red cell parameters between heterozygous HbE with and without α⁰-thalassemia trait and also determine their appropriated cut-off points for differentiating these two groups. High performance liquid chromatography analysis results and mean levels of red blood cell (RBC) parameters, including RBC count, total Hb, hematocrit, MCV, MCH and MCHC of heterozygous HbE with α⁰-thalassemia trait (n?=?183) and without α⁰-thalassemia trait (n?=?1437) were reviewed and compared. The α⁰-thalassemia status in these samples was detected by real-time PCR with SYBR Green1 and high resolution melting analysis. Mean levels of HbE, total Hb, MCV, MCH and MCHC of heterozygous HbE with α⁰-thalassemia trait were significantly lower than those of heterozygous HbE without α⁰-thalassemia trait (P?<?0.001). In addition, HbE level at a cut-off value of?<?24% was superior to MCV (<?80 fL) and MCH (<?27 pg) for differentiating the heterozygous HbE with and without α⁰-thalassemia trait with 100% sensitivity and 87.2% specificity. Despite certain limitations of this study like missing RDW and reticulocyte counts, and not testing for α⁺-thalassemia and Hb Constant Spring, we conclude that the HbE level at a cut-off point of?<?24% is a useful marker for initial discrimination between heterozygous HbE with and without α⁰-thalassemia trait.     

Adsorptive Depletion of α4 Integrin<Superscript>hi</Superscript>- and CX<Subscript>3</Subscript>CR1<Superscript>hi</Superscript>-Expressing Proinflammatory Monocytes in Patients with Ulcerative Colitis

Background  

Two main functionally distinct monocytes phenotypes are known: the CD14^hiCD16⁻ “classical” and the CD14⁺CD16⁺ “proinflammatory” phenotypes. The latter phenotype is elevated in patients with ulcerative colitis (UC) and is suspected to have a major role in the immunopathogenesis of UC.     

Compound heterozygosity for Hb Korle-Bu (β<Superscript>73</Superscript>; Asp-Asn) and Hb E (β<Superscript>26</Superscript>; Glu-Lys) with a 3.7-kb deletional α-thalassemia in Thai patients

Hemoglobin (Hb) Korle-Bu (beta73; Asp-Asn) is the most frequent of the rare beta-chain variants in the population of West Africa whereas Hb E (beta26; Glu-Lys) is common among the Southeast Asian population. We report a hitherto undescribed condition in which these two beta-chain variants co-segregate. The proband was a 19-year-old Thai pregnant woman in her second trimester of pregnancy who visited our thalassemia screening unit. Cellulose acetate electrophoresis and high-performance liquid chromatography (HPLC) analysis of Hb detected one abnormal Hb in addition to the Hb E. Analysis of DNA sequences revealed a GAT-AAT mutation at codon 73 in trans to a GAG-AAG mutation at codon 26 of the beta-globin gene. Polymerase chain reaction (PCR) analysis of the alpha-globin gene cluster of the patient detected a 3.7-kb deletional alpha-thalassemia 2. Family study identified that her mother had the same genotype and her father was a simple Hb E carrier. The hematological data of these unusual cases of hemoglobinopathy are presented and compared with a simple heterozygote for Hb Korle-Bu found in another unrelated Thai family. beta-Globin gene haplotype linked to the Thai beta(Korle-Bu) and a simple DNA assay based on allele-specific PCR for rapid diagnosis of Hb Korle-Bu are also described.     

Multipeptide-coupled nanoparticles induce tolerance in ‘humanised’ HLA-transgenic mice and inhibit diabetogenic CD8<Superscript>+</Superscript> T cell responses in type 1 diabetes

Aims/hypothesis

Induction of antigen-specific immunological tolerance may provide an attractive immunotherapy in the NOD mouse model but the conditions that lead to the successful translation to human type 1 diabetes are limited. In this study, we covalently linked 500 nm carboxylated polystyrene beads (PSB) with a mixture of immunodominant HLA-A*02:01-restricted epitopes (peptides-PSB) that may have high clinical relevance in humans as they promote immune tolerance; we then investigated the effect of the nanoparticle–peptide complexes on T cell tolerance.

Methods

PSB-coupled mixtures of HLA-A*02:01-restricted epitopes were administered to HHD II mice via intravenous injection. The effects on delaying the course of the disease were verified in NOD.β2m ^null HHD mice. The diabetogenic HLA-A*02:01-restricted cytotoxic lymphocyte (CTL) responses to treatment with peptides-PSB were validated in individuals with type 1 diabetes.

Results

We showed that peptides-PSB could induce antigen-specific tolerance in HHD II mice. The protective immunological mechanisms were mediated through the function of CD4⁺CD25⁺ regulatory T cells, suppressive T cell activation and T cell anergy. Furthermore, the peptides-PSB induced an activation and accumulation of regulatory T cells and CD11c⁺ dendritic cells through a rapid production of CD169⁺ macrophage-derived C-C motif chemokine 22 (CCL22). Peptides-PSB also prevented diabetes in ‘humanised’ NOD.β2m ^null HHD mice and suppressed pathogenic CTL responses in people with type 1 diabetes.

Conclusions/interpretation

Our findings demonstrate for the first time the potential for using multipeptide-PSB complexes to induce T cell tolerance and halt the autoimmune process. These findings represent a promising platform for an antigen-specific tolerance strategy in type 1 diabetes and highlight a mechanism through which metallophilic macrophages mediate the early cell–cell interactions required for peptides-PSB-induced immune tolerance.

     

Effect of dietary β carotene on cerebral aneurysm and subarachnoid haemorrhage in the brain apo E<Superscript>−/−</Superscript> mice

Atherosclerosis will lead to stenosis/occlusion in the lumen of various arteries of living body. This can lead various conditions including myocardial infarction, cerebral infarction/aneurysm and peripheral artery disease. Ang II is believed to be an important regulatory peptide involved in maintaining cardiovascular homeostasis and pathogenesis of various cardiovascular diseases. Matrix metalloproteinase’s (MMPs), adhesion molecules and plasminogen systems are involved in the inflammatory reaction of various blood vessels as well as pathogenesis of cerebro vasuclar disease in apo E^−/− mice during angiotensin II injection. The present study analyses the role of ang II in development of cerebral aneurysm and also evaluated the mRNA levels of MMPs, adhesion molecules, plasminogen systems and peroxisome proliferators-associated receptors in the brain of apo E^−/− mouse during the progression of cerebral aneurysm and ischemic conditions. Also, this study evaluates the role of dietary β carotene on cerebrovascular disease. Serum total cholesterol (TC), Low density lipoprotein (LDL) and triglyceride (TG) levels were significantly increased in angiotensin II treated animals and further β carotene supplementation reduces TC but does not affect the triglyceride and LDL levels. Circulating levels of macrophages were significantly increased in angiotensin treated animals and further beta carotene supplementation significantly reduced the circulating macrophages. Cerebro meningeous aneurysm, subarachnoid haemorrhage, multiple foci of infarction, necrosis and infiltration of inflammatory cells were observed in the cerebral hemispheres of ang II treated animals, however, infarction size were reduced and no aneurysm, inflammatory foci was observed in β carotene treated animals. Real time analysis showed down regulation of mRNA levels of MMP 2, uPA, PAI, PPAR-A, MCSF1 and up regulation of tPA and MCP-1 in the brain during the progression of cerebral aneurysm and β carotene supplementation to bring to normal expression levels of all the candidate genes for cerebrovascular diseases. Based on above results, Ang II may induced cerebral aneurysm, ischemia/infarction on brain through RAS system by down regulating the mRNA levels of MMP 2, uPA, PAI, PPAR-A, MCSF1 and up regulating tPA and MCP-1 and β carotene attenuates the disease condition and bring down to normal expression levels of above genes.     

Regulation of p70<Superscript>S6k</Superscript>, GSK-3β, and calcineurin in rat striated muscle during aging

In this study we compared the content and phosphorylation levels of several molecules believed to regulate muscle hypertrophy and fiber type changes in the extensor digitorum longus (EDL), soleus, diaphragm, and heart of adult (6 months), aged (30 months), and very aged (36 months) Fischer 344 × Brown Norway rats. With aging, the mass of the EDL and soleus decreased significantly (~38% and ~36%, respectively), the diaphragm’s mass remained unchanged while the mass of the heart increased (~35%). Western blotting demonstrated that calcineurin (CnA), the 70-kDa ribosomal S6 kinase (p70^S6k), glycogen synthase kinase-3β (GSK-3β), and the phosphorylated forms of GSK-3β and p70^S6k (p-GSK-3β^Ser9 and p-p70^S6kThr389) were regulated differently with aging and between muscle types. Total p70^S6k, GSK-3β, and p-GSK-3β^Ser9 decreased in the aged-atrophic EDL and soleus while p-p70^S6kThr389 increased. Although total p70^S6k content diminished in the continuously active diaphragm, phosphorylation of p70^S6k remained unchanged. Conversely, the expression of GSK-3β and p-GSK-3β^Ser9 increased in the diaphragm. With aging, the amount of p-p70^S6kThr389 decreased ~56% in the heart while p-GSK-3β ^Ser9 increased ~193%. Interestingly, CnA content remained unchanged in the diaphragm, increased ~204% in the EDL, and decreased ~30% and ~65% with aging in the soleus and heart, respectively. These results indicate remarkable differences in the regulation of molecules thought to govern protein synthesis and changes in contractile protein expression.     

Phagocytosis of co-developing neutrophil progenitors by dendritic cells in a culture of human CD34<Superscript>+</Superscript> cells with granulocyte colony-stimulating factor and tumor necrosis factor-α

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the differentiation of CD34(+) cells toward dendritic cells (DCs). We have previously shown that DCs are co-generated from human CD34(+) cells during erythroid or megakaryocytic differentiation in the presence of TNF-alpha, and those DCs are able to stimulate autologous T cell proliferation. The aim of this study was to learn whether the co-stimulation of granulocyte colony-stimulating factor (G-CSF) and TNF-alpha would generate neutrophil progenitors and DCs together from human CD34(+) cells, and if this was the case, to clarify the phenotypic and functional characteristics of these DCs. When highly purified human CD34(+) cells were cultured for 7 days with G-CSF alone, the generated cells predominantly expressed a granulocyte marker, CD15, and then differentiated into neutrophils after 14 days of culture. The addition of TNF-alpha with G-CSF markedly decreased the number of CD15(+) cells without affecting the total number of cells during 7 days of culture. Almost one third of the generated cells were positive for CD11c and CD123. Furthermore, CD11c(+) cells were found to phagocytose CD15(+) cells and were able to induce allogeneic, but not autologous, T cell proliferation in the mixed lymphocyte reaction (MLR). On the other hand, the CD11c(+) cells generated by TNF-alpha and cytokines capable of inducing erythroid differentiation were able to stimulate autologous T cells. There was a difference in the expression of CD80, CD83 and CD86 among CD11c(+) cells induced by G-CSF plus TNF-alpha and those generated by interleukin-3, stem cell factor, and erythropoietin plus TNF-alpha. These results indicate that the co-stimulation of human CD34(+) cells with G-CSF and TNF-alpha induces the phagocytosis of co-developing neutrophil progenitors by DCs, and the stimulatory effects of these DCs on autologous T cells is different from that of DCs generated from CD34(+) cells during erythroid differentiation.     

Why Can <Emphasis Type="Italic">dl</Emphasis>-Sotalol Prolong the QT Interval In Vivo Despite Its Weak Inhibitory Effect on hERG K<Superscript>+</Superscript> Channels In Vitro? Electrophysiological and Pharmacokinetic Analysis with the Halothane-Anesthetized Guinea Pig Model

In order to bridge the gap of action of dl-sotalol between the human ether-a-go-go-related gene (hERG) K⁺ channel inhibition in vitro and QT-interval prolongation in vivo, its electropharmacological effect and pharmacokinetic property were simultaneously studied in comparison with those of 10 drugs having potential to prolong the QT interval (positive drugs: bepridil, haloperidol, dl-sotalol, terfenadine, thioridazine, moxifloxacin, pimozide, sparfloxacin, diphenhydramine, imipramine and ketoconazole) and four drugs lacking such property (negative drugs: enalapril, phenytoin, propranolol or verapamil) with the halothane-anesthetized guinea pig model. A dose of each drug that caused 10 % prolongation of Fridericia-corrected QT interval (QTcF) was calculated, which was compared with respective known hERG K⁺ IC₅₀ value and currently obtained heart/plasma concentration ratio. Each positive drug prolonged the QTcF in a dose-related manner, whereas such effect was not observed by the negative drugs. Drugs with more potent hERG K⁺ channel inhibition showed higher heart/plasma concentration ratio, resulting in more potent QTcF prolongation in vivo. The potency of dl-sotalol for QTcF prolongation was flat middle, although its hERG K⁺ channel inhibitory property as well as heart/plasma concentration ratio was the smallest among the positive drugs, which may be partly explained by its lack of binding to plasma protein.     

K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca<Superscript>2+</Superscript> release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes

K201 has previously been shown to reduce diastolic contractions in vivo during β-adrenergic stimulation and elevated extracellular calcium concentration ([Ca²⁺]_o). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca²⁺ release and contractile events in isolated rat cardiomyocytes during β-adrenergic stimulation and elevated [Ca²⁺]_o. Parallel experiments using confocal microscopy examined spontaneous diastolic Ca²⁺ release events at an enhanced spatiotemporal resolution. 1.0 μmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca²⁺]_o significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca²⁺ transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca²⁺ release. Myofilament sensitivity to Ca²⁺ was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca²⁺ waves (2–3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 μmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 μmol/L K201 can reduce the probability of spontaneous diastolic Ca²⁺ release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo.