Antibody-mediated delivery of a viral MHC-I epitope into the cytosol of target tumor cells repurposes virus-specific CD8+ T cells for cancer immunotherapy.

BACKGROUND: Redirecting pre-existing virus-specific cytotoxic CD8+ T lymphocytes (CTLs) to tumors by simulating a viral infection of the tumor cells has great potential for cancer immunotherapy. However, this strategy is limited by lack of amenable method for viral antigen delivery into the cytosol of target tumors. Here, we addressed the limit by developing a CD8+ T cell epitope-delivering antibody, termed a TEDbody, which was engineered to deliver a viral MHC-I epitope peptide into the cytosol of target tumor cells by fusion with a tumor-specific cytosol-penetrating antibody. METHODS: To direct human cytomegalovirus (CMV)-specific CTLs against tumors, we designed a series of TEDbodies carrying various CMV pp65 antigen-derived peptides. CMV-specific CTLs from blood of CMV-seropositive healthy donors were expanded for use in in vitro and in vivo experiments. Comprehensive cellular assays were performed to determine the presentation mechanism of TEDbody-mediated CMV peptide-MHC-I complex (CMV-pMHCI) on the surface of target tumor cells and the recognition and lysis by CMV-specific CTLs. In vivo CMV-pMHCI presentation and antitumor efficacy of TEDbody were evaluated in immunodeficient mice bearing human tumors. RESULTS: TEDbody delivered the fused epitope peptides into target tumor cells to be intracellularly processed and surface displayed in the form of CMV-pMHCI, leading to disguise target tumor cells as virally infected cells for recognition and lysis by CMV-specific CTLs. When systemically injected into tumor-bearing immunodeficient mice, TEDbody efficiently marked tumor cells with CMV-pMHCI to augment the proliferation and cytotoxic property of tumor-infiltrated CMV-specific CTLs, resulting in significant inhibition of the in vivo tumor growth by redirecting adoptively transferred CMV-specific CTLs. Further, combination of TEDbody with anti-OX40 agonistic antibody substantially enhanced the in vivo antitumor activity. CONCLUSION: Our study offers an effective technology for MHC-I antigen cytosolic delivery. TEDbody may thus have utility as a therapeutic cancer vaccine to redirect pre-existing anti-viral CTLs arising from previously exposed viral infections to attack tumors.

Probe-Free Identification of RNA Virus Variants with Point Mutations by Surface-Enhanced Raman Spectroscopy.

As observed in the COVID-19 pandemic, RNA viruses continue to rapidly evolve through mutations. In the absence of effective therapeutics, early detection of new severely pathogenic viruses and quarantine of infected people are critical for reducing the spread of the viral infections. However, conventional detection methods require a substantial amount of time to develop probes specific to new viruses, thereby impeding immediate response to the emergence of viral pathogens. In this study, we identified multiple types of viruses by obtaining the spectral fingerprint of their surface proteins with probe-free surface-enhanced Raman scattering (SERS). In addition, the SERS-based method can remarkably distinguish influenza virus variants with several surface protein point mutations from their parental strain. Principal component analysis (PCA) of the SERS spectra systematically captured the key Raman bands to distinguish the variants. Our results show that the combination of SERS and PCA can be a promising tool for rapid detection of newly emerging mutant viruses without a virus-specific probe.

Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for a Cytomegalovirus pp65-Derived Peptide Presented by HLA-A*02:01.

Human cytomegalovirus (CMV) infection is widespread among adults (60-90%) and is usually undetected in healthy individuals without symptoms but can cause severe diseases in immunocompromised hosts. T-cell receptor (TCR)-like antibodies (Abs), which recognize complex antigens (peptide-MHC complex, pMHC) composed of MHC molecules with embedded short peptides derived from intracellular proteins, including pathogenic viral proteins, can serve as diagnostic and/or therapeutic agents. In this study, we aimed to engineer a TCR-like Ab specific for pMHC comprising a CMV pp65 protein-derived peptide (495NLVPMVATV503; hereafter, CMVpp65495-503) in complex with MHC-I molecule human leukocyte antigen (HLA)-A*02:01 (CMVpp65495-503/HLA-A*02:01) to increase affinity by sequential mutagenesis of complementarity-determining regions using yeast surface display technology. Compared with the parental Ab, the final generated Ab (C1-17) showed ~67-fold enhanced binding affinity (KD ≈ 5.2 nM) for the soluble pMHC, thereby detecting the cell surface-displayed CMVpp65495-503/HLA-A*02:01 complex with high sensitivity and exquisite specificity. Thus, the new high-affinity TCR-like Ab may be used for the detection and treatment of CMV infection.

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling.

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-ß and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Alteration of gammaretroviral vector integration patterns by insertion of histone and leucine zipper into integrase.

Retroviral vectors show long-term gene expression in gene therapy through the integration of transgenes into the human cell genome. Murine leukemia virus (MLV), a well-studied gammaretrovirus, has been often used as a representative retroviral vector. However, frequent integrations of MLV-based vectors into transcriptional start sites (TSSs) could lead to the activation of oncogenes by enhancer effects of the genetic components within the vectors. Therefore, the MLV integration preference for TSSs limits its wider use in clinical applications. To reduce the integration preference of MLV-based vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted histones and leucine zippers, having DNA-binding property, into internal sites of MLV integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild-type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer MLV-based retroviral vectors for use in clinical applications.

Design and synthesis of sulfonamidophenylethylamides as novel cardiac myosin activator.

The sulfonamidophenylethylamide analogues were explored for finding novel and potent cardiac myosin activators. Among them, N-(4-(N,N-dimethylsulfamoyl)phenethyl-N-methyl-5-phenylpentanamide (13, CMA at 10 µM = 48.5%; FS = 26.21%; EF = 15.28%) and its isomer, 4-(4-(N,N-dimethylsulfamoyl)phenyl-N-methyl-N-(3-phenylpropyl)butanamide (27, CMA at 10 µM = 55.0%; FS = 24.69%; EF = 14.08%) proved to be efficient cardiac myosin activators both in in vitro and in vivo studies. Compounds 13 (88.2 + 3.1% at 5 µM) and 27 (46.5 + 2.8% at 5 µM) showed positive inotropic effect in isolated rat ventricular myocytes. The potent compounds 13 and 27 were highly selective for cardiac myosin over skeletal and smooth muscle myosin, and therefore these potent and selective amide derivatives could be considered a new class of cardiac myosin activators for the treatment of systolic heart failure.

Shear stress enhances Ca2+ sparks through Nox2-dependent mitochondrial reactive oxygen species generation in rat ventricular myocytes.

Shear stress enhances diastolic and systolic Ca2+ concentration in ventricular myocytes. Here, using confocal Ca2+ imaging in rat ventricular myocytes, we assessed the effects of shear stress (~16dyn/cm2) on the frequency of spontaneous Ca2+ sparks and explored the mechanism underlying shear-mediated Ca2+ spark regulation. The frequency of Ca2+ sparks was immediately increased by shear stress (by ~80%), and increased further (by ~150%) during prolonged exposure (20s). The 2-D size and duration of individual sparks were increased by shear stimulation. Inhibition of nitric oxide synthase (NOS) only partially attenuated the prolonged shear-mediated enhancement in spark frequency. Pretreatment with antioxidants significantly attenuated the short- and long-term effects of shear on spark frequency. Microtubule or nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) inhibition abolished the immediate shear-induced increase in spark frequency and suppressed the effects of prolonged exposure to shear stress by ~70%. Scavenging of mitochondrial reactive oxygen species (ROS) and mitochondrial uncoupling also abolished the effect of short-term shear on spark occurrence, and markedly reduced (by ~80%) the effects of prolonged shear. Mitochondrial ROS levels increased under shear; this was eliminated by blocking Nox2. Sarcoplasmic reticulum Ca2+ content was increased only by prolonged shear. Our data suggest that shear stress enhances ventricular spark frequency mainly via ROS generated from mitochondria through Nox2, and that NOS and higher sarcoplasmic reticulum Ca2+ concentrations may also contribute to the enhancement of Ca2+ sparks under shear stress. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Ca2+ Signaling Triggered by Shear-Autocrine P2X Receptor Pathway in Rat Atrial Myocytes.

BACKGROUND/AIMS: The atrium is exposed to high shear stress during heart failure and valvular diseases. We aimed to understand atrial shear-induced Ca2+ signaling and its underlying mechanisms. METHODS: Pressurized micro-flow was applied to single rat atrial myocytes, and Ca2+ signal, membrane potential, and ATP release were assessed using confocal imaging, patch clamp technique, and luciferin-luciferase assay, respectively. RESULTS: Shear stress (∼16 dyn/cm2) induced global Ca2+ waves (∼0.1 events/s) from the periphery to the center of cells in a transverse direction ("T-wave"; ∼145 µm/s). Pharmacological interventions and simultaneous recording of membrane potential and Ca2+ demonstrated that shear-induced T-waves resulted from action potential (AP)-triggered Ca2+ release from the sarcoplasmic reticulum. T-waves were not sensitive to inhibitors of known shear signaling mechanisms except connexin hemichannels and ATP release. Shear stress caused ATP release from these myocytes (∼1.1x10-17 moles/unit membrane, µm2); ATP release was increased by enhancement of connexin hemichannels and suppressed by inhibition of the hemichannels, but not affected by inhibitors of other ATP release pathways. Blockade of P2X receptor, but not pannexin or the Na+-Ca2+ exchanger, eliminated shear-induced T-wave initiation. CONCLUSION: Our data suggest that shear stress triggers APs and concomitant Ca2+ signaling via activation of P2X receptors by connexin hemichannel-mediated ATP release in atrial myocytes.

Role of inositol 1,4,5-trisphosphate receptor type 1 in ATP-induced nuclear Ca2+ signal and hypertrophy in atrial myocytes.

Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is expressed in atrial muscle, but not in ventricle, and they are abundant in the perinucleus. We investigated the role of IP3R1 in the regulations of local Ca2+ signal and cell size in HL-1 atrial myocytes under stimulation by IP3-generating chemical messenger, ATP. Assessment of nuclear and cytosolic Ca2+ signal using confocal Ca2+ imaging revealed that IP3 generation by ATP (1 mM) induced monophasic nuclear Ca2+ increase, followed by cytosolic Ca2+ oscillation. Genetic knock-down (KD) of IP3R1 eliminated the monophasic nuclear Ca2+ signal and slowed the cytosolic Ca2+ oscillation upon ATP exposure. Prolonged application of ATP as well as other known hypertrophic agonists (endothelin-1 and phenylephrine) increased cell size in wild-type cells, but not in IP3R1 KD cells. Our data indicate that IP3R1 mediates sustained elevation in nuclear Ca2+ level and facilitates cytosolic Ca2+ oscillation upon external ATP increase, and further suggests possible role of nuclear IP3R1 in atrial hypertrophy.

Regulation of cardiac calcium by mechanotransduction: Role of mitochondria.

Myocardium is subjected to a variety of forces with each contraction, such as stretch, afterload, and shear stress, and adapts to those mechanical stimuli. These mechanical stimuli increase in heart failure, valvular heart disease and hypertension that are clinically associated with arrhythmia and myocyte remodeling. To understand cellular and molecular basis of mechanical stress-mediated cardiac dysfunction and remodeling, several experimental approaches have been successfully used in single cardiac myocytes. In this review, we will briefly summarize the current knowledge about the responses of cardiac myocytes to mechanical stimuli and underlying mechanisms in the context of Ca2+ signaling, with focusing on the role of mitochondria in these mechanotransductions. Recent evidence suggests that mechanotransduction, associated with mitochondrial metabolism, significantly alters Ca2+ signaling and ionic homeostasis in cardiac myocytes under shear stress or prolonged stretch, and that it may play a key role in the pathogenesis of heart failure.

Retrospective Genetic Analysis of 200 Cases of Sudden Infant Death Syndrome and Its Relationship with Long QT Syndrome in Korea.

BACKGROUND: There has been a campaign by the National Education on Sleeping Habits and Living Environment, to reduce the incidence of sudden infant death syndrome (SIDS). However, more than 100 infants die suddenly and unexplainably before the age of 1 year in Korea. Long QT syndrome (LQTS), an inheritable cardiac disease, has been reported to likely be associated with up to 14% of SIDS cases. However, genetic studies of the association between SIDS and LQTS have not yet been conducted in Korea. METHODS: We conducted genetic analysis using genomic DNA extracted from paraffin-embedded tissue blocks from 200 SIDS cases autopsied between 2005 and 2013. We analyzed the following genetic mutations associated with LQTS, KCNQ1, SCN5A, KCNE1, KCNE2, KCNJ2, and CAV3. RESULTS: Of the 200 SIDS cases, 58% involved male infants (116 male and 84 female infants, respectively), the mean age was 140 days (median, 107 days; range, 24-270 days), and they were all of Asian-Korean ethnicity. SIDS IA category criteria comprised 45 cases (22.5%) while the rest were SIDS IB. Fifteen infants (7.5%) had R1193Q in SCN5A, of doubtful pathogenicity, and no pathogenic LQTS variants were observed. CONCLUSION: This genetic investigation of LQTS in SIDS showed a low diagnostic yield. These findings suggest that LQTS molecular autopsy could be cautiously conducted in selected cases with family involvement to improve the available genetic counseling information. Meanwhile, a national SIDS registry should be established to document and evaluate the genetic risk of SIDS in Korea.

Signaling Pathway for Endothelin-1- and Phenylephrine-Induced cAMP Response Element Binding Protein Activation in Rat Ventricular Myocytes: Role of Inositol 1,4,5-Trisphosphate Receptors and CaMKII.

BACKGROUND/AIMS: Endothelin-1 (ET-1) and the α₁-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. METHODS: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. RESULTS: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca2+-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca2+ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca2+ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP₃R) blockers, or type 2 IP₃R (IP₃R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP₃R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca2+ buffering or to Gö6976, the inhibitor of Ca2+-dependent PKC and protein kinase D (PKD). CONCLUSION: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP₃R2/Ca2+-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP₃R2 and Ca2+-independent PKC, in ventricular myocytes.

Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5-trisphosphate receptors and Ca(2+) release.

KEY POINTS: During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress-sensitive membrane current in atrial myocytes using the whole-cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release in subsarcolemmal domains of atrial myocytes. Ca(2+) -mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability. ABSTRACT: Atrial myocytes are subjected to shear stress during the cardiac cycle under physiological or pathological conditions. The ionic currents regulated by shear stress remain poorly understood. We report the characteristics, molecular identity and activation mechanism of the shear stress-sensitive current (Ishear ) in rat atrial myocytes. A shear stress of ∼16 dyn cm(-2) was applied to single myocytes using a pressurized microflow system, and the current was measured by whole-cell patch clamp. In symmetrical CsCl solutions with minimal concentrations of internal EGTA, Ishear showed an outwardly rectifying current-voltage relationship (reversal at -2 mV). The current was conducted primarily (∼80%) by monovalent cations but not Ca(2+) . It was suppressed by intracellular Ca(2+) buffering at a fixed physiological level, inhibitors of transient receptor potential melastatin subfamily 4 (TRPM4), intracellular introduction of TRPM4 antibodies or knockdown of TRPM4 expression, suggesting that TRPM4 carries most of this current. A notable reduction in Ishear occurred upon inhibition of Ca(2+) release through the ryanodine receptors or inositol 1,4,5-trisphosphate receptors (IP3 R) and upon depletion of sarcoplasmic reticulum Ca(2+) . In type 2 IP3 R (IP3 R2) knockout atrial myocytes, Ishear was 10-20% of that in wild-type myocytes. Immunocytochemistry and proximity ligation assays revealed that TRPM4 and IP3 R2 were expressed at peripheral sites with co-localization, although they are not localized within 40 nm. Peripheral localization of TRPM4 was intact in IP3 R2 knockout cells. The data obtained in the present study suggest that shear stress activates TRPM4 current by triggering Ca(2+) release from the IP3 R2 in the peripheral domains of atrial myocytes.

Silica nanoparticles inhibit brown adipocyte differentiation via regulation of p38 phosphorylation.

Nanoparticles are of great interest due to their wide variety of biomedical and bioengineering applications. However, they affect cellular differentiation and/or intracellular signaling when applied and exposed to target organisms or cells. The brown adipocyte is a cell type important in energy homeostasis and thus closely related to obesity. In this study, we assessed the effects of silica nanoparticles (SNPs) on brown adipocyte differentiation. The results clearly showed that brown adipocyte differentiation was significantly repressed by exposure to SNPs. The brown adipocyte-specific genes as well as mitochondrial content were also markedly reduced. Additionally, SNPs led to suppressed p38 phosphorylation during brown adipocyte differentiation. These effects depend on the size of SNPs. Taken together, these results lead us to suggest that SNP has anti-brown adipogenic effect in a size-dependent manner via regulation of p38 phosphorylation.

Vacuolar H+ -ATPase c protects glial cell death induced by sodium nitroprusside under glutathione-depleted condition.

We examined the role of the c subunit (ATP6L) of vacuolar H(+) -ATPase and its molecular mechanisms in glial cell death induced by sodium nitroprusside (SNP). ATP6L siRNA-transfected cells treated with SNP showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, but reduction of ATP6L did not affect the regulation of lysosomal pH in analyses with lysosomal pH-dependent fluorescence probes. Photodegraded SNP and ferrous sulfate induced cytotoxicity with the same pattern as that of SNP, but SNAP and potassium cyanide did not show activity. Pretreatment of the transfected cells with deferoxamine (DFO) reduced ROS production and significantly inhibited the cytotoxicity, which indicates that primarily iron rather than nitric oxide or cyanide from SNP contributes to cell death. Involvement of apoptotic processes in the cells was not shown. Pretreatment with JNK or p38 chemical inhibitor significantly inhibited the cytotoxicity, and we also confirmed that the MAPKs were activated in the cells by immunoblot analysis. Significant increase of LC3-II conversion was observed in the cells, and the conversions were inhibited by cotransfection of the MAPK siRNAs and pretreatment with DFO. Introduction of Atg5 siRNA inhibited the cytotoxicity and inhibited the activation of MAPKs and the conversion of LC3. We finally confirmed autophagic cell death and involvement of MAPKs by observation of autophagic vacuoles via electron microscopy. These data suggest that ATP6L has a protective role against SNP-induced autophagic cell death via inhibition of JNK and p38 in GSH-depleted glial cells.

Effects of potassium on expression of renal sodium transporters in salt-sensitive hypertensive rats induced by uninephrectomy.

Dietary potassium is an important modulator of systemic blood pressure (BP). The purpose of this study was to determine whether dietary potassium is associated with an altered abundance of major renal sodium transporters that may contribute to the modulation of systemic BP. A unilateral nephrectomy (uNx) was performed in male Sprague-Dawley rats, and the rats were fed a normal-salt diet (0.3% NaCl) for 4 wk. Thereafter, the rats were fed a high-salt (HS) diet (3% NaCl) for the entire experimental period. The potassium-repleted (HS+KCl) group was given a mixed solution of 1% KCl as a substitute for drinking water. We examined the changes in the abundance of major renal sodium transporters and the expression of mRNA of With-No-Lysine (WNK) kinases sequentially at 1 and 3 wk. The systolic BP of the HS+KCl group was decreased compared with the HS group (140.3 ± 2.97 vs. 150.9 ± 4.04 mmHg at 1 wk; 180.3 ± 1.76 vs. 207.7 ± 6.21 mmHg at 3 wk). The protein abundances of type 3 Na(+)/H(+) exchanger (NHE3) and Na(+)-Cl(-) cotransporter (NCC) in the HS+KCl group were significantly decreased (53 and 45% of the HS group at 1 wk, respectively; 19 and 8% of HS group at 3 wk). WNK4 mRNA expression was significantly increased in the HS+KCl group (1.4-fold of control at 1 wk and 1.9-fold of control at 3 wk). The downregulation of NHE3 and NCC may contribute to the BP-attenuating effect of dietary potassium associated with increased urinary sodium excretion.

Suppression of L-type Ca2+ current by fluid pressure in rat ventricular myocytes: possible role of Cl(-)-OH(-) exchange.

The application of fluid pressure (FP) in ventricular myocytes using pressurized fluid flow inhibits L-type Ca(2+) current (I(Ca)), with approximately 80% of this effect coming through the enhancement of Ca(2+) releases from the sarcoplasmic reticulum. In the present study, we explored the remaining mechanisms for the inhibition of I(Ca) by FP. Since FP significantly increases H(+) concentration and H(+) is known to inhibit I(Ca), we examined whether pH regulation plays a role in the inhibitory effect by FP on I(Ca). A flow of pressurized (∼16.3 dyne/cm(2)) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes for which the I(Ca) was monitored using whole-cell patch-clamp under HEPES-buffered conditions. Extracellular application of the alkalizing agent, NH(4)Cl (20 mM), enhanced I(Ca) by ∼34% in the control conditions while increasing I(Ca) significantly less (by ∼21%) in FP-pretreated myocytes, suggesting an inhibition of the effect of NH(4)Cl on I(Ca) possibly by FP-induced acidosis. Application of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 500µM), which blocks Cl(-)-HCO(3)(-) exchange but not Cl(-)-OH(-) exchange, did not alter the inhibitory effect of FP on I(Ca). Replacement of external Cl(-) with aspartate attenuated the inhibitory effect of FP on I(Ca). In highly Ca(2+)-buffered cells, where Ca(2+)-dependent inhibition of I(Ca) was minimized, the external Cl(-) removal eliminated the inhibitory effect of FP on I(Ca). These results suggest that the decrease of I(Ca) in the presence of FP is at least partly caused by intracellular acidosis via activation of Cl(-)-OH(-) exchange in rat ventricular myocytes.

Chrysosplenol C increases contraction in rat ventricular myocytes.

Chrysosplenol C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) is a flavone contained in several medicinal plants including Miliusa balansae and Pterocaulon sphacelatum. This compound is known to have an antiviral effect and show cytotoxic activity in several cell lines. In the present study, we explored the effect of chrysosplenol C on contractility in isolated adult rat ventricular myocytes. Chrysosplenol C was isolated from M. balansae, and cell shortenings were measured in field-stimulated single myocytes using a video edge detection method at room temperature. Chrysosplenol C was found to increase cell shortenings in a dose-dependent manner with a half-maximal effective concentration of 45 ± 7.8 µM. Maximal effect of chrysosplenol C, approximately 185% of control, was observed at ≥80 µM. The positive inotropic effect caused by chrysosplenol C was reversible. Time-to-peak contraction and time-to-relengthening were significantly increased by chrysosplenol C. The velocity of cell shortening was slightly accelerated, whereas that of relaxation was not altered by chrysosplenol C. The chrysosplenol C­induced positive inotropic effect was not inhibited by propranolol posttreatment or H-89 pretreatment, suggesting that chrysosplenol C increased contraction independently of ß-adrenergic receptor stimulation and protein kinase A. Our findings are the first to demonstrate that chrysosplenol C is a positive inotropic agent in cardiac myocytes.

Comparison of vascular calcification scoring systems using plain radiographs to predict vascular stiffness in peritoneal dialysis patients.

AIM: Vascular stiffness is associated with cardiovascular mortality in dialysis patients and related with vascular calcification and microvascular inflammation. The objective of this study is to compare predictability of two different vascular calcification scoring systems using plain radiographs in peritoneal dialysis (PD) patients. METHODS: Vascular stiffness was represented by heart-to-femoral pulse wave velocity (hfPWV) in our 79 PD patients. Peripheral vascular calcification score (PVCS) and abdominal aortic calcification score (AACS) were measured from plain radiographs. Microvascular inflammation was represented by peritoneal protein clearance (PPC). Regression analysis and the receiver operating characteristic (ROC) curve analysis were used for analysis. RESULTS: The hfPWV revealed correlation with PVCS and AACS independently. In the ROC curve analysis, area under the curve (AUC) of PVC score was 0.7119 (P = 0.006), and AUC of AACS were 0.6960 (P = 0.011). After multiple linear regression analysis, PVCS remained as a predictor of vascular stiffness (R(2) = 0.579, ß = 0.210, P = 0.038). The combination of PVCS and PPC exhibited a trend toward better predictability for vascular stiffness (AUC 0.7738, P = 0.001) than any of the two parameters alone. CONCLUSION: It is assumed that the PVCS system is more predictable for vascular stiffness in our study. Moreover, the combination of PVCS and PPC might be more useful as a screening test for vascular stiffness.

A decline in renal function is associated with loss of bone mass in Korean postmenopausal women with mild renal dysfunction.

This study was conducted to assess the relationship between estimated glomerular filtration rate (eGFR) and bone mineral density (BMD) in Korean postmenopausal women with mild renal dysfunction. A total of 328 postmenopausal women who underwent BMD measurement during health check-up was investigated. BMD was measured in lumbar spine (L1-L4), femoral neck, total proximal femur and femoral trochanteric areas by dual energy radiography absorptiometry and renal function was estimated by eGFR using Cockcroft-Gault equation. Of the 328 subjects, 317 (96.6%) had an eGFR ≥60 mL/min/1.73 m(2). By using simple linear regression analysis, age, height, weight and eGFR were significantly associated with BMD for the 4 aforementioned anatomic sites, while serum levels of creatinine and blood urea nitrogen did not influence BMD. When multiple regression analyses were applied, age and body weight still had significant associations with BMD at 4 different anatomic sites (P < 0.001). A significant association of eGFR with BMD remained in the lumbar spine, femoral neck and proximal total femur (P < 0.05) but not in the trochanteric area (P = 0.300). Our study suggests that a decline of renal function is associated with lower BMD in the lumbar spine, femoral neck and total proximal femur areas in Korean menopausal women with mild renal dysfunction.