Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

Roles of L-type Ca2+ and delayed-rectifier K+ currents in sinoatrial node pacemaking: insights from stability and bifurcation analyses of a mathematical model

To elucidate the dynamical mechanisms of the sinoatrial (SA) node pacemaker activity, we investigated the roles of L-type Ca2+ (ICa,L) and delayed-rectifier K+ (IKr) currents in pacemaking by stability and bifurcation analyses of our rabbit SA node model (Kurata Y, Hisatome I, Imanishi S, and Shibamoto T. Am J Physiol Heart Circ Physiol 283: H2074-H2101, 2002). Equilibrium points (EPs), periodic orbits, stability of EPs, and Hopf bifurcation points were calculated as functions of conductance or gating time constants of the currents for constructing bifurcation diagrams. Structural stability (robustness) of the system was also evaluated by computing stability and dynamics during applications of constant bias currents (Ibias). Blocking ICa,L or IKr caused stabilization of an EP and cessation of pacemaking via a Hopf bifurcation. The unstable zero-current potential region determined with Ibias applications, where spontaneous oscillations appear, shrunk and finally disappeared as ICa,L diminished, but shrunk little when IKr was eliminated. The reduced system, including no time-dependent current except ICa,L, exhibited pacemaker activity. These results suggest that ICa,L is responsible for EP instability and pacemaker generation, whereas IKr is not necessarily required for constructing a pacemaker cell system. We further explored the effects of various K+ currents with different kinetics on stability and dynamics of the model cell. The original IKr of delayed activation and inward rectification appeared to be most favorable for generating large-amplitude oscillations with stable frequency, suggesting that IKr acts as an oscillation amplifier and frequency stabilizer. IKr may also play an important role in preventing bifurcation to quiescence of the system.     

Coordinate induction of AMP deaminase in human atrium with mitochondrial DNA deletion

Despite the heteroplasmic lower population of mitochondrial (mt) DNA deletion, mtDNA deletion is significantly related to the loss of atrial adenine nucleotides. To elucidate its mechanism, we examined the frequency of a 7.4-kb mtDNA deletion, the concentration of adenine nucleotides, and the activity of AMP catabolic enzymes in 10 human right atria obtained from cardiac surgery, using quantitative PCR, HPLC, and immunoprecipitations. The atrial concentrations of ATP, ADP, AMP, and the total adenine nucleotides were significantly lower in patients with deletion than those in patients without deletion, despite the lower frequency of their deletion. The activities of total AMP deaminase (AMPD), liver-type (AMPD 2), and heart-type isoform (AMPD 3) were significantly higher in patients with deletion than in patients without deletion, although there was no significant difference in the cytosolic 5(')-nucleotidase among them. In conclusion, mtDNA deletion coordinately induces AMP deaminase to contribute to the loss of atrial adenine nucleotides through degrading AMP excessively.     

Impairment of ubiquitin-proteasome system by E334K cMyBPC modifies channel proteins, leading to electrophysiological dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K_v1.5, Na_v1.5, Hcn4, Ca_v3.2, Ca_v1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca²⁺ transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca²⁺ transients were recapitulated by a simulation model. Although a Ca²⁺ antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca²⁺ transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.     

High Uric Acid Induces Insulin Resistance in Cardiomyocytes In Vitro and In Vivo

Clinical studies have shown hyperuricemia strongly associated with insulin resistance as well as cardiovascular disease. Direct evidence of how high uric acid (HUA) affects insulin resistance in cardiomyocytes, but the pathological mechanism of HUA associated with cardiovascular disease remains to be clarified. We aimed to examine the effect of HUA on insulin sensitivity in cardiomyocytes and on insulin resistance in hyperuricemic mouse model. We exposed primary cardiomyocytes and a rat cardiomyocyte cell line, H9c2 cardiomyocytes, to HUA, then quantified glucose uptake with a fluorescent glucose analog, 2-NBDG, after insulin challenge and detected reactive oxygen species (ROS) production. Western blot analysis was used to examine the levels of insulin receptor (IR), phosphorylated insulin receptor substrate 1 (IRS1, Ser307) and phospho-Akt (Ser473). We monitored the impact of HUA on insulin resistance, insulin signaling and IR, phospho-IRS1 (Ser307) and phospho-Akt levels in myocardial tissue of an acute hyperuricemia mouse model established by potassium oxonate treatment. HUA inhibited insulin-induced glucose uptake in H9c2 and primary cardiomyocytes. It increased ROS production; pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, reversed HUA-inhibited glucose uptake induced by insulin. HUA exposure directly increased the phospho-IRS1 (Ser307) response to insulin and inhibited that of phospho-Akt in H9C2 cardiomyocytes, which was blocked by NAC. Furthermore, the acute hyperuricemic mice model showed impaired glucose tolerance and insulin tolerance accompanied by increased phospho-IRS1 (Ser307) and inhibited phospho-Akt response to insulin in myocardial tissues. HUA inhibited insulin signaling and induced insulin resistance in cardiomyocytes in vitro and in vivo, which is a novel potential mechanism of hyperuricemic-related cardiovascular disease.     

Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.     

A TSH/dibutyryl cAMP activated Cl-/I- channel in FRTL-5 cells.

An iodide (I) and chloride (Cl) channel has been identified in the continuously cultured FRTL-5 thyroid cell line using a cell attached patch clamp technique. The channel is activated by TSH and dibutyryladenosine cyclic monophosphate (Bt2-cAMP) but not by phorbol 12-myristate 13-acetate (TPA). Gluconate can not replace chloride or iodide and the channel is impermeable to Na+,K+ and tetraethylammonium ions. The current-voltage relationship demonstrates that the single channel current is a linear function of the clamp voltage. Single channel currents reversed at a pipette potential close to 0 mV. The mean single channel conductance was 60 pS for Cl- and 50 pS for I-. From the I-V relationship there was a strong outward rectification with Cl-, and a complete block with I-, in the single channel current above +40 mV. The feature of the channel is manifested in the single channel records by four distinct, equally spaced conductance levels. We suggest the channel is important for the transport of I and Cl ions across the apical membrane into the colloid space and is important for hormone synthesis and follicle formation.     

Therapeutic effect of adenoviral-mediated hepatocyte growth factor gene administration on TNBS-induced colitis in mice

Inflammatory bowel disease is incurable and relapsing disease. In order to clarify the effect of HGF gene therapy for inflammatory bowel disease, the adenoviral-mediated HGF gene was intrarectally administered into TNBS-colitis-induced Balb/c mice. Adenoviral-mediated gene delivery targetted its expression mainly to intestinal epithelial cells. Mucosal damage of HGF-treated intestine was significantly improved, and compared with LacZ-treated and saline administered mice (P<0.05, each). The mice treated with intrarectal administration of pAxCAHGF showed an increased average of body weight in comparison with that of pAxCALacZ-treated and saline-treated mice (P<0.05, each). The PCNA-positive cells in pAxCALacZ-treated mice were 44.7+/-4.9%, 51.7+/-6.6%, and 53.9+/-4.5% at 10, 15, and 21 days after TNBS administration, however those in pAxCAHGF-treated mice were increased to 74.3+/-5.1%, 67.1+/-2.6%, and 69.2+/-4.6% (P<0.05, each). The TUNEL-positive cells in pAxCALacZ-treated mice were 13.3+/-5.2%, 11.5+/-2.1%, and 7.2+/-5.2%, respectively. However, those in pAxCAHGF-treated mice at 10, 15, and 21 days were significantly decreased to 5.4+/-1.8%, 3.8+/-1.3%, and 5.7+/-2.8% (P<0.05, respectively). Expression of ERK1/2 was stronger in pAxCAHGF mice than in pAxCALacZ. These data suggest that adenoviral-mediated HGF gene therapy via an intrarectal route is a promising therapy for inflammatory bowel disease.     

Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (K(m): 365+/-42 microm). The transport was Na(+)-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; urate [corrected] is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1).