Hideo Otani

Drug-induced long-QT syndrome associated with a subclinical SCN5A mutation.

Background—Subclinical mutations in genes associated with the congenital long-QT syndromes (LQTS) have been suggested as a risk factor for drug-induced LQTS and accompanying life-threatening arrhythmias. Recent studies have identified genetic variants of the cardiac K+ channel genes predisposing affected individuals to acquired LQTS. We have identified a novel Na+ channel mutation in an individual who exhibited drug-induced LQTS. Methods and Results—An elderly Japanese woman with documented QT prolongation and torsade de pointes during treatment with the prokinetic drug cisapride underwent mutational analysis of LQTS-related genes. A novel missense mutation (L1825P) was identified within the C-terminus region of the cardiac Na+ channel (SCN5A). The L1825P channel heterologously expressed in tsA-201 cells showed Na+ current with slow decay and a prominent tetrodotoxin-sensitive noninactivating component, similar to the gain-of-function phenotype most commonly observed for SCN5A-associated congenital LQTS (LQT3). In addition, L1825P exhibited loss of function Na+ channel features characteristic of Brugada syndrome. Peak Na+ current density observed in cells expressing L1825P was significantly diminished, and the voltage dependence of activation and inactivation was shifted toward more positive and negative potentials, respectively. Conclusions—This study demonstrates that subclinical mutations in the LQTS-related gene SCN5A may predispose certain individuals to drug-induced cardiac arrhythmias.

Elevated basic fibroblast growth factor in pericardial fluid of patients with unstable angina

BACKGROUND Collateral growth is induced by chemical signals from the ischemic myocardium. We hypothesized that angiogenic growth factors are produced by cardiac tissue; they are diffusible, more concentrated in pericardial fluids, and are increased by myocardial ischemia. METHODS AND RESULTS With the use of an enzyme-linked immunosorbent assay, we measured the concentrations of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in pericardial fluids of 12 patients with unstable angina (group 1) and of 8 patients with nonischemic heart diseases (group 2). The levels of protein in pericardial fluids were quite comparable between the two groups (34 +/- 2 versus 32 +/- 4 mg/mL). The concentration of bFGF in pericardial fluids in group 1 was 2036 +/- 357 pg/mL, significantly (P < .001) higher than the 289 +/- 72 pg/mL in group 2. The amount of bFGF per milligram of protein was also significantly (P < .05) higher in group 1 than in group 2 (67 +/- 15 versus 12 +/- 4 pg/mg). The concentration of VEGF in pericandial fluids tended to be higher in group 1, but the difference was statistically insignificant (39 +/- 7 versus 22 +/- 6 pg/mL). The amount of VEGF per milligram of protein was 1.2 +/- 0.3 pg/mg in group 1, similar to the 0.8 +/- 0.4 pg/mg in group 2. CONCLUSIONS This finding provides new evidence that bFGF plays an important role in mediating collateral growth in humans.

Novel KCNJ2 Mutation in Familial Periodic Paralysis With Ventricular Dysrhythmia

Background—Mutations in the KCNJ2 gene, which codes cardiac and skeletal inward rectifying K+ channels (Kir2.1), produce Andersen’s syndrome, which is characterized by periodic paralysis, cardiac arrhythmia, and dysmorphic features. Methods and Results—In 3 Japanese family members with periodic paralysis, ventricular arrhythmias, and marked QT prolongation, polymerase chain reaction/single-strand conformation polymorphism/DNA sequencing identified a novel, heterozygous, missense mutation in KCNJ2, Thr192Ala (T192A), which was located in the putative cytoplasmic chain after the second transmembrane region M2. Using the Xenopus oocyte expression system, we found that the T192A mutant was nonfunctional in the homomeric condition. Coinjection with the wild-type gene reduced the current amplitude, showing a weak dominant-negative effect. Conclusions—T192, which is located in the phosphatidylinositol-4,5-bisphosphate binding site and also the region necessary for Kir2.1 multimerization, is a highly conserved amino acid residue among inward-rectifier channels. We suggest that the T192A mutation resulted in the observed electrical phenotype.

Coordinate interaction between ATP-sensitive K+ channel and Na+, K+-ATPase modulates ischemic preconditioning

BACKGROUND We reported that digoxin abolishes the infarct size (IS)-limiting effect of ischemic preconditioning (IPC). Because ATP-sensitive K+ (KATP) channels are involved in IPC, we studied whether Na+,K+-ATPase and KATP channels functionally interact, thereby modulating IPC. METHODS AND RESULTS Rabbits received 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. IPC was elicited by 5 minutes of occlusion followed by 10 minutes of reperfusion. The IS, expressed as a percentage of the area at risk, was 40.2+/-2.8% in control and 39.8+/-5.0% in digoxin pretreatment rabbits. Both IPC and pretreatment with cromakalim, a KATP channel opener, reduced IS to 11.8+/-1.8% and 13.4+/-2.6% (P<0. 05 versus control). Digoxin abolished the reduction in IS induced by IPC (33.5+/-3.3%), whereas it did not change that induced by cromakalim (18.8+/-3.0%). In patch-clamp experiments, digoxin was found to inhibit the opening of KATP channels in single ventricular myocytes in which ATP depletion had been induced by metabolic stress. In contrast, digoxin had little effect on the channel opening induced by cromakalim. Moreover, the inhibitory action of digoxin on channel activities was dependent on subsarcolemmal ATP concentration. CONCLUSIONS The IS-limiting effect of IPC is modulated by an interaction between KATP channels and Na+,K+-ATPase through subsarcolemmal ATP.

Cytoplasmic terminus domains of Kir6.x confer different nucleotide‐dependent gating on the ATP‐sensitive K+ channel

1 In order to investigate the structural basis for the nucleotide‐dependent gating of ATP‐sensitive K+ channels (KATP), Kir6.1 (uKATP‐1), Kir6.2 (BIR1) and chimeric channels were co‐expressed with a common subtype of sulphonylurea receptor, SUR1, in COS7 cells. Representing the amino terminal domain‐transmembrane domain‐carboxyl‐terminal domain of Kir6.1 as 1–1–1 and of Kir6.2 as 2‐2‐2, chimeric Kir6.x channels were constructed by swapping the amino and/or carboxyl terminal domains between Kir6.1 and Kir6.2 to give the chimeric x‐1‐x channels 1‐1‐2, 2‐1‐1 and 2‐1‐2, and the chimeric x‐2‐x channels 2‐2‐1, 1‐2‐2 and 1‐2‐1. 2 Inside‐out patch clamp experiments revealed that both wild‐type Kir6.1 and Kir6.2 formed inwardly rectifying K+ channels. Single‐channel conductances were 36.3 and 66.1 pS, respectively. Chimeric x‐1‐x channels, whose transmembrane domain was that of Kir6.1, showed similar ion‐pore properties to wild‐type Kir6.1. Likewise, chimeric x‐2‐x channels had similar ion‐pore properties to wild‐type Kir6.2. 3 Wild‐type Kir6.1 and Kir6.2 possessed distinct gating properties towards intracellular nucleotides. The activity of Kir6.1 was entirely dependent on Mg2+ and nucleotide diphosphates (NDPs) such as UDP. In contrast, Kir6.2 was activated upon excision of patch membrane. When Kir6.2 underwent rundown, UDP reactivated the channel. 4 In order to eliminate UDP dependence from Kir6.1, it was necessary to replace both N‐ and C‐termini; chimera 2‐1‐2 opened in UDP‐free conditions. With Kir6.2, substitution of the N‐terminus with that of Kir6.1 conferred UDP dependence on chimeras 1‐2‐2 and 1‐2‐1. Chimera 2‐2‐1 opened in UDP‐free conditions, but UDP potentiated the channel activity by > 20‐fold. 5 The kinetics of UDP‐dependent activation were significantly different between Kir6.1 and Kir6.2. Kir6.1 maximally activated by UDP was sensitive to intracellular ATP, although its ATP sensitivity was significantly lower than that of Kir6.2 measured in identical conditions. The kinetics of UDP‐dependent activation and ATP sensitivity could be transferred between Kir6.1 and Kir6.2 only when both N‐ and C‐termini were replaced. We therefore concluded that nucleotide‐dependent gating was regulated by the N‐ and C‐terminal domains irrespective of the transmembrane domains.

Cystic fibrosis transmembrane conductance regulator mediates sulphonylurea block of the inwardly rectifying K+ channel Kir6.1

1 Recombinant ATP‐sensitive K+ channels (KATP channels) were heterologously expressed in the NIH3T3 mouse cell line, and the electrophysiological properties were studied using patch‐clamp techniques. 2 The NIH3T3 cell lines transfected with the inwardly rectifying K+ channel Kir6.1 alone or with both Kir6.1 and cystic fibrosis transmembrane conductance regulator (CFTR) exhibited time‐independent K+ currents with weak inward rectification. In contrast, no measurable K+ conductance was observed in mock‐transfected cells or in cells transfected with CFTR alone. Regardless of co‐transfection with Kir6.1, the transfection with CFTR produced a Cl− conductance that was activated by cell dialysis with cAMP (1 mM). The conductance was reversibly suppressed by glibenclamide (30 μM). 3 Whole‐cell currents at +60 mV were blocked in a concentration‐dependent manner by Ba2+ ions with similar IC50 values: 89.3 ± 23.3 μM (Kir6.1 alone) and 67.3 ± 24.9 μM (Kir6.1‐CFTR). 4 The currents recorded from Kir6.1‐transfected cells were not affected by glibenclamide, whereas glibenclamide did inhibit the conductance expressed in cells co‐transfected with CFTR (IC50= 35.9 ± 6.6 μM). 5 In the cell‐attached mode with a 150 mM K+ pipette solution, both Kir6.1‐ and Kir6.1‐CFTR‐transfected cells displayed a class of K+ channels showing weak inward rectification and a slope conductance of 50.7 ± 1.0 and 52.4 ± 4.9 pS, respectively. 6 In the inside‐out mode, the single‐channel currents recorded from both types of cells were not inhibited by intracellular ATP (1 mM). However, glibenclamide was found to block the single‐channel activities in the co‐transfected cells.

Probucol does not affect lipoprotein metabolism in macrophages of Watanabe heritable hyperlipidemic rabbits.

We recently reported that the antioxidant action of probucol inhibited the progression of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits. In this study, we investigated another possible action of probucol: its action as an antiatherogenic agent on macrophages. When WHHL rabbit peritoneal macrophages were pre-incubated in vitro with probucol and then incubated with several atherogenic lipoproteins, the incorporation of the lipoproteins was not significantly prevented. In the case of mouse peritoneal macrophages, pre-incubation with probucol showed slight, although not statistically significant, changes in the amount of lipoprotein incorporations. We also used macrophages obtained from mice and WHHL rabbits fed with probucol, but the amount of uptake of lipoproteins by these cells was not less than that by control macrophages. Furthermore, to investigate the incorporation of atherogenic lipoproteins into these cells, we prepared probucol-containing macrophages; however, probucol in macrophages failed to prevent the uptake of such lipoproteins. In conclusion, probucol did not prevent foam cell transformation of macrophages of WHHL rabbit or mice directly, and the effect of probucol against atherogenesis in WHHL rabbits was due mainly to its inhibitory effect on the oxidative modification of low density lipoprotein, as previously reported.

Regulation of apolipoprotein B secretion in hepatocytes from Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia

Regulation of apolipoprotein B-100 (apo B) secretion in the liver in familial hypercholesterolemia (FH) remains largely unknown. In a previous study, we developed a rabbit hepatocyte culture system and investigated a cellular mechanism which regulates apo B secretion from hepatocytes in response to cellular cholesteryl ester contents [18]. Using this system, in the present study, we investigated regulation of apo B secretion in hepatocytes from the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for FH. Incubation with low density lipoproteins (LDL) at concentrations of 50 or 200 micrograms/ml, which increased both cellular cholesteryl ester and apo B secretion significantly in normal rabbit hepatocytes, did not cause such increases in WHHL rabbit hepatocytes. However, when WHHL rabbit hepatocytes were incubated with LDL at a concentration of 500 micrograms/ml, a significant increase in cellular cholesteryl ester and apo B secretion was observed. The effect of the increase in cellular level of cholesteryl ester upon apo B secretion in WHHL rabbit hepatocytes was compatible with that in normal rabbit hepatocytes. Indeed, when WHHL rabbit hepatocytes were incubated with LDL at 1680 micrograms/ml, a concentration comparable to that of WHHL rabbit plasma, the amount of LDL degradation, cellular cholesteryl ester level, and level of apo B secretion were the same as those in normal rabbit hepatocytes that were incubated with LDL at 160 micrograms/ml, a concentration comparable to that of normal rabbit plasma. In summary, our present study suggests that, at a steady state with such a high plasma concentration of LDL, the hepatic cholesterol content in WHHL rabbits could be set at the same level as in normal rabbits. It was also shown that cellular cholesteryl ester contents had the same regulatory effect on apo B secretion in WHHL rabbit hepatocytes as in normal rabbit hepatocytes. Therefore, we conclude that in the presence of the genetic defect of the LDL receptor, plasma cholesterol in WHHL rabbit is maintained at a concentration such that apo B secretion is similar to that in normal rabbit.

Characterization of Low Density Lipoprotein Receptors in Normal and Watanabe Heritable Hyperlipidemic Rabbits

Lipid-laden foam cells are a characteristic in the early stage of atherosclerotic lesions. Many foam cells in these lesions preserve the characteristics of macrophages.’ BetaVLDL, which accumulate in the plasma of patients with type I11 hyperlipoproteinemia and cholesterol-fed animals, is known to transform macrophages into foam cells. Homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, like their human counterparts with familial hypercholesterolemia, have not only extraordinary high LDL levels, but also a substantially increased concentration of VLDL remnants, which are similar to PVLDL.2 In the present study, we investigated the uptake mechanism of P-VLDL by peritoneal macrophages and fibroblasts obtained from normal and WHHl rabbits. When incubated with peritoneal macrophages from normal and WHHL rabbits, [‘251]P-VLDL was degraded by both macrophages with saturation kinetics. At the saturation, [‘251]P-VLDL degraded by WHHL macrophages was about 30% that by normal rabbit macrophages. Normal rabbit macrophages degraded I2’I-LDL with a saturable, high affinity mechanism. However, WHHL rabbit macrophages did little. These results indicated that WHHL rabbit macrophages took up P-VLDL despite the lack of LDL receptor activity. The LDL receptor gene of WHHL rabbits has a small in-flame deletion of 12 nucleotides which results in the loss of four amino acids from the cysteine-rich repeat of the ligand binding d ~ m a i n . ~ Due to its defect, the abnormal LDL receptors of WHHL rabbit fibroblasts do not recognize apo B 100 in LDL particles. However, the precursor of the mutant LDL receptor from WHHL rabbit adrenal gland bind P-VLDL on the nitrocellulose blot.3 When incubated with fibroblasts from WHHL rabbit, [ I2’I]PVLDL was degraded in a saturable, high affinity mechanism. As P-VLDL uptake in fibroblasts is mediated exclusively by the LDL receptors, the result strongly suggested