Regulation of the cardiac delayed rectifier K^+ channel by membrane PIP_2 and its physiological significance

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2003～2004課題番号: 15590184研究代表者: 松浦 博（滋賀医科大学・医学部・教授）研究分担者: 林 維光（滋賀医科大学・医学部・助手）研究分担者: 豊田 太（滋賀医科大学・医学部・助手

Gradient-based parameter optimization method to determine membrane ionic current composition in human induced pluripotent stem cell-derived cardiomyocytes

Premature cardiac myocytes derived from human induced pluripotent stem cells (hiPSC-CMs) show heterogeneous action potentials (APs), probably due to different expression patterns of membrane ionic currents. We developed a method for determining expression patterns of functional channels in terms of whole-cell ionic conductance (Gx) using individual spontaneous AP configurations. It has been suggested that apparently identical AP configurations can be obtained using different sets of ionic currents in mathematical models of cardiac membrane excitation. If so, the inverse problem of Gx estimation might not be solved. We computationally tested the feasibility of the gradient-based optimization method. For a realistic examination, conventional 'cell-specific models' were prepared by superimposing the model output of AP on each experimental AP recorded by conventional manual adjustment of Gxs of the baseline model. Gxs of 4–6 major ionic currents of the 'cell-specific models' were randomized within a range of ± 5–15% and used as an initial parameter set for the gradient-based automatic Gxs recovery by decreasing the mean square error (MSE) between the target and model output. Plotting all data points of the MSE–Gx relationship during optimization revealed progressive convergence of the randomized population of Gxs to the original value of the cell-specific model with decreasing MSE. The absence of any other local minimum in the global search space was confirmed by mapping the MSE by randomizing Gxs over a range of 0.1–10 times the control. No additional local minimum MSE was obvious in the whole parameter space, in addition to the global minimum of MSE at the default model parameter

Regulation of the cardiac delayed rectifier K^+ channel by membrane phosphoinositides

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2001～2002課題番号: 13670042研究代表者: 松浦 博（滋賀医科大学・医学部・教授）研究分担者: 林 維光（滋賀医科大学・医学部・助手）研究分担者: 豊田 太（滋賀医科大学・医学部・助手

Molecular basis for the regulation of the cardiac delayed rectifier K^+ channel by membrane PIP_2

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2005～2006課題番号: 17590185研究代表者: 松浦 博（滋賀医科大学・医学部・教授）研究分担者: 林 維光（滋賀医科大学・医学部・助手）研究分担者: 豊田 太（滋賀医科大学・医学部・助手

The G-protein–gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation

A hERG mutation E1039X produced a synergistic lesion on I _Ks together with KCNQ1-R174C mutation in a LQTS family with three compound mutations

Congenital long QT syndrome (LQTS) caused by compound mutations is usually associated with more severe clinical phenotypes. We identified a LQTS family harboring three compound mutations in different genes (KCNQ1-R174C, hERG-E1039X and SCN5A-E428K). KCNQ1-R174C, hERG-E1039X and SCN5A-E428K mutations and/or relevant wild-type (WT) cDNAs were respectively expressed in mammalian cells. IKs-like, IKr-like, INa-like currents and the functional interaction between KCNQ1-R174C and hERG-E1039X channels were studied using patch-clamp and immunocytochemistry techniques. (1) Expression of KCNQ1-R174C alone showed no IKs. Co-expression of KCNQ1-WT + KCNQ1-R174C caused a loss-of-function in IKs and blunted the activation of IKs in response to isoproterenol. (2) Expression of hERG-E1039X alone and co-expression of hERG-WT + hERG-E1039X negatively shifted inactivation curves and decelerated the recovery time from inactivation. (3) Expression of SCN5A-E428K increased peak INa, but had no effect on late INa. (4) IKs and IKr interact, and hERG-E1039X caused a loss-of-function in IKs. (5) Immunocytochemical studies indicated that KCNQ1-R174C is trafficking defective and hERG-E1039X is defective in biosynthesis/degradation, but the abnormities were rescued by co-expression with WT. Thus, KCNQ1-R174C and hERG-E1039X disrupted IKs and IKr functions, respectively. The synergistic lesion, caused by KCNQ1-R174C and hERG-E1039X in IKs, is very likely why patients showed more severe phenotypes in the compound mutation case

Increased CaV1.2 late current by a CACNA1C p.R412M variant causes an atypical Timothy syndrome without syndactyly

Timothy syndrome (TS) is a rare pleiotropic disorder associated with long QT syndrome, syndactyly, dysmorphic features, and neurological symptoms. Several variants in exon 8 or 8a of CACNA1C, a gene encoding the α-subunit of voltage-gated Ca2+ channels (Cav1.2), are known to cause classical TS. We identified a p.R412M (exon 9) variant in an atypical TS case. The aim of this study was to examine the functional effects of CACNA1C p.R412M on CaV1.2 in comparison with those of p.G406R. The index patient was a 2-month-old female infant who suffered from a cardio-pulmonary arrest in association with prolonged QT intervals. She showed dysmorphic facial features and developmental delay, but not syndactyly. Interestingly, she also presented recurrent seizures from 4 months. Genetic tests identified a novel heterozygous CACNA1C variant, p.R412M. Using heterologous expression system with HEK-293 cells, analyses with whole-cell patch-clamp technique revealed that p.R412M caused late Ca2+ currents by significantly delaying CaV1.2 channel inactivation, consistent with the underlying mechanisms of classical TS. A novel CACNA1C variant, p.R412M, was found to be associated with atypical TS through the same mechanism as p.G406R, the variant responsible for classical TS

Deregulation in the oil industry : lessons from the U.S. experience for Japan

Thesis (M.S.)--Massachusetts Institute of Technology, Sloan School of Management, 1996.Includes bibliographical references (leaves 119-121).by Futoshi Toyoda.M.S

寒冷馴化動物における心機能の長期的調節に関する電気生理学的研究

京都大学0048新制・課程博士博士(農学)甲第8531号農博第1140号新制||農||807(附属図書館)学位論文||H12||N3434(農学部図書室)UT51-2000-J40京都大学大学院農学研究科応用生物科学専攻(主査)教授 宮崎 昭, 教授 矢野 秀雄, 教授 宮本 元学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA