Abstract Parameter optimization (PO) methods to determine the ionic current composition of experimental cardiac action potential (AP) waveform have been developed using a computer model of cardiac ...membrane excitation. However, it was suggested that fitting a single AP record in the PO method was not always successful in providing a unique answer because of a shortage of information. We found that the PO method worked perfectly if the PO method was applied to a pair of a control AP and a model output AP in which a single ionic current out of six current species, such as I Kr , I CaL , I Na , I Ks , I Kur or I bNSC was partially blocked in silico. When the target was replaced by a pair of experimental control and I Kr -blocked records of APs generated spontaneously in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the simultaneous fitting of the two waveforms by the PO method was hampered to some extent by the irregular slow fluctuations in the V m recording and/or sporadic alteration in AP configurations in the hiPSC-CMs. This technical problem was largely removed by selecting stable segments of the records for the PO method. Moreover, the PO method was made fail-proof by running iteratively in identifying the optimized parameter set to reconstruct both the control and the I Kr -blocked AP waveforms. In the lead potential analysis, the quantitative ionic mechanisms deduced from the optimized parameter set were totally consistent with the qualitative view of ionic mechanisms of AP so far described in physiological literature.
Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in ...pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both β1- and β2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β2-adrenoceptor stimulation than β1-adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under β-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.
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•The IKs channel was expressed on the membrane of pulmonary vein cardiomyocytes (PVC).•β- and α1-adrenoceptor stimulation enhanced IKs in PVC via the PKA and PKC pathway.•The IKs was more potentiated by β2-adrenoceptor stimulation than β1-adrenoceptor.•The β-adrenoceptor enhanced firing rate was markedly reduced by IKs inhibition.
The sustained inward Na
+
current (
I
st
) identified in the sinoatrial node (SAN) cell has been suggested to play a pivotal role in cardiac pacemaking. However, the composition of cells in the SAN ...is heterogeneous and cell-to-cell variability in the magnitude of
I
st
remains to be fully characterized. The present study investigated the current density of
I
st
in morphologically different types of pacemaker cells dissociated from guinea pig SAN.
I
st
was preferentially detected in spontaneously active spindle or spider-shaped cells, but was less well expressed in larger-sized elongated spindle-type cells and practically absent in clearly striated atrial-like cells, despite clear expression of the funny current (
I
f
). The current density of
I
st
in spindle and spider cells varied from 0.7 to 1.6 pA pF
−1
and was significantly reduced in non-beating cells with similar morphologies. By linear regression analysis, we identified a positive correlation between the current densities of
I
st
and the L-type Ca
2+
current (
I
Ca,L
), which was specifically observed in spindle and spider cells. These cells exhibited a more negative voltage for half maximal
I
Ca,L
activation than atrial-like cells, suggesting a variable ratio between Ca
V
1.2- and Ca
V
1.3-mediated
I
Ca,L
in SAN cells. Consistent single-cell transcript measurements confirmed a higher relative expression of Ca
V
1.3, which activates at more negative potentials, in spindle cells than in atrial-like cells. Taken together, these results can be interpreted as indicating that
I
st
plays a specific role in primary pacemaker cells and that its presence is closely correlated with functional levels of Ca
V
1.3-mediated
I
Ca,L
.
Objectives This study aims to address whether D85N, a KCNE1 polymorphism, is a gene variant that causes long QT syndrome (LQTS) phenotype. Background KCNE1 encodes the beta-subunit of cardiac ...voltage-gated K+ channels and causes LQTS, which is characterized by the prolongation of the QT interval and torsades de pointes, a lethal arrhythmia. D85N, a KCNE1 polymorphism, is known to be a functional variant associated with drug-induced LQTS. Methods In order to elucidate the prevalence and clinical significance of this polymorphism, we performed genetic screening in 317 LQTS probands. For comparison, we examined its presence in 496 healthy control subjects. We also conducted biophysical assays for the D85N variant in mammalian cells. Results The allele frequency for D85N carriers was 0.81% in healthy people. In contrast, among LQTS probands, there were 1 homozygous and 23 heterozygous carriers (allele frequency 3.9%). Seven of 23 heterozygous carriers had additional mutations in LQTS-related genes, and 3 female subjects had documented factors predisposing to the symptom. After excluding these probands, the D85N prevalence was significantly higher compared with control subjects (allele frequency 2.1%, p < 0.05). In a heterologous expression study with Chinese hamster ovarian cells, KCNE1-D85N was found to exert significant loss-of-function effects on both KCNQ1- and KCNH2-encoded channel currents. Conclusions The KCNE1-D85N polymorphism was significantly more frequent in our LQTS probands. The functional variant is a disease-causing gene variant of LQTS phenotype that functions by interacting with KCNH2 and KCNQ1. Since its allele frequency was ∼1% among control individuals, KCNE1-D85N may be a clinically important genetic variant.
Abstract
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 Ca
2+
channels (Ca
v
1.2), 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 Ca
V
1.2 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 Ca
2+
currents by significantly delaying Ca
V
1.2 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.
Abstract
The spontaneous activity of sinoatrial node (SAN) pacemaker cells is generated by a functional interplay between the activity of ionic currents of the plasma membrane and intracellular Ca
2+
...dynamics. The molecular correlate of a dihydropyridine (DHP)-sensitive sustained inward Na
+
current (
I
st
), a key player in SAN automaticity, is still unknown. Here we show that
I
st
and the L-type Ca
2+
current (
I
Ca,L
) share Ca
V
1.3 as a common molecular determinant. Patch-clamp recordings of mouse SAN cells showed that
I
st
is activated in the diastolic depolarization range, and displays Na
+
permeability and minimal inactivation and sensitivity to
I
Ca,L
activators and blockers. Both Ca
V
1.3-mediated
I
Ca,L
and
I
st
were abolished in Ca
V
1.3-deficient (Ca
V
1.3
−/−
) SAN cells but the Ca
V
1.2-mediated
I
Ca,L
current component was preserved. In SAN cells isolated from mice expressing DHP-insensitive Ca
V
1.2 channels (Ca
V
1.2
DHP−/−
),
I
st
and Ca
V
1.3-mediated
I
Ca,L
displayed overlapping sensitivity and concentration–response relationships to the DHP blocker nifedipine. Consistent with the hypothesis that Ca
V
1.3 rather than Ca
V
1.2 underlies
I
st
, a considerable fraction of
I
Ca,L
was resistant to nifedipine inhibition in Ca
V
1.2
DHP−/−
SAN cells. These findings identify Ca
V
1.3 channels as essential molecular components of the voltage-dependent, DHP-sensitive
I
st
Na
+
current in the SAN.
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 (G
) 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 G
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 G
s of the baseline model. G
s 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 G
s recovery by decreasing the mean square error (MSE) between the target and model output. Plotting all data points of the MSE-G
relationship during optimization revealed progressive convergence of the randomized population of G
s 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 G
s 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.
Pregnancy causes changes in the uterus, such as increased cell volume and altered water content. However, the mechanisms that protect the structure and maintain the function of uterine smooth muscle ...cells against these changes during pregnancy have not been clarified. This study focused on the volume-regulated anion channel (VRAC), which opens with cell swelling under low osmotic pressure and releases Cl– ions and various organic osmolytes to resist cell swelling and regulates a wide range of biological processes such as cell death. In this study, myometrial smooth muscle (MSM) tissues and cells (MSMCs) were collected from non-pregnant and pregnant mice. Using western blotting and immunocytochemistry, leucine-rich repeat containing protein 8A (LRRC8A), an essential membrane protein that constitutes part of the VRAC, was determined to be diffused throughout MSMCs including in the cell membrane. Patch-clamp experiments were performed to investigate the electrophysiology of swelling-induced Cl– currents (ICl, swell) mediated by the VRAC. No significant changes between non-pregnancy and pregnancy groups were observed in either the expression density of LRRC8A or the current density of ICl, swell, however the presence of LRRC8A on the cell membrane was significantly increased in the third trimester of pregnancy compared to the non-pregnancy. This study suggests that the VRAC may play a role, such as maintaining cellular homeostasis in the pregnant MSM.
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