Abstract Aims Phospholamban (PLB) regulates the cardiac Ca 2 + -ATPase (SERCA2a) in sarcoplasmic reticulum (SR). However, the localization of PLB at subcellular sites outside the SR and possible ...contributions to Ca 2 + cycling remain unknown. We examined the intracellular distribution of PLB and tested whether a pool of PLB exists in the nuclear envelope (NE) that might regulate perinuclear/nuclear Ca 2 + (nCa 2 + ) handling in cardiomyocytes (CMs). Methods and results Using confocal immunofluorescence microscopy and immunoblot analyses of CMs and CM nuclei, we discovered that PLB was highly concentrated in NE. Moreover, the ratio of PLB levels to SERCA levels was greater in NE than in SR. The increased levels of PLB in NE were a consistent finding using a range of antibodies, tissue samples, and species. To address a possible role in affecting Ca 2 + handling, we used Fluo-4 based confocal Ca 2 + imaging, with scan-lines across cytosol and nuclei, and evaluated the effects of PLB on cytosolic and nCa 2 + uptake and release in mouse CMs. In intact CMs, isoproterenol increased amplitude and decreased the decay time of Ca 2 + transients not only in cytosol but also in nuclear regions. In saponin-permeabilized mouse CMs (Ca 2 + i = 400 nM), we measured spontaneous Ca 2 + waves after specific reversal of PLB activity by addition of the Fab fragment of an anti-PLB monoclonal antibody (100 μg/ml). This highly selective immunological reagent enhanced Ca 2 + uptake (faster decay times) and Ca 2 + release (greater intensity) in both cytosol and across the nuclear regions. Conclusions Besides SR, PLB is concentrated in NE of CMs, and may be involved in modulation of nCa 2 + dynamics.
Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated ...K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.
Phospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope ...(NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling.
Fluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ (Ca2+i = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of Ca2+i. In WT mice, at 10 nM Ca2+i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19 ± 0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09 ± 0.02. At 50 nM Ca2+i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100 nM Ca2+i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice.
PLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.
•Phospholamban (PLB) is concentrated in nuclear envelope (NE) of cardiomyocytes (CMs).•The Fab fragment of PLB antibody increased the lumenal Ca inside the NE of CM nuclei.•Anti-PLB Fab increased Ca release in the nuclear regions of permeabilized CMs.•Anti-PLB Fab decreased Ca levels elevated by IP3 at rest in the nuclear regions.•PLB regulates nuclear Ca handling in CMs through mechanisms involving IP3R and RyR.
Female sex is a known risk factor for drug-induced long QT syndrome (diLQTS). We recently demonstrated a sex difference in apamin-sensitive small-conductance Ca
-activated K
current (I
) activation ...during β-adrenergic stimulation.
The purpose of this study was to test the hypothesis that there is a sex difference in I
in the rabbit models of diLQTS.
We evaluated the sex difference in ventricular repolarization in 15 male and 22 female Langendorff-perfused rabbit hearts with optical mapping techniques during atrial pacing. HMR1556 (slowly activating delayed rectifier K
current I
blocker), E4031 (rapidly activating delayed rectifier K
current I
blocker) and sea anemone toxin (ATX-II, late Na
current I
activator) were used to simulate types 1-3 long QT syndrome, respectively. Apamin, an I
blocker, was then added to determine the magnitude of further QT prolongation.
HMR1556, E4031, and ATX-II led to the prolongation of action potential duration at 80% repolarization (APD
) in both male and female ventricles at pacing cycle lengths of 300-400 ms. Apamin further prolonged APD
(pacing cycle length 350 ms) from 187.8±4.3 to 206.9±7.1 (P=.014) in HMR1556-treated, from 209.9±7.8 to 224.9±7.8 (P=.003) in E4031-treated, and from 174.3±3.3 to 188.1±3.0 (P=.0002) in ATX-II-treated female hearts. Apamin did not further prolong the APD
in male hearts. The Ca
transient duration (Ca
TD) was significantly longer in diLQTS than baseline but without sex differences. Apamin did not change Ca
TD.
We conclude that I
is abundantly increased in female but not in male ventricles with diLQTS. Increased I
helps preserve the repolarization reserve in female ventricles treated with I
and I
blockers or I
activators.
Apamin-sensitive small conductance calcium-activated K current (I
) is up-regulated during ventricular pacing and masks short-term cardiac memory (CM).
The purpose of this study was to determine the ...role of I
in long-term CM.
CM was created with 3-5 weeks of ventricular pacing and defined by a flat or inverted T wave off pacing. Epicardial optical mapping was performed in both paced and normal ventricles. Action potential duration (APD
) was determined during right atrial pacing. Ventricular stability was tested before and after I
blockade. Four paced hearts and 4 normal hearts were used for western blotting and histology.
There were no significant differences in either echocardiographic parameters or fibrosis levels between groups. Apamin induced more APD
prolongation in CM than in normal ventricles (mean 95% confidence interval: 9.6% 8.8%-10.5% vs 3.1% 1.9%-4.3%; P <.001). Apamin significantly lengthened APD
in the CM model at late activation sites, indicating significant I
up-regulation at those sites. The CM model also had altered Ca
handling, with the 50% Ca
transient duration and amplitude increased at distal sites compared to a proximal site (near the pacing site). After apamin, the CM model had increased ventricular fibrillation (VF) inducibility (paced vs control: 33/40 (82.5%) vs 7/20 (35%); P <.001) and longer VF durations (124 vs 26 seconds; P <.001).
Chronic ventricular pacing increases Ca
transients at late activation sites, which activates I
to maintain repolarization reserve. I
blockade increases VF vulnerability in chronically paced rabbit ventricles.
Ondansetron, a widely prescribed antiemetic, has been implicated in drug-induced long QT syndrome. Recent patch clamp experiments have shown that ondansetron inhibits the apamin-sensitive small ...conductance calcium-activated potassium current (I
).
The purpose of this study was to determine whether ondansetron causes action potential duration (APD) prolongation by I
inhibition.
Optical mapping was performed in rabbit hearts with pacing-induced heart failure (HF) and in normal hearts before and after ondansetron (100 nM) infusion. APD at 80% repolarization (APD
) and arrhythmia inducibility were determined. Additional studies with ondansetron were performed in normal hearts perfused with hypokalemic Tyrode's (2.4 mM) solution before or after apamin administration.
The corrected QT interval in HF was 326 ms (95% confidence interval CI 306-347 ms) at baseline and 364 ms (95% CI 351-378 ms) after ondansetron infusion (P < .001). Ondansetron significantly prolonged APD
in the HF group and promoted early afterdepolarizations, steepened the APD restitution curve, and increased ventricular vulnerability. Ventricular fibrillation was not inducible in HF ventricles at baseline, but after ondansetron infusion, ventricular fibrillation was induced in 5 of the 7 ventricles (P = .021). In hypokalemia, apamin prolonged APD
from 163 ms (95% CI 146-180 ms) to 180 ms (95% CI 156-204 ms) (P = .018). Subsequent administration of ondansetron failed to further prolong APD
(180 ms 95% CI 156-204 ms vs 179 ms 95% CI 165-194 ms; P = .789). The results were similar when ondansetron was administered first, followed by apamin.
Ondansetron is a specific I
blocker at therapeutic concentrations. Ondansetron may prolong the QT interval in HF by inhibiting small conductance calcium-activated potassium channels, which increases the vulnerability to ventricular arrhythmias.
The mechanisms of J wave syndrome (JWS) are incompletely understood. Here, we showed that the concomitant activation of small-conductance calcium-activated potassium (SK) current (IKAS) and ...inhibition of sodium current by cyclohexyl-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl-amine (CyPPA) recapitulate the phenotypes of JWS in Langendorff-perfused rabbit hearts. CyPPA induced significant J wave elevation and frequent spontaneous ventricular fibrillation (SVF), as well as sinus bradycardia, atrioventricular block, and intraventricular conduction delay. IKAS activation by CyPPA resulted in heterogeneous shortening of action potential (AP) duration (APD) and repolarization alternans. CyPPA inhibited cardiac sodium current (INa) and decelerated AP upstroke and intracellular calcium transient. SVFs were typically triggered by short-coupled premature ventricular contractions, initiated with phase 2 reentry and originated more frequently from the right than the left ventricles. Subsequent IKAS blockade by apamin reduced J wave elevation and eliminated SVF. β-Adrenergic stimulation was antiarrhythmic in CyPPA-induced electrical storm. Like CyPPA, hypothermia (32.0°C) also induced J wave elevation and SVF. It facilitated negative calcium-voltage coupling and phase 2 repolarization alternans with spatial and electromechanical discordance, which were ameliorated by apamin. These findings suggest that IKAS activation contributes to the development of JWS in rabbit ventricles.
The gating of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is primarily controlled by intracellular Ca(2+) and/or membrane depolarization. These channels play a role in the coupling of ...excitation-contraction and stimulus-secretion. A variety of structurally distinct compounds may influence the activity of these channels. Squamocin, an Annonaceous acetogenin, could interact with the BK(Ca) channel to increase the amplitude of Ca(2+)-activated K(+) current in coronary smooth muscle cells. Its stimulatory effect is related to intracellular Ca(2+) concentrations. In inside-out patches, application of ceramide to the bath suppressed the activity of BK(Ca) channels recorded from pituitary GH(3) cells and from retinal pigment epithelial cells. ICI-182,780, an estrogen receptor antagonist, was found to modulate BK(Ca)-channel activity in cultured endothelial cells and smooth muscle cells in a mechanism unlinked to the inhibition of estrogen receptors. Caffeic acid phenethyl ester (CAPE) and its analogy, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, could directly increase the activity of BK(Ca) channels in GH(3) cells. CAPE also reduced the frequency and amplitude of intracellular Ca(2+) oscillations in these cells. The CAPE-stimulated activity in BK(Ca) channels is thought to be unassociated with its inhibition of NF-kappaB activation. Cilostazol, an inhibitor of cyclic nucleotide phosphodiesterase, could stimulate BK(Ca) channel-activity and reduce the firing of action currents simultaneously in GH(3) cells. Therefore, the regulation by these compounds of BK(Ca) channels may in part be responsible for their regulatory actions on cell functions.
Bioactive lysophospholipids (LPLs) are released by blood cells and can modulate many cellular activities such as angiogenesis and cell survival. In this study, the effects of sphingosine-1-phosphate ...(S1P) and lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated Ca²⁺ current was inhibited by S1P and LPA pre-treatment in a concentration-dependent manner with IC₅₀s of 0.46 and 0.79 μmol/L, respectively. Inhibition was mostly reversible upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na⁺ current was inhibited by S1P, but not by LPA. However, recovery of Na⁺ channels from inactivation was slowed by both LPLs. The outward K⁺ current was also significantly reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to minimal injections of depolarizing current. Repetitive activity was dramatically reduced by LPLs. Consistent with the reduction in Ca²⁺ current, exocytosis elicited by a train of depolarizations and the ensuing endocytosis were both inhibited by LPL pre-treatments. These data demonstrate the interaction between immune and endocrine systems mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin cells.
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