Higher Na+-Ca2+ Exchanger Function and Triggered Activity Contribute to Male Predisposition to Atrial Fibrillation.

Male sex is one of the most important risk factors of atrial fibrillation (AF), with the incidence in men being almost double that in women. However, the reasons for this sex difference are unknown. Accordingly, in this study, we sought to determine whether there are sex differences in intracellular Ca2+ homeostasis in mouse atrial myocytes that might help explain male predisposition to AF. AF susceptibility was assessed in male (M) and female (F) mice (4-5 months old) using programmed electrical stimulation (EPS) protocols. Males were 50% more likely to develop AF. The Ca2+ transient amplitude was 28% higher in male atrial myocytes. Spontaneous systolic and diastolic Ca2+ releases, which are known sources of triggered activity, were significantly more frequent in males than females. The time to 90% decay of Ca2+ transient was faster in males. Males had 54% higher Na+-Ca2+ exchanger (NCX1) current density, and its expression was also more abundant. L-type Ca2+ current (ICaL) was recorded with and without BAPTA, a Ca2+ chelator. ICaL density was lower in males only in the absence of BAPTA, suggesting stronger Ca2+-dependent inactivation in males. CaV1.2 expression was similar between sexes. This study reports major sex differences in Ca2+ homeostasis in mouse atria, with larger Ca2+ transients and enhanced NCX1 function and expression in males resulting in more spontaneous Ca2+ releases. These sex differences may contribute to male susceptibility to AF by promoting triggered activity.

Zingerone Modulates Neuronal Voltage-Gated Na+ and L-Type Ca2+ Currents.

Zingerone (ZO), a nontoxic methoxyphenol, has been demonstrated to exert various important biological effects. However, its action on varying types of ionic currents and how they concert in neuronal cells remain incompletely understood. With the aid of patch clamp technology, we investigated the effects of ZO on the amplitude, gating, and hysteresis of plasmalemmal ionic currents from both pituitary tumor (GH3) cells and hippocampal (mHippoE-14) neurons. The exposure of the GH3 cells to ZO differentially diminished the peak and late components of the INa. Using a double ramp pulse, the amplitude of the INa(P) was measured, and the appearance of a hysteresis loop was observed. Moreover, ZO reversed the tefluthrin-mediated augmentation of the hysteretic strength of the INa(P) and led to a reduction in the ICa,L. As a double ramp pulse was applied, two types of voltage-dependent hysteresis loops were identified in the ICa,L, and the replacement with BaCl2-attenuated hysteresis of the ICa,L enhanced the ICa,L amplitude along with the current amplitude (i.e., the IBa). The hysteretic magnitude of the ICa,L activated by the double pulse was attenuated by ZO. The peak and late INa in the hippocampal mHippoE-14 neurons was also differentially inhibited by ZO. In addition to acting on the production of reactive oxygen species, ZO produced effects on multiple ionic currents demonstrated herein that, considered together, may significantly impact the functional activities of neuronal cells.

Effective Perturbations by Small-Molecule Modulators on Voltage-Dependent Hysteresis of Transmembrane Ionic Currents.

The non-linear voltage-dependent hysteresis (Hys(V)) of voltage-gated ionic currents can be robustly activated by the isosceles-triangular ramp voltage (Vramp) through digital-to-analog conversion. Perturbations on this Hys(V) behavior play a role in regulating membrane excitability in different excitable cells. A variety of small molecules may influence the strength of Hys(V) in different types of ionic currents elicited by long-lasting triangular Vramp. Pirfenidone, an anti-fibrotic drug, decreased the magnitude of Ih's Hys(V) activated by triangular Vramp, while dexmedetomidine, an agonist of α2-adrenoceptors, effectively suppressed Ih as well as diminished the Hys(V) strength of Ih. Oxaliplatin, a platinum-based anti-neoplastic drug, was noted to enhance the Ih's Hys(V) strength, which is thought to be linked to the occurrence of neuropathic pain, while honokiol, a hydroxylated biphenyl compound, decreased Ih's Hys(V). Cell exposure to lutein, a xanthophyll carotenoid, resulted in a reduction of Ih's Hys(V) magnitude. Moreover, with cell exposure to UCL-2077, SM-102, isoplumbagin, or plumbagin, the Hys(V) strength of erg-mediated K+ current activated by triangular Vramp was effectively diminished, whereas the presence of either remdesivir or QO-58 respectively decreased or increased Hys(V) magnitude of M-type K+ current. Zingerone, a methoxyphenol, was found to attenuate Hys(V) (with low- and high-threshold loops) of L-type Ca2+ current induced by long-lasting triangular Vramp. The Hys(V) properties of persistent Na+ current (INa(P)) evoked by triangular Vramp were characterized by a figure-of-eight (i.e., ∞) configuration with two distinct loops (i.e., low- and high-threshold loops). The presence of either tefluthrin, a pyrethroid insecticide, or t-butyl hydroperoxide, an oxidant, enhanced the Hys(V) strength of INa(P). However, further addition of dapagliflozin can reverse their augmenting effects in the Hys(V) magnitude of the current. Furthermore, the addition of esaxerenone, mirogabalin, or dapagliflozin was effective in inhibiting the strength of INa(P). Taken together, the observed perturbations by these small-molecule modulators on Hys(V) strength in different types of ionic currents evoked during triangular Vramp are expected to influence the functional activities (e.g., electrical behaviors) of different excitable cells in vitro or in vivo.

Selective changes in cytosolic ß-adrenergic cAMP signals and L-type Calcium Channel regulation by Phosphodiesterases during cardiac hypertrophy.

Background In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by ß-adrenergic receptors (ß-ARs), impacting notably the regulation of the L-type Ca2+ current (ICa,L). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and ICa,L is not known. Methods and Results CH was induced in rats by thoracic aortic banding over a time period of five weeks and was confirmed by anatomical measurements. Left ventricular myocytes (LVMs) were isolated from CH and sham-operated (SHAM) rats and transduced with an adenovirus encoding a Förster resonance energy transfer (FRET)-based cAMP biosensor or subjected to the whole-cell configuration of the patch-clamp technique to measure ICa,L. Aortic stenosis resulted in a 46% increase in heart weight to body weight ratio in CH compared to SHAM. In SHAM and CH LVMs, a short isoprenaline stimulation (Iso, 100 nM, 15 s) elicited a similar transient increase in cAMP with a half decay time (t1/2off) of ~50 s. In both groups, PDE4 inhibition with Ro 20-1724 (10 µM) markedly potentiated the amplitude and slowed the decline of the cAMP transient, this latter effect being more pronounced in SHAM (t1/2off ~ 250 s) than in CH (t1/2off ~ 150 s, P < 0.01). In contrast, PDE3 inhibition with cilostamide (1 µM) had no effect on the amplitude of the cAMP transient and a minimal effect on its recovery in SHAM, whereas it potentiated the amplitude and slowed the decay in CH (t1/2off ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in ICa,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated ICa,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of ICa,L in SHAM, both PDE3 and PDE4 contributed in CH. Conclusion These results identify selective alterations in cytosolic cAMP and ICa,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of ß-AR responses is shifted toward PDE3 during CH.

Alterations of Ca2+ signaling and Ca2+ release sites in cultured ventricular myocytes with intact internal Ca2+ storage.

Although cultured adult cardiac myocytes in combination with cell-level genetic modifications have been adopted for the study of protein function, the cellular alterations caused by the culture conditions themselves need to be clarified before we can interpret the effects of genetically altered proteins. We systematically compared the cellular morphology, global Ca2+ signaling, elementary Ca2+ release (sparks), and arrangement of ryanodine receptor (RyR) clusters in short-term (2 days)-cultured adult rat ventricular myocytes with those of freshly isolated myocytes. The transverse (t)-tubules were remarkably decreased (to ∼25%) by culture, and whole-cell capacitance was reduced by ∼35%. The magnitude of depolarization-induced Ca2+ transients decreased to ∼50%, and Ca2+ transient decay was slowed by culture. The culture did not affect sarcoplasmic reticulum (SR) Ca2+ loading. Therefore, fractional Ca2+ release was attenuated by culture. In the cultured cells, the L-type Ca2+ current (ICa) was smaller (∼50% of controls) and its inactivation was slower. In cultured myocytes, there were significantly fewer (∼50% of control) Ca2+ sparks, the local Ca2+ releases through RyR clusters, compared with in freshly isolated cells. Amplitude and kinetics (duration and time-to-peak) of individual sparks were similar, but they showed greater width in cultured cells. Immunolocalization analysis revealed that the cross-striation of RyRs distribution became weaker and less organized, and that the density of RyR clusters decreased in cultured myocytes. Our data suggest that the loss of t-tubules and generation of compromised Ca2+ transients and ICa in short-term adult ventricular cell culture are independent of SR Ca2+ loading status. In addition, the deteriorated arrangement of the RyR-clusters and their decreased density after short-term culture may be partly responsible for fewer Ca2+ sparks and a decrease in global Ca2+ release.

Warmer, faster, stronger: Ca2+ cycling in avian myocardium.

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca2+ content nor the cross-talk between channels underlying Ca2+-induced Ca2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail (Coturnix japonica). A trans-sarcolemmal Ca2+ current (ICa) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca2+ channels. The peak density of ICa was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steady-state SR Ca2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 µmol l-1 in atrial cells and 423.3±47.2 µmol l-1 in ventricular cells at 24°C. We observed SR Ca2+-dependent inactivation of ICa in ventricular myocytes, indicating cross-talk between sarcolemmal Ca2+ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high-efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure.

Safranal, an active constituent of saffron, ameliorates myocardial ischemia via reduction of oxidative stress and regulation of Ca2+ homeostasis.

Safranal (SFR) is the major constituent of saffron. The purpose of this study was to observe the effect of SFR on myocardial ischemia induced by isoprenaline (ISO) and to explore its possible mechanism. The myocardial ischemia rat model was established by subcutaneous injection of ISO (85 mg/kg/d) on the 8th and 9th day of the experiment. Serum creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured, as were changes in calcium concentration, reactive oxygen species (ROS) and cardiac morphology of the myocardial tissue. The effects of SFR on cell contraction, Ca2+ transient and L-type Ca2+ current (ICa-L) in isolated rat myocardial cells were measured using the Ion Optix detection system and the whole-cell patch-clamp technique. SFR can decrease the activity of serum CK, LDH and MDA, and increase the activity of serum SOD, reduce intracellular calcium concentration and the manufacture of ROS. In addition, SFR can improve changes in heart morphology. SFR can significantly inhibit contraction, Ca2+ transients and ICa-L in isolated ventricular myocytes. SFR has a cardioprotective role in ISO-induced MI rats, and the underling mechanism is related to the inhibition of oxidative stress, myocardial contractility, ICa-L and the regulation of Ca2+ homeostasis.

Mechanisms underlying the cardio-protection of total ginsenosides against myocardial ischemia in rats in vivo and in vitro: Possible involvement of L-type Ca2+ channels, contractility and Ca2+ homeostasis.

Here we aimed to observe the effects of total ginsenosides (TG) against isoproterenol (ISO) induced myocardial ischemia (MI) and to explore its underlying mechanisms based on L-type Ca2+ current (ICa-L), intracellular Ca2+ ([Ca2+]i) and contraction in isolated rat myocytes. Rat model of MI was induced by subcutaneously injection of ISO (85 mg/kg) for 2 consecutive days. J-point elevation, heart rate, serum levels of creatine kinase (CK) and lactated dehydrogenase (LDH), and heart morphology changes were observed. Influences of TG on ICa-L, [Ca2+]i and contraction in isolated rat myocytes were observed by the patch-clamp technique and IonOptix detection system. TG significantly reduced J-point elevation, heart rate, serum levels of CK and LDH, and improved heart pathologic morphology. TG decreased ICa-L in concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 31.65 µg/mL. TG (300 µg/mL) decreased ICa-L of normal and ischemic ventricular myocytes by 64.33 ± 1.28% and 61.29 ± 1.38% respectively. At 30 µg/mL, TG reduced Ca2+ transient by 21.67 ± 0.94% and cell shortening by 38.43 ± 6.49%. This study showed that TG displayed cardioprotective effects on ISO-induced MI rats and the underlying mechanisms may be related to inhibition of ICa-L, damping of [Ca2+]i and decrease of contractility.

Metabolic Inhibition Induces Transient Increase of L-type Ca2+ Current in Human and Rat Cardiac Myocytes.

Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during ß-adrenergic stimulation by isoprenaline, a non-selective agonist of ß-adrenergic receptors. The whole-cell patch⁻clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs.

Characterization of the Inhibitory Effect of Gastrodigenin and Gastrodin on M-type K+ Currents in Pituitary Cells and Hippocampal Neurons.

Gastrodigenin (HBA) and gastrodin (GAS) are phenolic ingredients found in Gastrodia elata Blume (GEB), a traditional Chinese herbal medicine. These compounds have been previously used to treat cognitive dysfunction, convulsion, and dizziness. However, at present, there is no available information regarding their potential ionic effects in electrically excitable cells. In the current study, the possible effects of HBA and GAS on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were investigated using the patch-clamp technique. The addition of HBA or GAS resulted in the differential inhibition of the M-type K+ current (IK(M)) density in a concentration-dependent manner in GH3 cells. HBA resulted in a slowing of the activation time course of IK(M), while GAS elevated it. HBA also mildly suppressed the density of erg-mediated or the delayed-rectifier K+ current in GH3 cells. Neither GAS nor HBA (10 µM) modified the voltage-gated Na+ current density, although they suppressed the L-type Ca2+ current density at the same concentration. In hippocampal mHippoE-14 neurons, HBA was effective at inhibiting IK(M) density as well as slowing the activation time course. Taken together, the present study provided the first evidence that HBA or GAS could act on cellular mechanisms, and could therefore potentially have a functional influence in various neurologic disorders.

Heterogeneous functional expression of the sustained inward Na+ current in guinea pig sinoatrial node cells.

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 Ca2+ 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 CaV1.2- and CaV1.3-mediated I Ca,L in SAN cells. Consistent single-cell transcript measurements confirmed a higher relative expression of CaV1.3, 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 CaV1.3-mediated I Ca,L.

Loss of caveolin-3-dependent regulation of ICa in rat ventricular myocytes in heart failure.

ß2-Adrenoceptors and L-type Ca2+ current ( ICa) redistribute from the t-tubules to the surface membrane of ventricular myocytes from failing hearts. The present study investigated the role of changes in caveolin-3 and PKA signaling, both of which have previously been implicated in this redistribution. ICa was recorded using the whole cell patch-clamp technique from ventricular myocytes isolated from the hearts of rats that had undergone either coronary artery ligation (CAL) or equivalent sham operation 18 wk earlier. ICa distribution between the surface and t-tubule membranes was determined using formamide-induced detubulation (DT). In sham myocytes, ß2-adrenoceptor stimulation increased ICa in intact but not DT myocytes; however, forskolin (to increase cAMP directly) and H-89 (to inhibit PKA) increased and decreased, respectively, ICa at both the surface and t-tubule membranes. C3SD peptide (which decreases binding to caveolin-3) inhibited ICa in intact but not DT myocytes but had no effect in the presence of H-89. In contrast, in CAL myocytes, ß2-adrenoceptor stimulation increased ICa in both intact and DT myocytes, but C3SD had no effect on ICa; forskolin and H-89 had similar effects as in sham myocytes. These data show the redistribution of ß2 -adrenoceptor activity and ICa in CAL myocytes and suggest constitutive stimulation of ICa by PKA in sham myocytes via concurrent caveolin-3-dependent (at the t-tubules) and caveolin-3-independent mechanisms, with the former being lost in CAL myocytes. NEW & NOTEWORTHY In ventricular myocytes from normal hearts, regulation of the L-type Ca2+ current by ß2-adrenoceptors and the constitutive regulation by caveolin-3 is localized to the t-tubules. In heart failure, the regulation of L-type Ca2+ current by ß2-adrenoceptors is redistributed to the surface membrane, and the constitutive regulation by caveolin-3 is lost.

Voltage dependence of the Ca2+ transient in endocardial and epicardial myocytes from the left ventricle of Goto-Kakizaki type 2 diabetic rats.

Diabetes mellitus is a major global health disorder and, currently, over 450 million people have diabetes with 90% suffering from type 2 diabetes. Left untreated, diabetes may lead to cardiovascular diseases which are a leading cause of death in diabetic patients. Calcium is the trigger and regulator of cardiac muscle contraction and derangement in cellular Ca2+ homeostasis, which can result in heart failure and sudden cardiac death. It is of paramount importance to investigate the regional involvement of Ca2+ in diabetes-induced cardiomyopathy. Therefore, the aim of this study was to investigate the voltage dependence of the Ca2+ transients in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of the Goto-Kakizaki (GK) rats, an experimental model of type 2 diabetes mellitus. Simultaneous measurement of L-type Ca2+ currents and Ca2+ transients was performed by whole-cell patch clamp techniques. GK rats displayed significantly increased heart weight, heart weight/body weight ratio, and non-fasting and fasting blood glucose compared to controls (CON). Although the voltage dependence of L-type Ca2+ current was unaltered, the voltage dependence of the Ca2+ transients was reduced to similar extents in EPI-GK and ENDO-GK compared to EPI-CON and ENDO-CON myocytes. TPK L-type Ca2+ current and Ca2+ transient were unaltered. THALF decay of L-type Ca2+ current was unaltered; however, THALF decay of the Ca2+ transient was shortened in ENDO and EPI myocytes from GK compared to CON rat hearts. In conclusion, the amplitude of L-type Ca2+ current was unaltered; however, the voltage dependence of the Ca2+ transient was reduced to similar extents in EPI and ENDO myocytes from GK rats compared to their respective controls, suggesting the possibility of dysfunctional sarcoplasmic reticulum Ca2+ transport in the GK diabetic rat hearts.

Important modifications by sugammadex, a modified γ-cyclodextrin, of ion currents in differentiated NSC-34 neuronal cells.

BACKGROUND: Sugammadex (SGX) is a modified γ-cyclodextrin used for reversal of steroidal neuromuscular blocking agents during general anesthesia. Despite its application in clinical use, whether SGX treatment exerts any effects on membrane ion currents in neurons remains largely unclear. In this study, effects of SGX treatment on ion currents, particularly on delayed-rectifier K+ current [I K(DR)], were extensively investigated in differentiated NSC-34 neuronal cells. RESULTS: After cells were exposed to SGX (30 µM), there was a reduction in the amplitude of I K(DR) followed by an apparent slowing in current activation in response to membrane depolarization. The challenge of cells with SGX produced a depolarized shift by 15 mV in the activation curve of I K(DR) accompanied by increased gating charge of this current. However, the inactivation curve of I K(DR) remained unchanged following SGX treatment, as compared with that in untreated cells. According to a minimal reaction scheme, the lengthening of activation time constant of I K(DR) caused by cell treatment with different SGX concentrations was quantitatively estimated with a dissociation constant of 17.5 µM, a value that is clinically achievable. Accumulative slowing in I K(DR) activation elicited by repetitive stimuli was enhanced in SGX-treated cells. SGX treatment did not alter the amplitude of voltage-gated Na+ currents. In SGX-treated cells, dexamethasone (30 µM), a synthetic glucocorticoid, produced little or no effect on L-type Ca2+ currents, although it effectively suppressed the amplitude of this current in untreated cells. CONCLUSIONS: The treatment of SGX may influence the amplitude and gating of I K(DR) and its actions could potentially contribute to functional activities of motor neurons if similar results were found in vivo.

Crocin, a carotenoid component of Crocus cativus, exerts inhibitory effects on L-type Ca(2+) current, Ca(2+) transient, and contractility in rat ventricular myocytes.

Crocin, a carotenoid component of Crocus sativus L. belonging to the Iridaceae family, has demonstrated cardioprotective effects. To investigate the cellular mechanisms of these cardioprotective effects, here we studied the influence of crocin on L-type Ca(2+)current (I(Ca-L)), intracellular Ca(2+) ([Ca(2+)]i), and contraction of isolated rat cardiomyocytes by using the whole-cell patch-clamp technique and video-based edge detection and dual excitation fluorescence photomultiplier systems. Crocin inhibited I(Ca-L) in a concentration-dependent manner with the half-maximal inhibitory concentration (IC50) of 45 µmol/L and the maximal inhibitory effect of 72.195% ± 1.54%. Neither current-voltage relationship of I(Ca-L), reversal potential of I(Ca-L), nor the activation/inactivation of I(Ca-L) was significantly changed. Crocin at 1 µmol/L reduced cell shortening by 44.64% ± 2.12% and the peak value of the Ca(2+) transient by 23.66% ± 4.52%. Crocin significantly reduced amplitudes of myocyte shortening and [Ca(2+)]i with an increase in the time to reach 10% of the peak (Tp) and a decrease in the time to 10% of the baseline (Tr). Thus, the cardioprotective effects of crocin may be attributed to the attenuation of [Ca(2+)]i through the inhibition of I(Ca-L) in rat cardiomyocytes and negative inotropic effects on myocardial contractility.

Atorvastatin blocks increased l-type Ca2+ current and cell injury elicited by angiotensin II via inhibiting oxide stress.

Thel-type Ca(2+)current (ICa,l) plays a crucial role in shaping action potential and is involved in cardiac arrhythmia. Statins have been demonstrated to contribute to anti-apoptotic and anti-arrhythmic effects in the heart. Here, we examined whether atorvastatin regulates theICa,land cell injury induced by angiotensin II (AngII) as well as the putative intracellular cascade responsible for the effects. Cultured neonatal rat ventricular myocytes were incubated with AngII for 24 h, and then cell injury and expression levels of Nox2/gp91(phox), p47(phox) ,and Cav1.2 were analyzed. In addition,ICa,lwas recorded using the whole-cell patch-clamp technique, and mechanisms of atorvastatin actions were also investigated. It was found that the number of apoptotic cardiomyocytes was increased and cell viability was significantly decreased after AngII administration. AngII also augmented the expressions of Nox2/gp91(phox)and p47(phox)compared with control cardiomyocytes. Exposure to AngII evokedICa,lin a voltage-dependent manner without affecting theI-Vrelationship. In addition, AngII enhanced membrane Cav1.2 expression. These effects were abolished in the presence of the reactive oxygen species (ROS) scavenger, manganese (III)-tetrakis 4-benzoic acid porphyrin [Mn(III)TBAP], or the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, atorvastatin. These results suggested that atorvastatin mediates cardioprotection against arrhythmias and cell injury by controlling the AngII-ROS cascade.

Adenylyl cyclase subtype-specific compartmentalization: differential regulation of L-type Ca2+ current in ventricular myocytes.

RATIONALE: Adenylyl cyclase (AC) represents one of the principal molecules in the ß-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. OBJECTIVE: We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. METHODS AND RESULTS: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for ß1-adrenergic receptor signaling-mediated enhancement of the L-type Ca(2+) current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. CONCLUSIONS: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of ß- adrenergic receptor and AC compartmentalization may also be important for ß-adrenergic receptor signaling in other cell types.

KCNE2 modulates cardiac L-type Ca(2+) channel.

KCNE2 plays an important role in maintaining cardiac electrical stability. Mutations in KCNE2 have been linked to long-QT syndrome (LQT6) and atrial fibrillation/short QT syndrome. It has been suggested that KCNE2 has the most promiscuity of function which can interact with multiple-subunits of voltage-dependent cation channels and modulate their functions. However, whether KCNE2 regulates voltage-dependent L-type Ca(2)(+) channel (LCC) remains unknown. This study investigated the possible role of KCNE2 in regulating cardiac LCCs and the pathophysiological relevance of this regulation. We found that overexpression of KCNE2 in Sprague-Dawley rat cardiomyocytes decreased L-type Ca(2+)current (ICa,L), whereas KCNE2 knockdown by RNA interference increased ICa,L. Upregulation of KCNE2 caused a slight positive shift of the voltage-dependent activation and a negative shift of the steady-state voltage-dependent inactivation, and slowed the recovery from inactivation of ICa,L, while knockdown of KCNE2 had the contrary effects. Similar regulation of ICa,L magnitude had been observed in transfected HEK 293 cells. Coimmunoprecipitation and colocalization assays in both cardiomyocytes and the transfected cell line suggest that Cav1.2 physically interacted with KCNE2. Deletion of the N-terminal inhibitory module (NTI) of Cav1.2 results in the large loss of KCNE2 regulation of ICa,L and interaction with Cav1.2. Furthermore, we found that the familial atrial fibrillation related KCNE2 mutation R27C enhanced the effect of KCNE2 on suppressing ICa,L. Taken together, our findings indicate that KCNE2 modulates ICa,L by regulating NTI function of Cav1.2. The KCNE2 mutation R27C may induce familial atrial fibrillation partially through enhancing the suppression of ICa,L.

The inotropic and arrhythmogenic effects of acutely increased late INa are associated with elevated ROS but not oxidation of PKARIα.

Background: Acute stimulation of the late sodium current (INaL) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient upon oxidative stress. Objectives: We aimed to investigate whether the increase of the systolic Ca2+ transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa through oxidized PKARIα. Methods: We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca2+ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and PKA target structures was studied using Western blot analysis. Results: ATX-II increased INaL to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations during Ca2+ transient decay and by diastolic SR Ca2+-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of ICa in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment. Conclusions: The inotropic and arrhythmogenic effects of acutely increased INaL are associated with elevated ROS, but do not involve oxidation of PKARIα.

Impaired Na⁺-dependent regulation of acetylcholine-activated inward-rectifier K⁺ current modulates action potential rate dependence in patients with chronic atrial fibrillation.

Shortened action-potential duration (APD) and blunted APD rate adaptation are hallmarks of chronic atrial fibrillation (cAF). Basal and muscarinic (M)-receptor-activated inward-rectifier K(+) currents (IK1 and IK,ACh, respectively) contribute to regulation of human atrial APD and are subject to cAF-dependent remodeling. Intracellular Na(+) ([Na(+)]i) enhances IK,ACh in experimental models but the effect of [Na(+)]i-dependent regulation of inward-rectifier K(+) currents on APD in human atrial myocytes is currently unknown. Here, we report a [Na(+)]i-dependent inhibition of outward IK1 in atrial myocytes from sinus rhythm (SR) or cAF patients. In contrast, IK,ACh activated by carbachol, a non-selective M-receptor agonist, increased with elevation of [Na(+)]i in SR. This [Na(+)]i-dependent IK,ACh regulation was absent in cAF. Including [Na(+)]i dependence of IK1 and IK,ACh in a recent computational model of the human atrial myocyte revealed that [Na(+)]i accumulation at fast rates inhibits IK1 and blunts physiological APD rate dependence in both groups. [Na(+)]i-dependent IK,ACh augmentation at fast rates increased APD rate dependence in SR, but not in cAF. These results identify impaired Na(+)-sensitivity of IK,ACh as one potential mechanism contributing to the blunted APD rate dependence in patients with cAF. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".