Channels In Rat Ventricular Myocytes Research Articles

Inhibitory Effects of Nobiletin on Voltage-Gated Na+ Channel in Rat Ventricular Myocytes Based on Electrophysiological Analysis and Molecular Docking Method.

Nobiletin (NOB) has attracted much attention owing to its outstanding bioactivities. This study aimed to investigate its anti-arrhythmic effect through electrophysiological and molecular docking studies. We assessed the anti-arrhythmic effects of NOB using aconitine-induced ventricular arrhythmia in a rat model and the electrophysiological effects of NOB on rat cardiomyocytes utilizing whole-cell patch-clamp techniques. Moreover, we investigated the binding characters of NOB with rNav1.5, rNav1.5/QQQ, and hNaV1.5 via docking analysis, comparing them with amiodarone and aconitine. NOB pretreatment delayed susceptibility to ventricular premature and ventricular tachycardia and decreased the incidence of fatal ventricular fibrillation. Whole-cell patch-clamp assays demonstrated that the peak current density of the voltage-gated Na+ channel current was reversibly reduced by NOB in a concentration-dependent manner. The steady-state activation and recovery curves were shifted in the positive direction along the voltage axis, and the steady-state inactivation curve was shifted in the negative direction along the voltage axis, as shown by gating kinetics. The molecular docking study showed NOB formed a π-π stacking interaction with rNav1.5 and rNav1.5/QQQ upon Phe-1762, which is the homolog to Phe-1760 in hNaV1.5 and plays an important role in antiarrhythmic action This study reveals that NOB may act as a class I sodium channel anti-arrhythmia agent.

Correction to: Inhibitory effects of aloperine on voltage-gated Na+ channels in rat ventricular myocytes

Correction to: Inhibitory effects of aloperine on voltage-gated Na+ channels in rat ventricular myocytes

Inhibitory effects of aloperine on voltage-gated Na+ channels in rat ventricular myocytes.

Aloperine (ALO), a quinolizidine alkaloid extracted from Sophora alopecuroides L., modulates hypertension, ventricular remodeling, and myocardial ischemia. However, few studies have evaluated the effects of ALO on other cardiovascular parameters. Accordingly, in this study, we used a rat model of aconitine-induced ventricular arrhythmia to assess the effects of ALO. Notably, ALO pretreatment delayed the onset of ventricular premature and ventricular tachycardia and reduced the incidence of fatal ventricular fibrillation. Moreover, whole-cell patch-clamp assays in rats' ventricular myocyte showed that ALO (3, 10, and 30 μM) significantly reduced the peak sodium current density of voltage-gated Na+ channel currents (INa) in a concentration-dependent manner. The gating kinetics characteristics showed that the steady-state activation and recovery curve were shifted in positive direction along the voltage axis, respectively, and the steady-state inactivation curve was shifted in negative direction along the voltage axis, i.e., which was similar to the inhibitory effects of amiodarone. These results indicated that ALO had anti-arrhythmic effects, partly attributed to INa inhibition. ALO may act as a class I sodium channel anti-arrhythmia agent.

Effects of thrombospondin-4 on voltage-gated ion channels in rat ventricular myocytes

Effects of thrombospondin-4 on voltage-gated ion channels in rat ventricular myocytes

Alterations of contractions and L-type Ca2+ currents by murrayafoline-A in rat ventricular myocytes

We examined the effects of murrayafoline-A (1-methoxy-3-methylcarbazole, Mu-A), which is isolated from the dried roots of Glycosmis stenocarpa, on cell shortenings and L-type Ca2+ currents (ICa,L) in rat ventricular myocytes. Cell shortenings and ICa,L were measured using the video edge detection method and patch-clamp techniques, respectively. Mu-A transiently increased cell shortenings in a concentration-dependent manner with an EC50 of ~20μM. The maximal effect of Mu-A, approximately 175% of the control, was observed at ≥100μM. The positive inotropic effect of Mu-A (25μM) reached a maximum after ~2-min exposures, and then decayed after a ~1-min steady-state. During the Mu-A-induced positive inotropy, the rate of contraction was accelerated, whereas the rate of relaxation was not significantly altered. To understand the possible mechanism for the Mu-A-induced positive inotropy, the ICa,L was assessed. Mu-A transiently enhanced the ICa,L. Concentration-dependence of the increase in ICa,L by Mu-A was similar to that of positive inotropic effect of Mu-A. The maximal effect of Mu-A (25μM) on ICa,L was observed at 2−3min after the application of Mu-A. A partial inhibition of ICa,L using verapamil (1μM) induced a right shift of concentration-response curve of the positive inotropic effect of Mu-A and significantly attenuated the effect. These results suggest that Mu-A may transiently enhance contractility, at least in part, by increasing the Ca2+ influx through the L-type Ca2+ channels in rat ventricular myocytes.

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes.

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. In the presence of 1-10 μM AEA, suppression of both Na(+) and L-type Ca(2+) channels was observed. Inhibition of Na(+) channels was voltage and Pertussis toxin (PTX) - independent. Radioligand-binding studies indicated that specific binding of [(3) H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd . Further studies on L-type Ca(2+) channels indicated that AEA potently inhibited these channels (IC50 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba(2+) currents. MetAEA also inhibited Na(+) and L-type Ca(2+) currents. Radioligand studies indicated that specific binding of [(3) H]isradipine, was inhibited significantly by metAEA. (10 μM), changing Bmax but not Kd . Results indicate that AEA inhibited the function of voltage-dependent Na(+) and L-type Ca(2+) channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation.

Activation of Small Conductance Calcium-Activated Potassium Channels by Sarcoplasmic Reticulum Calcium Release Attenuates Delayed Afterdepolarizations in Ventricular Myocytes

Small conductance calcium-activated potassium (SK) channels are upregulated in ventricular myocytes from human patients and animal models of heart failure (HF). However, their activation mechanism and function in ventricular myocytes remain elusive. We overexpressed SK2 channels in adult rat ventricular myocytes using adenovirus gene transfer to test the hypotheses that activation of SK channels in ventricular myocytes requires calcium release from sarcoplasmic reticulum (SR), and that upregulation of SK currents contributes to reducing triggered activity. Simultaneous voltage clamp and confocal calcium imaging experiments in SK2-overexpressing cells demonstrated that depolarizing voltage steps resulted in transient outward currents sensitive to the specific SK channel inhibitor apamin. SR calcium release induced by rapid application of 10 mM caffeine evoked repolarizing SK currents, whereas complete exhaustion of SR calcium stores eliminated SK currents in response to depolarizing voltage steps, despite intact calcium influx through L-type calcium channels. Furthermore, apamin-sensitive SK currents were activated by pro-arrhythmic global spontaneous SR calcium release events (calcium waves, SCWs). Current-clamp experiments demonstrated that SK overexpression reduced the amplitude of delayed afterdepolarizations (DADs) resulting from SCWs and shortened action potential duration (APD). Immuno-localization studies revealed that overexpressed SK channels were distributed both at external sarcolemmal membranes and along the Z-lines, resembling the distribution of endogenous SK channels. In summary, SR calcium release is both necessary and sufficient for the activation of SK channels in rat ventricular myocytes. SK currents contribute to repolarization during action potentials and attenuate DADs driven by SCWs. Thus, SK upregulation in HF may have an anti-arrhythmic effect by shortening APD and reducing triggered activity.

Sarcoplasmic reticulum Ca²⁺ release is both necessary and sufficient for SK channel activation in ventricular myocytes.

SK channels are upregulated in human patients and animal models of heart failure (HF). However, their activation mechanism and function in ventricular myocytes remain poorly understood. We aim to test the hypotheses that activation of SK channels in ventricular myocytes requires Ca(2+) release from sarcoplasmic reticulum (SR) and that SK currents contribute to reducing triggered activity. SK2 channels were overexpressed in adult rat ventricular myocytes using adenovirus gene transfer. Simultaneous patch clamp and confocal Ca(2+) imaging experiments in SK2-overexpressing cells demonstrated that depolarizations resulted in Ca(2+)-dependent outward currents sensitive to SK inhibitor apamin. SR Ca(2+) release induced by rapid application of 10 mM caffeine evoked repolarizing SK currents, whereas complete depletion of SR Ca(2+) content eliminated SK currents in response to depolarizations, despite intact Ca(2+) influx through L-type Ca(2+) channels. Furthermore, voltage-clamp experiments showed that SK channels can be activated by global spontaneous SR Ca(2+) release events Ca(2+) waves (SCWs). Current-clamp experiments revealed that SK overexpression reduces the amplitude of delayed afterdepolarizations (DADs) resulting from SCWs and shortens action potential duration. Immunolocalization studies showed that overexpressed SK channels are distributed both at external sarcolemmal membranes and along the Z-lines, resembling the distribution of endogenous SK channels. In summary, SR Ca(2+) release is both necessary and sufficient for the activation of SK channels in rat ventricular myocytes. SK currents contribute to repolarization during action potentials and attenuate DADs driven by SCWs. Thus SK upregulation in HF may have an anti-arrhythmic effect by reducing triggered activity.

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

The inhibitory effect of BIM (I) on L-type Ca2+ channels in rat ventricular cells

We investigated the effect of a specific protein kinase C (PKC) inhibitor, bisindolylmaleimide I [BIM (I)], on L-type Ca2+ channels in rat ventricular myocytes. BIM (I) alone inhibited the L-type Ca2+ current in a concentration-dependent manner, with a Kd value of 3.31±0.25μM, and a Hill coefficient of 2.34±0.23. Inhibition was immediate after applying BIM (I) in the bath solution and then it partially washed out. The steady-state activation curve was not altered by applying 3μM BIM (I), but the steady-state inactivation curve shifted to a more negative potential with a change in the slope factor. Other PKC inhibitors, PKC-IP and chelerythrine, showed no significant effects either on the L-type Ca2+ current or on the inhibitory effect of BIM (I) on the L-type Ca2+ current. The results suggest that the inhibitory effect of BIM (I) on the L-type Ca2+ current is independent of the PKC pathway. Thus, our results should be considered in studies using BIM (I) to inhibit PKC activity and ion channel modulation.

Effects of Taurine–Magnesium Coordination Compound on Ionic Channels in Rat Ventricular Myocytes of Arrhythmia Induced by Ouabain

Taurine-magnesium coordination compound (TMCC) has anti-arrhythmic effects. The aim of the present study was to explore the targets of the anti-arrhythmic effect of TMCC and the electrophysiological effects of TMCC on ouabain-induced arrhythmias in rat ventricular myocytes. Sodium current (I(Na)), L-type calcium current (I(ca, L)), and transient outward potassium current (I(to)) were measured and analyzed using whole-cell patch-clamp recording technique in normal rat cardiac myocytes and rat ventricular myocytes of arrhythmia induced by ouabain. In isolated ventricular myocytes, I(Na) and I(to) were blocked by TMCC (100, 200, 400 μM) in a concentration-dependent manner, and the effects of TMCC (400 μM) were equal to that of amiodarone. However, I (ca, L) was moderately increased by TMCC (400 μM) while significantly decreased by amiodarone. Ouabain (5 μM) significantly decreased sodium, L-type calcium, and transient outward potassium currents. TMCC (100 μM) relieved abnormal sodium currents induced by ouabain through facilitation of steady-state inactivation. TMCC (200 and 400 μM) relieved abnormal L-type calcium currents induced by ouabain through facilitation of steady-state activation and retardation of steady-state inactivation. TMCC failed to further inhibit abnormal transient outward potassium currents induced by ouabain. However, amiodarone inhibited the decreasing sodium, L-type calcium, and transient outward potassium currents further. These data suggest that I(Na), I(ca, L), and I(to) may be the targets of the antiarrhythmic effect of TMCC, which can antagonize ouabain-induced changes of ionic currents in rat ventricular myocytes.

Wide Spectrum of Inhibitory Effects of Sertraline on Cardiac Ion Channels

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In these experiments, we have used the whole cell patch clamp technique to examine the effects of sertraline on the major cardiac ion channels expressed in HEK293 cells and the native voltage-gated Ca2+ channels in rat ventricular myocytes. According to the results, sertraline is a potent blocker of cardiac K+ channels, such as hERG, IKs and IK1. The rank order of inhibitory potency was hERG >IK1> IKs with IC50 values of 0.7, 10.5, and 15.2 µM, respectively. In addition to K+ channels, sertraline also inhibited INa and ICa, and the IC50 values are 6.1 and 2.6 µM, respectively. Modification of these ion channels by sertraline could induce changes of the cardiac action potential duration and QT interval, and might result in cardiac arrhythmia.

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of Ito and augmentation of KATP channels

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.

The inhibitory effect of Ca 2+-activated K + channel activator, BMS on L-type Ca 2+ channels in rat ventricular myocytes

Aims We investigated the effects of BMS-204352 (BMS), a big-conductance calcium-activated potassium (BK Ca) channel activator, on L-type Ca 2+ channels. Main methods Electrophysiological recordings were performed in isolated rat ventricular myocytes. Whole-cell configuration was used. Key findings BMS caused inhibition of the Ca 2+ current in a dose-dependent manner, with K d of 6.00 ± 0.67 μM and a Hill coefficient of 1.33 ± 0.18. BMS did not affect the steady-state activation of L-type Ca 2+ channels. However, for those in steady-state inactivation, BMS shifted the half-maximal potential ( V 1/2) by − 11 mV, but the slope value ( k) was not altered. Iberiotoxin, inhibitor of membrane BK Ca channels and paxilline, inhibitor of mitochondrial BK Ca channel did not affect the inhibitory effect of BMS on L-type Ca 2+ channels. Pretreatment with inhibitors of protein kinase A (PKA), protein kinase C (PKC), and protein kinase G (PKG) did not significantly alter the inhibitory effect of BMS on L-type Ca 2+ current. The presence of a selective β-adrenergic receptor agonist, isoproterenol did not affect the inhibitory effect of BMS on L-type Ca 2+ current. Based on these results, we concluded that the inhibition of L-type Ca 2+ channels by BMS is independent of the inhibition of BK Ca channels or intracellular signaling pathways. Significance It is important to take BMS-204352 (BMS) effects on L-type Ca 2+ channels into consideration when using BMS as a BK Ca channel activator or therapeutic target in ventricular myocytes.

Hyposmotic challenge modulates function of L-type calcium channel in rat ventricular myocytes through protein kinase C

To study the effects and mechanisms by which hyposmotic challenge modulate function of L-type calcium current (I(Ca,L)) in rat ventricular myocytes. The whole-cell patch-clamp techniques were used to record I(Ca,L) in rat ventricular myocytes. Hyposmotic challenge(∼220 mosmol/L) induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease. I(Ca,L) increased by 19.1%±6.1% after short exposure (within 3 min) to hyposmotic solution. On the contrary, long hyposmotic challenge (10 min) decreased I(Ca,L) to 78.1%±11.0% of control, caused the inactivation of I(Ca,L), and shifted the steady-state inactivation curve of I(Ca,L) to the right. The decreased I(Ca,L) induced by hyposmotic swelling was reversed by isoproterenol or protein kinase A (PKA) activator foskolin. Hyposmotic swelling also reduced the stimulated I(Ca,L) by isoproterenol or foskolin. PKA inhibitor H-89 abolished swelling-induced transient increase of I(Ca,L), but did not affect the swelling-induced sustained decrease of I(Ca,L). NO donor SNAP and protein kinase G (PKG) inhibitor Rp-8-Br-PET-cGMPS did not interfere with swelling-induced biphasic changes of I(Ca,L). Protein kinase C (PKC) activator PMA decreased I(Ca,L) and hyposmotic solution with PMA reverted the decreased I(Ca,L) by PMA. PKC inhibitor BIM prevented the swelling-induced biphasic changes of I(Ca,L). Hyposmotic challenge induced biphasic changes of I(Ca,L), a transient increase followed by a sustained decrease, in rat ventricular myocytes through PKC pathway, but not PKG pathway. PKA system could be responsible for the transient increase of I(Ca,L) during short exposure to hyposmotic solution.

Epigallocatechin-3-Gallate Protects Na+ Channels in Rat Ventricular Myocytes Against Sulfite

Sulfite (bisulfite/sulfite) can affect voltage-gated sodium (Na(+)) channels (VGSC) in a concentration-dependent manner in isolated rat ventricular myocytes. In this study, the effect of epigallocatechin-3-gallate (EGCG) on VGSC in isolated ventricular myocytes was studied. Ventricular myocytes were exposed to 10 microM bisulfite/sulfite for 10 min, and EGCG was then administered in different concentrations (10, 30, 50 microg ml(-1)). Decreased activity of superoxide dismutase, catalase (CAT) and glutathione peroxidase (GPx) was observed after bisulfite/sulfite exposure, with significant increase in Na(+) currents (I (Na)) and alterations in half-activation voltage and half-inactivation voltage. Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)), hydroxyl (OH(.)), and superoxide anion (O (2) (.-) ) were increased. After EGCG treatment, activity of the aforementioned enzymes increased while the ROS level decreased. The effects progressed with increasing amounts of EGCG, up to a level similar to blank control at the dose of 50 microg ml(-1) EGCG, EGCG also reduced the I (Na) and reversed the alterations in half-activation voltage and half-inactivation voltage. In conclusion, EGCG could protect Na(+) channels in rat ventricular myocytes against the oxidative damage induced by sulfite as a scavenger of the ROS.

Constitutive CaMKII activity regulates Na + channel in rat ventricular myocytes

The cardiac voltage-gated Na + channel controls the upstroke of action potential and membrane excitability. The Na + channel associates with Ca 2+/CaM-dependent protein kinase (CaMKII), but the role of CaMKII on Na + channel activity in the resting state is not clear. In this report, we investigated whether CaMKII constitutively regulates Na + currents ( I Na), independent of Ca 2+ influx in rat ventricular myocytes using patch clamp technique. CaMKII inhibition (by KN93 or autocamtide-related inhibitory peptide) caused a negative shift in I Na steady-state inactivation and delayed recovery from slow inactivation, limiting channel availability. The reduction of I Na was 29.47 ± 3.01% at a holding potential ( V h) of − 120 mV and it increased to 77.70 ± 7.92% when V h was − 70 mV, suggesting that near the resting membrane potential, three-quarters of I Na depends on CaMKII action. CaMKII inhibition also enhanced intermediate inactivation, as well as delayed recovery from fast inactivation, and decreased late I Na. KN92, an inactive analog of KN93, had no effect on I Na. Using an antibody against phosphorylated (activated) CaMKII, we found that constitutively active CaMKII co-immunoprecipitated with Na + channels under resting conditions. CaMKII inhibitors reduced the level of phosphorylated CaMKII, which correlated with the degree of reduction in channel availability. These data suggest that CaMKII in an active form contributes to regulating I Na. Finally, we observed a drastic reduction in the upstroke velocity of action potentials upon CaMKII inhibition. In conclusion, CaMKII constitutively regulates cardiac Na + channel and this regulatory mechanism is important for the maintenance of Na + channel characteristics under physiological conditions.

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca2+-activated K+ (mitoBKCa) channel activator NS 1619 on L-type Ca2+ channels in rat ventricular myocytes. NS 1619 inhibited the Ca2+ current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BKCa channel, mitoBKCa channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca2+ current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective β-adrenoreceptor agonist, reduced the Ca2+ current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca2+ current independent of the mitoBKCa channel and protein kinases. Since NS 1619 is widely used to study mitoBKCa channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.

Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts

Cardiac voltage-gated Ca 2+ channels regulate the intracellular Ca 2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca 2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive Ca V1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant Ca V1.2 channel (Ca V1.2DHP i) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca 2+ current ( I Ca) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC 50 of 66 nM and complete block at 250 nM. In contrast, I Ca in cells infected with AdCa V1.2DHP i was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 μM diltiazem. In order to study Ca V1.2DHP i in isolation, myocytes infected with AdCa V1.2DHP i were incubated with nisoldipine. Under this condition the cells expressed a large I Ca (12 pA/pF) and displayed Ca 2+ transients during field stimulation. Furthermore, addition of 2 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased I Ba in the AdCa V1.2DHP i-infected cells. A Cd 2+-sensitive I Ba was also recorded in cardiac fibroblasts infected with AdCa V1.2DHP i. Thus, expression of Ca V1.2DHP i will provide an important tool in studies of cardiac myocyte and fibroblast function.

VISUALIZING Ca2+ SPARKS AND SUBSTRUCTURE OF Ca2+ WAVES BY TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY (TIRFM)

Based on near-field optical theory, total internal reflection fluorescence microscope shows a novel character that its picture has great signal-to-noise ratio and high temporal resolution achieved by high quality CCD camera. This allows us to analyze the spatiotemporal details of local Ca 2+ dynamics within the nanoscale microdomain surrounding different Ca 2+ channels. We have recently constructed a versatile objective TIRFM equipped with a high numerical aperture (NA = 1.45) objective. Using fluo-4 as the Ca 2+ indicator, we visualized the near-membrane profiles of Ca 2+ waves and elementary Ca 2+ sparks generated by Ca 2+ release channels in rat ventricular myocytes. Different from those detected using conventional or confocal microscopy, Ca 2+ waves observed with TIRFM exhibited fine inhomogenous substructures. The propagation of Ca 2+ waves with anfractuous routes of spark recruitment is much more complicated than previously imagined. We believe that TIRFM will provide a unique tool for dissecting the microscopic mechanisms of intracellular Ca 2+ signaling.