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(Circulation Research. 2005;96:64.)
© 2005 American Heart Association, Inc.

Cellular Biology

Role of Nitric Oxide in Ca²⁺ Sensitivity of the Slowly Activating Delayed Rectifier K⁺ Current in Cardiac Myocytes

Chang-Xi Bai, Iyuki Namekata, Junko Kurokawa, Hikaru Tanaka, Koki Shigenobu, Tetsushi Furukawa

From the Department of Bio-informational Pharmacology (C.-X.B., J.K., T.F.), Medical Research Institute, Tokyo Medical and Dental University, Tokyo; and the Department of Pharmacology (I.N., H.T., K.S.), Toho University School of Pharmaceutical Sciences, Funabashi city, Chiba, Japan.

Correspondence to Tetsushi Furukawa, MD, Department of Bio-informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail t_furukawa.bip{at}mri.tmd.ac.jp

	Abstract

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Sarcolemmal Ca²⁺ entry is a vital step for contraction of cardiomyocytes, but Ca²⁺ overload is harmful and may trigger arrhythmias and/or apoptosis. To maintain the amount of Ca²⁺ entry within an appropriate range, cardiomyocytes have feedback systems that tightly regulate ion channel activities in response to the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), thereby regulating Ca²⁺ entry. In guinea pig ventricular myocytes, Ca²⁺ ionophore, A23187, induced suppression of the L-type Ca²⁺ currents (I_Ca,L) and enhancement of the slowly activating delayed rectifier K⁺ currents (I_Ks). At a low stimulation rate, I_Ca,L suppression and I_Ks enhancement contributed to the A23187-induced APD shortening with a similar magnitude, whereas at a high stimulation rate, I_Ks enhancement dominantly contributed to APD shortening. I_Ks enhancement induced by A23187 was attributable to actions of nitric oxide (NO), because they were inhibited by an inhibitor of NO synthase (NOS) and by a NO scavenger. A23187-induced alterations of APD and I_Ks were strongly suppressed by a NOS3 inhibitor, but barely affected by a NOS1 inhibitor, suggesting that NOS3 was responsible for NO release in this phenomenon. Inhibition of calmodulin (CaM), but not Akt, blocked the enhancement of I_Ks by A23187. Thus, CaM-dependent NOS3 activation confers the selective Ca²⁺-sensitivity on I_Ks. Ca²⁺-induced I_Ks enhancement and resultant APD shortening potentially act as a physiological regulatory mechanism of Ca²⁺ recycling, because they were observed at a physiological range of [Ca²⁺]_i in cardiac myocytes and are induced by physiologically relevant Ca²⁺ loading, such as digitalis application and rise in extracellular Ca²⁺ concentration.

Key Words: ion channels • nitric oxide synthases • calmodulin • protein-protein interaction • caveolin

	Introduction

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To maintain the amount of Ca²⁺ entry within a narrow range, cardiomyocytes develop feedback systems that tightly regulate ion channel activities and thereby Ca²⁺ entry in response to the changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The elevation of [Ca²⁺]_i directly regulates Ca²⁺ entry pathway, the L-type Ca²⁺ channel.^1–3 It also alters the duration of action potential plateau and regulates the duration where the L-type Ca²⁺ channel can open. The slope of action potential plateau is mainly dependent on the balance between L-type Ca²⁺ channel currents (I_Ca,L) and delayed rectifier K⁺ currents. Delayed rectifier K⁺ currents consist of two components: the rapidly activating component (I_Kr) that is sensitive to a class III antiarrhythmic drug E-4031 and the slowly activating component (I_Ks) that is E-4031-resistant.⁴ Elevations in [Ca²⁺]_i selectively enhances the I_Ks component.^4,5 The range of [Ca²⁺]_i that regulates I_Ks (between 10^–8 and 10^–6 mol/L) is in the range that alters during the cycle of contraction and relaxation of cardiac myocytes.^5,6 Thus, the Ca²⁺ sensitivity of I_Ks may potentially act as a physiological feedback system to regulate [Ca²⁺]_i.

In a previous article, we showed that I_Ks enhancement by elevation of [Ca²⁺]_i was inhibited by a calmodulin (CaM) inhibitor, but not by an inhibitor of CaM-dependent kinase II.⁶ Although this finding suggests that the allosteric regulation of CaM is crucial for Ca²⁺-sensitive I_Ks alterations, the detailed underlying mechanism remains unknown. Recently, we found that I_Ks was enhanced by nitric oxide (NO) via a cGMP-independent mechanism.⁷ Because allosteric interaction of Ca²⁺/CaM complex with NO synthase (NOS) is a major mechanism of NOS activation and NO release,^8,9 we tested in the present study if NO plays an important role in the Ca²⁺-sensitive modulation of I_Ks in cardiac myocytes.

	Materials and Methods

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The investigation was conducted in accordance with the rules and regulations of the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.

Quantification of [Ca²⁺]_i
Quantification of [Ca²⁺]_i was performed as described previously.¹⁰ (See the Expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.) Briefly, guinea pig ventricular myocytes were loaded with 10 µmol/L indo-1-AM (Dojin), and Ca²⁺ transients were elicited by field-stimulation at 1 Hz. Cells were excited at 360 nm from a Xenon lamp and the emission bands, 395 to 415 nm and 470 to 490 nm, were separated (W-VIEW system, Hamamatsu Photonics), detected by a high-speed cooled CCD camera (HISCA, Hamamatsu Photonics), and ratio of the intensity of two emission bands was calculated (Aquacosmos software, Hamamatsu Photonics).

Patch Clamp
Single ventricular myocytes were harvested from hearts of adult guinea pigs (n=41, white Hartrey; Saitama Experimental Animal Supply Co Ltd, Saitama, Japan).¹¹ Action potentials and membrane currents were recorded with an Axopatch 200B amplifier (Axon Instruments) using the perforated configuration of the patch-clamp technique with Amphotericin B (Sigma-Aldrich), in a current-clamp mode and a voltage-clamp mode. Action potentials were elicited by passing depolarizing current pulses (<2 ms in duration at a rate of 1 Hz) of suprathreshold intensity. I_Ks were elicited by 3.5-second depolarizing pulses to various test potentials between –30 and +50 mV in 10-mV increments at 0.1 Hz from a holding potential of –40 mV, and amplitudes of tail I_Ks were measured by extrapolating from exponential fits. Compositions of solutions used are described in the online data supplement. All experiments were performed at 36±1°C. The averaged series resistance was 15.7±1.7 M, the capacitance time constant was 2.5±0.3 ms, and the membrane capacitance was 150±13 pF.

Immunoprecipitation and Immunoblotting
Cardiomyocytes were isolated from adult guinea pigs (n=3),¹² and immunoprecipitation and immunoblotting were performed as described previously (see online data supplement).¹³ Briefly, cell lysates were immunoprecipitated with a monoclonal anti-NOS3 antibody (Zymed), followed by immunoblot analysis with an anti-NOS3 antibody (Zymed), an anti-caveolin-3 (Cav-3) antibody (N-18, Santa Cruz Biotechnology), or an anti-CaM antibody (FL-149, Santa Cruz Biotechnology) followed by incubation with a horseradish peroxidase-conjugated anti-mouse IgG (DAKO Japan Co Ltd) or an anti-rabbit IgG (DAKO Japan Co Ltd). Proteins were detected using the advance enhanced chemiluminescence system (Amersham Bioscience).

Reagents
Chromanol 293 B was supplied by Hoechst. E-4031 was purchased from Eisai Co Ltd, A23187, W7 and W5 from Wako, SH-6 from Merck, digoxin from Nacalai tesque, and all other reagents from Sigma-Aldrich. A23187 was prepared as a 10 mmol/L stock solution in ethanol. Stock solutions of E-4031 (5 mmol/L), W7 (10 mmol/L), W5 (10 mmol/L), S-methylisothiourea (SMTU; 10 mmol/L), N-acetyl-L-cysteine (L-NAC; 100 mmol/L), S-methyl-L-thiocitrulline (SMTC; 5 mmol/L), L-N₅-(L-lminoethyl)ornithine (L-NIO; 1 mmol/L), and sodium nitroprusside (SNP; 1 mol/L) were prepared in distilled water, and nisoldipine (10 mmol/L), SH-6 (20 mmol/L), and 2-(4-morpholinyl)-8-phenyl-1-(4H)-benzopyran-4-one hydrochloride (LY-294,002; 60 mmol/L) stock solutions in dimethylsulfoxide. They were diluted in the bath solution to achieve the desired concentration.

Data Analysis
All values are presented as mean±SE. Statistical significance was determined by repeated-measure one-way analysis of variance (ANOVA) followed by Scheffé F test. Values P<0.05 are considered to be significant.

	Results

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Elevation of [Ca²⁺]_i by A23187 Shortened Action Potential Duration and Enhanced I_Ks
Addition of A23187 to the bath solution increased [Ca²⁺]_i both in the basal level and at the peak of Ca²⁺ transient (Figure 1A). The basal [Ca²⁺]_i was 0.12±0.01 µmol/L (n=10) in the control state, 0.16±0.03 µmol/L (n=5) in the presence of A23187 at 0.1 µmol/L (P=NS versus control), and 0.22±0.02 µmol/L (n=5) at 2 µmol/L of A23187 (P<0.05 versus control). The peak [Ca²⁺]_i was 0.97±0.03 µmol/L (n=10) in the control state, 1.24±0.03 µmol/L (n=5) at 0.1 µmol/L of A23187 (P<0.05 versus control), and 1.52±0.09 µmol/L (n=5) at 2 µmol/L of A23187 (P<0.05 versus control).

View larger version (22K): [in this window] [in a new window]   Figure 1. Effects of A23187 on [Ca2+]i, APD, and IKs. A, Effects of A23187 on [Ca2+]i. a and b, Representative Ca2+ transient elicited in the control state (a) and in the presence of A23187 at 2 µmol/L (b). c, Averaged [Ca2+]i in the basal state (closed bars) and at the peak of Ca2+ transient (open bars) in the control state, and in the presence of A23187 at 0.1 µmol/L and at 2 µmol/L. In this and following figures, numbers in parentheses represent number of experiments. *P<0.05, #P=NS. B, A23187 (2 µmol/L) application reversibly shortened APD. Left panel, Averaged APD20 (closed symbols) or APD90 (open symbols), elicited at 1 Hz, normalized to the initial value in 5 experiments. Closed and open circles are data without drug application (control), and closed and open triangles are data for A23187 application. Right panel, Representative action potential traces before (a), during (b), and after (c) application of A23187. C, A23187 (2 µmol/L) reversibly enhanced IKs. Left panel, Averaged tail IKs amplitude normalized to the initial value in 5 experiments. Open circles are data without drug application (control), and closed circles are data for A23187 application. Standard error bars are omitted to avoid crowdedness. Right panel, Representative current traces recorded before (a), during (b), and after (c) application of A23187.

Application of A23187 induced a dose-dependent shortening of action potential duration and enhancement of I_Ks, which attained a maximal value at a concentration of 2 µmol/L of A23187 (where APD₂₀ shortened by 49%, APD₉₀ by 20%, and I_Ks amplitude increased by 31%) (see online data supplement). These values are comparable to magnitude of Ca²⁺-induced enhancement of I_Ks recorded in the excised giant-patch recording.⁶ Thus, in the following experiments, we used A23187 at this concentration (2 µmol/L). Once electrical access in the perforated patch configuration was established, there was a slight time-dependent reduction in APD that occurred without drug application. Application of A23187 (2 µmol/L) reversibly shortened both APD₂₀ ( in Figure 1B) and APD₉₀ ( in Figure 1B) with a significantly greater magnitude than time-dependent reduction in APD without drug application ( and in Figure 1B) (n=5, P<0.01). Treatment with A23187 (2 µmol/L) for 15 minutes shortened APD₂₀ by 49.4±3.3% (Figure 1B, n=5, P<0.01 versus spontaneous APD₂₀ shortening), and APD₉₀ by 19.2±3.6% (Figure 1B, n=5, P<0.01 versus spontaneous APD₂₀ shortening). The tail amplitude of I_Ks in the control cells exhibited a time-dependent decrease ( in Figure 1C). Application of A23187 (2 µmol/L) reversibly increased tail I_Ks by 31.2±2.3% (Figure 1C, n=5, P<0.01 versus spontaneous time-dependent reduction in I_Ks). Addition of EGTA (10 mmol/L), a Ca²⁺ chelator, to the pipette solution prevented shortening of APD and enhancement of tail I_Ks amplitude by A23187 (see online data supplement). These results indicate that the effects of extracellularly applied A23187 on APD and I_Ks are dependent on the elevation in [Ca²⁺]_i.

Relative Contribution of I_Ks and I_Ca,L to Ca²⁺-Induced APD Shortening
I_Ca,L amplitude is diminished by elevation in [Ca²⁺]_i via the Ca²⁺-induced Ca²⁺ inactivation mechanism, which also induces APD shortening.^2,3 To assess relative contribution of Ca²⁺-induced I_Ks enhancement and I_Ca,L inactivation to the APD shortening induced by A23187, we used a I_Ks channel blocker, chromanol 293B, and/or a I_Ca,L channel blocker, nisoldipine, at supramaximal concentrations. Action potentials were continuously elicited at a stimulation rate of 0.1, 0.4, 1, and 4 Hz, and after shortening of APD by A23187 (2 µmol/L) had reached a pseudo-steady state, chromanol 293B (30 µmol/L), nisoldipine (3 µmol/L), or a combination of chromanol 293B (30 µmol/L) and nisoldipine (3 µmol/L) was added in the presence of A23187 (2 µmol/L). At 0.1 Hz, chromanol 293B alone or nisoldipine alone partially reversed A23187-induced APD₂₀ and APD₉₀ shortening with a similar magnitude (left panel in Figure 2A): APD₂₀ shortening was reversed by 49.4±3.3% by chromanol 293B and by 48.9±3.1% by nisoldipine, (n=5, P=NS) (Figure 2B), and APD₉₀ shortening was reversed by 54.5±3.4% by chromanol 293B and by 42.7±3.0% by nisoldipine (n=5, P=NS) (Figure 2C). A combination of chromanol 293B and nisoldipine completely reversed A23187-induced APD shortening to the control levels (Figure 2B and 2C). When stimulation rate was increased to 0.4, 1, and then 4 Hz, the magnitude of APD shortening was progressively increased with progressive increases in the fraction of APD shortening reversed by chromanol 293B, leaving the fraction reversed by nisoldipine constant. At 4 Hz, chromanol 293B reversed APD₂₀ shortening by 74.8±3.1% and APD₉₀ shortening by 79.8±3.3%, and nisoldipine reversed APD₂₀ shortening by 23.6±3.1% and APD₉₀ shortening by 20.0±3.1% (right panel in Figure 2A through 2C). Thus, at a low stimulation rate both Ca²⁺-induced I_Ks enhancement and Ca²⁺-induced I_Ca,L contributed to the Ca²⁺-induced APD shortening with a similar magnitude. However, at a high stimulation rate, contribution of I_Ks enhancement was significantly greater than contribution of I_Ca,L suppression.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effects of IKs blocker and ICa,L blocker on A23187-induced APD shortening. A, Representative traces of action potentials in the control state (), in the presence of A23187 (2 µmol/L) (), A23187 (2 µmol/L) with chromanol 293B (30 µmol/L) (), A23187 (2 µmol/L) with nisoldipine (3 µmol/L) (), or A23187 (2 µmol/L) with a combination of chromanol 293B (30 µmol/L) and nisoldipine (3 µmol/L) () at a stimulation rate of 0.1 Hz (left panel) and 4 Hz (right panel). B and C, Percent of the shortening of APD20 (B) and APD90 (C) by A23187 in 5 experiments. Horizontal axis represents stimulation rate. Symbols represent data in the control state, and symbols , , , and represent APD in the presence of A23187 (2 µmol/L) with nisoldipine (3 µmol/L), chromanol 293B, and a combination of nisoldipine (3 µmol/L) and chromanol 293B (30 µmol/L), which were normalized by the control APD.

Roles of NO in APD Shortening and I_Ks Enhancement by A23187
Ca²⁺ is a well-known activator of NOS.^8,9 Recently, we reported that NO increased I_Ks possibly by a mechanism dependent on direct S-nitrosylation of the channel protein,⁶ which prompted us to examine potential roles of NO in the A23187-induced enhancement of I_Ks. After A23187 (2 µmol/L) had shortened APD and enhanced I_Ks, we applied a nonspecific NOS inhibitor, SMTU (1 µmol/L). Effects of A23187 (2 µmol/L) on APD were partially, but significantly reversed by SMTU (Figure 3A), and those on I_Ks were fully reversed back to the control levels (Figure 3B). Application of a NO scavenger, L-NAC (1 mmol/L), also reversed enhancement of I_Ks induced by A23187 to the control levels (Figure 3B). Effects of A23187 on the gating properties of I_Ks were also similar to those by a NO donor, SNP, which provide supporting evidence that A23187 enhances I_Ks by causing the production of NO (see online data supplement).

View larger version (32K): [in this window] [in a new window]   Figure 3. NO and NOS3 play a role in the A23187-induced APD shortening and IKs enhancement. A, NOS inhibitor, SMTU (1 µmol/L), reversed APD shortening by A23187. a, Time course of changes in APD, and b depicts averaged percent changes in APD in the presence of A23187 alone (black bars) and in the presence of A23187 and SMTU (hatched bars). *P<0.05. B, NOS inhibitor, SMTU (1 µmol/L) (a), and a NO scavenger, L-NAC (1 mmol/L) (b), reversed the IKs enhancement by A23187. a and b, Time course of changes in IKs, and c depicts averaged percent changes in IKs in the presence of A23187 alone (black bars) and in the presence of A23187 and SMTU or L-NAC (hatched bars). *P<0.05. C, NOS1 inhibitor, SMTC (3 µmol/L), did not reverse IKs enhancement by A23187 (panel a). However, a NOS3 inhibitor, L-NIO (1 µmol/L), did reverse IKs enhancement by A23187 (b). c, Averaged percent changes in IKs in the presence of A23187 alone (black bars) and in the presence of A23187 and SMTC or L-NIO (hatched bars). *P<0.05, #P=NS.

NOS3 Is Responsible for A23187-Induced NO Release
Increase in I_Ks occurs rapidly after application of A23187, which argues against activation of inducible NOS (NOS2). Therefore, we determined whether A23187 activated NOS1 or NOS3 to cause enhancement of I_Ks. We used 2 different NOS inhibitors, SMTC, which has a significantly lower IC₅₀ for NOS1 (0.31 µmol/L) than for NOS3 (5.4 µmol/L),¹⁴ and L-NIO, which has a significantly lower IC₅₀ for NOS3 (0.5 µmol/L) than for NOS1 (3.9 µmol/L).¹⁵ Application of SMTC (3 µmol/L) did not alter enhancement of tail I_Ks amplitude by A23187 (a and c in Figure 3C), whereas L-NIO (1 µmol/L) reversed A23187-enhanced I_Ks to the initial levels (b and c in Figure 3C). These findings strongly suggest that effects of A23187 on APD and I_Ks occur via NO released from NOS3.

NOS3 Activation Is Dependent on CaM
NOS3 is activated via at least two distinct pathways, a Ca²⁺-dependent pathway involving Ca²⁺-binding protein CaM or a phosphorylation-dependent pathway involving Ser/Thr kinase Akt.^16,17 We determined how A23187 activated NOS3 by using a CaM inhibitor (W7) and an Akt inhibitor (SH-6). W7 (10 µmol/L) reversed enhancement of I_Ks by A23187 back to the initial levels (a in Figure 4A and 4B). A chemically related but inactive analog of W7, W5, did not alter A23187-enhanced I_Ks (b in Figure 4A and 4B). Neither SH-6 (10 µmol/L) nor LY-294,002 (30 µmol/L), an inhibitor of PI-3 kinase (an upstream activator of Akt), altered A23187-enhanced tail I_Ks (c and d in Figure 4A and 4B). Similarly, application of W7 (10 µmol/L) reversed shortening of APD₂₀ and APD₉₀ by A23187 (2 µmol/L) (a in Figure 4C and 4D). In contrast, SH-6 (10 µmol/L) application did not reverse shortening of APD₂₀ or APD₉₀ by A23187 (b in Figure 4C and 4D).

View larger version (21K): [in this window] [in a new window]   Figure 4. CaM, but not Akt, is involved in A23187-induced IKs enhancement and APD shortening. A, CaM inhibitor, W7 (10 µmol/L), reversed IKs enhancement by A23187 (a). However, an inactive analog of W7, W5 (10 µmol/L) (b), an Akt inhibitor, SH-6 (10 µmol/L) (c), and a PI3-kinase inhibitor, LY-294,002 (30 µmol/L) (d), did not reverse IKs enhancement by A23187. B, Percent changes in IKs in the presence of A23187 alone (black bars) or in the presence of A23187 with W7, W5, SH-6, or LY-294,002 (hatched bars). *P<0.05, #P=NS. C, W7 (10 µmol/L) reversed APD shortening (a), but SH-6 (10 µmol/L) did not (b). D, Percent changes in APD in the presence of A23187 alone (black bars) or in the presence of A23187 with W7 or SH-6 (hatched bars). *P<0.05, #P=NS.

In cardiomyocytes, NOS3 localizes into caveolae and interacts with muscle-specific caveolin, Cav-3.^18,19 Dissociation of NOS3 from Cav-3 is a mandatory step for NOS3 activation.²⁰ Using cardiac myocyte lysates, we performed immunoprecipitation using a NOS3 antibody to determine its relative associations with Cav-3 or CaM. In the control condition, immunoblot with an anti–Cav-3 antibody or an anti-CaM antibody demonstrated that NOS3 was predominantly associated with Cav-3 but not with CaM (lane 1 in Figure 5A and 5B). In contrast, cells treated with A23187 (2 µmol/L) for 20 minutes reduced association of NOS3 with Cav-3 and increased its association with CaM (lane 2 in Figure 5A and 5B). W7 (10 µmol/L) prevented changes in NOS3 association caused by A23187 (2 µmol/L) (lane 3 in Figure 5A and 5B), whereas SH-6 (10 µmol/L) did not alter changes in NOS3 association caused by A23187 (2 µmol/L) (lane 4 in Figure 5A and 5B). These data indicate that A23187 causes NOS3 to switch its binding partner from Cav-3 to CaM.

View larger version (69K): [in this window] [in a new window]   Figure 5. Effects of A23187 on interactions of NOS3 with Cav-3 and CaM. A and B, Cell lysates immunoprecipitated with an anti-NOS3 antibody were analyzed using immunoblotting with an anti–Cav-3 antibody (A), an anti-CaM antibody (B), or an anti-NOS3 antibody (bottom in A and B). Lanes 1 and 5, control; lanes 2 and 6, A23187 (2 µmol/L); lanes 3 and 7, A23187 (2 µmol/L) with W7 (10 µmol/L); lanes 4 and 8, A23187 (2 µmol/L) with SH-6 (10 µmol/L). Lanes 5 to 8 are data for preabsorption experiments. Arrowheads indicate immunoreactivity of Cav-3 or CaM. C and D, Densitometric analyses of Cav-3 immunodensity (C) and CaM immunodensity (D) in 3 experiments normalized to NOS3 immunodensity. Longitudinal axis represents arbitrary unit, in which immunodensity of Cav-3 or CaM was normalized by immunodensity of NOS3.

Effects of Ca²⁺ Loading by Digoxin and Rise in Extracellular Ca²⁺ Concentration on APD and I_Ks
Finally to examine if physiologically relevant Ca²⁺-loading also shorten APD and enhance I_Ks, we examined effects of digoxin and rise in extracellular Ca²⁺ concentration ([Ca²⁺]_o). Application of digoxin at 0.2 µmol/L to the bath solution did not significantly change basal [Ca²⁺]_i (0.10±0.01 µmol/L in the control state [n=6] and 0.13±0.02 µmol/L in the presence of digoxin at 0.2 µmol/L [n=6]; P=NS), whereas peak [Ca²⁺]_i was increased from 0.98±0.06 µmol/L (n=6) in the control state to 1.47±0.08 µmol/L (n=6) (P<0.05). Digoxin at 2 µmol/L increased both basal [Ca²⁺]_i from 0.10±0.01 µmol/L (n=6) in the control state to 0.20±0.01 µmol/L (n=5) (P<0.05) and peak [Ca²⁺]_i from 0.98±0.06 µmol/L (n=6) to 1.96±0.09 µmol/L (n=5) (P<0.05) (Figure 6A). Digoxin at 0.2 µmol/L shortened APD₂₀ by 28.0±3.9% and APD₉₀ by 12.6±3.1% relative to the control, and digoxin at 2 µmol/L shortened APD₂₀ by 58.8±3.4% and APD₉₀ by 24.0±3.4% relative to the control (Figure 6B and online data supplement). L-NAC (1 mmol/L) partially, but significantly, reversed APD shortening by digoxin (Figure 6B and online data supplement). Digoxin at 0.2 µmol/L increased I_Ks amplitude by 17.4±3.1%, and digoxin at 2 µmol/L by 37.7±7.1% relative to the control (Figure 6B and online data supplement). I_Ks enhancement by digoxin was fully reversed by application of L-NAC (1 mmol/L) (Figure 6B and online data supplement).

View larger version (33K): [in this window] [in a new window]   Figure 6. Effects of digoxin and rise in [Ca2+]o on [Ca2+]i, APD, and IKs. A, Effects of digoxin on [Ca2+]i. a and b, Representative Ca2+ transient elicited at 1 Hz in the control state (a) and in the presence of digoxin at 2 µmol/L (b). c, Averaged [Ca2+]i in the basal state (closed bars) and at the peak of Ca2+ transient (open bars) in the control state, and in the presence of digoxin at 0.2 µmol/L and at 2 µmol/L. *P<0.05, #P=NS. B, Digoxin shortened APD and enhanced IKs. Averaged % changes in APD20, APD90, and IKs by digoxin at 0.2 µmol/L or at 2 µmol/L alone (closed bars), or by digoxin with L-NAC (1 mmol/L) (hatched bars). *P<0.05. C, Rise in [Ca2+]o shortened APD and enhanced IKs. Averaged percent changes in APD20, APD90, and IKs by rise in [Ca2+]o from the control value (1.8 mmol/L) to 2.4 mmol/L (closed bars), to 3.0 µmol/L (open bars), and to 3.0 mmol/L in the presence of L-NAC (hatched bars). *P<0.05.

Rise in [Ca²⁺]_o from the control state (1.8 mmol/L) to 2.4 mmol/L shortened APD₂₀ by 27.2±3.9% and APD₉₀ by 17.4±3.0%, and to 3.0 mmol/L shortened APD₂₀ by 45.4±3.8% and APD₉₀ by 26.7±3.2%, which were partially, but significantly reversed by L-NAC (1 mmol/L) (Figure 6C and online data supplement). Rise in [Ca²⁺]_o from 1.8 mmol/L to 2.4 mmol/L, and to 3.0 mmol/L enhanced I_Ks by 27.9±8.3% and 43.8±11.2%, respectively, which were fully reversed by L-NAC (1 mmol/L) (Figure 6C and online data supplement).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
NO has been shown to play important roles in the physiological regulation of Ca²⁺ cycling of cardiomyocytes.²¹ In this study, we demonstrate that Ca²⁺ regulation of I_Ks channel is another critical NO-dependent Ca²⁺ cycling system based on the following findings: (1) a NO donor mimicked Ca²⁺-induced I_Ks enhancement; (2) a NOS inhibitor abolished I_Ks enhancement by Ca²⁺; (3) a NO scavenger also abolished Ca²⁺-induced I_Ks enhancement; and (4) changes in I_Ks kinetics caused by a NO donor were very similar to those by Ca²⁺.

To examine the mechanism underlying Ca²⁺-sensitivity of I_Ks, we increased [Ca²⁺]_i by using a Ca²⁺-ionophore, A23187, and recorded whole-cell I_Ks in the perforated patch configuration. This recording configuration is preferred to study Ca²⁺ regulation of I_Ks, because it prevents wash-out of the intracellular signal transduction machinery. The peak [Ca²⁺]_i was increased from 0.97±0.03 µmol/L in the control state to 1.24±0.03 µmol/L by A23187 at 0.1 µmol/L and to 1.52±0.09 µmol/L by A23187 at 2 µmol/L, whereas APD and I_Ks were barely affected by A23187 at 0.1 µmol/L and maximally changed by A23187 at 2 µmol/L. Thus, the threshold of [Ca²⁺]_i to modulate APD and I_Ks appears to be between 1.24 and 1.52 µmol/L, which is well within the range where [Ca²⁺]_i physiological varies in cardiac myocytes. The changes in [Ca²⁺]_i reached the stable level at 5 minutes after application of A23187 (see online data supplement), which is similar to the time course of changes in APD and I_Ks by A23187, further supporting that effects of APD and I_Ks by A23187 were attributable to changes in [Ca²⁺]_i.

In addition to Ca²⁺-induced enhancement of I_Ks, rise in [Ca²⁺]_i causes Ca²⁺-induced inactivation of I_Ca,L.^13,14 To understand the feedback system of Ca²⁺ entry, it is important to know relative contributions of Ca²⁺-induced I_Ks enhancement and I_Ca,L inactivation to the Ca²⁺-induced APD shortening. In the present study, at a low stimulation rate, both I_Ks enhancement and I_Ca,L inactivation contribute to the Ca²⁺-induced APD shortening with a similar magnitude. However, at a high stimulation rate, A23187-induced APD shortening was reversed by 75% to 80% with chromanol 293B and only by 20% to 25% with nisoldipine. Thus, at a high stimulation rate, Ca²⁺-induced I_Ks enhancement plays a dominant role in APD shortening. A23187-induced APD shortening was dependent on stimulation rate: the higher the stimulation was, the greater the A23187-induced APD shortening was. This result is consistent with a simulation study showing that I_Ks underlies rate-dependent adaptation of APD at fast heart rates.²² Fast pacing results in short diastolic intervals that prevent complete deactivation of I_Ks, resulting in build-up of instantaneous I_Ks repolarizing current.²² In our study, elevated I_Ks components at higher [Ca²⁺]_i may cause additional accumulation of the repolarizing current, resulting in the greater APD shortening in the presence of A23187.

We have previously demonstrated that I_Ca,L is suppressed by NO via a cGMP-dependent pathway.¹¹ However, NO-dependent modulation of I_Ca,L appears not to play a significant role in the Ca²⁺-induced I_Ca,L inactivation in our experiments, because neither a NO scavenger, L-NAC, nor a NOS inhibitor, SMTU, reversed the A23187-induced I_Ca,L suppression (see online data supplement). Because I_Ca,L channel inactivation is underlined by direct interaction of Ca²⁺/CaM with the I_Ca,L channels^2,3 that is situated in a pathway upstream of Ca²⁺/CaM-dependent activation of NOS3, effects of NO on the I_Ca,L channels may be masked by a direct effect of Ca²⁺/CaM. The finding that L-NAC and SMTU reversed A23187-induced APD shortening only partially may further implicate that a fraction of APD shortening caused by I_Ca,L inactivation is insensitive to NO modulators.

It is also critical to know whether Ca²⁺-induced I_Ks enhancement and resultant APD shortening act in physiological and/or pathological conditions of the hearts. To address this question, we used physiologically relevant Ca²⁺ loading such as digoxin and rise in [Ca²⁺]_o. Digoxin, a selective blocker of Na⁺/K⁺-ATPase is known to increase [Ca²⁺]_i by indirectly enhancing activity of Na⁺-Ca²⁺ exchanger, and in fact in our study, digoxin increased both basal and peak [Ca²⁺]_i. Both application of digoxin and rise in [Ca²⁺]_o induced shortening of APD and enhancement of I_Ks. APD shortening was partially reversed by a NO scavenger, L-NAC, and I_Ks enhancement was fully reversed by L-NAC. Thus, Ca²⁺-induced I_Ks enhancement and resultant APD shortening appear to be one of the physiological and pathological regulators of I_Ks, among other well-known regulators, such as ß-adrenergic regulation.^23,24

Because Ca²⁺ enters in cells during the action potential plateau, changes in APD may affect the amount of Ca²⁺ entry, and thereby [Ca²⁺]_i.²⁵ Indeed, Padmala and Demir reported that the 40% reduction in APD results in 60% reduction in Ca²⁺ entry through I_Ca,L, 23% reduction in peak [Ca²⁺]_i, and 18% reduction in diastolic [Ca²⁺]_i.²⁶ Thus, Ca²⁺-induced APD shortening potentially acts as a negative feedback system to maintain [Ca²⁺]_i within an appropriate range.

Cardiomyocytes express both NOS1 and NOS3.^19,21 The finding that L-NIO, a relatively specific NOS3 inhibitor completely abolished I_Ks enhancement by A23187, whereas SMTC, a relatively specific NOS1 inhibitor, barely affected it, strongly suggests that the activation of NOS3, rather than NOS1, is involved in the I_Ks enhancement. NOS3 is activated via at least two distinct pathways, a CaM-dependent pathway and an Akt-dependent pathway.^17,18 In the present study, Ca²⁺-dependent I_Ks enhancement was blocked by a CaM inhibitor, W7, but was barely affected by an Akt inhibitor, SH-6, indicating that a CaM-dependent activation of NOS3 participates in this phenomenon. We have previously reported that I_Ks is enhanced by intracellular Ca²⁺, which may be consequences of allosteric interaction of CaM rather than phosphorylation by CaM kinase II, because a CaM inhibitor, W7, but not a CaM kinase II inhibitor, KN62, inhibited the Ca²⁺-induced I_Ks enhancement.⁵ This hypothesis is supported by our immunoprecipitation-immunoblotting data that the elevation of [Ca²⁺]_i by A23187 switched the protein-protein interaction from NOS3-Cav-3 to NOS3-CaM. In cardiomyocytes, NOS1 and NOS3 have opposing actions in Ca²⁺ cycling: NO released from NOS3 decreases [Ca²⁺]_i by suppressing the I_Ca,L channel activities, whereas NO released from NOS1 increases [Ca²⁺]_i by activating ryanodine receptor type 2.^19,21 The opposing actions of NOS1 and NOS3 in Ca²⁺ cycling are critically dependent on their spatial confinement with effecter molecules: the ryanodine receptor type 2 and NOS 1 on endoplasmic reticulum and the I_Ca,L channel and NOS3 on sarcolemma.^19,21 The Ca²⁺-induced enhancement of I_Ks can be observed in cell-free excised patch configuration,⁶ implicating that the spatial confinement of NOS3 with the I_Ks channel also takes place. Interestingly, the subunit of the I_Ks channel, KCNQ1, has two potential binding sites for Ca²⁺-free CaM (apo-CaM), an IQ motif and a motif, in its intracellular C-terminal region.²⁷ Yeast two-hybrid assay also suggests that KCNQ1 associates with apo-CaM.²⁷ Thus, the changes in protein-protein interactions may occur in the membrane-delimited microdomains that involve Cav-3, NOS3, CaM, and KCNQ1. Because of participation of Cav-3, the microdomains are likely to be in the caveola. Although the I_Ks channel has been suggested to colocalize with the ß₂-adrenergic receptor that accumulates in caveolae,²⁸ the direct demonstration of localization of components of the I_Ks channel, KCNQ1 and KCNE1, with the caveolae remains to be shown.

	Acknowledgments

 
This work was supported in part by Research Grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and a research grant from the Takeda Science Foundation. We thank Dr Brian Delisle (University of Wisconsin) for checking English, K. Yoshida for the technical assistance, and A. Sugai for the secretarial services.

	Footnotes

 
Original received July 14, 2004; revision received November 9, 2004; accepted November 17, 2004.

	References

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