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Am J Physiol Heart Circ Physiol 294: H285-H292, 2008. First published November 2, 2007; doi:10.1152/ajpheart.00824.2007
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Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its β-adrenergic enhancement in ventricular myocytes

Hiroto Tsujikawa,1 Yumei Song,1 Makino Watanabe,1 Haruko Masumiya,1 Sachin A. Gupte,2 Rikuo Ochi,1,2 and Takao Okada1

1Department of Physiology, Juntendo University School of Medicine, Tokyo, Japan; and 2Department of Physiology, New York Medical College, Valhalla, New York

Submitted 16 July 2007 ; accepted in final form 29 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae, where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl-β-cyclodextrin (MβCD), we studied the role of cholesterol in the modulation of L-type Ca2+ currents (ICa,L). MβCD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM MβCD for 10 min, a positive shift in membrane potential at half-maximal activation (V0.5) from –8 to –2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased ICa,L approximately threefold and caused a negative shift in V0.5 in control cells, but it did not increase ICa,L in MβCD-treated myocytes, nor did it shift V0.5. The effect of MβCD (10 or 30 mM) was concentration dependent: 30 mM MβCD suppressed the ISO-induced increase in ICa,L more effectively than 10 mM MβCD. MβCD dialysis also abolished the increase in ICa,L elicited by forskolin or dibutyryl cAMP, but not that elicited by (–)BAY K 8644. External application of MβCD-cholesterol complex to rat myocytes attenuated the MβCD-mediated inhibition of the ISO-induced increase of ICa,L. Biochemical analysis confirmed that the myocytes' cholesterol content was diminished by MβCD and increased by MβCD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal ICa,L and β-adrenergic cAMP/PKA-mediated increases in ICa,L. We suggest that cholesterol affects the structural coupling between L-type Ca2+ channels and adjacent regulatory proteins.

lipid raft; adenosine 3',5'-cyclic monophosphate; protein kinase A; phosphorylation

L-TYPE CALCIUM CURRENTS (ICa,L) play an important role in excitation-contraction coupling in cardiac myocytes, and the mechanism by which β-adrenergic agonists (e.g., epinephrine and norepinephrine) increase myocardial ICa,L amplitude has been extensively studied (23, 31). These agonists bind to G protein-coupled β-adrenergic receptors (β-ARs) to accelerate production of cAMP by adenylyl cyclase (AC). This in turn leads to phosphorylation of the {alpha}1C (CaV1.2) pore-containing primary subunit (5, 20) and the β2-subunit (4) of the L-type Ca2+ channel by cAMP-activated PKA in a process that is assisted by A kinase-anchoring protein (AKAP) tethered to the CaV1.2 (19). The fact that basal ICa,L is increased by phosphatase blockers indicates that the L-type Ca2+ channel is under dynamic control involving phosphorylation by kinases and dephosphorylation by phosphatases, even in the absence of humoral stimulation (10).

Lipid rafts are microdomains within the plasmalemma that are characterized by high densities of cholesterol and sphingomyelin; caveolae, a subset of lipid rafts, also contain the scaffold protein caveolin (36). Recent biochemical and histochemical studies have shown that G protein-coupled receptor signaling proteins and target proteins form macromolecular complexes within lipid rafts (21, 40, 41). By increasing the messenger concentration and enabling direct protein-to-protein interactions, this colocalization ensures that signaling is specific and fast. β-ARs, G proteins, AC, PKA regulatory subunit II (PKARII), and caveolin-3 localize to caveolae in neonatal rat cardiac myocytes (38). In adult rat ventricular myocytes, β1- and β2-ARs are present within caveolae, colocalizing with G proteins, AC, and caveolin-3 (16). In addition, CaV1.2 has been shown to colocalize with β2-ARs, PKARII, and phosphatase 2A (PP2A) within caveolae in mouse cardiomyocytes (1).

Methyl-β-cyclodextrin (MβCD) increases cellular cholesterol efflux by depleting cholesterol from membranes (7, 24). MβCD has often been used to study the role of cholesterol in ion channel function and signal integration within lipid rafts. In cardiac myocytes, several ion channels have been identified within lipid rafts, which are involved in their functional regulation (29). For example, KV1.5 channels are present within lipid rafts, and disruption of the rafts by cholesterol depletion induces a hyperpolarizing shift in the voltage-dependent activation and inactivation of the channels (30). Depletion of cholesterol also increases the hyperpolarization-activated current through hyperpolarization-activated cyclic nucleotide-gated channels in sinoatrial node cells (2). Conversely, the loading of cholesterol into the plasma membrane caused by hypercholesterolemia suppresses inwardly rectifying K+ channels in endothelial cells (12). In neonatal mouse cardiomyocytes, disruption of caveolae abolishes the increase of ICa,L induced by β2-adrenergic stimulation without affecting the β1-induced increase (1).

The primary aim of the present study was to assess the involvement of cholesterol and lipid rafts in modulating basal ICa,L and the increase in ICa,L induced by β-adrenergic stimulation in adult cardiac myocytes by examining the effects of MβCD-mediated cholesterol depletion. Because application of MβCD to mechanically skinned skeletal muscle fibers is known to suppress excitation-contraction coupling more rapidly than its external application to intact bundles of fibers (28), we applied MβCD to ventricular myocytes by dialysis from the pipette solution together with BAPTA, which blocks activation of β2-AR-coupled Ca2+-sensitive type VIII AC (20, 41). We found that cholesterol depletion significantly modulates basal ICa,L and completely abolishes the β-adrenergic cAMP/PKA-mediated increase of ICa,L.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study was conducted in accordance with the guiding principles for the care and use of animals in the field of physiological sciences of the Physiological Society of Japan (34). The protocol was approved by our university's Animal Experimentation Committee.

Isolation of ventricular myocytes. Single ventricular myocytes were isolated from rabbit or rat hearts. Japanese White rabbits (1.6 kg) or Wistar rats (300 g) were deeply anesthetized by injection of pentobarbitone sodium (40 mg/kg). The hearts were then quickly excised and perfused using a Langendorff apparatus, first with normal Tyrode solution for a few minutes and then with nominally Ca2+-free Tyrode solution for 5 min, collagenase solution for 25 min, and Kraft-Brühe (KB) solution for 5 min. All solutions were saturated with 100% O2 and warmed to 37°C. After digestion, the ventricles were cut into small pieces with scissors, and rabbit cardiomyocytes were dissociated in KB solution in a shaking water bath, while the rat cells were dissociated by trituration with a fire-polished Pasteur pipette. The KB solution was then filtered through a coarse metal sieve to obtain the dissociated myocytes. After the cells were precipitated to the bottom of a beaker, they were aspirated into a Pasteur pipette, dispersed into normal Tyrode solution in another beaker, and incubated for 15 min at 37°C. The cell-containing beaker was removed from the bath and kept at room temperature. The cells were used within 8 h.

Patch-clamp procedures. Membrane currents were recorded using the whole cell patch-clamp technique in the ruptured membrane configuration (15) with an Axopatch 1-D or Axopatch 200B patch-clamp amplifier (MDS Analytical Technologies, Toronto, ON, Canada). For rabbit myocytes, patch pipettes were pulled from plain hematocrit capillary tubes with ~3-M{Omega} resistance when filled with pipette solution. For rat cells, the pipettes were pulled from borosilicate glass capillary tubes with ~6-M{Omega} resistance. The liquid junction potential between the pipette solution and normal Tyrode solution was compensated. The pipette capacitance was compensated after formation of a gigaohm seal. Data sampling and command voltage pulse generation were conducted using Digidata interface and pCLAMP software (MDS Analytical Technologies). Membrane currents were filtered using a Bessel filter at 1 kHz and digitized at 5 kHz. ICa,L were evoked at a rate of 0.2 Hz by application of 300-ms depolarizing pulses in 10-mV increments from –40 to +50 mV or from –20 to +20 mV after 55-ms prepulses to –40 mV that were imposed after a 20-ms ramp pulse from the holding potential of –80 mV. Peak ICa,L amplitude was estimated as the difference between the negative peak of the current and the current at the end of the depolarization pulse. The conductance of the L-type Ca2+ channel (GCa,L) was estimated from the peak ICa,L amplitude and the membrane potential (V) according to the following equation: GCa,L = ICa,L/(V – Erev), where Erev is reversal potential of ICa,L (+53 mV). GCa,L was fitted by the Boltzmann equation: GCa,L = GCa,L,max/[1 + exp(V0.5 – V)/k], where GCa,L,max is maximal GCa,L, V0.5 is the potential at which GCa,L is activated to 50% of GCa,L,max, and k is a slope parameter. The membrane capacitance was determined by integrating the capacitance transient elicited by a 5-mV hyperpolarizing pulse from a holding potential of –40 mV. The mean membrane capacitance in each group was ~100 pF. All experiments were conducted at room temperature (22–25°C).

Experimental protocol. We started whole cell clamp by rupturing the patch membrane with a short negative-pressure pulse in normal Tyrode solution. After estimation of the membrane capacitance and compensation of the series resistance, the external solution was switched to Cs+-containing Tyrode (Cs+-Tyrode) solution. Basal ICa,L were recorded every 1 or 2 min using 5 or 10 different pulses beginning 2 min after establishment of the whole cell clamp and continuing for 9 or 11 min. Inasmuch as ~1 min was required to record a set of currents for a current-voltage (I-V) curve, the recording times are approximate. MβCD was usually dialyzed intracellularly from the pipette solution but was also applied from bath solution by switching from normal Tyrode to MβCD-containing Cs+-Tyrode solution soon after initiation of whole cell clamp. After the last series of basal ICa,L (grouped as control values at 10 min) was recorded, isoproterenol (ISO), forskolin (FSK), DBcAMP, or (–) BAY K 8644 (BAY K) was introduced, and its effect on ICa,L was examined after ~3 min.

Estimation of cellular cholestrol content. Cholestrol content of isolated rat ventricular myocytes was measured spectrophotometrically using a cholesterol quantitation kit (Cholesterol/Cholesteryl Ester Quantitation Kit, Bio Vision, Mountain View, CA). Aliquots of myocytes from each heart were incubated for 10 min at room temperature in Cs+-Tyrode solution, with or without 10 mM MβCD or 5 mM cholesterol-conjugated MβCD. The cells were then homogenized in chloroform-Triton X-100, a cholesterol probe was added, and the cholesterol content was estimated in a colorimetric assay, according to the manufacturer's protocol. The protein content of each sample was estimated using a Bradford protein assay, and the cholesterol concentration was expressed as micrograms per milligram of protein.

Solutions and drugs. The composition of normal Tyrode solution was (mM) 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5 HEPES, and 11 glucose (pH was adjusted to 7.40 with NaOH). Collagenase solution contained 1 mg/ml collagenase [type I, Sigma-Aldrich, St. Louis, MO (for rabbit); type 2, Worthington, Lakewood, NJ (for rat)] in low-Ca2+ (18 µM) Tyrode solution. KB solution contained (mM) 25 KCl, 10 KH2PO4, 70 glutamic acid, 10 oxalic acid, 10 taurine, 0.5 EGTA-Tris, 5 HEPES, and 11 glucose (pH was adjusted to 7.20 with KOH). For the Cs+-Tyrode solution, KCl was replaced with 5.4 mM CsCl in the normal Tyrode solution. The Cs+-Tyrode solution used with rat ventricular myocytes contained 20 mM tetraethylammonium chloride replacing equimolar NaCl. The pipette solution contained (mM) 135 CsCl, 1.0 MgCl2, 5.0 Mg-ATP, 5.0 BAPTA, and 5 HEPES 5 (pH was adjusted to 7.30 with CsOH). When MβCD or MβCD-cholesterol complex was added, the concentration of NaCl (for external application) or CsCl in pipette solution (for dialysis) was decreased to keep the osmolality constant. MβCD, cholesterol-conjugated MβCD (water-soluble cholesterol), ISO, FSK, DBcAMP, IBMX, and BAY K were purchased from Sigma-Aldrich.

Data analysis and statistics. Recorded membrane currents were analyzed using pCLAMP (MDS Analytical Technologies) and Igor Pro (version 6, WaveMetrics, Portland, OR) software. Statistical analysis was carried out using Prism software (version 5, GraphPad Software, San Diego, CA). Analyses included two-way (Figs. 1, 2, and 3) and one-way (Fig. 5, Table 1) ANOVA followed by post hoc Bonferroni's tests, and paired t-tests were used to evaluate cholesterol content. Values are means ± SE. P < 0.05 was considered significant.


Figure 1
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  		Fig. 1. Effect of methyl-β-cyclodextrin (MβCD) dialysis on time-dependent changes of L-type Ca2+ current (ICa,L) in rabbit ventricular myocytes. A: typical current traces elicited by depolarization to 0 and +20 mV 3 and 9 min after initiation of whole cell clamp without (a) and with (b) 30 mM MβCD in pipette solution. Arrowhead (in b) indicates peak of ICa,L at 9 min. B: time-dependent changes in peak ICa,L density after initiation of whole cell clamp. Peak current densities of ICa,L elicited by pulses to 0 and +20 mV in the presence and absence of MβCD are shown (n = 21–31 for control and 12–25 for MβCD). C: current-voltage (I-V) relationships at ~3 and ~10 min in the absence (a, n = 24–31) and presence (b, n = 14–25) of 30 mM MβCD. *P < 0.001 between 3- and 10-min values at each voltage, except +20 mV in b. #P < 0.05 between 10-min values in a and b at +10 mV, P < 0.001 at +20 mV, and P < 0.01 at +30 mV.

 

Figure 2
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  		Fig. 2. Suppression by cholesterol depletion of isoproterenol (ISO)-induced increase in ICa,L in rabbit ventricular myocytes. A: ICa,L elicited by depolarization to –10, 0, 10, and 20 mV before (black line, after 10 min of dialysis) and after application of ISO (gray line) with (a) and without (b) 30 mM MβCD in pipette solution. Background current at –40 mV increased slightly with time and in the presence of MβCD (arrow). B: I-V relationship before and after ISO without (a, n = 16) and with (b, n = 9) 30 mM MβCD in pipette solution. *P < 0.001 from –20 to +30 mV and P < 0.05 at +40 mV between values obtained before and after ISO at each voltage.

 

Figure 3
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  		Fig. 3. Effect of cholesterol depletion on conductance of L-type Ca2+ channel (GCa,L). GCa,L-voltage (V) relationships reflect time-dependent change in ICa,L density in Fig. 1 (A) and effect of ISO in Fig. 2 (B). GCa,L was calculated from mean current density and V at reversal potential (Erev) of +53 mV. Aa and Ba: absolute values of GCa,L. In Aa, *P < 0.001, 3 min vs. 10 min; #P < 0.001, control vs. MβCD at 10 min. In Ba, *P < 0.001, before vs. after ISO in control; #P < 0.05, control vs. MβCD before ISO. Ab and Bb: GCa,L normalized by maximal mean value. In Ab, *P < 0.05 (–10 mV), P < 0.001 (0 mV), and P < 0.01 (+10 mV), 3 min vs. 10 min in the presence of MβCD; #P < 0.001, control vs. MβCD at 10 min. In Bb, *P < 0.01, before vs. after ISO; #P < 0.05, control vs. MβCD before ISO. GCa,L-V relationships were fitted by the Boltzmann equation. GCa,L,max (in nS/pF) = 0.23 at 3 min vs. 0.14 at 10 min (Aa, control), 0.25 at 3 min and 0.22 at 10 min (Aa, MβCD), 0.15 before ISO and 0.38 after ISO (Ba, control), and 0.25 before ISO and 0.23 after ISO (Ba, MβCD); membrane potential at half-maximal activation (V0.5) (in mV) = –10.5 at 3 min and –9.4 at 10 min (Aa,b control), –8.2 at 3 min and –2.2 at 10 min (Aa and Ab, MβCD), –8.1 before ISO and –16.4 after ISO (Ba and Bb, control), and 1.1 before ISO and 2.5 after ISO (Ba and Bb, MβCD); and slope parameter (k) = 4.5 at 3 min and 4.8 at 10 min (Aa and Ab, control), 5.5 at 3 min and 8.1 at 10 min (Aa and Ab, MβCD), 5.5 before ISO and 5.1 after ISO (Ba and Bb, control), and 8.8 before ISO and 12.3 after ISO (Ba and Bb, MβCD).

 

Figure 5
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  		Fig. 5. Effects of forskolin (FSK), dibutyryl cAMP (DBcAMP), and BAY K 8644 (BAY K) on ICa,L in rabbit ventricular myocytes. A: representative original ICa,L traces recorded before (black line) and after (gray line) application of modulators [FSK (a), DBcAMP with 1 mM IBMX (b), or BAY K (c)] with (top traces, depolarization to 0 mV) and without (bottom traces, depolarization to +10 mV but 0 mV for BAY K) 30 mM MβCD in pipette. Each modulator was applied 10 min after dialysis with MβCD. B: statistical comparison of modulator-induced increases in ICa,L. Maximal ICa,L amplitudes were normalized to the amplitude recorded before application of the respective modulator. For FSK (MβCD –) and FSK (MβCD +), n = 8; for DBcAMP (MβCD –), n = 9; for DBcAMP (MβCD +) and BAY K (MβCD –), n = 7; for BAY K (MβCD +), n = 5. ***P < 0.001 vs. ICa,L increase in the absence of MβCD. NS, not significant.

 

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  		Table 1. Effect of MβCD and MβCD-cholesterol complex on β-adrenergic enhancement of ICa,L in ventricular myocytes

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of cholesterol depletion on basal ICa,L. MβCD (30 mM) was applied to the interior of isolated rabbit ventricular myocytes by dialysis from the pipette solution, and the effect of the gradual removal of cholesterol on ICa,L was examined by recording a set of ICa,L every 2 min (Fig. 1). Because of the rundown typically seen with ICa,L, in the absence and presence of MβCD, peak ICa,L elicited by depolarization to 0 mV declined by 30–40% over a 10-min period from the maximum at 3 min (Fig. 1, A and B). There was no appreciable change in the time course of inactivation during that period, however. In the presence of MβCD, the peak ICa,L amplitude elicited by a pulse to +20 mV was reduced to a lesser degree than that elicited by a pulse to 0 mV (Fig. 1B), although the reductions were similar in the absence of MβCD (control). The I-V relationship for ICa,L illustrates the voltage-dependent modulation of ICa,L activation by MβCD (Fig. 1C). The rundown of ICa,L occurred with only a small shift in the I-V relationship under control conditions but was accompanied by a clear positive shift in the presence of MβCD. Starting from similar I-V relationships, the current density was significantly larger at positive potentials between +10 and +30 mV after depletion of membrane cholesterol than under time-matched control conditions. The largest increase occurred at +20 mV. Current density was increased by 42% at 10 min in the presence of MβCD [4.78 ± 0.19 pA/pF (control, n = 31) vs. 6.79 ± 0.31 pA/pF (MβCD, n = 25)].

The maximal ICa,L density elicited by depolarization to 0 or +10 mV at 10 min under control conditions was 6.77 ± 0.29 pA/pF (n = 41). Dialysis of 30 mM MβCD did not affect the maximal ICa,L density at 3 min, but it apparently decelerated the channel's rundown, so that the maximal value was 8.03 ± 0.15 pA/pF at 10 min (n = 36, P < 0.05 vs. time-matched control).

Effect of cholesterol depletion on ISO-induced increase of ICa,L. ISO (1 µM) was applied to the rabbit ventricular myocytes ~10 min after initiation of the whole cell clamp, when the recording of basal ICa,L was complete. ISO markedly enhanced ICa,L in control cells but did not elicit a similar increase in myocytes dialyzed with 30 mM MβCD (Fig. 2A). In seven of nine challenges, the I-V relationships showed that ISO slightly reduced ICa,L amplitude in cholesterol-depleted myocytes, probably due to rundown. In the remaining two challenges, however, it caused a transient 30–40% increase 3 min after application, which was followed by time-dependent suppression of the increase in ICa,L amplitude. I-V relationships determined from the averaged ICa,L densities showed that, in control cells, ISO produced a nearly threefold increase in peak current with a clear negative shift of the I-V curve, but it had no significant effect on ICa,L density in cholesterol-depleted myocytes (Fig. 2B).

The background current often increased with time, and the increase was greater in the presence of MβCD: –0.14 ± 0.04 vs. –0.27 ± 0.08 pA/pF at 3 min and –0.76 ± 0.13 (P < 0.001 vs. 3 min) vs. –1.47 ± 0.31 pA/pF at 10 min (P < 0. 0.001 vs. 3 min, P < 0.05 vs. control) for control (n = 30) and 30 mM MβCD (n = 24), respectively. Background current also increased with application of ISO: –1.33 ± 0.19 pA/pF for control (n = 18) and –2.67 ± 0.32 pA/pF for 30 mM MβCD (n = 15, P < 0.01 vs. 10 min and P < 0.001 vs. control).

Effect of time, ISO, and MβCD on GCa,L-V relationship. We next analyzed the changes in GCa,L underlying the time- and ISO-dependent changes in ICa,L in Figs. 1 and 2. Figure 3 shows the GCa,L-V relationships in the absence and presence of 30 mM MβCD and their modulation by time and ISO. During whole cell clamp under control conditions, GCa,L,max declined by 39% between minutes 3 and 10, without a significant shift in the GCa,L-V relationship. This is illustrated by the close superposition of the normalized curves (Fig. 3Ab). V0.5 was –10.5 and –9.4 mV at 3 and 10 min, respectively. In cholesterol-depleted cells, a 14% reduction in GCa,L,max over the same period was accompanied by a significant positive shift in the GCa,L-V curve and a reduction in V0.5 from –8.2 to –2.2 mV. ISO increased GCa,L,max 2.5-fold, with a negative shift in V0.5 from –8.1 to –16.4 mV in control cells, but produced neither an increase of GCa,L,max nor a shift in V0.5 in cholesterol-depleted myocytes. In addition, the slope of the curve was less steep in the cholesterol-depleted cells than in the time-matched controls.

Changes in cellular cholesterol content elicited by MβCD and MβCD-cholesterol complex. The effect of MβCD or MβCD-cholesterol complex on membrane cholesterol content was examined biochemically using rat ventricular myocytes exposed to these agents for 10 min at room temperature. We found that 10 mM MβCD significantly reduced the cellular cholesterol content from 10.5 ± 1.4 (control) to 5.0 ± 0.6 µg/mg protein (n = 6, P < 0.01 vs. control), whereas 5 mM MβCD-cholesterol complex increased the cholesterol content to 16.5 ± 1.6 µg/mg protein (n = 5, P < 0.01 vs. control).

Dose-dependent effects of increasing and reducing membrane cholesterol on the β-adrenergic increase in ICa,L. Table 1 shows the dose dependence of MβCD-induced inhibition of β-adrenergic increase in ICa,L, as well as its dependence on which side of the membrane it is applied. In rabbit ventricular myocytes, ISO increased the maximal ICa,L density threefold in time-matched controls, and the small reduction in the ISO-induced increase elicited by dialysis or superfusion of 10 mM MβCD was not statistically significant. However, increasing the concentration of dialyzed MβCD to 30 mM completely suppressed the ISO-induced increase of ICa,L. Bath application of 30 mM MβCD caused a smaller but still significant reduction in the increase in maximal ICa,L density elicited by ISO. Loading cholesterol into the membrane by dialysis of 15 mM MβCD-cholesterol complex did not significantly affect the ISO-induced increase in ICa,L density. In rat ventricular myocytes, dialysis of 30 mM MβCD suppressed the ISO-induced increase of ICa,L. This suppression was not affected by simultaneous bath application of 5 mM MβCD-cholesterol complex during the dialysis, although in the presence of 15 mM MβCD-cholesterol, ISO induced a clear increase of ICa,L in 30 mM MβCD-dialyzed myocytes (Fig. 4, Table 1). These procedures used to manipulate membrane cholesterol had only a slight effect on maximal density of basal ICa,L.


Figure 4
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  		Fig. 4. MβCD-mediated inhibition of ISO-induced increase in ICa,L and its reduction by MβCD-cholesterol complex in rat ventricular myocytes. Representative traces show ICa,L elicited by depolarization to 0 mV. Currents were elicited after 10 min of dialysis, before (black traces) and after (gray traces) ISO application. A: control. B: after dialysis of 30 mM MβCD. C: after dialysis of MβCD with 15 mM MβCD-cholesterol complex in external solution.

 
Effects of cholesterol depletion on the increases in ICa,L elicited by several modulators. We also examined the effects of FSK and DBcAMP on ICa,L in normal and cholesterol-depleted rabbit ventricular myocytes with the aim of identifying the signaling step that, when disrupted by cholesterol depletion, results in suppression of the β-adrenergic increase of ICa,L (Fig. 5). In time-matched controls, FSK (10 µM) applied 10 min after initiation of the whole cell clamp robustly increased ICa,L in a manner similar to ISO. On the other hand, it failed to cause a significant increase of ICa,L after depletion of cholesterol by dialysis of 30 mM MβCD for 10 min. Application of DBcAMP (3 mM), a membrane-permeable cAMP analog, together with IBMX (1 mM), a nonspecific phosphodiesterase inhibitor, caused a nearly threefold increase in ICa,L in the time-matched controls, but again there was no significant increase after the cholesterol depletion. By contrast, application of BAY K (1 µM) increased ICa,L to the same extent before and after cholesterol depletion.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cholesterol depletion shifted the voltage-dependent activation of basal ICa,L in a positive direction and suppressed the cAMP-mediated enhancement of ICa,L elicited by ISO, FSK, or a membrane-permeant cAMP analog.

Dialysis of MβCD to deplete membrane cholesterol. MβCD is a cyclic oligosaccharide with a hydrophilic surface and a hydrophobic cavity that directly extracts cholesterol from the membrane, sequestering it (7). Judging from the efficiency with which it suppressed the β-adrenergic increase in ICa,L, MβCD appears to decrease membrane cholesterol content more rapidly via the internal side of the membrane than the external side (Table 1). Since cholesterol molecules readily "flip-flop" between the inner and outer membrane leaflets with half times on the order of seconds (26), MβCD would be expected to deplete membrane cholesterol from both leaflets irrespective of its side of application. Nonetheless, cholesterols surrounding macromolecules were removed more efficiently by close apposition of MβCD to the internal leaflet of the lipid bilayer. Perhaps because dialyzed MβCD also depletes cholesterol from organelle membranes, it reduces replenishment of plasmalemmal cholesterol mediated by internal trafficking. External application of MβCD-cholesterol complex during dialysis of MβCD clearly reduced the dialysis-induced inhibition of β-adrenergic increase in ICa,L (Fig. 4, Table 1). This strongly suggests that the effects of dialyzed MβCD reflect, at least in part, cholesterol depletion from the plasmalemma.

Mechanism of basal ICa,L modulation by cholesterol depletion. Because of rundown, the maximal amplitude of basal ICa,L declined by ~30~40% over time, which typically occurs during whole cell ICa,L recordings (31). Although rundown occurred in the presence and absence of MβCD, altering cholesterol levels within the lipid raft and nonraft regions of the plasmalemma can affect channel function by modifying the surrounding lipid environment. For instance, loading cholesterol using MβCD-cholesterol complex markedly inhibits ICa,L in gallbladder smooth muscle cells (22) and reversibly inhibits ICa,L by ~30% in coronary vascular smooth muscle, without affecting the I-V relationships (3). We found that, in the presence of MβCD, the I-V relationship for ICa,L was time dependently shifted in a positive direction and ICa,L density became larger at positive potentials. Consistent with that finding, the I-V relationship for ICa,L was previously shown to be shifted positively by cholesterol depletion in freshly isolated fetal skeletal muscles (35). Basal myocardial ICa,L are sustained by phosphorylation of L-type Ca2+ channels and reflect the coordinated activities of kinases and phosphatases, even in the absence of humoral stimulation (9, 10). In cardiac myocytes, dephosphorylation of CaV1.2 is primarily catalyzed by phosphatase 1 (PP1) and phosphatase 2A (PP2A) (17). Furthermore, the majority of cardiac CaV1.2 expressed in HEK-293 cells has a distinct PP2A binding site, which enables PP2A to continuously catalyze the dephosphorylation of the channel (8). The phosphatase-mediated regulation of basal L-type Ca2+ channel phosphorylation in murine ventricular myocytes is reflected by the prominent increase of basal ICa,L and the negative shift in the I-V relationship induced by calyculin, a PP1/PP2A blocker, in the absence of humoral modulators (9, 10). Similarly, activation of PP2A also downregulates ICa,L and causes a positive shift in the I-V relationship in atrial myocytes from patients with chronic atrial fibrillation (6).

Taken together, the findings summarized above suggest that the shift in the I-V relationship for basal ICa,L induced by cholesterol depletion, as well as the significant increase in current density at positive potentials, may be explained by the combined effects of predominant phosphatase activity, reflecting protein kinase suppression, and an increase of the number of functional L-type Ca2+ channels, reflecting removal of the suppressive effect of cholesterol.

Mechanism of inhibition of β-adrenergic increases in ICa,L by cholesterol depletion. β1- and β2-ARs distribute to discrete membrane domains, forming macromolecular complexes with downstream signaling proteins in cardiac myocytes (40, 42, 43). The formation of macromolecular signaling complexes within lipid rafts allows specific and highly efficient signal transduction. In adult murine ventricular myocytes, stimulation of β1-ARs induces a large and diffuse increase in cAMP, whereas β2-AR stimulation induces a smaller, localized increase in cAMP (32). In neonatal mouse cardiomyocytes, disruption of lipid raft/caveolae by 10 mM MβCD suppresses β2-AR-mediated increases in ICa,L without affecting β1-AR signaling (1). β2-AR-coupled type VIII AC is dependent on Ca2+ influx, either through voltage-gated Ca2+ channels or capacitative Ca2+ entry (11, 41). Consequently, β2-AR-mediated CaV1.2 phosphorylation and the resultant increase in ICa,L are suppressed by BAPTA, whereas the more influential β1-AR-mediated ICa,L increase is resistant to BAPTA in adult rat ventricular myocytes (20). The suppression of ISO-induced increase of ICa,L elicited by cholesterol depletion in the presence of 5 mM BAPTA thus suggests that cholesterol depletion inhibits β1-AR-mediated phosphorylation of CaV1.2 channels in rabbit and rat ventricular myocytes. Because most cholesterol within a cell is localized to plasma membrane (27), we would expect that the 50% decrease in cellular cholesterol content induced by 10 mM MβCD would reflect a similar depletion of membrane cholesterol. We hypothesize that dialysis of 30 mM MβCD depleted membrane cholesterol even more extensively, even affecting the noncaveolar membrane, resulting in suppression of the β1-adrenergic increase of ICa,L.

The small increases in the background current after the application of ISO or FSK in the presence of MβCD were similar to those seen in the time-matched controls. This suggests that the β1-AR-mediated, cAMP-regulated Cl– current (18) was also elicited by ISO and FSK in cholesterol-depleted myocytes. On the other hand, the increased background current could be due to an increase in the nonspecific leak current, reflecting the greater membrane fragility (39), or due to unidentified currents through channels and exchangers/transporters. Irrespective of the upstream uncoupling of the β-adrenergic signal transduction, the marked suppression of the DBcAMP-induced increase in ICa,L elicited by MβCD dialysis strongly suggests that cholesterol depletion suppresses PKA-catalyzed phosphorylation of L-type Ca2+ channels.

The CaV1.2 channel and AKAP, as well as β-adrenergic signaling molecules such as G protein-coupled receptors and AC, colocalize in the cholesterol-rich, lipid raft/caveolae of cardiac myocytes (1, 16). AKAPs are required for the β-adrenergic enhancement of cardiac ICa,L (14). To phosphorylate CaV1.2, PKARII is tethered to the distal COOH terminus of CaV1.2 via AKAP15, which, in ventricular myocytes, is anchored to the COOH terminus by a leucine zipper-like motif (19). In addition, AKAP15/18 is anchored to the lipid raft by myristoylation and palmitoylation of its NH2 terminus (13, 37), which suggests that, because it stabilizes the membrane structure, the cholesterol localized in the lipid raft is crucial for the close coupling between CaV1.2 and AKAP. The fact that BAY K 8644, a dihydropyridine Ca2+ agonist that directly binds to CaV1.2 and increases ICa,L independently of PKA-catalyzed phosphorylation (25, 33), increased ICa,L to a similar extent in cholesterol-depleted myocytes and time-matched controls suggests that CaV1.2 is modulated by dihydropyridine agents, even after cholesterol depletion.

Finally, our findings suggest that, by affecting the cholesterol content in the plasmalemma, hypocholesterolemia may affect L-type Ca2+ channel function indirectly by modulating its phosphorylation.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
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This work was supported by grants from the Japanese Ministry of Education, Science, Sports, Culture, and Technology and the Promotion and Mutual Aid for Private Schools of Japan.


   ACKNOWLEDGMENTS
 
Present addresses: Y. Song, Department of Pediatrics and Immunology, University of Washington and Children's Hospital and Regional Medical Center, Seattle, WA 98195; H. Masumiya, Department of Physiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Ochi, Dept. of Physiology, New York Medical College, Basic Science Bldg., Valhalla, NY 10595 (e-mail: rikuoochi{at}optonline.net)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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