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(Circulation Research. 2000;86:30.)
© 2000 American Heart Association, Inc.

Cellular Biology

Microtubule Disruption Modulates Ca²⁺ Signaling in Rat Cardiac Myocytes

A. M. Gómez, B. G. Kerfant, G. Vassort

From Physiopathologie Cardiovasculaire, INSERM U-390, Montpellier, France.

Correspondence to Ana M. Gómez, INSERM U-390, CHU Arnaud de Villeneuve, 34295 Montpellier, France. E-mail agomez{at}u390.montp.inserm.fr

	Abstract

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Abstract—Microtubules have been shown to alter contraction in cardiac myocytes through changes in cellular stiffness. However, an effect on excitation-contraction coupling has not been examined. Here we analyze the effects of microtubule disruption by 1 µmol/L colchicine on calcium currents (I_Ca) and [Ca²⁺]_i transients in rat ventricular myocytes. I_Ca was studied using the whole-cell patch-clamp technique. Colchicine treatment increased I_Ca density (peak values, -4.6±0.4 and -9.1±1.3 pA/pF in 11 control and 12 colchicine-treated myocytes, respectively; P<0.05). I_Ca inactivation was well fitted by a biexponential function. The slow component of inactivation was unchanged, whereas the fast component was accelerated after colchicine treatment (at -10 mV, 11.8±1.0 versus 6.7±1.0 ms in control versus colchicine-treated cells; P<0.005). [Ca²⁺]_i transients were analyzed by fluo-3 epifluorescence simultaneously with I_Ca. Peak [Ca²⁺]_i transients were significantly increased in cardiac myocytes treated with colchicine. The values of F/F₀ at 0 mV were 1.1±0.02 in 9 control cells and 1.4±0.1 in 11 colchicine-treated cells (P<0.05). ß-Adrenergic stimulation with 1 µmol/L isoproterenol increased both I_Ca and [Ca²⁺]_i transient in control cells. However, no significant change was induced by isoproterenol on colchicine-treated cells. Colchicine and isoproterenol effects were similar and not additive. Inhibition of adenylyl cyclase by 200 µmol/L 2'-deoxyadenosine 3'-monophosphate blunted the colchicine effect. We suggest that ß-adrenergic stimulation and microtubule disruption share a common pathway to enhance I_Ca and [Ca²⁺]_i transient.

Key Words: heart • Ca²⁺ current • microtubule • Ca²⁺ transient • ß-adrenergic stimulation

	Introduction

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Microtubules constitute one of the main cytoskeletal components, together with actin and intermediate filaments. The microtubule network is dynamic, composed by the self-association of ,ß-tubulin dimers. Thus, by polymerization and depolymerization, the cell can change the amount of microtubules at constant tubulin amount. The presence of microtubules in the cardiac myocytes is well known, but its role in physiology and pathology is thought to be purely mechanical. In this regard, it has been shown that in pressure-overload cardiac hypertrophy, there is an increase in the microtubule network, which would be responsible for the contractile dysfunction in hypertrophied cells.¹ In this elegant work, Tsutsui et al¹ studied right ventricular cardiac myocytes isolated from cats subjected to pulmonary artery constriction. Under these experimental conditions, hypertrophied cells presented an increased number of microtubules and contracted weakly. When treated with the depolymerizing agent colchicine, hypertrophied myocytes contracted normally. Tsutsui et al¹ concluded that the contractile defect of hypertrophied cells is due to an increase in stiffness and viscosity on the cell imposed by the increased microtubule network triggered by the pressure overload.² However, it is also possible that microtubule polymerization and depolymerization play other roles in addition to the mechanical one. In this regard, we have recently shown that heart failure after pressure-overload cardiac hypertrophy induces a dysfunction of the excitation-contraction (EC) coupling.³ This alteration can account for the decreased contractile function found in this animal model. Because microtubules are increased in the weakly contracting myocytes after pressure-overload cardiac hypertrophy,^{4 5} this cytoskeletal abnormality might be in part responsible for the contractile dysfunction observed in pressure overload–induced heart failure.

Electrical excitation during an action potential activates sarcolemmal Ca²⁺ channels. Ca²⁺ influx that follows opening of these channels induces a local elevation of [Ca²⁺]_i around the sarcoplasmic reticulum (SR) Ca²⁺ channels, or ryanodine receptors (RyRs). Activation of RyRs by Ca²⁺ triggers the SR Ca²⁺ release that would be able to activate contractile fibrils and contraction.

In this study, we analyzed the effects of microtubule depolymerization on the 2 main components (Ca²⁺ current and SR Ca²⁺ release) of EC coupling, and we found that microtubule depolymerization increases them both. Moreover, this effect is blocked by inhibition of adenylyl cyclase. Thus, besides a mechanical role, microtubules seem to be important modulators of calcium signaling and, hence, cardiac function.

	Materials and Methods

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Cardiac ventricular myocytes were isolated from adult male Wistar rats (275 to 325 g) as previously described.⁶

Cardiomyocytes were fixed and immunolabeled with anti–ß-tubulin and a fluorescence secondary antibody (C. Frederick and W.J. Lederer, unpublished data, 1996; see online-only supplementary information, http://www.circresaha.org). Cells were viewed using a confocal microscope Zeiss LSM 510 fitted with an argon laser (488-nm wavelength). Emission was collected through a low-pass filter at 505 nm. Parameters were first adjusted with a control cell and maintained constant to examine all cells.

Electrophysiology
The whole-cell mode of the patch-clamp technique⁷ was used to study L-type Ca²⁺ current (I_Ca). Myocytes were perfused with HEPES solution containing, in mmol/L, NaCl 140, MgCl₂ 0.5, CsCl 5, glucose 5.5, HEPES 5, and CaCl₂ 1.8 (pH set to 7.4 with NaOH). Myocytes were voltage-clamped (Axopatch 200A, Axon instruments) with a suction pipette filled with a solution containing, in mmol/L, CsCl 130, MgCl₂ 1, NaH₂PO₄ 1, Na₂ phosphocreatine 3.6, MgATP 5, HEPES 10, and fluo-3 (pentapotassium salt) 0.1 (pH fixed at 7.2 with CsOH). In some experiments, 200 µmol/L 2'-deoxyadenosine 3'-monophosphate (2'd3'AMP) was added to the pipette solution. Pipettes had tip resistances of 0.9 to 1.2 M. Capacitance and series resistance were electronically compensated to 60%. I_Ca was elicited as previously explained.³

Fluorescence
Cells were loaded with the fluorescence-Ca²⁺ dye fluo-3 (Molecular Probes) either by diffusion of its salt form through the patch pipette or by using its acetoxymethyl ester derivative as previously described.⁶ This second method was used in the experiments conducted to estimate the SR Ca²⁺ load.

Fluo-3–loaded cells were excited with a xenon lamp at 460- to 490-nm wavelength through an epifluorescence attachment. Emission fluorescence (520 nm) was detected with a photomultiplier tube. Microscope and fluorescence equipment were from Nikon France. The signal was then amplified and low-pass filtered at 100 kHz (Fern Development).

I_Ca and fluorescence signals were simultaneously digitized (Digidata 1200, Axon instruments) and acquired at sampling rate of 100 µs using pClamp 7.

Statistics
Data are presented as mean±SEM. An unpaired Student t test was performed to compare control and colchicine-treated cells or control and paclitaxel (Taxol)–treated cells, whereas a paired Student t test was used to test the isoproterenol (ISO) effect. P<0.05 was considered significant.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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The role of microtubules in EC coupling was investigated by analyzing the effect of colchicine, a substance known to induce microtubule depolymerization, on Ca²⁺ current and SR Ca²⁺ release.

To be sure that colchicine treatment was effectively disrupting microtubules in our hands, some cells were fixed for immunofluorescence assay. Control myocytes and cells exposed to 1 µmol/L colchicine at different times were marked with anti–ß-tubulin and fluorescein-conjugated secondary antibody as described in Materials and Methods. To allow comparison between different cell groups, we used the same parameters in the microscope configuration to visualize all cells. After 30 minutes and for up to 4 hours, microtubules were effectively disrupted. Because we started doing patch-clamp experiments after 1.5 hours of colchicine treatment, we chose to present images and data of immunofluorescence after 2 hours colchicine treatment. Figure 1A shows examples of 1 cardiac myocyte not exposed to colchicine (left) and after 2 hours of colchicine treatment (right). The observed filamentous structures in control myocytes were absent in colchicine-treated cells. Because all cells were marked in the same way and images were taken under the same conditions, we measured the averaged fluorescence in each cell, which is correlated with the number of microtubules. Fluorescence values were 61.4±3.4 versus 38.3±3.3 in 14 control versus 19 colchicine-treated myocytes, P<0.0001.

View larger version (20K): [in this window] [in a new window]   Figure 1. Colchicine treatment increases ICa and [Ca2+]i transient in adult rat myocytes. A, Immunofluorescence confocal images of ß-tubulin–labeled rat cardiac myocytes, incubated in the presence (right, •) or absence (left, ) of 1 µmol/L colchicine for 2 hours at room temperature (25°C). B, Top, Voltage protocol. Middle, Representative traces of [Ca2+]i transients recorded at 0 mV in a control (left, ) and a colchicine-treated (right, •) myocyte. The fluorescent signal (F) was normalized to the signal before depolarization (F0). Dotted line represents unity. Bottom, Representative ICa traces recorded simultaneously. Dotted line represents 0 current level. C, Current and fluorescence/voltage relationships of mean ICa density (bottom) and [Ca2+]i transient (top) recorded in 11 control cells () and 12 colchicine-treated cells (•). Lines are drawn by eye. Colchicine treatment significantly increased ICa current density and [Ca2+]i transient (*P<0.05). ICa and [Ca2+]i transient were elicited by 100-ms depolarization at various potentials, ranging from -50 to +60 mV, every 10 seconds. Holding potential was -80 mV.

Ca²⁺ Current and [Ca²⁺]_i Transient
The effect of disrupting microtubules on L-type calcium current, I_Ca, was analyzed in rat ventricular cells under whole-cell patch clamp. I_Ca was elicited by applying 100-ms depolarizing pulses from -50 to +60 mV every 10 seconds from a holding potential of -80 mV. To allow steady-state Ca²⁺ load of the SR, 4 steps to 0 mV were applied at 1 Hz between test pulses. Sodium current was inactivated by predepolarization to -50 mV (achieved by a 500-ms ramp followed by maintaining at this potential for 100 ms) before each test pulse.³

Colchicine was first dissolved in DMSO and then added to an aliquot of cells. DMSO concentration in the cell suspension was 0.01%. This concentration of DMSO did not induce any significant change in I_Ca (data not shown). Experiments were made 1.5 to 4 hours after colchicine addition.

I_Ca recorded in cells treated with 1 µmol/L colchicine was markedly increased when compared with I_Ca recorded in cells in control conditions (Figure 1B, bottom). To avoid error in pooling data from different-sized myocytes, we normalized the I_Ca amplitude by the cell capacitance, to get I_Ca density. Cell capacitance was of similar magnitude in control cells and in cells that were incubated with colchicine (172.0±15.0 [n=13] versus 156.9±23.4 pF [n=12] in control versus colchicine). The current density/voltage relationship of I_Ca is shown in Figure 1C (bottom). Microtubule disruption induced an increase in I_Ca that is statistically significant from -10 to +20 mV. To check whether or not the observed effect was due to a direct effect of colchicine, some control cells were patch-clamped and perfused with 1 µmol/L colchicine. Direct perfusion of myocytes up to 5 minutes with colchicine was without effect on I_Ca (data not shown).

Cardiac myocytes contraction arises when the increase in [Ca²⁺]_i triggered by Ca²⁺ influx through Ca²⁺ channel activates neighboring RyRs. The opening of RyRs provokes a large Ca²⁺ release that activates contractile fibrils. Microtubule disassembly increased I_Ca; thus, we addressed the issue of whether microtubules could modulate [Ca²⁺]_i transient. Figure 1B, top, shows representative fluorescence traces recorded simultaneously with I_Ca in a control and a colchicine-treated cell. Colchicine treatment markedly increased [Ca²⁺]_i transient. Comparison of average data are shown in Figure 1C (top). This result was expected, because I_Ca, which triggers Ca²⁺ release, is increased under these conditions (Figure 1C, bottom).

I_Ca and [Ca²⁺]_i Transient Kinetics
This is the first time to our knowledge that an effect of microtubules on I_Ca at the whole-cell level is shown. There is, however, a study in single Ca²⁺ current.⁸ In this study, performed in embryonic chick ventricle cells, colchicine increased the inactivation of single-channel current. We further analyzed the effect of colchicine on I_Ca kinetics. As observed in the current records (see Figures 1B and 5A), I_Ca inactivation seems faster in colchicine-treated myocytes. To quantify this observation, current decay was fitted by the following biexponential equation: y=C+A_fastxe^-t/_fast+A_slowxe^-t/_slow

View larger version (24K): [in this window] [in a new window]   Figure 5. ß-Adrenergic stimulation is ineffective on rat cardiac myocytes after microtubule disassembly. A, Sample traces of ICa and [Ca2+]i transients (represented as F/F0, measured as in Figure 1) at 0 mV before (C) and after 1 µmol/L ISO application, in control and colchicine (Colchi)–treated cells. Above the traces in the control myocyte, the voltage protocol is represented. Dotted lines represent 0 current level for ICa traces and a fluorescence ratio level of 1 for F/F0. B, Effect of 1 µmol/L ISO in ICa in control myocytes (white bar) and myocytes previously treated with colchicine (black bar). In each cell, the values of ICa at 0 mV after ISO application (ICa, ISO) were divided by the values of ICa at 0 mV before ISO application (ICa, C). Values in graph represent ICa, ISO/ICa, C (n=4). A similar rationing is made for the peak fluorescent transients (F/F0)ISO/(F/F0)C for control myocytes (white bar) and myocytes previously treated with colchicine (black bar); n=5. **P<0.005 of ISO values compared with values before ISO application.

where C is a constant, A_fast and A_slow are the maximal amplitude of the fast and slow components respectively, and _fast and _slow are the time constant of the fast and slow components, respectively. The slow time constant of I_Ca inactivation was unaltered by microtubule disruption (Figure 2A, right). The fast component of I_Ca inactivation, _fast, was shorter after microtubule disruption (at -10 mV, 11.8±1.0 versus 6.7±1.0 ms in control [n=11] versus colchicine-treated cells [n=12], P<0.005) (Figure 2A, left).

View larger version (19K): [in this window] [in a new window]   Figure 2. Effect of colchicine treatment on ICa kinetics. A, Bar graph of the time constants of ICa inactivation, obtained by fitting the decay of each trace to an exponential function of second order. The fast (fast) and slow (slow) inactivation time constants at -10 mV are plotted on the graph at left and right, respectively. Control myocytes are represented by the white bar (n=11), and myocytes treated with colchicine (Colchi) are represented by the black bar (n=12, P<0.005). B, Steady-state inactivation of 8 control () and 4 colchicine-treated myocytes (•) are plotted against the predepolarization voltage. A slowly depolarizing ramp was applied from -80 to -50 mV to inactivate INa. The membrane potential was immediately changed to the predepolarization voltage for 1 second to produce a steady-state inactivation of ICa before stepping to 0 mV for 100 ms to assess ICa. Data were fitted to a Boltzmann function and are plotted as a dashed line for control myocytes and a solid line for colchicine-treated myocytes.

I_Ca steady-state inactivation was analyzed by applying voltage steps at 0 mV preceded by a 1-second predepolarization to different potentials ranging from -50 to +50 mV. The stimulation frequency was 0.1 Hz. The current elicited during each test potential was normalized to the current obtained during the test potential that was preceded by predepolarization at -50 mV. Normalized currents are plotted against predepolarization voltages in Figure 2B. By fitting to a Boltzmann function, we obtained the following voltages of half-inactivation, which were similar in both myocyte groups: –36.2±1.4 mV for control cells (n=8) and –32.4±1.6 mV after colchicine treatment (n=4).

[Ca²⁺]_i transient kinetics were then examined. The time from start of the depolarizing pulse to peak of the [Ca²⁺]_i transient was not statistically different in both cell groups (Figure 3A). However, decay time of the [Ca²⁺]_i transient was accelerated. The [Ca²⁺]_i transient decay could be well fitted to a single exponential function. The [Ca²⁺]_i transient decay was significantly faster in cells treated with colchicine than in control cells (Figure 3B).

View larger version (15K): [in this window] [in a new window]   Figure 3. Microtubule disruption accelerates [Ca2+]i transient decay. A, Bar graph showing the time to peak of the fluorescence signal in control (n=9) and colchicine-treated (n=11) myocytes at 0 mV. Time to peak was measured as time from start of depolarizing pulse to maximal fluorescence. B, Rate of decay constant of the [Ca2+]i transient (decay) at 0 mV obtained by fitting the fluorescence trace to a single exponential. White bar, control cells (n=9); black bar, colchicine-treated cells (n=11). *P<0.05.

The acceleration of the [Ca²⁺]_i transient decay could be due to an acceleration of the SR Ca²⁺-ATPase activity. If this were true, SR Ca²⁺ content could be elevated. The increased amount of Ca²⁺ entry though Ca²⁺ channels observed after colchicine treatment (Figure 1) could also account for differences in the SR Ca²⁺ load. After steady-state field stimulation at 1 Hz for 2 minutes, the amplitude of the fluorescence transient obtained by a rapid application of 10 mmol/L caffeine was used to assess SR Ca²⁺ content.⁶ Figure 4A shows representative traces of caffeine-induced fluorescence transients in a control (open circles) and a colchicine-treated (closed circles) cell. After colchicine treatment, SR Ca²⁺ content was increased. On average, the amplitude of the caffeine-induced [Ca²⁺]_i transient (F/F₀) was 2.4±0.3 in 10 control myocytes versus 4.0±0.6 in 9 colchicine-treated myocytes (P<0.05). To check whether or not the observed increase in [Ca²⁺]_i transient (Figure 2) was due to an increase in the activating I_Ca, we analyzed the "gain" function estimated by normalizing the rate of Ca²⁺ release to the integral of I_Ca, which we called calcium-induced calcium release (CICR) gain. If colchicine treatment alters EC coupling, CICR gain should be modified. Figure 4B shows that CICR gain is voltage-dependent and not significantly different after colchicine treatment. It appears that microtubule disruption does not affect coupling but rather induces changes in other aspects of EC coupling such as I_Ca and [Ca²⁺]_i transient.

View larger version (16K): [in this window] [in a new window]   Figure 4. Colchicine treatment increases SR Ca2+ content but does not alter release gain function. A, Example fluorescence traces obtained as a response of rapid application of 10 mmol/L caffeine on a control cell () and a colchicine-treated cell (•). F/F0 was obtained as explained in Figure 1. B, CICR gain function obtained as the rate of Ca2+ release per ICa and plotted as voltage function. CICR gain=(rate of release)/ICa. Rate of release=(peak F/F0)/time to peak. The rate of Ca2+ release was estimated as the peak F/F0 divided by the time from depolarization to peak fluorescence. The rate of release obtained in this way was divided by the integral of ICa (pC/pF). CICR gain is voltage-dependent and is not different for control () and colchicine-treated cells (•).

Effect of ß-Adrenergic Stimulation
In cardiac tissues, ß-adrenergic stimulation is known to increase cAMP that will activate phosphorylation by protein kinase A (PKA). It has been suggested that microtubules modulate the ß-adrenergic response in rat cardiac hypertrophy.⁹ Forskolin, a direct activator of PKA, is ineffective after microtubule disruption on colonic epithelia.¹⁰ These observations led us to test the effects of PKA activation by a ß-agonist on control cells and cells after microtubule disruption. After measuring I_Ca and [Ca²⁺]_i transient, we added to the bath solution 1 µmol/L ISO. ISO induced a marked increase in both I_Ca and [Ca²⁺]_i transient in control myocytes. However, only a weak increase in I_Ca and [Ca²⁺]_i transient could be registered after ISO application in the cells treated with colchicine (Figure 5A). Moreover, despite the difference in I_Ca in both cell groups, after ISO treatment I_Ca became similar in control and colchicine-treated myocytes.

Figure 5B summarizes the effect of ISO application on I_Ca and [Ca²⁺]_i transient at 0 mV in control cells and in cells treated with colchicine. For each cell, the values obtained after ISO application are divided by the values before ISO application (unit=no ISO effect). In control cells, there is a 76.5% increase in I_Ca and 83.5% in [Ca²⁺]_i transient (F/F₀). However, the ß-adrenergic agonist had no more significant effects in cardiac cells after microtubule disruption by colchicine.

Effect of Taxol on I_Ca
As a result of microtubule disruption, the amount of free tubulin increases within the cell. It is possible that the observed effect on Ca²⁺ signaling after colchicine treatment is due to the diminution of polymerized tubulin or to an increase on free tubulin. To check this possibility, the microtubule-stabilizing agent Taxol was used. Taxol was first dissolved in DMSO as colchicine. The final DMSO concentration was 0.01%.

Because the changes in Ca²⁺ signaling are mainly due to the increase on I_Ca, we analyzed I_Ca density in cells incubated with 10 µmol/L Taxol for at least 1.5 hours, which stabilized microtubules. Figure 6A shows confocal images of a control (left) and Taxol-treated (right) myocyte labeled with anti–ß-tubulin antibody, as explained in Materials and Methods. Averaged fluorescence levels in Taxol-treated cells were higher than in control cells: 61.4±3.4 (control, n=14) versus 93.2±6.0 (Taxol, n=31); P<0.01. Figure 6B shows voltage dependence of I_Ca density recorded in control cells (left, open circles) and in Taxol-treated myocytes (right, open triangles). Both curves were not statistically different, as recently reported.¹¹ After 1 µmol/L ISO addition, both groups of cells responded in a similar manner (control, left, closed circles; and Taxol-treated cells, right, closed triangles).

View larger version (27K): [in this window] [in a new window]   Figure 6. Calcium current and ß-adrenergic response are unaffected after microtubule stabilization by Taxol. A, Immunofluorescence confocal images of ß-tubulin–labeled rat cardiomyocytes, incubated in the presence (right, ) or absence (left, ) of 10 µmol/L Taxol for 2 hours at room temperature (25°C). B, ICa density-voltage relationships in control (left, ) and 10 µmol/L Taxol-treated cells (right, ). Cells were incubated with Taxol for at least 1.5 hours before experiments. Taxol-treated myocytes did not show significant differences on ICa density compared with control myocytes. Both cell groups responded in a similar way to 1 µmol/L ISO (corresponding closed symbols).

Effect of Blocking Adenylyl Cyclase
Our findings using colchicine are quite similar to the effects of ß-adrenergic stimulation, with increased I_Ca and [Ca²⁺]_i transient, as well as acceleration of [Ca²⁺]_i transient decay time. In this regard, it has been reported in rat cerebral cortex that free tubulin stimulates adenylyl cyclase.¹² To analyze the involvement of adenylyl cyclase in the signal pathway under our experimental conditions, we tested the effect of the adenylyl cyclase inhibitor 2'd3'AMP on the whole-cell I_Ca enhancement after colchicine treatment. Cardiac myocytes were incubated with colchicine as earlier, and I_Ca was analyzed by the patch-clamp technique. Figure 7A shows I_Ca density at 0 mV over time after whole-cell configuration achievement in the presence of 200 µmol/L 2'd3'AMP in the internal solution, on a control myocyte (open circles) and a colchicine-treated myocyte (closed circles). Shortly after breaking into the cell, I_Ca was markedly higher on the colchicine-treated cell (Figure 7Aa) but became similar to the control myocyte with adenylyl cyclase dialysis (Figure 7Ab). Data are summarized in Figure 7B; whereas 2,d3'AMP had no statistically significant effect on the control myocytes, it did block the I_Ca increase after colchicine treatment.

View larger version (26K): [in this window] [in a new window]   Figure 7. Adenylyl cyclase antagonist blocks colchicine effect on ICa. A, Time course of adenylyl cyclase inhibition on ICa in a control () and a colchicine-treated (•) cell. In both cases, 200 µmol/L 2'd3'AMP was included in the patch pipette. After whole-cell configuration achievement, 200-ms–step depolarizations to 0 mV were applied every 20 seconds. a, The current trace at 1 minute after opening the seal is larger in a colchicine-treated (•) than in a control () cell. b, After 6-minute intracellular application of 2'd3'AMP, ICa became similar in both cells. B, Averaged data of ICa density at 0 mV after stabilization of ICa (6 minutes after whole-cell configuration achievement) in control cells (white, n=11; hatched bars, n=6) and colchicine-treated cells (black, n=12; crossed bars, n=9) in control (white and black bars) and in the internal solution supplemented with 200 µmol/L 2'd3'AMP (hatched and crossed bars).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present data show that microtubules are able to modulate EC coupling by modulating Ca²⁺ current and consequently [Ca²⁺]_i transient. Particularly, microtubule disassembly induces an increase in both I_Ca and [Ca²⁺]_i transient and accelerates both I_Ca inactivation and [Ca²⁺]_i transient decay. Thus, the effects of disrupting microtubules appear rather similar to ß-adrenergic stimulation. Moreover, after microtubule disruption, the effects of ISO on I_Ca and [Ca²⁺]_i transient are blunted. Furthermore, by blocking adenylyl cyclase activity we can reverse colchicine effects. These results show that colchicine and ISO actions share an inotropic effect involving activation of adenylyl cyclase.

Since 1993, Tsutsui et al^{1 13} and Tagawa et al⁴ have demonstrated that colchicine treatment can increase contraction in hypertrophied cardiac myocytes in which contraction was decreased. The suggested mechanism was a decrease in stiffness and viscosity, consequent to the decrease in microtubules.² A passive effect of the microtubule network is a plausible mechanism; however, the effect that these authors observed with colchicine treatment in hypertrophied cells might also be explained by the increase in I_Ca and SR Ca²⁺ release that we report in this study, although they fail to see an effect on normal cells.¹ We do not discard, however, a concomitant action on the cellular viscous load. In fact, these authors also observed a decrease in contraction after microtubule stabilization with Taxol¹ that so far can only be explained by an increase in cell viscosity and stiffness, because we failed to observe significant modification on I_Ca by Taxol (Figure 6). Returning to the cardiac effect of microtubule disruption, it has been shown that colchicine treatment accelerates the beating frequency in neonatal cardiac cells.^{14 15} This effect could also be explained by the increase in I_Ca that we report. However, after shorter periods of colchicine treatment than ours, some authors did not find an effect on contraction in either control or hypertrophied myocytes.^{16 17}

Cytoskeleton, and in particular microtubules, can bind several proteins, probably including ionic channels.¹⁸ Galli and DeFelice⁸ have found that colchicine modifies Ca²⁺ channel inactivation, but a direct effect of colchicine was discarded, because they failed to see an effect on excised patches. In a similar way, we observed that the fast component of I_Ca inactivation is accelerated in colchicine-treated cells. This first inactivation phase is dependent on Ca²⁺. Because sarcolemmal Ca²⁺channel and RyR are close to each other in a restricted space,¹⁹ a bigger Ca²⁺release by the RyR could increase the Ca²⁺-induced inactivation of the sarcolemmal Ca²⁺channel.²⁰ After colchicine treatment, as a result of a bigger triggering I_Ca, we obtained a larger [Ca²⁺]_i transient. The faster inactivation of I_Ca that we observed (see Figure 2A) can be the result of the increase in [Ca²⁺]_i transient induced after colchicine treatment (Figure 1).

To comprehend our results, one might consider the peculiar feature of microtubules. Microtubules are formed by the self-assembly of ,ß-tubulin dimers that polymerize and depolymerize dynamically.¹⁸ ,ß-Tubulin dimer is a GTP-binding protein with amino acid homologies and significant functional similarities to the G proteins.²¹ Moreover, in the neuronal system, relatively high-affinity binding between dimeric tubulin and the subunits of G_s, G_i1, and G_q have been reported,^{22 23} whereas assembled microtubules bind G protein quite weakly. It has also been observed that the tubulin dimer, also called free tubulin, causes stimulation of adenylyl cyclase in rat cerebral cortex membranes.¹² This effect results from a direct transfer of nucleotide from the exchangeable GTP-binding site of tubulin to the G_s protein.^{12 24} These findings strongly suggest that the increase in I_Ca and [Ca²⁺]_i transient presently reported in cardiac myocytes might be due to the activation of adenylyl cyclase by tubulin dimers. As a matter of fact, in cardiac tissues, cAMP-dependent activation of PKA has various effects on EC coupling. Among them, a phosphorylation of the L-type Ca²⁺ channel induces an increase in I_Ca (see Figure 1). There is also a phosphorylation of phospholamban that will result in an acceleration of the SR Ca²⁺ pump (see Figure 3B) and an increase in the SR Ca²⁺ load (Figure 4A). Moreover, RyRs can also be phosphorylated, modulating in this way their sensitivity to Ca²⁺.²⁵ We thus suspected that microtubule disruption increases both I_Ca and SR Ca²⁺ release by increasing free tubulin, which leads to adenylyl cyclase activation.

This hypothesis was supported by the observation that, after microtubule disruption, the ß-adrenergic stimulatory effect of I_Ca and [Ca²⁺]_i transient are blunted. The lack of additivity suggests that both the ß-adrenergic and the colchicine effects occur through a similar pathway. Two previous experimental reports are in line with this hypothesis. First, after microtubule disruption by colchicine, the cAMP-dependent Cl^- secretion is no longer sensitive to forskolin, whereas the Cl^-- secretory response of colonic epithelia is still Ca²⁺-dependent.¹⁰ Second, the forskolin-induced relocation of CFTR on T84 cells is blocked by the microtubule-disrupting agent nocodazole.²⁶ Furthermore, Limas and Limas²⁷ have suggested that microtubules can fix ß-adrenergic receptors in the membranes and that after colchicine treatment, the fraction of ß-receptors in internal vesicles compared with sarcolemma was increased. This possibility could explain the decrease in ISO effect that we observed; however, it cannot account for the increase in I_Ca and Ca²⁺ transient after colchicine treatment. In fact, their observation could rather be interpreted as ß-adrenergic receptor endocytosis, a secondary phase of agonist-independent phosphorylation and receptor desensitization mediated by PKA.²⁸ In our experiments, if free tubulin activates adenylyl cyclase and consequently PKA, as well as ISO application, it would be possible that once PKA is activated by increase in tubulin dimer and phosphorylation is induced, further activation of PKA by ISO would seem ineffective. We thus repeated the experiments in the presence of 2'd3'AMP, an inhibitor of the adenylyl cyclase that interacts with the purine site of the cyclase.²⁹ In line with our hypothesis, blocking the adenylyl cyclase reversed the microtubule disruption effect on I_Ca (Figure 7).

In conclusion, we show that microtubules can modulate calcium signaling in cardiac cells. We suggest that the microtubule disruption–increased level of soluble tubulin dimers activates G_s protein and leads to activation of the adenylyl cyclase. This effect triggers the cascade that leads to an increase in I_Ca and [Ca²⁺]_i transient and, in the end, to an increase in contraction. Moreover, this mechanism could help to explain, at least in part, the alterations in heart contraction observed in several pathologies in which changes in microtubules are reported.^{1 30}

	Acknowledgments

 
This work has been supported by a grant from Association Française contre les Myopathies (MNM 6086, 1997). We are indebted to Jean-Pierre Bénitah for help with the manuscript. We thank W. Jonathan Lederer and Paco Lorente for comments on the paper and Eva Le Charpentier for technical assistance. We also thank Cecilia Frederick for advice on the immunolabeling experiments. A.M. Gómez is supported by the Centre National de la Recherche Scientifique.

Received September 1, 1999; accepted October 19, 1999.

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