© 2001 American Heart Association, Inc.
Research Commentary |
Modulation of Ca2+ Signaling by Microtubule Disruption in Rat Ventricular Myocytes and Its Dependence on the Ruptured Patch-Clamp Configuration
From the School of Biomedical Sciences (S.C.C., E.W.), University of Leeds, Leeds, UK; Nutrition, Croissance, Cancer EA2103 (J.-Y.G.), University of Tours, Tours, France.
Correspondence to Dr S. Calaghan, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. E-mail s.c.calaghan{at}leeds.ac.uk
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Abstract |
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Abstract—In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al (Circ Res. 2000;86:30–36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased ICa,L density and [Ca2+]i transient amplitude and depressed the response of these parameters to the ß-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 µmol/L colchicine had no effect on shortening or [Ca2+]i transient amplitude or on the amplitude of ICa,L in perforated patch-clamped cells, under basal conditions and after stimulation with 1 µmol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal ICa,L was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, ICa,L did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca2+ signaling or the response to ß stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates ICa,L and attenuates the response to ß stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.
Key Words: cardiac myocyte • contraction • calcium signaling • microtubules • colchicine
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microtubules are hollow protein cylinders of
- and ß-tubulin heterodimers. Although
they form a major component of the cardiac cell cytoskeleton, their
functional role within the myocyte remains unclear. Microtubules have
been implicated in pathological conditions including
ischemia,1 cardiac
hypertrophy, and failure.2 In
some (but not all) cardiac hypertrophies, microtubules have been shown
to be
proliferated.3 4 5 6
Such microtubule proliferation is associated with a depression of
cardiac contractile
function,3 4 5 6 7
which has been shown to be normalized by the microtubule-disrupter
colchicine,3 4 5 6
thereby directly implicating microtubules in the modulation of
contraction. It has been suggested that microtubule proliferation may
modify contraction by increasing sarcomeric
load,3 although contractile
depression associated with chemical proliferation of the microtubules
by taxol is accompanied by a decrease in the amplitude of the
intracellular
[Ca2+]i
transient.7 In the absence of prior hypertrophic proliferation, a number of studies have shown that microtubule disruption by colchicine does not increase contractility in either multicellular preparations or single myocytes in the rat,5 8 9 10 cat,3 4 6 11 12 guinea pig,13 and dog.14 However, the recent study by Gomez et al15 published in Circulation Research showed that in ruptured patch-clamped rat ventricular myocytes, disruption of microtubules by colchicine increased ICa,L density and the amplitude of the [Ca2+]i transient and depressed the response of these parameters to the ß-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by free tubulin within the cell. Given the involvement of microtubules in some models of hypertrophic contractile dysfunction and the central role of ß-adrenergic stimulation in cardiac function, the work by Gomez et al15 raises interesting questions about the possible role of microtubules in the heart. However, the increase in [Ca2+]i and ICa,L seen by Gomez et al15 appears inconsistent with the lack of contractile effect of colchicine reported by others.
To date, no single study has measured the response of contraction, [Ca2+]i, and ICa,L to colchicine or the effect of colchicine on the response of these parameters to ß-adrenergic stimulation. We have undertaken such an investigation, and our data allow us to reconcile the apparent inconsistencies between Gomez et al15 and previous contractile studies. Disruption of microtubules by colchicine had no effect on Ca2+ signaling in intact cells or under perforated patch-clamp conditions but appeared to make cells more sensitive to some aspect of the ruptured patch-clamp configuration, resulting in activation of ICa,L and attenuation of the response to ß-adrenergic stimulation. Our observations suggest that this effect on Ca2+ signaling is unlikely to be associated with an increase in free tubulin, and alternative interpretations are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Single ventricular myocytes were isolated enzymatically from hearts of male Wistar rats (200 to 250 g) as described previously.16 Rats supplied by Central Biomedical Services at the University of Leeds were used in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act of 1986.
Immunofluorescence Confocal Microscopy
The microtubules were labeled with monoclonal
antibody to ß-tubulin (1:200; Sigma Chemical Co) and FITC-conjugated
donkey anti-mouse IgG (1:50; Jackson), as described by Howarth et
al.7 Sections of 
2 µm
thickness taken longitudinally through the cell at the level of the
nucleus were imaged using confocal laser microscopy (Leica True
Confocal Scanner SP). Settings were identical for imaging of vehicle-
and colchicine-treated cells.
Measurement of Contraction and
[Ca2+]i
Contraction and
[Ca2+]i were
measured in cells loaded with fura-2-AM (Molecular
Probes).17 Cells were
electrically stimulated at 0.5 Hz, and cell shortening was monitored
using a video edge-detection system (Crescent Electronics). Myocytes
were alternately illuminated by 340 and 380 nm light, and the
fluorescent emission at 510 nm was recorded. The ratio of the emitted
fluorescence at the two excitation wavelengths (340/380 ratio) was
calculated to provide an index of
[Ca2+]i.
Experiments were performed at 22°C to 25°C.
Perforated and Ruptured Patch-Clamp
Studies
For perforated patch-clamp studies, pipettes were
filled with PP solution (see Solutions) containing 400 µg/mL
amphotericin B. Access resistance using this configuration was
13.8±0.7 M
, and time to peak of
ICa,L,
an indicator of good access, was 7.5±0.5 ms. For ruptured patch
experiments, pipettes were filled with RP solution (see Solutions).
Cell capacitance and series resistance were electronically compensated
by 60%. Pipette tip resistance was 2 to 3 M.
ICa,L
was elicited from a holding potential of -80 mV by a 300-ms step
depolarization to -40 mV (to inactivate
INa),
followed by a 300-ms depolarization to 0 mV (at a stimulation frequency
of 0.5 Hz). Current-voltage relationships for
ICa,L
were measured using a conventional protocol that was preceded at each
potential by 4 priming depolarizations to 0 mV. In ruptured patch
experiments, cell size was also recorded from a video image of the cell
at selected time points after establishment of the whole-cell
configuration.
Solutions
HEPES-based bathing solution contained (mmol/L) NaCl
113, HEPES 5, Na2HPO4 1,
MgSO4 · 7H2O 1, KCl 5,
CaCl2 1, glucose 10, sodium acetate 20, insulin
5 U/L; pH 7.4. PP pipette solution contained (mmol/L) KCl 10, NaCl 10,
MgCl2 · 6H2O 1,
CaCl2 1, potassium glutamate 110, HEPES 5; pH
7.2 with KOH. RP pipette solution contained (mmol/L) KCl 130,
MgCl2 · 6H2O 1,
NaH2PO4 1,
Na2 phosphocreatine 3.6, MgATP 5, HEPES 10, EGTA
0.1; pH 7.2 with KOH. Stock solutions of colchicine were dissolved in
methanol. The final concentration of methanol in colchicine-containing
solutions was 0.01% or 0.1%. Cells were exposed to colchicine (1 or
10 µmol/L) or vehicle (0.01% or 0.1% vol/vol) for 2 hours and then
studied within a further 2-hour period. All experiments were conducted
in the presence of colchicine or vehicle as appropriate. Myocytes from
each heart were always used to study the effects of both vehicle and
colchicine in parallel. Contraction,
[Ca2+]i, and
ICa,L
were measured under basal conditions and at steady state after
perfusion with 1 µmol/L isoproterenol.
Statistics
Results are expressed as mean±SEM of
n observations. Statistical
analysis was performed using the Student’s
t test (paired and unpaired) or
2-way repeated-measures ANOVA followed by Bonferroni
t test for multiple
comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of Colchicine on Microtubules
Immunofluorescence techniques were used to establish the effect of colchicine on the microtubules in rat ventricular myocytes. Figure 1
shows the effect of 2-hour exposure of myocytes to
10 µmol/L colchicine. Fluorescence intensity in sections taken at the
level of the nucleus in colchicine-treated cells was reduced to 57% of
that recorded in vehicle-treated cells
(P<0.01) confirming that, in
our study, colchicine caused disruption of the
microtubules.
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Effect of Colchicine on Contraction and
[Ca2+]i
Initially, a study of the effect of colchicine (at the
two concentrations used most widely in the literature) on contraction
and [Ca2+]i in
myocytes with intact sarcolemma was carried out. Exposure of rat
ventricular myocytes to 1 and 10 µmol/L colchicine for 2 hours had no
effect on either the magnitude or kinetics of cell shortening
(Table,
top). Diastolic
[Ca2+]i was
significantly higher (P<0.01)
in cells exposed to 10 µmol/L colchicine; however, at neither
concentration of colchicine did we observe any significant effect on
[Ca2+]i transient
amplitude
(Table,
bottom). The only effect of colchicine on transient kinetics was a
reduction (P<0.01) in the
t1/2 of
the transient at 10 µmol/L colchicine
(Table,
bottom).
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Additional studies examining the effect of colchicine
on the response to ß-adrenergic stimulation were then performed.
Figure 2 shows the effect of 10 µmol/L colchicine on the
contractile response to the ß-adrenergic agonist isoproterenol.
Colchicine did not modify the effect of isoproterenol on either the
magnitude or kinetics of contraction. Neither did colchicine affect the
increase in [Ca2+]i
transient amplitude with isoproterenol
(Figure 3). In this series of experiments, the only effect of
colchicine on the
[Ca2+]i transient
was an attenuation of the increase in time to peak of the transient
observed in the presence of isoproterenol
(Figure 3).
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) and in the presence of isoproterenol (•). Shortening under basal conditions with colchicine is normalized to that under basal conditions in the vehicle-treated cell for ease of comparison. B, Shortening expressed as a percentage of resting cell length. C, Time to peak of the contraction. D, Time to half-relaxation. Open bars are under basal conditions, and hatched bars are in the presence of isoproterenol. All bars are mean+SEM of 11 to 12 observations. *P<0.05, **P<0.01, ***P<0.001 compared with respective group under basal conditions (paired Student’s t test). There were no significant differences between colchicine- and vehicle-treated cells.
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Effect of Colchicine on
ICa,L
The effect of colchicine on
ICa,L
was measured using both amphotericin perforated patch-clamp and
ruptured patch-clamp techniques. Basal
ICa,L
was similar in perforated patch-clamped cells in the presence of
vehicle and colchicine (8.7±1.5 versus 10.0±1.9 pA/pF; n=8), and, in
both groups of cells,
ICa,L
increased 2-fold (P<0.05)
in response to isoproterenol
(Figure 4C). However, in ruptured patch-clamped myocytes,
basal
ICa,L
was significantly higher
(P<0.05) in colchicine-treated
cells (11.8±2.0; n=6) than in vehicle-treated cells (6.4±0.7; n=6).
Furthermore, in the ruptured patch-clamp configuration, although we
observed a significant increase in
ICa,L in
vehicle-treated cells in response to isoproterenol, in cells treated
with colchicine, we saw no significant increase
(P>0.05) in
ICa,L
after exposure to isoproterenol
(Figure 4D). By contrast to the study of Gomez et
al15 performed in the
ruptured patch configuration, using perforated patch there was no
difference (P>0.05) in
current-voltage relationships under basal conditions between
vehicle-treated cells and colchicine-treated cells
(Figure 5). There was a tendency for the
ICa,L
response to isoproterenol to be enhanced in cells treated with
colchicine; however, this difference was not significant (2-way
repeated-measures ANOVA).
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Figure 6 summarizes the effects of colchicine on the
response of contraction,
[Ca2+]i transient,
ICa,L
measured in perforated patch, and
ICa,L
measured in ruptured patch configuration to isoproterenol. This figure
shows clearly that in cells with an intact sarcolemma (measurements of
contraction,
[Ca2+]i transient,
and ICaL
under perforated patch-clamp conditions), colchicine had no effect on
the ratio of isoproterenol-stimulated to basal values of the
parameters. In fact there is a (nonsignificant) tendency for this ratio
to be increased in colchicine-treated cells
(P>0.05). However, in ruptured
patch-clamped cells, the ratio of isoproterenol-stimulated to basal
value is significantly reduced
(P<0.05) in colchicine-treated
cells compared with vehicle-treated cells.
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Effect of Colchicine on Cell Size in Ruptured
Patch Configuration
The ruptured patch configuration may cause
dialysis of the cell with the contents of the microelectrode; this can
lead to dilution of intracellular contents and alterations in cell
shape. To test whether an interaction between the effects of colchicine
and a change in cell shape might account for the results obtained in
ruptured patch, cell size was recorded immediately and at 8 to 15
minutes after rupture of the membrane patch (at 10.4±1.2 and 13.3±1.1
minutes in vehicle- and colchicine-treated cells, respectively;
P>0.05). In vehicle-treated
cells, neither resting cell length (105±8 versus 104±7 µm; n=5) nor
cell width (18.1±2.9 versus 17.5±2.6 µm) changed during the course
of the experiment (P>0.05). In
colchicine-treated cells, although there was no significant change
(P>0.05) in resting cell
length (99.0±3.1 versus 97.8±3.0 µm; n=6), cell width was
significantly smaller
(P<0.001) at the later time
point (25.2±1.5 versus 22.8±1.4
µm).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When the dynamic network of microtubules within the cardiac cell is proliferated either pathologically (in some models of pressure-overload hypertrophy) or chemically (by taxol), contractility is reduced.3 4 5 6 7 However, in the absence of prior hypertrophic proliferation, evidence strongly suggests that microtubule disruption by colchicine does not modulate contraction.3 4 5 6 8 9 10 11 12 13 14 However, Gomez et al15 recently presented data in Circulation Research showing that in ruptured patch-clamped myocytes, microtubule disruption by colchicine can increase ICa,L and the [Ca2+]i transient, effects that would be expected to increase contraction, unless myofilament sensitivity was concurrently depressed (for review, see Calaghan and White18 ). We have addressed the apparent inconsistency highlighted by the findings of Gomez et al15 by undertaking a comprehensive investigation of the effect of colchicine on contraction, [Ca2+]i, and ICa,L.
The ability of colchicine to disrupt microtubules under the conditions of our experiments was confirmed by the use of immunofluorescence confocal microscopy. We saw a 43% reduction in immunofluorescence intensity in cells treated with 10 µmol/L colchicine for 2 hours; this degree of reduction in immunofluorescence intensity is comparable with the 38% reduction observed in the study of Gomez et al15 after a 2-hour incubation of rat ventricular myocytes with 1 µmol/L colchicine.
Our findings that microtubule disruption had no effect on the amplitude of the [Ca2+]i transient or contraction in intact cells nor on the amplitude of ICa,L under perforated patch-clamp conditions are consistent with those of many contractile studies reported in the literature.3 4 5 6 8 9 10 11 12 13 14 In addition, we saw no effect of colchicine on the change in amplitude of contraction, [Ca2+]i transient (see Palmer et al19 ), or ICa,L in response to the ß-adrenoceptor agonist isoproterenol. We conclude that in cells with an intact sarcolemma and cytosol, microtubule disruption by colchicine does not activate adenylyl cyclase. These results contrast with measurements of [Ca2+]i transients and ICa,L obtained by Gomez et al15 in ruptured patch-clamped cells.
However, our own observations on ICa,L under ruptured patch conditions agree with those of Gomez et al.15 In cells treated with colchicine, basal ICa,L is enhanced and the response to isoproterenol is blunted. Therefore, it appears that these data and those of Gomez et al15 are related to some aspect of the ruptured patch-clamp configuration. Our use of K+-based pipette solution for the ruptured patch experiments allows us to exclude the possibility that Cs+-based solutions15 contributed to these effects.20
Gomez et al15 proposed that, on microtubule disruption, adenylyl cyclase is activated via nucleotide exchange to Gs protein by accumulation of free tubulin within the cell. Our lack of response to colchicine in intact cells and under perforated patch conditions argues against this explanation. In ruptured patch conditions, inhibitors of adenylyl cyclase blunt the response to colchicine15 suggesting that the combination of microtubule disruption and the ruptured patch configuration work together to activate adenylyl cyclase. This configuration can cause more dialysis of the intracellular milieu with the contents of the recording microelectrode than is seen in the perforated patch configuration.21 22 Indeed run-down of ICa,L in ruptured patch-clamped cells is a common observation that has been related to intracellular dialysis.23 One possible mechanism that could account for the effect of colchicine in ruptured patch-clamped cells is the loss, through dialysis, of cellular tubulin (or a tubulin-Gi complex) resulting in decreased cellular activity of Gi. In support of this hypothesis, there is evidence that tubulin can activate Gi24 25 and, in the cardiac cell, Gi is in excess of Gs.26 In our experiments, colchicine appeared to oppose the effect of the ruptured configuration on ICa,L run-down.
The ruptured patch-clamp configuration may also be associated with changes in cell shape, and an alternative explanation to account for the effect of colchicine on ruptured patch-clamped myocytes is based on our observation that disruption of the microtubular network by colchicine was associated with a reduction in cell width under ruptured patch conditions. Shrinkage of rat cardiac myocytes has been shown to activate adenylyl cyclase.27 Alterations in cell size might reflect changes in stress on the sarcolemma and t-tubular system, and there is evidence that adenylyl cyclase is mechanosensitive.28 29 Early time-dependent potentiation of ICa,L after establishment of the whole-cell configuration in ruptured patch has been ascribed to mechanical effects of this configuration.30
There are several reports in the literature of pathological conditions (including ischemia, hypothyroidism, and chronic pressure-overload hypertrophy)19 31 32 in which the cardiac microtubule network is diminished and in which cellular levels of tubulin may be decreased. Based on our observations and re-interpretation of the findings of Gomez et al,15 the possibility that the loss of cellular tubulin activates adenylyl cyclase or that microtubule disruption renders adenylyl cyclase more susceptible to mechanical stimulation has important implications for such conditions.
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Acknowledgments |
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This work was sponsored by the British Heart Foundation and a Wellcome Trust Biomedical Collaborative Grant.
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Footnotes |
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Original received October 12, 2000; revision received February 7, 2001; accepted February 7, 2001.
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