1 Renal Unit, Massachusetts General Hospital East, Charlestown 02129; 3 Division of Experimental Medicine, Brigham and Women's Hospital, and 2 Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The actin cytoskeleton is an important contributor to the
modulation of the cell function. However, little is known about the
regulatory role of this supermolecular structure in the membrane events
that take place in the heart. In this report, the regulation of cardiac
myocyte function by actin filament organization was investigated in
neonatal mouse cardiac myocytes (NMCM) from both wild-type mice and
mice genetically devoid of the actin filament severing protein gelsolin
(Gsn/
). Cardiac L-type calcium channel currents
(ICa) were
assessed using the whole cell voltage-clamp technique. Addition of the
actin filament stabilizer phalloidin to wild-type NMCM increased
ICa by 227% over
control conditions. The basal
ICa of
Gsn
/
NMCM was 300% higher than wild-type controls. This
increase was completely reversed by intracellular perfusion of the
Gsn
/
NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn
/
or phalloidin-dialyzed
wild-type NMCM with cytochalasin D (CD) decreased the enhanced
ICa by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase ICa,
whereas actin filament disruption with CD or dialysis of
Gsn
/
NMCM with gelsolin decrease
ICa. We conclude
that cardiac L-type calcium channel regulation is tightly controlled by
actin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.
neonatal mouse cardiac myocytes; cytochalasin D; inactivation
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INTRODUCTION |
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THE CELL'S CYTOSKELETON is an intracellular superstructure comprised of microfilaments of actin and associated proteins, microtubules, and intermediate filaments. The actin cytoskeleton, in particular, is involved in providing structural support and a functional role in cell motility (36, 37). Recent evidence indicates, however, that cytoskeletal components also regulate membrane transport events (for a recent review see Ref. 20). The actin cytoskeleton has been implicated in the regulation of epithelial sodium channels (5, 31), including ENaC (2), potassium channels in nonexcitable (4) and excitable cells (28), and anion channels such as cystic fibrosis transmembrane conductance regulator (10, 32). The actin cytoskeleton may also be involved in the regulation of voltage-dependent channels. In neurons, for example, actin-based microfilamental and tubulin-based microtubular cytoskeletons have both been implicated in the regulation of sodium (34) and calcium (21, 22) channel activity, respectively.
The proper regulation of sodium and calcium channels in cardiac tissues is also critically dependent on the various components of the cell's cytoskeleton. Microtubules, for example, regulate L-type calcium currents (ICa) in isolated chick myocytes (13). Other studies also demonstrated that disruption of the actin cytoskeleton elicits a decrease of the whole cell sodium currents in skeletal (14) and cardiac (39) myocytes. The role of actin in regulating L-type calcium channels of mammalian cardiac myocytes, however, is still largely unknown.
In this study, therefore, the role of actin filament organization in
the regulation of
ICa in neonatal
mouse cardiac myocytes (NMCM) was assessed. Cardiac myocytes obtained
from mice genetically devoid of the actin-severing protein, gelsolin
(Gsn/
), displayed an
ICa significantly
larger than controls, which returned to control values by either
treatment with cytochalasin D (CD) or intracellular dialysis with
exogenous gelsolin. The effect of actin filament stabilization on
ICa was confirmed
in wild-type NMCM by disruption of actin filaments using CD, which
resulted in a decrease in the ICa compared with
control values, whereas phalloidin, which promotes actin filament
stabilization, resulted in an increase in
ICa.
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MATERIALS AND METHODS |
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Primary cultures of neonatal mouse cardiac myocytes. Primary cultures of NMCM were obtained with procedural modifications to a commercial isolation kit originally developed for neonatal rat ventricular myocytes (Worthington Biochemicals, Freehold, NJ). Briefly, beating hearts were harvested from <24-h-old neonatal mice (C57BL/6) and immediately placed in a calcium- and magnesium-free Hanks' balanced salt Solution (HBSS; Worthington). Hearts were minced and subjected to trypsin (100 µg/ml in HBSS) digestion for 16-18 h at 4°C. Trypsin digestion was stopped by addition of trypsin inhibitor (Worthington). Further collagenase digestion (type II collagenase, 150 U/ml; Worthington) was conducted at 37°C on a shaking bath for 45 min. Cell clumps were flushed through a pipette, centrifuged, and washed with fresh Leibovitz L-15 medium. Cell pellets were resuspended in Ham's F-10 medium with L-glutamine (BioWhittaker, Walkersville, MD) also containing 5% bovine serum and 10% horse serum (BioWhittaker). Cells were seeded onto glass coverslips and allowed to grow at 37°C in an incubator gassed with 5% CO2. Healthy (beating) cells were observed after 24 h in culture and were usually healthy for up to 1 wk, with no apparent electrical differences at the various times in culture. All experiments were performed on cells after at least 24 h but <5 days in culture.
Gelsolin knockout mice. Gelsolin null
(Gsn/
) mice were generated by targeted gene disruption of
the gelsolin gene, as previously reported (40). Gsn
/
mice
in mixed strain backgrounds have normal developmental and reproductive function.
Whole cell currents. Patch pipettes
were made with WPI-150 glass capillaries (World Precision Instruments),
fire polished, and filled with the following solution (in mM): 125 CsCl, 20 tetraethylammonium chloride, 10 HEPES, 5 MgATP, and 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid at pH 7.3 with CsOH. The bathing solution consisted of (in mM) 140 NaCl, 5.0 CsCl, 2.0 CaCl2, 1.0 MgCl2, and 10 HEPES at pH 7.4 with
NaOH. CsCl was substituted for KCl to eliminate potassium channel
activity. Actual currents and step potentials were obtained and driven
with a Dagan 3900 (Dagan, Minneapolis, MN). Signals were filtered at 2 kHz with an eight-pole Bessel filter (Frequency Devices, Haverhill,
MA), and data were stored in a hard disk of a personal computer to be
analyzed with pCLAMP 6.0.3 (Axon Instruments, Foster City, CA).
ICa
current-voltage relationships were obtained by applying 200-ms, 10-mV
voltage steps between 50 and 70 mV, starting from a holding
potential of
50 mV. The
ICa was
determined by subtracting the peak inward (negative) currents from the
currents measured at 190 ms. We have found that this methodology
effectively eliminates contamination of
ICa by
voltage-activated sodium channels in NMCM (27). NMCM in culture are
largely round in shape, and the whole cell capacitace of wild-type and
Gsn
/
NMCM were similar (42.5 ± 6.9 pF,
n = 11, vs. 35.8 ± 4.6 pF, n = 5, P < 0.5); therefore, no correction
for whole cell currents was conducted.
Actin cytoskeleton imaging in NMCM. Cytochemical labeling of the actin cytoskeleton was performed as previously described (23). NMCM plated as above were fixed with 4% paraformaldehyde in PBS for 40 min at room temperature, followed by cell permeabilization with 0.1% Triton X-100 for 5 min, and then incubated with PBS containing 1% BSA to block nonspecific binding (10 min). Fluorescein isothiocyanate-phalloidin (Sigma) was diluted 1:100 in PBS (13 µM) and then placed on each coverslip for 45 min at room temperature. After they were extensively washed in PBS, the coverslips were mounted in Vectashield anti-fading medium (Vector Labs, Burlingame, CA) diluted 1:1 in 0.3 M Tris base, pH 8.9, sealed, and examined with a Nikon FXA fluorescence microscope. Color images from representative cells were captured using an Optronics 3-bit charge-coupled device color camera (Optronics Engineering, Goleta, CA) and IP Lab Spectrum (Scanalytics, Vianna, VA) acquisition and analysis software running on a Power PC 8500. Images were imported as TIFF files into Adobe Photoshop 3.0.4 for size reduction and printing on a Tektronic Phaser 440 dye-sublimation color printer.
Drugs and chemicals. The salts used in the pipette and bathing solutions were obtained from Sigma Chemical (St. Louis, MO). CD (Sigma) was dissolved in DMSO and used at a final concentration of 167 µM. The final concentration of DMSO added to the bath was 1.6%, which caused a slight (<5%), yet not statistically significant increase in ICa (data not shown). Phalloidin (Sigma) was dissolved in water and used at a final concentration of 13 µM. Gelsolin (1 mg/ml in PBS; Cytoskeleton, Boulder, CO) was further dissolved in intracellular saline and used at a final concentration of 600 nM. These concentrations were chosen as they resulted in maximal regulatory effects on the actin cytoskeleton and are consistent with previous reports in neurons (21, 28).
Calculations and statistical analysis. Statistical significance was obtained by unpaired t-test comparison of sample groups of similar size (33). Average data values were expressed as means ± SE, where n indicates the number of experiments (cells) analyzed. Statistical significance was accepted as P < 0.05.
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RESULTS |
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Effect of changes in actin filament organization on
ICa of wild-type NMCM.
To assess the role of actin filament organization on the
ICa of NMCM,
cultured wild-type cardiac myocytes were treated with the actin
filament stabilizer phalloidin (13 µM) by diffusion to the
cytoplasmic compartment from the patch pipette. Phalloidin-dialyzed NMCM had a peak
ICa of 667 ± 165 pA/cell
(n = 12; Fig
1A),
which was 227% higher than control NMCM (
204 ± 33 pA/cell,
n = 13, P < 0.01; Fig.
1A).
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ICa from gelsolin null
NMCM.
To confirm that the changes in
ICa observed with
phalloidin and CD in the wild-type NMCM were a direct result of changes
in the organization of the actin cytoskeleton, the role of actin filament organization was further assessed on NMCM obtained from Gsn/
mice (40). Consistent with its role as an actin
filament-severing protein, and results in Gsn
/
fibroblasts (1, 40), the lack of gelsolin in the Gsn
/
NMCM was accompanied by a dramatic stabilization of the polymerized
actin networks, as shown by the phalloidin labeling of stress fibers in
cultured NMCM (Fig. 3). The peak ICa of untreated
Gsn
/
NMCM was 298% higher than that of control cells
(
812 ± 119 pA/cell, n = 14, vs.
204 ± 33 pA/cell, n = 13, P < 0.001; Fig.
4, A and
B). A further indication that the enhanced ICa of
the Gsn
/
NMCM was due to a stabilized actin cytoskeleton
was confirmed by different treatments applied to these cells. First,
Gsn
/
cells were perfused intracellularly with exogenous
gelsolin (600 nM). Under these conditions, peak ICa was
significantly lower compared with untreated Gsn
/
NMCM (
812 ± 119 pA/cell, n = 14, vs.
95 ± 49 pA/cell, n = 5, P < 0.01; Fig. 4,
A and
B). Second, similar results were
obtained when Gsn
/
NMCM were treated with CD (167 µM),
which resulted in a comparatively similar (84%; Fig.
4C) decrease in the peak ICa to that
obtained with exogenous gelsolin (
812 ± 119 pA/cell, n = 14, vs.
131 ± 73 pA/cell, n = 4, P < 0.01; Fig.
4C).
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DISCUSSION |
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The data in this report demonstrate that actin filament organization is an important determinant of ICa in cardiac myocytes. Two conditions were used to stabilize the actin cytoskeleton: 1) intracellular treatment with the actin filament stabilizer phalloidin and 2) the genetic lack of the actin-severing protein gelsolin. Phalloidin binds to actin filaments, thereby stabilizing them and resulting in a net increase in actin filaments (6, 7). Gelsolin is a calcium-dependent actin filament-severing protein that severs actin filaments, nucleates actin filament assembly, and caps the fast-growing end of actin filaments (26, 41). After activation, the predominant effect of gelsolin in most cells is to sever actin filaments and reduce actin filament content and/or rigidity. CD also binds to the fast-growing end of actin filaments, resulting in a net decrease in actin filament content in most cell types, and thus resembles the intracellular effect of gelsolin.
Actin filament stabilization of wild-type NMCM with phalloidin resulted in an increase in ICa, which was reversed on addition CD. Addition of CD to untreated cells also reduced basal ICa. A comparison of rundown kinetics revealed that, after CD addition, ICa decreased as fast in the phalloidin-treated NMCM as in the untreated cells, indicating the dominant effect of CD treatment under these experimental conditions. The slightly faster rate of rundown before CD in the phalloidin-dialyzed NMCM compared with control NMCM (Fig. 2), however, may be attributed to the higher peak ICa. An increase in intracellular calcium has been shown to increase calcium channel rundown (18). Thus a higher ICa would result in more calcium entry into the cell, thereby increasing the rate of current rundown. Nonetheless, the ICa of the phalloidin-dialyzed NMCM decreased substantially faster following CD treatment. These observations are consistent with an increase in calcium channel rundown (inactivation) by the shortening of F-actin.
Parallel observations were made using Gsn/
NMCM (40).
Increased F-actin content was observed in these cells, similar to what
has been seen in Gsn
/
fibroblasts (1, 40), consistent with the activity of gelsolin in reducing F-actin content (17, 24). The
basal ICa of
Gsn
/
NMCM were comparable to those of the
phalloidin-treated wild-type NMCM (values were slightly higher, but
differences were not statistically significant). Intracellular perfusion of Gsn
/
NMCM with exogenous gelsolin
significantly reversed the enhanced
ICa. Further, CD
also reduced the
ICa significantly in Gsn
/
NMCM. Thus the data on the Gsn
/
NMCM confirmed the regulatory role of the actin filament organization
on ICa.
Gelsolin plays an important role in the dynamics of actin filament
organization in multiple cells, as most clearly shown by analysis of
cells and tissues derived from the gelsolin null mice (1, 9, 40).
Motility is markedly reduced and altered in Gsn/
fibroblasts, which have increased the F-actin content and reduced
ruffling activity and pinocytosis. Gelsolin function may also have an
effect on membrane-associated events, including ion channel function.
By modifying the length of actin filaments, stoichiometric complexes of
actin-gelsolin have been observed to regulate epithelial ion channel
function (2, 5). Moreover, gelsolin appears to have a critical function
in the downregulation of voltage-dependent calcium channels in neurons
(12), which is reflected in the fact that Gsn
/
mice are
prone to seizure and ischemic stroke (9).
The cell's cytoskeleton has also been shown to regulate calcium channel kinetics (11, 21, 22). Johnson and Byerly (21, 22), for example, have provided direct evidence for a regulatory role of both microtubules and actin filaments in ICa inactivation in snail neurons. In those studies, Johnson and collaborators showed that cytoskeletal modifiers such as the disrupters colchicine and CD, and stabilizers such as taxol and phalloidin, increased and decreased ICa inactivation, respectively.
The encompassed data are most consistent with a scenario in which,
under physiological conditions, the calcium-induced activation of
gelsolin modifies the kinetic response of
ICa by shifting
the inactivation rate of cardiac L-type calcium channels toward a more
rapidly inactivating state. Intracellular calcium is known to
inactivate calcium channels in a mechanism that requires calcium binding to site(s) located at the inner membrane surface (8, 18, 19,
35). Various studies have also forwarded the idea that the
calcium-mediated
ICa inactivation
requires regulatory calcium binding site(s) outside the channel pore
itself (8, 18, 19). This has been recently confirmed by studies where various -subunits of the L-type calcium channel were expressed in
Xenopus oocytes. Although the
regulatory site(s) could be very close to the internal mouth of the
channel (42), the actual site for intracellular calcium regulation is
still largely unknown. However, potential sites may be associated with
the cytoskeleton (21). Indeed, cardiac cell function is altered in
humans expressing either mutated forms of actin (30) or the altered
expression of cardiac and smooth muscle actin in the heart (25).
Mutations in the actin-binding proteins dystrophin,
-tropomyosin,
and cardiac troponin T all cause cardiomyopathies, leading to
congestive heart failure (29, 38). Thus the actin cytoskeleton may be
an important target for the regulation of a normal contractile cell
response. Furthermore, evidence is mounting to support the idea that
the actin cytoskeleton is also essential for other cellular functions, including signal transduction (15, 16, 20) and ion transport regulation
(3, 5). A functional link between the actin cytoskeleton and the L-type
calcium channels may thus provide a feedback mechanism that regulates
basal cell function independent of external signals.
In conclusion, we have demonstrated that the lack of endogenous gelsolin increases ICa in cultured NMCM. Our data indicate that gelsolin is important in regulating calcium channel inactivation, via modulation of the actin cytoskeleton with net actin filament depolymerization. The data confirm the important function of the actin cytoskeleton in helping maintain channel activity by stabilizing actin filament organization. Disruption of the actin cytoskeleton with CD decreases ICa. The data in this report indicate that ICa is a relevant target for actin filament organization in cardiac myocytes.
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ACKNOWLEDGEMENTS |
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We are extremely grateful to George R. Jackson, Jr., for excellent technical support.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. F. Cantiello, Renal Unit, 8th Floor, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail: cantiello{at}helix.mgh.harvard.edu).
Received 6 August 1999; accepted in final form 10 September 1999.
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