RAPID COMMUNICATION
Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels

Alan S. Lader1,2, David J. Kwiatkowski2,3, and Horacio F. Cantiello1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   A: effect of phalloidin and cytochalasin D (CD) on calcium currents of neonatal mouse cardiac myocytes (NMCM). Calcium channel currents (ICa) from wild-type NMCM were obtained under control conditions (), after intracellular dialysis with phalloidin (13 µM, triangle ), and phalloidin + cytochalasin D (CD; 167 µM, black-triangle). Data are means ± SE of 12, 13, and 4 experiments, respectively. B: representative tracings indicate control (top), in the presence of phalloidin alone (middle), and phalloidin + CD (bottom). Dashed line indicates zero current. Step potentials were applied for 200 ms between -50 and 70 mV from a holding potential of -50 mV. Tracings shown indicate 1st 120 ms of current tracings. C, top: addition of CD (167 µM) reduced basal ICa. Representative tracings were obtained as a step potential to 0 mV from a holding potential of -50 mV. Bottom: CD had a similar effect on phalloidin-treated cells. Dashed line indicates zero current. Tracings shown are 1st 120 ms of current tracings. D: peak ICa were obtained as in A for control conditions (left) and after CD treatment (167 µM; right), in absence (open bars) or presence of phalloidin (13 µM, shaded bars). Data are means ± SE of 13 and 12 experiments under basal conditions and 12 and 4 experiments after CD for control and phalloidin treatment, respectively. * P < 0.01, statistical significance between control and phalloidin-dialyzed cells. **,*** Statistical significance (P < 0.01) before and after addition of CD (not statistically different from each other).

To further assess the regulatory role of actin cytoskeleton organization on ICa, actin filament disruption was also evaluated on control and phalloidin-treated NMCM. Addition of CD (167 µM) decreased the peak ICa of phalloidin-dialyzed NMCM by 87% (-667 ± 165 pA/cell, n = 12 vs. -68 ± 27 pA/cell, n = 4, P < 0.01; Fig. 1A), with a maximal effect achieved after 1.5 ± 0.8 min (n = 4). Addition of CD (167 µM) alone to the bath also resulted in a significant decrease (~90%) in the peak basal ICa of control NMCM (-204 ± 33 pA/cell, n = 13, vs. -21 ± 10 pA/cell, n = 12, P < 0.001, Fig. 1B, C and D).

A comparison of the rundown kinetics showed that the ICa from CD-treated NMCM decreased much faster than the control cells (Fig. 1, B and C, and Fig. 2). The ICa of untreated control myocytes remained relatively stable, decreasing by only 20% of the initial peak ICa in 6 min (Fig. 2). Addition of CD (167 µM), however, dramatically increased the rundown of the peak ICa. Interestingly, a similar phenomenon was also observed after addition of CD to the phalloidin-treated cells, such that ICa in both the control and phalloidin-treated myocytes ran down at similar rates (Fig. 2). The ICa of both the control and phalloidin-treated cells decreased by 90% within 2 min following treatment with CD, although the magnitude of the total currents was highly different (Fig. 1D).


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Fig. 2.   Time-dependent ICa rundown in wild-type NMCM. Peak calcium currents were followed for several minutes after whole cell breaking. Values expressed as percentage of maximal ICa. Values were obtained for representative untreated wild-type myocytes (, n = 4), after addition of CD (167 µM, open circle , n = 5), and phalloidin + CD (black-diamond , n = 4). Arrow, addition of CD.

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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Fig. 3.   Actin labeling in neonatal mouse cardiac myocytes. NMCM from wild-type (A) and Gsn-/- (B) mice were labeled with fluorescein isothiocyanate-phalloidin to assess structural differences in the actin cytoskeleton. As shown by these representative cells, fluorescent images of wild-type NMCM showed less bundled actin than Gsn-/- NMCM.






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Fig. 4.   A: effect of gelsolin and CD on calcium currents of Gsn-/- NMCM. ICa were obtained under control conditions before (triangle ), either in presence of exogenous gelsolin in patch pipette (600 nM, ), or after addition of CD (167 µM, black-triangle). Data are means ± SE for 14, 5, and 4 experiments, respectively. B: tracings indicate ICa from Gsn-/- control (top), in presence of gelsolin (600 nM, middle), and after addition of CD (167 µM, bottom). Dashed line indicates zero level. Data are representative tracings of 14, 4, and 5 experiments, respectively. Inset: representative tracings at 0 mV indicate Gsn-/- control and gelsolin-treated NMCM. Gelsolin addition from pipette (600 nM) largely decreased whole cell currents. Dashed line indicates zero current. Tracings shown indicate 1st 120 ms of current tracings. C: peak ICa were obtained as in Fig. 1A for Gsn-/- control (left), gelsolin dialyzed (600 nM, middle), and after addition of CD (167 µM, right). Data are means ± SE of 14, 5, and 4 experiments, respectively. *,** Statistical significance (P < 0.01) with respect to control conditions (not statistically different from each other).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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, alpha -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.


    ACKNOWLEDGEMENTS

We are extremely grateful to George R. Jackson, Jr., for excellent technical support.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 277(6):C1277-C1283
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