Mechanical Stimulation Regulates Voltage-Gated Potassium Currents in Cardiac Microvascular Endothelial Cells

Isolation and Culture of CMECs

CMECs were isolated and characterized as described previously.¹⁴ Briefly, hearts were removed from adult rats (weight, 180 to 200 g), mounted on a Langendorff-type column, and perfused for 5 minutes with Krebs solution composed of (in mmol/L) NaCl 118, KCl 4.7, CaCl₂ 1.25, MgSO₄ 1.2, KH₂PO₄ 1.2, and NaHCO₃ 25, saturated with 5% CO2/95% O₂, at pH 7.4. After perfusion, the outer one-fourth of the left ventricle free wall and septum were dissected away to remove epicardial arteries and larger penetrating vessels. The remaining tissue was minced in 0.2% collagenase (type B, Boehringer-Mannheim Biochemicals) and incubated for 30 minutes at 37°C in a shaking bath. Trypsin (0.02%, Sigma Chemical Co) was then added, and the tissue sheared 10 times over a period of 30 minutes. Dissociated cells were filtered through a 100 μm mesh filter, washed with Ca²⁺-free solution, and centrifuged at 100g for 5 minutes. CMECs were resuspended in DMEM supplemented with 20% fetal bovine serum and antibiotics (penicillin, streptomycin, and fungiozone). The CMECs were plated on laminin-coated dishes at a density of ≈2.5×10³ cells/cm². After 2 hours, attached cells were washed with DMEM to allow differential adhesion.¹⁵ Cultures were maintained in a humidified atmosphere of 5% CO₂ at 37°C and grown to confluency (6 to 7 days) before use in the stretch experiments. Differential uptake of acetylated low-density lipoprotein, determined by fluorescence-activated cell sorting, indicated that the cultures contained >90% endothelial cells.¹⁴

Application of Static and Cyclic Stretch

For stretch application, cells were treated with trypsin and plated on silastic membranes.¹⁶ Membranes were coated with 1% albumin and fixed with 1% glutaraldehyde. After allowing 24 hours for cell attachment, the silastic membranes were subjected to varying degrees of stretch (5%, 10%, and 15%) with the use of adjustable brackets. When compared with cyclic stretch, the advantage of applying static stretch was that it required a simple system that could be precisely set and maintained. In addition, the static stretch device was small enough to fit into the recording chambers. This feature allowed stretch to be maintained during the measurement of membrane currents. In one set of experiments, silastic membranes were attached to a mechanical stimulator and subjected to 24 hours of cyclic stretch applied at 0.5 Hz with 10% elongation. To reduce cell proliferation during the 24 hours of static and cyclic stretch, the serum concentration in the media was reduced to 3%. Nonstretched cells were cultured in an identical manner, with the exception that force was not applied to the silastic membranes. Pairs of nonstretched and stretched cultures were examined on the same experimental day to limit culture-to-culture variations in ion-channel density.

Recording Procedure and Measurement of K⁺ Currents

The patch-clamp method¹⁷ was used to record whole-cell CMEC currents with L/M EPC-7 (Adams and List Associates) and Axopatch 200 (Axon Instruments) amplifiers. Pipettes were made from Gold Seal Accu-fill 90 Micropets (Clay Adams Inc) and had resistances of 2 to 4 MΩ when filled with internal solution. Unless stated otherwise, all experiments were conducted on isolated, noncoupled CMECs at room temperature (22°C to 24°C). Cells were placed in an external solution consisting of (in mmol/L) NaCl 132, KCl 5, MgCl₂ 2, CaCl₂ 1, dextrose 5, and HEPES 5, pH 7.4 (with NaOH) (280 mOsm.) The internal solution consisted of (in mmol/L) KCl 50, K-glutamate 60, MgCl₂ 2, CaCl₂ 1, EGTA 11, ATP 3, and HEPES 10, pH 7.3 (with KOH) ([K⁺]=140 mmol/L) (280 mOsm). The ratio of EGTA/CaCl₂ in this solution sets the free intracellular Ca²⁺ concentration to ≈10 nmol/L.¹⁸ A reference electrode made from a Ag-AgCl pellet was connected to the bath with an agar salt bridge saturated with external solution. Data were adjusted for liquid junction potentials that occurred between the pipette solution and bath solution and between the reference electrode and the bath.¹⁹ Offset potentials, created at the junction of the internal/external solution interface, were measured at the beginning and end of the experiments and were between −5 and +5 mV.

Membrane currents were recorded with 12-bit analog/digital converters (Axon Instruments). Data were sampled at 10 kHz, filtered at 2 kHz with a low-pass Bessel filter (Frequency Devices), and stored with the use of personal computers (Northgate and Dell). Series resistance was compensated to provide the fastest possible capacity transient without producing oscillations. With this procedure, >50% of the series resistance could be compensated. Linear leak and capacity transients were removed from test currents with records obtained during 4 hyperpolarizing pulses from the holding potential of –80 to −100 mV. These records were averaged and subtracted from the test currents. Use of this protocol was justified because voltage- and/or time-dependent conductances were not present at these potentials. For a typical set of cultures, the membrane capacity of the nonstretched CMECs ranged from 17 to 40 pF with a mean±SE of 29±1 pF (n=24 cells). The capacity of the stretched cells was 29±2 pF (n=24 cells).

All experiments were conducted at room temperature (22°C to 24°C). Averaged values presented are means±SE. Where appropriate, statistical significance was estimated with the use of Student’s t test for unpaired observations.

Materials and Drugs

DMEM, 4-aminopyridine, tetraethlyammonium, phorbol 12-myristate 13-acetate (PMA), and pertussis toxin (PTX) were purchased from Sigma Chemical Co.. Charybdotoxin (CTX) was obtained from Alomone Laboratories LTD (Jerusalem, Israel).

Stretch-Induced Increases in the CMEC I_K

The top left panel of Figure 1⇓ shows an example of membrane currents obtained from a CMEC during voltage steps applied from a holding potential −80 mV to various potentials. Voltage steps to potentials more positive than −30 mV resulted in the activation of outward K⁺ currents. The time-dependent and voltage-dependent properties of the CMEC current were similar to those of a family of delayed rectifier K⁺ currents found in a large number of cell types.^{3 20 21} The CMEC current was identified as a K⁺ current on the basis of 3 criteria. First, the reversal potential of the tail currents −73±2 mV (n=5 cells), obtained during deactivation of the current, was close to the theoretical K⁺ equilibrium potential (−83 mV) under the conditions of the experiments ([K⁺]₀=5 mmol/L, [K⁺]₀=140 mmol/L). Second, the CMEC current was inhibited by a 500 μmol/L concentration of the K⁺ channel blocker 4-aminopyridine (95±1% decrease at 0 mV, n=4 cells) and during substitution of external NaCl with TEA chloride(Figure 5⇓). Finally, use of a CsCl internal solution resulted in a complete elimination of the outward current.¹⁴

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Figure 1.

Stretch augments voltage-gated K⁺ currents in CMECs. Top panel: Currents recorded during voltage steps, given in 20 mV increments, applied from a holding potential of −80 mV to potentials ranging from −20 to +40 mV. Currents were recorded from a nonstretched cell (left) and a cell exposed to 10% static stretch for 24 hours (right). Cells F912C01 & F912C27. Bottom panel: Current vs voltage relationship for I_K measured in the top panel and normalized to the cell membrane capacities.

To determine the effect of mechanical stimulation on ion channels, CMECs were stretched by 5%, 10%, and 15% and compared with control, nonstretched cells cultured under identical conditions. In initial experiments, membrane currents were measured during voltage steps to −100 mV and −60 mV to determine whether stretch was activating time-independent and voltage-independent background currents that might occur as a result of the opening of mechano-sensitive ion channels.^{9 10} Current densities were −0.9±0.1 pA/pF (−100 mV) and −0.8±0.1 pA/pF (−60 mV) in the nonstretched CMECs and −1.1±0.1 pA/pF (−100 mV) and −1.0±0.1 pA/pF (−60 mV) in the stretched CMECs (n=16 cells). However, when a stretched cell was compared with a nonstretched cell of similar membrane capacity, 24 hours of stretch resulted in a dramatic increase in the size of the delayed rectifier K⁺ current (I_K) (Figure 1⇑). Figures 2⇓ and 3⇓ summarize the results of I_K measurements obtained from >160 CMECs recorded during stretched and nonstretched conditions. Stretched-induced increases of 97% and 355% (at +30 mV) in the peak amplitude I_K were measured in cells stretched at 5% and 10%, respectively (Figure 2⇓, left panel). Increases in I_K were also observed in cultures stretched at 15% for 24 hours (Figure 2⇓, left panel). However, this increase (106%) was significantly less than that measured during 10% stretch (P<0.05). The higher incidence of cellular damage in the 15% stretched cultures may have accounted, at least in part, for this decline in current amplitude. The mean value of I_K increased after 6 hours of stretch and was significant at 12 hours (Figure 2⇓, right panel). Measurements were not taken after 24 hours of stretch because of difficulties in finding single, noncoupled cells at later times. Application of cyclic stretch also increased the amplitude of I_K. The results plotted in Figure 3⇓ compare the effects of 10% static stretch and 10% cyclic stretch on I_K measured at 0, +30, and +50 mV. There was no significant difference (P>0.05) at any voltage in the size of I_K recorded after 24 hours of static and cyclic stretch.

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Figure 2.

Effect of static stretch on CMEC K⁺ currents. Left panel: Peak I_K density measured at +30 mV in nonstretched CMECs (open bars, n=48 total) and in cells exposed to 5%, 10%, and 15% stretch for 24 hours (hatched bars, n=49 total). Right panel: Peak I_K density measured at 0 mV in nonstretched CMECs (open bars, n=44 total) and in cells exposed to 10% stretch for 6, 12, and 24 hours (hatched bars, n=43 total). The * indicates a significant difference (P<0.05) between the stretched and nonstretched cells.

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Figure 3.

Comparison of static and cyclic stretch-induced changes in CMEC K⁺ currents. Left panel: Peak I_K density measured at 0, +30, and +50 mV in nonstretched CMECs (open bars, n=13) and in cells exposed to 10% static stretch for 24 hours (hatched bars, n=14). Right panel: Peak I_K density measured at 0, +30, and +50 mV in nonstretched CMECs (open bars, n=7) and in cells exposed to 10% cyclic stretch for 24 hours (hatched bars, n=10). The * indicates a significant difference (P<0.05) between the stretched and nonstretched cells.

Voltage Dependence of I_K Activation in Stretched and Nonstretched CMECs

The results displayed in Figures 1 to 3⇑⇑⇑ indicate that mechanical stimulation produces a strong augmentation in the size of the CMEC I_K. One possible explanation for these results could be that stretch up-regulates the expression of K⁺ channels in the endothelial cells. With respect to those channels present in the nonstretched cells, these channels might represent the same or possibly a different class of K⁺ channels. For this reason, it was important to compare the voltage-dependent and pharmacological properties of I_K in the stretched and nonstretched cells.

The normalized conductance for the currents measured in the stretched and nonstretched cells is plotted as a function of the test voltage in Figure 4⇓. The continuous lines represent the best fits of the data points to the Boltzmann equation (see Figure 4⇓ legend). The half-maximal voltage (V_1/2) required for activation was +34 mV in the nonstretched cells and decreased to −5 mV in cells stretched at 10% for 24 hours. In addition, the parameter k, which reflects the slope of the fitted curve, decreased from 20 (nonstretched cells) to 8 (stretched cells). The activation curve for I_K in cells stretched by 5% was intermediate to that of the 10% stretched and nonstretched cells with a V_1/2 of +10 mV (Figure 4⇓). In this case, k decreased to 15.

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Figure 4.

Stretch shifts the voltage-dependence of CMEC K⁺ current activation to more negative potentials. Activation curves obtained in nonstretched CMECs (n=6)and cells exposed to 5% (n=5) and 10% (n=5) static stretch for 24 hours. Conductance was determined by dividing the peak current amplitude at each potential by the driving force for K⁺, (V_m - E_K). The continuous lines represents the best fits of the Boltzmann equation, g_K=g_Kmax {1 + exp[-(V_m-V_1/2)/k]}, where V_1/2 is the half-maximal voltage required for activation and k gives the steepness of the voltage-dependence, to the data points. Fitted parameters for nonstretched, 5% stretched, and 10% stretched cells were respectively: V_1/2=34, 10, and −5 mV, k=20, 15, and 8.

Pharmacology of I_K in Stretched and Nonstretched CMECs

Both tetraethylammonium (TEA) and charybdotoxin (CTX) serve as useful pharmacological agents for characterizing voltage-gated K⁺ channels.^{3 20 21} As indicated in the top panel of Figure 5⇓, relatively high concentrations of TEA were required to block the CMEC I_K. Substitution of the 132 mmol/L NaCl in the external solution with 132 mmol/L TEA chloride reduced I_K in the nonstretched cells by 88±4% (n= 4 cells, 0 mV) (Figure 5⇓, left panel). A similar decrease in I_K was recorded in the presence of TEA for CMECs stretched at 10% (% change=83±4%, n= 4 cells) (Figure 5⇓, right panel). A smaller decrease in I_K (35% to 40%) was measured after the addition of 25 mmol/L TEA (results not shown). CTX was also an effective blocker of I_K in the stretched CMECs. In 5 cells examined, 50 nmol/L CTX inhibited I_K by 88±2% at 0 mV (Figure 6⇓, right panel). Block of I_K occurred in a voltage-dependent manner with a significantly smaller reduction in I_K recorded at +50 mV. Surprisingly, I_K measured in the nonstretched CMECs displayed a reduced sensitivity to block by CTX, with an overall decrease of 21±7% at 0 mV (n= 8 cells) (Figure 6⇓, left panel) recorded after toxin application. This change was significantly different than the inhibition recorded in the stretched CMECs (P<0.05).

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Figure 5.

Block of the CMEC K⁺ current by TEA. Top panel: I_K measured in a nonstretched (left) and stretched (right) cell during a voltage step to +50 mV, before (CON) and after the substitution of 132 mmol/L NaCl with 132 mmol/L TEA chloride(TEA). Cells FD10C23 & FD11C08. Bottom panel: Percent inhibition of I_K caused by TEA in nonstretched (n=4) and stretched (n=4) cells.

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Figure 6.

Block of the CMEC K⁺ current by CTX. Top panel: I_K measured in a nonstretched (left) and stretched (right) cell during a voltage step to 0 mV before (CON) and after the addition of 50 nmol/L CTX. Cells F206C15 & F202C02. Bottom panel: Percent inhibition of I_K caused by CTX in nonstretched (n=8) and stretched (n=5) cells.

Effect of PTX and PMA on Stretch-Induced Increases in I_K

Previous studies have demonstrated that mechanical stimulation of endothelial cells effects G protein-coupled, signal transduction pathways²² and protein kinase C-regulated proteins.²³ As a first step in determining the mechanism of stretch-induced augmentation of I_K, cell cultures were preincubated with either PTX (1 μg/mL), to inhibit the G_i and G_o proteins, or PMA (500 nmol/L), to down-regulate protein kinase C. Exposure of the CMECs to PTX during the 24-hour period of stretch had no effect on the stretch-induced increase in I_K (Figure 7⇓, left panel). Twenty four hours of treatment with PMA also did not prevent stretch-induced increases in I_K. Surprisingly, in the presence of PMA, there was a significant augmentation in the size of I_K at all measured voltages (Figure 7⇓, left panel). This effect on I_K was associated with a stimulation of cell proliferation and an increase in the number of cellular processes projecting from the cell bodies.

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Figure 7.

Pretreatment of cells with PTX or PMA does not prevent stretch-induced increases in I_K. Left panel: Peak I_K density measured at +30 mV in cells exposed to 10% stretch for 24 hours in the absence (open bars, n=16) or presence of either PTX (1 μg/mL) (left hatched bar, n=8) or PMA (500 nmol/L) (right hatched bar, n=10). The * indicates a significant difference (P<0.05) between PMA-treated and untreated cells. Right panel: Effect of stretch on cell proliferation. Cells were seeded at a density of 30,000 cells per dish and counted after 48 hours either under control conditions (open bar, n=5 experiments) or after 24 hours of 10% static stretch (hatched bar, n=5 experiments). The * indicates a significant difference (P<0.05) between the number of stretched and nonstretched cells.

Effect of Stretch on I_K in Culture Medium Containing 3% and 15% Serum

As shown in the right panel of Figure 7⇑, 24 hours of static stretch stimulated endothelial cell proliferation. Unlike the increase in cell proliferation observed during PMA treatment, this action was not associated with any change in cell structure. Because stretch induces both an increase in I_K density and a stimulation in endothelial cell proliferation, it was determined that changes in K⁺ channel density might be dependent on cell growth. Figure 8⇓ summarizes the results of experiments in which the CMECs were grown in a culture medium containing 15% serum. Under these conditions, the endothelial cells proliferated to an extent greater than that measured in 3% serum, and the application of 10% static stretch did not significantly change the rate of proliferation (results not shown). As shown in Figure 8⇓, 24 hours of static stretch produced a significant increase in the I_K density in cells grown in 15% serum. This increase was not as large as that measured in cells grown in 3% serum (summarized in Figure 8⇓) due to the augmented basal current level recorded in the CMECs grown in 15% serum. This suggests that multiple factors (stretch, serum concentration, proliferative state, etc) can regulate the I_K density.

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Figure 8.

Effect of static stretch on K⁺ currents in CMECs grown in a culture medium supplemented with either 3% or 15% serum. Peak I_K density measured at +30 mV in nonstretched CMECs grown in the presence of either 3% or 15% serum (open bars, n=29 total) and in the corresponding cells exposed to 10% static stretch for 24 hours (hatched bars, n=30 total). The * indicates a significant difference (P<0.05) between the stretched and nonstretched cells grown in the presence of similar concentrations of serum.

Mechanically Induced Changes in Endothelial Ion Channels

Vascular endothelial cells respond to 2 different types of mechanical force: shear stress that results from blood flow, and circumferential wall stretch that results from transmural pressure. The goal of the present study was to determine the effects of mechanical stretch on K⁺ currents in CMECs. Although mechano-sensitive ion channels have been identified in endothelial cells under conditions that produce small and transient changes in cell shape,^{7 9 10} the effect of 24 hours of mechanical stimulation on endothelial electrical activity is not known. Previous studies have demonstrated that both stretch and shear stress regulate endothelial ion channels.^{9 10 11 12} Lansman et al⁹ identified a 40 pS ion channel that is activated during deformation of membrane patches in aortic endothelial cells. A similar nonselective cation channel with a permeability for Ca²⁺, K⁺, and Na⁺ is present in cerebral capillary endothelial cells.¹⁰ A whole-cell, time-independent, background current corresponding to the opening of these channels could not be measured in CMECs stretched for 6, 12 or 24 hours. Because mechano-sensitive ion channels are known to inactivate during continuous stretch,²⁴ it is not surprising that mechanically induced currents, if present in the CMECs, were not recorded in the chronically stretched cells.

The major finding of the present study is that stretch increases the size of a voltage-gated K⁺ current. The different activation parameters and charybdotoxin-sensitivity of I_K in the nonstretched and stretched cells suggest that a different type of K⁺ channel may be up-regulated by stretch. The voltage-gated K⁺ channel (Kv) gene family is represented by a large and diverse number of α subunits that compose the pore of the channel.²⁵ Expression of individual rKv1.2, rKv1.4, and rKv1.5 subunits results in time-dependent outward K⁺ currents with a V_1/2s of −20 to 0 mV, moderate sensitivities to CTX (K_d=10 to 100 nmol/L), and relative insensitivities to TEA (K_d>50 mmol/L).²⁵ These properties are quite similar to I_K measured in the stretched cells and may suggest that one or more of these subunits is preferentially up-regulated by mechanical stimulation. Olesen et el¹¹ demonstrated that shear stress activates a whole-cell inward rectifier K⁺ current in bovine aortic endothelial cells. The inward rectifier was found to vary in size as a function of the amount of shear stress, desensitize slowly with time, and recover on cessation of flow.¹¹ The Ca²⁺-sensitivity of inward rectifier channel,¹² and the requirement of G proteins in the shear stress activation process,²⁶ indicate that this channel differs from the classic inward rectifier found in endothelial cells.^{27 28} The shear stress–activated inward rectifier also clearly differs from I_K recorded in the CMECs and suggests that the Kv family of proteins represent a hereto unknown target of mechanically induced regulation. This idea is supported by the recent finding that cyclic stretch increases the expression of Kv1.5 channels in cultured cardiac ventricular myoctyes.²⁹

Mechanically Induced Changes in Endothelial Intracellular Signaling

Mechanical forces modulate a number of signal transduction pathways in endothelial cells including heterotrimer G proteins,²² cAMP,³⁰ intracellular Ca,^{2 31} protein kinase C (PKC),²³ mitogen-activated protein (MAP) kinases,³² and tyrosine kinases.^{33 34} In addition to the direct modulatory effects on cellular proteins, stimulation of these pathways leads to the activation of multiple gene transcription factors.³⁵ Pretreatment of the CMECs with either (PTX), to inhibit G_i or G_o, or PMA, to down-regulate PKC,³⁶ was ineffective in preventing stretch-induced increases in I_K. In actuality, PMA potentiated−stretch induced increases in I_K (Figure 7⇑). However, since PKC activity was not measured directly, we can only infer that this enzyme was down-regulated. Previous studies have shown that acute stimulation of PKC by phorbol esters inhibits Kv channels including the cloned Kv1.3 and Kv1.4 channels.^{37 38 39} Thus, by down-regulating PKC, chronic treatment of the cells with PMA may relieve a basal inhibition of I_K caused through protein phosphorylation. Alternatively, PMA treatment may result in the induction of genes encoding for Kv channels. As observed with the CMECs, phorbol esters induce morphological changes and proliferation in both macrovascular and microvascular endothelial cells.⁴⁰ These actions are associated with changes in the expression of adhesion molecules including E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1.⁴⁰ In addition, treatment of cancer cells with mitogens such as concanavalin A, lipopolysaccharides, and interleukins cause large increases in the expression of K⁺ channels.⁴¹ Further experiments with specific inhibitors of PKC and tyrosine kinases will be necessary to understand the role of these signaling proteins in the stretch-induced increase in I_K.

Relevance of the Study to Cardiovascular Physiology

The expression of K⁺ channels varies greatly between different types of endothelial cells.^{4 5} In macrovascular endothelial cells, the inward rectifier K⁺ channel is the main contributor in setting the resting membrane potential (−50 to −70 mV).^{42 43} In contrast, Cl^- channels and Kv channels play a more dominant role in setting the resting potential in microvascular endothelial cells such as the CMECs, which lack inward rectifier currents at physiological [K⁺]⁴⁴ (our unpublished results). This is consistent with the more positive resting potential (−10 to −40 mV) measured in the microvascular cells.^{14 44} By the modulation of the K⁺ conductance of the cell and thus the resting potential, mechanical stimulation should increase Ca²⁺ influx and the release of vasoactive substances such as nitric oxide, endothelin, and prostaglandins from the cells. In addition, increases in K⁺ channel density may be important in mediating endothelium responses to stimulating agents such as histamine and thrombin.

Recent evidence suggests that K⁺ channels are involved in the regulatory steps of cell proliferation.⁴¹ Experiments that demonstrate that K⁺ channel blockers inhibit cell division in neuroblastoma, melanoma, and breast cancer cells,^{45 46 47} imply that there is a relationship between the internal [K⁺] and the proliferative activity of the cell. In the cardiovascular system, mechanical forces have been shown to stimulate capillary angiogenesis under conditions that produce mild-to-moderate vessel wall stretch.^{48 49} Because microvascular endothelial cell proliferation is a critical step in angiogenesis, it will be important to access the role of voltage-gated K⁺ channels in this process. Stretch-induced increases in CMEC K⁺ currents may contribute to capillary growth during periods of mechanical perturbation. Future experiments will be required to determine whether stimulation of endothelial cell proliferation or ion channel expression is the primary initiator of these vessel changes.