Ras Reduces L-Type Calcium Channel Current in Cardiac Myocytes
Corrective Effects of L-Channels and SERCA2 on [Ca2+]i Regulation and Cell Morphology
Abstract—Heart failure is associated with dysregulation of intracellular calcium ([Ca2+]i), reduction in myofibrils, and increased activation of Ras, a regulator of signal-transduction pathways. To evaluate the potential effects of Ras on [Ca2+]i, we expressed constitutively active Ras (Ha-RasV12) in cardiac myocytes and monitored [Ca2+]i via fluorescence and electrophysiological techniques. Ha-RasV12 reduced the magnitude of the contractile calcium transients. Unexpectedly, however, calcium loading of the sarcoplasmic reticulum was increased, suggesting that Ha-RasV12 introduces a defect in excitation-calcium release coupling. Consistent with this idea, L-channel calcium currents were reduced by Ha-RasV12, which also downregulated the activity of the L-channel gene promoter. Coexpression of L-channels and SERCA2 largely corrected Ha-RasV12–induced dysregulation of [Ca2+]i. Furthermore, whereas Ha-RasV12 downregulated myofibrils, this effect was blocked by coexpression of L-channels. These results suggest that Ras downregulates L-channel expression, which may play a pathophysiological role in cardiac disease.
In enlarged, failing hearts, there is often prolongation of the decay phase of the contractile Ca2+ transient, increased end-diastolic intracellular calcium ([Ca2+]i), and diminished systolic [Ca2+]i.1 2 3 The defects in [Ca2+]i regulation may relate to reduced expression of Ca2+ regulatory proteins, such as SERCA2.4 5 Expression of L-type calcium channels may also be reduced,6 7 8 9 although this is not a universal finding.10 Because SERCA2 is responsible for sarcoplasmic reticulum (SR) Ca2+ reuptake after systole, reductions in SERCA2 may prolong decay of the Ca2+ transient. Because Ca2+-induced Ca2+ release, which is the main source of [Ca2+]i driving the transient, depends on L-channel–mediated Ca2+ influx, reductions in L-channel density could conceivably reduce systolic [Ca2+]i. Additionally, myofibril density is reduced in heart failure,11 but the factors responsible for this are unknown.
Hypertension and other conditions that lead to heart failure increase expression of Ras.12 13 Also, cardiac targeting of constitutively active Ras leads to conditions similar to hypertrophic cardiomyopathy in transgenic mice, including myocyte enlargement, prolonged relaxation, and tissue disorganization.14 Dominant-negative mutants of Ras inhibit the expression of genes that are upregulated during hypertrophy.15 Thus, Ras may participate in cardiac enlargement and failure.
Ras regulates various signal-transduction pathways, including the extracellular signal–regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) cascades.16 17 18 Ras may also generally upregulate the activity of the cardiac gene transcriptional machinery.19 Although the interaction of Ras with cardiac signaling pathways has been extensively studied, less is known about how Ras alters myocyte function. Previously, we observed that expression of constitutively active Ras (Ha-RasV12) diminished and prolonged the contractile calcium transients.20 Ha-RasV12 also diminished the expression of SERCA2 and striated myofibrils. However, whereas coexpression of SERCA2 restored the kinetics of the calcium transient, transient magnitude remained reduced, suggesting that Ras modifies [Ca2+]i through additional, non–SERCA2-related mechanisms. The goal of the present study was to elucidate the mechanisms by which Ras modifies [Ca2+]i and cell morphology and to determine potential strategies to ameliorate these effects. Our results suggest that Ha-RasV12 reduces L-channel current in cardiac myocytes, most likely by downregulating L-channel expression. Additionally, we provide evidence that L-channels stabilize cardiac myofibril organization, which may represent a previously unsuspected role for L-channels in maintaining cardiac myocyte structure.
Materials and Methods
Neonatal rat ventricular myocytes were prepared and transfected by electroporation, as described elsewhere.20 After plating, myocytes were placed in serum-free maintenance media (DMEM supplemented with 1 nmol/L l-3,3′,5-triiodothyronine, sodium [Calbiochem], 5 μg/mL apo-transferrin [Sigma], 1 μg/mL insulin [Sigma], and 0.1 nmol/L selenium [Sigma]). Cells were kept in maintenance media for 48 hours before analysis, unless noted. For all single-cell analysis, an expression plasmid for green fluorescent protein (GFP) was included in the transfections to allow identification of transfected cells.
Expression Constructs and Transfections
The expression plasmids that encode Ha-RasV12, GFP, the dominant-negative mutant of MEK (dnMEK) (MAPKK1-Ala-217), and SERCA2, and the control plasmids pCMV6 and pCH110-LacZ have been previously described.20 Plasmids encoding L-type channel subunits pcDNA3-1α1C, pcDNA3-1Zeoα2, and pcDNA3-1Hydro β2A were from G. Varadi (University of Cincinnati, Cincinnati, Ohio). The L-channel promoter-luciferase construct 1.7Lch α1C-luc, which contains 1.7 kb of the 5′ flanking sequence of the rat α1C gene ligated upstream from firefly luciferase, was prepared from GenBank AF221551 and was independently generated but highly homologous to that reported.21
Measurement of Indo-1 Fluorescence in Transfected Myocytes
Measurement was performed as previously described.21 Although indo-1 fluorescence is related to free-Ca2+ concentration by well-established equilibrium relationships, exact calibration in vivo is problematic. Results are thus presented as the ratio of indo-1 emission fluorescence recorded at 405 and 485 nm.
Rapid Application of Caffeine and KCl to Indo-1–Loaded Cardiac Myocytes
In certain experiments, indo-1 measurements were made during the addition of either caffeine or KCl. To do this, coverslips containing indo-1–loaded myocytes were placed in a Biophysica chamber containing 1 mL of Tyrode solution, which was mounted on the stage of the fluorescence recording station. During the experimental runs, 1 mL of a solution containing a 2× concentration of test compound made up in Tyrode buffer was rapidly pipetted into the chamber. This resulted in complete mixing of the test compound within 0.3 seconds (mixing time was estimated in trials using indo-1 solutions).
Reporter Enzyme Assays
Luciferase and β-galactosidase were assayed as previously described.18
Whole-cell calcium currents were recorded with a List EPC-7 patch clamp from transfected myocytes after 48 to 72 hours in serum-free media. The bathing solution was normal Tyrode (1 mmol/L calcium) with Cs+ substituted for K+ for the purposes of blocking inward rectifier K+ currents. The internal solution in the pipette contained 3 mmol/L Na2ATP, 3.5 mmol/L MgCl2, and 5 mmol/L HEPES with 120 mmol/L Cs aspartate and 20 mmol/L CsCl to block outward K+ current. Solution pH was adjusted with CsOH. Patch-clamping experiments were performed using either conventional whole-cell recording or β-escin.22 Equivalent results were obtained with both methods.
Quantification of Cell Size and Myofibril Density
Myocytes were stained for actin using Texas Red–conjugated phalloidin and photographed at ×1000 magnification. The photomicrographs were scanned, and the images were optimized for contrast using Adobe Photoshop. To quantify myofibril density, a procedure was adapted from a technique used to quantify ultrastructural features in electron micrographs.23 A calibrated grid (lines spaced an equivalent of 4.3 μm apart) was superimposed on the images. Each image was printed, and cell size and myofibril density were estimated in a blinded fashion by counting the number of grid intersection points that overlaid each cell and the percentage of grid points that overlaid striated myofibrils.
Ha-RasV12 Introduces a Defect in Excitation-Calcium Release Coupling
Expression of Ha-RasV12 in cardiac myocytes reduces SERCA2 expression and the magnitude of contractile Ca2+ transients.20 Because SERCA2 controls Ca2+ uptake into the SR, it was conceivable that Ha-RasV12 might reduce SR calcium loading. To test this idea, myocytes were transfected with the control plasmid, pCMV6, or the expression plasmid for Ha-RasV12 (4 μg/transfection cuvette, throughout the study), cultured in the absence of serum, and then loaded with indo-1. The myocytes were electrically paced at 0.3 Hz for ≈15 seconds, which was sufficient for the Ca2+ transients to reach a steady state in magnitude. Relatively few myocytes exhibited spontaneous contractions, and these were excluded from the study. Pacing was then stopped, the myocytes were switched to nominally calcium-free Tyrode buffer, and caffeine was rapidly added. Consistent with previous results, systolic [Ca2+]i, as represented by Rsys and transient magnitude, was reduced during pacing by Ha-RasV12 (Figure 1A⇓ and Table⇓, experiment 1). Unexpectedly, however, the response to caffeine was increased. This result suggests that the SR is well loaded with Ca2+ in Ha-RasV12–expressing myocytes. Although it is of interest that the caffeine response was increased by Ha-Ras,V12 this aspect of the result was not pursued. Instead, attention was focused on the implication that Ha-RasV12 may introduce a defect in excitation-calcium release coupling, because Rsys and transient magnitude were diminished in myocytes in which the SR was well loaded with Ca2+.
Ha-RasV12 Slows Calcium Responses to K+ Depolarization
Excitation-contraction coupling requires Ca2+ influx through voltage-dependent L-type Ca2+ channels and Ca2+-induced Ca2+ release from the SR. To address which of these processes may be altered by Ha-RasV12, myocytes were depolarized with elevated K+, which leads to L-channel–mediated Ca2+ influx.24 The protocol was similar to above, except that Ca2+-containing Tyrode buffer was used throughout. Rsys was again diminished by Ha-RasV12 during the pacing period (traces not shown, but see the Table⇑, experiment 2). Depolarization with elevated K+ led to an abrupt increase [Ca2+]i in control cells, but Ha-RasV12–expressing myocytes exhibited a slower, smaller response (Figure 1B⇑, Table⇑, experiment 2). This suggests that Ha-RasV12 may reduce depolarization-induced Ca2+ influx.
L-type Ca2+ Currents Are Reduced by Ha-RasV12
Whole-cell patch-clamp experiments were conducted to directly test the effect of Ha-RasV12 on L-channel conductance. Control myocytes displayed large Ca2+ currents (Figure 2A⇓, trace 1), characterized by a rapidly initiating, long-lasting, inward current. Consistent with L-channel behavior, 3 mmol/L cobalt completely blocked these currents (Figure 2A⇓, trace 2). In contrast, Ha-RasV12–expressing myocytes exhibited severely reduced inward Ca2+ currents (Figures 2B⇓ and 2C⇓).
Ha-RasV12–expressing cells also exhibited more leak current than controls, but it is unlikely that this obscured the observation of L-channel currents in the Ha-RasV12–expressing myocytes. The leak current was not blocked by cobalt (Figure 2B⇑, trace 1 versus 2). Thus, the Co2+-sensitive currents, obtained by the subtractive procedure, are corrected for leak current, and, as illustrated (Figure 2B⇑, trace 3), these were smaller in Ha-RasV12–expressing myocytes (data from 1 of 2 Co2+-subtracted control cells and 1 of 2 Co2+-substracted Ha-RasV12–expressing myocytes are shown). In a second strategy, the normally linear leak-current measured at +60 mV, which is near the reversal potential for Ca2+, was used to calculate leak at the lesser depolarization steps, and these values were subtracted from the original currents. Figure 2C⇑, traces 1 and 2, represent this leak-correction procedure applied to a Ha-RasV12–expressing myocyte. Some leak nonlinearity induced by Ha-RasV12 expression causes this method to slightly overestimate L-type Ca2+ current. Nevertheless, after leak correction (via cobalt for 2 control and 2 Ha-RasV12–transfected myocytes or via linear leak subtraction for the remaining cells), maximum L-type channel currents averaged ≈6.5 pA/pF for controls versus 2.5 pA/pF (Figure 3⇓) for the Ha-RasV12–expressing myocytes. For every voltage from −30 mV to +60 mV, inward Ca2+ current was significantly reduced by Ha-RasV12.
Membrane capacitance averaged 2.1-fold higher for Ha-RasV12–expressing myocytes. However, because of high variability, this difference did not achieve statistical significance. It was often impossible to voltage clamp the largest Ha-RasV12–expressing myocytes. Membrane-capacitance data may thus underestimate the hypertrophic effect of Ha-RasV12.
L-Channel Promoter Activity Is Reduced by Ha-RasV12
To test if Ha-RasV12 may regulate L-channel expression, myocytes were transfected with a construct containing the promoter region of the rat α1C L-channel gene (1.7Lch α1C-luc). This construct demonstrates reporter-gene expression in cultured cardiac and vascular smooth muscle cell lines (N.S., unpublished data, April 1999) similar to the virtually identical 2-kb 5′ flanking sequence described by other studies.21 The myocytes were also cotransfected with a plasmid featuring the SV40 promoter upstream of β-galactosidase (pCH110, 12 μg/cuvette) as a general indicator of transcriptional activity. Certain myocytes were also cotransfected with dnMEK (45 μg/cuvette), which blocks the activation of ERK-MAPK by Ha-RasV12. Luciferase expression was not significantly changed by Ha-RasV12 or dnMEK (Figure 4A⇓); however, the combination of Ha-RasV12 and dnMEK stimulated luciferase by 2.5-fold. β-Galactosidase was increased 6-fold by Ha-RasV12. dnMEK did not influence β-galactosidase expression but partially inhibited the effects of Ha-RasV12 on β-galactosidase (Figure 4B⇓). For luciferase normalized to β-galactosidase, a commonly used index of promoter activity, Ha-RasV12 had a strong negative effect, diminishing this ratio to 20% of control (Figure 4C⇓). dnMEK had no effect on luciferase or β-galactosidase but largely reversed the inhibitory effects of Ha-RasV12. These results suggest that Ha-RasV12 diminishes L-channel promoter activity through the Raf-MEK-ERK pathway, which may reduce transcription of the rat α1C gene.
Overexpression of L-Channels Corrects Ha-RasV12–Induced Reductions in Ca2+ Transient Magnitude
To test if expressing L-channels would correct Ha-RasV12–induced dysregulation of [Ca2+]i, myocytes were transfected with plasmids encoding human L-channel subunits α1, α2, and β2.25 In this experiment, Ha-RasV12 diminished Rsys and transient magnitude and increased tdecay (Figure 5A⇓ and Table⇑, experiment 3). The human L-channels had no effect on diastolic [Ca2+]i (Rdia) but significantly increased Rsys and magnitude. Tdecay increased somewhat, but this did not reach statistical significance. Coexpression of L-channels in the presence of Ha-RasV12 increased both Rdia and Rsys. Importantly, transient magnitudes for these myocytes were similar to controls. Thus, the inhibitory effects of Ha-RasV12 on transient magnitude were corrected by coexpressing L-channels. These myocytes exhibited large tdecay (Figure 5A⇓ and Table⇑), which is likely attributable to Ha-RasV12–induced reduction in SERCA2 expression leading to slow Ca2+ reuptake into the SR.
Overexpression of L-Channels Plus SERCA2 Corrects [Ca2+]i Regulation
Although coexpressing L-channels corrected Ha-RasV12 effects on Rsys and magnitude, tdecay was elevated. To test if coexpressing SERCA2 might correct this remaining defect, SERCA2 was also coexpressed. In this experiment, Ha-RasV12 significantly increased Rdia, diminished Rsys, reduced transient magnitude, and increased tdecay (Figure 5B⇑ and Table⇑, experiment 4). For myocytes expressing L-channels plus SERCA2, Rdia was slightly increased, but this did not reach statistical significance. Notably, Rsys was dramatically increased, but tdecay remained similar to the controls. For myocytes expressing Ha-RasV12 plus L-channels plus SERCA2, Rdia was significantly increased but Rsys was virtually identical to the control myocytes. The magnitudes of the transients and the tdecay were also similar to the control cells. These data demonstrate that the dysregulating effect of Ha-RasV12 on [Ca2+]i can be largely corrected by coexpressing L-channels and SERCA2. The Ha-RasV12–induced alteration in caffeine-induced calcium release was also corrected (Table⇑, experiment 4).
Expression of L-Channel Subunits Increases Myofibril Density
Ha-RasV12–expressing myocytes are enlarged compared with control cells and reduced in myofibril density.21 To determine whether expression of the L-channel subunits would affect cell morphology or modulate morphological effects of Ha-RasV12, myocytes were stained for actin to visualize the striated myofibrils. Myofibril density and cell size were then evaluated.
Expression of L-channels increased myofibril density by almost 2-fold compared with control cells (from 35% to 63%, Figures 6⇓ and 7A⇓). Expression of Ha-RasV12 had the opposite effect, strongly reducing the occurrence of striated myofibrils. Myocytes expressing Ha-RasV12 plus L-channels featured a mean myofibril density that was virtually identical to the controls, and certain cells from this group expressed a remarkably high density of myofibrils (Figure 6⇓). Thus, expression of L-channels significantly increased myofibril expression and prevented the decrease in myofibril expression associated with Ha-RasV12 expression.
Control myocytes averaged 737±60 μm2 in size. Myocytes expressing the L-channel subunits averaged 1160±126 μm2 in size, suggesting a hypertrophic effect, but this difference was not statistically significant in this multigroup comparison (Figure 7B⇑). Consistent with previous studies, Ha-RasV12 increased cell size by almost 6-fold. Myocytes coexpressing Ha-RasV12 plus L-channels were identical in size to Ha-RasV12–expressing myocytes. Thus, expression of L-channels did not modify the overall growth response to Ha-RasV12.
A separate experiment was conducted that included SERCA2. Myocytes expressing L-channels plus SERCA2 were very similar to myocytes expressing L-channels alone (1178±168 μm2 cell size, 62±6% myofibril density, mean±SE, n=6). Although not completely definitive, these data suggest that SERCA2 expression may be less directly related to myofibril density than is L-channel expression.
In this study, experiments conducted using indo-1, caffeine, and elevated K+ led to the hypothesis that Ha-RasV12 may reduce depolarization-induced calcium influx. This hypothesis was confirmed through use of patch-clamp recording techniques. The reduction of L-channel current by Ha-RasV12 could conceivably result from either a disruption in L-channel function or reduced L-channel expression. Consistent with the latter possibility, Ha-RasV12 reduced the activity of the L-channel α1C subunit gene promoter; therefore, it is likely that Ha-RasV12 reduces transcription of the cardiac L-channel gene.
Although it is unusual for Ha-RasV12 to downregulate expression of a cardiac protein, there is precedent for this phenomenon. Phenylephrine and phorbol esters, which activate Ras in cardiac myocytes, also reduce L-channel expression,26 and Ha-RasV12 reduces T-type calcium channel density in Swiss 3T3 fibroblasts.27 28 For most genes tested in cardiac myocytes,19 29 transcriptional activity is upregulated by Ha-RasV12. This was also true in this study for β-galactosidase expression, which was upregulated by Ha-RasV12 to a similar degree as the general increase in cell size. Thus, whereas Ha-RasV12 likely leads to an overall increase in transcription, the activity of the α1C gene promoter may not be increased in a proportional manner. Thus, in relation to total cellular protein, L-channel expression is likely to be reduced. Notably, Ha-RasV12 similarly diminishes the activity of the SERCA2 gene promoter.30 Both SERCA2 and L-channels are often downregulated in cardiac hypertrophy and failure. Excessive activation of the Ras-Raf-MEK-ERK pathway in response to stress might be responsible for this, perhaps producing cells that are less representative of differentiated cardiac myocytes.
Although dnMEK by itself had no effect on L-channel promoter activity, dnMEK increased promoter activity in the presence of Ha-RasV12. This would not occur if the L-channel promoter were simply refractory toward Ha-RasV12. These data suggest that Ha-RasV12 may have simultaneous positive and negative effects on L-channel transcription, with the Raf-MEK-ERK cascade acting to reduce promoter activity, as has been proposed for the regulation of ANF transcription.31 Our understanding of L-channel promoter activity is, admittedly, still incomplete, because this promoter region has just recently been identified. It is notable that dnMEK corrects the effects of Ha-RasV12 on both L-channel promoter activity and calcium transient magnitude.21 Coexpressing L-channels and SERCA2 had a similar, corrective effect on Ha-RasV12–induced dysregulation of [Ca2+]i regulation as coexpressing dnMEK.21 Thus, we have identified two deleterious effects of Ha-RasV12 on Ca2+-regulatory proteins (downregulation of SERCA2 and L-channels) and have demonstrated that these defects can be alleviated by gene-transfection techniques.
The cardiac action potential is often altered in heart failure.9 Although it is beyond the scope of the present study, it is likely that Ha-RasV12 may also affect expression of other pathologically relevant ion channels.
The increase in myofibril density in myocytes coexpressing L-channels is similar, in certain respects, to transgenic mice featuring cardiac-targeted overexpression of the α1c L-channel subunit. The size of the cardiac myocytes in this model is increased without a compromise in contractility,32 indicating that expression of the contractile apparatus is also upregulated. The basis for this is likely to involve increased [Ca2+]i in cardiac myocytes that overexpress L-channels. Recent evidence also suggests the intriguing possibility of potentially stabilizing interactions between L-channels and myofibrils. In skeletal muscle cells, L-channels interact with integrin α7,33 and cardiac myofibrils may be stabilized by integrin-mediated effects at the Z-disk structure.34 Thus, L-channels might conceivably play a structural role in maintaining the integrity of the cardiac contractile apparatus. Elucidating the relationships between L-channel expression and myofibril density will likely improve our understanding of the cardiac myocyte phenotype and may provide a basis for developing novel therapeutic approaches to treating myofibril disorganization.
This work was supported by National Institutes of Health Grants HL-54030 (to P.M.M.), NL/HL-25073 (to C.C.G.), HL-63975 (to C.C.G.), and AR-41526 (to P.T.P.). P.D.H. was the recipient of an American Heart Association, California affiliate, predoctoral fellowship. We acknowledge Cathy Andrews, Donna Thuerauf, and Joshua Martindale for technical assistance.
Original received May 10, 2000; resubmission received October 17, 2000; revised resubmission received November 14, 2000; accepted November 16, 2000.
- © 2001 American Heart Association, Inc.
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