Persistent Activation of a Swelling-Activated Cation Current in Ventricular Myocytes From Dogs With Tachycardia-Induced Congestive Heart Failure

From the Departments of Internal Medicine (H.F.C.) and Physiology (H.F.C., C.M.B.), Medical College of Virginia, Virginia Commonwealth University, Richmond, Va, and the Division of Cardiology (B.S.S.), Veterans Affairs Medical Center, West Roxbury, and Harvard University, Boston, Mass.

Correspondence to Henry F. Clemo, MD, PhD, Department of Physiology, Medical College of Virginia, PO Box 980551, Richmond, VA 23298-0551. E-mail hclemo{at}hsc.vcu.edu

	Abstract

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Abstract—The hypothesis that cellular hypertrophy in congestive heart failure (CHF) modulates mechanosensitive (ie, swelling- or stretch-activated) channels was tested. Digital video microscopy and amphotericin–perforated-patch voltage clamp were used to measure cell volume and ion currents in ventricular myocytes isolated from normal dogs and dogs with rapid ventricular pacing–induced CHF. In normal myocytes, osmotic swelling in 0.9x to 0.6x isosmotic solution (296 mOsm/L) was required to elicit an inwardly rectifying swelling-activated cation current (I_Cir,swell) that reversed near –60 mV and was inhibited by 10 µmol/L Gd³⁺, a mechanosensitive channel blocker. Block of I_Cir,swell by Gd³⁺ simultaneously reduced the volume of normal cells in hyposmotic solutions by up to 10%, but Gd³⁺ had no effect on volume in isosmotic solution. In contrast, I_Cir,swell was persistently activated under isosmotic conditions in CHF myocytes, and Gd³⁺ decreased cell volume by 8%. Osmotic shrinkage in 1.1x to 1.5x isosmotic solution inhibited both I_Cir,swell and Gd³⁺-induced cell shrinkage in CHF cells, whereas osmotic swelling only slightly increased I_Cir,swell. The K_0.5 and Hill coefficient for Gd³⁺ block of I_Cir,swell and Gd³⁺-induced cell shrinkage were estimated as 2.0 µmol/L and 1.9, respectively, for both normal and CHF cells. In both groups, the effects of Gd³⁺ on current and volume were blocked by replacing bath Na⁺ and K⁺ and were linearly related with varying Gd³⁺ concentration and the degree of cell swelling. CHF thus altered the set point for and caused persistent activation of I_Cir,swell. This current may contribute to dysrhythmias, hypertrophy, and altered contractile function in CHF and may be a novel target for therapy.

Key Words: arrhythmia • cardiomyopathy • cardiac edema • cell size • ion channel gating

	Introduction

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Details of the complex chain of events leading to CHF and the accompanying cellular hypertrophy, contractile dysfunction, and dysrhythmias remain incompletely understood.^{1 2 3 4} It is clear, however, that hemodynamic disturbances capable of initiating CHF place unnatural stresses on cardiac myocytes. The resulting cellular remodeling may further alter mechanical forces detected by the cytoskeleton and cell membrane. The mechanical nature of the primary event and the ensuing hypertrophy suggest that modulation of SACs^{5 6 7} and the processes underlying cell volume regulation^{8 9} might play a role in the pathophysiology of CHF.

A variety of SACs have been identified in myocardial cells. Mechanical deformation or swelling activates poorly selective cation channels,^{10 11 12 13 14} K⁺ channels,^{15 16 17} and Cl^- channels.^{18 19 20 21} In addition, the function of a number of voltage-dependent channels, including the delayed rectifier^{22 23 24} and L-type Ca²⁺ channel,^{22 25} are mechanosensitive, although there is disagreement about the details.

Several cation SACs are blocked by micromolar concentrations of the trivalent lanthanide gadolinium (Gd³⁺).^{13 14 26 27 28} The efficacy of Gd³⁺ as an inhibitor of stretch-induced release of atrial natriuretic peptide from rat atria,²⁹ the generation of ventricular premature beats by rapid inflation of a balloon in canine ventricles,^{30 31} and afterdepolarizations, premature beats, and poststretch augmentation of contractile force in rat atria³² argue that activation of cation SACs are responsible for these stretch-induced phenomena. We found that Gd³⁺ also decreases the magnitude of osmotic swelling in intact rabbit ventricular myocytes but that it does not affect cell volume under isosmotic conditions, when SACs are thought to be closed.³³ The same effects of Gd³⁺ on cell volume are observed in perforated-patch voltage-clamp studies and are due to block of I_Cir,swell, a swelling-activated poorly selective cation current (P_K/P_Na, 6) that exhibits inward rectification.¹⁴ The primary effect of Gd³⁺ is on current rather than cell volume because the onset of block of I_Cir,swell precedes cell shrinkage. I_Cir,swell is a depolarizing current at the normal resting E_m and is postulated to contribute to stretch-induced dysrhythmias and other stretch-dependent events.

The purpose of the present study was to evaluate whether SAC behavior and the regulation of cell volume are transformed in a canine dilated cardiomyopathy model of CHF produced by several weeks of rapid ventricular pacing. This model has been extensively characterized in both dogs and pigs.³⁴ Chronic tachycardia produces biventricular dilation with eccentric cellular hypertrophy, decreases contractility, downregulates the response to catecholamines, elevates plasma catecholamines, atrial natriuretic peptide, renin, and endothelin-1, and decreases the density of the myocardial collagen network. All of these features are hallmarks of dilated cardiomyopathy in humans.^{35 36 37 38} Thus, the model is an appropriate starting point for evaluating the role of SACs in CHF.

The present study found that I_Cir,swell was persistently activated in isosmotic media in ventricular myocytes isolated from dogs with CHF; osmotic swelling increased I_Cir,swell slightly, whereas osmotic shrinkage inhibited the current. In contrast, I_Cir,swell was detected in normal canine myocytes only after osmotic swelling. Moreover, block of I_Cir,swell by Gd³⁺ in CHF and normal myocytes caused a proportional cell shrinkage. These data are consistent with the idea that elongated myocytes from dogs in congestive failure behave as if they are stretched. Cation influx via I_Cir,swell may contribute to cell hypertrophy and dysrhythmias as well as modulate contractile function. Consequently, SACs are a novel target for therapeutic approaches.

	Materials and Methods

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Heart Failure Model
Adult mongrel dogs underwent transvenous insertion of a permanent pacemaker and were paced from the right ventricular apex at 235 bpm for 4 to 6 weeks. Retrograde ventriculoatrial conduction was absent during pacing. Chronic rapid pacing produced clinical evidence of CHF in all dogs as manifested by variable degrees of anorexia, lethargy, exercise intolerance, ascites, tachypnea, and/or muscle wasting. Two-dimensional echocardiography was performed at baseline and serially during the period of rapid ventricular pacing. Ventricular dysfunction was documented by a fall in left ventricular ejection fraction from 0.65±0.06 to 0.23±0.09 (P<0.0001), a >3-fold increase in left ventricular end-systolic volume from 19.5±7.9 to 61.4±15.3 mL (P<0.0001), and a 50% increase in left ventricular end-diastolic volume from 50.3±11.9 to 75.5±13.9 mL (P<0.0001).

Cell Isolation
Dogs (normal, n=4; CHF, n=6) were anesthetized with intravenous sodium thiopental (30 mg/kg) followed by -chloralose (10 mg/kg) and were intubated. A median sternotomy was made, and sections of the right and left ventricular apices were removed and washed in a nominally Ca²⁺-free Tyrode's solution gassed with 100% O₂. Right and left ventricular sections were divided into 1-mm² pieces with a scalpel and then dispersed separately using an albumin-containing collagenase-pronase solution described previously.^{39 40} The tissue was placed in enzyme-containing flasks in a shaker bath and agitated for 10 minutes at 37°C. After filtration through a 250-µm nylon mesh to remove incompletely digested material, cells were washed twice and stored in a modified Kraft-Brühe solution containing (mmol/L) KOH 132, glutamic acid 120, KCl 2.5, KH₂PO₄ 10, MgSO₄ 1.8, K₂EGTA 0.5, glucose 11, taurine 10, and HEPES 10 (pH 7.2, 295 mOsm/L). Typical yields were 30% to 50% Ca²⁺-tolerant rod-shaped cells. Myocytes were used within 6 hours of harvesting, and only quiescent cells with regular striations and no evidence of membrane blebbing were selected for study. Only quiescent cells were chosen to eliminate myocytes that might have been damaged in isolation. This procedure may eliminate CHF cells that were most severely injured in vivo from the study because it excludes cells that become spontaneously active in vivo, and cells severely injured in vivo may not survive isolation.

Experimental Solutions
Cells were placed in a glass-bottomed chamber (0.3 mL) and superfused with room temperature (22°C) bathing solution at 3 mL/min. Solution changes were complete within 10 seconds, as estimated from the liquid junction potential of a microelectrode. The standard bathing solution contained (mmol/L) NaCl 65, KCl 5, CaSO₄ 2.5, MgSO₄ 0.5, HEPES 5, glucose 10, and mannitol 17 to 283 (pH 7.4). The reduced, fixed NaCl concentration permitted adjustment of osmolarity with mannitol at a constant ionic strength. To evaluate the role of physiological monovalent cations, Na⁺ and K⁺ in the bathing media were replaced by equimolar amounts of NMDG in some experiments. An osmolarity of 296 mOsm/L was taken as isosmotic (1T). Osmolarity ranged from 178 to 266 mOsm/L in hyposmotic solutions (0.6T to 0.9T) and was 444 mOsm/L in hyperosmotic solution (1.5T). Osmolarity routinely was checked with a freezing-point depression osmometer (Osmette S, Precision Systems).

Voltage-Clamp Technique
Patch electrodes with a tip diameter of 3 to 4 µm and a resistance of 0.5 to 1 M were made from borosilicate capillary tubing (7740 glass with filament; outer diameter, 1.5 mm; inner diameter, 1.12 mm; Glass Co of America). The standard electrode filling solution contained (mmol/L) potassium aspartate 120, KCl 10, NaCl 10, MgSO₄ 3, and HEPES 10 (pH 7.1). In addition, a Na⁺- and K⁺-free pipette solution was made by replacing Na⁺ and K⁺ salts with Cs⁺ salts for experiments in which bath Na⁺ and K⁺ were replaced with NMDG.

The amphotericin–perforated-patch voltage-clamp technique was used for all studies to avoid unpredictable cell swelling and changes in membrane currents that often slowly occur with ruptured patches.^{14 19} Amphotericin B (Sigma Chemical Co) was freshly dissolved in DMSO (Sigma) and then diluted in electrode filling solution to give final amphotericin and DMSO concentrations of 100 µg/mL and 0.2% (vol/vol), respectively. After dipping the pipette tip into amphotericin-free solution for 2 seconds, pipettes were backfilled with ionophore, and gigaseals were formed as rapidly as possible. Access resistance decreased to 7 to 10 M within 20 minutes of seal formation, and then experimental protocols were begun.

An Axoclamp 200A amplifier (Axon, Foster City, CA) was used to measure whole-cell currents. Voltage-clamp protocols, electrophysiological and video data acquisition, and off-line data analysis were managed with a suite of custom programs written in ASYST (Keithley). To study quasi–steady-state currents that may contribute to the regulation of cell volume, slow voltage ramps (28 mV/s) were applied. The voltage was stepped from –80 to +40 mV for 20 milliseconds, ramped to –100 mV over 5 seconds, and after 10 milliseconds, ramped back to +40 mV over 5 seconds. Currents elicited by the depolarizing and hyperpolarizing legs of the ramp were virtually identical and were averaged to cancel the small capacitive current (see Figure 1D). This ramp protocol is the same as previously used to study I_Cir,swell in rabbit ventricular myocytes¹⁴ and may underestimate any swelling-activated components of the slowly activated delayed rectifier current I_Ks. Ramp currents were digitized at 1 kHz after low-pass filtering at 200 Hz. Membrane capacitance was calculated from the integral of the current transient in response to 10-mV hyperpolarizing pulses. Reported values of E_m were corrected by +3.8 and +2.1 mV, the liquid junction potentials (bath-pipette),⁴¹ for the standard and Na⁺- and K⁺-free solution pairs, respectively. The bath was grounded via a 3 mol/L KCl agar bridge.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of osmotic swelling on whole-cell currents recorded by amphotericin–perforated-patch method from a normal myocyte. A, I-V relationships in isosmotic (1T) and hyposmotic (0.6T) solutions are shown. Traces are average of hyperpolarizing and depolarizing ramps. Osmotic swelling increased both inward and outward currents. B, Gd3+ (10 µmol/L), a blocker of stretch-activated cation channels, markedly attenuated the swelling-activated current in 0.6T solution but had little effect on current in 1T solution. C, Gd3+-sensitive difference currents calculated as current in Gd3+-free minus that in Gd3+-containing 1T and 0.6T solutions are shown. The swelling- activated Gd3+-sensitive current (0.6T) inwardly rectified with an Erev of -63 mV and was denoted ICir,swell. D, Diagram of voltage ramp (28 mV/s) protocol. Difference between currents elicited in 1T solution by hyperpolarizing (RH) and depolarizing (RD) ramps is shown at high gain.

Determination of Relative Cell Volume
Methods for determining relative cell volume have been described previously.^{39 40} An inverted microscope (Diaphot, Nikon, Inc) equipped with Hoffman modulation optics (x40; numerical aperture, 0.55) and a high-resolution TV camera (CCD72, Dage-MTI) coupled to a video frame-grabber (Targa-M8, Truevision) was used to image myocytes. Images were captured on-line each time a ramp protocol was performed. A combination of commercial (MOCHA, SPSS) and custom (ASYST, Keithley) programs was used to determine cell width, length, and the planar area of the image.

Changes in cell width and thickness on exposure to anisosmotic solutions are proportional.⁴² With each cell used as its own control, relative cell volume was calculated as follows: vol_t/vol_c= (area_txwidth_t)/(area_cxwidth_c), where t and c refer to test (eg, 0.6T) and control (1T) solutions, respectively. The value of relative cell volume is independent of assumptions regarding the geometric shape of the cross section of the myocyte as long as the shape does not change. These methods provide estimates of relative cell volume that are reproducible to <1%.^{39 40}

Statistics
Data are reported as mean±SEM, and n represents the number of cells, unless otherwise noted. Mean current densities were expressed in pA/pF to account for differences in cell membrane area. When multiple comparisons were made, ANOVA and the Bonferroni method for group comparisons were used. For simple comparisons, a Student t test was performed. Comparison of the sensitivity of normal and CHF cells to Gd³⁺ was evaluated by a 2-way ANOVA, including an interaction term. All statistical analyses were conducted with SigmaStat 2.0 (SPSS), and curve-fitting was performed in SigmaPlot 4.0 (SPSS). Least squares methods were used to determine lines of best fit, and nonlinear fitting used the Marquardt-Levenberg algorithm.

	Results

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Characteristics of Ventricular Myocytes From Normal and CHF Dogs
Morphological and electrophysiological characteristics of ventricular myocytes isolated from normal and CHF dogs and selected for study are summarized in Table 1. Chronic rapid pacing caused ventricular myocytes to enlarge. The planar area of the image of the myocyte and the estimated cell volume were 19.8% and 23.9% greater, respectively, for cells from CHF dogs than for those from normal animals. These morphological changes reflected mainly a 15.7% increase in cell length, whereas cell width did not change significantly. Concomitantly, membrane capacitance increased 19.2%, reflecting an extension of sarcolemmal membrane area. Because the myocytes studied here were selected as being appropriate for both patch clamp and imaging, they may not represent an unbiased sample of myocardial cells. Moreover, the volumes of cells studied in vitro may not be equal to their volumes in vivo.

Swelling-Activated Current in Normal Canine Ventricular Myocytes
A swelling-activated, poorly selective cation current that inwardly rectifies and is blocked by Gd³⁺ is found in rabbit ventricular myocytes.¹⁴ This current is called I_Cir,swell. Osmotic cell swelling elicited a similar current in normal canine ventricular myocytes. Figure 1A illustrates the I-V relationship in isosmotic solution (1T) and after exposure to hyposmotic solution (0.6T) for 5 minutes. Hyposmotic solution augmented the inwardly rectifying current at negative potentials, and an outwardly rectifying current developed at potentials positive to –65 mV. These changes in the I-V relationship completely reversed on returning to 1T solution.

After recovery in 1T solution, the effect of 10 µmol/L Gd³⁺ was tested in 1T solution and after swelling the myocytes in 0.6T solution (Figure 1B). The Gd³⁺-sensitive difference currents in 1T and 0.6T solutions, calculated as the current in Gd³⁺-free solution minus that in the presence of 10 µmol/L Gd³⁺, are depicted in Figure 1C. During osmotic swelling, Gd³⁺ blocked an inwardly rectifying current that reversed near –60 mV, whereas the difference current was negligible in 1T solution. The similarity of these results to those in rabbit myocytes¹⁴ was striking and suggested that I_Cir,swell also was present in normal canine ventricular myocytes; evidence that the inwardly rectifying Gd³⁺-sensitive current is a cation current is presented later (see Figure 9). In addition, Gd³⁺ did not block the outwardly rectifying current caused by swelling in 0.6T. This Gd³⁺-resistant current was nearly abolished by replacement of Cl^- with methanesulfonate and by 9-anthracene carboxylic acid,⁴³ suggesting that it is mainly the swelling-activated anion current, I_Cl,swell, which has been previously described in atrial, ventricular, and sinoatrial nodal myocytes from dogs^{18 19} and other species,^{20 21 44} including humans.^{45 46}

View larger version (20K): [in this window] [in a new window]   Figure 9. Gd3+-sensitive current is cation dependent in normal (A and B, n=4) and CHF (C and D, n=4) myocytes. Na+ and K+ were replaced with NMDG in the bath and by Cs+ in the pipette, and the effect of 10 µmol/L Gd3+ was studied in 1T and 0.6T solution in normal myocytes and 1T and 1.5T solution in CHF myocytes. After replacing physiological monovalent cations, the Gd3+-sensitive current (A and C) and Gd3+-induced cell shrinkage (B and D) were negligible in both normal and CHF cells. Times (a to d) of recording of I-V relationships are indicated.

Figure 2 shows that I_Cir,swell was graded by the amount of cell swelling in normal canine myocytes. I-V relationships and relative cell volume were recorded simultaneously in selected hyposmotic (0.6T to 0.9T), isosmotic (1T), and hyperosmotic (1.5T) solutions, and as described previously,¹⁴ I_Cir,swell was measured as the difference between the I-V relationships ±10 µmol/L Gd³⁺ at each osmolarity (Figure 2A). Gd³⁺ did not affect the I-V relationship in 1T solution or after cell shrinkage in 1.5T solution. On the other hand, even after only modestly swelling myocytes in 0.9T solution to a relative cell volume of 1.088±0.026 (P<0.005, Figure 2B), I_Cir,swell was already obvious (-0.25±0.05 pA/pF, P<0.001). I_Cir,swell increased on additional swelling but reached a maximum in 0.7T solution at a volume of 1.268±0.029, despite the fact that myocytes swelled further in 0.6T solution.

View larger version (22K): [in this window] [in a new window]   Figure 2. Gd3+-sensitive current, ICir,swell, and Gd3+-induced cell shrinkage were observed in normal myocytes after osmotic swelling in 0.6T to 0.9T hyposmotic solutions but not in isosmotic (1T) or hyperosmotic (1.5T) solutions. A and B, Average Gd3+-sensitive currents (A) and average cell volumes in the absence and presence of 10 µmol/L Gd3+ (B). As bath osmolarity was decreased, Gd3+-sensitive current and Gd3+-induced reduction of cell volume increased. Maximum responses were obtained at 0.7T. In this and subsequent figures, time 0 for relative cell volume corresponds to sealing of the amphotericin-filled pipette on the cell. Myocyte volume in 1T solution did not change during the 20-minute period of patch perforation. C, Gd3+-sensitive current at -80 mV and Gd3+-induced cell shrinkage plotted vs bath relative osmolarity (n=6 cells).

Gd³⁺ not only blocked I_Cir,swell, it also decreased the extent of osmotic cell swelling (Figure 2B). For example, cells swelled to 1.378±0.018 in 0.6T solution but only 1.267±0.027 in 0.6T solution+Gd³⁺ (P<0.01). The effects of Gd³⁺ on membrane current and cell volume are summarized in Figure 2C, where Gd³⁺-sensitive current at –80 mV and Gd³⁺-induced shrinkage are plotted against superfusate osmolarity. Both effects of Gd³⁺ were apparent and saturated over the same range of osmolarities.

Persistent Activation of I_Cir,swell in CHF Myocytes
Ventricular diastolic wall stress is increased in tachycardia-induced CHF.^{47 48} This raises the possibility that activation of I_Cir,swell in situ contributes to the pathophysiology of CHF. The situation may be different for single cells isolated from CHF dogs, however. The distending forces present during diastole in the intact animal are absent in isolated myocytes under isosmotic conditions. On the other hand, the CHF-induced increase in cell volume may be equivalent to an isosmotic stretch and thereby lead to activation of I_Cir,swell. To investigate this hypothesis, Gd³⁺-sensitive I_Cir,swell and cell volumes of myocytes isolated from dogs in CHF were measured simultaneously at osmolarities ranging from 0.6T to 1.5T.

Dissection of the Gd³⁺-sensitive current in a CHF cell is illustrated in Figure 3. I-V relationships were recorded in the absence (Figure 3A) and presence (Figure 3B) of 10 µmol/L Gd³⁺ in both 1T solution and after osmotic shrinkage in 1.5T solution, and the Gd³⁺-sensitive difference currents were calculated (Figure 3C). In contrast to normal cells wherein swelling was necessary to elicit a Gd³⁺-sensitive current, the CHF myocyte exhibited a large, inwardly rectifying, Gd³⁺-sensitive current in 1T solution that was abolished by osmotic shrinkage. Figure 4 shows averaged data for CHF cells in 1T and hyposmotic solutions. The Gd³⁺-sensitive current in CHF cells in 1T solution was –1.21±0.35 pA/pF at –80 mV (P<0.01) in 1T solution, but cell swelling in successively more hyposmotic solutions (0.9T to 0.6T) only slightly increased the Gd³⁺-sensitive current further (eg, –1.50±0.27 pA/pF at –80 mV in 0.8T solution). Simultaneously, 10 µmol/L Gd³⁺ reduced relative cell volume in 1T solution by 9.1%, from 0.997±0.004 to 0.906±0.010 (Figure 4B, P<0.001), and significantly reduced cell volume in each of the hyposmotic solutions. Figure 4C emphasizes, however, that neither Gd³⁺-sensitive current at –80 mV nor Gd³⁺-induced cell shrinkage was significantly affected by osmotic swelling of CHF myocytes. In addition to the data presented in Figure 4, protocols depicted in Figures 5 and 7 also included measurements of current and volume in 1T solution. Combining all experiments (30 cells from 6 CHF dogs), the current blocked by 10 µmol/L Gd³⁺ in 1T solution was –1.07±0.16 pA/pF at –80 mV (P<0.001), and Gd³⁺ decreased cell volume by 7.8±0.7% (P<0.001).

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of osmotic swelling on whole-cell currents in a CHF myocyte. A and B, Currents under isosmotic (1T) and hyperosmotic (1.5T) conditions in the absence (A) and presence (B) of 10 µmol/L Gd3+. Osmotic shrinkage reduced both inward and outward currents. C, Gd3+-sensitive difference currents obtained by digitally subtracting the curves in panels A and B. A Gd3+-sensitive, inwardly rectifying current was present in CHF cells under isosmotic conditions (1T) and was abolished by osmotic shrinkage (1.5T).

View larger version (20K): [in this window] [in a new window]   Figure 4. ICir,swell was chronically activated in isolated CHF myocytes. A and B, Average Gd3+-sensitive currents (A) and average cell volumes in the absence and presence of 10 µmol/L Gd3+ (B) in 1T to 0.6T solutions. In 1T solution, ICir,swell was 1.21±0.35 pA/pF at -80 mV, and Gd3+ caused a 9.1% decrease of cell volume. Osmotic swelling (0.9T to 0.6T) did not significantly alter the response to Gd3+. C, Gd3+-sensitive current at -80 mV and Gd3+-induced cell shrinkage plotted vs bath relative osmolarity (n=12 cells).

View larger version (21K): [in this window] [in a new window]   Figure 5. Osmotic shrinkage abolished ICir,swell in CHF myocytes. A and B, Average Gd3+-sensitive currents (A) and average cell volumes in the absence and presence of 10 µmol/L Gd3+ (B) in 1T to 1.5T solutions. ICir,swell decreased as bath osmolarity was increased. At -80 mV, ICir,swell was not significantly different from 0 in 1.3T. Osmotic shrinkage also reduced the effect of Gd3+ on cell volume. C, Gd3+-sensitive current at -80 mV and Gd3+-induced cell shrinkage plotted vs bath relative osmolarity (n=12 cells).

View larger version (24K): [in this window] [in a new window]   Figure 7. Dose dependence of Gd3+-sensitive current and Gd3+-induced cell shrinkage in normal (n=6) and CHF (n=6) myocytes. Normal cells were osmotically swollen (0.6T) to activate ICir,swell, and CHF cells were studied in isosmotic solution (1T). Cells were exposed to each concentration of Gd3+ for 5 minutes, a time sufficient to obtain the steady-state response.14 A and C, Average Gd3+-sensitive currents with 1 to 30 µmol/L Gd3+ in normal (A) and CHF (C) myocytes. B and D, Concurrent measurements of cell volumes of normal (B) and CHF (D) cells.

Observation of Gd³⁺-sensitive current and Gd³⁺-induced cell shrinkage in CHF myocytes cells under isosmotic conditions is in stark contrast to the results in normal myocytes (compare with Figure 2). If I_Cir,swell is persistently activated by CHF because CHF cells are "swollen" even in 1T solution and if the membrane and cytoskeletal elements thereby are "stretched," then graded osmotic shrinkage should relieve the stress and turn off I_Cir,swell in a graded fashion. To test this prediction, responses to Gd³⁺ in isosmotic solution (1T) were compared with those in a series of hyperosmotic solutions (1.1T to 1.5T). As shown in Figure 5A, graded shrinkage of CHF cells led to a graded reduction of the Gd³⁺-sensitive current. I_Cir,swell was undetectable at osmolarities >1.4T. Along with the inhibition of I_Cir,swell, the ability of 10 µmol/L Gd³⁺ to reduce cell volume also was diminished by cell shrinkage (Figure 5B). Gd³⁺-sensitive changes in current and cell volume in 1T to 1.5T solutions are summarized in Figure 5C.

Cell Volume Dependence of the Gd³⁺ Sensitivity of Normal and CHF Myocytes
The responses of normal and CHF myocytes are directly compared in Figure 6, in which the Gd³⁺-sensitive current at –80 mV (Figure 6A) and the Gd³⁺-induced cell shrinkage (Figure 6B) are plotted as functions of cell volume before application of 10 µmol/L Gd³⁺ in 0.6T to 1.5T solutions. CHF cells (solid circles) remained Gd³⁺ sensitive even when they were shrunken to a relative cell volume of 0.861±0.014 in 1.3T solution, whereas normal cells (open squares) became Gd³⁺ sensitive only after swelling to 1.079±0.024 in 0.9T solution. The maximum response to Gd³⁺ was observed at volumes of 1.075±0.012 (0.9T) in CHF cells and 1.272±0.025 (0.7T) in normal cells, respectively. Thus, the set point for Gd³⁺ sensitivity appeared to be shifted to a relative cell volume 0.2 lower in CHF than in normal myocytes. The Gd³⁺-sensitive current and volume change, when expressed as a percentage of the maximal Gd³⁺-induced change, were proportional over the entire range of osmolarities explored (Figure 6C). This relationship suggests a tight coupling between the block of current and the reduction of cell volume.

View larger version (16K): [in this window] [in a new window]   Figure 6. Gd3+-sensitive current and Gd3+-induced cell shrinkage depend on cell volume. Data were taken from Figure 2 (normal cells, ) and Figures 4 and 5 (CHF cells, ). A and B, Gd3+-sensitive current at -80 mV (A) and Gd3+-induced cell shrinkage (B) plotted as functions of relative cell volume before adding Gd3+ (volumes normalized to that in 1T Gd3+-free solution). Maximum responses to Gd3+ were observed at relative cell volumes of 1.075±0.012 (0.9T) and 1.272±0.025 (0.7T) in CHF and normal cells, respectively. C, Approximately linear relationship between Gd3+-induced block of current and Gd3+-induced reduction of cell volume.

The preceding figures combine data obtained from myocytes isolated from both ventricles. The Gd³⁺-sensitive current and Gd³⁺-induced cell shrinkage in right and left ventricular myocytes are compared in Table 2 for both normal and CHF cells. No difference was detected in the effects of Gd³⁺ on right and left ventricular myocytes.

Dose Dependence of Effect of Gd³⁺ on Cell Volume and Current
Block of I_Cir,swell by Gd³⁺ and the resulting volume changes were dose dependent. Normal cells first were swollen in 0.6T to activate I_Cir,swell and then were exposed for 5-minute periods to successively higher doses of Gd³⁺ (1 to 30 µmol/L) in 0.6T solution. As the Gd³⁺ concentration was progressively increased, the magnitude of the blocked current increased significantly (Figure 7A), and relative cell volume decreased significantly (Figure 7B). The dose dependence of the effects of Gd³⁺ was also examined in CHF cells bathed in isosmotic solution, conditions under which I_Cir,swell was already activated. As for normal cells, exposing CHF cells to successively higher Gd³⁺ concentrations for 5-minute periods resulted in stepwise block of I_Cir,swell (Figure 7C) and decrease of cell volume (Figure 7D).

Dose-response curves for Gd³⁺-sensitive current at –80 mV and cell volume are shown in Figure 8A for normal cells and in Figure 8C for CHF cells. To compare the present results with each other and with previous work,¹⁴ data from Figure 7 were fitted to the Hill equation, and the responses were plotted, with the fitted maximum taken as 1. The K_0.5 and Hill coefficient for current and volume estimated from the fitted curves were 2.0±0.2 µmol/L and 1.9±0.4 and 2.1±0.3 µmol/L and 1.8±0.3, respectively, in normal cells and 2.0±0.2 µmol/L and 1.9±0.4 and 1.9±0.2 µmol/L and 2.0±0.3, respectively, in CHF cells. Cooperative binding and a sigmoidal dose-response relationship for Gd³⁺ were reported previously.^{14 26} To compare the responses of normal and CHF cells, a two-way ANOVA with factors cell type, dose, and cell typexdose was performed for Gd³⁺-induced block of current and cell shrinkage (Figure 8A and 8C). F-ratio tests on the interaction term indicated that the current (P=0.725) and volume (P=0.804) responses of normal and CHF cells were indistinguishable. Moreover, the fit parameters were similar to those obtained in rabbit myocytes.¹⁴ On the basis of these fits, 10 µmol/L Gd³⁺ is expected to block >90% of the Gd³⁺-sensitive current and cause >90% of the maximum Gd³⁺-induced shrinkage in both normal and CHF cells.

View larger version (19K): [in this window] [in a new window]   Figure 8. Comparison of Gd3+-sensitive current and Gd3+- induced cell shrinkage for normal (A and B) and CHF (C and D) myocytes after 5-minute exposures to Gd3+. Data from Figure 7 are replotted with responses (R) expressed as a fraction of the fitted maximum response (Rmax). Dose-response relationships for block of current at -80 mV () and reduction of volume () in normal (A) and CHF (C) cells were fitted to the Hill equation, R/Rmax=Cn/(K0.5n+Cn), with a K0.5 of 2 µmol/L and Hill coefficient, n, of 1.9 (solid line) estimated from the fits to all 4 dose-response relationships. Fitted relationships are extrapolated to 0.1 and 100 µmol/L to illustrate doses expected to give 0 and maximal responses. Fractional block of current at by Gd3+ (Gd3+ current) was linearly related to fractional Gd3+-induced cell shrinkage (Gd3+ volume) for normal (B) and CHF cells (D). See text for further details.

The excellent correlations between the effects of Gd³⁺ on current and volume in both normal and CHF cells are emphasized in Figure 8B and 8D, in which responses were expressed as a fraction of the fitted maximum response. The relationships were described by straight lines with slopes and intercepts not significantly different from 1 and 0, respectively, as previously found for normal rabbit ventricular cells.¹⁴ This is consistent with the idea that I_Cir,swell is one factor regulating cell volume.

Ionic Basis of Gd³⁺-Sensitive Current and Volume Changes
I_Cir,swell in rabbit ventricular myocytes is a poorly selective cation current with a P_K/P_Na of 6.¹⁴ If physiological monovalent cations such as Na⁺ and K⁺ constitute the majority of the Gd³⁺-sensitive current described thus far in myocytes from normal and CHF dogs, removal of these ions from the bath and electrode solutions should markedly attenuate both the Gd³⁺-sensitive current and the Gd³⁺-induced cell shrinkage. To test this prediction, Na⁺ and K⁺ were replaced with NMDG in the bath solution and with Cs⁺ in the pipette solution. In normal cells, removal of Na⁺ and K⁺ virtually eliminated the Gd³⁺-sensitive current elicited by cell swelling in 0.6T solution (Figure 9A, curve b–d), and as before, Gd³⁺ did not affect the current in 1T (curve a–c). Similarly, removal of Na⁺ and K⁺ eliminated the Gd³⁺-sensitive current seen in CHF cells in isosmotic solution (Figure 9C, curve a–c), and as before, the current remained insensitive to Gd³⁺ after osmotic shrinkage in 1.5T solution (curve b–d). The experiments in 1T solution for normal cells and in 1.5T solution for CHF cells demonstrate that Gd³⁺ has no effect on the remaining membrane currents under conditions previously shown to block I_Cir,swell. Cation replacement also abolished the Gd³⁺-induced shrinkage of normal cells in 0.6T solution (Figure 9B) and of CHF cells in 1T solution (Figure 9D). These data verified that I_Cir,swell in both normal and CHF canine myocytes was carried by cations. Based on the E_rev for I_Cir,swell (–61.5±3.3 mV in normal cells and –60.4±3.5 mV in CHF cells in 0.6T solution, Figures 2 and 4, respectively) and the bath and pipette Na⁺ and K⁺ concentrations, the constant field P_K/P_Na ratios were 8.3±1.6 in normal cells (n=6) and 8.5±2.0 in CHF cells (n=12). The calculation of P_K/P_Na assumes that Na⁺ and K⁺ were the only charge carriers, a reasonable approximation, because removing Na⁺ and K⁺ eliminated the current. A small Ca²⁺ component cannot be ruled out, however. The contribution of Ca²⁺ to I_Cir,swell was not investigated, although it is known that other cation SACs in the heart are permeant to Ca²⁺.¹¹

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A stretch-activated current, I_Cir,swell, was persistently activated under isosmotic conditions (1T) in ventricular myocytes isolated from dogs with tachycardia-induced CHF; the myocytes behaved as if they were swollen even in 1T solution. Graded osmotic swelling caused graded activation of I_Cir,swell, a poorly selective, inwardly rectifying cation current that is blocked by Gd³⁺.¹⁴ Although Gd³⁺ is not perfectly selective, its lack of effect in normal cells in 1T solution suggests that Gd³⁺ primarily blocks swelling-activated steady-state currents at the concentrations used. I_Cir,swell was 88% activated in 1T solution in CHF myocytes, and osmotic shrinkage with 1.3T solution to a relative cell volume of 0.861 was required to inhibit the current fully. In myocytes obtained from normal dogs, I_Cir,swell was absent in 1T. The maximum I_Cir,swell current density evoked was not significantly different in normal and CHF cells. Because membrane capacitance was 20% greater in the CHF cells, this means that the magnitude of fully activated of I_Cir,swell in CHF cells exceeded that in normal cells by the same factor. Thus, as sarcolemmal membrane area expanded, there was a proportional increase in the number of I_Cir,swell channels that could be activated by cell swelling, their open probability on activation, or both.

Cellular hypertrophy led to persistent activation of I_Cir,swell. At the same time, I_Cir,swell can explain in part the greater volume of CHF than normal myocytes. Hypertrophy requires both protein synthesis and a net gain of osmolytes to increase cell volume. I_Cir,swell was an inwardly directed current at the resting E_m (E_rev, –60 mV) carried by an influx of Na⁺ and K⁺.¹⁴ Its block by 10 µmol/L Gd³⁺ reduced the volume of CHF cells in 1T by 8%, but Gd³⁺ did not affect the volume of normal myocytes in 1T in which I_Cir,swell was not observed. The requirement for macroscopic electroneutrality dictates that a flux of anions must accompany a flux of cation sufficient to alter cell volume. This is likely to be carried by I_Cl,swell (see Figure 1), and preliminary experiments indicate that I_Cl,swell also is persistently activated in congestive failure.⁴³

Characteristics of I_Cir,swell
I_Cir,swell was only recently discovered.¹⁴ The characteristics of the current in both normal and CHF dog ventricular myocytes were virtually identical to those previously reported for rabbit myocytes.¹⁴ I_Cir,swell in both species exhibited (1) graded activation on stretch, (2) inward rectification, (3) dependence on bath K⁺ and Na⁺ with E_rev near –60 mV, (4) inhibition by Gd³⁺ with K_0.5 of 2 µmol/L and a Hill coefficient of 2, and (5) block of I_Cir,swell by Gd³⁺, leading to a proportional cell shrinkage of up to 10%. In rabbit myocytes, I_Cir,swell is unaffected by replacement of Cl^- with methanesulfonate and is insensitive to 0.2 mmol/L Ba²⁺, a blocker of the background K⁺ current, I_K1.

The major difference between I_Cir,swell in normal and CHF myocytes appeared to be the set point for its activation, as measured in terms of solution osmolarity or relative cell volume (ie, the volume of a cell relative to the volume of the same cell in 1T solution). However, the volume of CHF cells was estimated to be 23.9% greater than that of normal cells in 1T solution. If normal myocytes were osmotically swollen by this amount, the current density of I_Cir,swell observed was the maximum that could be elicited compared with the 81% activation of I_Cir,swell in CHF cells of the same absolute volume (see Figure 6). Similarly, osmotically shrinking CHF cells to a relative cell volume of 0.861 in 1.3T solution to turn off I_Cir,swell is equivalent to a normal cell volume of 1.067 (ie, 1.239x0.861), a volume at which I_Cir,swell was turned on <15% in normal cells. This suggests that the shift in the set point for activation of I_Cir,swell in CHF cells can be accounted for by assuming that myocytes "remember" their preCHF volume and thus are "swollen" even in isosmotic solution. This idea is appealing; it suggests why osmotic shrinkage reverses CHF-induced activation of I_Cir,swell, but it is likely too simple to fully explain the results. One problem is the difference in how cell volume increased. Osmotic swelling caused a much greater increase in cell width than length, as noted previously,^{18 19 42} whereas the present study and others^{49 50} showed that chronic tachycardia increases cell length much more than width. If a common mechanism underlies activation of I_Cir,swell in both cases, it must account for these differences in how cell volume enlarges. Distortions of cell width and length may have common effects on the modulation of protein function by the cytoskeleton because cytoskeletal elements are said to be interconnected in a tension-stabilized (ie, tensegrity) network.⁵¹ Thus, the radial and longitudinal arrangement of cytoskeletal elements are coupled.

It is yet to be established whether I_Cir,swell is persistently activated in other forms of hypertrophy and CHF. Preliminary data in a rabbit aortic regurgitation CHF model, which combines features of both pressure and volume overload, suggest that I_Cir,swell is persistently activated in this case also (H.F. Clemo and C.M. Baumgarten, unpublished data, 1997). If increased cell width, as occurs with osmotic swelling, is a sufficient stimulus, then activation of I_Cir,swell is expected in pressure-overload hypertrophies, such as those associated with arterial coarctation. Pressure overload induces an unambiguous increase in myocyte width.^{52 53 54} Furthermore, the width of myocytes from patients with dilated cardiomyopathy is increased.^{55 56}

Mechanism of Activation of I_Cir,swell in CHF
The mechanism by which CHF or cell swelling activates I_Cir,swell remains undefined. Evidence in other systems indicates that various ion channels and transport proteins are linked to the actin cytoskeleton by ankyrin and/or spectrin and suggests that the cytoskeleton can regulate their function directly or via second messengers (for review, see References 57 and 58^{57 58} ). The influence of the cytoskeleton appears to be tissue and transport protein specific, however, so specific predictions for CHF are difficult to make. Nevertheless, the complex cell biology of CHF offers several candidate mechanisms for why I_Cir,swell was activated here. Cytoskeletal architecture is altered in tachycardia-induced cardiomyopathy, and levels of mRNA for -tubulin and ß- and -actin are increased.⁵⁹ In the dilated human heart, the arrangement of titin and -actinin is altered⁶⁰ ; tubulin, desmin, and vimentin are increased; and the cytoskeleton is disorganized.⁶¹ One or more of these might contribute to the activation of I_Cir,swell. Alternative candidates for regulating I_Cir,swell include the multiple second-messenger cascades activated by stretch, hypertrophy, and failure.^{4 62 63 64}

Pathophysiological Consequences of Persistent Activation of I_Cir,swell
Mechanoelectrical coupling influences cardiac electrical activity in a variety of experimental situations and has long been postulated on indirect grounds to contribute to dysrhythmias and mechanical dysfunction in humans.^{65 66} I_Cir,swell was inward at resting E_m, and its chronic activation can explain the 5- to 7-mV depolarization of resting E_m observed in tachycardia-induced dilated cardiomyopathy^{47 67} and would be expected to prolong terminal repolarization. This current is not the only one altered in CHF, however. Alterations in the shape of the I-V relationship and magnitude of I_K1 were noted previously,^{68 69} but these studies did not distinguish between I_K1 and SAC currents. On the other hand, I_Cir,swell cannot explain the CHF-induced prolongation of action potential duration measured at 50% repolarization.^{47 67 68 69} Recently, this has been attributed to inhibition of the transient outward current,^{68 70} but effects of hypertrophy on the delayed rectifier and the L-type Ca²⁺ current may also play a role in action potential prolongation.

The putative role of SACs in arrhythmias associated with CHF is of potential importance. Detailed intramural mapping studies show that ventricular tachyarrhythmias arise focally rather than by reentrant mechanisms in models of CHF.⁷¹ Furthermore, rapid pacing–induced CHF promotes sustained atrial tachycardias that have a focal mechanism presumably related to delayed afterdepolarizations.^{72 73} Gd³⁺-sensitive delayed afterdepolarizations and triggered activity are induced by acute stretch,^{30 31 32} and it is reasonable to think that activation of the Gd³⁺-sensitive inward current I_Cir,swell contributes to both these phenomena and to tachyarrhythmias in CHF. Moreover, I_Cir,swell favors delayed afterdepolarizations by overloading of the Ca²⁺ stores (see below).

I_Cir,swell poorly discriminated between K⁺ and Na⁺ (P_K/P_Na, 8), which implies that Na⁺ is the main charge carrier of the inward current with physiological ion gradients. Based on current density, capacitive membrane area, cell volume, and permeability ratio, the magnitude of Na⁺ influx in CHF cells at –80 mV is sufficient to increase intracellular Na⁺ concentration by 1.5 mmol/L per minute. This is a substantial Na⁺ load. Increased intracellular Na⁺ is expected in turn to increase intracellular Ca²⁺ and Ca²⁺ stores via the Na⁺-Ca²⁺ exchanger. In addition, poorly selective cation SACs are permeant to Ca²⁺ also.¹¹ Consequently, persistent activation of I_Cir,swell may affect contractile performance.

It remains to be established whether persistent I_Cir,swell contributes to the cellular remodeling observed with chronic tachycardia or is the result of remodeling. Na⁺ loading of cells long has been thought to contribute to cell growth. For example, stretch-dependent increased synthesis of actin and myosin heavy chain in adult ventricle is correlated with the rate of ²² Na⁺ uptake⁷⁴ ; streptomycin, a blocker of certain mechanosensitive channels,²⁸ reduced protein synthesis, whereas enhancing Na⁺ entry with monensin or veratridine augmented it. One then might postulate that Gd³⁺ would prevent stretch-induced changes in gene expression. This hypothesis was tested by Sadoshima et al,²⁷ who found that up to 50 µmol/L Gd³⁺ did not alter the expression of immediate-early genes including c-jun, Erg, c-fos, JE, or c-myc or protein synthesis in a neonatal rat myocyte culture system. These authors concluded that Gd³⁺-sensitive SACs are not part of the signal transduction pathway. However, a methodological problem confounds their interpretation. Gene expression and protein synthesis were studied in a modified DMEM/F-12 (1:1) medium, which is phosphate-bicarbonate–buffered. Published stability constants⁷⁵ indicate that effectively all of the Gd³⁺ would have been complexed by these anions and unavailable to block SACs. Thus, a role for Gd³⁺-sensitive SACs in the modulation of gene expression and protein synthesis remains a viable hypothesis.

Limitations of the Study
The canine tachycardia-induced cardiomyopathy model used in the present study has been well characterized and mirrors many of the pathophysiological changes of congestive heart failure in humans.³⁴ On the other hand, the changes reverse on cessation of rapid ventricular pacing, whereas in humans, most cardiomyopathies are irreversible. Also of note is that tachycardia-induced cardiomyopathy is a volume-overload model, leading to eccentric cellular and biventricular hypertrophy. Many clinical cardiomyopathies are due to pressure or the combined effects of pressure and volume overload.

Another concern is that the same bath and electrode solutions were used for both normal and CHF cells. This ignores complex changes in the extracellular and intracellular milieu that occur as a result of rapid pacing and the development of CHF in vivo. Such changes may modulate myocyte currents and cell volume. Even for control cells, myocyte volume may not be identical in vitro and in vivo. Furthermore, there is some variability in the reported effects of rapid pacing on several of the morphometric parameters measured here. We found a 16% increase in cell length, no significant change in cell width, and a 19% increase in membrane capacitance in the small population of cells studied. Spinale and coworkers^{49 50 76 77} reported that cell length increases in tachycardia-induced CHF but that cell diameter either does not change^{76 77} or increases⁴⁹ in the pig and decreases in the dog.⁵⁰ However, others found that canine myocyte width and length both increase.⁷⁸ Membrane capacitance also previously was found to increase in a pig chronic tachycardia model,⁴⁷ but Kääb et al⁶⁸ failed to see a change in membrane capacitance in a dog model.

In summary, the present results demonstrate that I_Cir,swell is persistently activated in tachycardia-induced CHF in the dog. The requirements for current activation and its characteristics suggest that I_Cir,swell is likely to be activated in multiple forms of cardiac hypertrophy and failure and that it may contribute to dysrhythmias and altered contractile function. To the extent that it does, I_Cir,swell may represent a novel target for therapeutic interventions.

	Selected Abbreviations and Acronyms

 

CHF = congestive heart failure Em = membrane potential Erev = reversal potential I-V = current-voltage ICir,swell = swelling-activated inwardly rectifying cation current ICl,swell = swelling-activated Cl- current IK1 = background K+ current K0.5 = concentration giving half-maximal response NMDG = N-methyl-D-glucamine PK/PNa = K+/Na+ permeability ratio SAC = swelling- or stretch-activated channel T = relative osmolarity (xisosmotic)

	Acknowledgments

 
This study was supported by National Heart, Lung, and Blood Institute grants HL-02798 (Dr Clemo) and HL-46764 (Dr Baumgarten) and a Merit Review Award from the Department of Veterans Affairs (Dr Stambler).

	Footnotes

 
Presented previously in abstract form (Circulation. 1995;92[suppl I]:I-504).

Received December 8, 1997; accepted April 29, 1998.

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