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(Circulation Research. 1996;78:650-659.)
© 1996 American Heart Association, Inc.

Articles

Enhancement of the L-Type Ca²⁺ Current by Mechanical Stimulation in Single Rabbit Cardiac Myocytes

Naoki Matsuda, Nobuhisa Hagiwara, Morio Shoda, Hiroshi Kasanuki, Saichi Hosoda

From the Heart Institute of Japan, Tokyo Women's Medical College.

Correspondence to Nobuhisa Hagiwara, MD, The Heart Institute of Japan, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan.

	Abstract

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Abstract Anion conductance is known to be activated by mechanical stimulation, such as osmotic cell swelling or cell inflation via the patch pipette, of canine or rabbit cardiac myocytes. The effects of mechanical stimulation on time-dependent currents, however, remain unsettled. Using the whole-cell voltage-clamp method, we have found that mechanical stimuli enhance the L-type Ca²⁺ current (I_Ca,L) in rabbit cardiac myocytes. At every membrane potential, I_Ca,L was reversibly increased by osmotic cell swelling and by cell inflation caused by applying a positive pressure of 10 to 15 cm H₂O via the patch pipette. I_Ca,L was increased during cell inflation by 37±21% (mean±SD, n=17) in atrial cells and by 37±8% (n=7) in sinoatrial node cells in solution containing 2 mmol/L Ca²⁺. The current-voltage relationship, the inactivation time constant, the steady state inactivation curve, and the conductance properties of I_Ca,L were all virtually unaffected by mechanical stimulation except for the open probability, which appears to increase. The increase in I_Ca,L was not dependent on protein kinase A, since an inhibitor peptide of cAMP-dependent protein kinase failed to prevent the increase in I_Ca,L during mechanical stimuli (n=5). The increase in I_Ca,L caused by cell inflation was unaffected by the chelation of intracellular Ca²⁺ by the addition of 10 mmol/L EGTA or 10 mmol/L BAPTA to the pipette solution, suggesting that the effect was not mediated by changes in intracellular Ca²⁺. Thus, mechanical stimulation due to cell swelling or inflation may itself directly increase I_Ca,L in rabbit cardiac myocytes.

Key Words: mechanical stimulation • atrial cells • L-type Ca²⁺ current • whole-cell voltage-clamp method • sinoatrial node cells

	Introduction

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Swelling of cardiac cells is recognized as an important pathogenic factor in pathophysiological conditions such as ischemic heart disease.^{1 2} However, possible changes in membrane currents during cell volume alterations or morphological changes have not been fully examined. Recently, anion conductance was found to be activated in canine and rabbit cardiac myocytes by cell swelling or by inflating the cells via the patch pipette.^{3 4 5} It was thought probable that membrane stretch itself activates anion conductance in cardiac myocytes. In addition, cation channels activated by the stretch induced by osmotic cell swelling or by applying negative pressure via the patch pipette have been described in rat atrial myocytes⁶ and tissue-cultured chick heart cells.⁷ These authors reported that the channels were nonselective cation channels that could carry either monovalent or divalent cations, such as Na⁺, K⁺, and Ca²⁺. These anion and cation channels most probably have negligible basal activity in the absence of membrane stretch. Both these swelling- and stretch-activated anion and cation currents show time-independent characteristics.

To date, the effects of mechanical stimulation on time-dependent currents have not been described in the literature, except for those affecting I_K in guinea pig ventricular cells.⁸ Since the action potential configuration of cardiac muscle cells consists mainly of time-dependent currents, modulation of such currents during cell swelling or membrane stretch might well contribute to changes in action potential configuration and to the genesis of arrhythmia in pathophysiological conditions. For this reason, we thought it important to test thoroughly for the effects of mechanical stimulation on time-dependent currents in cardiac myocytes. Indeed, in pulmonary arterial smooth muscle cells, the verapamil-sensitive pathway for Ca²⁺ influx appears to be activated by membrane stretch,⁹ and I_Ca,L in rat basilar artery is increased by cell inflation or hypotonic external solution.¹⁰ These results seemed to us to increase the likelihood that I_Ca,L might be stretch-sensitive in cardiac myocytes.

In the present study, using both osmotic cell swelling and cell inflation in single sinoatrial node and atrial cells of the rabbit with the whole-cell voltage-clamp method, we found that I_Ca,L was indeed enhanced by such mechanical stimuli. This effect was independent both of channel phosphorylation by cAMP-dependent protein kinase and of any dialyzing effects on intracellular components such as Ca²⁺, Mg²⁺, and ATP of the type previously reported in guinea pig ventricular myocytes.^{11 12 13 14} We have some evidence that the effect is achieved via an increase in the open probability of the L-type Ca²⁺ channel.

	Materials and Methods

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Preparation of Single Cells
The methods of cell isolation using enzymes were the same as described elsewhere.^{5 15} In brief, an albino rabbit was deeply anesthetized with an intravenous injection of sodium pentobarbital (40 mg/kg) under heparinization (300 U/kg). The heart was dissected out, and the sinoatrial node and right atrial region were isolated, cut into pieces, and incubated in warm Tyrode's solution for 10 minutes. The strips were transferred to a nominally Ca²⁺-free Tyrode's solution containing 0.8 mg/mL collagenase (Yakult) with 0.5 mg/mL elastase (type II-A, Sigma Chemical Co) for 60 minutes at 37°C. The collagenase was then washed out by rinsing with high-K⁺ low-Cl^- solution,¹⁶ and the digested tissue was stored in the same solution.

Electrical Recordings
The whole-cell voltage-clamp method used was the same as that described previously.^{5 15 17} The amplifier (TM-1000, ACT ME Laboratory) was used in conjunction with a 100 M feedback resistor, and series resistance was partially compensated. The I-V signals were stored on a video recorder (S-6000, Victor), with a PCM convertor system (RP-880, NF Electronic Instruments) being used for off-line computer analysis (PC 9801 RA, NEC). The current signals were fed from the video recorder to the computer via a 2.5-kHz eight-pole Bessel-type low-pass filter. The liquid junction potential (-7.5 mV) between the pipette and the bathing solutions was corrected. Experiments were performed at 36°C to 37°C. To examine the effect of mechanical stimulation on membrane current in the whole-cell configuration, we used either (1) inflation of the cell by applying positive pressure via the pipette or (2) osmotic cell swelling. Changes in cell size were displayed on a video monitor (C1846-03, Hamamatsu Photonics) and recorded with a VHS video recorder (SR-1750, Victor) through a CCD camera (C3077, Hamamatsu Photonics) mounted on the inverted microscope (Diaphoto, Nikon).⁵ The cross-sectional area of each cell was measured by counting the pixels contained within the cell boundary, which was traced around the cell's perimeter (Percept scope, C3160, Hamamatsu Photonics). The final cross-sectional area measured after its increase by osmotic cell swelling or by applying a positive pressure via the patch pipette was expressed as a percentage of the control. We then compared the changes in I_Ca,L with the relative changes in cross-sectional area.

Ensemble Noise Analysis
The ensemble noise analysis developed by Sigworth¹⁸ was used to examine the effect of mechanical stimulation on I_Ca,L. Ensembles of consecutive currents were elicited by a series of identical pulses to +10 mV from a holding potential of -40 mV. Currents were sampled at 0.2-ms intervals after being filtered at 2.5 kHz. Mean current and variance were computed for each time point. To avoid the effect of the capacitive transient, the start of the analysis was delayed until the inactivation phase of the currents. It has been shown that the mean current at time t, I(t), and the variance of the current, ²(t), are represented as follows:

(1)

(2)

where i is the unitary current, N is the number of channels, and p(t) is the probability of a channel being open at time t. Therefore, the relation between the variance and the mean current was fitted by the following parabolic curve:

(3)

The values for N and i were obtained by a least-squares curve-fitting procedure. The probability (p) was then calculated from Equation 1.

Solutions
The external solutions were made up as follows: Where stated, osmolarity was measured with an osmometer (Auto and Stat OM 6030, Kyoto Dai-ichi Kagaku), and for each measurement, values from three solutions were averaged. Normal Tyrode's solution contained (mmol/L) NaCl 136.9, KCl 5.4, CaCl₂ 1.8, NaH₂PO₄ 0.33, glucose 5, and HEPES 5 (pH adjusted to 7.4 with NaOH). The standard external solution contained (mmol/L) NaCl 150, CaCl₂ 2, HEPES 5, and SITS 1 (Tokyo Kasei) to block the stretch-activated Cl^- current⁵ (pH 7.4 with NaOH), and K⁺ was not included. In some experiments, the 2 mmol/L CaCl₂ was replaced equimolarly with BaCl₂. The isotonic external solution contained (mmol/L) NaCl 60, CaCl₂ 2, HEPES 5, mannitol 150, and SITS 1 (284±3 mOsm/kg). When the effect of hypotonic cell swelling on I_Ca,L was to be examined, mannitol was removed from the isotonic external solution, and the osmolarity was adjusted to 60% (172±8 mOsm/kg). The 50 mmol/L Ca²⁺ external solution contained (mmol/L) CaCl₂ 50, mannitol 150, HEPES 5, and SITS 1 (292±6 mOsm/kg). A hypotonic 50 mmol/L Ca²⁺ solution (195±6 mOsm/kg) was made by removing mannitol from the above solution.

The composition of the standard Cs⁺-rich internal solution was (mmol/L) CsOH 120, CsCl 20, aspartic acid 100, EGTA 10 (Sigma), MgCl₂ 2, MgATP 5, creatine phosphate dipotassium 5, and HEPES 5 (pH 7.4 with CsOH, 295±8 mOsm/kg). To produce a hypertonic (140%) pipette solution, 100 mmol/L mannitol was added to the standard internal solution (415±10 mOsm/kg). For recording I_K and I_h, Cs⁺ was replaced equimolarly with K⁺. In some experiments, the 10 mmol/L EGTA was replaced equimolarly with BAPTA (Sigma).

Drugs
Forskolin (Sigma) was dissolved in ethanol and prepared as a 10 mmol/L stock solution. cAMP (Sigma) and cAMP-dependent PKI (PKI_5-24, American Peptide Co) were directly dissolved in the pipette solution. H-89 (Seikagaku Kogyo) was dissolved in dimethyl sulfoxide as a 10 mmol/L stock solution. H-8 (Seikagaku Kogyo) was dissolved in distilled water as a 10 mmol/L stock solution. Nifedipine (Sigma) was dissolved in ethanol as a 10 mmol/L stock solution. Nisoldipine was kindly provided by Bayer Yakuhin Ltd (Osaka, Japan) and was dissolved in ethanol as a 10 mmol/L stock solution. A paired Student's t test was used to evaluate the statistical significance of differences between means. Values of P<.05 were considered to indicate statistical significance. All statistical data are given as mean±SD.

	Results

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Current Changes During Hypotonic Stress in the Presence of a Ca²⁺ Channel Blocker
To test for the existence of stretch-activated Ca²⁺-permeable currents of the type already described in various cell species, including cardiac myocytes,^{6 7 19 20} we first examined the effect of hypotonic external solution on the membrane current using conditions similar to those described previously in human epithelial cells.²⁰ Thus, we measured the current changes during superfusion of sinoatrial node cells with hypotonic external solution containing 50 mmol/L Ca²⁺ with 2 µmol/L nifedipine (Fig 1A). Nifedipine is known to block the L-type Ca²⁺ channel, but it does not affect stretch-activated cation-selective channels.²¹ Under our conditions, the I-V relationship was almost linear in the control isotonic external solution, and the membrane conductance was 0.63 nS (0.72±0.08 nS, n=7) at +40 mV (Fig 1B, 1 [tracing a]). Superfusing the cell with the hypotonic external solution increased the membrane conductance to 5.31 nS (6.38±1.12 nS, n=7) within 5 minutes, concomitant with osmotic cell swelling (Fig 1B, 1 [tracing b]). The curve produced by subtraction of the control values (tracing a) from those recorded in hypotonic external solution (tracing b) intersected the voltage axis at -37 mV (-36.9±4.2 mV, n=7; Fig 1B, 2 [tracing b-a]). The distribution of Cl^-, which was 100 mmol/L in the external solution and 24 mmol/L in the pipette solution, gave a Cl^- reversal potential of -37.2 mV, a value close to the reversal potential obtained in 50 mmol/L Ca²⁺ hypotonic external solution. The stilbene-derived Cl^- channel blocker SITS (1 mmol/L) blocked this current very effectively, as shown in Fig 1A (tracing c). In five of five cells, SITS significantly inhibited the swelling-activated current, as also described in our previous report.⁵ These results indicate that the hypotonic external solution containing 50 mmol/L Ca²⁺ with nifedipine activated only a swelling-induced Cl^- current,^{3 4 5} and under our study conditions, we failed to detect the stretch-activated Ca²⁺-permeable currents previously described in cardiac myocytes.^{6 7}

View larger version (20K): [in this window] [in a new window]   Figure 1. Current changes during hypotonic stress in the presence of nifedipine. Sinoatrial node cells were superfused with an external solution containing 50 mmol/L Ca2+ with 2 µmol/L nifedipine. A, Slow-speed chart record of current changes in response to ramp clamp pulses. Ramp pulses of ±70-mV (0.93-V/s) amplitude were applied every 10 seconds from a holding potential of -30 mV. Six minutes after superfusion with hypotonic solution, 1 mmol/L SITS was applied in the external solution, as indicated above the current tracing. B, I-V relationships in isotonic external solution (1, tracing a), in hypotonic solution (1, tracing b), and 3.5 minutes after application of 1 mmol/L SITS (1, tracing c). In this cell, the membrane conductance was increased from 0.63 (tracing a) to 5.31 nS (tracing b) at +40 mV. The reversal potential for the current difference between tracings a and b (2, tracing b-a) was -37 mV.

Osmotic Cell Swelling Enhances I_Ca,L
To examine the effect of hypotonic cell swelling on I_Ca, we superfused 50 mmol/L Ca²⁺ solution without nifedipine. The external solution contained 1 mmol/L SITS to block the stretch-activated Cl^- current⁵ ; this concentration did not affect the control amplitude of I_Ca (result not illustrated). Under these conditions, I_Ca was elicited every 10 seconds by constant depolarizing step pulses to +20 mV from a holding potential of -40 mV (Fig 2A). After the peak inward current had reached a steady state value, the isotonic external solution was replaced by a hypotonic external solution. Cell swelling occurred within 2 minutes in the cell illustrated in Fig 2, and the peak inward current increased gradually from 1450 to 1950 pA (ie, by 34.5%) at +20 mV (Fig 2B [tracing b]). Such an increase in I_Ca was observed in all cells examined, with the mean increase being 31.6±8.5% at +20 mV (n=5). Similar results were achieved with hypotonic external solution containing 2 mmol/L Ca²⁺; in that case, I_Ca was increased by 32.8±12.6% at 0 mV from a holding potential of -40 mV (n=5; results not illustrated).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of hypotonic external solution on ICa in a sinoatrial node cell. Currents were elicited by constant depolarizing step pulses from a holding potential of -40 to +20 mV in external solution containing 50 mmol/L Ca2+ without nifedipine. The stretch-activated Cl- current was blocked by 1 mmol/L SITS. A, A chart record of current changes. After the change to hypotonic solution from isotonic solution, the peak inward current gradually increased. B, Current tracings evoked by step pulses to +20 mV in control (tracing a) and in hypotonic solution (tracing b). On changing the bathing solution from hypotonic back to isotonic, the peak inward current returned to the control value (tracing c).

To determine whether the inward current enhanced by osmotic cell swelling was a dihydropyridine-sensitive Ca²⁺ current (I_Ca,L), we added 2 µmol/L nifedipine or 2 µmol/L nisoldipine to the external solution. The current changes, of the type shown in Fig 2, were completely abolished by application of either nifedipine (n=3) or nisoldipine (n=3), indicating that the current enhanced by hypotonic cell swelling was indeed I_Ca,L, as previously described in cardiac myocytes.^{22 23 24}

We then checked the osmotic effect on I_Ca,L using a hypertonic pipette solution instead of the hypotonic external solution (Fig 3). After disruption of the cell membrane, a negative pressure was applied via the patch pipette to prevent cell swelling. When the I_Ca,L elicited by depolarizing pulses to 0 mV from a holding potential of -40 mV had reached a steady state value, the control I-V relationship was determined (Fig 3C). After the end of the period of negative pressure, the inward current gradually increased and reached a new steady state value within 3 minutes (concomitant with cell swelling by 15% in terms of cross-sectional area), which was similar to that obtained using hypotonic external solution. In five experiments using the hypertonic pipette solution, I_Ca,L was increased by 30.5±6.2% in 2 mmol/L Ca²⁺ at 0 mV.

View larger version (20K): [in this window] [in a new window]   Figure 3. Increase in ICa,L on osmotic cell swelling caused by the use of hypertonic pipette solution in an atrial cell. A, Negative pressure of -20 cm H2O was applied, as indicated above the chart record, via the patch pipette to record the control I-V relationship in standard external solution containing 2 mmol/L Ca2+. B, The peak inward currents at -10 mV (B, 1) and 0 mV (B, 2) were increased from 250 and 375 pA to 375 and 525 pA, respectively, in this particular cell. C, I-V relationships for the peak inward currents before () and during () osmotic cell swelling. The inward current was increased by osmotic cell swelling at every level of membrane potential.

Since use of either a hypotonic external solution or a hypertonic pipette solution resulted in an increase in I_Ca,L concomitant with cell swelling, it appeared probable that membrane stretch, per se, enhances I_Ca,L. To attempt to confirm this, we then used another method for stretching the cell membrane, namely, cell inflation; the following results were obtained by applying positive pressure via the patch pipette, a method previously used by Hagiwara et al.⁵

Increase in I_Ca,L on Inflation of the Cell
Fig 4 illustrates the effect of inflating the cell on I_Ca,L. After the I-V relationship for the control I_Ca,L had been determined using various depolarizing pulses from a holding potential of -40 mV, a positive pressure of 10 cm H₂O was applied via the patch pipette. The resulting inflation of the cell was accompanied by a 20% increase in the membrane's relative cross-sectional area and by an increase in the amplitude of the peak inward current (Fig 4A [tracing b]). The time course of the current response to cell inflation could be followed reliably on the chart recorder (Fig 4A): the peak inward current began to increase immediately upon inflation of the cell and reached its maximum value within 1 minute. The inward current then remained constant while the pipette pressure was maintained at a steady level by closing a stopcock for 3 minutes. Subsequent deflation of the cell by application of a negative pressure via the patch pipette (-30 cm H₂O) returned the amplitude of the inward current to its control level. Fig 4B illustrates current tracings recorded at 0 mV before inflation and while inflation was maintained; the I-V relationships in these two conditions are illustrated in Fig 4C. On average, the membrane current at 0 mV increased from 473.5±248.4 to 650.6±342.6 pA (ie, by 37.1±21.2%; n=17) on inflation of atrial cells and from 261.4±49.1 to 357.9±61.9 pA (ie, by 37.4±8.3%; n=7) in sinoatrial node cells. The incremental increase in membrane cross-sectional area was 18.5±5.5% in the two types of cell (n=24). The increase in inward current on inflation of the cell was also abolished by the application of 2 µmol/L nifedipine (n=5) or 2 µmol/L nisoldipine (n=3) in both types of cell, indicating that it was indeed I_Ca,L that was enhanced by the cell inflation.¹⁰

View larger version (19K): [in this window] [in a new window]   Figure 4. Current changes during inflation and deflation of a rabbit atrial cell in the presence of 2 mmol/L Ca2+. A, Chart record of current changes in response to step pulses. The duration of inflation and of deflation is indicated above the chart record. B, Current tracings elicited by depolarizing step pulses to 0 mV from a holding potential of -40 mV, before (tracing a) and during (tracing b) inflation of the cell. The amplitude of the peak inward current increased from 390 pA (tracing a) to 640 pA (tracing b) on inflation of this particular cell (a 64% increase). C, I-V relationships for the peak inward currents before () and during () the inflation of the cell shown in panel A.

A similar response to cell inflation was observed when Ba²⁺ was used as the charge carrier for I_Ca,L. The same protocol was repeated in experiments on atrial cells using external solution containing 2 mmol/L Ba²⁺. The amplitude of the control inward current at 0 mV was greater when Ba²⁺ was in the external solution (for 2 mmol/L Ca²⁺, 473.5±248.4 pA, n=17; for 2 mmol/L Ba²⁺, 1305.2±950.5 pA, n=10), and the inactivation time constant was much slower than that obtained with 2 mmol/L Ca²⁺ (time courses are illustrated in Fig 7). In the presence of 2 mmol/L Ba²⁺, cell inflation increased the inward current by 34.1% at 0 mV (29.7±8.5%, n=10), which is similar to the increase obtained in the presence of 2 mmol/L Ca²⁺.

View larger version (19K): [in this window] [in a new window]   Figure 7. Kinetic properties of ICa,L before and during cell inflation. A, 1, Superimposed current tracings taken before and during inflation at a depolarizing test potential from -40 to 0 mV in the presence of 2 mmol/L Ca2+. A, 2, Inactivation time course of ICa,L before and during cell inflation. The plots were obtained from the data shown in panel A, 1. B, Current tracings (1) and plots of inactivation time course (2) before and during cell inflation in the presence of 2 mmol/L Ba2+. C and D, The steady state inactivation of ICa,L before and during inflation of atrial cells. Panel C shows superimposed current tracings (1, control tracings; 2, tracings during cell inflation) elicited by test pulses to 0 mV from various holding potentials between -70 and -10 mV. Panel D shows steady state inactivation curves for ICa,L before (solid line) and during (dotted line) inflation. The data were obtained from six different experiments. indicates control data; , data taken during cell inflation.

We also checked the effect of cell inflation on other time-dependent currents, namely, I_K and I_h, in sinoatrial node cells. Fig 5 illustrates the effect of cell inflation on I_Ca,L, I_K, and I_h in a sinoatrial node cell. We used a two-pulse protocol so that we could investigate simultaneously the changes evoked by cell inflation in I_Ca,L and I_K with a depolarizing step pulse of +10 mV (first step) and in I_h with a hyperpolarization pulse of -100 mV from a holding potential of -40 mV (second step). The inflation of the cell increased I_Ca,L from 545 pA (Fig 5B, 2 [tracing a]) to 765 pA (Fig 5B, 2 [tracing b]), an increase of 40.4% in this cell (overall by 35.6±3.8%, n=6). By contrast, I_K and I_h were hardly affected by the present stimuli in any cell (n=6, Fig 5B, 1). These results suggest that I_Ca,L was selectively enhanced by mechanical stimulation in rabbit cardiac myocytes.

View larger version (21K): [in this window] [in a new window]   Figure 5. Effect of cell inflation on other time-dependent currents. A, Chart record of current changes in response to double-step pulses in normal Tyrode's solution containing 1 mmol/L SITS. The pipette contained K+-rich internal solution. B, 1, Current tracings elicited by the two-pulse protocol. From a holding potential of -40 mV, a depolarizing pulse was applied to +10 mV for 300 ms (first step), followed by a return to the holding potential for 500 ms and then a hyperpolarizing pulse to -100 mV for 300 ms (second step); this was done before (tracing a) and after (tracing b) inflation of the sinoatrial node cell. Pulse protocol is illustrated below the current tracings. Both IK and Ih were virtually unaffected by cell inflation. B, 2, Fast-sweep current record evoked by the first, depolarizing, pulse shown in panel B, 1. ICa,L was increased by 40.4% in this particular cell.

Reproducibility of the Increase in I_Ca,L on Cell Inflation
Next, we checked the reproducibility of the change in I_Ca,L evoked by cell inflation. In the experiment shown in Fig 6, we performed cell inflation three times over a 15-minute experimental period. Although, as expected, I_Ca declined with time under our whole-cell conditions, as shown in tracings c and e of Fig 6A (the "rundown" phenomenon^{13 25 26} ), cell inflation was still able to increase the amplitude of I_Ca,L during this rundown (Fig 6B [tracings b, d, and f]). Because both osmotic cell swelling and cell inflation increased I_Ca,L reproducibility, the evoked increase in I_Ca,L seems unlikely to be caused by a dialysis of the intracellular medium, involving compounds such as ATP or another nucleotide, of the kind previously described in guinea pig ventricular myocytes.^{14 27}

View larger version (12K): [in this window] [in a new window]   Figure 6. Reproducibility of changes in ICa,L during cell inflation in a sinoatrial node cell. A, Slow-speed chart record of current changes in response to step pulses from -40 to 0 mV in standard external solution. Cell inflation (*) was performed three times during a 15-minute experimental period. B, Superimposed current tracings taken before and during inflation of the cell. Tracings a and b relate to the first inflation; c and d, to the second inflation; and e and f, to the third inflation.

Kinetic Properties of I_Ca,L During Mechanical Stimulation
To check whether mechanical stimulation affects the kinetic properties of I_Ca,L, we analyzed the inactivation time course and the steady state inactivation curve for I_Ca,L. Fig 7A shows an example of the inactivation time course at 0 mV in the presence of 2 mmol/L Ca²⁺. It was composed of two components: a slow component, with a time constant in that example of 37.2 ms (_s, 43.7±7.7 ms overall [n=15]), and a fast component, with a time constant of 7.1 ms (_f, 9.9±2.2 ms [n=15]), under control conditions. These values were almost the same as those determined while the cell was inflated (_s, 37.1 ms, 43.1±5.0 ms [n=15]; _f, 7.0 ms, 10.5±1.9 ms [n=15]). The inactivation time constant was also unchanged by cell inflation in the presence of 2 mmol/L Ba²⁺. Fig 7B, 2, illustrates the inactivation time course before and during cell inflation in 2 mmol/L Ba²⁺. The time constants in that example were 80 and 82 ms, respectively (overall, 85.1±5.0 versus 92.4±7.8 ms [n=5]). Consequently, we concluded that the inactivation time course of I_Ca,L was unaffected by mechanical stimulation.

To determine the steady state inactivation curve of I_Ca,L, the membrane potential was held at various levels between -70 and -10 mV, and depolarizing test pulses to 0 mV were applied (Fig 7C). The peak amplitudes of I_Ca,L were normalized, plotted against the membrane potential (Fig 7D), and fitted to a Boltzmann equation as follows:

(4)

where y is the inactivation parameter, V_m is the membrane potential, V_0.5 is the potential required to give a half value, and s is the slope factor. The slope factor in the control condition was 4.5 mV, and V_0.5 was -29.4 mV; during cell inflation, the corresponding values were 4.4 and -31.7 mV, respectively. These results showed that the steady state inactivation curve was also essentially unchanged by cell inflation. Consequently, we concluded that the kinetic properties of I_Ca,L were unaffected by mechanical stimulation.

Conductance Properties of I_Ca,L During Mechanical Stimulation
The increase in I_Ca,L induced by mechanical stimulation could depend on any or all of three factors: the number of functional channels, the probability of channels being open, and the unit amplitude of the current. To examine the effect of mechanical stimuli on these factors, we applied ensemble noise analysis.¹⁸ Mean current and variance records under control conditions and during inflation in 50 mmol/L Ca²⁺ are illustrated in Fig 8A and 8B. To estimate the number of functional channels, open probability, and unit amplitude for each record, plots of variance against mean current were fitted by parabolic curves given by Equation 3, with values for unit amplitude and functional channel number of 0.63 pA and 5692, respectively, in the control condition and 0.65 pA and 5950, respectively, during the inflation of the cell (Fig 8C). The calculated open probability was, therefore, .24 in the control condition and .34 during inflation. In five different experiments in 50 mmol/L Ca²⁺, neither unit amplitude, which is indicated by the initial slope of the relationship, nor functional channel number showed a significant difference between the two conditions. Unit amplitude was 0.61±0.02 pA and the number of functional channels was 5528±1718 in the control condition. The corresponding values were 0.62±0.03 pA and 5676±1449 after inflation (not significant in each case). In contrast, open probability increased from .27±.05 to .36±.03 (P<.005) with cell inflation. Although these values were obtained at limited amplitude from the current records, they indicate that whereas unit amplitude and functional channel number remained unchanged, open probability was increased by some 33% with the present mechanical stimulation.

View larger version (14K): [in this window] [in a new window]   Figure 8. A and B, Ensemble noise analysis of ICa,L before and during cell inflation. Ensembles of currents were elicited by a series of identical pulses to +10 mV from a holding potential of -40 mV (18 current recordings in the control and 15 recordings after inflation). Mean current (A) and variance (B) are shown as functions of time. C, Variance (2) plotted against mean current (I).

Regulatory Mechanism Influencing I_Ca,L on Mechanical Stimulation
Several intracellular processes are known to regulate I_Ca,L: in particular, cAMP-dependent phosphorylation is known to modulate I_Ca,L in cardiac myocytes.^{28 29} To determine whether our mechanical stimulation was activating I_Ca,L via cAMP-dependent phosphorylation, we examined the effects of PKIs on the present enhancement of I_Ca,L during cell inflation.

PKI_5-24, the specific peptide inhibitor of PKA is the most potent inhibitor known of this kinase, and it suppresses PKA-regulated Cl^- channel activity in guinea pig ventricular myocytes.³⁰ Therefore, we examined the effect of cell inflation on I_Ca,L with 50 µmol/L PKI in the pipette solution (Fig 9A and 9B). In control experiments, the enhancement of I_Ca,L by an application of 0.1 µmol/L isoproterenol was inhibited with 50 µmol/L PKI in the pipette solution. However, inflation of the cell still increased I_Ca,L in the presence of PKI (by 33.1±11.3%, n=5). We also found that cell inflation still increased I_Ca,L in the presence of 20 µmol/L H-8 (n=5) or 5 µmol/L H-89 (n=3), both nonspecific blockers of protein kinases³¹ (results not illustrated). When 10 µmol/L forskolin was added to the bathing solution, this alone increased I_Ca,L from 310 pA (Fig 9C [tracing a]) to 625 pA (Fig 9C [tracing b]); this was an increase of 102% in that cell. However, inflation of that cell further increased I_Ca,L by 31% (Fig 9C [tracing c]), which was similar to the control increase in I_Ca,L in the absence of forskolin (overall: control, 37.1±21.2% [n=17]; forskolin, 32.6±5.0% [n=5]) in atrial cells. Similar findings were made using a pipette solution containing 50 µmol/L cAMP: although cytoplasmic application of 50 µmol/L cAMP is known to induce maximal phosphorylation of the Ca²⁺ channel through PKA,¹¹ cell inflation still increased I_Ca,L by 31.7±12.5% (n=5; results not illustrated). Together, these results suggest that the effect of mechanical stimulation on I_Ca,L is independent of cAMP-dependent phosphorylation.

View larger version (13K): [in this window] [in a new window]   Figure 9. The effects of PKI and forskolin during cell inflation. A, Chart record of current changes in response to test pulses. The pipette solution contained 50 µmol/L PKI. After 5-minute disruption of the patch membrane, the cell was inflated via the patch pipette in the presence of 50 µmol/L PKI. B, Superimposed current tracings at 0 mV from a holding potential of -40 mV before (tracing a) and during (tracing b) inflation. C, Superimposed current tracings at 0 mV from a holding potential of -40 mV, in the control standard external solution (tracing a), after administration of 10 µmol/L forskolin (tracing b), and during cell inflation (tracing c). Superfusing the cell with solution containing 10 µmol/L forskolin caused an increase in the amplitude of ICa,L of 102% (tracing b), and 5 minutes after application of forskolin, the positive pressure applied through the patch pipette (cell inflation) further increased ICa,L by 31% (tracing c).

It is well known that I_Ca,L is modulated by the cytoplasmic free Ca²⁺ concentration: an increase in free Ca²⁺ can both decrease I_Ca,L by increasing inactivation and increase it by another mechanism.^{12 13 32} Therefore, it was important to check whether the effect of our mechanical stimulation on I_Ca,L might be exerted through processes dependent on intracellular Ca²⁺. When we used a pipette solution containing 10 mmol/L BAPTA, which is an even more potent Ca²⁺-buffering agent than EGTA, the increase in I_Ca,L evoked by cell inflation was unchanged. Fig 10 shows the effect of cell inflation on I_Ca,L with 10 mmol/L BAPTA in the pipette solution. In that cell, the amplitude of I_Ca,L under control conditions at 0 mV was 900 pA (Fig 10A, 2 [tracing c]), and it was increased to 1260 pA by cell inflation (tracing d, increased by 40%). Overall, the amplitude of I_Ca,L was increased by 37.1±8.6% in eight atrial cells with 10 mmol/L BAPTA in the pipette solution. Furthermore, the increase in I_Ca,L was unaffected by the presence of 1 to 10 µmol/L ryanodine in the external solution (results not illustrated). All these findings support the view that the increase in I_Ca,L evoked by mechanical stimulation was not mediated via processes dependent on intracellular Ca²⁺.

View larger version (11K): [in this window] [in a new window]   Figure 10. Increase in ICa,L evoked by cell inflation with 10 mmol/L BAPTA in the pipette solution. A, Superimposed current tracings elicited by depolarizing pulses from -40 to -10 mV (1) and 0 mV (2). The amplitude of ICa,L was increased by 42% (tracing b) and 40% (tracing d). ICa,L was increased to a similar extent in the presence of 10 mmol/L EGTA. B, I-V relationships for ICa,L before () and during () the inflation of the cell shown in panel A.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increase in the Amplitude of I_Ca,L on Mechanical Stimulation of the Cell Membrane
We have shown that I_Ca is increased by osmotic cell swelling and by cell inflation in single cells from the rabbit atrium and sinoatrial node. The increased membrane current is carried by both Ca²⁺ and Ba²⁺ and is abolished by the application of nifedipine or nisoldipine. These results indicate that the change in membrane conductance does indeed represent I_Ca,L.²³ A dihydropyridine-insensitive stretch-activated I_Ca was not found, and the stretch-activated Cl^- current was blocked by the application of 1 mmol/L SITS in the same experiments (Fig 1).⁵

Stretch-activated channels that open in response to a change in mechanical stimulation have been widely observed in various types of cells: in plants, in yeast, and in animal cells, including cardiac myocytes.¹⁹ When recording stretch-activated channels under voltage-clamp conditions, several methods have been used to induce membrane stretch. Membrane patches have been stressed by negative or positive pressure exerted via the patch pipette during single-channel recording.¹⁹ In the whole-cell configuration, inflation of the cell by applying positive pressure and osmotic cell swelling have both been used for studying stretch-activated currents.^{5 10 19 20 33 34} In the present study, we found that enhancement of I_Ca,L was induced by osmotic cell swelling achieved by using either hypotonic external or hypertonic pipette solutions (Figs 2 and 3). The increase in I_Ca,L was also evoked using a cell inflation method, as shown in Figs 4 through 10. The effectiveness of three different mechanical stimuli in the present study lends strong support to the idea that the effects are mediated by mechanical stretch of the cell membrane, per se. The quantitative relationship between the degree of stretch and the increase in I_Ca,L was not systematically examined in the present study; however, an increase of 5% in the relative cross-sectional area was sufficient to enhance I_Ca,L in our rabbit cardiac myocytes, as it was sufficient to enhance the anion conductance in canine and rabbit cardiac myocytes.^{4 5}

Recently, a novel cation-selective mechanosensitive ion channel that is permeable to Na⁺, K⁺, or Ca²⁺ was found in atrial myocytes of the newborn rat and in tissue-cultured chick heart cells.^{6 7} This mechanism does not appear to exist in our cells, since hypotonic stress in the presence of 50 mmol/L Ca²⁺ with 2 µmol/L nifedipine did not induce a stretch-activated Ca²⁺-mediated current. Despite this, under the same conditions, we could observe the described changes in Cl^- current and I_Ca,L (Figs 1 and 2). It is noteworthy in this regard that mechanosensitive channels are known to appear, or increase in density, during the culturing of cells.¹⁹ Thus, the difference between our results and those of others might be due to the different specimens used, ie, freshly isolated rather than tissue-cultured cardiac myocytes.⁷

Site of Action of Mechanical Stimuli on I_Ca,L
Several intracellular processes are known to regulate ion channels in cardiac myocytes. ß-Adrenergic stimulation of heart cells results in an increase in both I_Ca,L and the delayed rectifier K⁺ current and in an activation of the Cl^- channel.^{11 35 36} ß-Adrenergic agonists activate adenylate cyclase to produce cAMP. This in turn dissociates the inactive PKA to form an active catalytic subunit, which finally phosphorylates the Ca²⁺ channel.³⁷ The increase in I_Ca,L during cell inflation occurred with 10 µmol/L forskolin or 50 µmol/L cAMP in the pipette solution. Since this is the dose of cAMP needed to induce a maximal production of the active catalytic subunit,³⁶ the present increase in I_Ca,L evoked by cell inflation is unlikely to be dependent on PKA-regulated phosphorylation. Furthermore, I_Ca,L was increased to a similar degree whether in the presence or absence of the specific peptide inhibitor of PKA. All these results support the view that the modulation of I_Ca,L seen during mechanical stimulation in the present experiments was independent of PKA.

Recently, a phosphorylation-independent modulation of I_Ca,L by Mg²⁺- nucleotide complexes was described by O'Rourke et al.¹⁴ However, the pipette solution in the present experiments contained 5 mmol/L MgATP, which is a much higher concentration than that needed to affect I_Ca,L in their guinea pig ventricular myocytes (<58 µmol/L).¹⁴ Thus, the reproducible nature of our current changes (Fig 6), also observed with osmotic cell swelling (Figs 2 and 3), suggests that the increase in I_Ca,L in our experiments was due to a direct effect on the Ca²⁺ channel and not to any dialyzing effect leading to a raised intracellular medium involving compounds such as ATP or Mg²⁺.^{14 27 38}

The intracellular free Ca²⁺ concentration has been known for some time to regulate I_Ca,L in various excitable cells.^{12 13 32 39 40} Recently, Romanin et al⁴¹ reported that single L-type Ca²⁺ channel activity was markedly decreased, concomitant with an increase in cytoplasmic Ca²⁺, in guinea pig ventricular myocytes. The higher cytoplasmic Ca²⁺ reduced the availability of functionally active channels and consequently resulted in an overall suppression of I_Ca,L. On the other hand, it has been reported that flash photolysis of intracellular Ca²⁺ increased I_Ca,L through a Ca²⁺-dependent phosphorylation process in frog atrial and guinea pig ventricular myocytes.^{12 32} Therefore, if the intracellular Ca²⁺ did change during our mechanical stimulation, such changes might be expected to have modulated the amplitude of I_Ca,L. However, we can exclude the possibility that the present increase in I_Ca,L was secondary to changes in cytoplasmic Ca²⁺. First, the concentration of Ca²⁺ in the pipette solution was buffered with 10 mmol/L EGTA, which would keep the intracellular Ca²⁺ at a subnanomolar level. Second, the increase in I_Ca,L was unchanged when we used a pipette solution containing 10 mmol/L BAPTA, which binds intracellular Ca²⁺ even more tightly than does EGTA (Fig 10). Finally, it is noteworthy that the inactivation time constant of I_Ca,L was hardly affected by mechanical stimulation (Fig 7). This result is inconsistent with the idea of a significant role for changes in cytoplasmic Ca²⁺, since cytoplasmic Ca²⁺ appears to modulate Ca²⁺ channel inactivation in guinea pig ventricular myocytes.⁴¹ Together, our findings effectively exclude the possibility that changes in intracellular Ca²⁺ mediated the enhancement of I_Ca,L in the present experiments.

The mechanisms underlying the enhancement of I_Ca,L by mechanical stimuli in cardiac myocytes cannot be conclusively deduced from the present experiments; however, some insight into the mechanism can be gained from the ensemble noise analysis. The results of this analysis suggest that cell inflation increases the open probability of the functional L-type Ca²⁺ channel without effecting significant changes in the unit amplitude or the number of functional channels. Since the PKA-regulated phosphorylation process and changes in the intracellular milieu were apparently not causally related to the regulation of I_Ca,L during cell inflation, it seems likely that stretching of the membrane may itself directly affect the open probability of the L-type Ca²⁺ channel. However, single-channel recording during membrane stretch will be essential to prove or disprove this suggestion.

Functional Significance of the Stretch Sensitivity of I_Ca,L
The type of sarcolemmal stretch that would have occurred in the present experiments resulted from osmotic swelling or inflation of the cells. Therefore, this membrane stretch may be different in some respects from the types of stretch that occur in the working heart. However, it is known that the verapamil-sensitive pathway for Ca²⁺ influx is enhanced by physiological stretch in pulmonary arterial smooth muscle cells⁹ and that I_Ca,L in rat basilar artery is increased by both osmotic cell swelling and cell inflation.¹⁰ This supports the possibility that the enhancement of I_Ca,L seen in the present study may contribute to the effects of physiological membrane stretch in the intact heart. In addition, cell swelling occurs in pathophysiological conditions, such as ischemic heart disease.^{1 2} It has been reported that tissue osmolarity in the ischemic myocardium increased by 40 mOsm/kg in the isolated porcine heart, which increased tissue water volume by an average of 17%.¹ This being so, an increase in I_Ca,L during cell swelling of the type described here might well contribute to the regulation of Ca²⁺ influx under such conditions.

In a series of studies of pacemaker activity, it has been shown that stretch of the sinus node tissue produces a positive chronotropic effect.^{42 43} Direct stretch of the sinoatrial node region increases the spontaneous heart rate, whereas release of such stretching results in a slowing of pacemaker activity. However, the ionic mechanisms underlying such a stretch-induced positive chronotropic effect remain unclear. Recently, we have identified a stretch-activated Cl^- current in sinus node and atrial cells by inflating the cell via the patch electrode.⁵ An activation of this time-independent Cl^- current was proposed as one of the ionic mechanisms underlying the positive chronotropic effect during membrane stretch. In mammalian sinoatrial node cells, however, deactivation of I_K and activation of I_Ca, as well as of I_h, are all considered to be responsible for the generation of pacemaker activity.⁴⁴ Therefore, changes in I_Ca,L in response to mechanical stimulation might, while not being entirely responsible, at least contribute to the change in pacemaker activity following mechanical stretch of sinoatrial node cells.

	Selected Abbreviations and Acronyms

 

f, s = fast and slow time constants H-8 = N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride H-89 = N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide I-V = current-voltage ICa = Ca2+ current ICa,L = L-type Ca2+ current Ih = hyperpolarization-activated current IK = delayed outward K+ current PKA = cAMP-dependent protein kinase PKI = protein kinase inhibitor

	Acknowledgments

 
This study was supported by a research grant from the Ministry of Education, Science, and Culture of Japan.

	Footnotes

 
Previously presented in part in Abstracts of the XXXII Congress of the International Union of Physiological Sciences, Glasgow, Scotland, August 1, 1993.

Received March 27, 1995; accepted December 14, 1995.

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