QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.121111v1 321/3/921    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Olson, M. L. Articles by Kargacin, G. J. Search for Related Content PubMed PubMed Citation Articles by Olson, M. L. Articles by Kargacin, G. J.

CARDIOVASCULAR

Effects of Phloretin and Phloridzin on Ca²⁺ Handling, the Action Potential, and Ion Currents in Rat Ventricular Myocytes

Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada (M.L.O., M.E.K., G.J.K.); and Department of Physiology, Queen's University, Kingston, Ontario, Canada (C.A.W.)

Received February 8, 2007; accepted March 20, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The effects of the phytoestrogens phloretin and phloridzin on Ca²⁺ handling, cell shortening, the action potential, and Ca²⁺ and K⁺ currents in freshly isolated cardiac myocytes from rat ventricle were examined. Phloretin increased the amplitude and area and decreased the rate of decline of electrically evoked Ca²⁺ transients in the myocytes. These effects were accompanied by an increase in the Ca²⁺ load of the sarcoplasmic reticulum, as determined by the area of caffeine-evoked Ca²⁺ transients. An increase in the extent of shortening of the myocytes in response to electrically evoked action potentials was also observed in the presence of phloretin. To further examine possible mechanisms contributing to the observed changes in Ca²⁺ handling and contractility, the effects of phloretin on the cardiac action potential and plasma membrane Ca²⁺ and K⁺ currents were examined. Phloretin markedly increased the action potential duration in the myocytes, and it inhibited the Ca²⁺-independent transient outward K⁺ current (I_to). The inwardly rectifying K⁺ current, the sustained outward delayed rectifier K⁺ current, and L-type Ca²⁺ currents were not significantly different in the presence and absence of phloretin, nor was there any evidence that the Na⁺/Ca²⁺ exchanger was affected. The effects of phloretin on Ca²⁺ handling in the myocytes are consistent with its effects on I_to. Phloridzin did not significantly alter the amplitude or area of electrically evoked Ca²⁺ transients in the myocytes, nor did it have detectable effects on the sarcoplasmic reticulum Ca²⁺ load, cell shortening, or the action potential.

In a previous study (Olson et al., 2006), we found that phloretin and phloridzin reduce the maximal velocity of Ca²⁺ uptake into the cardiac muscle sarcoplasmic reticulum (SR). The effects of these compounds on the SR are due primarily to their inhibitory effects on the sarcoplasmic reticulum/endoplasmic reticulum Ca²⁺ ATPase (SERCA). Phloretin is lipophilic (Bechinger and Seelig, 1991), and phloridzin is transported across cell membranes by the sodium-dependent glucose transporter found in many cell types, including cardiac myocytes (Walle and Walle, 2003; Zhou et al., 2003). These characteristics, and our results showing SERCA inhibition, suggest that phloretin and phloridzin could affect Ca²⁺ regulation and contraction in intact cardiac myocytes. To test this possibility, we examined the effects of phloretin and phloridzin on electrically evoked Ca²⁺ transients, the SR Ca²⁺ load, and cell shortening of intact rat ventricular cardiac myocytes. The results of these experiments further motivated us to examine the effects of phloretin and phloridzin on the action potential and whole-cell K⁺ and Ca²⁺ currents in these cells.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Isolation of Rat Cardiac Myocytes. Myocytes were isolated from adult male Sprague-Dawley rats as described previously (Ward and Giles, 1997). In brief, rats (200–225 g body weight) were killed in accordance with protocols approved by the Canadian Council on Animal Care. Hearts were removed, and they were mounted on a Langendorff apparatus and perfused for 5 min through an aortic cannula with Tyrode's solution containing 1 mM Ca²⁺ (perfusion rate was 10 ml/min; all solutions were bubbled with 100% O₂ and kept at 37°C). Hearts were then perfused in nominally Ca²⁺-free Tyrode's solution for 5 min followed by a 7-min perfusion in nominally Ca²⁺-free Tyrode's solution with 0.02 mg/ml collagenase (Yakult, Tokyo, Japan) and 0.004 mg/ml protease (type XIV; Sigma-Aldrich, St. Louis, MO). The right ventricular wall was then removed, and it was minced in 10 ml of Tyrode's solution with 0.5 mg/ml collagenase, 0.1 mg/ml protease, and 3 mg/ml bovine serum albumin (BSA; Sigma-Aldrich) and gently shaken for 20 to 40 min in a water bath at 37°C. Aliquots of suspended cells were removed from the preparation at 5-min intervals, added to tubes with Kraftbrühe solution, and stored at 4°C until use. Recordings were made from quiescent single rod-shaped cells. Experiments were done at room temperature (20–23°C).

Measurement of Intracellular Ca²⁺ Transients in Single Rat Cardiac Myocytes. To measure Ca²⁺ transients in response to electrical field stimulation, freshly isolated myocytes were allowed to adhere for 20 min to the bottom of a recording chamber on the stage of an inverted microscope. The extracellular Kraftbrühe storage solution was then replaced with Tyrode's solution containing 1 mM Ca²⁺ by perfusing the media through the chamber for 5 min at a rate of 0.5 ml/min. Before each experiment, the background fluorescence and light scatter of the cells was determined, and Ca²⁺ transients were recorded using a SPEX CMX fluorometer (fura-2 experiments; SPEX, Edison, NJ) or an SFX2 microfluorometer (fluo-3 experiments; Solamere Technologies, Salt Lake City, UT). Cells were visualized through a 100x oil immersion objective. The acetoxymethyl ester (AM) form of fura-2 or fluo-3 (2 µg/ml fura-2/AM or 1 µg/ml fluo-3/AM in Tyrode's solution containing 1 mM Ca²⁺ and 1 mg/ml BSA) was then added to the chamber and incubated with the myocytes for 10 min. Excess dye was removed by perfusing the chamber for 5 min with Tyrode's solution (with 1 mM Ca²⁺). The cells were then left for 15 min (with continuous superfusion) to allow cleavage of the AM moiety.

For the experiments using fura-2, fluorescence was excited at 340 and 380 nm, and emission was recorded at 510 nm; 340/380 fluorescence ratios were determined every 10 ms. For the experiments with fluo-3, fluorescence was excited at 480 nm, and emission was monitored at 505 nm. Cells were field stimulated at a frequency of 1 Hz through electrodes on either side of the chamber. Cells were first field stimulated for 5 min to allow the SR to develop a constant extent of Ca²⁺ loading and the Ca²⁺ transients produced after 5 min of continuous field stimulation were recorded for 1 min. Phoretin, phloridzin, or ethanol (vehicle control) was then added under continued field stimulation.

Before analysis, Ca²⁺ transients were corrected for background fluorescence and light scatter. Individual peak heights for each Ca²⁺ transient were determined after subtraction of the fluorescence signal during diastole; 10 to 20 individual consecutive transients were averaged to obtain the mean transient amplitude and area under the transient. Changes in transient amplitude or area are expressed as percentage of control.

Assessment of Sarcoplasmic Reticulum Ca²⁺ Content. The content of the sarcoplasmic reticulum Ca²⁺ store was assessed through caffeine exposure (Kargacin et al., 2000, and references therein). Stores were first loaded by continuously field stimulating the cells at 1 Hz for 5 min. Field stimulation was then stopped, and within 3 s, 10 mM caffeine was applied using a rapid solution exchanger. The area of the resulting caffeine-evoked Ca²⁺ transient was then determined. Field stimulation was started again after the caffeine-evoked Ca²⁺ transient returned to baseline, and then phloretin, phloridzin, or ethanol only was added to the superfusion buffer for 10 min (with continuous field stimulation). Field stimulation was then stopped and a second caffeine pulse was then applied within 3 s, and the area under the resulting transient was compared with the area under the transient evoked by caffeine during the first (control) pulse. Experiments were also conducted in which two control Ca²⁺ transients were evoked by caffeine in buffer without ethanol, phloretin, or phloridzin to ensure that there were no significant changes in amplitude and/or area without the addition of drug (also see Kargacin et al., 2000).

Measurement of Cell Shortening. The extent of unloaded shortening of ventricular myocytes was determined and quantified by an edge-detection device (Crescent Electronics, Crescent, CO) that tracked changes in cell length. Shortening was elicited in response to action potentials applied to the cell under current-clamp conditions (see below) through a patch pipette. The extent of unloaded shortening was determined by averaging cell lengths measured from a train of five action potentials delivered at a frequency of 1 Hz. Unloaded cell shortening was calculated as the fractional change of cell length relative to diastolic values before, and after, exposure to phloretin, phloridzin, or ethanol (control). The exposure time (10 min) required to obtain the maximal effects was determined empirically.

Electrical Recording. Patch pipettes were pulled (P-97 puller; Sutter Instrument Company, Novato, CA) from borosilicate glass and polished on a microforge (MF-200; WPI, Sarasota, FL). Pipettes, when filled with internal recording solution (see below), had resistances of 2 to 3 M. For electrical recording, aliquots of isolated myocytes in suspension were transferred to a chamber on the stage of a Nikon (TE300) inverted microscope, and the cells were allowed to settle to the bottom of the chamber. The chamber was perfused at a rate of 2 ml/min (bath solution was exchanged approximately every 6 s). When used, phloretin was added to the superfusion buffer, and, after 10 min of superfusion, current recordings were made. The exposure time (10 min) required to obtain the maximal effects of phloretin was determined empirically.

The Ca²⁺-independent transient outward K⁺ current (I_to), sustained outward delayed rectifier K⁺ current (I_sus), and inwardly rectifying K⁺ current (I_K1) were recorded using the ruptured patch whole-cell configuration. Whole-cell K⁺ currents were evoked from a holding potential of –80 mV by 500-ms test pulses, in 10-mV increments, to voltages between –120 and +50 mV at a rate of 0.2 Hz.

To determine I_to, whole-cell currents were first recorded with the above-mentioned voltage protocol, and they were then recorded when 100-ms prepulses to –40 mV (to voltage-inactivate I_to) were applied immediately before the 500-ms test pulses. Off-line subtraction of currents recorded using these protocols yielded I_to.I_K1 was measured using the same whole-cell protocol in the absence and presence of 200 µMBa²⁺ (Ba²⁺ selectively blocks I_K1). Steady-state I_K1 current values at the end of the 500-ms test pulses were analyzed.

To determine I_sus, currents were recorded in the presence of 200 µMBa²⁺, and they were evoked by 500-ms test pulses at voltages between –120 and +50 mV following 100-ms prepulses to –40 mV from a holding potential of –80 mV. The steady-state current at the end of the 500-ms test pulse is I_sus and was used in the analysis.

To measure the voltage dependence of I_to inactivation, voltage-clamped cells were held at a holding potential of –80 mV and stepped for 5 s to conditioning voltages between –120 and –10 mV, in 5-mV increments. A test pulse was then applied by stepping to +50 mV for 1 s to activate and record the remaining noninactivated current. Results are expressed relative to the maximal currents elicited during the test pulses.

Reactivation kinetics of I_to was examined using a standard paired pulse protocol. From a holding potential of –110 mV, I_to was activated during 500-ms steps to +50 mV (yielding current P₁). The cells were then returned to the holding potential of –110 mV for set durations ranging from 5 to 600 ms. This was then followed by a second voltage step to +50 mV (yielding current P₂). The current ratio, P₂/P₁, was then calculated for sets of currents. The fractional recovery of I_to (P₂/P₁) as a function of time was determined from the peak currents obtained after the different recovery periods at –110 mV.

L-type Ca²⁺ currents (I_Ca,L) were recorded in Cs⁺ buffer (see below) to block K⁺ currents using the perforated patch technique. I_Ca,L was elicited by a 1-s ramp from –80 to –40 mV (to voltage inactivate Na⁺ currents) followed by a 300-ms step to 10 mV, to activate I_Ca,L. Phloretin (20 µM) was added, and the voltage protocol was recorded every 2 min for a total of 10 min.

Action potentials were elicited in current-clamp mode through amphotericin B perforated patches. Currents (800 pA; 5-ms duration) were injected at a frequency of 1 Hz, filtered at 1 kHz, and sampled at 5 kHz. Five consecutive individual action potentials were averaged for analysis. Action potential durations were measured as the time required to reach 90% repolarization.

Analysis of Patch-Clamp Experiments. Whole-cell patch-clamp currents were recorded with an Axopatch 1-D amplifier (Molecular Devices, Sunnyvale, CA) using p-CLAMP 8.0 Digidata 1200 data acquisition and analysis software (Molecular Devices). Currents are expressed as current densities (pA/pF), and they were filtered at 1 kHz, sampled at 5 kHz, and corrected for cell capacitance before analysis. Recordings were also corrected for junction potentials (typically 9–11 mV).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Ca2+ transients recorded from electrically stimulated freshly isolated cardiac myocytes in the absence and presence of phloretin or phloridzin. A, transients recorded from an isolated myocyte before (black trace) and 10 min after the addition of 20 µM phloretin to the superfusion buffer (red trace). B and C, summary of measurements of the amplitude (B) and area (C) of electrically evoked Ca2+ transients in freshly isolated ventricular myocytes in the absence of, and 10 min after, exposure to ethanol, phloretin, or phloridzin. Mean amplitude and area of the transients are expressed relative to mean amplitude and area of transients evoked before the addition of 20 µM phloretin, 100 µM phloridzin, or ethanol only to the superfusion buffer. Myocytes were electrically stimulated at 1 Hz. Amplitude and area in the presence of phloretin were significantly greater than those measured when ethanol only was added to the superfusion buffer (p < 0.03; n = 6 for all experiments); amplitude and area values in the presence of phloridzin were not significantly different from those measured when ethanol only was added to the buffer.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Effects of Phloretin and Phloridzin on Ca²⁺ Transients of Rat Cardiac Myocytes. Figure 1A shows trains of Ca²⁺ transients recorded from a single isolated rat ventricular myocyte in response to field stimulation at 1 Hz in the absence of phloretin and 10 min after starting superfusion of the recording chamber with 20 µM phloretin. The amplitudes of the Ca²⁺ transients recorded in the presence of phloretin were clearly greater than the amplitudes recorded in its absence; however, the diastolic Ca²⁺ level was not altered by phloretin. Figure 1B summarizes the results of experiments similar to those shown in Fig. 1A. Ca²⁺ transient amplitudes were significantly increased in the presence of phloretin over the amplitudes recorded before the addition of phloretin to the superfusion buffer or when ethanol alone was added to the buffer. The area of the electrically evoked Ca²⁺ transients was also significantly greater in the presence of phloretin (Fig. 1C). Phloridzin (100 µM) seemed to decreased the amplitude and area of the Ca²⁺ transients over those measured when the myocytes were superfused with ethanol only; however, this trend was not significant (Fig. 1, B and C). The rate constant for decay of electrically evoked Ca²⁺ transients in the presence of phloretin was significantly increased (from 169 ± 12 ms in the absence to 200 ± 10 ms in the presence of phloretin; p = 0.0018; n = 8). The rate constants for decay were not significantly different in the presence and absence of phloridzin or ethanol.

The concentration of phloridzin (100 µM) used in the experiments described above had a maximal inhibitory effect on the velocity of SR Ca²⁺ uptake in our previous study (Olson et al., 2006). The concentration of phloretin (20 µM) used in the experiments in Fig. 1 was chosen because this concentration of phloretin inhibited SR Ca²⁺ uptake to approximately the same extent as 100 µM phloridzin. The phloretin concentration, however, is lower than that shown in our previous work to maximally inhibit Ca²⁺ uptake into cardiac SR vesicles. Therefore, we attempted to examine the effects of higher phloretin concentrations on Ca²⁺ transients in intact myocytes; however, when myocytes were superfused with buffer containing concentrations of phloretin greater than 20 µM, the cells hypercontracted once electrical stimulation was started, and they did not relax again. For this reason, we used 20 µM phloretin and 100 µM phloridzin for the remaining experiments reported here.

The experiments described above were done using fluo-3 to record the Ca²⁺ transients. Because fluo-3 is a nonratiometric indicator, it is possible that motion artifacts or changes in cell size due to cell shortening (see below) could have affected the transients recorded. To rule out this possibility, electrically evoked Ca²⁺ transients were also recorded using the ratiometric Ca²⁺ indicator fura-2. The results (data not shown) obtained with fura-2 were similar to those recorded with fluo-3.

Assessment of the SR Ca²⁺ Load in the Presence of Phloretin and Phloridzin. To assess the Ca²⁺ load within the SR of cardiac myocytes upon exposure to phloridzin and phloretin, we examined the effects of caffeine on isolated myocytes before, and after, the addition of phloretin or phloridzin to the superfusion buffer. The SR Ca²⁺ load of myocytes, as assessed by the area of the Ca²⁺ transients elicited in response to 10 mM caffeine, was significantly increased after 10-min exposure to 20 µM phloretin; transient area in the presence of phloretin was 202 ± 56% of control (p = 0.03; n = 8). In contrast, the SR Ca²⁺ load of the myocytes was not significantly different from control in the presence of 100 µM phloridzin or ethanol alone.

As described above, we noted an increase in the time constant for the decline of electrically evoked Ca²⁺ transients in the presence of 20 µM phloretin. This is consistent with an inhibition of SR Ca²⁺ uptake and/or Ca²⁺ extrusion through the Na⁺/Ca²⁺ exchanger; however, it is difficult to unambiguously interpret these results. The maximal cytosolic [Ca²⁺] values recorded in the myocytes in response to electrical stimulation were different in the presence and absence of phloretin, and it is known that the maximal [Ca²⁺] that SERCA is exposed to can affect its subsequent function (Bers and Berlin, 1995). The maximal intracellular [Ca²⁺] values measured in response to caffeine varied more from cell to cell than those initiated by electrical stimulation. This offered us an opportunity to compare the rate of decline of caffeine-evoked Ca²⁺ transients from different cells when the maximal [Ca²⁺] levels reached in control transients were similar in magnitude to those reached in other cells in the presence of phloretin. In analyzing 12 such transients in which the maximal Ca²⁺ levels were not significantly different from one another (five transients from control experiments and seven transients recorded in the presence of 20 µM phloretin), we did not detect any differences in the rate constants for the decline of the transients (determined from exponential fits to the declining phases of the transients; data not shown). This suggests that neither SERCA-mediated SR Ca²⁺ uptake nor Ca²⁺ extrusion by the Na⁺/Ca⁺ exchanger was measurably affected in the presence of phloretin in intact myocytes.

Measurement of Cell Shortening and the Cardiac Action Potential in the Presence and Absence of Phloretin and Phloridzin. The effects of phloretin on electrically evoked Ca²⁺ transients in isolated ventricular myocytes would be expected to be associated with an increase in cell shortening in response to contractile stimuli. The results in Fig. 2 show that, after a 10-min exposure to 20 µM phloretin, the extent of cell shortening in response to action potentials (initiated through an amphotericin B perforated patch) was increased approximately 4-fold over the extent of shortening recorded in its absence. Phloridzin at 100 µM did not have a detectable effect on shortening (Fig. 2B).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effects of phloretin and phloridzin on unloaded shortening of isolated cardiac myocytes. A, results of individual experiments. Percentage of unloaded cell shortening of rat ventricular myocytes is shown before (control) and 10 min after the addition of 20 µM phloretin to the superfusion buffer. Results from individual cells are connected by lines; mean shortening before phloretin addition was 3.0 ± 0.3%, and mean shortening after phloretin exposure was 11.9 ± 0.4%. B, summary of results of experiments similar to those shown in A for phloretin and phloridzin expressed relative to the shortening recorded before (open bars) and after exposure to phloretin (upward diagonal shading) or phloridzin (downward diagonal shading). Shortening was evoked by action potentials delivered in current-clamp mode as described under Materials and Methods. Shortening in the presence of phloretin was significantly different from control (p = 0.02; n = 6); shortening in the presence of phloridzin was not significantly different from control (p = 0.6; n = 3); shortening in the presence of ethanol alone was not significantly different from control (p = 0.1; n = 3; data not shown).

To determine the mechanisms responsible for the effects of phloretin on the Ca²⁺ transients and contractility of ventricular myocytes, we examined the action potentials of these cells in current-clamp mode. Representative action potentials recorded from isolated rat ventricular myocytes in the absence and presence of 20 µM phloretin or 100 µM phloridzin are shown in Fig. 3. As can be seen in the figure, phloretin greatly increased the action potential duration. The results of analysis of action potentials from six individual myocytes (Fig. 4) revealed that the mean action potential duration increased from 31 ± 5 ms before the addition of phloretin to 165 ± 18 ms after the addition (significantly different at p < 0.01; n = 6). As expected from the Ca²⁺ transient data, the action potential duration was not significantly different in the presence and absence of phloridzin (Fig. 4B; also see Fig. 3B).

View larger version (7K): [in this window] [in a new window]   Fig. 3. Ventricular action potentials recorded in the presence and absence of phloretin or phloridzin. A, top trace, action potential recorded from a rat ventricular myocyte before the addition of phloretin; bottom trace, action potential recorded 10 min after the addition of 20 µM phloretin to the superfusion buffer. B, action potentials recorded in the absence (upper trace) and after the addition (lower trace) of 100 µM phloridzin to the superfusion buffer. Action potentials were elicited as described under Materials and Methods.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Summary of effects of phloretin and phloridzin on the action potential duration. A, action potential duration for individual cardiac myocytes before, and after, 10-min exposure of the myocytes to 20 µM phloretin. B, mean action potential durations in the absence (white bars) and presence (shaded bars) of 20 µM phloretin or 100 µM phloridzin in the superfusion buffer. Action potential durations were significantly increased in the presence of phloretin (p = 4 x 10–4; n = 6), but they were not significantly different from control when phloridzin (p = 0.9; n = 3) or ethanol only (p = 0.4; n = 3; data not shown) was added to the superfusion buffer. Action potentials were elicited as described under Materials and Methods.

Whole-Cell Ca²⁺ and K⁺ Currents in the Absence and Presence of Phloretin. The results presented above indicate that the effects of phloretin on Ca²⁺ handling and cell shortening in ventricular myocytes are accompanied by significant changes in the action potential elicited from these cells. The changes in action potential that were recorded in the presence of phloretin (Fig. 4) are similar to those reported by others (Zhang et al., 1994; Bogdanov et al., 1998; Biliczki et al., 2002; He et al., 2003) in response to the inhibition of cardiac cell K⁺ currents. An increase in L-type Ca²⁺ current would also be expected to increase action potential duration. We therefore examined the effects of phloretin on individual whole-cell Ca²⁺ and K⁺ currents in isolated myocytes.

L-type Ca²⁺ currents were measured using the voltage protocol described under Materials and Methods. Peak inward currents at 10 mV were recorded at 2-min intervals for 10 min, and they are expressed relative to the current values at time t = 0 following the addition of vehicle (four experiments) or phloretin (four experiments). There were no significant differences in the L-type Ca²⁺ currents recorded in the presence and absence of phloretin at any of the time points examined (Fig. 5).

View larger version (13K): [in this window] [in a new window]   Fig. 5. Summary of L-type Ca2+ current measurements recorded from cardiac myocytes in the presence and absence of phloretin. Peak inward currents at 10 mV were recorded every 2 min according to the protocol described under Materials and Methods. Results are expressed relative to the currents at t = 0 for the vehicle (filled squares; n = 4) and after exposure to 20 µM phloretin (open circles; n = 5).

Representative whole-cell current traces recorded before the dissection of individual K⁺ currents are shown in (Fig. 6A). Current traces obtained after prepulses to –40 mV to eliminate I_to are shown in Fig. 6B, and currents recorded in the presence of 200 µM BaCl₂ to eliminate I_K1 are shown in Fig. 6C. The effects of 20 µM phloretin on these currents are shown in Fig. 6, D to F. From comparison of the traces in Fig. 6, A and C, with those in Fig. 6, D and F, it is clear that phloretin has a marked effect on the peak whole-cell outward currents but that it does not seem to affect the sustained phase of the currents. The peak whole-cell currents recorded after I_to was eliminated, however, do not seem to be affected by phloretin (compare Fig. 6, B and E).

View larger version (30K): [in this window] [in a new window]   Fig. 6. Representative families of current traces illustrating the protocols used to isolate Ito and IK1 in isolated rat cardiac myocytes and the effects of phloretin on these currents. A and D, whole-cell currents obtained from a holding potential of –80 mV by applying 500-ms voltage pulses to potentials between –120 and +50 mV in 10-mV increments. A, currents before the addition of 20 µM phloretin to the superfusion buffer, D, currents after phloretin addition. B and E, currents obtained with the same protocol as described in A, except that a 100-ms prepulse to –40 mV to inactivate Ito was included in the protocol before the 500-ms test pulses. B, currents before the addition of phloretin. E, currents after the addition of 20 µM phloretin to the superfusion buffer. C and F, currents obtained with the same protocol as in described in A except that 200 µM BaCl2 was included in the superfusion buffer to block IK1; C, currents before addition of phloretin; F, currents after the addition of 20 µM phloretin. Currents were measured before and after 10-min exposure to phloretin. Note that only every other current trace is plotted for clarity.

Effects of Phloretin on Individual Ventricular K⁺ Currents. Figure 7 shows individual whole-cell K⁺ currents obtained as described in Materials and Methods. Representative I_to currents in the absence (Fig. 7A) and presence of 20 µM phloretin (Fig. 7D) indicate that peak I_to is reduced by 20 µM phloretin; however, I_K1 (Fig. 7, B and E) and I_sus (Fig. 7, C and F) do not seem to be altered by phloretin. These results were confirmed when the current-voltage relationships of the three currents were examined. Figure 8A shows that I_to was significantly lower in the presence of phloretin at all potentials above –30 mV. At +50 mV, I_to was reduced from a mean control value of 27 ± 3 to 6 ± 1 pA/pF (p < 0.05; n = 6) in the presence of 20 µM phloretin. I_K1 (Fig. 8B) was not altered by 20 µM phloretin; mean I_K1 at –120 mV was –10.3 ± 1.7 and –8.8 ± 0.7 pA/pF (p = 0.45; n = 6) in the absence and presence of phloretin, respectively. I_sus (Fig. 8C) in the presence of 20 µM phloretin seemed to be slightly greater at potentials more negative than –70 mV and reduced at positive membrane potentials; however, the current values were not significantly different from control. Mean I_sus at +50 mV was 7.7 ± 0.6 and 6.8 ± 0.5 pA/pF (n = 6), respectively, in the absence and presence of phloretin. These values were not significantly different (p = 0.25), nor were the values obtained at negative membrane potentials; mean I_sus at –120 mV was –1.3 ± 0.6 and –3.0 ± 0.9 pA/pF (p = 0.13; n = 6), respectively, in the absence and presence of phloretin.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effects of phloretin on the three primary K+ currents in rat cardiac myocytes. A and D, Ito. A, Ito recorded before the addition of phloretin to the superfusion buffer. D, Ito recorded after the addition of 20 µM phloretin to the buffer. B and E, IK1. B, IK1 recorded before the addition of phloretin to the superfusion buffer. E, IK1 recorded after the addition of 20 µM phloretin to the buffer. C and F, Isus. C, Isus recorded before the addition of phloretin to the superfusion buffer. F, Isus recorded after the addition of 20 µM phloretin to the buffer. Currents were measured before and after 10-min exposure to phloretin; currents were isolated using the protocols described under Materials and Methods.

View larger version (10K): [in this window] [in a new window]   Fig. 8. Current-voltage relationships for Ito, IK1, and Isus in the presence and absence of phloretin. A, current-voltage relationship of Ito in the absence (open circles) and presence (closed circles) of 20 µM phloretin; asterisks indicate significant difference (p < 0.05; n = 6 for each measurement). Line on the phloretin trace was obtained by multiplying each data point of the control trace by the mean ratio of phloretin current to control current. B, current-voltage relationship for IK1: open circles, absence of phloretin; and closed circles, presence of 20 µM phloretin. C, current voltage relationship for Isus: open circles, absence of phloretin; and closed circles, presence of 20 µM phloretin. Currents were measured before and after 10-min exposure to phloretin. Results are normalized to cell capacitance, and they are plotted as mean ± S.E.

Effects of Phloretin on the Inactivation and Reactivation of I_to. The effects of phloretin on voltage-dependent inactivation of I_to is shown in Fig. 9A. The available I_to at +50 mV is plotted as a function of conditioning voltage for conditioning voltages between –120 and –10 mV. The available I_to was significantly lower than control in the presence of phloretin for conditioning potentials between –80 and –60 mV, and it was significantly higher than control for conditioning potentials between –40 and –30 mV. Fitting the inactivation curves with a Boltzmann-type function indicated that the primary effect of phloretin on inactivation was to decrease the sensitivity of inactivation to changes in voltage (Table 1). There were no significant differences in the reactivation curves for I_to (Fig. 9B) measured in the presence and absence of phloretin; comparison of individual curve fits showed that the time constant for recovery from inactivation was 17.3 ± 1.1 ms (n = 3) in control experiments and 17.2 ± 1.5 ms (n = 3) in the presence of phloretin.

View larger version (14K): [in this window] [in a new window]   Fig. 9. Inactivation and reactivation of Ito in the presence and absence of phloretin. A, inactivation of Ito as a function of voltage: open circles, absence of phloretin; and closed circles, presence of 20 µM phloretin. Available Ito current was determined as described under Materials and Methods; * indicate mean values that are significantly different from control (p < 0.05; n = 3). The solid lines are fits of the data to a function of the form: fractional current = 1 – A/(1 + e(Vm – V)/k) (Table 1). B, reactivation of Ito as a function of time: open circles, currents recorded in the absence of phloretin; and closed circles, currents recorded in the presence of 20 µM phloretin. Fractional reactivation was determined as described under Materials and Methods. Mean values in phloretin were not significantly different from controls (n = 3 for all measurements). Currents in A and B were measured before and after 10-min exposure to phloretin. Results are normalized to cell capacitance, and they are plotted as mean ± S.E.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The main findings of this study are as follows. 1) Phloretin increases the area and amplitude of electrically evoked Ca²⁺ transients, increases cell shortening, and increases the SR Ca²⁺ load in rat ventricular myocytes. Phloretin also increases the duration of action potentials evoked from these cells under current-clamp conditions and specifically inhibits the Ca²⁺-independent transient outward K⁺ current of the cells. 2) Phloridzin did not have significant effects on the area or amplitude of electrically evoked Ca²⁺ transients, nor did it measurably change the SR Ca²⁺ load, the extent of unloaded shortening, or the action potential of the myocytes.

In a previous study (Olson et al., 2006), we reported that both phloretin, at concentrations of 20 µM or greater, and phloridzin, at concentrations greater that 50 µM, inhibit Ca²⁺ uptake into isolated cardiac SR vesicles. From these results, one might predict that electrically evoked Ca²⁺ transients, the SR Ca²⁺ load and unloaded cell shortening would be decreased in intact cardiac myocytes in the presence of either compound at the concentrations used in our experiments. Our experiments, however, did not reveal any significant effects of phloridzin on intact cardiac myocytes, and the effects of phloretin are generally opposite of what one would predict from the study of its effects on SR function.

A recent study (Walle and Walle, 2003) showed that phloridzin is transported across cell membranes by the sodium-dependent glucose transporter found in many cell types, including cardiac myocytes (Zhou et al., 2003). Our failure to detect an effect of phloridzin on intact myocytes could be a result of the transport mechanism not allowing enough phloridzin to enter the cells to significantly affect any of the parameters that we measured. In this regard, it should be noted that SR Ca²⁺ uptake velocity was only reduced to a maximal extent of approximately 20% by phloridzin in our previous experiments (Olson et al., 2006). Thus, a limited transport of phloridzin into the myocytes could reduce the extent of it effects to an undetectable level. It is also possible that any inhibitory effects on Ca²⁺ handling were balanced by agonistic effects on other mechanisms.

Our results showing that phloretin increases cell shortening, Ca²⁺ transient amplitude, and the SR Ca²⁺ load in intact cardiac myocytes are consistent with a prolongation of the action potential through a mechanism that involves specific inhibition of I_to. The effects of phloretin on I_to develop relatively slowly (maximal effect after 10 min), suggesting that they may be use-dependent or that they may require phloretin to insert into, or cross, the plasma membrane. Although determination of the time constants for the decline of electrically evoked Ca²⁺ transients suggests that Ca²⁺ removal processes might be affected by phloretin, this was not confirmed when caffeine-evoked transients in control conditions were compared with transients recorded in the presence of phloretin when the transients declined from similar starting Ca²⁺ levels. This suggests that neither the Na⁺/Ca²⁺ exchanger nor SERCA function was measurably affected by phloretin in our experiments with intact myocytes. It should be noted, however, that the effects of phloretin on intact cardiac myocytes in these experiments were examined at a phloretin concentration of 20 µM. At this concentration, phloretin was shown to have only a minimal inhibitory effect on the rate of SR Ca²⁺ uptake (10% inhibition) in our previous study (Olson et al., 2006); thus, an effect on SR function would probably be masked by the more robust effect on I_to and the cardiac action potential. We were unable to examine the effects of higher concentrations of phloretin in the present study because, as noted above, at concentrations above 20 µM, the isolated ventricular myocytes hypercontracted and did not relax again when they were electrically stimulated.

To our knowledge, the present study is the first to examine the actions of phloretin on mammalian cardiac muscle K⁺ channels, and it is the first to show inhibition of I_to. Phloretin, however, is known to affect some other types of K⁺ channels. It has been shown to increase the current through mouse and human large-conductance Ca²⁺-activated K⁺ channels expressed in Xenopus laevis oocytes or in human embryonic kidney 293 cells (Gribkoff et al., 1996). It also opens Ca²⁺-activated K⁺ channels in myelinated nerve fibers (Koh et al., 1994) and in human glioma cells in culture (Ransom and Sontheimer, 2001). In addition to agonistic effects on Ca²⁺-activated K⁺ channels, phloretin has also been reported to block two types of delayed-rectifier K⁺ channels (I and F channels) in myelinated nerve fibers from X. laevis (Klusemann and Meves, 1992; Koh et al., 1994) and the fast K⁺ channel in frog nerve fibers (Klusemann and Meves, 1991).

Although phloretin increases inactivation of I_to relative to control at conditioning voltages between –80 and –60 mV, this cannot completely explain the effects of phloretin on I_to in the voltage-clamp experiments. When cells are held at a holding potential of –80 mV, one would expect phloretin to reduce I_to by approximately 20%, based on its effect on voltage-dependent inactivation (Fig. 9). In the voltage-clamp experiments, however, I_to was reduced by 80% in the presence of 20 µM phloretin (Fig. 8).

Analysis of the inhibition of I_to in the presence of phloretin revealed that the extent of inhibition was independent of voltage; the mean value of I_to in the presence 20 µM phloretin was 21.6% of its value in its absence. The solid line on the phloretin trace in Fig. 8 shows the value of I_to predicted at each test voltage when the control values of I_to were reduced by the mean percentage of inhibition determined from all test voltages. What makes these findings even more intriguing is that although we observed a near 80% reduction of I_to, this concentration of phloretin had little or no effect on other K⁺ currents in these cells (Fig. 8), suggesting a relative specificity for I_to. The observed action potential prolongation and positive inotropic effect is also consistent with I_to inhibition, probably as a consequence of increased Ca²⁺ influx through L-type Ca²⁺ channels due to a delay in repolarization (for review, see Sah et al., 2003).

As noted above, phytoestrogens have been shown in clinical studies to reduce the risk of cardiovascular disease (Clarkson, 2002; de Kleijn et al., 2002), although there is little experimental information pertaining to the mechanisms by which this might occur. The results of the present study suggest that there could be risks associated with high levels of phloridzin or phloretin consumption. Rats ingesting the equivalent of 22 mg of phloretin in a single feeding (either directly as phloretin or as phloridzin) were found to have plasma phloretin levels of 70 µM (approximately 10% of this total was in the form of free phloretin) after 10 h (Crespy et al., 2001). These levels are of the same order of magnitude as that of the concentration shown to affect the contractility and Ca²⁺ handling in cardiac myocytes in the present study. The phloretin/phloridzin content of apple pulp depends upon variety, but it may be as high as 20 mg/kg. The content of apple peel is higher; it may reach levels of 400 mg/kg (Escarpa and Gonzalez, 1998). Thus, it is unlikely that the ingestion of phloretin through the normal consumption of apples or apple products could affect cardiac contractility; however, dietary supplements with concentrated amounts could pose a health risk. Further assessment of this possibility seems warranted if such products are contemplated or become available.

	Acknowledgements

 
We thank Gail McMartin, Teresa Emmett, and Dragana Ponjevic for technical support.

	Footnotes

 
This work was supported by the Heart and Stroke Foundation of Alberta, Heart and Stroke Foundation of Ontario Grant T5234, and the Canadian Institutes of Health Research. M.L.O. was supported by a studentship from the Heart and Stroke Foundation of Canada and by a travel award from the University of Calgary.

C.A.W. and G.J.K. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121111.

ABBREVIATIONS: SR, sarcoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca²⁺ ATPase; BSA, bovine serum albumin; AM, acetoxymethyl ester; I_to, Ca²⁺-independent transient outward K⁺ current; I_sus, sustained outward delayed rectifier K⁺ current; I_K1, inwardly rectifying K⁺ current, I_Ca,L, L-type Ca²⁺ current.

Address correspondence to: Dr. Gary J. Kargacin, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. E-mail kargacin{at}ucalgary.ca

	References

Top Abstract Materials and Methods Results Discussion References

 

Alvarado F and Crane RK (1964) Studies on the mechanism of intestinal absorption of sugars. VII. Phenylglycoside transport and its possible relationship to phlorizin inhibition of the active transport of sugars by the small intestine. Biochim Biophys Acta 93: 116–135.[Medline]

Apkon M and Nerbonne JM (1991) Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes. J Gen Physiol 97: 973–1011.[Abstract/Free Full Text]

Bechinger B and Seelig J (1991) Interaction of electric dipoles with phospholipid head groups. A 2H and 31P NMR study of phloretin and phloretin analogues in phosphatidylcholine membranes. Biochemistry 30: 3923–3929.[CrossRef][Medline]

Bers DM and Berlin JR (1995) Kinetics of [Ca]i decline in cardiac myocytes depend on peak [Ca]i. Am J Physiol 268: C271–C277.[Medline]

Biliczki P, Virag L, Iost N, Papp JG, and Varro A (2002) Interaction of different potassium channels in cardiac repolarization in dog ventricular preparations: role of repolarization reserve. Br J Pharmacol 137: 361–368.[CrossRef][Medline]

Bogdanov KY, Spurgeon HA, Vinogradova TM, and Lakatta EG (1998) Modulation of the transient outward current in adult rat ventricular myocytes by polyunsaturated fatty acids. Am J Physiol 274: H571–H579.[Medline]

Boyer J and Liu RH (2004) Apple phytochemicals and their health benefits. Nutr J 3: 5.[CrossRef][Medline]

Clarkson TB (2002) Soy, soy phytoestrogens and cardiovascular disease. J Nutr 132: 566S–569S.[Abstract/Free Full Text]

Crespy V, Morand C, Besson C, Manach C, Demigne C, and Remesy C (2001) Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats. J Nutr 131: 2109–2114.[Abstract/Free Full Text]

de Kleijn MJ, van der Schouw YT, Wilson PW, Grobbee DE, and Jacques PF (2002) Dietary intake of phytoestrogens is associated with a favorable metabolic cardiovascular risk profile in postmenopausal U.S. women: the Framingham study. J Nutr 132: 276–282.[Abstract/Free Full Text]

Escarpa A and Gonzalez MC (1998) High-performance liquid chromatography with diode-array detection for the determination of phenolic compounds in peel and pulp from different apple varieties. J Chromatogr A 823: 331–337.[CrossRef][Medline]

Fedida D, Wible B, Wang Z, Fermini B, Faust F, Nattel S, and Brown AM (1993) Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current. Circ Res 73: 210–216.[Abstract]

Gribkoff VK, Lum-Ragan JT, Boissard CG, Post-Munson DJ, Meanwell NA, Starrett JE Jr, Kozlowski ES, Romine JL, Trojnacki JT, McKay MC, et al. (1996) Effects of channel modulators on cloned large-conductance calcium-activated potassium channels. Mol Pharmacol 50: 206–217.[Abstract]

He J, Kargacin ME, Kargacin GJ, and Ward CA (2003) Tamoxifen inhibits Na+ and K+ currents in rat ventricular myocytes. Am J Physiol 285: H661–H668.

Kargacin ME, Ali Z, Ward CA, Pollock NS, and Kargacin GJ (2000) Tamoxifen inhibits Ca²⁺ uptake by the cardiac sarcoplasmic reticulum. Pflueg Arch 440: 573–579.[Medline]

Klusemann J and Meves H (1991) Phloretin affects the fast potassium channels in frog nerve fibres. Eur Biophys J 20: 79–86.[Medline]

Klusemann J and Meves H (1992) The effect of phloretin on single potassium channels in myelinated nerve. Eur Biophys J 21: 93–97.[Medline]

Koh DS, Reid G, and Vogel W (1994) Activating effect of the flavonoid phloretin on Ca(²⁺)-activated K⁺ channels in myelinated nerve fibers of Xenopus laevis [corrected]. Neurosci Lett 165: 167–170.[CrossRef][Medline]

Li GR, Feng J, Yue L, and Carrier M (1998) Transmural heterogeneity of action potentials and I to 1 in myocytes isolated from the human right ventricle. Am J Physiol 275: H369–H377.[Medline]

Lopatin AN and Nichols CG (2001) Inward rectifiers in the heart: an update on I(K1). J Mol Cell Cardiol 33: 625–638.[CrossRef][Medline]

Olson ML, Kargacin ME, Honeyman TW, Ward CA, and Kargacin GJ (2006) Effects of phytoestrogens on sarcoplasmic/endoplasmic reticulum calcium ATPase 2a and Ca²⁺ uptake into cardiac sarcoplasmic reticulum. J Pharmacol Exp Ther 316: 628–635.[Abstract/Free Full Text]

Ransom CB and Sontheimer H (2001) BK channels in human glioma cells. J Neurophysiol 85: 790–803.[Abstract/Free Full Text]

Rossetti L, Smith D, Shulman GI, Papachristou D, and DeFronzo RA (1987) Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J Clin Invest 79: 1510–1515.[Medline]

Sah R, Ramirez RJ, Oudit GY, Gidrewicz D, Trivieri MG, Zobel C, and Backx PH (2003) Regulation of cardiac excitation-contraction coupling by action potential repolarization: role of the transient outward potassium current (I(to)). J Physiol 546: 5–18.[Abstract/Free Full Text]

Thiyagarajah P, Kuttan SC, Lim SC, Teo TS, and Das NP (1991) Effect of myricetin and other flavonoids on the liver plasma membrane Ca²⁺ pump. Kinetics and structure-function relationships. Biochem Pharmacol 41: 669–675.[CrossRef][Medline]

Walle T and Walle UK (2003) The beta-D-glucoside and sodium-dependent glucose transporter 1 (SGLT1)-inhibitor phloridzin is transported by both SGLT1 and multidrug resistance-associated proteins 1/2. Drug Metab Dispos 31: 1288–1291.[Abstract/Free Full Text]

Wang Z, Fermini B, and Nattel S (1993) Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents. Circ Res 73: 1061–1076.[Abstract/Free Full Text]

Ward CA and Giles WR (1997) Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J Physiol 500: 631–642.[Medline]

Zhang ZH, Boutjdir M, and el-Sherif N (1994) Ketanserin inhibits depolarization-activated outward potassium current in rat ventricular myocytes. Circ Res 75: 711–721.[Abstract/Free Full Text]

Zhou L, Cryan EV, D'Andrea MR, Belkowski S, Conway BR, and Demarest KT (2003) Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1). J Cell Biochem 90: 339–346.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.121111v1 321/3/921    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Olson, M. L. Articles by Kargacin, G. J. Search for Related Content PubMed PubMed Citation Articles by Olson, M. L. Articles by Kargacin, G. J.