AJP - Heart AJP: Renal Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H1948-H1955, 2008. First published February 22, 2008; doi:10.1152/ajpheart.01209.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/H1948    most recent
01209.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Borchert, G. H.
Right arrow Articles by Escriba, P. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Borchert, G. H.
Right arrow Articles by Escriba, P. V.

2-Hydroxyoleic acid affects cardiomyocyte [Ca2+]i transient and contractility in a region-dependent manner

Gudrun H. Borchert,1,2 Mike Giggey,3 Frantisek Kolar,2 Tak Ming Wong,4 Peter H. Backx,3 and Pablo V. Escriba1

1Laboratory of Molecular and Cellular Biomedicine, University of the Balearic Islands, Palma de Mallorca, Spain; 2Institute of Physiology, Academy of Sciences of the Czech Republic and Centre for Cardiovascular Research, Prague, Czech Republic; 3Department of Physiology and Medicine, University of Toronto, Heart and Stroke/Richard-Lewar-Centre of Excellence, Toronto, Canada; and 4Department of Physiology, The University of Hong Kong, Hong Kong, China

Submitted 18 October 2007 ; accepted in final form 20 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Monounsaturated fatty acids such as oleic acid are cardioprotective, modify the physicochemical properties of cardiomyocyte membranes, and affect the electrical stability of these cells by regulating the conductance of ion channels. We have designed a nonhydrolysable oleic acid derivative, 2-hydroxyoleic acid (2-OHOA), which regulates membrane lipid structure and cell signaling, resulting in beneficial cardiovascular effects. We previously demonstrated that 2-OHOA induces PKA activation and PKC{alpha} translocation to the membrane; both pathways are thought to regulate transient outward K+ current (Ito) depending on the stimulus and the species used. This study was designed to investigate the effect of 2-OHOA on isolated cardiomyocytes. We examined the dose- and time-dependent effect of 2-OHOA on cytosolic Ca2+ concentration ([Ca2+]i) transient and contraction of myocytes isolated from different parts of the rat ventricular myocardium. Although this drug had no effect on [Ca2+]i transient and cell shortening in myocytes isolated from the septum, it increased (up to 95%) [Ca2+]i transient and cell shortening in subpopulations of myocytes from the right and left ventricles. The pattern of the effects of 2-OHOA was similar to that observed following the application of the Ito blocker 4-aminopyridine, suggesting that the drug may act on this channel. Unlike the effect of 2-OHOA on [Ca2+]i transient and cell shortening, PKC{alpha} translocation to membranes was not region specific. Thus 2-OHOA-induced effects on [Ca2+]i transients and cell shortening are likely related to reductions in Ito function, but PKC{alpha} translocation does not seem to play a role. The present results indicate that 2-OHOA selectively increases myocyte inotropic responsiveness, which could underlie its beneficial cardiovascular effects.

calcium; contractile function; fatty acid; protein kinase C; signal transduction

UNSATURATED FATTY ACIDS have been shown to be cardioprotective (1, 2, 6, 7, 13, 19, 23, 24, 26, 27, 46). They can alter the physicochemical properties of plasma membranes, thereby modulating ion channel function and altering electrical stability (7, 12, 27, 46). These effects of unsaturated fatty acids are potentially relevant for cardiovascular health since the plasma membrane composition is altered in hypertensive subjects (17, 43, 46) who are susceptible to arrhythmias (12). The type and abundance of membrane lipid species are regulated by dietary fat intake, which thus influences the properties of the membrane (18). In addition, current therapies targeting lipids can reverse or prevent heart diseases. In this context, the regulation of membrane lipid composition by drug administration has been shown to be an alternative approach to treat cardiovascular and other pathologies in clinical practice (16).

We have previously shown that cis-monounsaturated fatty acids, such as oleic acid and 2-hydroxyoleic acid (2-OHOA), can affect membrane lipid structure and cell signaling, leading to blood pressure reductions in hypertensive humans and animals [spontaneous hypertensive rats (SHRs)] (1, 2, 17, 28). In contrast, closely related fatty acids (e.g., elaidic acid, the trans-isomer of oleic acid, and stearic acid) with slight structural differences with respect to 2-OHOA do not significantly change blood pressure. In a previous study (2), we demonstrated that 2-OHOA strongly increased the expression and activity of protein kinase A (PKA) and decreased blood pressure in SHRs. This blood pressure lowering effect was partially reversed in vivo (by about 60%) upon administration of the PKA inhibitor Rp-8-bromo-cAMP (2). Although this result demonstrates the physiological relevance of this signaling pathway, it also suggests that there might be additional mechanisms regulating the cardiovascular action of 2-OHOA, such as Rho kinase, protein kinase C (PKC), or other G protein-associated pathways (1, 2, 28, 45). On the other hand, oleic acid exhibits an inhibitory effect on transient outward K+ current (Ito) in the human right atrium (12). Blockade of Ito by oleic acid (12) or by {alpha}1-adrenoceptor stimulation has been shown to be PKC independent (9, 12, 22).

The present study was designed to investigate the molecular basis underlying the cardiovascular effects of 2-OHOA (2) using isolated rat heart myocytes from various ventricular regions. Previous studies have shown structural, functional, and metabolic differences between the right and left ventricles (31) as well as between regions within the ventricle itself (3, 8, 10, 11, 15, 25, 29, 30, 35, 38–40). Therefore, we investigated left ventricular (LV), right ventricular (RV), and septum myocytes (LVM, RVM, and SEPM, respectively) separately. Since there are no studies available about the effect of 2-OHOA on isolated myocytes, we first examined potential toxic effects by evaluating cell viability. We then studied cytosolic Ca2+ concentration ([Ca2+]i) transient as well as cell shortening at different time points and 2-OHOA concentrations. The main conclusion from our experiments is that 2-OHOA increases [Ca2+]i transient and cell contraction in defined subpopulations of ventricular myocytes likely by inhibiting Ito in a PKC{alpha}-independent manner. These results may in part explain the beneficial cardiovascular effects of this drug.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The study was conducted in accordance with the guidelines of and approved by the Care and Use of Laboratory Animals Committee of the University of Toronto and conducted according to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Chemicals were obtained from Sigma (Hamburg, Germany) unless indicated otherwise.

Isolation of rat ventricular myocytes. Hearts from male Sprague-Dawley rats (250–400 g, Charles River) were perfused through the aorta, first for about 3–5 min with Ca2+-supplemented Tyrode solution containing (in mmol/l) 140 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 1 CaCl2, and 10 D-glucose (pH 7.4) at 37°C (37) and then with Ca2+-free Tyrode solution for 5 min before digestion with 30 ml of the same solution containing collagenase (type II, 20 mg, 700 units/mg; Yakult, Tokyo, Japan) and protease (type XIV, 0.2 mg/ml) for 7–9 min. Collagenase was subsequently removed by perfusion with Ca2+-free Tyrode solution at pH 7.4 for 5 min. All the solutions were gassed with 100% O2 for 5 min before use. The atria and blood vessels were then removed, and free RV and LV walls and septum were dissected. Cells were dissociated by gentle mechanical shaking. Isolated myocytes were then incubated in Ca2+-free Tyrode solutions at room temperature for 30 min. The concentration of Ca2+ was gradually increased up to 1.25 mM during the next 40 min. Only Ca2+-tolerant, quiescent, rod-shaped myocytes with clear cross-striations were selected for measurement of [Ca2+]i transient and cell shortening. Myocytes isolated from the same part of each heart were randomly divided into two groups (control or treated) to minimize interexperimental variations.

Primary cell culture. Isolated LVM, RVM and SEPM were cultured in 50% Dulbecco's modified Eagles medium and 50% Nutrient Mixture F12HAM, containing 0.2% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin. Myocytes were kept in a CO2 incubator (95% room air-5% CO2, 28°C) in the presence or absence (control) of 10 or 50 µM 2-OHOA for 1, 8, or 20 h.

In a separate series of experiments, myocytes were incubated with 2 mM 4-aminopyridine (4-AP) for 15 min before commencing the protocol to block Ito. Only RVM and SEPM were analyzed for this set of experiments to avoid uninterpretable results due to the heterogeneous distribution of Ito in the LV wall. RVM and SEPM were treated as follows: 1) control myocytes (treated with vehicle for 1 h); 2) myocytes treated with 50 µM 2-OHOA for 1 h; 3) myocytes treated with 2 mM 4-AP for 1 h, 4) myocytes pretreated (15 min) with 2 mM 4-AP followed by treatment with 50 µM 2-OHOA for 1 h.

Cell viability. The proportion of viable cells was determined by trypan blue exclusion (44). Typically, 50–100 myocytes were counted in duplicates from six to eight independent experiments.

Measurements of [Ca2+]i transient and cell shortening. Myocytes were mounted in a chamber on the stage of an inverted microscope (Olympus IX50, New York, NY) and superfused with Tyrode solution. Myocytes were field stimulated with 1 Hz [pulse length of 5 ms, 60 V, and 0.05-ms delay using Grass stimulator (Grass Telefactor, West Warwick, RI)]. [Ca2+]i transients were measured in myocytes loaded with 10 µmol/l indo-1-AM (Molecular Probes, Burlington, ON, Canada) and excited at 360 nm. The ratio of fluorescence intensity between light emission at 405 and 495 nm was used to measure [Ca2+]i transient, after subtracting the background fluorescence (37). This ratio was used to estimate the [Ca2+]i transients in electrically stimulated myocytes and under resting conditions. The resting [Ca2+]i, and the amplitude and maximal velocities of the [Ca2+]i transient were used to evaluate the effect of 2-OHOA.

Cell shortening was recorded using a video-edge detector coupled to a high-frequency (240 Hz) charge-coupled Phillips-800 camera (Crescent Electronics, Salt Lake City, Utah) and analyzed as described previously (37). Percent cell shortening and maximum velocities of shortening and relaxation were calculated using custom-designed software.

The criterion to allot each myocyte to positively responding, negatively responding, or nonresponding subpopulations was based on a comparison of its [Ca2+]i transient or cell shortening after 2-OHOA treatment with a corresponding mean value calculated for vehicle-treated (control) LVM, SEPM, and RVM in each experiment. Cells were designated as positively responding or negatively responding when their amplitude of [Ca2+]i transient or cell shortening was higher or lower, respectively, than the 95% confidence interval of the control group. The remaining cells were designated as nonresponding.

For [Ca2+]i transient and cell shortening measurements, 1,000 data points/s were collected. Results are presented as means ± SE from 12–20 myocytes in each group out of four independent experiments.

PKC{alpha} translocation. After isolation, myocytes from RV, LV, and septum were separated into four groups as described in Primary cell culture. After the treatment, myocytes were harvested and stored at –80°C until use. Positively, negatively, and nonresponding cells could not be separated for this type of experiment because they only can be differentiated after electrical stimulation. Myocyte cytosolic or membrane (lysate) proteins (100 µg) were fractionated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked overnight at 4°C in PBS (Gibco, Carlsbad, CA) containing 5% nonfat dry milk, 0.5% BSA, and 0.1% Tween 20. Polyvinylidene fluoride membranes were then incubated with the primary antibody (anti-PKC{alpha}, 1:200; anti-GAPDH, 1:10,000, or anti-calsequestrin, 1:1,000) for 1 h. Anti-GAPDH was obtained from Ambion Europe (Cambridgeshire, UK), and the remaining antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). After the primary antibody was removed, the membranes were washed three times for 10 min each with PBS and then incubated with horseradish peroxidase-linked secondary antibody for 1 h at room temperature. Immunoreactivity was detected using the enhanced chemiluminescence (ECL) Western Blot detection system followed by exposure to ECL hyperfilm (Amersham Bioscience, Buckinghamshire, UK). Films were scanned at a resolution of 300 dpi, and the immunoreactivity for PKC was normalized to GAPDH and calsequestrin levels as loading controls for cytosolic and membrane fractions, respectively. Finally, the membrane-to-cytosol ratios of PKC{alpha} were calculated. Results are expressed as means ± SE of six independent experiments.

Statistical analysis. Data are expressed as means ± SE of the indicated number of experiments. For statistical analysis, one-way ANOVA with Bonferroni post hoc means comparison was used. Differences were considered statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We studied viability, [Ca2+]i transient and cell shortening in LVM, RVM, and SEPM after 1, 8, and 20 h under control conditions and after treatment with 10 and 50 µM 2-OHOA. Since these two concentrations exhibited similar effects, we present here only data obtained with the higher concentration.

Cell viability. We first examined the potential toxicity of 2-OHOA on cardiac myocytes by monitoring their survival. Myocyte viability was not altered by exposure to 2-OHOA at any time point investigated. The percentage of rod-shaped cells under control conditions was 42% and 50% for LV and RV, respectively, with no changes after the treatment.

[Ca2+]i transient. The resting [Ca2+]i was not significantly different between LVM, RVM, and SEPM under control conditions, regardless of the cell culture time (Table 1). After 1 h incubation with 50 µM 2-OHOA, the SEPM showed a significantly lower resting [Ca2+]i compared with control SEPM. Interestingly, SEPM and RVM showed similar resting [Ca2+]i that were significantly higher than those of LVM after 8 h of treatment, whereas after 20 h of treatment with 2-OHOA, SEPM and LVM exhibited similar resting [Ca2+]i that were different from the bimodal response of RVM with a significant decrease in one subpopulation of cells (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Resting [Ca2+]i, [Ca2+]i transient, and cell shortening under control conditions and after 50 µM 2-OHOA

 
Under control conditions, the amplitude of [Ca2+]i transient in LVM and SEPM was significantly different from that in RVM. SEPM showed the highest and RVM the lowest amplitudes of [Ca2+]i transient (P < 0.005, Table 1). 2-OHOA did not induce significant changes in SEPM at any time point investigated. In contrast, in LVM and RVM, the treatment induced heterogeneous effects, indicating the existence of distinct myocyte populations. One population of LVM (54.4%) appeared to increase the amplitude of [Ca2+]i transient, whereas in the other LVM population (45.6%), this parameter was rather negatively modulated by the drug treatment. Likewise, one RVM population (61.1%) responded to 2-OHOA treatment with an increase of [Ca2+]i transient, whereas the other RVM population (38.9%) did not show significant changes (Figs. 1 and 2). The amplitude of [Ca2+]i transient was significantly different for positively responding myocytes from the LV and RV compared with negatively responding (LV) or nonresponding (RV) myocytes (P < 0.005) under all experimental conditions used. The amplitude of [Ca2+]i transient of positively responding LVM and RVM was similar, although the control values for RVM were significantly lower. For negatively responding LVM and nonresponding RVM, the amplitude of [Ca2+]i transient was similar to that of RVM under control conditions (Table 1).


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 1. Examples of original recordings of the cytosolic Ca2+ concentration ([Ca2+]i) transient (left) and cell shortening (right) from left ventricular (LV) myocytes (LVM) after 8 h cell culture. A: control myocytes (vehicle treated). B: 2-hydroxyoleic acid (2-OHOA)-treated (50 µM) myocytes, positive response. C: 2-OHOA-treated (50 µM) myocytes, negative response. For [Ca2+]i transient, recordings are shown as fluorescence ratio of 405 to 495 nm (405/495 nm) with the baseline deducted before the calculation of the ratio.

 

Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 2. Distribution of values of the amplitude of [Ca2+]i transient (fluorescence ratio, 405/495 nm) individual myocytes. Treatment with 2-OHOA (50 in µM) induced an increase [LVM and right ventricular (RV) myocyte (RVM)], a decrease (LVM) or no change [RVM and septum myocyte (SEPM)].

 
The effect of 2-OHOA on the amplitude of [Ca2+]i transient was significant after 1 h and persisted up to 20 h (Table 1 and Fig. 3), but the greatest effect was observed after 8 h of 2-OHOA treatment in the positively responding subpopulation of myocytes, reaching a maximum of 148.6 ± 6.3% and 194.6 ± 11.7% for LVM and RVM, respectively. Indeed, under all experimental conditions used, the percent increase in [Ca2+]i transient was significantly greater in RVM than in LVM (P < 0.05). On the other hand, the second LVM population showed a reduced amplitude of [Ca2+]i transient after 1, 8, and 20 h of 2-OHOA treatments (Table 1 and Fig. 3).


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 3. Amplitude of [Ca2+]i transient and cell shortening after treatment with 50 µM 2-OHOA in LVM, SEPM, and RVM. Values (% of control) are means ± SE of 12–20 myocytes out of 4 individual experiments. Dotted line, control value (100%); black columns, positive response; white columns, negative or no response. *P < 0.05 and ***P < 0.005, treated vs. corresponding control.

 
The velocities of [Ca2+]i transient rise and decline were similar in LV, septum, and RV control myocytes. Interestingly, although the amplitude of the [Ca2+]i transient decreased with the time of cell culture, the maximal velocities of the [Ca2+]i transient did not change in myocytes cultured for 20 h. Changes of these parameters after 2-OHOA treatment essentially followed those of the amplitude of the [Ca2+]i transient (data not shown).

Cell shortening. As described for the [Ca2+]i transient, cell shortening occurred to a lesser extent in RVM under control conditions, being significantly different from that of LVM and SEPM (Table 1). In fact, the effects of 2-OHOA on myocyte shortening followed a similar pattern to that of [Ca2+]i transient, reflecting the presence of different populations of LVM and RVM. Thus cell contraction increased significantly in certain ventricular myocyte subpopulations after 2-OHOA treatment (51.1% and 63.8% of LVM and RVM, respectively), whereas 36.2% of RVM and all SEPM (100%) remained unaffected and 48.9% of LVM showed significant decreases (Table 1, and Figs. 1 and 3). Incubation with 2-OHOA for 1 h augmented cell shortening significantly in positively responding LVM and RVM to 137.0 ± 6.4% and 142.9 ± 7.2%, respectively. Longer exposure (8 h) significantly increased these values to 167.2 ± 8.4% in LVM and 195.5 ± 14.2% in RVM. As described for [Ca2+]i transients, RVM displayed a significantly greater shortening (P < 0.05) than LVM after 1, 8, and 20 h of 2-OHOA treatment (Table 1 and Fig. 3).

Interestingly, the maximum velocities of cell shortening and relaxation were slower in RVM than in LVM and SEPM under control conditions, but no difference was observed between the LVM and SEPM. Exposure to 2-OHOA led to a similar pattern of changes in these parameters as described for cell shortening (data not shown).

Effects of 4-AP. The heterogeneous response of myocytes to 2-OHOA and the proportion of cells responding in the LV and RV suggested that the two cell populations may correspond to subepi- and subendocardial cells and, therefore, 2-OHOA may act by blocking Ito. For this reason, we investigated whether 2 mM 4-AP affects the response to 50 µM 2-OHOA in RVM and SEPM (Fig. 4A). The effect of 4-AP alone on RVM and SEPM cell shortening was similar to that of 2-OHOA, and the distribution of positively responding and nonresponding RVM was the same as reported following the exposure to 2-OHOA. Moreover, no additional effects were observed when the two drugs were administered together.


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 4. A: effects of 4-aminopyridine (4-AP) and 2-OHOA on cell shortening in SEPM and RVM. Data are means ± SE of 12–20 myocytes from 4 individual experiments. B: Western blot analysis of PKC{alpha}: cytosolic fraction and the solubilized membrane fraction were used, and the ratios of the membrane-to-cytosolic immunoreactivity are shown in LVM, SEPM, and RVM. Myocytes were treated for 1 h with either 2 mM 4-AP or 50 µM 2-OHOA with or without 15 min of 4-AP preincubation. Data are means ± SE from 6 individual experiments. White columns, control myocytes; vertically stripped columns, 2-OHOA-treated myocytes; horizontally stripped columns, 4-AP-treated myocytes; cross-checked columns, 4-AP- and 2-OHOA-treated myocytes. *P < 0.05 and ***P < 0.005, treated vs. corresponding control.

 
PKC{alpha} translocation to membranes. The subcellular distribution (between cytosolic and membrane fraction) of PKC{alpha} was examined to determine whether the previously described translocation of PKC{alpha} (1, 28) was associated with the effect of 2-OHOA on [Ca2+]i transient and cell shortening. The membrane-to-cytosol ratio of the PKC{alpha} immunoreactivity was significantly and similarly increased in LVM, SEPM, and RVM (Fig. 4B) after the incubation of myocytes with either 2-OHOA or 4-AP compared with the ratio observed under control conditions. An additive effect of the combined two drugs was not observed.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
2-OHOA, a nonhydrolysable oleic acid derivative, is currently under pharmaceutical investigation as an agent for treatment of hypertension and other cardiovascular diseases. However, very few studies are available about the molecular mechanism underlying cardiovascular effects of this drug. As improved cardiac performance is known to be associated with increases of [Ca2+]i transient and contractile function of cardiomyocytes (14, 36, 41), this study was designed to determine the effects of 2-OHOA on these functional indexes. Our results show for the first time that 2-OHOA increases [Ca2+]i transient and cell shortening in isolated cardiomyocytes from the LV and the RV but not from the septum. Moreover, the cardiomyocyte subpopulations from the LV and RV did not respond uniformly to the drug. Based on our results and the literary data available, we suggest that the observed effects of 2-OHOA are due to the inhibition of Ito channels.

The existence of heterogeneous populations of myocytes between ventricles as well as within the ventricle itself has been reported previously; it is based on molecular differences in membrane composition, e.g., those underlying the heterogeneity of action potential duration in subepicardial and subendocardial cells (3, 8, 10, 11, 15, 25, 29, 30, 32, 35, 38–40). The expression of Ito channels gradually decreases from the subepicardial to the subendocardial layers in the LV and RV, whereas the SEP exhibits much lower expression as demonstrated in the dog (35). The total amount of Ca2+ entering the cell via L-type Ca2+ channel per pulse increases as Ito falls (38). Thus, when Ito channels are reduced or missing (as in SEPM membranes), 2-OHOA does not exert any effect on [Ca2+]i transient and cell shortening as demonstrated in our study. The existence of subpopulations of RVM and LVM that did not positively respond to 2-OHOA is in agreement with the observation that subendocardial cells express much fewer Ito channels than subepicardial cells (35). Moreover, we found that 2-OHOA had greater effects on [Ca2+]i transient and contractility of the positively responding population of RVM compared with that of LVM. This difference could be attributed to a greater expression of Ito channels in RVM (11, 30, 35). In line with these observations, LVM and RVM that responded positively to 2-OHOA showed the same maximum amplitude of [Ca2+]i transient, although control values for RVM were significantly lower. Interestingly, [Ca2+]i transients of SEPM under control conditions were similar to those of 2-OHOA-treated positively responding LVM and RVM. Hence, when Ito is blocked (as it is presumably in our experiments after 2-OHOA treatment) or nearly absent (as in SEPM), [Ca2+]i transients are similar.

The view that Ito channel is a target of 2-OHOA is further supported by our observation that 4-AP, a potent Ito inhibitor (7, 9, 12, 22, 25, 32), increased cell shortening similarly as 2-OHOA, and no synergistic effect was observed when cells were treated with 4-AP and 2-OHOA simultaneously. In line with this view, data from another study (12) showed that the 2-OHOA analog, oleic acid, inhibits Ito, although it has no effect on other K+ currents (12).

The mechanism of Ito inhibition by 2-OHOA is not clear, and several possibilities should be taken into consideration. The greater effect of 2-OHOA on [Ca2+]i transient and cell shortening after 8 h compared with 1-h treatment suggests the existence of a rapid component of 2-OHOA action associated with changes in membrane lipid structure (28) and/or direct action on Ito (12) and the second slow component possibly causing channel phosphorylation or other modifications (22).

Oleic acid modulates the channel in its open state, so that Ito inhibition is likely due to the modification of membrane lipid structure (12). We have shown previously that low concentrations of oleic acid (up to 100 µM) induced profound changes in membrane lipid structure (28). Treatment with oleic acid (12), and related molecules (9, 22), modulates in vivo the adrenoceptor-PKA pathway by activating G{alpha}s proteins, as well as increasing cAMP levels, which results in PKA activation (1, 2) and leads to Ito channel phosphorylation and current reduction (22). Indeed, treatment with 2-OHOA induces increases in the expression and/or activity of the G{alpha}s protein and PKA in rat LV membranes (1, 2). Thus the effect of 2-OHOA on Ito is likely produced via the regulation of the membrane lipid structure (as described in Ref. 12), in a similar way to the action of oleic acid on G-proteins (45). This is supported by the observation that the fluorescence signal of indo-1-labeled RVM under control conditions was increased after the treatment with 2-OHOA. It suggests that the exposure of myocytes to 2-OHOA likely changed the membrane properties and allowed the dye to enter the myocytes more easily.

It has been shown that the inhibition of PKA in vivo reverses the hypotensive action of 2-OHOA by 60%, which demonstrates the involvement of the PKA pathway in the effect of the drug (2), but it also suggests that other pathways may play a role. Since previous experiments demonstrated translocation of PKC{alpha} to the membrane fraction in LV after whole animal treatment with 2-OHOA (1), we examined this enzyme in a more detail. The PKC{alpha} translocation shown in the present study represents the average from all myocytes of the respective ventricular part (both responding or nonresponding to 2-OHOA), because the distinct subpopulations of myocytes can only be distinguished according to their response to electrical stimulation. We observed a similar degree of translocation of PKC{alpha} in LVM, SEPM and RVM after exposure to 2-OHOA, 4-AP, or the combination of the two drugs. These results led us to conclude that PKC{alpha} pathway is unlikely to be involved in the stimulatory effect of 2-OHOA on [Ca2+]i transient and cell shortening in LVM and RVM. The present data are in agreement with previous studies, showing the failure of the PKC inhibitor staurosporine to suppress Ito blockade induced by oleic acid in human right atrium (12). Although some studies suggest that PKC may regulate this channel (4, 33), PKC inhibitors (in contrast with PKA inhibitors) failed to reduce the Ito-blocking effects of methoxamine or phenylephrine (9, 22), which supports the view that PKC is not involved in the regulation of Ito.

In conclusion, this study shows positive effects of 2-OHOA on [Ca2+]i transient and cell shortening in distinct subpopulations of LVM and RVM. The pattern of the effects of 2-OHOA was similar to that observed following the application of the Ito blocker 4-AP, suggesting that the drug may act on this channel. Unlike the effect of 2-OHOA on [Ca2+]i transient and cell shortening, PKC{alpha} translocation to membranes was not region specific. Thus 2-OHOA-induced effects on [Ca2+]i transient and cell shortening are unlikely related to PKC{alpha} translocation. The present results indicate that 2-OHOA selectively increases myocyte inotropic responsiveness, which could underlie its beneficial cardiovascular effects. The regulation of cellular activity by modulating the membrane lipid structure is a new promising field in molecular medicine (16).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a Ministero de Education, Cultura y Deporte, (Madrid, Spain) Grant SAB2002-079 (to G. H. Borchert); the Ministerio de Educación y Ciencia (Spain) Grants SAF2003-00232 and SAF2004-05249; and the Conselleria d'Economia, Hisenda i Innovació del Govern Balear Grants PRIB2004-10131 and PCTIB-2005GC4-07 (to P. V. Escriba); the Ministry of Education (Czech Republic) Grant 1M0510; and the Agency of the Czech Republic Grant 305/07/1008 (to G. H. Borchert and F. Kolar). The Canadian Institute for Health Research supported M. Giggey.


   ACKNOWLEDGMENTS
 
We are grateful to Chi Pui Mok and Alan Prendergast for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. H. Borchert, Inst. of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic (e-mail: gudrunhborchert{at}compuserve.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alemany R, Teres S, Baamonde C, Benet M, Vögler O, Escriba PV. 2-Hydroxyoleic acid: a new hypotensive molecule. Hypertension 43: 249–254, 2004.[Abstract/Free Full Text]
  2. Alemany R, Vögler O, Teres S, Egea C, Baamonde C, Barcelo F, Delgado C, Jakobs KH, Escriba PV. Antihypertensive action of 2-hydroxyoleic acid in SHRs via modulation of the protein kinase A pathway and Rho kinase. J Lipid Res 47: 1762–1770, 2006.[Abstract/Free Full Text]
  3. Antzelevitch C. Cardiac repolarization. The long and short of it. Europace 7, Suppl 2: 3–9, 2005.[Free Full Text]
  4. Apkon M, Nerbonne JM. {alpha}1-Adrenergic agonists selectively suppress voltage-dependent K+ currents in rat ventricular myocytes. Proc Natl Acad Sci USA 85: 8756–8760, 1988.[Abstract/Free Full Text]
  5. Barcelo F, Prades J, Funari SS, Frau J, Alemany R, Escriba PV. The hypotensive drug 2-hydroxyoleic acid modifies the structural properties of model membranes. Mol Membr Biol 21: 261–268, 2004.[CrossRef][Web of Science][Medline]
  6. Billman GE, Hallaq H, Leaf A. Prevention of ischemia induced-ventricular fibrillation by {omega}3-fatty acids. Proc Natl Acad Sci USA 91: 4427–4430, 1994.[Abstract/Free Full Text]
  7. Bogdanov KY, Spurgeon HA, Vinogradova TM, Lakatta EG. Modulation of transient outward current in adult rat ventricular myocytes by polyunsaturated fatty acids. Am J Physiol Heart Circ Physiol 274: H571–H579, 1998.[Abstract/Free Full Text]
  8. Brahamajothi MV, Campbell DL, Rasmusson RL, Morales MJ, Trimmer JS, Nerbonne M, Strauss HC. Distinct transient outward potassium current (Ito) phenotypes and distribution in fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes. J Gen Physiol 113: 581–600, 1999.[Abstract/Free Full Text]
  9. Braun AP, Fedida D, Clark RB, Giles WR. Intracellular mechanisms for {alpha}1-adrenergic regulation of the transient outward current in rabbit atrial myocytes. J Physiol 431: 689–712, 1990.[Abstract/Free Full Text]
  10. Capasso JM, Li P, Anversa P. Cytosolic calcium transients in myocytes isolated from rats with ischemic heart failure. Am J Physiol Heart Circ Physiol 265: H1953–H1964, 1993.[Abstract/Free Full Text]
  11. Casis O, Iriarte M, Gallego M, Sanchez-Chapula JA. Differences in regional distribution of K+ current densities in rat ventricle. Life Sci 63: 391–400, 1998.[CrossRef][Web of Science][Medline]
  12. Crumb WJ, Munfakh N, Heck HA, Harrison LH. Fatty acid block of the transient outward current in adult human atrium. J Pharmacol Exp Ther 289: 386–391, 1999.[Abstract/Free Full Text]
  13. Dameron DS, Van Wagoner DR, Moravec CS, Bond M. Arachidonic acid and endothelin potentiate Ca2+ transients in rat cardiac myocytes via inhibition of distinct K+ channels. J Biol Chem 268: 27335–27344, 1993.[Abstract/Free Full Text]
  14. De Waard MC, van der Velden J, Bito V, Ozdemir S, Biesmans L, Boontje NM, Dekkers DH, Schoonderwoerd K, Schuurbies HC, de Crom R, Stienen GJ, Sipido KR, Lamers JM, Duncker DJ. Early exercise training normalizes myofilament function and attenuates left ventricular pump dysfunction in mice with a large myocardial infarction. Circ Res 100: 1079–1088, 2007.[Abstract/Free Full Text]
  15. Di Diego JM, Sun ZQ, Antzelevitch C. Ito and the action potential notch are smaller in left vs. right canine ventricular epicardium. Am J Physiol Heart Circ Physiol 271: H548–H561, 1996.[Abstract/Free Full Text]
  16. Escriba PV. Membrane-lipid therapy: a new approach in molecular medicine. Trends Mol Med 12: 34–43, 2006.[CrossRef][Web of Science][Medline]
  17. Escriba PV, Sanchez-Dominguez JM, Alemany R, Perona JS, Ruiz-Gutiérrez V. Alterations of lipids, G-proteins, and PKC in cell membranes of elderly hypertensives. Hypertension 41: 176–182, 2003.[Abstract/Free Full Text]
  18. Escudero A, Montilla JC, Garcia JM, Sanchez-Quevedo MC, Periago JL, Hortelano P, Suarez MD. Effect of dietary (n-9), (n-6) and (n-3) fatty acids on membrane lipid composition and morphology of rat erytrocytes. Biochim Biophys Acta 1394: 65–73, 1998.[Medline]
  19. Ferrara LA, Raimondi AS, d'Episcopo L, Guida L, Dello Russo A, Marotta T. Olive oil and reduced need for antihypertensive medications. Arch Intern Med 160: 837–842, 2000.[Abstract/Free Full Text]
  20. Foulon V, Sniekers M, Huysmans E, Asselberghs S, Mahieu V, Mannaerts GP, Van Veldhoven PP, Casteels M. Breakdown of 2-hydroxylated straight chain fatty acids via peroxisomal 2-hydroxyphytanoyl-CoA lyase: a revised pathway for the alpha-oxidation of straight chain fatty acids. J Biol Chem 280: 9802–9812, 2005.[Abstract/Free Full Text]
  21. Funari SS, Barcelo F, Escriba PV. Effects of oleic acid and its congeners, eliadic and stearic acids, on the structural properties of phosphatidylethanolamine membranes. J Lipid Res 44: 567–575, 2003.[Abstract/Free Full Text]
  22. Gallego M, Setien R, Puebla L, Boyano-Adanez Mdel C, Arilla E, Casis O. {alpha}1-Adrenoceptors stimulate a G{alpha}s protein and reduce the transient outward K+ current via a cAMP/PKA-mediated pathway in the rat heart. Am J Physiol Cell Physiol 288: C577–C585, 2005.[Abstract/Free Full Text]
  23. Jude S, Bedut S, Roger S, Pinault M, Chameroux P, White E, Le Guennec JY. Peroxidation of docosahexaenoic acid is responsible for its effects on Ito and Iss in rat ventricular myocytes. Br J Pharmacol 139: 816–822, 2003.[CrossRef][Web of Science][Medline]
  24. Kang JX, Leaf A. Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins. Proc Natl Acad Sci USA 93: 3542–3546, 1996.[Abstract/Free Full Text]
  25. Kondo RP, Dederko DA, Teutsch C, Chrast J, Catalucci D, Chien KR, Giles WR. Comparison of contraction and calcium handling between right and left ventricular myocytes from adult mouse heart: a role for repolarization waveform. J Physiol 571: 131–146, 2006.[Abstract/Free Full Text]
  26. Leaf A, Kang JX, Xiao YF, Billman GE. Clinical prevention of sudden cardiac death by n-3 polyunsaturated fatty acids and mechanism of prevention of arrhythmias by n-3 fish oils. Circulation 107: 2646–2652, 2003.[Free Full Text]
  27. Leaf A, Xiao YF, Kang JX. Interactions of n-3 fatty acids with ion channels in excitable tissue. Prostaglandins Leukot Essent Fatty Acids 67: 113–120, 2002.[CrossRef][Web of Science][Medline]
  28. Martinez J, Vögler O, Casas J, Barcelo F, Alemany R, Prades J, Nagy T, Baamonde C, Kasprzyk PG, Teres S, Saus C, Escriba PV. Membrane structure modulation, protein kinase C{alpha} activation and anticancer activity of Minerval. Mol Pharmacol 67: 531–540, 2005.[Abstract/Free Full Text]
  29. McCrossan ZA, Billeter R, White E. Transmural changes in size, contractile and electrical properties of SHR in left ventricular myocytes during compensated hypertrophy. Cardiovasc Res 63: 283–292, 2004.[Abstract/Free Full Text]
  30. Nerbonne JM. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol 525: 285–298, 2000.[Abstract/Free Full Text]
  31. Ostadal B, Mirejovska E, Hurych J, Pelouch V, Prochazka J. Effect of intermittent high altitude hypoxia on the synthesis of collagenous and non-collagenous proteins of the right and left ventricular myocardium. Cardiovasc Res 12: 303–308, 1978.[Abstract/Free Full Text]
  32. Patel SP, Campbell DL. Transient outward potassium current, ‘Ito’, phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms. J Physiol 569: 7–39, 2005.[Abstract/Free Full Text]
  33. Po SS, Wu RC, Juang GJ, Kong W, Tomaselli GF. Mechanism of {alpha}-adrenergic regulation of expressed hKV4.3 currents. Am J Physiol Heart Circ Physiol 281: H2518–H2527, 2001.[Abstract/Free Full Text]
  34. Prades J, Funari SS, Escriba PV, Barcelo F. Effects of unsaturated fatty acids and triacylglycerols on phosphatidylethanolamine membrane structure. J Lipid Res 44: 1720–1727, 2003.[Abstract/Free Full Text]
  35. Rosati B, Grau F, Rodriguez S, Li H, Nerbonne JM, McKinnon D. Concordant expression of KCHIP2 mRNA, protein and transient outward current throughout the canine ventricle. J Physiol 548: 815–822, 2003.[Abstract/Free Full Text]
  36. Sabbah HN, Haddad W, Mika Y, Nass O, Aviv R, Sharov VG, Maltsev V, Felzen B, Undrovinas AI, Goldstein S, Darvish N, Ben-Haim SA. Cardiac contractility modulation with the impulse dynamics signal: studies in dogs with chronic heart failure. Heart Fail Rev 6: 45–53, 2001.[CrossRef][Medline]
  37. Sah R, Ramirez RJ, Kaprielian R, Backx PH. Alterations in action potential profile enhance excitation-contraction coupling in rat cardiac myocytes. J Physiol 533: 201–214, 2001.[Abstract/Free Full Text]
  38. Sah R, Ramirez RJ, Oudit GY, Gidrewicz D, Trivieri MG, Zobel C, Backx PH. Regulation of cardiac excitation-contraction coupling by action potential repolarization: role of the transient outward potassium current (Ito). J Physiol 546: 5–18, 2003.[Abstract/Free Full Text]
  39. Sathish V, Xu A, Karmazyn M, Sims SM, Narayanan N. Mechanistic basis of difference in calcium handling properties of sarcoplasmic reticulum in the right and left ventricles of normal rat myocardium. Am J Physiol Heart Circ Physiol 291: H88–H96, 2006.[Abstract/Free Full Text]
  40. Shipsey SJ, Bryant SM, Hart G. Effects of hypertrophy on regional action potential characteristics in rat left ventricle. Circulation 96: 2061–2068, 1997.[Abstract/Free Full Text]
  41. Suarez J, Gloss B, Belke DD, Hu Y, Scott B, Dieterle T, Kim YK, Valencik ML, McDonald JA, Dillmann WH. Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy. Am J Physiol Heart Circ Physiol 287: H2164–H2172, 2004.[Abstract/Free Full Text]
  42. Visioli F, Caruso D, Grande S, Bosisio R, Villa M, Galli G, Sirtori S, Galli C. Virgin Olive Oil Study (VOLOS): vasoprotective potential of extra virgin olive oil in mildly dislipidemic patients. Eur J Nutr 44: 121–127, 2005.[CrossRef][Web of Science][Medline]
  43. Vögler O, Casas J, Capo D, Nagy T, Borchert G, Martorell G, Escriba PV. The Gβ{gamma}-dimer drives the interaction of the heterotrimeric Gi proteins with nonlamelar membrane structures. J Biol Chem 279: 36540–36545, 2004.[Abstract/Free Full Text]
  44. Wu S, Li HY, Wong TM. Cardioprotection of preconditioning by metabolic inhibition in the rat ventricular myocyte. Involvement of {kappa}-opioid receptor. Circ Res 84: 1388–1395, 1999.[Abstract/Free Full Text]
  45. Yang Q, Alemany R, Casas J, Kitajka K, Lanier SM, Escriba PV. Influence of the membrane lipid structure on signal processing via G protein-coupled receptors. Mol Pharmacol 68: 210–217, 2005.[Abstract/Free Full Text]
  46. Zicha J, Kunes J, Devycnk MA. Abnormalities of membrane function and lipid metabolism in hypertension. Am J Hypertens 12: 315–331, 1999.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/H1948    most recent
01209.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Borchert, G. H.
Right arrow Articles by Escriba, P. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Borchert, G. H.
Right arrow Articles by Escriba, P. V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.