PageArticles 1 of 26 in PresS. Am J Physiol Heart Circ Physiol (February 22, 2008). doi:10.1152/ajpheart.01209.2007 1
2-Hydroxyoleic acid affects cardiomyocyte [Ca2+]i transient and contractility in a region-dependent manner
Gudrun H. Borchert1,2, Mike Giggey3, Frantisek Kolar2, Tak Ming Wong4, Peter H. Backx3, Pablo V. Escriba1
1
Laboratory 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; 4
Department of Physiology, The University of Hong Kong, Hong Kong, China
Correspondence to Gudrun H. Borchert, PhD, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic phone: +420.241062559, fax: +420.241062125; Email:
[email protected]
Short title: 2-OHOA increases Ca2+-transients and cell shortening
Number of words: 5945
Keywords: calcium, contractile function, fatty acid, cardiomyocytes, protein kinase C, signal transduction
Copyright Information Copyright © 2008 by the American Physiological Society.
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ABSTRACT Monounsaturated fatty acids such as oleic acid are cardioprotective, modify the physico-chemical properties of cardiomyocyte membranes and affect the electrical stability of these cells by regulating the conductance of ion channels. We have designed a non-hydrolysable oleic acid derivative, 2hydroxyoleic 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 translocation to the membrane; both pathways are thought to regulate 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 2OHOA on cytosolic [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 sub-populations of myocytes from 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 transients and cell shortening, PKC
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 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.
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INTRODUCTION
Unsaturated fatty acids have been shown to be cardioprotective (1, 2, 6, 7, 13, 19, 23, 24, 26, 27, 46). They can alter the physico-chemical 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, 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 2hydroxyoleic acid (2-OHOA), can affect membrane lipid structure and cell signaling, leading to blood pressure reductions in hypertensive humans and animals (spontaneous hypertensive rats (SHR)) (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), which led to decreased blood pressure in SHR rats. This blood pressure lowering effect was partially reversed in vivo (by about 60 %) upon administration of the PKA inhibitor Rp-8Br-cAMP (2). While 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 potassium current (Ito) in the human right atrium (12).
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Blockade of Ito by oleic acid (12) or by
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 (LVM), right ventricular (RVM) and septum myocytes (SEPM) separately. There are no studies available about the effect of 2-OHOA on isolated myocytes, so that we first examined potential toxic effects by evaluating cell viability. Then we studied [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 sub-populations of ventricular myocytes likely by inhibiting Ito in a PKC -independent manner. These results may in part explain the beneficial cardiovascular effects of this drug.
MATERIALS AND METHODS The study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals Committee of the University of Toronto and 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’s solution (mmol/L): 140 NaCl, 5.4 KCl, 10 Hepes, 1 MgCl2, 1 CaCl2 and 10 D-glucose (pH
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7.4) at 37°C (37) and then with Ca2+-free Tyrode’s solution for 5 min prior to digestion with the same solution containing collagenase (Type II, 20 mg, 700 units/mg; Yakult, Tokio, Japan) and protease (Type XIV, 0.2 mg/ml) for 7–9 min. Collagenase was subsequently removed by perfusion with Ca2+-free Tyrode’s solution at pH 7.4 for 5 min. All the solutions were gassed with 100 % O2 for 5 min prior to use. Then, atria and blood vessels were 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’s 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 cytosolic [Ca2+]i transients and cell shortening. Myocytes isolated from the same part of each heart were randomly divided into two groups (control or treated) to minimize inter-experimental 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 Ng/ml streptomycin. Myocytes were kept in a CO2 incubator (95 % air/ 5 % CO2, 28°C) in the presence or absence (control) of 10 or 50 NM 2-OHOA for 1, 8 or 20 h. In a separate series of experiments, myocytes were incubated with 2 mM 4-AP for 15 min before commencing the protocol described above to block Ito. Only RVM and SEPM were analyzed for this set of experiments to avoid un-interpretable 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 NM 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 NM 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 6-8 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 Copyright Information
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with Tyrode’s 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). Intracellular [Ca2+]i transients were measured in myocytes loaded with 10 µmol/L indo-1-AM (Molecular Probes, Burlington, Ontario, Canada) and excited at 360 nm. The ratio of fluorescence intensity between light emission at 405 nm and 495 nm was used to measure [Ca2+]i transients, 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 level, 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-, negatively- or non-responding 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 mean ± SE from 12-20 myocytes in each group out of 4 independent experiments. PKC translocation. After isolation, myocytes from RV, LV and septum were separated into 4 groups as described above. After the treatment, myocytes were harvested and stored at -80°C until use. Positively, negatively and non-responding cells could not be separated for this type of experiments because they only can be differentiated after electrical stimulation. Myocyte cytosolic Copyright Information
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or membrane (lysate) proteins (100 Ng) were fractionated on 10 % SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked overnight at 4°C in PBS (Gibco, Carlsbad, California) containing 5 % non-fat dry milk, 0.5 % bovine serum albumin and 0.1 % Tween 20. PVDF membranes were then incubated with the primary antibody (anti-PKC 1:200, anti-GAPDH 1:10,000, or anti-calsequestrin 1:1,000) for 1 h. Anti-GAPDH was obtained from Ambion Europe Ltd (Cambridgeshire, United Kingdom) and the remaining antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). After removing the primary antibody, the membranes were washed three times for 10 min each with phosphate buffered saline (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, United Kingdom). 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 were calculated. Results are expressed as mean ± SE of 6 independent experiments. Statistical analysis. Data are expressed as mean values ± 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
