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Am J Physiol Heart Circ Physiol 294: H2775-H2784, 2008. First published April 25, 2008; doi:10.1152/ajpheart.00269.2008
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A cardioprotective role for platelet-activating factor through NOS-dependent S-nitrosylation

Peter J. Leary,1,* Surender Rajasekaran,1,* R. Ray Morrison,1 Elaine I. Tuomanen,2 Thomas K. Chin,3 and Polly A. Hofmann4

1Division of Critical Care Medicine and 2Department of Infectious Diseases, St. Jude Children's Research Hospital, and Departments of 3Pediatrics and 4Physiology, University of Tennessee Health Sciences Center, Memphis, Tennessee

Submitted 12 March 2008 ; accepted in final form 18 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Controversy exists as to whether platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, can actually protect the heart from postischemic injury. To determine whether endogenous activation of the PAF receptor is cardioprotective, we examined postischemic functional recovery in isolated hearts from wild-type and PAF receptor-knockout mice. Postischemic function was reduced in hearts with targeted deletion of the PAF receptor and in wild-type hearts treated with a PAF receptor antagonist. Furthermore, perfusion with picomolar concentrations of PAF improved postischemic function in hearts from wild-type mice. To elucidate the mechanism of a PAF-mediated cardioprotective effect, we employed a model of intracellular Ca2+ overload and loss of function in nonischemic ventricular myocytes. We found that PAF receptor activation attenuates the time-dependent loss of shortening and increases in intracellular Ca2+ transients in Ca2+-overloaded myocytes. These protective effects of PAF depend on nitric oxide, but not activation of cGMP. In addition, we found that reversible S-nitrosylation of myocardial proteins must occur in order for PAF to moderate Ca2+ overload and loss of myocyte function. Thus our data are consistent with the hypothesis that low-level PAF receptor activation initiates nitric oxide-induced S-nitrosylation of Ca2+-handling proteins, e.g., L-type Ca2+ channels, to attenuate Ca2+ overload during ischemia-reperfusion in the heart. Since inhibition of the PAF protective pathway reduces myocardial postischemic function, our results raise concern that clinical therapies for inflammatory diseases that lead to complete blockade of the PAF receptor may eliminate a significant, endogenous cardioprotective pathway.

cardioprotection; calcium handling; ischemia-reperfusion injury

PLATELET-ACTIVATING FACTOR (PAF) is a phospholipid best known for its ability to mediate proinflammatory effects. PAF can also induce systemic hypotension, coronary vasoconstriction, and cardiac arrhythmias (17). Furthermore, ischemic hearts exposed to nanomolar concentrations of PAF during reperfusion have decreased myocardial functional recovery and an increase in infarct size (7). Consistent with these findings is the observation that PAF receptor antagonists protect the heart from ischemia-reperfusion (I/R) damage (1, 3, 7). However, one study demonstrated reductions in I/R-induced dysfunction and infarct size in hearts pretreated with picomolar concentrations of PAF (18). This finding is consistent with the observation that PAF receptor antagonists do not always reduce myocardial infarct size (4, 18). Thus the first aim of the present study was to use PAF receptor agonists and antagonists and hearts with a targeted deletion of the PAF receptor to definitively establish whether low levels of PAF receptor activation attenuate I/R-induced damage in the heart.

Most of the effects of PAF are mediated through a dedicated G protein-coupled receptor. PAF receptor activation has been linked to increases in phosphatidylinositol (3,4,5)-triphosphate and production of nitric oxide (NO) (1, 2, 24). Increased NO production has been shown to alter intracellular Ca2+ handling in cardiac myocytes, and NO-dependent decreases in L-type Ca2+ channel activity have been shown to correlate to a decrease in I/R injury in hearts (6, 10, 29). These observations led us to hypothesize that a cardioprotective effect of PAF may be mediated through an NO-induced reduction in intracellular Ca2+ overload during and after myocardial I/R. To test for a relationship between PAF and intracellular Ca2+ homeostasis, we employed a nonischemic, cardiac myocyte model of Ca2+ overload and contractile dysfunction. This model allowed us to address the second aim of the present study, which was to determine whether PAF protects cells from contractile dysfunction through an NO-dependent decrease in intracellular Ca2+ overload.

NO effects can be cGMP dependent or cGMP independent. One of the increasingly studied alternatives to the NO-cGMP pathway is posttranslational S-nitrosylation of proteins. For example, knocking out neuronal NO synthase (NOS1) reduces S-nitrosylation of the ryanodine receptor on the sarcoplasmic reticulum, which leads to diastolic Ca2+ leak in cardiac myocytes (9). Furthermore, NO-induced S-nitrosylation of the L-type Ca2+ channel correlates with gender-dependent reduction in I/R injury in ventricular myocytes (29). Thus the third aim of the present study was to investigate the role of cGMP and S-nitrosylation in PAF-dependent cardioprotection from I/R injury.

If low-level release of PAF acts in a paracrine or autocrine fashion to activate PAF receptors on cardiac myocytes and initiate an endogenous protective pathway during I/R, then a better understanding of such a pathway may ultimately lead to the development of novel clinical therapies. Furthermore, if such a pathway exists, consideration needs to be given to the possibility that therapies used to dramatically reduce PAF in inflammatory diseases may have unintended consequences to the heart. With this in mind, the present study explores a possible protective role for PAF receptor activation in limiting myocardial I/R-induced dysfunction, examines changes in Ca2+ handling as one potential end target of a PAF-mediated cardioprotection, and characterizes the relative contribution of cGMP and S-nitrosylation to PAF-dependent NO effects.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal procedures were approved by the Animal Care and Use Committees at the University of Tennessee Health Science Center and St. Jude Children's Research Hospital. Male and female 12- to 16-wk-old C57BL/6 wild-type or PAF receptor-knockout mice were utilized. Generation of mice lacking the PAF receptor is extensively described by Radin et al. (21). PAF receptor knockout was confirmed using PCR (21). Male and female Wistar rats were purchased from Harlan. All animals were allowed free access to standard laboratory chow and water.

Pharmacological agents. PAF was used at 0.1 pM, since this concentration does not alter myocardial contractility (18). The PAF receptor antagonist BN-52021 was used at 1 µM, inasmuch as a similar concentration of BN-52021 blocks the PAF-induced release of atrial natriuretic factor from rat hearts (22). Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-BrcGMPs), a cell-permeable cGMP antagonist, and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, were used at 50 µM and 0.4 µM, respectively. Control experiments (see RESULTS) were done to ensure that these concentrations of Rp-8-BrcGMPs and ODQ inhibit known cGMP effects in ventricular myocytes. N-nitro-L-arginine (L-NNA), a competitive inhibitor of NOS with selectivity for the neuronal and endothelial isoforms, was used at 0.1 µM. The L-NNA inhibitor constant is 0.09 µM for neuronal NOS, 0.02 µM for endothelial NOS, and >25 µM for inducible NOS (23). 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium (DEA-NO), an NO donor, was used at 1 nM, since at this concentration it has no effect on twitch amplitude (15). DEA-NO was also used at 100 µM to inhibit shortening of cardiac myocytes, as described by Layland et al. (15). A stock of DEA-NO was kept in aliquots at –80°C and brought to room temperature 5 min before use. Sodium ascorbate, a reducing agent for S-nitrosylation, was used at 1 mM, inasmuch as this concentration it is not toxic to cells over a 1-h exposure time (26). Ascorbate was made immediately before use. Agents were purchased from Sigma Chemical or BIOMOL.

Langendorff-perfused hearts. Mice were anesthetized with pentobarbital sodium, and the excised heart was perfused in a retrograde fashion with a modified Krebs bicarbonate buffer (in mM: 119 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 1.2 KH2PO4, 11 glucose, 2.5 CaCl2, 0.5 pyruvate, and 0.5 EDTA). Krebs buffer was maintained at 37°C and pH 7.4 by bubbling with 95% O2-5% CO2. Perfusion was at a constant pressure of 80 mmHg and constant temperature of 37°C. Hearts were immersed in a water-jacketed perfusate bath maintained at 37°C. Aortic pressure was monitored using a pressure transducer (CAPTO SP844, ADInstruments) in-line with the aortic cannula. A 0.6-mm-diameter cellophane balloon was inserted into the left ventricle and connected to a pressure transducer to monitor contractile function. Balloon volume was adjusted to maintain a left ventricular diastolic pressure (LVDP) of 2–5 mmHg. External pacing at 7 Hz was set at a voltage 20% greater than threshold. Systolic pressure, end-diastolic pressure (EDP), temperature, ±dP/dt, and aortic pressure were constantly recorded (Power Lab, ADInstruments). LVDP was calculated as the difference between systolic pressure and diastolic pressure. Coronary effluent rate was measured every 10 min.

After instrumentation, baseline measurements were obtained for 30 min. Hearts that were unable to maintain LVDP >90 mmHg during this equilibration period were excluded from data analysis. After equilibration, global ischemia for 15 or 20 min was produced by clamping the aortic cannula and simultaneously bubbling 95% N2-5% CO2 into the organ bath. Pacing was discontinued during global ischemia. At the end of ischemia, hearts were reperfused for 30 min. In some experiments, hearts were treated with 0.1 pM PAF or 1 µM BN-52021 during equilibration and reperfusion. PAF and BN-52021 were delivered to the perfusate immediately above the aorta at a rate of 0.02 ml/min by a syringe pump.

Detection of S-nitrosylation. A separate set of Langendorff-perfused hearts were used to detect S-nitrosylation. Wild-type mouse hearts were initially perfused with 100 µM L-NNA for 30 min, 10 µM ODQ for 10 min, or vehicle. Subsequently, hearts were perfused for 10 min with 0.1 pM PAF, 100 µM sodium ascorbate, PAF + L-NNA, PAF + ODQ, PAF + ascorbate, or vehicle. Hearts were then subjected to 20 min of ischemia followed by 5 min of reperfusion, and ventricles were homogenized in 1 ml of HEN buffer [250 mM HEPES (pH 7.7), 1 mM EDTA, and 0.1 mM neocuproine]. Homogenates were centrifuged at 2,000 g for 10 min at 4°C, and 250 µl of the supernatant were mixed with 800 µl of HEN buffer containing freshly prepared S-methyl methanethiosulfonate [10% (vol/vol) in N,N-dimethylformamide] and SDS [25% (vol/vol)] to final concentrations of 0.1% and 2.5%, respectively. This mixture was incubated at 50°C for 20 min. Proteins were then precipitated with 10 ml of chilled acetone at –20°C for 15 min. After centrifugation at 2,000 g for 10 min, the pellet was carefully separated and resuspended in 180 µl of HEN buffer containing 1% SDS. For biotinylation of cysteines, 60 µl of biotin-HPDP (2.5 mg/ml in Me2SO) and 10 µl of freshly prepared sodium ascorbate in HEN buffer were added, and the samples were incubated for 1 h. Samples were stored with an equal volume of Laemmli buffer without DTT at –20°C. Processing of the tissue and labeling reactions were conducted in the dark. This protocol was based on that of Jaffrey and Snyder (11). For detection of protein biotinylation, protein concentration in stored samples was equalized to 2.5 µg/µl, and 20 µl of each sample were run on an 8% polyacrylamide gel and then immunoblotted with an anti-biotin antibody (1:4,000 dilution).

Isolation of rat ventricular myocytes. Ventricular myocytes were isolated using a modification of the procedure of Liu and Hofmann (16). Briefly, hearts were cannulated and perfused at 37°C with Ca2+-free Ringer solution containing 0.5 mM EDTA for 2 min and then with Ringer solution containing 0.001 g/ml type II collagenase (Worthington). Collagenase perfusion continued until the initial coronary effluent drip rate doubled (~25 min). Ventricles were then chopped into two pieces and sequentially incubated with Ca2+-free Ringer solution containing 1) 0.05% BSA with 0.0005 g/ml collagenase for 5 min and 2) 0.1% BSA for 5 min. Any remaining pieces of tissue were triturated, and released cells were transferred to a Ca2+-free Ringer solution containing 0.1% BSA for 10 min. Cells were then exposed to Ringer solution containing 1.25 mM CaCl2 and 0.1% BSA, and photomicrographs were obtained. Isolations containing a <50% yield of rectangular-shaped myocytes relative to total myocytes + myoballs were not used. Ringer solution contained (in mM) 118 NaCl, 4.8 KCl, 2 KH2PO4, 5 pyruvate, 25 HEPES (pH 7.4), 3.4 MgCl2, and 7.5 glucose.

Unloaded shortening of ventricular myocytes. Cells in Ringer solution containing 1.25 mM CaCl2 and 0.1% BSA were placed in a field stimulation chamber mounted on an inverted microscope. In the absence of electrical stimulation, noncontracting myocytes with a cell length ≥100 µm and a length-to-width ratio >5 were selected for use. Cells were then stimulated with a voltage 1.5 times threshold at 0.5 Hz. A charge-coupled device video camera (model JE7326, Javelin) was used to collect images, and the output was displayed on a monitor and stored. Cell shortening was measured using a video edge detector (Crescent Electronics, Crescent, CO). Unloaded fractional shortening was determined from the average of peak change in cell length relative to total cell length at rest from a train of five contractions at the time point indicated. Fractional shortening was measured at room temperature.

A PAF dose-response curve was obtained by increasing PAF concentration every 5 min and measuring peak fractional shortening. No more than four different PAF concentrations were tested per cell to avoid rundown in fractional shortening. These experiments were done in Ringer solution containing 1.25 mM CaCl2. Data were expressed as percent change in peak fractional shortening (PS) compared with fractional shortening obtained in the absence of PAF.

High extracellular Ca2+ challenge experiments were carried out in cells pretreated with 1.25 mM CaCl2 Ringer solution containing 0.1 pM PAF, 1 µM BN-52021, 0.4 µM ODQ, 50 µM Rp-8-BrcGMPs, 0.1 µM L-NNA, 1 nM DEA-NO, 5 mM sodium ascorbate, 0.1% ethanol (vehicle), or a combination of these agents. The cells were pretreated for 5 min with pacing, and cells demonstrating >10% variability of PS over this period were discarded. After a 5-min exposure, baseline PS was recorded for use in the normalization of data. Bathing Ca2+ was then increased to 6 mM CaCl2 for 10 min with pacing, and PS was recorded only at 1, 5, and 10 min. At the end of 10 min of exposure to 6 mM CaCl2, the bath extracellular Ca2+ was reduced to 2.1 mM CaCl2 without a change in the concentration of the pharmacological agent(s). PS was monitored over the subsequent 20 min. Data are presented as percent change in PS compared with fractional shortening obtained immediately before exposure to high Ca2+. It should be noted that after the protective effect of PAF was established using PAF and the PAF receptor antagonist BN-52021, cells from isolations that did not demonstrate a robust protective effect of PAF were not used (3 of a total of 24 isolations).

Measurement of intracellular Ca2+. Myocytes were loaded with 5 µM fura 2-AM (Invitrogen, Carlsbad, CA) in Ringer solution for 15 min at room temperature, washed three times in Ringer solution containing 1.25 mM CaCl2 and 0.1% BSA, and allowed to sit for ≥20 min to ensure deesterification. Cells were then stored on ice to lessen compartmentalization.

Fura-loaded cells were incubated for 5 min with 0.1 µM L-NNA, 0.4 µM ODQ, 5 mM ascorbate, or vehicle, placed in a low-volume chamber on the stage of an inverted microscope, and paced. Fluorescence was recorded at 510 nm after excitation from light passed through a 340- or 380-nm filter using a dual-excitation fluorescence photomultiplier system (IonOptix, Milton, MA). Fluorescence ratios were compared to give relative Ca2+ changes between groups (see below). All measurements were conducted at room temperature. Fluorescence was measured before and after PAF or vehicle exposure and after a high-Ca2+ challenge. The Ca2+ transients of cells paced at 0.5 Hz in a solution containing 1.25 mM Ca2+ were recorded. Pacing was then stopped, and 0.1 pM PAF or vehicle was added to the bath for a 5-min period of equilibration. Pacing was resumed, and Ca2+ transients were again recorded. Cells were then challenged by an immediate increase in extracellular Ca2+ to 4 mM.

To ensure consistency of response over the period of data collection, the first and last cell of the day were exposed to 20 mM caffeine. The peak amplitude of the Ca2+ transient induced by caffeine changed by <10% from the first to the last cell in all isolations. Furthermore, fluorescence adjacent to cells was monitored and found to be consistent and minimal between isolations over the entire period of data collection; i.e., none of the isolations had cells with a higher-than-normal release of fura into the bathing solution. Finally, neither the addition of caffeine nor high extracellular Ca2+ altered the fluorescence of the extracellular bathing medium.

Statistical analysis. Values are means ± SE. Statistical significance was assumed for P < 0.05. Data were analyzed by a standard ANOVA followed by Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PAF effects on isolated heart postischemic recovery. Isolated hearts from wild-type mice were equilibrated with 0.1 pM PAF or vehicle for 30 min, and LVDP and EDP were measured (Fig. 1). PAF had no effect on preischemic LVDP or EDP. After the 30-min equilibration period, hearts were subjected to 20 min of global ischemia and 30 min of reperfusion. Postischemic LVDP was significantly greater in PAF- than in vehicle-treated hearts (74.6 ± 4.0% vs. 46.1 ± 6.2%; Fig. 1A). Consistent with improved postischemic LVDP, EDP was lower in PAF- than in vehicle-treated hearts (Fig. 1B).


Figure 1
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  		Fig. 1. Effect of 0.1 pM platelet-activating factor (PAF) on isolated heart left ventricular developed pressure (LVDP; A) and end-diastolic pressure (EDP; B) before and after 20 min of global ischemia. LVDP is expressed relative to preischemic baseline. Values are means ± SE of 8 hearts in each group. *P < 0.05.

 
Hearts from wild-type mice were equilibrated with 1 µM BN-52021, a PAF receptor antagonist, or vehicle for 30 min, and LVDP and EDP were measured (Fig. 2). BN-52021 had no effect on preischemic LVDP or EDP. After the 30-min equilibration, hearts were subjected to 15 min of global ischemia and 30 min of reperfusion. Postischemic LVDP was significantly reduced in BN-52021- compared with vehicle-treated hearts (42.0 ± 4.7% vs. 71.0 ± 4.6%; Fig. 2A). Consistent with impaired postischemic LVDP, EDP was significantly higher in BN-52021- than in vehicle-treated hearts (Fig. 2B).


Figure 2
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  		Fig. 2. Effect of 1 µM BN-52021, a PAF receptor antagonist, on isolated heart LVDP (A) and EDP (B) before and after 15 min of global ischemia. LVDP is expressed relative to preischemic baseline. Values are means ± SE of 8 hearts in each group. *P < 0.05.

 
Functional recovery from global ischemia was evaluated in hearts from PAF receptor-knockout mice (Fig. 3). Deletion of the PAF receptor did not significantly alter preischemic LVDP (127.3 ± 2.5 and 133.3 ± 10.4 mmHg in wild-type and PAF receptor-knockout hearts, respectively, n = 8). However, after 20 min of global ischemia and 30 min of reperfusion, LVDP was significantly reduced and EDP was significantly higher in PAF receptor-knockout hearts than in hearts from wild-type mice (Fig. 3). Furthermore, postischemic troponin I content in the myocardial effluent was significantly greater in PAF receptor-knockout hearts than in hearts from wild-type mice (Fig. 3C). Before ischemia, troponin I released into the myocardial effluent was not different [10.1 ± 1.9 (n = 6) and 6.8 ± 1.3 (n = 7) ng/g in wild-type and PAF receptor-knockout hearts, respectively].


Figure 3
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  		Fig. 3. Effect of PAF receptor deletion on isolated heart LVDP (A), EDP (B), and postischemic cardiac effluent troponin I (TnI) content (C). After equilibration, hearts from PAF receptor-knockout (KO) and wild-type mice were subjected to 20 min of global ischemia followed by 30 min of reperfusion. Values are means ± SE of 8 (LVDP and EDP) and 6–7 (TnI) hearts in each group. *P < 0.05.

 
PAF effects on ventricular myocyte shortening. Isolated ventricular myocytes were exposed to increasing concentrations of PAF, and the peak shortening was determined. PAF at 0.01–1 pM had no effect on peak shortening (Fig. 4A). Exposure to ≥10 pM PAF led to a progressive decrease in peak shortening. To minimize the potentially confounding influence of this negative inotropic effect on data interpretation, 0.1 pM PAF was used in all subsequent experiments.


Figure 4
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  		Fig. 4. Ventricular myocyte shortening as a function of extracellular PAF concentration (A) or after Ca2+ overload in myocytes treated with a PAF receptor agonist and/or antagonist (B). A: for a given PAF concentration, change in peak shortening was normalized to peak shortening in the absence of PAF. Values are means ± SE from 4–5 cells. {dagger}P < 0.05 vs. absence of PAF. B: ventricular myocytes were pretreated with vehicle, 0.1 pM PAF, 1 µM BN-52021 (a PAF receptor antagonist), or PAF + BN-52021. Myocyte shortening was continually monitored during 10 min of exposure to Ringer solution containing 6 mM Ca2+ (high Ca2+; 0–10 min) and on return to Ringer solution containing 2 mM Ca2+ (10–30 min on x-axis). Peak shortening at a given time was normalized to peak shortening immediately before high-Ca2+ exposure (time 0) for each cell. Values are means ± SE. *P < 0.05 vs. vehicle. Significant differences were also observed at 15–30 min between BN-52021- and vehicle-treated cells.

 
Isolated ventricular myocytes were subjected to Ca2+ overload by exposure of cells to 6 mM extracellular Ca2+ for 10 min followed by a return to a physiological Ca2+ (Fig. 4B) to mimic the rise in intracellular Ca2+ that occurs during and after I/R. Cells were pretreated with vehicle, PAF, and/or the PAF receptor antagonist BN-52021. During the 10 min of high extracellular Ca2+ exposure, the functional response of the cells was highly variable and did not reach a statistically significant difference between groups. At 20 min after Ca2+ challenge, peak shortening decreased 64.3 ± 4.1% in the vehicle-treated cells. PAF reduced myocyte dysfunction, with a loss in peak shortening of only 14.6 ± 2.4% at 20 min. Coexposure to PAF and the PAF receptor antagonist BN-52021 blocked the PAF protective effect, with a 77.4 ± 6.5% decrease in peak shortening at 20 min after Ca2+ challenge. In the absence of PAF, loss of peak shortening was significantly greater in BN-52021 than in vehicle-treated cells: 83.4 ± 10.6% vs. 64.3 ± 4.1% decrease in peak shortening at 20 min after Ca2+ challenge.

NO, but not cGMP, involvement in PAF-dependent protection. The protective effect of PAF was blocked by the addition of L-NNA, a competitive arginine inhibitor of NOS (Fig. 5A). Peak shortening in PAF-treated cells decreased 19.4 ± 4.5%, whereas loss of peak shortening in cells treated with L-NNA and L-NNA + PAF was 82.0 ± 4.6% and 76.3 ± 5.7%, respectively. DEA-NO, an NO donor, mimicked the protective effect of PAF when cells were challenged with a Ca2+ overload (Fig. 5B). Exposure of cells to 1 nM DEA-NO before Ca2+ overload led to a 43.1 ± 4.0% decrease in peak shortening compared with vehicle-treated cells, which lost 81.4 ± 4.9% of peak shortening at 20 min after Ca2+ challenge. Previous studies demonstrated that 1 nM DEA-NO is below the concentration of the negative inotropic effect of DEA-NO (15).


Figure 5
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  		Fig. 5. Effect of various inhibitors on PAF and nitric oxide (NO) donor-induced protection from Ca2+ overload. A: effect of pretreatment with vehicle, 0.1 pM PAF, and/or 0.1 µM N-nitro-L-arginine [L-NNA, an inhibitor of NO synthase (NOS1 and NOS3)] on peak shortening in Ca2+-challenged ventricular myocytes. B: effect of pretreatment with vehicle, 1 nM 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium (DEA-NO, an NO donor), and/or 0.4 µM 1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ, an inhibitor of soluble guanylyl cyclase) on peak shortening in Ca2+-challenged ventricular myocytes. C: effect of pretreatment with vehicle, 0.1 pM PAF, and/or 0.4 µM ODQ on peak shortening in Ca2+-challenged ventricular myocytes. D: effect of pretreatment with vehicle, 0.1 pM PAF, and/or 50 µM Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp-8-BrcGMPs, a cGMP antagonist) on peak shortening in Ca2+-challenged ventricular myocytes. Shortening was monitored during 10 min of exposure to Ringer solution containing 6 mM Ca2+ (high Ca2+; 0–10 min) and on return to Ringer solution containing 2 mM Ca2+ (10–30 min). Peak shortening at a given time was normalized to peak shortening immediately before high-Ca2+ exposure (time 0) for each cell. Values are means ± SE. *P < 0.05 vs. vehicle.

 
The PAF and NO protective effects appear to be independent of cGMP. The DEA-NO protective effect was not blocked by pretreatment with ODQ, an inhibitor of soluble guanylyl cyclase (Fig. 5B). The PAF-induced protection against loss of peak shortening after a Ca2+ challenge was also not effected by ODQ (Fig. 5C). Decay of peak shortening in cells treated with PAF or PAF + ODQ was not statistically different (23.3 ± 3.0% and 13.5 ± 7.3%, respectively). Finally, the PAF-induced protection against loss of peak shortening was not affected by Rp-8-BrcGMPs, a cell-permeable cGMP antagonist (Fig. 5D). Decay of peak shortening in cells treated with PAF and PAF + Rp-8-BrcGMPs was not statistically different (15.0 ± 3.7% and 2.0 ± 4.2%, respectively).

Control experiments were done to ensure that ODQ and Rp-8-BrcGMPs were inhibiting cGMP (Fig. 5). Previous studies demonstrated that cGMP activation accounts for the negative inotropic effect of 100 µM DEA-NO (15). First, we confirmed that 100 µM DEA-NO reduces shortening of ventricular myocytes bathed in a Ringer solution containing 1.25 mM CaCl2. In this non-Ca2+-overloaded control, DEA-NO alone caused an immediate decrease in peak shortening of 69.5 ± 11.1% (n = 6) compared with vehicle-treated myocytes (n = 4). This effect was abolished by 4 min of pretreatment with 0.4 µM ODQ, such that peak shortening decreased only 3.7 ± 6.9% in cells treated with ODQ + DEA-NO (n = 6). A similar ability to block the negative inotropic effect of DEA-NO using 50 µM Rp-8-BrcGMPs was seen in three cells (data not shown).

NO dependent S-nitrosylation as a mechanism of PAF protection. NO-dependent S-nitrosylation of proteins was examined as a possible mechanism of PAF-induced protection. Hearts from wild-type mice were perfused with PAF, L-NNA, ODQ, and/or the S-nitrosylating reducing agent sodium ascorbate. All hearts then underwent 20 min of ischemia followed by 5 min of reperfusion. To monitor the S-nitrosylation of cysteine moieties on proteins, nitrosylated groups were converted to stable biotinylations. A protein with a molecular mass of ~220 kDa, consistent with the mass of the {alpha}1-subunit of the L-type Ca2+ channel, was biotinylated/nitrosylated by PAF (Fig. 6A). L-NNA, an inhibitor of NOS, reduced PAF-dependent biotinylation/nitrosylation of the 220-kDa protein (Fig. 6A). ODQ, an inhibitor of guanylyl cyclase, did not reduce PAF-dependent biotinylation/nitrosylation of the 220-kDa protein (Fig. 6A). Sodium ascorbate blocked PAF-dependent biotinylation/nitrosylation of the 220-kDa protein (Fig. 6A). Furthermore, nonischemic ventricular myocytes pretreated with ascorbate and then exposed to PAF demonstrated a loss of PAF-induced protection in the high extracellular Ca2+ challenge model (Fig. 6B). Ascorbate + PAF led to an 84.7 ± 4.1% decrease in peak shortening compared PAF alone, which caused a 26.6 ± 5.0% decrease in peak shortening at 20 min after Ca2+ challenge.


Figure 6
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  		Fig. 6. Protein nitrosylation in hearts subjected to ischemia-reperfusion (I/R) after pretreatment with PAF and inhibitors to purported mediators of PAF (A) and effect of ascorbate-induced denitrosylation on PAF protection in myocytes (B). A: to determine extent of protein nitrosylation, isolated mouse hearts were perfused with 0.1 pM PAF, 100 µM L-NNA, 10 µM ODQ, 100 µM sodium ascorbate (Asc), or a combination of these agents or left untreated (control) and subjected to 20 min of global ischemia followed by 5 min of reperfusion. Proteins from heart homogenates were then processed for conversion of nitrosylated cysteines to biotinylated cysteines. A ~220-kDa biotin-containing/nitrosylated protein is shown. Numbers in treatment group label denote unique hearts. For all groups, similar results were obtained in ≥3 hearts. B: to determine effect of denitrosylation on the protective effect of PAF, ventricular myocytes were pretreated with vehicle, 0.1 pM PAF, and/or 5 mM ascorbate. Peak shortening was continually monitored during 10 min of exposure to Ringer solution containing 6 mM Ca2+ (high Ca2+; 0–10 min) and on return to Ringer solution containing 2.1 mM Ca2+ (10–30 min). Peak shortening at a given time was normalized to peak shortening immediately before high Ca2+ exposure (time 0) for each cell. Values are means ± SE. *P < 0.05 vs. vehicle.

 
Attenuation of the Ca2+ transient as a mechanism of PAF-dependent protection. A PAF-dependent improvement in intracellular Ca2+ handling was examined as a possible mechanism of PAF-induced protection. We determined basal and peak Ca2+ transients in rat ventricular myocytes treated with PAF or vehicle and then challenged with an increase in extracellular Ca2+ from 1.25 to 4 mM (Fig. 7). PAF reduced the rise in basal Ca2+ and the increase in peak Ca2+ transient after exposure to 4 mM CaCl2 compared with vehicle-treated myocytes (Fig. 7). L-NNA-mediated inhibition of NOS blocked the ability of PAF to attenuate Ca2+ overload during exposure to high extracellular Ca2+ (Fig. 7). However, ODQ-mediated inhibition of guanylyl cyclase did not block the ability of PAF to reduce Ca2+ overload (Fig. 8). Furthermore, ascrobate-mediated denitrosylation blocked the ability of PAF to reduce Ca2+ overload (Fig. 8). These data are consistent with mediation of PAF-dependent attenuation of Ca2+ overload by a cGMP-independent, NO-induced nitrosylation of Ca2+-handling protein(s).


Figure 7
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  		Fig. 7. Effect of high extracellular Ca2+ on intracellular Ca2+ in adult rat ventricular myocytes pretreated with vehicle, 0.1 pM PAF, and/or 0.1 µM L-NNA. A: representative fura ratio (absorbance at 340 nm to absorbance at 380 nm) traces. B: cumulative data of baseline and peak amplitude intracellular Ca2+. Extracellular Ca2+ was increased from 1.25 to 4 mM (arrow). Values obtained in high extracellular Ca2+ solution were normalized to the fura ratio of the cell bathed in lower-Ca2+ Ringer solution. Values are means ± SE. *P < 0.05 vs. vehicle.

 

Figure 8
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  		Fig. 8. Effect of high extracellular Ca2+ on intracellular free Ca2+ in adult rat ventricular myocytes pretreated with 0.4 µM ODQ, ODQ + 0.1 pM PAF, 5 mM ascorbate, or ascorbate + PAF. A: representative fura traces. B: cumulative data of baseline and peak amplitude intracellular Ca2+. Extracellular Ca2+ was increased from 1.25 to 4 mM (arrow). Values obtained in high extracellular Ca2+ solution were normalized to the fura ratio of the cell bathed in lower-Ca2+ Ringer solution. Values are means ± SE. *P < 0.05 vs. ODQ baseline.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study supports the hypothesis that low-level activation of the PAF receptor reduces injury to the heart due to I/R. This effect appears to occur through an NO-dependent decrease in intracellular Ca2+ overload in ventricular myocytes. Furthermore, our studies indicate the PAF-NO protection from Ca2+ overload-induced damage is mediated by an increase in S-nitrosylation of protein(s) responsible for Ca2+ homeostasis.

Various cell types within the heart release PAF during myocardial I/R (27). Prolonged ischemia and the resulting high concentrations of PAF are thought to promote postischemic injury through a direct negative inotropic effect of PAF on the heart and PAF-induced coronary vasoconstriction, as well as arrhythmogenic effects (27). Consistent with the idea that PAF is harmful to the heart, PAF receptor antagonists have been shown to reduce myocardial postischemic contractile dysfunction and infarct size (1, 3, 7, 27). However, not all studies demonstrate that PAF receptor antagonists reduce myocardial infarct size (4, 18). Furthermore, Penna et al. (18) showed that preischemic treatment with 20 pM PAF protects the heart from reperfusion damage and that PAF released during brief cycles of I/R improves postischemic recovery. This suggests that concentrations of PAF in the picomolar range, such as seen with brief periods of ischemia, exercise or at the onset of inflammation, may initiate cellular protective pathways. In the present study, we confirmed that picomolar concentrations of PAF improve myocardial postischemic function. We went on to demonstrate that PAF receptor antagonists given before a brief period of ischemia increase myocardial postischemic dysfunction. Furthermore, we showed that targeted deletion of the PAF receptor reduces the recovery of hearts from I/R. Thus basal-to-low levels of PAF receptor activation are cardioprotective. This finding suggests that a cautious approach may be needed in the clinical use of PAF inhibitors and PAF receptor antagonists in inflammatory diseases (8, 13, 20, 30). Complete inhibition of PAF receptor activation may block endogenous PAF protective mechanism(s), rendering the heart susceptible to ischemic damage.

The mechanism(s) by which low concentrations of PAF improve postischemic myocardial recovery has not previously been studied. However, Pietsch et al. (19) demonstrated that low concentrations of PAF decrease intracellular peak Ca2+ concentration in ventricular myocytes. Since decreasing Ca2+ overload during I/R reduces injury to the heart (14, 28), we hypothesized that PAF-dependent protection was due to attenuation of Ca2+ overload associated with I/R. To address this hypothesis, we induced Ca2+ overload by exposing nonischemic myocytes to a high extracellular Ca2+ concentration. The resulting acute increase in intracellular Ca2+ led to a rapid loss of function of ventricular myocytes. Utilizing this model, we demonstrated that PAF protects from Ca2+-dependent loss of function and that the PAF receptor antagonist BN-52021 blocks this protection in ventricular myocytes. Consistent with these observations, PAF significantly reduced basal Ca2+ and peak intracellular Ca2+ in the Ca2+ overload model. From these studies, we concluded that cardioprotective effects of PAF are likely mediated through Ca2+ homeostatic mechanisms.

To further explore the basis of PAF protection, we next tested the hypothesis that PAF-dependent protection from the loss of function in Ca2+ overload was due to NOS activation in myocytes. PAF stimulation was previously shown to induce phosphorylation and activation of NOS3 in endothelial cells and ventricular myocytes (1, 24). In addition, cardiac-specific NOS3 overexpression improves postischemia function in I/R hearts (5), whereas targeted deletion of NOS3 or pharmacological inhibition of NOS3 increases the area of infarct and reduces LVDP in I/R hearts (12, 29). We found that inhibition of NOS1 and NOS3 blocked PAF-dependent protection and that increase of NO production mimicked the PAF protection from Ca2+ overload-induced loss of function in myocytes. Furthermore, inhibition of NOS blocked the PAF-dependent decrease in resting and peak intracellular Ca2+ levels in the Ca2+ overload model. We also demonstrated that cGMP was not involved in the PAF-NO-dependent protection from loss of function or the PAF-induced decrease in resting Ca2+. Thus PAF is protective through NO production and a cGMP-independent decrease in intracellular Ca2+ overload.

One cGMP-independent pathway that can mediate NO effects is reversible S-nitrosylation of cysteines. NO has been shown to S-nitrosylate cysteines on the ryanodine receptor (9, 32) and the L-type Ca2+ channel in ventricular myocytes (29). S-nitrosylation of the ryanodine receptor sensitizes the cell to Ca2+-induced Ca2+ release (32), whereas hyponitrosylation of the ryanodine receptor leads to leak of Ca2+ from the sarcoplasmic reticulum during diastole (9). S-nitrosylation of the L-type Ca2+ channel has been shown to decrease Ca2+ current and the Ca2+ transient (29). We found that PAF receptor activation consistently increased the S-nitrosylation of a ~220-kDa protein. This is similar to the reported size of the pore-forming {alpha}1-subunit of L-type Ca2+ channels (25). However, the present study does not prove that the ~220-kDa S-nitrosylated protein is the L-type Ca2+ channel but, rather, that S-nitrosylation is an important PAF mechanism of action. We demonstrated that PAF-dependent nitrosylation of the ~220-kDa protein was blocked by NOS inhibition, but not inhibition of soluble guanylyl cyclase. Brief treatments with ascorbate, which is known to reduce S-nitrosylated cysteines to thiols, blocked PAF-dependent nitrosylation. Furthermore, ascorbate blocked the protection afforded by PAF with respect to the Ca2+ overload-induced loss-of-function increase in resting Ca2+ levels in myocytes. These data are consistent with the hypothesis that PAF-dependent nitrosylation of Ca2+-handling proteins is cardioprotective.

Limitations of the present study should be noted. One potential limitation is that targeted deletion of the PAF receptor (Fig. 3) could have unidentified consequences during embryonic development that reduce the ability of the adult heart to recover from I/R. However, I/R-induced dysfunction was increased in hearts with the PAF receptor deletion and wild-type hearts treated with a PAF receptor antagonist. This suggests that the poor performance in these two independent models is due to the shared absence of PAF receptor responsiveness in the heart. Another concern is the comparison of data from hearts with different levels of ischemic insult, inasmuch as the pathways activated in control hearts with an 80% postischemic recovery may not be identical to those activated in hearts with a 40% postischemic recovery. However, findings of these studies (Figs. 1–3) are consistent with the overall hypothesis that activation of the PAF receptor is cardioprotective. It should also be noted that Ca2+ overload is only one of the mechanisms responsible for injury in I/R (Figs. 4–7). Thus our studies cannot exclude the possibility that PAF-dependent protection in I/R may also attenuate damage due to other effects, such as a decrease in free radical production. Finally, the biotin switch method of determining nitrosylation and use of ascorbate to block nitrosylation have limits (Fig. 6). The biotin assay is useful when nanomole per milligram protein levels of S-nitrosylation are present (33). Thus S-nitrosylated proteins expressed at low levels or that have low levels of nitrosylation would not be identified by this technique. Furthermore, ascorbate is a broad-spectrum reducing agent. However, ascorbate efficiently reduces S-nitrosylated groups, and no other nontoxic, cell-permeable agent with a higher degree of selectivity is available.

The present study demonstrates that picomolar concentrations of PAF protect against myocardial dysfunction, and PAF-induced cardioprotection likely involves an NO-dependent nitrosylation of Ca2+-handling proteins, such as L-type Ca2+ channels, to attenuate Ca2+ overload during and after ischemia. Complete inhibition or antagonism of this PAF protective pathway reduces myocardial postischemic functional recovery. This raises concerns that clinical treatments that either reduce circulating PAF concentrations to undetectable levels or elicit complete PAF receptor antagonism, such as called for in recent human studies on inflammation and anaphylaxis (31), may unintentionally lead to the loss of a PAF-dependent endogenous protective mechanism in the heart.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants HL-48839 (P. A. Hofmann), AI-27913 (to E. I. Tuomanen), and HL-74001 (R. R. Morrison) and by the American Lebanese-Syrian-Associated Charities.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Z. Fan (Department of Physiology, University of Tennessee Health Science Center) for allowing us to use his IonOptix system, Geli Gao (St. Jude Children's Research Hospital) for genotyping and animal husbandry, and Julie Groff (St. Jude Children's Research Hospital) for help with graphics.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Hofmann, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (e-mail: phofmann{at}physio1.utmem.edu)

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.

* P. J. Leary and S. Rajasekaran contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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