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Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart

Departments of ¹Physiology and ²Medicine, University of South Alabama College of Medicine, Mobile, Alabama; ³Department of Cardiology, Ernst-Moritz-Arndt Universität, Greifswald, Germany; and ⁴Department of Pharmacology, University of Vermont, Burlington, Vermont

Submitted 26 August 2004 ; accepted in final form 2 December 2004

	ABSTRACT

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Bradykinin and acetylcholine (ACh) trigger preconditioning by ATP-sensitive K⁺ (K_ATP) channel-dependent production of reactive oxygen species (ROS). Recent evidence suggests that ROS production may in turn be influenced by cGMP-dependent protein kinase (PKG). This study utilized DT-2 and DT-3 peptides, highly specific membrane-permeable blockers of PKG. Rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 µM) caused a 49.9 ± 5.9% and 46.8 ± 1.7% increase in ROS production, respectively (P < 0.005 vs. untreated cells). Coincubation with DT-3 (250 nM) abolished both the ACh- and bradykinin-induced ROS signal, whereas a nonpermeable form of the peptide (W45) had no effect on ACh-induced ROS production. DT-3 was unable to block ROS production from diazoxide (100 µM), a selective opener of mitochondrial K_ATP channels, suggesting that these channels are downstream of PKG. DT-2 (125 nM) also prevented ACh from triggering ROS production. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (100 µM), a cGMP analog and potent direct activator of PKG, increased ROS production of cardiomyocytes by 44.7 ± 7.1% (P < 0.001 vs. untreated cells). This increase was blocked by DT-2. Neither DT-2 nor DT-3 could block the anti-infarct effect of bradykinin in isolated rabbit hearts. Studies with fluorescent-tagged DT-3 revealed that it was confined to endothelial cells and never reached the myocytes. We conclude that both bradykinin and ACh trigger ROS generation by a pathway that includes PKG. Although the peptides may be inappropriate for a whole heart model, they are likely to become important tool drugs for elucidation of signal transduction pathways in cell preparations.

protein kinase G; reactive oxygen species

W45 (LRK₅H) is a highly specific peptide blocker of PKG that was designed to bind to a unique amino acid sequence of PKG and, therefore, to selectively antagonize this kinase (4, 5). To make the peptide cell permeant, W45 was fused to either a Drosophila antennapedia homeodomain (RQIKIWFQNRRMKWKK) or human immunodeficiency virus-1 tat protein (YGRKKRRQRRRPP), thus resulting in rapid translocation of the peptide across cell membranes. Hence, the resulting peptides, DT-3 and DT-2, respectively, are highly specific PKG blockers that can be used in intact cells. To resolve the issue of PKG involvement in the generation of ROS and triggering of preconditioning, we tested whether either of these peptides would block ROS production in rabbit cardiomyocytes stimulated by either of the preconditioning mimetics BK and acetylcholine (ACh) or the highly specific PKG activator 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP). We also tested whether DT-3 and DT-2 might be used in whole heart studies by measuring their effects on the infarct-sparing effect of BK. We determined whether we could load ventricular muscle cells of the intact heart with DT-3 when a fluorescein-labeled version of the peptide was introduced into the coronary perfusate.

	METHODS

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This study was performed in accordance with The Guide for the Care and Use of Laboratory Animals (10).

Adult rabbit myocytes. Rabbit ventricular myocytes were isolated as described previously in detail (2). Briefly, hearts of New Zealand White rabbits were excised and retrogradely perfused with calcium-free Krebs-Henseleit-HEPES buffer containing collagenase (type 2, Worthington; Lakewood, NJ) (200 U/ml) at 37°C. Viable myocytes were separated by repetitive slow-speed centrifugation and made calcium tolerant by stepwise restoration of calcium in the medium to 1.25 mM. Usually, 30–35 million viable, calcium-tolerant cells were extracted per heart.

Immediately after the isolation and separation procedure, cells were plated on laminin-coated 24-well plates (Becton Dickinson; Bedford, MA) using creatine (5 mM)-, L-carnitine (2 mM)-, and taurine (5 mM)-supplemented medium 199 (CCT-medium 199) as described by Piper et al. (13) and Mitcheson et al. (9). Penicillin (100 U/ml) and streptomycin (100 µg/ml) were added as antibiotics. Cells were stored in incubators at 37°C in air enriched with 5% CO₂. The first medium change was performed after 3–4 h; afterward, cells were undisturbed and allowed to equilibrate for at least 18 h.

Experimental design. Each experiment started with a change of medium for 10 min. The medium was then removed and replaced with one containing the drug or drug plus blocker (if required) and reduced MitoTracker red (1 µM) as a dye. This reduced form of the probe is nonfluorescent. When the probe is oxidized by ROS, it then becomes fluorescent. The oxidized product is bound to thiol groups and proteins within mitochondria. After an incubation with MitoTracker red for 15 min, cells were washed twice with fresh MitoTracker-free CCT medium. The wash served to remove the unbound and thus voltage-dependent pool of dye held in the cells. After cells were washed, fluorescence becomes stable for at least 30 min.

In experiments in which the effect of the peptides W45 (8.2 µM), DT-2 (125 nM), and DT-3 (250 nM) on ROS production by ACh (250 µM), BK (400 nM), diazoxide (100 µM), or 8-pCPT-cGMP (100 µM) was to be examined, the blocker was present in the medium during the10-min period before the addition of MitoTracker red and the agonist. Previous investigations have documented that the described protocol of a timed incubation followed by washing permits reliable measurement of ROS generation and minimizes any possible influence of changing mitochondrial transmembrane potential (6, 12).

Measurement of ROS production. Experiments were designed such that four different conditions were always simultaneously evaluated. Mitochondrial ROS generation was analyzed by measuring the fluorescence of at least 40 individual rod-shaped cells that were randomly selected within each well. The average fluorescence for the selected cells in each well was computed and compared with the average single cell fluorescence in the respective control well in the same chamber. Thus the treated cells were only compared with untreated cells of the same age and isolation and stained with the same MitoTracker red lot. Single cell fluorescence was quantified as described previously (6). Each set of experiments was repeated five to eight times on different days with cells of different ages. Approximately 200–400 typical rod-shaped cells contributed data for each experiment.

Fluorescent images. To determine whether DT-3 enters the myocytes in the above cell model or reaches the myocardium in the isolated heart model described below, fluorescein-tagged W45 (Flu-W45) and fluorescein-tagged DT-3 (Flu-DT-3, 250 nM) were used. In the cell model, the myocytes were treated for 30 min with either Flu-W45 or Flu-DT-3. Cell fluorescence was measured at 488 nm using a Leica TCS SP2 (Leica Microsystems; Exton, PA) confocal laser scanning microscope.

In the whole heart model, isolated Langendorff-perfused rabbit hearts were treated for 10 min with Flu-DT-3. The hearts were then removed and frozen in OCT compound and sectioned into slices with a thickness of 60 µm. Nuclear staining was performed using propidium iodide. Cell fluorescence was measured as above.

Isolated heart model. As previously described (3), a 2-0 silk suture was passed around a branch of the left coronary artery of New Zealand White rabbits to form a snare by passing the ends of the thread through a small vinyl tube. The heart was rapidly excised, mounted on a Langendorff apparatus by the aortic root, and perfused with oxygenated, warmed Krebs buffer. Perfusion pressure was set at 75 mmHg by adjusting the height of the reservoir. A fluid-filled latex balloon was inserted into the left ventricle and inflated to set an end-diastolic pressure of 5 mmHg. All hearts were allowed to equilibrate for 30 min before the protocols were started.

For the infarct studies, four groups of hearts were evaluated. All hearts were subjected to 30 min of regional ischemia and then reperfused for 2 h. Control hearts received no treatment. The second group of hearts was treated with BK (400 nM) for 5 min followed by 10 min of washout before the long ischemia. The third and fourth groups were also treated with BK. In addition, either DT-3 (250nM) or DT-2 (125nM) was added to the perfusate for 20 min before BK treatment.

Infarct size measurement. As previously detailed (3), the risk zone was delineated with 2- to 9-µm diameter green fluorescent microspheres (Duke Scientific; Palo Alto, CA) injected into the aortic perfusate and infarction of left ventricular slices with triphenyltetrazolium chloride staining. The areas of infarct and risk zone for each slice were determined by planimetry and volumes calculated by multiplying each area by slice thickness and summing them for each heart. Infarct size is expressed as a percentage of the risk zone.

Chemicals. All drugs required for cell isolation and culture were purchased from Sigma (St. Louis, MO). Reduced MitoTracker red was purchased from Molecular Probes (Eugene, OR). 8-pCPT-cGMP was obtained from BIOLOG (Life Science; Bremen, Germany). LRK₅H (W45), RQIKIWFQNRRMKWKK-LRK₅H (DT-3), YGRKKRRQRRRPP-LRK₅H (DT-2), fluorescein-C--Ala- LRK₅H (Flu-W45), and fluorescein-C--Ala-RQIKIWFQNRRMKWKK-LRK₅H (Flu-DT-3) were prepared as previously detailed (4).

Data analysis. Fluorescence measurements provide data in arbitrary units. To remove the variability caused by different MitoTracker red lots, cell age, and environmental conditions, average cell fluorescence was calculated and compared with that of simultaneously studied control cells as described above. Therefore, fluorescence data are provided as a percentage of the respective control (means ± SE). To further minimize the possible influence of these variables on the data, ANOVA for repeated measures with Tukey's post hoc testing was used to evaluate differences in mean fluorescence of the groups within the same experiment. Baseline hemodynamic variables and risk zone and infarct size data among groups were compared by one-way ANOVA with Tukey's post hoc testing. A value of P < 0.05 was considered significant.

	RESULTS

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Isolated ventricular myocytes. When observed with a confocal microscope, rapid uptake of the fluorescein marker by isolated ventricular myocytes was noted after the addition of Flu-DT-3 to the bathing medium. As expected, fluorescence could not be found inside the myocytes after their incubation in Flu-W45, which lacks the membrane translocation component.

When isolated myocytes were treated with either of the receptor agonists ACh or BK, a significant increase in ROS generation was seen (48.4 ± 11.0% and 36.6 ± 7.8%, P = 0.003 and P < 0.001, respectively; Figs. 1 and 2). This ROS burst triggered by either ACh or BK could be blocked by DT-3. DT-3 alone had no effect on fluorescence. In contrast, preincubation with W45 did not block ACh-induced ROS production (45.3 ± 15.1%, P = 0.022; Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 1. ROS production as measured by cell fluorescence after incubation of cardiomyocytes in reduced MitoTracker red. Treatment of myocytes with acetylcholine (ACh) increased ROS generation compared with the baseline fluorescence of untreated cells. Cotreatment with the specific cell-permeable PKG blocking peptide DT-3 totally abolished the ACh-induced ROS generation. Treatment with DT-3 alone had no effect on cell fluorescence.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Exposure of adult rabbit cardiomyocytes to bradykinin (BK) increased ROS generation, whereas coincubation with DT-3 blocked this increase of ROS. DT-3 alone had no effect.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Cotreatment of adult rabbit ventricular myocytes with ACh and the nonmembrane-permanent form of the PKG blocking peptide W45 did not block the increase of cell fluorescence seen with ACh treatment alone. Treatment with W45 alone had no effect.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Exposure of adult rabbit cardiomyocytes to ACh increased ROS production, but coincubation with DT-2 blocked the increase. DT-2 alone had no effect.

View larger version (11K): [in this window] [in a new window]   Fig. 5. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) stimulated ROS production by adult rabbit ventricular myocytes, but this enhanced ROS production was blocked by DT-2.

View larger version (12K): [in this window] [in a new window]   Fig. 6. The direct ATP-sensitive K+ channel opener diazoxide significantly increased cell fluorescence, and this increase was not blocked by DT-3. *P < 0.001 vs. untreated cells; P = 0.002 vs. DT-3.

View larger version (107K): [in this window] [in a new window]   Fig. 7. Representative isolated rabbit heart section stained with propidium iodide for nuclear staining (red) after fluorescein-tagged DT-3 (green) infusion.

Baseline hemodynamics were similar in the control and all experimental groups. Both DT-3 and DT-2 caused coronary vasoconstriction and depression of left ventricular developed pressure, although this effect was much more profound with DT-2. Generally DT-3 depressed developed pressure to 50–75 mmHg with appreciable rebound after discontinuation of the infusion and washout. Developed pressure during DT-2 infusion fell to 25–50 mmHg. Although there was also rebound in most hearts after completion of the infusion, two hearts remained severely depressed with developed pressures of 30 and 45 mmHg.

Risk zone volumes were comparable in all groups. In untreated control hearts, 29.8 ± 2.4% of the region at risk infarcted after 30 min of ischemia followed by 2 h of reperfusion. As expected, pretreatment with BK for 5 min reduced infarct size to 13.9 ± 1.5% (P < 0.001 vs. control) (Fig. 8). The cardioprotective effect of BK could not be blocked by cotreatment with DT-3 (16.9 ± 3.0% infarction, P = 0.02 vs. control).

View larger version (15K): [in this window] [in a new window]   Fig. 8. Effect of BK on infarct size expressed as a percentage of the risk zone. Pretreatment with 5 min of BK before the long ischemia was protective. Infusion of DT-3 immediately before BK did not block the protective effect. Infarction in the DT-2 group was not different from that in control hearts suggesting that BK's protection had been blocked. However, the two hearts with the largest infarcts had a marked and prolonged depression of coronary flow and left ventricular function after cessation of peptide infusion. If these two hearts are eliminated, then there is significantly less infarction in the BK + DT-2 -treated hearts than in control hearts. *P < 0.005 and **P < 0.02 vs. control.

	DISCUSSION

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In these studies, DT-3 was observed to block ROS formation from both BK and ACh in rabbit cardiomyocytes, whereas DT-2 abolished ROS production by ACh and 8-pCPT-cGMP, a direct activator of PKG, thus providing the strongest evidence to date that activation of PKG is an essential step in this process. These results further support our hypothesis that PKG carries BK's signal to open the mitoK_ATP channel. Prior investigation has documented that 8-pCPT-cGMP in isolated buffer-perfused intact rabbit hearts decreased infarction, whereas this infarct-sparing action could be aborted by 5-hydroxy-decanoate (15), thus establishing that both PKG and the mitoK_ATP channel were in the same signaling pathway and further suggesting that PKG was upstream of the K_ATP channel. Because DT-3 did not block the increased ROS formation after exposure of cardiomyocytes to the mitoK_ATP channel opener diazoxide, we now have confirming evidence that PKG is upstream of the K_ATP channel in this signaling pathway. On the other hand, protection from BK could not be blocked by the peptides in an intact heart when they were introduced into the coronary perfusate. Our fluorescence studies indicate that the failure was related to the inability of DT-3 to penetrate to the ventricular myocytes, thus resolving the challenge to our underlying hypothesis.

We have proposed that BK and ACh precondition myocardium through a complex pathway that includes activation of phosphatidylinositol 3-kinase and Akt (7, 11, 14). In our scheme, Akt then activates eNOS, which produces NO. NO in turn activates guanylyl cyclase, producing cGMP, and finally cGMP activates PKG (11, 15). Unfortunately, PKG activation is not the only way that NO or cGMP can alter conditions within a cell. NO can react with oxygen to form ROS such as peroxynitrite, which could possibly trigger the preconditioned state directly. The most likely effect of NO, however, would be to activate guanylyl cyclase and raise cGMP levels in the cell. Indeed, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a very selective inhibitor of guanylyl cyclase, inhibited ROS production by BK in cardiomyocytes as well as BK's anti-infarct effect in intact hearts (11). Although cGMP is known to activate PKG, it, too, has other cellular targets. It can inhibit phosphodiesterase 3 and thus raise cAMP levels (1, 19), directly stimulate PKA (16), and open cGMP-dependent ion channels (17, 21). Therefore, it cannot be assumed that PKG is part of all signaling pathways involving guanylyl cyclase. The best way to prove PKG involvement is to demonstrate that the response in question is aborted by a highly selective blocker of PKG. To that end, we have shown that 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cGMPS), a cell-permeant cGMP-based inhibitor, could block ROS production from BK in our cardiomyocyte model (11). However, the selectivity of the compound has been questioned. The present study extends the prior observation by showing that the highly specific peptide inhibitors could block ROS production from both G protein-coupled receptor agonists and the cGMP analog 8-pCPT-cGMP.

There is strong circumstantial evidence supporting PKG in BK's protective mechanism. The guanylyl cyclase blocker ODQ eliminated BK's protection, indicating that cGMP is involved (11). Conversely, 8-pCPT-cGMP, a cell-permeable cGMP analog and direct activator of PKG, mimicked BK's anti-infarct effect (15). Nevertheless, it has been surprisingly difficult to confirm PKG's involvement in preconditioning's anti-infarct effect because of lack of a suitable inhibitor that can be used in the intact heart. Economic concerns preclude the use of the cell-permeant cyclic nucleotide-based inhibitors. The other inhibitors are either toxic or lack specificity. To that end, we attempted to use the highly selective, nontoxic peptide PKG antagonist DT-3. We infused DT-3 into the coronary perfusate for 20 min at the same concentration as was used successfully in the cardiomyocytes (250 nM). However, DT-3 failed to block BK's protection. When we infused a fluorescein-labeled version of the peptide, we found that it did not penetrate the tissue beyond the blood vessel walls. Thus it is likely that failure of the peptide to block BK’s anti-infarct effect was due to its failure to load the cardiomyocytes. Because of the amino acid sequence of these peptides, they are susceptible to tryptic degradation. It is possible that absence of effect of the peptides in intact organs could be the result of enzymatic destruction in the interstitium. However, this would not explain the absence of fluorescence outside of the endothelial layer.

DT-3 is thought to remain intact in the cell, and thus in theory the peptide should be able to move in and out of cells and thus penetrate beyond the capillary endothelial layer. Indeed, Dostmann et al. (4) saw Flu-DT-3 penetrate into the smooth muscle layers of 150-µm-diameter rat brain arteries when it was introduced from the adventitial side. Introduction from the luminal side as was done in the present study was not tested. Perhaps a higher concentration of DT-3 would have provided adequate loading of the ventricular myocytes.

We also examined the effect of DT-2 in the intact heart. This peptide linked to the more potent HIV-1 tat protein to promote transmembrane passage caused intense coronary vasoconstriction, which would be an expected consequence of PKG blockade in the vasculature. Vasoconstriction of isolated cerebral arteries by DT-2 has previously been reported (18). The fall in coronary perfusion resulted in marked deterioration of left ventricular function. This hemodynamic effect also makes this peptide problematic for intact heart studies. In general, DT-2 was also unable to block BK's cardioprotective effect. However, a definite conclusion regarding DT-2's effect was confounded by the adverse hemodynamic profile and the likelihood that some hearts were exposed to prolonged ischemia, which itself would have increased infarct size.

In summary, receptor agonists trigger the preconditioned state by generating ROS, which we can detect in our isolated cardiomyocytes. DT-3, a highly selective inhibitor of PKG, prevented BK- and ACh- but not diazoxide-induced ROS formation in rabbit cardiomyocytes, and a second PKG peptide inhibitor, DT-2, blocked the enhanced ROS production mediated by ACh and 8-pCPT-cGMP. These observations provide strong evidence that these agonists trigger preconditioning by a pathway that includes PKG located upstream of the mitoK_ATP channel. Attempts at loading a whole heart with the DT-3 peptide failed because the peptide seemed to concentrate in the vasculature and did not reach the cardiomyocytes. This observation and adverse hemodynamic effects related to coronary vasoconstriction make it unlikely that these peptides can be used in intact hearts to study PKG involvement in signaling pathways. However, these peptides are potent tools for study of signal transduction pathways in isolated cells and should prove to be invaluable tools for future studies of PKG.

	ACKNOWLEDGMENTS

 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-20648, HL-50699, and HL-68891 and the Totman Trust for Medical Research.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. V. Cohen, Dept. of Physiology, MSB 3050, Univ. of South Alabama College of Medicine, Mobile, AL 36688 (E-mail: mcohen{at}usouthal.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.

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