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RECEPTORS AND SIGNAL TRANSDUCTION
Cardiovascular Biology Research Group, Department of Pharmacology, National University of Singapore, Singapore
Submitted 7 August 2007 ; accepted in final form 15 November 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PKC
, and PKC
to membrane fraction but only translocation of PKC and PKC was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca2+] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca2+ removal via both accelerating uptake of Ca2+ into sarcoplasmic reticulum and enhancing Ca2+ extrusion through Na+/Ca2+ exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca2+ overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKC, PKC, and PKC in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca2+ handling. protein kinase C isoforms; ischemia and reperfusion; cardioprotection; ATP-sensitive potassium channel
Protein kinase C (PKC) also correlates closely with cardioprotection. Numerous studies have documented a central role of PKC in the cardioprotection through various mechanisms like ischemic preconditioning (IP) (13, 15, 24) and anesthetic preconditioning (28, 31). The PKC family consists of at least 10 isoforms, among which PKC, PKC, and PKC are the prominent isoforms expressed in the heart (12). Upon stimuli, PKC isoforms translocate from the cytosol to subcellular membrane regions, a process associated with their activation. Such translocation has been deemed as a hallmark of PKC activation (12). However, it is completely unknown whether SP can stimulate PKC activation and what the even downstream effects are.
PKC activation has been reported to play a role in regulating intracellular calcium handling (10, 25, 26). Under the physiological condition, intracellular calcium concentration ([Ca2+]i) is sophisticatedly regulated by several proteins present in the sarcolemmal and sarcoplasmic reticulum (SR) membranes. Upon the arrival of action potential, Ca2+ influxes through the L-type Ca2+ channel and triggers the opening of the ryanodine receptor (RyR), resulting in further release of Ca2+ from the SR, which accomplishes the sharp [Ca2+]i elevation required for myofibril contraction (7). In the rat cardiomyocytes, >90% of the Ca2+ after contraction is immediately reuptaken by SR via sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), whereas the remaining Ca2+ is pumped out of the cell via Na+/Ca2+ exchanger (NCX) (2).
However, the well-controlled intracellular Ca2+ homeostasis is vulnerably disrupted with the advent of ischemia and reperfusion insults. During ischemia, excessive Ca2+ accumulates in the cytosol (19) and leads to a series of severe damages upon reperfusion. For example, once the myofilaments are reenergized by reperfusion, they contract intensely in an extreme and sustained manner (hypercontracture) due to overstimulation of calcium on the contractile apparatus (23). In single cardiomyocytes, such hypercontracture causes irreversible shortening of the cell length. In tissues, it causes a disruptive change in the myocardium termed contraction band necrosis (6). Under this circumstance, a faster clearing of excessive Ca2+ from cytosol is therapeutically important because it would potentially attenuate Ca2+ overloading during ischemia challenge, reduce the myocyte hypercontracture (1), and preserve the cardiac function. Since PKC is implicated in the intracellular Ca2+ handling, it is worthwhile investigating whether H2S produces any effect on Ca2+ handling, given PKC is activated after SP.
Taken together, there is no information so far available about the effect of H2S on PKC and how the cardioprotective signals are transduced toward and forward. Thus in the present study, we intended to provide a close inspection on PKC and its upstream and downstream connections in the signal transduction pathway of H2S preconditioning.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drugs and chemicals. Fura 2-AM, type II collagenase, protease, lactate, 2-deoxyglucose (2-DOG), sodium dithionite (Na2S2O4), sodium hydrosulfide (NaHS), caffeine, trypan blue dye, laminin, medium 199, lactate dehydrogenase (LDH) assay kit, and chelerythrine chloride were purchased from Sigma Chemical. Glibenclamide was obtained from Tocris Cookson. All chemicals were dissolved in deionized water except fura 2-AM and glibenclamide, which were dissolved in DMSO at a final concentration <0.1% (wt/vol).
Cardiac myocytes preparation. Cardiac myocytes were isolated from the hearts of adult male Sprague-Dawley rats using a collagenase perfusion method as described before (17). The heart was quickly excised from Pentobarbitone-anesthetized rats (250–300 g body weight), mounted via the aorta on a Langendorff apparatus, and retrogradely perfused with Ca2+-free Tyrode buffer at 37°C for 5 min. The heart was then perfused for another 25–30 min with circulating Ca2+-free Tyrode solution containing 0.84 mg/ml collagenase (type II) and 0.28 mg/ml protease. Thereafter, the ventricular tissue was cut into fragments and shaken gently to dissociate cardiac myocytes in Ca2+-Tyrode solution. The cell suspension was filtered, centrifuged, and washed three times. More than 80% of the cells were rod shaped and impermeable to trypan blue. The Ca2+ concentration of the Tyrode solution was then increased gradually to 1.25 mM in 45 min. Cells were allowed to stabilize for 30 min at room temperature.
Induction of simulated ischemia. Severe ischemia was induced by ischemia buffer containing (mM) 20 2-DOG (an inhibitor of glycolysis), 5 sodium lactate, 20 Na2S2O4 (an oxygen scavenger), 137 NaCl, 15.8 KCl, 0.49 MgCl2, 0.9 CaCl2, and 4 HEPES. The pH was adjusted to 6.6 to mimic acidosis. For simulation of ischemia preconditioning (IP), the concentrations of 2-DOG and Na2S2O4 were halved.
Experimental protocol. Myocytes were subjected to H2S preconditioning (SP) by incubation with 100 µM NaHS for 30 min. The concentration was adopted based on our previous study in which a maximum protection was observed at 100 µM (17). For ischemia preconditioning (IP) that was used as a reference model for PKC translocation, myocytes were subjected to simulated ischemia preconditioning as described above. The control group (VP) did not receive any pretreatment. Cells in all groups were washed several times before being cultured with Dulbecco's modified Eagle's medium (DMEM) in a CO2 incubator for 20 h. Samples were then harvested for Western blot analysis or intracellular Ca2+ transient recording. Or the cells were subjected to severe ischemia with ischemia buffer for 10 min after the 20-h culture, followed by 10 min reperfusion with normal medium, after which cell viability and cellular injury were assessed. Resting Ca2+ elevation was traced real-time during ischemia for examination on cytosolic Ca2+ accumulation. Cell length was compared between before ischemia and after the onset of reperfusion for evaluation on hypercontracture. To study the involvement of PKC, the PKC inhibitor chelerythrine chloride (3 µM) was added into the cell medium 15 min before and during SP preconditioning (Fig. 1A). To study the sequence of signaling events between PKC activation and KATP opening, cells were treated with glibenclamide (10 µM, a blocker of KATP channel) 15 min before and during SP.
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LDH assay. LDH release was measured after 10 min reperfusion as a cellular injury index (16). Both culture medium and cell lysates (prepared with lysis buffer containing 1% Triton-X100) were collected for determination of LDH activity. LDH assay was performed using a commercially available kit (Sigma). The assay was based on the reduction of NAD catalyzed by LDH. The reduced NAD (NADH) was utilized in the stoichiometric conversion of a tetrazolium dye. The absorbance at a wavelength of 490 nm was measured spectrophotometrically with a microplate reader (Tecon Systems). The background absorbance at 690 nm was subtracted from the absorbance at 490 nm. The results were presented as LDH released into the medium in terms of percentage of the total LDH activity (medium + cell lysate), normalized to 100% for VP group.
Measurement of [Ca2+]i. Ventricular myocytes were incubated with fura 2-AM (4 µM) for 35 min. The loaded cells were transferred to a superfusion chamber on the stage of an inverted microscope (Nikon TS100), which was coupled with a dual-wavelength excitation spectrofluorometer (Photon Technology International). The myocytes were perfused with Krebs bicarbonate buffer containing (in mM) 118 NaCl, 5 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.25 CaCl2, 25 NaHCO3, and 11 glucose; pH 7.4. To generate electrically induced [Ca2+]i transients (E[Ca2+]i), myocytes were stimulated at 0.2 Hz with a stimulator(Grass S88), whereas the caffeine-induced [Ca2+]i transients (C[Ca2+]i) were generated by adding 10 mM caffeine directly to the incubation buffer. Resting Ca2+ level was recorded without any above stimulation during ischemia challenge. Fluorescence signals obtained at 340 nm (F340) and at 380 nm (F380) excitation wavelengths were recorded and stored in a computer for data processing. The F340-to-F380 ratio was used to represent [Ca2+]i changes in the myocytes.
Measurement of cell length. Cardiomyocytes were placed on the stage of an inverted microscope (Nikon TE2000-S). The cell image was taken with a digital camera (Nikon DS-5M-L1) connected to the microscope with a x20 objective and analyzed with NIS-documentation software (Nikon).
Cell fractionation and Western blot analysis.
A cell fractionation technique was adopted from the literature (11, 30). After 20 h of incubation, cardiomyocytes were lysed with 150 µl ice-cold lysis buffer containing 125 mM NaCl, 25 mM Tris (pH 7.5), 5 mM EDTA, 1% NP-40, and protease inhibitors and shaken on ice for 1 h. The cell lysate was centrifuged at 1,000 g at 4°C for 10 min for rough partition between cytosolic and membrane fractions. The supernatant was recentrifuged at 16,000 g at 4°C for 15 min to get rid of contaminating pellet materials and collected as cytosolic fraction. The initial pellets were resuspended in 100 µl cell lysis buffer containing 1% Triton X-100 and shaken on ice for another 60 min and were then centrifuged at 16,000 g at 4°C for 15 min. The second supernatant was collected as membrane fraction. Epitopes were exposed by boiling the protein samples at 90°C water for 5 min. Each fraction was analyzed for protein content by the Bradford assay. Equal amounts of protein were loaded and electrophoresed with 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was probed with antibody against PKC (Santa Cruz Biotechnology), PKC, and PKC (Cell Signaling Technology). Immunoreactivity was detected using an ECL advance Western blot detection kit (Amersham Biosciences).
Statistical analysis. Values presented are means ± SE. Statistic comparisons were performed by one-way ANOVA and Bonferroni for post hoc analysis. The significance level was set at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LDH release was measured as an index of cellular injury (16), which was presented as the ratio of the medium LDH activity over the total LDH activity (medium ± intracellular) and normalized to 100% of control. As shown in Fig. 1C, SP significantly reduced the LDH release induced by severe ischemia, and this effect was reversed by pretreatment with chelerythrine (3 µM), which itself had no effect on LDH release.
Effect of SP on translocation of PKC isoforms.
To determine the activated PKC isoforms in the delayed phase of cardioprotection induced by SP and IP, subcellular distributions of three main PKC isoforms present in the heart, , , and , were examined 20 h after SP and IP with Western blotting experiments. As shown in Fig. 2, A–C, SP induced all three isoforms of PKC translocation from cytosol to membrane. The membrane-to-cytosol ratios of PKC-, , and increased approximately twofolds in SP (Fig. 2, D–F). Interestingly, IP only induced translocation of PKC and PKC, but had no effect on PKC translocation. These data suggest that the SP and IP may employ different subsets of PKC isoforms to mediate their cardioprotection.
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but did not affect translocation of PKC and PKC, suggesting that only PKC among the three isoforms examined is downstream to KATP channel in the signaling pathway of SP.
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90% Ca2+ from the cytosol (2). Thus half-decay time (t50) and 90% decay time (t90) of E[Ca2+]i were measured as indicators of SR uptake rate. As shown in Fig. 4, both t50 and t90 were significantly shortened by SP and IP when compared with those observed in VP. Cotreatment with 3 µM chelerythrine or 10 µM glibenclamide during SP reversed this effect, suggesting that SP accelerated the rate of SR-Ca2+ uptake through a KATP-PKC pathway.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PKC, and PKC are the three main isoforms expressed in adult cardiomyocytes and also the most important ones involved in cardioprotection of ischemic preconditioning (9, 20). For this reason, we examined the effect of SP on these three isoforms before ischemia insults, which itself can stimulate PKC translocation. We found that H2S preconditioning motivated translocation of the three isoforms, PKC, PKC, and PKC, to membrane fraction at 20 h after preconditioning. Such translocation before ischemia attack may act as an essential step to switch the cells into a state tolerant to ischemia insults and failure of such translocation results in the loss of cardioprotection as what we observed in the presence of PKC inhibitor (Fig. 1).
Individual PKC isozymes are believed to mediate characteristic cell functions, as upon stimuli they are directed to distinct subcellular membrane regions by isozyme-specific receptors for activated C kinase (RACK) (12). By binding to their specific RACKs, the activated isozymes are anchored close to their particular substrates. In the present study, we employed IP as a reference model due to its recognized stimulatory effect on PKC. Intriguingly, we found that IP only promoted PKC and PKC translocation but not that of PKC. The discrepancy between SP and IP suggests that they may employ different subsets of PKC isoforms to convey their cardioprotective signals to respective subcellular regions, affording probably similar but not identical cardioprotection.
PKC and KATP.
In our previous study, KATP channel has been shown to be involved in the late phase of cardioprotection induced by SP (17). Since H2S has been proposed to have a direct effect on KATP channels (27), it raises the question whether SP-induced activation is secondary to the opening of KATP channels. Unexpectedly, we observed that blockade of KATP channel only diminished the SP-induced translocation of PKC but failed to affect the translocation of PKC and PKC to a noticeable extent. Thus KATP channel opening may only be necessary for PKC activation in the SP signaling pathway.
It is until recently that individual PKC isoforms were found located differently in relation to KATP channel in the cardioprotection signaling pathway. Hassouna et al. (8) demonstrated that PKC is located upstream, whereas PKC is downstream to mitochondrial KATP channel in IP signaling pathway. This implies a considerable diversity regarding PKC activation in the cardioprotective signaling mechanisms despite the similarity of key players. PKC can also be activated by other signaling molecules like NO or Ca2+ (14, 18). More studies are warranted to test whether SP-induced activation of PKC and PKC are through provoking release of these signaling molecules.
PKC and intracellular Ca2+ handling. Of great importance, we addressed the mechanism in this study how the SP-activated PKC mediates the cardioprotection. By monitoring the resting Ca2+ level in single cardiomyocytes, we observed that SP lowered elevation in [Ca2+]i during ischemia in a PKC-dependent manner. Such a timely rectification on elevated [Ca2+]i during ischemia challenge could be therapeutically important, as uncontrolled elevation in [Ca2+]i could induce irreversible injuries like mitochondria dysfunction (36), membrane degradation, and contractile derangement (37). If the ischemia is followed by reperfusion, the myocytes will exhibit hypercontracture at the onset of reperfusion due to massive stimulation on the contractile machinery by accumulated Ca2+ (16). In perfused myocardium, this hypercontracture is manifested by contraction band necrosis (17). Even if the necrotic myocardium can be replaced by scar tissues in a subsequent remodeling process, the akinetic fibrotic tissue will impair pumping function of the heart and when substantial enough will lead to heart failure (38).
To further corroborate the effect of H2S on resting Ca2+ during ischemia, we examined the myocyte hypercontracture at the onset of reperfusion. Indeed, SP reduced the development of myocyte hypercontracture through a PKC-dependent pathway. These beneficial effects triggered by SP and mediated by PKC could, in turn, at least partly account for the cardioprotection observed in cell viability and cellular injury tests (Fig. 1). It is also predictable that this limitation on the development of Ca2+ overloading and hypercontracture in single cells would achieve further significant benefits by preserving contractile functions in the intact heart.
A previous (1) study has demonstrated an effective approach to attenuate myocyte hypercontracture by increasing SERCA activity. Since SR uptake through SERCA presents the dominant route for Ca2+ removal in cardiomyocytes, it is plausible that this reduced hypercontracture is due to a faster Ca2+ clearing from cytosol before reperfusion. Enlightened by this finding, we examined the SR-Ca2+ uptake rate as well as the minor mechanism for Ca2+ removal; i.e., extrusion via NCX. We found that SP accelerated the clearing rate through both of these routes. Again, all these beneficial effects induced by SP were reversed by inhibition of PKC, implying that PKC may phosphorylate these calcium-handling proteins and improve their function.
In conclusion, the present study significantly advanced our understanding on the SP-induced cardioprotection by delineating the essential role of PKC in the context of signaling pathway (Fig. 7). The results demonstrate that SP activates PKC, PKC, and PKC in cardiomyocytes, among which only activation of PKC is secondary to the KATP channel opening. Such PKC activation intervenes in the development of Ca2+ overloading and myocyte hypercontracture induced by ischemia-reperfusion insults by facilitating cytosolic Ca2+ clearing through SERCA and NCX.
Limitation and perspective. The findings in our study beg more research into these issues. First, H2S alone is sufficient to activate three PKC isoforms, but the inhibitor chelerythrine could not distinguish the one or ones that are necessary for the genesis of the late phase of cardioprotection. It is more likely that different isoforms act on different substrates at various subcellular sites and afford the protection from diverse aspects (12). Assigning specific roles to each PKC isoform needs isoform-specific antagonists with explicit selectivity. Yet some redundant signaling pathways may place additional complexity and difficulty. Second, the signaling pathway mapped in this study is by no means the only signaling chain headed by H2S. Taking PKC as a nodal point, it has a spectrum of triggers that could vary from NO, adenosine, to free radicals, whereas its targets could range from mitogen-activated protein kinases, heat shock proteins, and mitochondria proteins (21). It is more likely to be a signal network rather than single pathways that translate the extracellular stimulus of H2S into final protection. Finally, the cardiomyocytes model is advantageous in monitoring intracellular ionic changes and avoiding confounding effects of other cell types in the heart. However, as always, it needs further corroboration in intact heart model or animal model to extrapolate these findings to clinical trials.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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200–500 cells each. *P < 0.05 vs. VP; +P < 0.05 vs. SP. C: LDH release. Values were presented as supernatant LDH activity/total LDH activities (supernatant + cell lysate) normalized to 100% of VP group (control); n = 7 experiments. Values are means ± SE; **P < 0.01 vs. VP, +P < 0.05 vs. SP.
