Cyclic GMP-dependent Protein Kinase Iα Attenuates Necrosis and Apoptosis Following Ischemia/Reoxygenation in Adult Cardiomyocyte*

Cyclic GMP-dependent protein kinases protein kinase G (PKG) Iα and PKGIβ are major mediators of cGMP signaling in the cardiovascular system. PKGIα is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIα against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIα or catalytically inactive mutant hPKGIαK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of KATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 μm), HMR1098 (30 μm), or glibenclamide (50 μm), the respective blockers of mitochondrial, sarcolemmal, or both types of KATP channels prior to SI. The necrosis observed in 33.7 ± 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 ± 0.8% by PKGIα (mean ± S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 ± 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 ± 0.6% by PKGIα (mean ± S.E., n = 7, p < 0.001). In addition, PKGIα inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIαK390A mutant showed no protection. PKGIα enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIα-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIα-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIα.

Apoptotic cell death in cardiac myocytes is well recognized to be responsible for myocardial infarction following ischemia/ reperfusion injury (1), hypertrophy (2), and development of heart failure (3). Preconditioning is a cardioprotective phenomenon whereby repeated brief episodes of ischemia protect the myocardium from more prolonged periods of ischemia and future myocardial infarction as well as stunning (4). A number of receptors and intracellular signaling pathways have been identified that play an essential role in the cardioprotective effect of preconditioning. Endogenously released agents including adenosine, norepinephrine, opioids, free radicals, and bradykinin are involved in preconditioning (5,6). Several pharmacological interventions mimic this preconditioning-like cardioprotective effect (6,7). In recent years, there has been considerable interest in the role of the NO-cGMP-protein kinase G (PKG) 2 pathway in protection of the heart against ischemia/ reperfusion injury (8). Inhibition of cGMP-specific phosphodiesterase 5A with sildenafil citrate (Viagra) induced protective effects against ischemia/reperfusion injury in the intact heart and adult cardiomyocytes (9 -13). Conceptually, sildenafil inhibits the enzymatic hydrolysis of cGMP, which in turn maintains the tissue accumulation of cGMP, leading to downstream protective mechanisms involving PKG activation and opening of mitochondrial ATP-sensitive potassium (mitoK ATP ) channels (6). It has been shown that sildenafil induces preconditioning through NO generated from endothelial and/or inducible nitric-oxide synthase (eNOS/iNOS) and via activation of protein kinase C and opening of the mitoK ATP channels (9,10,12). Phosphodiesterase 5A inhibition also attenuated cell death resulting from necrosis and apoptosis by increasing the Bcl-2/ Bax ratio through NO signaling (13).
A recent study also showed that phosphodiesterase 5A inhibition by sildenafil generates a potent anti-hypertrophic effect by enhancing PKGI activity, without increasing total cGMP in mice (14). Adenoviral gene transfer of PKGI␤ selectively enhanced antihypertrophic effects of NO, without increasing apoptosis (15). Qin et al. (16) showed that exogenous NO triggers preconditioning effect by stimulation of guanylyl cyclase to make cGMP, activation of PKG, opening of mitoK ATP channels, and production of reactive oxygen species (ROS). Peptide blockers of PKG inhibited the ROS generation by pharmacological preconditioning agents such as acetylcholine and bradykinin in cardiomyocytes (17). Nevertheless, the direct role of PKGI␣ and the downstream signaling pathways that lead to protection of cardiomyocytes against apoptosis after ischemia/reoxygenation injury remain to be elucidated.
PKGI has a number of effects that may be relevant to its regulation of apoptosis. PKGI can modulate gene expression in a variety of cell types by either stimulating or inhibiting extracel-lular signal-regulated kinase (ERK1/ 2)/mitogen-activated protein kinase (MAPK) (18,19). PKGI has been shown to suppress cell proliferation by inhibiting the Ras/MAPK pathway in baby hamster kidney cells transfected with PKGI␤ (20). PKGI also induced the expression of MAPK phosphatase-1, which reverses activation of the MAPK pathway (20). PKGI␣ induced ERK1/2 by activating MAPK/ERK kinase in smooth muscle cells (21) but activated p38 MAPK in fibroblasts (22). In human colon cancer cells, PKG activated the MEKK1-SEK1-JNK1 pathway, by directly phosphorylating and activating MEKK1 (23).
Although PKG effects on cell survival/proliferation pathways have been observed in various cells types, the mechanism of PKG attenuation of ischemia/reoxygenation injury in the heart is not clear. The present study was designed to examine whether the expression of PKGI␣ protects cardiomyocytes from necrosis and apoptosis following simulated ischemia and reoxygenation. We also examined whether opening of mitoK ATP channel, ROS generation and activation of multiple signaling pathways including Akt, NOS, and MAPKs are essential to PKG-mediated cellular protection. Our findings define mechanisms by which PKGI␣ inhibits necrosis and apoptosis signaling pathways to protect cardiomyocytes from ischemia/reperfusion injury.

Isolation of Ventricular Cardiomyocytes and Adenoviral Infection-
Adult male Wistar rats (300 g) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. Ventricular cardiomyocytes were isolated using an enzymatic technique as previously reported (13). The freshly isolated cardiomyocytes were plated with Medium 199 containing 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 5 mM glucose, 0.1 M insulin, and 1% penicillin-streptomycin. After 1 h of plating, the myocytes were infected with adenoviral vectors containing hPKGI␣ (Ad.PKGI␣) (24) or catalytically inactive hPKGI␣K390A (25) in serum-free growth medium for 24 h. The cells were routinely infected with the viruses at a concentration of 1 ϫ 10 3 particles/cell. Simulated Ischemia/Reoxygenation Protocol-After 24 h of adenoviral infection, the cells were subjected to simulated ischemia (SI) for 90 min by replacing the cell medium with an "ischemia buffer" that contained 118 mM NaCl, 24 mM NaHCO 3 , 1.0 mM NaH 2 PO 4 , 2.5 mM CaCl 2 -2H 2 O, 1.2 mM MgCl 2 , 20 mM sodium lactate, 16 mM KCl, 10 mM 2-deoxyglucose (pH adjusted to 6.2) as reported previously (13). The cells were incubated at 37°C in tri-gas incubator adjusting 1-2% O 2 and 5% CO 2 during the entire SI period. RO was accomplished by replacing the ischemic buffer with normal cell medium under normoxic conditions. Cell necrosis and apoptosis were assessed after 2 or 18 h of reoxygenation, respectively.
Evaluation of Cell Viability and Apoptosis-Trypan blue exclusion assay and lactate dehydrogenase release into the medium were used to assess cell necrosis (13). Cardiomyocyte apoptosis was analyzed by TUNEL staining as reported previously (13).
Inhibitor Studies-Each experiment was started with a change of medium in the wells. To evaluate the involvement of K ATP channels in PKG1␣-mediated protection, Ad.P-KGI␣-infected cells were treated for 30 min before SI-RO with 5-hydroxydecanoate (5-HD, 100 M), HMR1098 (30 M), or glibenclamide (Glib, 50 M), the respective blockers of mitochondrial, sarcolemmal, or sarcolemmal/mitoK ATP channels. To test the effect of a ROS scavenger on PKG-induced protection, a subgroup of cells were treated with N-(2 mercaptopropionyl)glycine (MPG, 1 mM) for 30 min prior to SI-RO. Other subgroups of cells were treated similarly with the NOS inhibitor L-nitro-amino-methylester (L-NAME, 100 M), PI 3-kinase inhibitors wortmannin (100 nM) and LY-294002 (10 M), the ERK inhibitor PD-98059 (25 M), and the JNK inhibitor SP600125 (10 M).
Western Blot Analysis-Western blots were performed as described previously (13). The blots were incubated with rabbit polyclonal primary antibody or mouse monoclonal antibody at a dilution of 1:1000 for each of the respective proteins, i.e. PKGI␣, nNOS, iNOS, eNOS, Bcl-2, Bax, pAkt, Akt, pERK, ERK1, pp38, p38, pJNK, JNK, and actin (all purchased from Santa Cruz) for 2 h. The membranes were then incubated with antirabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000 dilution; Amersham Biosciences) for 1 h. The blots were developed using a chemiluminescent system, and the bands were scanned and quantified by densitometric analysis.
Data Analysis and Statistics-The data are presented as the means Ϯ S.E. The difference between the groups was analyzed respectively with an unpaired t test or one-way analysis of variance followed by Student-Newman-Keul post-hoc test. p Ͻ 0.001 was considered to be statistically significant. Fig. 1A shows a typical preparation of our isolated adult rat cardiomyocytes. At least 90% of the cardiomyocytes had rod- shaped morphology. After SI (90 min)-RO (2 h), the percentage of necrotic, trypan blue-positive cardiomyocytes increased to 33.7 Ϯ 1.6 as compared with non-SI-RO controls (2.0 Ϯ 0.2) (n ϭ 7; p Ͻ 0.001; Fig. 1, A, B, and E). PKGI␣ expression in cardiomyocytes exposed to SI-RO reduced trypan blue-positive cardiomyocytes (n ϭ 7; p Ͻ 0.001 versus SI-RO alone; Fig. 1, C and E). The overexpression of catalytically inactive PKGI␣K390A failed to protect cells (Fig. 1,  D and E). Similarly, PKGI␣ overexpression attenuated the release of lactate dehydrogenase as compared with SI-RO alone, whereas cardiomyocytes overexpressing PKGI␣K390A did not (Fig. 1F).

PKGI␣-induced Cardioprotective Signaling
Effect of K ATP Channel Blockers and a ROS Scavenger on PKGI␣-induced Protection-Both Glib and 5-HD abolished PKGI␣ protection of cardiomyocytes exposed to SI-RO (p Ͻ 0.001 versus PKGI␣; n ϭ 3; Fig. 3A). Similarly, Glib and 5-HD blocked PKGI␣ protection against apoptosis, as demonstrated by an increase in TUNEL-positive cells (p Ͻ 0.001 versus PKGI␣; n ϭ 3; Fig. 3B). HMR1098 failed to block the protective effect of PKGI␣ against necrosis (Fig. 3A) as well as apoptosis (Fig. 3B) (p Ͼ 0.05 versus PKGI␣; n ϭ 3), ruling out a role of sarcolemmal K ATP channels in PKGI␣-induced protection. Glib, 5-HD, and HMR1098 had no effect on necrotic or apoptotic cell death after SI-RO in control or PKGI␣K390A-expressing cardiomyocytes, thereby ruling out any potential PKG-independent effects of these agents. MPG abolished protective effects of PKGI␣, i.e. increased the percentage of trypan blue-positive cells (Fig. 3A) and TUNEL-positive nuclei (Fig. 3B) after SI-RO, suggesting a role for ROS in PKGI-dependent cardiomyocyte protection.

PKGI␣ Increases Phosphorylation of Akt-Because phosphorylation of
Akt at Ser 473 is required for its full activation, we used an antibody that specifically recognized Akt phosphorylated at Ser 473 . PKGI␣ overexpression increased the phosphorylation of Akt as compared with control (without infection) and PKGI␣K390A in cardiomyocytes (Fig. 4, A and B). No further increases in Akt phosphorylation were observed when cardiomyocytes were subjected to SI and 15 or 30 min of reoxygenation following transfection with PKGI␣ (data not shown). Wortmannin and LY-294002 increased the percentage of trypan blue-positive cardiomyocytes from 18.1 Ϯ 0.3 (PKGI␣ overexpression group) to 33.5 Ϯ 0.6 and 34.7 Ϯ 0.9, respectively (n ϭ 3; p Ͻ 0.001; Fig. 4C). The number of TUNEL-positive nuclei was also increased from 6.8 Ϯ 0.8 (in PKGI␣ overexpression group) to 23.5 Ϯ 0.2 and 23.8 Ϯ 1.0 by wortmannin and LY-294002, respectively (n ϭ 3; p Ͻ 0.001; Fig.  4D). Wortmannin and LY-294002 had no effect on necrosis or apoptosis in control or PKGI␣K390Aoverexpressing cardiomyocytes exposed to SI-RO. Effect of PKGI␣ on Expression of NOS, Bcl-2, and Bax-Adenoviral transfection with PKGI␣ and PKGI␣K390A augmented the expression of PKGI␣ protein after 24 h (Fig. 5A). PKGI␣ overexpression clearly increased iNOS, eNOS, and Bcl-2 expression as compared with the control cells or cells infected with PKGI␣K390A (Fig. 5, A and B). However, Bax was significantly decreased with overexpression of PKGI␣ as compared with control and PKGI␣K390A-overexpressing cells (Fig.  5, A and B). No increase in nNOS was observed after PKGI␣ or PKGI␣K390A overexpression as compared with control cells (Fig. 5, A and B). Similar results were obtained when cardiomyocytes were subjected to SI and 15 or 30 min of reoxygenation following transfection with PKGI␣ (data not shown).
To demonstrate the effect of NOS expression on PKGI␣induced cardiomyocyte protection, we treated cells with the nonselective NOS inhibitor L-NAME before SI-RO. As shown in Fig. 6A, L-NAME increased the percentage of trypan blue-positive cardiomyocytes from 18.1 Ϯ 0.3 (PKGI␣-overexpressing group) to 31.8 Ϯ 1.2 (n ϭ 3; p Ͻ 0.001). The number of TUNEL-positive nuclei also increased from 6.8 Ϯ 0.8 in PKGI␣-overexpressing cardiomyocytes to 21.5 Ϯ 1.1 following pretreatment with L-NAME (n ϭ 3; p Ͻ 0.001; Fig. 6B). L-NAME had no effect on necrosis and apo- ptosis in control and PKGI␣K390A-overexpressing cardiomyocytes exposed to SI-RO, ruling out any PKG-independent effect of the drug. These results suggest that the protective effect of PKGI␣ is mediated by the NOS-dependent pathway.
Effect of PKGI␣ on Phosphorylation of MAPKs-ERK1 and JNK phosphorylation were increased in cardiomyocytes overexpressing PKGI␣ as compared with control and PKGI␣K390A-overexpressing cells (Fig. 7). However, p38 phosphorylation was not enhanced in cells overexpressing PKGI␣. The expression of total ERK1, JNK, and p38 was not altered with PKGI␣ or PKGI␣K390A. Similar to Akt, no further increases in ERK1 and JNK phosphorylation was observed when cardiomyocytes were subjected to SI and 15 or 30 min of reoxygenation following transfection with PKGI␣ (data not shown). To determine the relationship between PKGI␣-induced cytoprotection and activation of JNK and ERK, cardiomyocytes were treated with inhibitors of MAPKs. As shown in Fig. 8A, PD-98059 and SP600125 increased the percentage of trypan blue-positive cardiomyocytes from 19.6 Ϯ 0.4 in PKGI␣-overexpressing cardiomyocytes to 29.5 Ϯ 1.3 and 33.2 Ϯ 1.8, respectively (n ϭ 3, p Ͻ 0.001). In addition, PD-98059 and SP600125 increased TUNEL-positive nuclei from 6.6 Ϯ 0.4 in PKGI␣-overexpressing cardiomyocytes to 15.4 Ϯ 1.0 and 15.8 Ϯ 0.6, respectively (n ϭ 3; p Ͻ 0.001; Fig. 8B). These results suggest that phosphorylation of ERK and JNK were involved in PKGI␣-induced protection against SI-RO injury in cardiomyocytes.

DISCUSSION
PKG is a serine/threonine protein kinase and is one of the major intracellular receptors for cGMP. There are two types of PKG present in eukaryotic cells: type I and type II. The N terminus of PKG type I is encoded by two alternatively spliced exons that produce I␣ and I␤ isoforms (21,26,27). PKG is present in high concentrations in smooth muscle, platelets, cerebellum, hippocampus, dorsal root ganglia, neuromuscular end plate, and the kidney vasculature (18). Low levels have been identified in vascular endothelium, granulocytes, chondrocytes, and osteoclasts. The PKGI␣ isozyme is mainly found in lung, heart, platelets, and cerebellum. The I␤ Note that iNOS, eNOS, and Bcl-2 proteins were significantly increased, but Bax was decreased by PKGI␣ overexpression. DECEMBER 15, 2006 • VOLUME 281 • NUMBER 50 form is highly expressed with I␣ in smooth muscle, including uterus, vessels, intestine, and trachea (18,28). The activation of PKG phosphorylates many intracellular proteins and regulates important physiological functions such as relaxation of vascular smooth muscle, inhibition of cell differentiation and proliferation, and inhibition of platelet aggregation and apoptosis (18,29).

PKGI␣-induced Cardioprotective Signaling
Several studies have demonstrated that generation of NO from iNOS or eNOS and opening of the mitoK ATP channel plays an important role in ischemic as well as pharmacological preconditioning in the heart (30). Opening the mitoK ATP channel partially compensates the membrane potential, which enables additional protons to be pumped out to form a H ϩ electrochemical gradient for both ATP synthesis and Ca 2ϩ transport (31). Sarcolemmal channels are the premier sensors of the energy state of the myocyte and, when activated, shorten or interrupt the action potential (32). In times of metabolic stress, decreasing cellular electrical excitabil-ity could protect cells against injury. Han et al. (8) first suggested that K ATP channels are regulated by a PKG signaling pathway that induces ischemic preconditioning in cardiomyocytes. In this study, NO donors and PKG activators facilitated pinacidil-induced K ATP channel activities in a concentration-dependent manner, and a selective PKG inhibitor abrogated these effects. However, the effect of PKGI␣ and associated downstream signaling pathways in protection against SI-RO injury independent of confounding factors associated with preconditioning stimuli or drugs has never been investigated. Therefore, we used adenoviral transfection of PKGI␣ to evaluate its direct effect on protection of adult cardiomyocytes. Our results show that PKGI␣ induced protection of cardiomyocytes against SI-RO injury that was mediated by opening of mitoK ATP channels, because protection was abrogated by glibenclamide and 5-HD (Fig. 3). The protective effect of PKGI␣ was not inhibited by HMR1098, the selective blocker of sarcolemmal K ATP channel, thereby further supporting the role of mitoK ATP channels in PKGI␣-dependent protection. . NOS inhibitor blocks PKGI␣-induced cardiomyocyte protection. PKGI␣-overexpressing cardiomyocytes were treated with 100 M of the nonselective NOS inhibitor L-NAME for 30 min before SI-RO. A, cell necrosis is determined by trypan blue-positive cells after 90 min of SI and 2 h of RO. B, apoptotic nuclei identified using TUNEL assay after 90 min of SI and 18 h of RO. Note that L-NAME blocked cardiomyocyte protection by PKG (n ϭ 3; p Ͻ 0.001 versus control). L-NAME had no effect on necrosis or apoptosis in control and PKGI␣K390A-expressing cardiomyocytes exposed to SI-RO. FIGURE 7. PKGI␣ induces phosphorylation of ERK and JNK. A, representative Western blots showing expression levels of phospho-ERK, total ERK, phospho-p38, total p38, phospho-JNK, and total JNK in PKGI␣-or PKGI␣K390A-expressing cardiomyocytes. B, bar diagram showing the ratio of phospho-ERK to total ERK, phospho-p38 to total p38, and phospho-JNK to total JNK expression. Note that phospho-ERK and phospho-JNK were increased with PKG1␣ expression (n ϭ 3; p Ͻ 0.001 versus control).
Our results also showed that PKGI␣-induced protection against SI-RO was associated with enhanced phosphorylation of Akt, ERK1/2, and JNK. The inhibitors of PI 3-kinase (Fig. 4, C  and D), ERK, or JNK1 (Fig. 8) abolished PKGI␣-dependent protection against necrosis and apoptosis. These results suggest that PKGI␣-dependent myocyte protection relies upon multiple and possibly interrelated pathways involving activation of PI 3-kinase, Akt, ERK, and JNK1. PKGI␣ also increased the expression of iNOS, eNOS, and Bcl-2 and decreased expression of Bax (Fig. 5). The nonselective NOS inhibitor L-NAME significantly blocked PKG-dependent cardiomyocyte protection against SI-RO (Fig. 6). Previous studies have shown that PI 3-kinase plays an important role in preconditioning of the heart against ischemia/reperfusion injury (33). Many growth factors and hormones have been shown to exert their cellular function, including the activation of eNOS via the PI 3-kinase/Akt signaling pathway (34). The pharmacological activation of the antiapoptotic pro-survival kinase signaling cascades including PI 3-kinase-Akt and ERK by administration of growth factors at the time of reperfusion conferred cardioprotection by limiting both the apoptotic and necrotic components of cell death (35). Also, it has been shown that PI 3-kinase-Akt is activated and protects against ischemia/reperfusion injury by recruitment of downstream anti-apoptotic pathways of cell survival (36). These include phosphorylation and inactivation of pro-apoptotic proteins such as BAD (37), Bax (38,39), and caspases (40,41), as well as phosphorylation and activation of eNOS (42). In neuronal cells, NO has been shown to suppress apoptosis by inducing PKG activation, which induces phosphorylation of Akt and Bad (43). In the present study, PKGI␣ caused increased expression of Bcl-2, decreased expression of Bax, and activity of caspase-3, which may be mediated by the activation of PI 3-kinase-Akt.
Signaling pathways including the activation of protein kinase C and MAPKs (12,44), synthesis of iNOS, generation of NO, and opening of mitoK ATP channels have also been implicated in cardioprotection (45)(46)(47). PI 3-kinase activates Akt through phosphoinositide-dependent kinases (PDK-1 and -2), which in turn activate eNOS (42, 48 -51). The NO generation from eNOS stimulates guanylyl cyclase, which then activates PKG. MAPKs are important mediators of signal transduction from the cell surface to the nucleus, thereby regulating several cellular responses (21). Three distinct MAPKs have been identified in mammalian cells: ERK, JNK, and p38 kinase. ERK is involved in myocardial ischemic preconditioning (51)(52)(53)(54)(55) and antiapoptotic pathways (56). Our results show a significant increase of phosphorylation of ERK1 and JNK, but not p38, after overexpression of PKGI␣ as compared with the nontreated control and PKGI␣K390A-overexpressing cardiomyocytes (Fig. 7). Previous studies have also shown that PKG activated the JNK1 pathways via phosphorylation of MEKK1 in human colon cancer cells (23) and in cultured rat aortic smooth muscle cells (21). In the present study, we showed that ERK and JNK inhibitors completely abolished the protection against necrosis and apoptosis (Fig. 8), suggesting an essential role for these two kinases in PKGI␣-induced protection.
Our data show that MPG, a putative antioxidant blocked, the protective effect of PKGI␣ against necrosis and apoptosis following SI-RO (Fig. 3). These results suggest a possible role for redox signaling in the protective pathway(s) triggered by PKGI␣. The exact molecular target(s) of ROS by PKGI␣ is not clear from the present study. However it could possibly include activation of MAPKs, PI 3-kinase, and opening of mitoK ATP channels. It has been shown that the release of ROS following the opening of mitoK ATP channel trigger cardioprotection (57). It was suggested that opening of these channels allows potassium to enter the mitochondrial inner matrix, which then causes generation and release of ROS from the respiratory chain (58). ROS then acts as second messengers to activate a downstream pathway of protective kinases, including protein kinase C and possibly p38 MAPK, that finally converge on the cardioprotective end effector. The G i -coupled receptors, including those binding bradykinin and acetylcholine, have also been shown to mediate protection by opening mitoK ATP channels with the generation of ROS that act as second messengers to activate protein kinase C (17).
In summary, this study provides the first direct evidence that adenoviral expression of PKGI␣ in the absence of any stimulus (such as preconditioning or drugs) promotes anti-necrotic and anti-apoptotic effects following ischemia-reoxygenation injury in cardiomyocytes. Our data also show that opening of mitoK ATP channels; generation of ROS; phosphorylation of Akt, ERK, and JNK; increased expression of iNOS, eNOS, and Bcl-2; and decrease of Bax play an essential role in the protective effect of PKGI␣. Future studies are needed to further define interactions of the several signaling pathways that eventually lead to PKGI␣-dependent attenuation of necrosis and apoptosis in cardiomyocytes. Strategies for enhancing PKGI␣ preconditioning-like effects should be examined for their potential to expand therapeutic options for protecting the heart against ischemia/reperfusion injury.