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. Author manuscript; available in PMC: 2013 Sep 1.
Published in final edited form as: J Thromb Haemost. 2012 Sep;10(9):1736–1744. doi: 10.1111/j.1538-7836.2012.04833.x

Activated Protein C Modulates Cardiac Metabolism and Augments Autophagy in the Ischemic Heart

Robert Costa *, Alex Morrison *, Jingying Wang *, Chandrashekhara Manithody †, Ji Li *,1, Alireza R Rezaie †,1
PMCID: PMC3433592  NIHMSID: NIHMS388028  PMID: 22738025

Summary

Background

Modulation of the energy substrate metabolism may constitute a novel therapeutic intervention in the ischemic heart. AMP-activated protein kinase (AMPK) has emerged as a key regulator of favorable metabolic signaling pathways in response to myocardial ischemia. Recently, we demonstrated that activated protein C (APC) is cardioprotective against ischemia/reperfusion (I/R) injury by augmenting AMPK signaling.

Objectives

The objective of this study is to determine if the APC modulation of substrate metabolism contributes to its cardioprotective effect in I/R injury.

Methods

An ex vivo working mouse heart perfusion system was used to characterize the effect of wild-type APC and its signaling-proficient mutant, APC-2Cys (has dramatically reduced anticoagulant activity), on glucose transport in the ischemic heart.

Results

Both APC and APC-2Cys (0.2 μg/g) augment the ischemic stress-induced translocation of the glucose transporter (GLUT4) to the myocardial cell membrane, leading to increased glucose uptake and glucose oxidation in the ischemic heart (p<0.05 vs. vehicle). Both APC derivatives increased the autophagic flux in the heart following I/R. The activity of APC-2Cys in modulating these metabolic pathways was significantly higher than APC during I/R (p<0.05). Intriguingly, APC-2Cys, but not wild-type APC, attenuated the I/R-initiated fatty acid oxidation by 80% (p<0.01 vs. vehicle).

Conclusions

APC exerts a cardioprotective effect against I/R injury by preferentially enhancing the oxidation of glucose over fatty acids as energy substrates in the ischemic heart. Noting its significantly higher beneficial metabolic modulatory effect, APC-2Cys may be developed as a potential therapeutic drug for treating ischemic heart disease without risk of bleeding.

Keywords: APC, AMPK, GLUT4, Ischemia/reperfusion, Autophagy, Cardioprotection

Introduction

Myocardial ischemia is a condition that occurs when blood flow to the myocardium is reduced due to coronary atherosclerosis, coronary thrombosis, and the narrowing of arterioles in the heart. Current treatments are largely targeted at immediate restoration of blood flow to the heart by recanalization of the occluded coronary artery via the use of percutaneous coronary intervention, thrombolytics, and anticoagulants. These interventions, however, have the risk of exacerbating bleeding in patients who may already be at an increased risk due to other medications or conditions [1]. While the process of reperfusion of the heart aids in reducing the mortality rate of myocardial ischemia by up to 50% [2], the rapid restoration of coronary blood flow following myocardial ischemia can paradoxically induce the death of cardiac myocytes, a pathological condition known as reperfusion injury [3]. Reperfusion injury has been demonstrated to largely arise from the oxidative stress and no true therapies to date have been developed to alleviate it [4]. Therefore, there is an urgent need for novel therapeutic strategies that can limit myocardial ischemia/reperfusion (I/R) injury without increasing the risk of bleeding [5].

We recently demonstrated that recombinant activated protein C (APC) exerts a potent cardioprotective effect in the I/R injury through activation of AMP-activated protein kinase (AMPK) [6]. APC is a vitamin-K dependent serine protease that inhibits blood clotting through proteolytic degradation of procoagulant factors Va and VIIIa which are required cofactors for thrombin generation in plasma during the blood coagulation process [7,8]. In addition to its anticoagulant function, APC also elicits anti-inflammatory and cytoprotective signaling responses when it binds to endothelial protein C receptor (EPCR) to activate protease-activator receptor-1 (PAR-1) on vascular endothelium [9–11]. The anti-inflammatory and cytoprotective signaling properties of APC have been demonstrated to be independent of its anticoagulant activity [11]. Thus, in a recent study we demonstrated that a signaling-proficient APC mutant (APC-2Cys), which has dramatically reduced anticoagulant activity, ameliorated cardiac dysfunction under I/R conditions to an extent that was similar or even more effective than that observed with wild-type APC [6].

AMPK signaling has been shown to protect against myocardial ischemic injury through the regulation of metabolism in the heart by balancing the energy demand and supply in response to ischemic stress [3,12]. Activated AMPK can phosphorylate acetyl-CoA carboxylase (ACC), thereby inhibiting its activity in the fatty acid synthesis pathway [13]. Other downstream effects of AMPK pathways include glucose uptake [14,15], glycolysis [14], and fatty acid oxidation [16], all of which favor ATP production in order to supply sufficient energy for cell survival under stress conditions. Recently, AMPK-mediated autophagy has also been shown to play a possible protective role during myocardial ischemia [17]. Autophagy has emerged as an important mediator in the regulation of glucose homeostasis [18]. There is increasing evidence that enhancing glucose oxidation and inhibiting fatty acid oxidation in the ischemic heart has a beneficial effect for maintaining cardiac efficiency [19,20]. For this reason, novel therapeutics that can selectively target the up-regulation of glucose metabolism in the ischemic heart may be of high value. Whether the cardioprotective signaling function of APC contributes to modulation of energy substrate metabolism in the ischemic heart has never been investigated. We undertook this study to address this important question.

Materials and methods

Animals

8–12 weeks male C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were maintained on a 12 hour light/dark cycle in a controlled environment with water ad libitum. All animal protocols were approved by the Institutional Animal Care and Use Committee of State University of New York (SUNY) at Buffalo.

Ex vivo heart perfusion

Mice were anesthetized with pentobarbital sodium (60 mg/kg i.p.) and heparinized (100 units i.p.). Hearts were excised and retroperfused (4 mL/min) in the perfusion heart system (Radnoti Glass Technology Inc., Monrovia, CA) with 95% O2 and 5% CO2 in equilibrated Krebs-Henseleit buffer (KHB) containing 7 mM glucose, 1% BSA and 0.4 mM oleate. For the ex vivo ischemic model the buffer flow was stopped for 10 min, at which point hearts were reperfused with the same flow rate and buffer containing APC (20 nM), APC-2Cys (20 nM) [21], APC-E170A (20 nM) [22] or Protein C-2Cys (20 nM) [21]. The LabChart7 software from ADInstruments (Colorado Springs, CO) was used to monitor the heart rate and left ventricle pressure.

Cell surface GLUT4 labeling by Bio-LC-ATB-BGPA

Cell membrane GLUT4 labeling with 4,4′-O-[2-[2-[2-[2-[2-[6-(biotinylamino)hexanoyl]-amino]ethoxy]ethoxy]-ethoxy]-4-(1-azi-2,2,2,-trifluoroethyl)benzoyl]amino-1,3-propanediyl]bis-D-glucose (bio-LC-ATB-BGPA) was performed as described [23]. After perfusion, mouse hearts were flushed through aortic cannulation with 1 mL ice-cold glucose-free KHB and then perfused with the same buffer containing 300 μM bio-LC-ATB-BGPA. Hearts, infused with bio-LC-ATB-BGPA, were incubated at 4°C for 15 min. To enhance crosslink between bio-LC-ATB-BGPA and cell surface GLUT4, the left ventricle (LV) and right ventricle (RV) were cut sagittally and reactions were exposed to UV irradiation twice 5 min each. Heart tissues were then freeze-clamped and stored in −80ºC until further analysis.

To isolate cell surface GLUT4 protein, photolabeled cardiac tissues were homogenized in 250-μL of HEPES-EDTA-sucrose (HES) buffer containing 20 mM HEPES, 5 mM Na-EDTA, 255 mM sucrose, and protease inhibitor cocktail (Hoffmann-La Roche Inc., Indianapolis, IN). Tissue homogenates were added 250-μL of 4% Thesit/PBS, incubated on ice for 15 min, and then kept at 4°C for another 15 min. Tissue homogenates were centrifuged at 20,000g at 4°C for 30 min and pellets were discarded. 10-μL of the supernatant was taken to measure total protein concentrations. To isolate the photolabeled GLUT4, 400 μg total membrane protein were incubated with 100-μL streptavidin bound to 6% agarose beads (Pierce, Rockford, IL) overnight at 4°C. The steptavidin-agarose isolated labeled fraction of GLUT4 was washed extensively with PBS containing decreasing concentrations of Thesit (1, 0.1, and 0%). The labeled GLUT4 was then dissociated from streptavidin by boiling in the loading buffer for 30 min prior to analysis by SDS-PAGE.

Measurement of glucose uptake

Glucose uptake was analyzed in the Langendorff heart perfusion mode by measuring the production of 3H2O from D-[2-3H]-glucose, as previously described [12]. Briefly, the KHB buffer containing D-[2-3H]-glucose (50 μCi/L) was perfused into the isolated heart and the coronary effluent was sampled every 5 min. To separate the non-metabolized D-[2-3H]-glucose from 3H2O, ion exchange chromatography (Bio-Rad AG1–8X resin; Bio-Rad, Hercules, CA) was conducted by activating the resin with 1M sodium hydroxide. Resin columns were extensively washed with dH2O to make sure the pH is less than 8. A 500-μL coronary effluent sample was added to each column to allow binding of glucose to the resin. 3H2O was washed out by flushing the column with 2.5-mL dH2O. All flow was collected in scintillation vials, which were then subjected to radioactive counting. The rate of glucose uptake was calculated by the amount of 3H2O production.

Fatty acid/glucose oxidation analysis

Cardiac substrate metabolism was determined in the working heart model as described [12,24]. The working heart preload was set up at 15 cm H2O, and afterload at 80 cm H2O. The flow rate was kept at 15 mL/min. Mouse heart was first aortically cannulated in order to initiate Langendorff perfusion. The pulmonary vein was then cannulated and the working heart mode was started with the perfusion of [9,10-3H]-oleate and [U-14C] glucose. The heart function was monitored by pressure transducer connected to aortic outflow.

Fatty acid oxidation was determined by the production of 3H2O from [9,10-3H]-oleate. To separate 3H2O from [9,10-3H]-oleate, perfusate samples were filtered through the anion exchange resin (200–400 Bio-Rad AG1–2X resin; Bio-Rad, Hercules, CA) pretreated with 1M sodium hydroxide. Resin columns were extensively washed with dH2O to lower the pH below 8. A 400-μL sample was loaded on the column and eluted into scintillation vials with 2.5-mL of dH2O. A 10-mL of scintillation fluid was added to each vial and the samples were read. Glucose oxidation was determined by the production of 14CO2 from [U-14C] Glucose. The 14CO2 was captured using hyamine hydroxide, placed in scintillation vials, and 13-mL of scintillation fluid was added to each vial followed by reading the radioactive signal on a liquid scintillation counter.

Immunoblotting analysis

Immunoblotting was performed as described [6]. Heart homogenates were resolved by SDS-PAGE, and proteins were transferred onto polyvinylidene difluoride membranes. For reprobing, membranes were stripped with 50 mM Tris·HCl, 2% SDS, and 0.1M β-mercaptoethanol (pH 6.8). Rabbit polyclonal antibodies against phosphorylated Akt (Ser473), GLUT4, and LC3 were purchased from Cell Signaling Tech (Danvers, MA). Rabbit polyclonal antibodies against total Akt were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit secondary antibodies were purchased from Cell Signaling Tech.

Cellular redox measurement

The production of reactive oxygen species (ROS), by ex vivo hearts subjected to 10 min global ischemia and 20 min reperfusion, was inferred through measuring the GSH/GSSG ratio in the heart tissue using a Glutathione detection kit (Enzo Life Sciences, Farmingdale, NY) and a plate reader according to the manufacturer’s instruction.

Measurement of autophagy

Autophagy was investigated by analysis of the intracellular localization and processing of the microtubule-associated protein 1 light chain 3 (LC3) in the heart following I/R as described [25]. LC3 is synthesized as pro-LC3 which is cleaved by Atg4B to form LC3-I with the carboxyl terminal Gly exposed [25]. LC3-I is activated by Atg7 and conjugated to phosphatidylethanolamine (PE) which has been designated LC3-II [25]. LC3-II is used as a marker of autophagy because its lipidation and specific recruitment to autophagosomes increases its electrophoretic mobility on the gels compared with LC3-I [25]. To determine the amount of LC3-II, immunoblotting analysis was performed using antibody against LC3, and densities of LC3-II and LC3-I were measured by the NIH image software.

Statistical analysis

Data were expressed as means ± S.E. Data were analyzed using 1-way ANOVA to measure statistical significance. For single- and multi-factorial analyses, appropriate post-hoc test(s) were performed to measure individual group differences of interest. A p value of less than 0.05 was considered statistically significant.

Results

APC and APC-2Cys modulate GLUT4 translocation to the membrane

AMPK activation is known to protect against I/R injury via modulating energy substrate metabolism [26,27]. In light of our recent finding that the signaling function of APC elicits potent cardioprotective activity through activation of AMPK [6], we decided to investigate whether APC and the anticoagulant-defective mutant, APC-2Cys, can modulate glucose metabolism during I/R. The hearts from C57BL/6 mice were isolated and subjected to 10 min of global ischemia followed by 20 min of reperfusion. Isolated hearts were then infused with bio-LC-ATB-BGPA in order to label the pool of GLUT4 localized to the cell membrane. This strategy allowed quantification of the amount of GLUT4 translocated from intracellular vesicles to cell surface during ischemic stress. Results presented in Fig. 1A showed that both APC and APC-2Cys markedly increase the I/R-stimulated GLUT4 membrane accumulation compared to the I/R-vehicle group (p<0.05).

Fig. 1.

Fig. 1

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APC increases glucose uptake during I/R. (A) APC modulates GLUT4 translocation to the membrane. Immunoblotting analysis of the cell membrane-bound and total GLUT4 transporter in the heart tissues. Isolated C57BL/6 mouse hearts were subjected to 10 min global ischemia followed by 20 min reperfusion in the ex vivo working heart perfused system. Cell surface GLUT4 were labeled by the cell membrane impermeable compound bio-LC-ATB-BGPA. (B) C57BL/6 mouse hearts were isolated and perfused with D-[2-3H]-glucose labeled perfusion buffer in the ex vivo working heart perfused system. Isolated hearts were subjected to 10 min global ischemia followed by 20 min reperfusion. Perfusates were collected 5 min intervals during reperfusion and the production of 3H2O from D-[2-3H]-glucose were measured by scintillation counter. Values are means ± S.E., n=6 per group, *p<0.05 vs. control, †p<0.05 vs. I/R vehicle.

APC increases glucose uptake during I/R

Next, we investigated the functional significance of APC- and APC-2Cys-mediated enhancement in the membrane accumulation of GLUT4 during I/R. Glucose uptake was analyzed by collecting samples every 5 min and measuring the production of 3H2O from D-[2-3H]-glucose in the perfusate of an ex vivo working heart perfusion system. Both APC and APC-2Cys significantly enhanced the I/R-induced glucose uptake compared to the I/R-vehicle groups (Fig. 1B). Intriguingly, APC-2Cys demonstrated a significantly stronger augmentation of glucose uptake as compared to APC (p<0.01) (Fig. 1B). The capacity of APC-2Cys in mediating GLUT4 translocation to the cell surface was also slightly higher than that of wild-type APC, though the difference was not statistically significant (Fig. 1A).

Akt signaling is not involved in APC-mediated glucose transport during I/R

To understand the molecular basis for the APC-mediated glucose uptake during I/R, we examined the role of Akt signaling in the I/R-induced GLUT4 translocation and glucose uptake [28]. Akt is at the center of the insulin cascade and is required for the insulin-mediated glucose uptake by mediating GLUT4 translocation [29]. Isolated hearts were subjected to 10 min of ischemia followed by 20 min of reperfusion in the ex vivo working heart perfusion system and then phospho-Akt (Ser473) levels were assessed by immunoblotting. The results presented in Fig. 2 indicate that neither APC nor APC-2Cys activates Akt during I/R, suggesting that Akt is not involved in the APC modulation of glucose transport in the heart.

Fig. 2.

Fig. 2

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APC inhibits Akt signaling pathway in the heart during I/R. Isolated C57BL/6 mouse hearts were subjected to 10 min global ischemia followed by 20 min reperfusion in the ex vivo working heart perfused system. Immunoblotting analysis of Akt phosphorylation in the heart tissue homogenates was conducted using an antibody that recognizes phosphorylated levels of Akt at Ser473 (p-Akt) in the heart. Values are means ± S.E., n=5–7 per group, *p<0.05 vs. control, †p<0.05 vs. I/R vehicle.

Both APC and APC-2Cys modulate glucose oxidation during I/R

With APC increasing both GLUT4 translocation and glucose uptake in the heart during I/R, the next question we asked was whether or not this process is correlated with an increase in the cardiac glucose oxidation post reperfusion. Glucose oxidation was analyzed by measuring [14C]-glucose incorporation into 14CO2 released from the ex vivo working heart after 10 min of ischemia and 20 min of reperfusion. Results demonstrated that both APC and APC-2Cys markedly increased the rates of glucose oxidation in the I/R-stressed hearts (p<0.01) (Fig. 3A). Consistent with a more pronounced glucose uptake, APC-2Cys was more efficient than APC in glucose oxidation in the heart during I/R (p<0.01) (Fig. 3A). However, neither APC-E170A, which lacks cytoprotective signaling activities, nor PC-2Cys which has no catalytic activity, had an effect on ischemic glucose oxidation, suggesting that active-site dependent signaling function of APC is involved in modulating this metabolic process (Fig. 3A).

Fig. 3.

Fig. 3

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APC augments glucose oxidation in the heart during I/R. Glucose oxidation was analyzed by measuring [14C]-glucose incorporation into 14CO2 in the ex vivo C57BL/6 mouse hearts subjected to 10 min ischemia and 20 min reperfusion. The oleate oxidation was analyzed by measuring incorporation of [9,10-3H] oleate into 3H2O. Values are means ± S.E., n=5–6 per group, *p<0.05 vs. control, †p<0.05 vs. I/R vehicle, #p<0.01 vs. I/R APC.

Effects of APC derivatives on fatty acid oxidation

In light of significant effect of APC and APC-2Cys on glucose metabolism, we decided to investigate their impact on cardiac fatty acid oxidation, the major energy source for normal cardiac metabolism [30]. Fatty acid oxidation was measured by the production of 3H2O from [9,10-3H]-oleate in the ex vivo working hearts. Isolated hearts were subjected to 10 min of ischemia and 20 min of reperfusion. As presented in Fig. 3B, oleate oxidation was significantly up-regulated in the heart during I/R. Interestingly, only APC-2Cys significantly reduced the rate of oleate oxidation during I/R (Fig. 3B). None of other APC derivatives, including wild-type APC, the signaling defective APC-E170A, and the catalytically inactive zymogen protein C-2Cys (PC-2Cys) altered the level of oleate oxidation in the heart during I/R.

APC-2Cys improves intracellular redox in the heart during I/R

The I/R-induced acceleration of cardiac fatty acid oxidation has been shown to create more reactive oxygen species (ROS) as compared to glucose oxidation [31]. Therefore, we reasoned that a reduction in oleate oxidation may lead to a decrease in ROS generation, thereby improving the intracellular redox status in the heart during I/R. The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) has been used as an indicator of the intracellular redox status in the heart tissue [32]. Thus, we measured the GSH/GSSG ratio to assess the redox status of hearts subjected to 10 min of global ischemia and 20 min of reperfusion in the ex vivo working heart perfusion system. Results showed that I/R stress impairs the intracellular redox by decreasing the cardiac GSH/GSSG ratio (Fig. 4). APC-2Cys significantly improved the intracellular redox of the ischemic heart as indicated by an increase in the ratio of GSH to GSSG as compared to the I/R-vehicle group (Fig. 4). By contrast, neither APC nor APC-E170A had an effect on the GSH/GSSG ratio (Fig. 4). We also determined effects of APC derivatives on the intracellular redox of isolated cardiomyocytes. Results demonstrated that none of the APC derivatives can alter the intracellular redox under basal conditions (data not shown).

Fig. 4.

Fig. 4

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APC-2Cys improves intracellular redox status in the heart during I/R. The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was calculated as an index of the intracellular redox status. GSH/GSSG ratios were measured using a Glutathione detection kit. Values are means ± S.E., n=11 per group, *p<0.01 vs. control, †p<0.01 vs. I/R vehicle.

APC and APC-2Cys modulate autophagy in the heart during I/R

The importance of autophagy in the heart during I/R has been recognized by several studies [17,33]. More recently, there is evidence that autophagy is involved in modulation of glucose homeostasis in the skeletal muscle in response to energy stress like exercise [18]. Moreover, it has been demonstrated that AMPK signaling regulates autophagy [34–36]. Therefore, we tested the ability of APC to modulate the autophagic flux during I/R by measuring the LC3 II/LC3 I ratio as an indicator of autophagy [18]. Results demonstrated that I/R induces autophagy and that both APC and APC-2Cys can augment this process (Fig. 5). Intriguingly, APC-2Cys exhibited a more pronounced effect on modulating autophagy which may explain the basis for the observed higher activity of APC-2Cys in regulating cardiac glucose metabolism during I/R.

Fig. 5.

Fig. 5

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APC augments I/R-triggered autophagy in the heart. Isolated C57BL/6 mouse hearts were subjected to 10 min global ischemia followed by 20 min reperfusion in the ex vivo working heart perfused system. Autophagy was monitored by immunoblotting analysis of LC3-II and LC3-I in the heart tissue homogenates and defined as the ratio of LC3-II/LC3-I in the heart. Values are means ± S.E., n=6 per group, *p<0.05 vs. control, †p<0.05 vs. I/R vehicle.

Discussion

Previous studies have established a protective role for APC in limiting myocardial I/R injury [37–40]. We recently demonstrated that APC exerts its EPCR- and PAR-1-dependent cardioprotective effect through the up-regulation of AMPK signaling largely independent of its anticoagulant function [6]. In the present study, we show that APC increases glucose uptake and up-regulates GLUT4 translocation to the cell membrane, thereby modulating energy substrate metabolism in the ischemic heart in a beneficial manner so that ATP is generated primarily through the glucose oxidation during reperfusion. Interestingly, APC also increased the rate of autophagy and improved the intracellular redox status of the I/R-stressed heart. These results support recent interesting findings that autophagy contributes to intracellular glucose homeostasis under short-term stress conditions [18].

Activated AMPK, through phosphorylation of downstream substrates, initiates a number of biological events that culminates in modulating energy substrate metabolism and subsequent restoration of cardiomyocyte ATP levels [3,41]. In the ischemic heart, AMPK enhances glucose uptake via mediating the translocation of the glucose transporter GLUT4 from storage vesicles to the cell surface [12,42]. AMPK also stimulates glycolysis by directly activating phosphofructose kinase 2 (PFK-2) through phosphorylation at Ser466, which further increases the production of fructose 2,6-bisphophate, an allosteric activator of PFK-1 in the glycolytic pathway [43]. AMPK activation maintains an enhanced glucose uptake during initial reperfusion stage to improve cardiac contractile functions, as evidenced by transgenic mice expressing a kinase dead mutation exhibiting impaired glucose uptake and post-ischemic contractile function [12]. Results of this study now demonstrate that both APC and APC-2Cys increase the cell surface accumulation of GLUT4, thereby leading to augmentation of glucose uptake and glucose oxidation during I/R. These results, together with our previous findings indicate that both APC and APC-2Cys modulate cardiac glucose metabolism through the activation of the AMPK signaling pathway [6].

It has been demonstrated that insulin also can exert a cardioprotective effect during ischemic stress [15,44]. In this case, however, the cardioprotective effect of insulin is mediated through the phosphatidylinositol 3-kinase (PI3K)-dependent activation of Akt [15,44]. The insulin-dependent Akt phosphorylation has been shown to mediate the translocation of GLUT4 to the plasma membrane, thereby leading to the enhancement of glucose uptake and its oxidation by the I/R-stressed heart. The observation of this study that APC inhibited the phosphorylation of Akt suggests that the APC-mediated GLUT4 translocation in the ischemic heart is mediated by a different mechanism that is independent of Akt signaling. Actually, an inverse correlation has been observed between Akt and AMPK signaling in the ischemic heart [44]. APC exerts its cardioprotective and anti-inflammatory activities through the Gla-dependent interaction with EPCR [9], followed by its activation of PAR-1 [10,11], localized to membrane lipid-rafts [45]. Thrombin can also activate PAR-1 [11]. In this case, however, the activation of PAR-1 initiates a pro-inflammatory response [11]. We have demonstrated that the interaction of Gla-domain of APC with EPCR in the lipid-raft micoenvironment switches the signaling specificity of PAR-1 from a pro-inflammatory to a protective response [46]. Noting that both APC and APC-2Cys have normal affinity for EPCR and both proteases cleave PAR-1 with similar efficiency [40], the molecular basis for the significantly higher modulatory activity of APC-2Cys toward glucose metabolism remains unknown. Nevertheless, the active-site dependent signaling function of the protease was required for its ability to modulate glucose metabolism in the heart since neither the zymogen protein C-2Cys nor the signaling defective APC-E170A mutant exhibited a glucose modulatory effect in the I/R-stressed heart. It is worth noting that, in addition to EPCR and PAR-1, the protective signaling activity of APC also requires receptor crosstalk and/or protease interaction with a number of other G-protein (i.e., PAR-3 [47] and sphingosine 1-phosphate receptor 1 [11]) and non-G-protein coupled (i.e., apolipoprotein E receptor 2 [48] and Tie2 [49]) receptors. Thus, further studies will be required to determine whether possible differences in the binding affinity of APC and APC-2Cys with any one of these receptors contributes to their differential modulatory effect on substrate metabolism in the I/R-stressed heart.

It was interesting to note that, unlike its enhanced activity in modulating glucose metabolism, APC-2Cys markedly inhibited the oxidation of oleate in the heart during I/R. This function of APC-2Cys may be beneficial for the ischemic heart since during reperfusion a sudden increase in the oxygen level can lead to enhanced fatty acid oxidation and subsequent generation of more reactive oxygen species (ROS), which can cause greater cardiac damage [50]. There are reports suggesting that cardiac AMPK activation accelerates fatty acid oxidation in heart [13,51]. Similarly, results of the ex vivo working heart model in this study showed an increase in oleate oxidation during reperfusion after ischemia. Interestingly, while wild-type APC did not significantly affect oleate oxidation, APC-2Cys dramatically inhibited cardiac oleate oxidation during I/R. The molecular basis of the APC-2Cys-mediated inhibition of oleate oxidation may be due to its up regulation of glucose oxidation which can lead to a decreased fatty acid β-oxidation via the feedback inhibition of 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase [19]. Thus, the APC-2Cys-mediated increase in glucose oxidation can improve the coupling of glucose metabolism, thereby decreasing proton production and improving cardiac efficiency [19]. In line with the beneficial effect of enhanced glucose metabolism during I/R, several fatty acid β-oxidation inhibitors have been developed as therapeutic drugs and proven to be highly effective against ischemic heart disease. Two such drugs, trimetazidine and ranolazine, are both partial fatty acid β-oxidation inhibitors that reciprocally increase glucose oxidation [52,53]. Therefore, not withstanding the controversial nature of the question as to whether or not elevated glucose oxidation at the expense of reduced fatty acid oxidation in the ischemic heart is beneficial [54], APC-2Cys has potential therapeutic utility as a fatty acid oxidation inhibitor for treating ischemic heart disease without increasing the risk of bleeding that may be associated with the use of wild-type APC.

APC is a metal-ion binding protease which requires both Ca2+ and Na+ for its normal catalytic function [21]. APC-2Cys is a mutant in which the Ca2+-binding loop of the protease in the catalytic domain has been stabilized by an engineered disulfide bond [21]. Noting that the Ca2+ and Na+ binding loops of APC are allosterically linked, this construction strategy abrogates the requirement for the metal ions by the protease and also eliminates the anticoagulant function of APC without significantly affecting its cytoprotective signaling properties [21]. In light of the observation that APC-2Cys promoted cardiac glucose metabolism and inhibited fatty acid oxidation during reperfusion, we postulated that this property of the mutant may arise from a small fraction of the protease potentially existing at equilibrium with the reduced form of the protease in which the engineered cysteine residues are not engaged in a disulfide bond. In this case, this small fraction of free-cysteine containing protease could function as a sink for the ROS produced during I/R, thus leading to alterations in the lipid-raft redox signaling mechanism in the membrane of cardiomyocytes [55]. This possibility was tested by measuring glucose and oleate oxidation in the working heart model using a reperfusion buffer which contained 20 nM oxidized glutathione (GSSG), sufficient to quench the potential trace amount of free cysteine residues in the mutant protease. However, we found that the addition of GSSG to the buffer does not affect either oleate or glucose oxidation rates of the APC-2Cys groups, ruling out the possibility that free cysteine residues, if any, in APC-2Cys contributes to its unique metabolic modulatory properties. Further support for this hypothesis was provided the observation that the zymogen form of the mutant (PC-2Cys) had no modulatory effect on either glucose or fatty acid oxidation. Nevertheless, APC-2Cys did demonstrate a stronger anti-oxidative activity as evidenced by the increased ratio of GSH/GSSG during I/R, which could somehow contribute to modulation of the lipid-raft redox signaling system of the cell membrane, thereby differentially affecting glucose and fatty acid oxidation by the mutant protease.

Finally, autophagy, defined as a “self-eating”, has been recognized an important process in modulating glucose homeostasis under stress conditions [18,56]. Therefore, we tested the effect of APC and APC-2Cys on the stimulation of autophagy in the heart during I/R. Both APC and APC-2Cys were found to significantly augment I/R-triggered autophagy in the heart, with APC-2Cys demonstrating a significantly greater potency in modulating I/R-induced autophagy in the heart. This property of APC-2Cys may further contribute to the mutant protease’s stronger modulatory activity in glucose metabolism in the ischemic heart. Although autophagy is thought to be protective in the ischemic heart, recently it has been shown that excessive autophagy can also be detrimental [57], thus further studies are required to understand the mechanisms by which APC regulates autophagy in the ischemic heart and also to decipher the importance of autophagy in general during I/R injury.

Taken together, the results of this study suggest that the APC modulation of the energy substrate metabolism may exert a cardioprotective effect in the ischemic heart, thereby reducing the incidence of myocardial infarction. This observation may potentially lead to the development of APC-based novel therapeutics for better treatment of myocardial I/R injury in humans.

Acknowledgments

We would like to thank Audrey Rezaie for proofreading the manuscript. This work was supported by grants awarded by the National Heart, Lung, and Blood Institute of the National Institutes of Health HL 101917 and HL 68571 (A.R.R.); and by grants awarded by American Heart Association 0835169N, 12GRNT11620029 and American Diabetes Association 1-11-BS-92 (J.L.).

Footnotes

Disclosure of Conflict of Interests

There authors state that they have no conflict of interest

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