University of Groningen
Isoflurane induced eNOS signaling and cardioprotection
Baotic, Ines
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Publication date: 2018
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Baotic, I. (2018). Isoflurane induced eNOS signaling and cardioprotection: Preconditioning mechanisms under normal and hyperglycemic conditions. University of Groningen.
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General Discussion
and Future Perspectives
Main findings of the thesis
The investigations presented in this thesis reveal a central role of regulati-on of eNOS and ROS in the protective mechanisms cregulati-onferring cardiopro-tection by APC and in the attenuation thereof by diabetes and hyperglyce-mia . Such is evidenced by the following:
• Our investigation demonstrated that APC by isoflurane effectively re-duced myocardial ischemia and reperfusion injury in Wistar rats by a GTPCH-1–dependent mechanism . APC up-regulated the expression of GTPCH-1 and eNOS and enhanced production of NO in myocardium after reperfusion . The cardioprotective effects of APC were blocked by DAHP, a pharmacologic antagonist of GTPCH-1, and this inhibitor also abolished APC-induced increases in NO . Taken together with previo-us evidence, these findings substantiate that isoflurane stimulates a NO biosynthetic pathway furthermore identifying potential key regulatory points that may determine clinically relevant sensitivity versus resistan-ce to APC cardioprotective effects .
• In our endothelial cell – cardiomyocyte (EC-CM) co-culture model, EC contributed to isoflurane-induced protection of CM against hypoxia and reoxygenation injury and this protection was conferred by the increase in HIF1α and NO synthesis (Leucker, Bienengraeber et al. 2011). Further-more, the presence of EC delayed mitochondrial permeability transition pore (mPTP) opening in CM following APC, reflecting the preservation of mitochondrial integrity . Taken together, our data support the concept that EC stimulated by isoflurane produce important cardioprotective fa-ctors that may contribute to the protection of myocardium during ische-mia and reperfusion injury (Leucker, Bienengraeber et al . 2011) .
• By using a type 2 diabetic rat model with two different variants of mito-chondrial DNA, mtWistar and mtFHH, we demonstrated that differences in the mitochondrial genome modulate isoflurane-induced generation of ROS which translates into a differential effectiveness of APC to reduce ischemia–reperfusion injury (Muravyeva, Baotic et al . 2014) . The supe-rior protection by APC of diabetic mtWistar rats was accompanied by a lower oxidation of mitochondrial flavoproteins and delayed opening of the mPTP . Thus, mitochondrial ROS production and redox state were central in the signaling pathway modulated by diabetes and APC . • We demonstrated the Apolipoprotein A-1 mimetic, D-4F, to enhance
isoflurane-induced eNOS signaling and to restore APC mediated cardi-oprotection during acute hyperglycemia (AHG) in male C57BL/6J mice .
Potential mechanisms of D-4F restoring APC elicited cardioprotection were further investigated in HCAEC culture model during normogly-cemia and hyperglynormogly-cemia. D-4F reduced superoxide generation and enhanced caveolin-1 and eNOS caveolar compartmentalization and posttranslational eNOS modifications, thus restoring induction of NO production by isoflurane APC during AHG (Baotic, Ge et al . 2013) . To-gether, these results indicate that isoflurane increases bioavailability of NO by modulating intracellular compartmentalization and activation of eNOS within caveolae, a process that is blocked by high glucose con-centrations. Further, the adverse effects of AHG on APC were ameliora-ted by D-4F, an ApoA-1 mimetic.
Figure 1. Diagram depicting key elements of the pathways activated in
anesthet-ic-induced preconditioning with the results of the published investigations includ-ed into PhD thesis using the indicatinclud-ed blockers (Fig 2 from the Introduction with added results) . (Kikuchi, Dosenovic et al. 2015).
The APC exerts its actions both on endothelial cells (left) and cardiomyocytes (right), in-fluencing endothelial cell-to-cardiomyocyte interactions. In endothelial cells, APC induc-es a limited increase in production of ROS, ultimately rinduc-esulting in activation of eNOS and increased NO production. In cardiomyocytes, APC activates GPCR and multiple protec-tive signaling pathways towards mitochondria, furthermore directly or indirectly interacting with mitochondrial ETC complex I and/or complex III thus promoting a limited formation of ROS (‘signaling ROS’). Akt, protein kinase B; ERK, extracellular signal-regulated kinase; ETC, electron transport chain; GPCR, G protein-coupled receptors; GTPCH-1, guanosine triphosphate cyclohydrolase-1; HIF1α, hypoxia-inducible factor 1α; HSP90, heat shock
pro-tein 90; MAPK, mitogen- activated propro-tein kinase; Mito KATP, mitochondrial ATP-sensitive potassium channels; mPTP, mitochondrial permeability transition pore; PI3K, Phosphatidy-linositol 3-Kinase; RNS, reactive nitrogen species; Sarc KATP, sarcolemmal ATP-sensitive potassium channels; SUR, sulfonylurea receptor; VEGF, vascular endothelial growth factor. Inhibitors: DAHP, 2,4-diamino-6-hydroxypyrimidine (GTPCH-1 inhibitor); U0126 or PD98059 -two chemically distinct inhibitors of MEK (upstream kinase that phosphorylates ERK); NAC, N-acetylcysteine (ROS scavenger); GSK3β, Glycogen synthase kinase-3 beta (inhibition of
mPTP). Number in brackets indicates the chapter of PhD thesis in which result is presented.
The main results of the thesis are depicted in Figure 1.
In chapter 1, perioperative cardiovascular complications andhyperglyce-mia/diabetes as independent risks factors for increased CV complications during cardiac and non-cardiac surgery are introduced . Moreover, precon-ditioning with volatile anesthetics and its therapeutic application as phar-macological cardioprotection during I/R injury is briefly summarized, which is followed by a description of the underlying mechanisms of APC induced cardioprotection in experimental models. Further, the loss of cardioprote-ctive APC signaling by a hyperglycemic metabolic state in animal models is discussed . However, due to inconclusive results from clinical investigations on the cardioprotective effect of APC applied during cardiac surgery even in normoglycemic patients, data on APC in patients with hyperglycemia are lacking .
In chapter 2 “Isoflurane Favorably Modulates Guanosine Triphosphate
Cyclohydrolase-1 and Endothelial Nitric Oxide Synthase during Myocardial Ischemia and Reperfusion Injury in Rats” (Baotic, Weihrauch et al . 2015), we demonstrated that the cardioprotective effect of APC is elicited by NO production through a GTPCH-1–dependent mechanism . APC induced ti-me-dependent changes in eNOS derived NO production, related to an in-crease both in eNOS and GTPCH-1 gene and protein expressions after 60 and 90 min of reperfusion in rat heart (Baotic, Weihrauch et al . 2015) . The importance of this pathway was further evidenced by the loss of APC indu-ced cardioprotection and the inhibition of NO production by pretreatment with DAHP, a pharmacologic inhibitor of GTPCH-1 . Our results suggest that isoflurane may have enhanced eNOS coupling by a GTPCH-1 dependent mechanism; however, we did not assess this action in myocardium directly (Baotic, Weihrauch et al . 2015) . Overall, these data offer better insight in basic mechanisms of APC on NO biology that may determine clinically rele-vant sensitivity vs . resistance to this pharmacological preconditioning . The importance of GTPCH-1 in this setting is underscored by previous observa-tions that targeted overexpression of GTPCH-1 in endothelial cells (Leucker, Ge et al . 2013) or myocardium (Ge, Ionova et al . 2011) protects against I/R injury in vivo and in vitro. Further, GTPCH-1 expression and NO production are increased in Brown Norway rats that are resistant to myocardial infarcti-on compared with the ischemia-sensitive Dahl S rats (An, Du et al . 2009), and human genetic variants of GTPCH-1 may predict cardiovascular risk (Zhang, Rao et al . 2007) .
However, as we did not evaluate the GTPCH-1 role in APC during cardiac I/R injury in hyperglycemia, this still needs to be investigated . Nevertheless, re-cent reports support a crucial role of GTPCH-1 as an important modulator of cardiac dysfunction in diabetes . Wu et al . (2016) used transgenic mice with cardiomyocyte specific overexpression of GTPCH-1 or a specific inhibitor of GTPCH-1, DAHP, both in normoglycemic and hyperglycemic mice (strepto-zotocin induced T1DM) . Overall, their results demonstrate that GTPCH-1 is necessary for maintenance of normal cardiac histology and contractile fun-ction . Diabetes decreased the expression of cardiac GTPCH-1 protein witho-ut a significant change in GTPCH-1 mRNA levels, suggesting that the diabe-tes-induced decrease of GTPCH-1 protein is due to accelerated degradation rather than a decrease in biosynthesis . Also, diabetes resulted in a significant decrease in left ventricle (LV) wall thickness and contractile function, which was reduced by GTPCH-1 overexpression (Wu, Baumgardt et al . 2016) . The beneficial effects of GTPCH-1 overexpression on diabetic hearts was asso-ciated with an improvement of intracellular Ca2+ signaling as a result of
in-creased BH4 bioavailability, inin-creased levels of dimeric and phosphorylated nNOS and SR Ca2+ handling proteins, and decreases in p-p38 MAPK and O
2
-production (Wu, Baumgardt et al . 2016) . Although the beneficial effect of in-creased GTPCH-1 expression was found in the context of the development of diabetic cardiomyopathy rather than myocardial I/R injury, the findings clearly implicate a predominant role for GTPCH-1 in the mitigation of diabe-tes-induced cardiac injury . Moreover, while isoflurane increases the ratio of reduced (BH4) to oxidized (BH2) biopterin and NO production in endothelial cells (Amour, Brzezinska et al . 2010), APC induced BH4 increase is blocked by hyperglycemia producing loss of cardioprotection in vivo (Amour, Brzezin-ska et al . 2010) . In addition, overexpression of human GTPCH-1 gene profo-undly increased BH4 content in myocardium of transgenic mice and restored the protective effects of ischemic preconditioning during hyperglycemia (Ge, Ionova et al . 2011) . Moreover, in preliminary experiments we found BH4 con-centrations in reperfused rat myocardium to be below the limits of detection, a finding consistent with previous reports that BH4 levels were depleted in ischemic hearts (Dumitrescu, Biondi et al . 2007) . Although it was possible to detect BH4 in transgenic mouse hearts (Ge, Ionova et al . 2011) and endothe-lial cells (Amour, Brzezinska et al . 2009), these measurements were made in the absence of prolonged coronary artery occlusion and reperfusion as per-formed in the current investigation . Together, these studies strongly support that modulation of GTPCH-1 may limit myocardial injury both in the normal conditions and in the setting of diabetes/hyperglycemia .
Endocardial EC and capillary EC share an active blood-heart barrier and may influence neighboring CM through juxtacrine and paracrine signaling (Brutsaert 2003; Leucker and Jones 2014) . In chapter 3
“Endothelial–car-diomyocyte crosstalk enhances pharmacological cardioprotection” (Leuc-ker, Bienengraeber et al . 2011), we used an EC-CM co-culture model and isoflurane as a pharmacological stimulus to enhance EC protection of CM against hypoxia and reoxygenation (H/R) injury . Triggering of intracellular si-gnal transduction pathways culminating in the enhanced production of NO appears to be a central component of pharmacologically induced cardio-protection of CM . Although the endothelium is well recognized as a regula-tor of vascular tone by producing NO, little attention has been given to its potential importance in mediating cardioprotection (Leucker, Bienengraeber et al . 2011) . Studies investigating the protection against myocardial I/R injury have focused primarily on NO derived from a NOS isoform in CM (Jung, Pal-mer et al . 2000; Schulz, Kelm et al . 2004; Bloch and Janssens 2005), but our data indicate that EC are an important and underappreciated paracrine source of NO during APC induced cardioprotection . In our model, isoflura-ne enhanced the release of NO in EC and EC–CM co-culture resulting in a HIF1α dependent, sustained NO release during reoxygenation (Leucker, Bienengraeber et al . 2011) . It has been long known that hypoxia induces HIF1 activity through changes in HIF1α mRNA and protein levels in the he-art (Lee, Wolf et al . 2000; Jurgensen, Rosenberger et al . 2004), and that ge-netic variation at the human HIF1A locus influences the protective role of HIF1-dependent homeostatic mechanisms in the pathophysiology of ische-mic heart disease (Hlatky, Quertermous et al. 2007). Furthermore, an addi-tional mechanism of HIF1α dysregulation may include disrupted adenosine signaling provoked by (acute) cardiac adaptation to limited oxygen availa-bility . Previous research has identified the circadian rhythm protein Period2 (Per2) as a downstream factor in adenosine receptor A2b (Adora2b)-elicited cardioadaptive responses . In turn, Adora2b mediated stabilization of Per2 attenuated myocardial infarction by adapting metabolism toward more oxy-gen efficient utilization of carbohydrates (Eckle, Hartmann et al . 2012), de-pending on direct protein-protein interactions of Per2 with HIF1α, as found in Per2−/−mice with and without expression of a HIF1α reporter (Eltzschig,
Bonney et al. 2013). These results suggest a direct link between HIF1α and adenosine-mediated Per2 stabilization and cardiac metabolism during is-chemia (Eckle, Hartmann et al . 2012) . Moreover, this study may implicate circadian rhythm, i .e . time of the day, as a modulator of sensitivity to cardiac ischemia and APC .
Diabetes and hyperglycemia are known to cause EC dysfunction that mi-ght also affect CM-EC interactions and ultimately lead to ischemic cardiac injury (Nystrom, Nygren et al . 2005) . Therefore, our group used the co-cul-ture model to further explore EC-CM interactions during H/R injury under conditions of normoglycemia as compared with hyperglycemia (Leucker, Ge et al . 2013), since loss of NO bioactivity secondary to endothelial dys-function is probably one of the most important events contributing to the pathophysiology of type 2 DM (Brownlee, 2001; Du et al ., 2001)(Leucker and Jones 2014) . In addition to its potent vasodilatory effect, eNOS-derived NO has been demonstrated to be cardioprotective (Amour, Brzezinska et al. 2009; Frantz, Adamek et al. 2009; Szelid, Pokreisz et al. 2010). However, HG leads to a significant increase in oxidative stress, in turn oxidizing BH4 to enzymatically incompetent dihydrobiopterin, which competes with BH4 for eNOS binding (Schulz, Jansen et al . 2008; Amour, Brzezinska et al . 2010; Vladic, Ge et al . 2011) . When BH4 levels are inadequate, oxygen reduction by eNOS is uncoupled to L-arginine oxidation, resulting in the generation of the cardiotoxic mediator superoxide rather than NO (Vasquez-Vivar, Kalya-naraman et al . 1998; Vasquez-Vivar, Martasek et al . 2002; Vladic, Ge et al . 2011; Leucker, Ge et al . 2013) . In accord, in the EC-CM co-culture model, increased BH4 content in coronary vascular ECs exerted protective acti-ons on CMs during hypoxia and reoxygenation (H/R) via eNOS-derived NO . However, HG abolished the protective effects of ECs on CMs, which was overcome by increasing BH4 content in ECs by pharmacological and ge-netic approaches (Leucker, Ge et al . 2013) . Thus, BH4 is likely to play a key role in maintaining the physiological function of eNOS and eNOS derived NO production during H/R (Leucker, Ge et al . 2013) . Indeed, such notion is in accord with previous data from our group, showing that HG adverse-ly modulates NO signaling during pharmacological protection with volatile anesthetics by attenuating heat shock HSP 90 interactions with eNOS and by decreasing BH4 concentrations (Amour, Brzezinska et al . 2010) .
In chapter 4 “Cardioprotection during Diabetes, The Role of Mitochondrial
DNA” (Muravyeva, Baotic et al . 2014), we present a novel model of type 2 diabetes in rats with mtDNA variations, T2DNmtWistar, and T2DNmtFHH .
Myo-cardial infarct size was measured in Wistar, T2DNmtWistar, and T2DNmtFHH rats
with or without APC and in the presence or absence of the ROS scavenger, N-acetylcysteine (NAC) to determine if the mitochondrial genome is modu-lating susceptibility to I/R injury during diabetes and if modulation is ROS dependent (Muravyeva, Baotic et al . 2014) . Our data demonstrate the mitoc-hondrial genome to determine myocardial susceptibility to I/R injury as APC
reduced infarct size in T2DNmtWistar but not in T2DNmtFHH. Further, low-dose
NAC conferred cardiopotection during APC in T2DNmtFHH rats . In addition,
we investigated the influence of mitochondrial genome on mitochondrial re-dox state, ROS production and mPTP opening in isolated cardiomyocytes . Interestingly, APC elicited the largest ROS production in T2DNmtFHH . It has
been previously shown that the isoflurane/APC-induced generation of ROS is mediated by actions on complex I (Hirata, Shim et al . 2011) . Therefore, we speculate that the amino acid alterations in this ETC protein (modifications in ND2 and ND4, subunits of complex I in T2DNmtFHH (Sethumadhavan,
Va-squez-Vivar et al . 2012)) are responsible for the more pronounced effect of isoflurane on ROS generation during APC in T2DNmtFHH versus T2DNmtWistar
cardiomyocytes . In parallel with higher ROS generation, the redox state of flavoproteins was increased in T2DNmtFHH cardiomyocytes . As
isoflurane-in-duced flavoprotein oxidation was similar in Wistar and T2DNmtWistar
cardio-myocytes, the differences in isoflurane-induced changes in flavoprotein re-dox state between T2DNmtFHH versus T2DNmtWistar seem primarily related to
differences in mtDNA-coded ETC proteins . Consequently, these findings are in line with previous data showing that isoflurane exerts a direct action on ETC complexes (Sedlic, Pravdic et al. 2010). Furthermore, APC delayed mPTP opening in T2DNmtWistar and Wistar, but had no effect in T2DNmtFHH
car-diomyocytes, which is in line with previous in vivo evidence suggesting that prevention of mPTP opening represents an end-effector of volatile anesthe-tic-induced cardioprotection (Piriou, Chiari et al . 2004) .
The loss of the APC effect in T2DN rats upon the switch of mtDNA from Wistar to FHH is primarily related to enhanced isoflurane-induced ROS generation in T2DNmtFHH compared to T2DNmtWistar . Due to the excess ROS
production, APC is unable to mount cytoprotective pathways as high le-vels of ROS directly damages the cell and blocks preconditioning in
T2D-NmtFHH . A slight reduction of the high ROS levels by addition of NAC, still
allowing for a moderate increase in ROS, thus restores APC . Together with mitochondrial Ca2+ overload, ROS is also the main inducer of mPTP
ope-ning (Sedlic, Sepac et al . 2010) . Several studies have shown that mPTP opening may lead to additional ROS production, a phenomenon called ROS-induced ROS release, closing a vicious circle of self-sustained ROS production and mPTP opening (Zorov, Filburn et al. 2000; Zorov, Juhas-zova et al . 2006) . Moreover, irrespective of being caused by excess ROS or otherwise, mPTP opening is by itself an important initiator of cell de-ath (Baines 2009) . In accord, the interplay between ROS formation, mPTP opening and cell death in I/R cardiomyocytes is corroborated by
attenua-tion of cell death and excessive ROS producattenua-tion by partial mitochondrial depolarization (Sedlic, Sepac et al . 2010) . In hyperglycemia the opposite happens: excessive ROS production accelerates mPTP opening resulting in hyperpolarization and cell death, which may be attenuated by reducing ROS production by DNP (Sedlic, Muravyeva et al . 2017) . Likewise, hyper-glycemia-induced mitochondrial hyperpolarization attenuated APC-indu-ced mitochondrial depolarization, the delay in mPTP opening and cyto-protection (Sedlic, Muravyeva et al . 2017) . In line with these findings, a previous study showed APC also to be attenuated by the induction of mi-tochondrial hyperpolarization by pyruvate (Sedlic, Sepac et al . 2010) . Taken together, in diabetic T2DNmtFHH the excess ROS production, which
results in direct cellular damage and opening of mPTP, subsequently acti-vates cellular death pathways and off-sets APC effects .
In chapter 5 “Mitochondrial Bioenergetics in Diabetic Myocardium –
Im-plications for Protective Conditioning Strategies” we provide a review on mitochondrial bioenergetics in diabetic myocardium and implications for protective conditioning strategies . In the initial part we present normal mi-tochondrial function and how it depends on a highly complex interplay between protein content, substrate supply and its network morphology . Moreover, we briefly discuss actions of NO in mitochondria, whether from internal or external sources, NO has profound effects on mitochondrial fun-ction under physiological and pathological conditions affecting both OXP-HOS complexes and mitochondrial ion channels (Lacza, Pankotai et al . 2009) . While diabetes and acute hyperglycemia substantially alter mitoc-hondrial substrate use and bioenergetics and affects their morphology and nitroso-redox balance, those alterations collectively increase cardiomyocyte susceptibility to I/R injury. Further, by affecting mitochondria, diabetes and hyperglycemia confer a loss of cardioprotective conditioning signaling in heart . Based on our current understanding of the mechanisms underlying such mitochondrial changes, different strategies are proposed to restore mi-tochondrial bioenergetics and function and thus to restore the cardioprote-ctive effect of preconditioning strategies in diabetic myocardium .
While previous chapters focused on mechanisms by which AHG attenuates APC, chapter 6 “Apolipoprotein A-1 mimetic D-4F enhances
isoflurane-in-duced eNOS signaling and cardioprotection during acute hyperglycemia” (Baotic, Ge et al . 2013) investigates the potential of the apolipoprotein A-1 mimetic, D-4F, to reverse the effects of hyperglycemia on NO signaling (Bao-tic, Ge et al . 2013) . Our findings demonstrate that AHG impairs
isoflurane-sti-mulated eNOS activity and subsequent cardioprotection by interfering with lipid rafts . A subclass of membrane lipid rafts, caveolae, are distinguished by the presence of the scaffolding proteins Cav-1, Cav-2 and Cav-3 (Cohen, Combs et al . 2003) . Caveolin isoforms are differentially expressed in various cell types . Since Cav-1 is predominantly expressed in EC, Cav-1 was the focus of our investigation . In previous studies, Cav-1-/- mice have been
de-monstrated to be resistant to the cardioprotective effects of isoflurane (Patel, Tsutsumi et al. 2007). Furthermore, Cav-1 has been shown to regulate eNOS activity through protein-protein interactions localized in specific membrane domains (Feron and Kelly 2001). Our results show that during normoglyce-mic conditions, Cav-1 resided in heavy/noncaveolar fractions, while eNOS was equally distributed between caveolar and noncaveolar compartments with its Ser1177 phosphorylated form (p-eNOS) predominantly present in
ca-veolar fractions . Isoflurane significantly promoted the redistribution of eNOS and Cav-1 from noncaveolar to caveolar fractions and increased Ser1177
phosphorylation of eNOS (p-eNOS) exclusively in caveolar fractions . In con-trast, AHG increased the amount of eNOS, p-eNOS, and Cav-1 in noncave-olar fractions and abrogated isoflurane protein redistribution toward caveo-lae. Importantly, treatment with D-4F in AHG restored both basline caveolar compartmentalization of proteins and isoflurane-induced Cav-1, eNOS, and p-eNOS caveolar compartmentalization. Furthermore, D-4F enhanced flurane-stimulated eNOS homodimerization during AHG and enhanced iso-flurane mediated increase in eNOS protein dimer-to-monomer ratio . Thus, these results extend previous findings that lipid rafts are critical components during pharmacological cardioprotection (Patel, Tsutsumi et al . 2007; Tsut-sumi, Kawaraguchi et al. 2010), and identify D-4F as a pharmacological stra-tegy to attenuate negative effects of AHG (Baotic, Ge et al . 2013) .
Although the mechanism of action of D-4F also involves modulation of bioactive oxidized lipids at the intestine, vascular and cellular level, resul-ting in anti-at herogenic and anti-inflammatory properties in line with ot-her 4F compounds (Getz and Reardon 2014), its effectiveness in restoring APC in AH is likely conveyed by its antioxidant effects and a decrease in O2· formation . Our study confirms AHG to increase the formation of O
2 .
in endothelial cells, and although the intracellular source of ROS was not identified, previous data show that multiple enzymatic sources are involved, including mitochondria (Peterson, Husney et al . 2007) . Moreover, under conditions of oxidative stress and tetrahydrobiopterin deficiency, eNOS fails to produce NO and can itself generate O2 ., a condition referred to as eNOS
monomerization of the protein, a process exacerbated by high glucose and diabetes (Cai, Khoo et al . 2005; Baotic, Ge et al . 2013) . Our data show that D-4F ameliorated O2· formation during AHG irrespective of APC . Moreover,
during AHG, D-4F enhanced eNOS dimerization and NO production in the presence of isoflurane APC, consistent with an action of D-4F to promote coupled eNOS activity. D-4F induced preservation of eNOS coupling is con-sistent with previous evidence showing that the ApoA-1 mimetic L-4F pre-vents LDL induced uncoupling of eNOS, thus enhancing NO and decrea-sing O2·generation in endothelial cells (Ou, Ou et al . 2003) .
Future Perspectives
Harnessing Clinical Evidence for (in)effectiveness of APC
While APC has its cardioprotective effects in I/R injury in experimental mo-dels, clinical evidence on beneficial effects of volatile anesthetics applied prior to cardiac surgery is still elusive . This requires further research in evalu-ating predictors of poor outcome or variables interfering with the translation of APC-induced cardioprotection from the experimental setting to patients with cardiovascular risk factors undergoing cardiac surgery . One of the in-terfering variables is certainly AHG, as we clearly demonstrated the deva-stating effects of AHG on APC in vivo and in vitro . In addition, according to our clinical experience, patients show a high incidence of AHG during cardiac surgery, both in the diabetic and nondiabetic population . Indeed, a retrospective study by Doenst et al . observed that 26% of non-diabeti-cs and 47% of diabetinon-diabeti-cs had perioperative blood glucose values over 20 mmol/L, which was associated with a tripling of in-hospital mortality (Do-enst, Wijeysundera et al. 2005). Furthermore, AHG conferred a similar in-crease in mortality risk both in patients with and without diabetes . These results led the authors to conclude that hyperglycemia during CPB reflects a state of insulin resistance, which precipitates an adverse postoperative out-come, rather than hyperglycemia per se (Doenst, Wijeysundera et al . 2005) . Further, a prospective cohort study demonstrated severe intraoperative AHG present in about 50% of non-diabetic cardiosurgery patients and to be slightly higher in on-pump compared to off-pump operations (Azarfarin and Alizadeh Asl 2008). Factors contributing to AHG during cardiac ope-rations may constitute hypothermia and particular drugs, such as heparin (drastically increasing circulating FFA), which by acutely impairing the insulin signaling network may contribute to extreme insulin resistance
necessita-ting large doses of insulin to manage blood glucose levels . Consequently, it needs to be investigated whether outcome may be improved by taking insulin sensitivity into account (the rate of change in blood glucose), rather than sticking to the current protocols for insulin administration based solely on blood glucose levels . Alternatively, short-term metformin pretreatment ahead of APC could be beneficial, although metformin monotherapy does not reduce perioperative myocardial injury in nondiabetic patients undergoi-ng CABG surgery (El Messaoudi, Nederlof et al . 2015), nor preserves LV fun-ction in PCI-treated nondiabetic patients with STEMI (Lexis, van der Horst et al . 2014) . But most importantly from the perspective of this thesis, blood glucose levels should be reported and controlled in clinical APC studies . Importantly, the few studies on the clinical use of APC, which reported ab-sence of effect of volatile anesthetics vs intravenous anesthesia in cardiac surgery, did not report and evaluate the influence of AHG (Flier, Post et al. 2010; Landoni, Guarracino et al . 2014) . Possibly, a large variability in blo-od glucose/insulin resistance status in these studies in conjunction with a relative low number of patients has precluded identification of a beneficial APC effect (i .e . type II statistical error) . Thus, evaluating APC effects in the clinical setting still calls for studies with a higher number of patients, strin-gent management of blood glucose levels and more coherence in the type of cardiac surgery .
In addition, it would be of interest to evaluate the contribution of diabetes mellitus or AHG in APC to changes in cardiomyocyte bioenergetics and mi-tochondrial metabolic function in I/R injury . To this end, mimi-tochondrial oxy-gen consumption (rate of oxyoxy-gen consumption per milligram of left ventri-cular tissue, i .e . mitochondrial respiration in nmol O2/minute/mg of tissue) should be measured as an indicator of mitochondrial ATP production capa-city (preferably using different metabolic substrates) in patients undergoing CABG with and without diabetes and with and without APC . Such seems feasible, as during CPB-assisted cardiac operations a small piece of cardiac tissue can be removed when the right atrium is cannulated to connect the patient to the heart-lung machine as we have demonstrated in earlier stu-dies (Gelissen, Epema et al. 1996; Ruifrok, Westenbrink et al. 2010). From this tissue sample RNA should be isolated to assess levels of mitochondrial RNA (mt-RNA), particularly those of mt-RNA19 which has previously been identified to be affected by high glucose (Sanchez, Mercer et al . 2011) . In addition, such sample may serve to measure levels of the pentatricopeptide repeat (PPR) proteins domain 1 (PTCD1) . PTCD-1, which are mitochondrial RNA-binding proteins that are nuclearly encoded and critical for the
regu-lation of mt-RNA (expression of mtDNA) (Perks, Ferreira et al. 2017). Also, transmission electron microscopy (TEM) may be used to examine mitoc-hondrial morphology and cristae structure in heart tissue, further exploring the differences between normo- and hyperglycemic patients and their sus-ceptibility to I/R injury .
Potential strategies counteracting loss of APC efficacy in
diabetes/hyperglycemia
There is a pile of evidence from in vivo, ex vivo and in vitro studies indicating that changes in NO and mitochondrial bioenergetics underlie the suscepti-bility to myocardial injury during diabetes and AHG . Therefore, dysfunction in NO biology and mitochondrial bioenergetics are likely important contribu-tors to the loss of APC induced cardioprotection during hyperglycemia . In depth exploration of the mechanisms producing this dysfunction is impe-rative to identify effective and safe pharmacological agents for the reversal/ restoration of APC cardioprotective effect . The apolipoprotein A-1 mimetic, D-4F, seems highly promising, as it restored APC effectivity in AHG by incre-asing NO and limiting ROS production in cultured HCAEC, and conferred cardioprotection in an in vivo mouse model . However, there is need for ran-domized clinical trials to confirm this effect in patients with hyperglycemia/ diabetes .
The Adora2b-elicited cardioadaptive response constitutes an additional potential pathway of APC related cardioprotection that merits further inve-stigations with respect to hyperglycemia . As a first step, the influence of receptor stimulation and blockade (BAY 60-658310) should be studied in human coronary artery endothelial cells (HCAEC) by evaluating the binding of cAMP response element binding protein (CREB) to the protein Period 2 (PER2) promoter in normo- and hyperglycemic conditions with and without isoflurane exposure. Furthermore, how adenosine signaling and circadian rhythm protein PER2 play a role for the mechanism of APC mediated cardi-oprotection could be addressed by investigating infarct size in Per2-/- mice
in normo and AHG conditions . GTPCH-1 represents a third potential pat-hway that need further exploration . Given the beneficial effects of increased GTPCH-1 expression on I/R injury during diabetes or AHG, small molecules like oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA) that improved high glucose-impaired endothelial function via a PPARδ-mediated mecha-nism and through eNOS/Akt/NO pathway (Zhang, Jiang et al . 2017) should
be further explored in animal models of I/R injury as potential GTPCH-1 en-hancer .
A fourth route to explore to improve APC in the clinics during AHG or T2DM, although not investigated in this thesis, might be the administration of SGLT-2 inhibitors . This class of compounds has recently been shown to reduce risk on cardiovascular mortality in T2DM patients by 40% . This effect is lin-ked to an upregulation of ketone metabolism (3-hydroxybutirate, 3-OHB) – serving as a cardioprotective fuel substrate (Ferrannini, Mark et al. 2016; Taegtmeyer 2016) . So far, ketone body formation has not been taken into account in animal studies nor in clinical trials evaluating APC in normogly-cemia/AHG. Furthermore, the presence of heart failure has never been re-ported in APC clinical trials, despite its potentially major impact on ketone bodies levels (Gormsen, Svart et al . 2017) . In conjunction, it is of note that in our experimental studies animals were never food restricted prior to surgery (to mimic the usual clinical settings) . Potentially, this may be of additional influence on ketone body formation .
This thesis substantiates that effects on mitochondria and NO are corner-stones of the cardioprotective effect elicited by APC during I/R injury . More-over, modulation hereof by a range of cellular pathways involves the influen-cing of bioavailability of NO and function of mitochondria, as occurs during hyperglycemia and diabetes. Finally, it is demonstrated that negative effects of hyperglycemia on APC may be precluded efficiently by maintaining eNOS coupling, offering a therapeutic perspective . Although the beneficial effects of APC in animal models is undisputed, controversy still exists on the use of APC as cardioprotective strategy during cardiac surgeries because of nega-tive clinical studies . One explanation for the discrepancy may be the lack of documentation and control of the vast number of factors related to glucose metabolism . Such are not confined to overt diabetes, but also relate to effe-cts of fasting, co-medication, underlying disease and AHG during clinical trials . The efficacy of APC may thus heavily depend on correct alignment of factors which still largely needs to be disclosed. Future research evaluating metabolic factors influencing APC likely provide critical information for the successful translation of cardioprotection in clinical practice .
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