REVIEW published: 12 November 2020 doi: 10.3389/fphys.2020.602497 PCSK9 in Myocardial Infarction and Cardioprotection: Importance of Lipid Metabolism and Inflammation Ioanna Andreadou 1* , Maria Tsoumani 1 , Gemma Vilahur 2,3 , Ignatios Ikonomidis 4 , Lina Badimon 2,3,5 , Zoltán V. Varga 6,7 , Péter Ferdinandy 6,8 and Rainer Schulz 9* 1 Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece, 2 Cardiovascular Program-ICCC, Research Institute-Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain, 3 CIBERCV, Instituto Salud Carlos III, Madrid, Spain, 4 Second Cardiology Department, Attikon Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece, 5 Cardiovascular Research Chair, Autonomous University of Barcelona (UAB), Barcelona Spain, 6 Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary, 7 HCEMM-SU Cardiometabolic Immunology Research Group, Budapest, Hungary, 8 Pharmahungary Group, Szeged, Hungary, 9 Institute for Physiology, Justus-Liebig University Giessen, Giessen, Germany Extensive evidence from epidemiologic, genetic, and clinical intervention studies Edited by: has indisputably shown that elevated low-density lipoprotein cholesterol (LDL-C) Vincenza Cifarelli, Washington University in St. Louis, concentrations play a central role in the pathophysiology of atherosclerotic United States cardiovascular disease. Apart from LDL-C, also triglycerides independently modulate Reviewed by: cardiovascular risk. Reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9) Massimiliano Ruscica, has emerged as a therapeutic target for reducing plasma LDL-C, but it is also associated University of Milan, Italy Andrew Carley, with a reduction in triglyceride levels potentially through modulation of the expression The Ohio State University, of free fatty acid transporters. Preclinical data indicate that PCSK9 is up-regulated United States in the ischaemic heart and decreasing PCSK9 expression impacts on infarct size, *Correspondence: Ioanna Andreadou post infarct inflammation and remodeling as well as cardiac dysfunction following jandread@pharm.uoa.gr ischaemia/reperfusion. Clinical data support that notion in that PCSK9 inhibition is Rainer Schulz associated with reductions in the incidence of myocardial infarction, stroke, and Rainer.Schulz@physiologie.med.uni- giessen.de coronary revascularization and an improvement of endothelial function in subjects with increased cardiovascular risk. The aim of the current review is to summarize the Specialty section: This article was submitted to current knowledge on the importance of free fatty acid metabolism on myocardial Lipid and Fatty Acid Research, ischaemia/reperfusion injury and to provide an update on recent evidence on the role of a section of the journal hyperlipidemia and PCSK9 in myocardial infarction and cardioprotection. Frontiers in Physiology Received: 03 September 2020 Keywords: dyslipidemia, heart failure, ischaemia, LDL cholesterol, myocardial infarction, PCSK9, reperfusion Accepted: 26 October 2020 Published: 12 November 2020 Citation: INTRODUCTION Andreadou I, Tsoumani M, Vilahur G, Ikonomidis I, Badimon L, Triglycerides (TG) and Free Fatty Acids (FA) Metabolism During Varga ZV, Ferdinandy P and Schulz R Ischaemia/Reperfusion Injury (IRI) (2020) PCSK9 in Myocardial Infarction and Cardioprotection: Importance The heart possesses a remarkably high metabolic flexibility when it comes to substrate use for of Lipid Metabolism and Inflammation. adenosine triphosphate (ATP) production. At rest and during aerobic conditions, the oxidation of Front. Physiol. 11:602497. fatty acids (FA) highly contributes to total cardiac ATP production under physiological conditions, doi: 10.3389/fphys.2020.602497 followed by the oxidation of carbohydrates and ketone bodies (Pascual and Coleman, 2016). Frontiers in Physiology | www.frontiersin.org 1 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction In contrast, upon pathophysiological conditions with low myocardial infarction and ensuing development of heart failure oxygen availability, a metabolic shift toward higher oxidation (Schulze, 2009). In this regard, reperfusion after a prolonged and of carbohydrates occurs given that glucose is the most energy- severe ischaemic period triggers cardiac lipotoxicity which, in efficient myocardial substrate (Rosano et al., 2008). turn, impairs the tissue growth factor (TGF)β/TβRII/Smad2/3 Yet, if oxygen deprivation persists oxidative metabolism signaling pathway hindering the fibrotic reparative process of the is repressed and anaerobic glycolysis is activated in order evolving scar, leading to large infarcts and cardiac dysfunction to spare the use of limited oxygen. The mismatch between (Vilahur et al., 2012). glycolysis and glucose oxidation may result in the production Multiple experimental studies in rodents and in perfusion of lactate and protons and the consequent fall in the pH systems have demonstrated that cardiac metabolic perturbations which may impair cardiac contractility (Steenbergen et al., 1977). occur during both ischaemia and at reperfusion, and are Besides, proton removal and sodium and calcium homeostasis determined by energy substrate availability as well as the lead to ATP consumption which lowers cardiac efficiency regulation of substrate metabolism (Zuurbier et al., 2020). and may further deteriorate the cardiac function (Lopaschuk During ischaemia, the drop in oxygen supply dramatically et al., 2010). Recently, a study in rats have also revealed reduces mitochondrial oxidative processes, and anaerobic that the uncoupling between glycolysis and oxidation may glycolysis becomes an important source of ATP production contribute to the development of heart failure with preserved in order to maintain intracellular integrity and crucial cellular ejection fraction (Fillmore et al., 2018). Of note, however, functions. However, oxidation of the glycolysis end-product in the presence of free FA, lactate may improve cardiac pyruvate is impaired during ischaemia resulting in the efficiency by anaplerotic mechanisms including an enhanced accumulation of lactic acid and the subsequent decline in tricarboxylic acid cycle combustion and reduced glycolysis rate pH and ionic disturbances (H+ , Na+ , and Ca2+ ) (Dennis (Lopaschuk et al., 1993). et al., 1991). In this latter regard, the re-establishment of ion The major sources of FA are albumin-bound free FAs or homeostasis during ischaemia entails the use of a fraction FAs released from vascular lipoprotein lipase (LPL)-hydrolysis of of ATP, further aggravating cardiac dysfunction. Despite the circulating Triglycerides (TG)-rich lipoproteins (Figure 1). FAs metabolic switch during ischaemia from lactate uptake to may cross the sarcolemma by passive diffusion or by membrane- lactate production, FAs continue to be the predominant fuel associated proteins including fatty acid transport protein (FATP), for the heart. Catecholamine discharge secondary to ischaemia- fatty acid binding protein (FABP), and fatty acid translocase related peripheral sympathetic nervous system activation (FAT/CD36), the latter being regulated in part by proprotein promotes adipose tissue lipolysis with the ensuing increase in convertase subtilisin/kexin type 9 (PCSK9) (Van Der Vusse et al., plasma FA concentrations and myocardial delivery (Kantor 2000; Coort et al., 2004). Once translocated into cardiomyocytes, et al., 1999). In line with this phenomenon, ischaemia has FAs are converted into long-chain fatty acyl-CoA which may been shown to induce the transcription of genes encoding enter the mitochondria for β-oxidation or may be stored as for FA uptake, transport into mitochondria, and β-oxidation TG within lipid droplets which are adjacent to mitochondria (Djouadi et al., 1999; Rosano et al., 2008). Moreover, during (Figure 1). The heart contains significant amounts of TG within ischaemia, there is also an increase of CPT-1 activity through the lipid droplets in order to endogenously provide FA for AMP-dependent protein kinase (AMPK)–phosphorylation oxidative metabolism for cardiac function in time of energetic (Figure 1). CPT-1 activation, besides favoring FA uptake into needs. Lipid droplets can be catabolized by intracellular lipases the mitochondria, inhibits acetyl coenzyme A carboxylase (ACC) [adipose triglyceride lipase (ATGL) and hormone-sensitive lipase with the consequent reduction in the conversion of acetyl-CoA (HSL)] and acyltransferases and, in counterpart, preserved by to malonyl-CoA, a potent CPT-1 inhibitor. Hence, a decrease the action of cardiac perilipins (PLIN) 2 to 5, mainly PLIN5 in malonyl-CoA intracellular levels allows the entry of FA (Figure 1; Kimmel and Sztalryd, 2014; Barbosa et al., 2015). into the mitochondria for oxidation, allowing ATP generation Besides energy storage, TGs also serve as a transient buffer during myocardial ischaemia. A persistent reduction in oxygen to prevent aberrant metabolism of increased FA (Evans and supply may eventually overcome AMPK activity. As such, we Hauton, 2016). Yet, when TG buffer is exceeded because of have observed in a pig model of cardiac ischaemia (without excessive FA uptake or limited FA oxidation as occurs in several reperfusion) a reduction in AMPK activation in the jeopardized pathological conditions, FA overload leads to the formation of myocardium after severe and complete coronary occlusion toxic FA metabolites, such as ceramides, diacylglycerols, long- which was accompanied by enhanced detection of several chain acyl-CoAs, and acylcarnitines, which can interfere with markers of acute cardiac damage (Mendieta et al., 2020a,b). The cellular signaling pathways leading to apoptotic cell death. Taken temporal dynamics of AMPK activation during ischaemia in as a whole, alterations due to excess FA and TG accumulation preclinical animal models deserve to be further investigated. are thought to contribute to cardiac dysfunction, a term referred In summary, during ischaemia, despite an increase in the rate to as cardiac lipotoxicity (D’Souza et al., 2016). Myocardial of glycolysis, high circulating levels of FA in concurrence with lipotoxicity has been observed in metabolic disturbances such higher CPT1 activity and lower malonyl-CoA content renders as obesity and diabetes and its occurrence seems to precede FAs the main source for energy requirements. Yet, while this the development of cardiac dysfunction and adverse remodeling is true for mild ischaemia, severe ischaemia has also shown (McGavock et al., 2007; Ernande et al., 2010). Moreover, to inhibit FA β-oxidation secondary to the accumulation of cardiolipotoxicity has also been reported to occur in the setting of reducing equivalents NADH and FADH2 resulting in the Frontiers in Physiology | www.frontiersin.org 2 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction FIGURE 1 | Summary of the mechanisms of myocardial FA metabolism in the healthy heart covered in this review that includes mitochondrial FA oxidation or FA storage as TG within the lipid droplets. In red are highlighted the metabolic changes that occur during ischaemia that may lead to FA overloading and consequent lipotoxicity. LPL, lipoprotein lipase; FA, fatty acid; FATP1, fatty acid transport protein 1; FABP, fatty acid binding protein; ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase; HGL, human gastric lipase; PLIN, perilipin; CPT-1, Carnitine palmitoyltransferase-1; AMPK, AMP-dependent protein kinase; ACC, acetyl coenzyme A carboxylase; TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; ER, endoplasmic reticulum. accumulation of the FA metabolites stated above favoring CD36 is almost entirely responsible for the AMPK-mediated cardiolipotoxicity (Figure 1). stimulation of long chain FA uptake into cardiomyocytes (Habets During aerobic reperfusion of ischaemic myocardium, AMPK et al., 2007) and in turn, the interaction of FAs with CD36 activation markedly reduces malonyl-CoA levels allowing a regulates AMPK activity (Samovski et al., 2015). In humans, rapid recovery of FA β-oxidation to near pre-ischaemic values, CD36 is needed for normal cardiac FA uptake over a range yet, at expenses of pyruvate oxidation (Lopaschuk et al., of extracellular FA concentrations from low to slightly elevated 2010). This in concurrence with the further aggravation of (Hames et al., 2014) and mutations of the FAT/CD36 gene ionic disturbances (mainly Na+ and Ca2+ overload) due to with lack of CD36 protein expression reduces accumulation the rapid pH normalization upon reperfusion which primes of long chain FAs in the human heart (Tanaka et al., 2001). the myocardium for further damage culminating in the Even cardiomyocyte FA uptake–delivered through exosomes– impaired recovery of both cardiac function and efficiency depends on CD36 (Garcia et al., 2019). Thus, there is clear (Jaswal et al., 2011). evidence that CD36 is of utmost importance for free FA transport into cardiomyocytes. In rats, sarcolemmal FAT/CD36 expression is decreased Fatty Acid Translocase CD36 and PCSK9 during ischaemia which is accompanied by an almost complete in IRI loss of FA oxidation, with no change in intramyocardial lipid There is substantial molecular, biochemical and physiologic content. Following reperfusion, the decrease in the sarcolemmal evidence that long-chain FA transport involves a protein- FAT/CD36 persists, but the FA oxidation rate returns to mediated process. A number of FATP have been identified and pre-ischaemic levels resulting in a decrease in myocardial some of them are co-expressed in the same tissues. Among the triglyceride content (Heather et al., 2013). Genetic knockout proteins involved in FA uptake, FAT/CD36 and FABP appear of the FAT/CD36 protein reduces FFA oxidation in isolated to be key transporters. Besides its sarcolemmal localization, mice hearts. On aerobic reperfusion after ischaemia, cardiac CD36 is also stored in intracellular compartments that reversibly function of FAT/CD36 knockout hearts recover to the same translocate to the plasma membrane of the cardiomyocyte in extent as wild-type hearts (Kuang et al., 2004) or even show response to an increase in energy demand (Kim and Dyck, a reduced functional recovery (Irie et al., 2003) which can be 2016). In the heart, studies in FAT/CD36 null mice have rescued by adding medium-chain FAs which do not require revealed that this protein is key to regulate the increase in CD36 for myocardial uptake. 4–6 weeks following cardiomyocyte the rate of FA metabolism (Chabowski et al., 2007) and this specific ablation of CD36, hearts again have a decreased FA might be due to FAT/CD36 being a transporter itself or an uptake compared to controls and a significant reduction in the indirect regulator through interaction with other free FAT intramyocardial triacylglycerol content. In contrast to the global in the cell membrane (Carley and Kleinfeld, 2011). In mice, CD36 knockout, however, hearts with a cardiomyocyte specific Frontiers in Physiology | www.frontiersin.org 3 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction ablation of CD36 exhibit significantly improved functional a post-hoc analysis of three Phase II clinical trials revealed recovery following ischaemia and reperfusion. This improved equally modest reductions in plasma TG levels following functional recovery is associated with lower calculated proton alirocumab treatment in patients receiving background statin production prior to and following ischaemia compared to control therapy (Toth et al., 2016). The effect of PCSK9 inhibitors on hearts (Nagendran et al., 2013). A recently published review TG levels is contributed to the enhanced LDL-R-mediated article summarizes the effect of small molecular inhibitors catabolism of intermediate-density lipoproteins (IDLs) of enzymes in FA transport and metabolism pathways; here (Dijk et al., 2018). inhibition of FA oxidation in general reduces damage induced by ischaemia and reperfusion. The authors, however, point out that effects of FAs on cardiac ischaemia and reperfusion EFFECTS OF PCSK9 BEYOND LDL injury are critically dependent on the FA concentration, with REDUCTION: IMPACT ON IRI detrimental effects commonly only observed at rather high levels of FAs which are usually not observed in humans The majority of experimental studies have shown that during ischaemia and reperfusion episodes (for details, see hypercholesterolemia increases infarct size in animal Zuurbier et al., 2020). models. Decreased cardiac nitric oxide content, increased FAs oxidation is mainly driven by the lipolysis of TG stored in oxidative/nitrosative stress, enhanced apoptotic cell death the lipid droplets rather than through CD36 internalization (Kim and dramatic changes in the cardiac gene expression profile, and Dyck, 2016). Furthermore, specific cardiomyocyte deletion in presence of hypercholesterolemia, leads to myocardial of CD36 has shown to improve cardiac recovery post-myocardial dysfunction (Andreadou et al., 2017). infarction (MI) by redirecting cardiac metabolism toward glucose PCSK9 may not only play a crucial role in binding to the LDL- utilization (Nagendran et al., 2013). Yet, whereas CD36 decline R and increasing its endosomal and lysosomal degradation thus might be beneficial in the short-term post-ischaemia because of inhibiting its recycling to the cell surface but may also exhibit improved myocardial efficiency rates, levels of CD36 need to several, LDL-R-independent activities on a plethora of cell types, be restored upon glucose and intracellular TG storage depletion including endothelial cells, vascular smooth cells, immune cells in order to meet cardiac energy demands (Kim and Dyck, and cardiomyocytes (Figure 2). 2016). In this regard, taking into consideration that PCSK9, The role of PCSK9 regarding the function and adaptation which is found to be enhanced by hypoxia/reoxygenation (Yang of cardiomyocytes is under investigation. PCSK9 indirectly et al., 2020), has also shown to degrade CD36 beyond the affects cardiomyocytes by modulating the plasma concentrations LDL-R (Demers et al., 2015), PCSK9 inhibition might hold of low-density lipoprotein cholesterol (LDL-C) and oxidized promise as a therapeutic strategy to modulate myocardial CD36 LDL. Cardiomyocytes express LDL-R but it seems that low content and expression and consequently FA metabolism levels density lipoprotein receptor-related protein 1 (LRP1) and very in the setting of MI. low-density lipoprotein (VLDL) receptors are responsible for In this context, PCSK9 monoclonal antibodies modestly cholesterol accumulation in these cells (Cal et al., 2012). reduce plasma TG levels (Stoekenbroek et al., 2018b). When adult rabbit ventricular cardiomyocytes are exposed to Evolocumab treatment of 27,564 patients at high CVD risk LDL-C, calcium transients through transsarcolemmal calcium modestly reduced fasting plasma TG levels by 16.2% (Sabatine transport pathways are slightly increased suggesting that et al., 2017). Same reduction levels were observed for patients LDL-C may improve cardiac function. On the other hand, with mixed hyperlipidaemia (Rosenson et al., 2016). Similarly, in atrial-like cardiomyocytes (HL-1 cells), LDL-C decreases FIGURE 2 | Pleiotropic effects of increased PCSK9 expression during acute MI. SREBP-2, sterol response element binding protein 2; HNF1α, hepatocyte nuclear factor 1 α; LRP5, Low-density lipoprotein receptor-related protein 5; NF-κB, Nuclear Factor kappa-light-chain-enhancer of activated B cells; TLR4, Toll-like receptor 4; PCSK9, Proprotein convertase subtilisin/kexin type 9. Frontiers in Physiology | www.frontiersin.org 4 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction the expression of sarcoplasmatic reticulum (SR) calcium function and amplifying certain inflammatory reactions that ATPase (SERCA) 2a, ryanodine receptor 2 (RyR2), and may lead to progression of coronary atherosclerosis (Paciullo connexin-40 within 24 h causing a reduction in calcium et al., 2017; Ágg et al., 2018; Ricci et al., 2018; Kim et al., 2019; transients indicating a deterioration of cardiac function (Glerup Momtazi-Borojeni et al., 2019). et al., 2017). The participation of PCSK9 in these effects of The investigation on PCSK9 regulatory function in myocardial LDL-C was investigated in ventricular cardiomyocytes without ischaemia revealed that PCSK9 activated NF-κB signaling defining the intracellular targets of PCSK9. It was reported resulting in a more pronounced secretion of pro-inflammatory that adult terminally differentiated ventricular cardiomyocytes cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 constitutively express PCSK9 on the protein and mRNA (IL-6) and IL-1β by macrophages worsening the hypoxia- level and that oxLDL-dependent effects on cell shortening reoxygenation-induced injury in cardiomyocytes (Yang et al., can be antagonized by neutralizing PCSK9 in cardiomyocytes 2020). PCSK9 has been shown to promote inflammation in (Schlüter et al., 2017). atherosclerosis through TLR4/NF-κB pathway and increase PCSK9 exerts important cardiac effects. In 2014, it was firstly macrophage release of pro-inflammatory cytokines, TNF-α, IL-6, reported that along with hepatic PCSK9 mRNA expression, IL-1β, interferon-γ (IFN-γ), C-X-C motif ligand 2, and Monocyte the plasma PCSK9 concentration is drastically increased in Chemoattractant Protein-1 (Tang et al., 2017; Ricci et al., 2018; acute MI in rats. The up-regulation of PCSK9 is driven by Macchi et al., 2019). Specifically, IFN-γ promotes and exacerbates sterol response element binding protein 2 (SREBP-2) and atherosclerosis by affecting the lipid accumulation and foam hepatocyte nuclear factor 1 α (HNF1α), both of which are cell formation in the vascular wall and altering the cellular the predominate transcription factors for PCSK9 (Zhang et al., structure in the plaque (Luo et al., 2017). Other cytokines 2014). PCSK9 is also expressed in the myocardium and is such as IL-13 contribute to hypercholesterolemia associated with significantly up-regulated in hypoxia/reoxygenation-stimulated increased PCSK9 hepatic synthesis and enhanced circulating primary murine cardiomyocytes compared with cells grown PCSK9 levels (Low et al., 2020). The induction of PCSK9 in in normoxic environments (Ding et al., 2018; Yang et al., response to TNF-α is dependent in the suppressor of cytokine 2020). Again, in MI the maximum expression of PCSK9 was signaling 3 (SOCS3) factor. This factor has been implicated found in the border zone 1 week after left coronary artery in chronic-inflammation associated with ischaemia/reperfusion (LCA) ligation (Ding et al., 2018). The fate of cardiomyocytes injury (IRI). Transfection with small interfering RNA (siRNA) in the border zone of an infarction is crucial since although anti-signal transducer and activator of transcript 3 (STAT3), they experience hypoxic phases, they do not undergo cell inhibited the induction of PCSK9 by TNF-α suggesting the death immediately. Thus, the enlargement of the infarct or the involvement of the JAK/STAT pathway (Ruscica et al., 2016). limitation of its extent is defined in part by the border zone, The involvement of the inflammatory pathway of STAT3 therefore the border zone cardiomyocytes appear to be promising was confirmed in a recent publication showing that upon targets for therapeutic strategies during MI (Kuhn et al., 2020). STAT3 silencing, leptin and resistin (proinflammatory adipokines The expression of PCSK9 in cardiomyocytes was associated that mediate transcriptional induction of PCSK9), lose their with the development of autophagy in the ischaemic heart ability to activate PCSK9 (Macchi et al., 2020). Furthermore, (Ding et al., 2018). Autophagy is activated in cardiomyocytes PCSK9 has also shown to cooperate with LRP5 in TLR4/NFkB early in response to hypoxia, pro-inflammatory cytokines, and signaling; a decreased TLR4 protein expression levels and angiotensin II and its attenuation can limit ischaemic injury a decreased nuclear translocation of NFκB was detected in in experimental models of myocardial ischaemia highlighting PCSK9 silenced cells after lipid loading, indicating a down- a therapeutic target of PCSK9 inhibitors. Interestingly, hearts regulation of the TLR4/NFκB pathway, showing that LRP5 of patients who had died of acute MI (1–7 days post-MI) also facilitates macrophage lipid uptake and forms a complex with showed expression of PCSK9 and autophagy (LC3 expression) PCSK9 that up-regulates TLR4/NFκB favoring inflammation in the border zone-similar to the findings in the infarcted mouse (Badimon et al., 2020). Conversely, PCSK9 siRNA lead to a heart (Ding et al., 2018). reduction in inflammation by inhibiting NF-kB activation (Tang Besides expression of PCSK9 in the cardiomyocytes, et al., 2012). Recently, it was found that PCSK9 is expressed PCSK9 is secreted from the myocardium and is correlated and secreted from epicardial adipose tissue (EAT) affecting to infarct size and cardiac function (Ding et al., 2018). the coronary vessels and the myocardium. EAT inflammation Specifically, the sustained PCSK9 release during cardiac is associated with local PCSK9 expression, regardless of ischaemia and reperfusion induced serious cardiac deleterious circulating PCSK9 levels, suggesting that PCSK9 derived from effects including cell death and dysfunction (Ding et al., EAT could play an additional and independent risk to CVD 2018). Inflammatory stimuli such as NLRP3 inflammasome- (Dozio et al., 2020). dependent activation of interleukin 1 beta (IL-1β), are However, it has to be noted that in a recent secretome analysis, considered to be powerful inducers for PCSK9 secretion PCSK6 and not PCSK9 was secreted from and expressed in in both macrophages and tissues such as heart and aorta primary cardiomyocytes under hypoxic conditions as well as (Ding et al., 2020). Interestingly, PCSK9 itself might be an after MI in mice and humans (Kuhn et al., 2020). PCSK6 could important inflammatory mediator since PCSK9 might induce present a novel biomarker for cardiac fibrosis and might be a intracellular cholesterol accumulation in macrophages and promising future therapeutic target to reduce adverse remodeling several other immune cell types, increasing toll-like receptor after myocardial injury. Frontiers in Physiology | www.frontiersin.org 5 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction Taken together, these data suggest that the inhibition of PCSK9 whereas PCSK9 inhibition reduces ROS formation (Ding expression may suppress the inflammatory process and thereby et al., 2018). Likewise, in vitro studies have reported that ROS contribute to the treatment of myocardial ischaemia. stimulators enhanced PCSK9 expression whereas ROS inhibitors blocked PCSK9 expression. In summary, although very few studies have investigated the ROLE OF PCSK9 INHIBITION IN role of PCSK9 on myocardial infarct size in experimental animal MYOCARDIAL INFARCTION models, it seems that the pharmacological inhibition of PCSK9 results in cardioprotective effects. Experimental Studies on the Role of PCSK9 Inhibition in Myocardial Infarction Clinical Studies on the Role of PCSK9 In animal experiments, an increased concentration of LDL-C Inhibition in Myocardial Infarction due to dietary interventions is either associated with a Patients suffering from a MI are considered at very high risk reduced, unchanged or increased MI size (Andreadou et al., for subsequent cardiovascular (CV) events since the reported 2019). Explanations for a reduction in MI size by high-fat 5-year risk of recurrent MI or fatal coronary heart disease diet-induced metabolic alterations included activation of (CHD) for those aged >65 years is 22% (Go et al., 2014). Due salvage kinases (Poncelas et al., 2015) or maintained low to the high burden of recurrent CV events, clinicians have to pH during initial reperfusion following ischaemia (Inserte investigate new strategies for risk reduction in these patients. et al., 2019), while increased MI size in hypercholesterolemia A further, rapid LDL-C reduction within 2–4 weeks following is associated with increased formation of reactive oxygen an acute MI beyond current standard care, including high-dose species (ROS) during reperfusion (Andreadou et al., 2017, statins, seems an appealing option in order to reduce CV risk. 2019). Interestingly, even with reduced MI size functional Although statins decrease LDL-C levels by 1 mmol/L which outcome following ischaemia/reperfusion is worse in mice with is translated to an approximate 20% reduction in the rate of hypercholesterolemia (Pluijmert et al., 2019). Additionally, MI, the most potent hypolipidemic agents, PCSK9 inhibitors hypercholesterolemia has a profound negative effect on evolocumab and alirocumab that reduce LDL-C by >1 mmol/L cardioprotection of the ischaemic heart induced either by below statin-treated levels, could further reduce the risk of MI ischaemic conditioning or cardioprotective compounds (Trankle and Abbate, 2016). Low LDL-C levels are directly (Ferdinandy et al., 2014), that might relate to the complex correlated with reduced risk of atherosclerotic cardiovascular effects of cholesterol on myocardial redox homeostasis (Varga disease. The pharmacological inhibition of PCSK9 has led to et al., 2013), intercellular communication (Görbe et al., 2011), indisputable benefits in terms of LDL-C and CV-risk lowering. autophagy (Giricz et al., 2017), and transcriptome regulation Interestingly, measuring PCSK9 circulating levels has become (Ágg et al., 2018). important. Many studies support that pharmacological therapy, The role of PCSK9-inhibition or -absence in MI has been i.e., statins modulate PCSK9 gene expression and protein levels studied in in vivo acute MI models. In an experimental study providing a pointer to estimate possible alterations in the overall evaluating potential adverse effects of PCSK9 absence in acute response to lipid-lowering medications in patients at high CV risk MI, no significant differences in cardiac function [cardiac (Macchi et al., 2019). Thus, in August 2019, a joint consensus output, left ventricular end systolic or diastolic volume (LVESV, statement from the European Society of Cardiology (ESC) and LVEDV), stroke volume or ejection fraction] between wild type European Atherosclerosis Society (EAS) recommended a stricter and PCSK9 knockout (KO) mice were observed. In addition, LDL-C level target for patients with recent MI [below the the left ventricular wall in the infarct area was thicker in PCSK9 1.4 mmol/L (<55 mg/dL)]. In a recent study with 25,466 patients knockout mice, a finding that could point to a lower rate of who had attended a follow-up visit 6–10 weeks after an MI ventricular rupture and improved outcome in patients with big event, it was shown that half of this cohort were eligible to infarct sizes when treated with PCSK9 inhibitors (Winter et al., 2018; Table 1). Another study showed that PCSK9 inhibition PCSK9 inhibitors according to updated ESC/EAS guidelines resulted in an improved cardiac function and reduced infarct (Allahyari et al., 2020). With regard to safety, very low LDL- size. Specifically, the PCSK9 inhibitor, Pep2-8 trifluoroacetate C concentrations do not significantly increase risk of adverse at a dose of 10 µg/kg was administered at different time-points events (e.g., new-onset diabetes mellitus, elevations in hepatic in an acute MI model: before ischaemia, during ischaemia and transaminases, muscle symptoms and increase in neurocognitive at the onset of reperfusion. Pep2-8 trifluoroacetate exerted events) (Giugliano et al., 2017). Whether PCSK9 inhibition cardioprotective effects demonstrated by a significant reduction has beneficial effect specifically on patients with MI has to be in infarct size and improved LV function only when given 15 min established in clinical trials. Currently, we have limited data in prior the onset of ischaemia. The underlying cardioprotective this regard. Results from clinical studies have already shown mechanism was attributed to the ability of the PCSK9 inhibitor that PCSK9 inhibitors have different effects on type and size to attenuate cardiac mitochondrial dysfunction and fission and of myocardial infarcts. For example, evolocumab reduced the decrease the apoptotic process in the ischaemic myocardium risk of spontaneous and procedural MI, but had no effect (Palee et al., 2019; Table 1). The attenuation of mitochondrial on type 2 (myocardial oxygen supply demand mismatch) MI damage has also been attributed to a decreased ROS levels caused events. The benefit for evolocumab was consistent for myocardial by PCSK9 inhibition. As such, recombinant mouse PCSK9 infarcts generally considered more severe, including ST-elevation protein (mPCSK9) has shown to enhance ROS generation, myocardial infarction (STEMI) and those with larger elevations Frontiers in Physiology | www.frontiersin.org 6 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction TABLE 1 | Experimental and clinical studies regarding the role of PCSK9 in myocardial infarction. Study Model/Patients Protocol/Intervention Effect Winter et al., 2018 MI mice model: Permanent ligation PCSK9 deficiency No significant differences in cardiac function (cardiac of the LAD coronary artery output, left ventricular end systolic or diastolic volume (LVESV, LVEDV), stroke volume or ejection fraction), Thicker left ventricular wall in the infarct area Palee et al., 2019 IRI rat model Pep2−8 trifluoroacetate 10 µg/kg Reduction in infarct size (a) Before ischaemia Improvement in LV function only when it was given (b) During ischaemia before ischaemia (c) At the onset of reperfusion Trankle et al., 2019 Type 1 NSTEMI patients Randomization 1:1, double-blinded Significant reduction of LDL-C levels study with 2 groups: Neutral effects on inflammatory biomarkers (follow up (a) Alirocumab 150 mg subcutaneously at 14 days) within 24 h after NSTEMI (b) Placebo Gencer et al., 2020 Patients with prior MI Secondary analysis of FOURIER trial Significant reduction of the risk of the composite (a) Evolocumab, 140 mg every 2 weeks outcome of cardiovascular death, MI, stroke, unstable or 420 mg monthly angina, or coronary revascularization by 19% (b) Placebo Schwartz et al., ACS patients treated with statin Randomization, double-blinded study Significant reduction of the risk of a composite of 2018 (atorvastatin or rosuvastatin) at a with 2 groups: death from coronary heart disease, non-fatal MI, fatal high-intensity dose or at the (a) Alirocumab 75 mg subcutaneously or non-fatal ischemic stroke, or unstable angina maximum tolerated dose (b) Placebo requiring hospitalization Wiviott et al., 2020 Patients with stable atherosclerotic Pre-specified analysis of the FOURIER Reduction of Type 1 and Type 4 MIs disease receiving statin therapy trial (a) Evolocumab, 140 mg every 2 weeks or 420 mg monthly (b) Placebo White et al., 2019 Patients with recent ACS and Pre-specified analysis of the ODYSSEY Reduction of Type 1 and Type 2 MIs elevated LDL-C OUTCOMES trial (a) Alirocumab 75 mg every 2 weeks (b) Placebo ACS, Acute Coronary Syndrome; FOURIER, Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk, IRI, Ischaemia/Reperfusion Injury (IRI); LAD, Left Anterior Descending; LDL-C, Low-density lipoprotein cholesterol; MI, Myocardial Infarction; NSTEMI, non-ST elevation MI. of cardiac biomarkers (Wiviott et al., 2020). On the other be more impressive if evolocumab would be found effective hand, alirocumab added to intensive statin therapy was found in reducing the area and severity of MIs. It is known that to attenuate the risk of type 2 myocardial infarct events larger MIs are associated with increased risk, suggesting that (White et al., 2019). a robust reduction of larger MIs would lead over time to a Alirocumab may benefit patients who have had an MI and mortality reduction (Wiviott et al., 2020). Such an effect in are at very high risk of recurrent CV events since LDL-C target mortality has been confirmed over a longer duration of follow-up levels can be achieved, regardless of MI or ischemic stroke status (2.8 years) in a study that involved 18,924 ACS patients treated (Bruckert et al., 2019). Also, in these high-risk patients that with alirocumab subcutaneously at a dose of 75 mg (Schwartz were included in the study, alirocumab was well tolerated with et al., 2018; Table 1). a favorable safety profile [similar treatment-emergent adverse In a recent analysis of FOURIER trial, evolocumab reduced events (TEAEs) were observed among groups], regardless of the number of atherothrombotic (Type 1) MI and consistent previous history of MI/ischaemic stroke (Bruckert et al., 2019). reductions were seen for type 1 and PCI-related (Type 4) In a pilot study, it was shown that alirocumab administration MIs, STEMI, and NSTEMI, and those with higher troponin at the time of non-ST elevation MI (NSTEMI) significantly levels (Wiviott et al., 2020; Table 1). In a pre-specified analysis reduced LDL-C levels at 14 days but had neutral effects on from the ODYSSEY OUTCOMES trial, alirocumab added to inflammatory biomarkers such as high-sensitivity C-reactive intensive statin therapy during 2.8 years of follow-up reduced protein (hsCRP), IL-6, and TNF-a (Trankle et al., 2019; Table 1). the occurrence of both Type 1 and due to myocardial supply An important question that should be addressed is whether the and/or demand imbalance (Type 2) MIs in patients with recent PCSK9 inhibition has direct effect on CV outcomes in patients (1–12 months) ACS and elevated LDL-C. However, there was with MI. In a secondary analysis of the FOURIER trial including no apparent effect of alirocumab on reducing Type 4 MIs 5,711 patients with a recent MI, evolocumab significantly reduced which is mainly attributed to differences in patient populations’ the risk of the composite outcome of cardiovascular death, MI, enrollment (high-risk patients versus recent ACS patients) and stroke, unstable angina, or coronary revascularization by 19% duration of follow-up (2.8 versus 2.2 years in the FOURIER trial) (Gencer et al., 2020; Table 1). The results for MI patients would (White et al., 2019; Table 1). Frontiers in Physiology | www.frontiersin.org 7 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction In a recent meta-analysis of 67 randomized controlled trials increase in the risk of death, a detailed description of the effect (RCTs) including 259,429 participants PCSK9 inhibitors plus of PCSK9 in infarct size and cardiac function is very important. statin significantly reduced the risk of non-fatal MI (RR 0.82, 95% The results of clinical trials indicate that PCSK9 inhibitors are CI 0.72–0.93, p = 0.003) or stroke (RR 0.74, 95% CI 0.65–0.85, beneficial across multiple MI subtypes. p < 0.001) (Chaiyasothi et al., 2019). However, the effect of PCSK9 inhibitors on cardioprotective Inclisiran, a long acting synthetic siRNA that targets hepatic cellular mechanisms needs to be tested to reveal if these group PCSK9 synthesis, produces similar reductions in LDL-C as of drugs may promote cardioprotection (Ferdinandy et al., 2014) PCSK9 monoclonal antibodies (Board et al., 2020; Landmesser or possess some hidden cardiotoxic effect (Ferdinandy et al., et al., 2020). In a recent meta-analysis of three RCTs comparing 2019). Experimental studies would help to shed light on these cardiovascular outcomes in patients with hypercholesterolemia open questions. Future studies should be conducted in order treated with maximally tolerated dose statins with or without to examine different agents, doses, time of administration for additional lipid-lowering therapy (n = 3,783 patients), it was more conclusive results regarding the role on PCSK9 inhibitors found that there was no statistically significant difference in the in cardiac IRI and cardioprotection from it. risk of MI in patients randomized to inclisiran compared with placebo (Asbeutah et al., 2020). However, ORION-4 trial has been designed as a “cardiovascular outcomes trial” and will assess AUTHOR CONTRIBUTIONS the effects of inclisiran on clinical outcomes in approximately 15,000 subjects with atherosclerotic cardiovascular disease. The All authors contributed to the content of the manuscript, have results of this study will shed light to the effect of inclisiran read and approved the final version of the manuscript. IA and RS on the MI (Stoekenbroek et al., 2018a). Increased Lipoprotein finally revised the manuscript. (Lp) (a) levels are prothrombotic and associated with MI. PCSK9 inhibitors are the only drugs reducing increased Lp(a) levels by 25 to 30% (Shapiro et al., 2019). Kinetic studies ACKNOWLEDGMENTS have revealed the mechanism of action of evolocumab and alirocumab lower plasma Lp(a) concentration. Evolocumab PF and ZV was supported by the National Research, decreases the production (as monotherapy) and increases Development and Innovation Office of Hungary (National clearance (in combination with atorvastatin) of Lp(a) particles. Heart Program NVKP 16-1-2016-0017); by “Research Excellence Increased clearance of Lp(a) with PCSK9 inhibition may relate Program of the National Research, Development and Innovation to supraphysiological up-regulation of hepatic LDL-Rs and/or Office of the Ministry of Innovation and Technology in Hungary decreased competition of Lp(a) with LDL particles for LDL- (TKP/ITM/NKFIH),” by the Higher Education Institutional R uptake (Watts et al., 2018). Alirocumab efficiently reduces Excellence Program of the Ministry of Human Capacities in circulating apoB100 levels primarily by enhancing its catabolism Hungary, within the framework of the Therapeutic Development and apo(a) levels primarily by lowering its production (Croyal thematic program of the Semmelweis University and by the et al., 2018). Future studies should examine if these new therapies European Union’s Horizon 2020 research and innovation that lower Lp(a) could reduce the risk for future MI. The program under grant agreement No 739593. Further support was reduction of Lp(a) by PCSK9 inhibitors is not always in parallel given by Plan Nacional de Salud (PNS) [PGC2018-094025-B-I00 with the respective reduction of LDL-C, a finding suggesting to GV and SAF2016-76819-R to LB] from the Spanish Ministry of a direct effect of PCSK9 on reducing Lp(a) production and Science and Innovation and funds FEDER “Una Manera de Hacer not necessarily through an effective reduction of LDL-C levels Europa”; and CIBERCV (to LB). We also thank the support of the (Shapiro et al., 2019). Generalitat of Catalunya (Secretaria d’Universitats i Recerca del In conclusion, the provided data suggests that PCSK9 Departament d’Economia i Coneixement de la Generalitat, 2017 inhibition has a beneficial role in patients with MI. The SGR 1480) and the Fundación Investigación Cardiovascular- mechanism by which this occurs is under investigation. It Fundación Jesus Serra for their continuous support. IA was has been postulated that better outcomes may derive from a supported by the European Union (ERDF) and Greek national rapid LDL-C reduction (stabilizing the LDL-R), a diminishment funds through the Operational Program “Competitiveness, of the pro-inflammatory signaling by potentially reducing Entrepreneurship and Innovation,” under the call “Strengthening those LDL particles susceptible to oxidation or through Lp(a) Research and Innovation Infrastructures” (project code: decline, and/or enhanced removal of the pro-inflammatory 5002803). IA and MT were supported by Kleon Tsetis Foundation apoptotic debris. Scientific Research & Culture. This article is based upon work from COST Action EUCARDIOPROTECTION CA16225 supported by COST (European Cooperation in Science CONCLUSION and Technology). PF is the vice-chair, RS and IA are work package leaders and GV is Management Committee substitute Hypercholesterolemia has profound negative effect on of COST Action EUCARDIOPROTECTION CA16225. cardioprotection and PCSK9 has emerged as a therapeutic The authors wish to acknowledge SERVIER Medical Art target for reducing plasma LDL-C. Taking into consideration (https://smart.servier.com/http://www.servier.fr) for use of their that MIs of increasing size are associated with a significant medical art kits when making the illustrations in the article. Frontiers in Physiology | www.frontiersin.org 8 November 2020 | Volume 11 | Article 602497 Andreadou et al. Role of PCSK9 in Myocardial Infarction REFERENCES Dennis, S. C., Gevers, W., and Opie, L. H. (1991). Protons in ischemia: Where do they come from, Where do they go to? J. Mol. Cell. Cardiol. 23, 1077–1086. Ágg, B., Baranyai, T., Makkos, A., Veto, B., Faragó, N., Zvara, Á., et al. 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R., Bayer, and AstraZeneca, has a Research Grant of AstraZeneca, speaker fees of Lilly, et al. (2016). Effect of alirocumab on specific lipoprotein non-high-density MSD-Boerhinger and AstraZeneca and to have founded the Spin-off Glycardial lipoprotein cholesterol and subfractions as measured by the vertical auto profile Diagnostics SL. GV has a Research Grant of AstraZeneca and is founder of the method: analysis of 3 randomized trials versus placebo. Lipids Health Dis. 15, Spin-off Glycardial Diagnostics SL. IA, II, and MT declare that they have not a 28. potential conflict of interest. Trankle, C., and Abbate, A. (2016). PCSK9 inhibition in acute myocardial infarction: a novel opportunity. Transl. Med. 6:e135. doi: 10.4172/2161-1025. The remaining authors declare that the research was conducted in the absence of 1000e135 any commercial or financial relationships that could be construed as a potential Trankle, C. R., Wohlford, G., Buckley, L. F., Kadariya, D., Ravindra, K., Markley, conflict of interest. R., et al. (2019). Alirocumab in acute myocardial infarction: results from the virginia commonwealth university alirocumab response trial (VCU-AlirocRT). Copyright © 2020 Andreadou, Tsoumani, Vilahur, Ikonomidis, Badimon, Varga, J. Cardiovasc. Pharmacol. 74, 266–269. doi: 10.1097/FJC.0000000000000706 Ferdinandy and Schulz. This is an open-access article distributed under the terms Van Der Vusse, G. J., Van Bilsen, M., and Glatz, J. F. C. (2000). Cardiac fatty of the Creative Commons Attribution License (CC BY). The use, distribution or acid uptake and transport in health and disease. Cardiovasc. Res. 45, 279–293. reproduction in other forums is permitted, provided the original author(s) and the doi: 10.1016/S0008-6363(99)00263-1 copyright owner(s) are credited and that the original publication in this journal Varga, Z. V., Kupai, K., Szucs, G., Gáspár, R., Pálóczi, J., Faragó, N., is cited, in accordance with accepted academic practice. No use, distribution or et al. (2013). MicroRNA-25-dependent up-regulation of NADPH oxidase 4 reproduction is permitted which does not comply with these terms. Frontiers in Physiology | www.frontiersin.org 11 November 2020 | Volume 11 | Article 602497