Interaction of Cardiovascular Nonmodifiable Risk Factors, Comorbidities and Comedications With Ischemia/Reperfusion Injury and Cardioprotection by Pharmacological Treatments and Ischemic Conditioning

1. Ischemia-Related

The “conditioning” phenomena describe the IS reduction after sustained I/R by brief nonlethal periods of I/R, which are performed either before (pre), during (per), or after (post) the sustained ischemia followed by reperfusion. Whereas in ischemic preconditioning (PreC) or ischemic postconditioning (PostC) the heart itself is subjected to the conditioning I/R, in remote ischemic conditioning (RIC) organs or tissues other than the heart (e.g., skeletal muscle) undergo I/R, and this “conditioning at a distance” reduces myocardial IS (for review see Hausenloy et al., 2017; Heusch et al., 2015). As with ischemic PreC, the benefits of RIC appear to be biphasic with a short initial window of protection during the first 12 hours, followed by a period of loss of protection and finally a longer “delayed” or “second” window of protection lasting as long as 72 hours after RIC exposure (Madias, 2015). When using leg ischemia to induce RIC in mice, leg temperature is decisive for cardioprotection, and leg hypothermia abrogates protection (Penna et al., 2022). The importance of different conditioning strategies to improve patients’ outcome is highlighted in Section III.

Repeated administration of RIC over weeks or months (long-term RIC), has been investigated for improving several aspects of cardiovascular health (for review see Epps et al., 2016; Chong et al., 2019). A seminal experimental study demonstrated that RIC repeated daily for 28 days after MI protected against adverse LV remodeling and increased survival in a rat model even though IS was not reduced compared with the single RIC treatment (Wei et al., 2011). This was further supported by a rat study showing survival benefit even when RIC treatment was commenced 4 weeks after MI (Yamaguchi et al., 2015). In a lipopolysaccharide-induced mouse model mimicking bacterial sepsis, RIC reduced circulating and myocardial inflammatory mediators and led to improved LV function, cardiac output, and survival (Honda et al., 2019). Moreover, repeated RIC provided an additional 7 days survival benefit beyond a single-occasion treatment (Honda et al., 2019).

2. Drug-Related

Elucidation of some mechanisms involved in cardioprotection induced by ischemic conditioning strategies has identified a number of signaling pathways, many of which have been targeted by pharmacological agents applied either before the sustained ischemia (pharmacological PreC) or just at the onset of reperfusion (pharmacological PostC) to reduce myocardial IRI (for review see Heusch, 2015, 2020; Calabrese, 2016b; Torregroza et al., 2020). While results from experimental studies were encouraging, translation to the clinical setting again failed to meet the expectations (for review see Heusch, 2017; Roth et al., 2021 and Sections III and V for details).

Apart from ischemic and pharmacological PreC and PostC, another clinically applicable possibility to precondition the heart against IRI is exercise (for review see Wojcik et al., 2018). Recent clinical investigations confirm that exercise may precondition the human heart (for review see Quindry and Franklin, 2021). Low-load blood flow restricted resistance exercise (Bøtker, Lassen, et al., 2018) improved functional capacity, quality of life, and muscle mitochondrial function in patients (Groennebaek et al., 2019). The mechanisms responsible for exercise-induced cardioprotection include maintenance of endothelial nitric oxide synthase (NOS) coupling (Santana et al., 2018), increased release of circulating hormones (Lu and Pan, 2017; Otaka et al., 2018; Bo et al., 2021), as well as extracellular vesicles (Görgens et al., 2015; Bei et al., 2017), finally leading to preserved mitochondrial dynamics (Ghahremani et al., 2018; Yuan et al., 2018) in cardiomyocytes.

1. Cardiomyocyte Death

To develop strategies to protect the heart from IRI, it is important to define the precise mechanisms by which cardiomyocytes die. Necrosis is known to play a major role in myocardial IRI. Apoptosis is a form of programmed cell death mediated by caspases and characterized by cell shrinkage, chromatin condensation, and blebbing (budding) of the plasma membrane. Despite early studies showing evidence of apoptosis in the heart following I/R, its contribution remains controversial. The protein machinery required for apoptosis is expressed at very low levels in the healthy adult heart, which suggests that the signs of apoptosis that can be detected may be due to cardiac cells other than cardiomyocytes (e.g., fibroblasts, endothelium, leukocytes) (reviewed in Davidson et al., 2020).

Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism in the case of mitochondrial permeability transition pore (mPTP) opening leading to necrosis or as part of the signaling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy (for in-depth review see Gatica et al., 2022). More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described (for review see Davidson et al., 2020).

Necrosis was previously believed to always be an uncontrolled pathway of cell death. However, it is now evident that there are controlled forms of necrosis, of which necroptosis and pyroptosis are of particular relevance in the heart. Necroptosis involves the recruitment of cytosolic adaptor proteins to complex I, an increase in plasma membrane permeability, re-localization of phosphorylated mixed-lineage kinase domain-like pseudokinase to the plasma membrane, and receptor-interacting-protein 3 activation (Zhang et al., 2016). In the heart, receptor-interacting-protein 3 also causes activation of calmodulin-dependent protein kinase II (Zhang et al., 2016). Necroptotic proteins are also present in the human failing heart due to MI (Szobi et al., 2017). Necroptosis clearly contributes to IRI, because either pharmacological inhibition or deletion of key proteins is cardioprotective (Smith et al., 2007; Newton et al., 2016). Limited evidence suggests that conditioning strategies can reduce necroptosis. For example, ischemic PreC is associated with inhibition of translocation of mixed-lineage kinase domain-like pseudokinase within the plasma membrane, and ischemic PreC is ineffective in hearts where necroptosis is already inhibited (Szobi et al., 2018).

Pyroptosis is a type of programmed necrosis that can be activated in the heart in response to injury. One major difference between pyroptosis and other forms of necrosis is that the proteins involved in pyroptosis are expressed at low levels in the healthy heart, and, as such, in healthy hearts, the contribution of pyroptosis to infarction occurring in the first few hours of reperfusion following ischemia may be limited. However, damage-associated molecular patterns such as interleukin (IL)-1β increase the expression of proteins of the inflammatory and innate immune systems including nucleotide-binding and oligomerization domain-like receptor domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing caspase recruitment domains, and caspase-1, which make up a complex called the NLRP3 inflammasome (Kawaguchi et al., 2011). Following this “priming” stimulus, a stress such as I/R causes rapid assembly and activation of the NLRP3 inflammasome, leading to cleavage and activation of IL-1β and IL-18. Activated caspase-1 also cleaves the protein gasdermin D, which forms cytosolic membrane pores, the lethal and defining feature of pyroptotic cell death (Shi et al., 2015).

Most acute I/R experiments use IS (relative to ischemic area at risk) as a hard endpoint, measured using either tetrazolium staining (postmortem) or late gadolinium cardiac magnetic resonance imaging (MRI) (Bøtker, Hausenloy, et al., 2018). The levels of cardiac proteins such as troponin released by necrotic cells into the blood or perfusate may be used as a supporting measurement. Cardiac contractile function is sometimes used as endpoint but is less robust and less meaningful in the acute period since cardioprotective strategies such as ischemic PreC may have inconsistent effects on ventricular function (Kloner and Jennings, 2001; Gelpi et al., 2002). Since ventricular fibrillation contributes to deaths following MI, another relevant endpoint measurement, which is a target for ischemic PreC, is cardiac arrhythmia (Hagar et al., 1991).

2. Coronary Microvascular Obstruction

Another target for cardioprotection that has been somewhat overlooked in acute myocardial IRI is the coronary circulation (Heusch, 2016, 2019a,; Hausenloy, Chilian, et al., 2019). The function in health and disease of the coronary circulation including the microvasculature is pivotal to our understanding of the complex processes and interactions among myocardial ischemic injury, reperfusion injury, and cardioprotection (Kaski et al., 2018). The putative initiating mechanism in approximately 97% of patients who experience an acute coronary syndrome is plaque erosion or rupture (type I MI), and there is abundant evidence that the response of the microvasculature plays a crucial role in determining the clinical course and final outcome. Moreover, type II MI, which results from changes in systemic hemodynamics and their impact on the matching of coronary blood flow and myocardial metabolism, occurs usually in the presence of significant epicardial coronary atherosclerosis (Ibanez et al., 2018; Thygesen et al., 2018). Finally, MI in the absence of obstructive (<50% diameter reduction on angiography) coronary artery disease still involves in most cases structural or functional alterations of the epicardial or more distal coronary microcirculation (Ibanez et al., 2018; Thygesen et al., 2018). Whereas obstruction of the coronary circulation and reduction of coronary blood flow causes myocardial ischemia (Heusch, 2019b), it is the reopening of the coronary circulation and restoration of coronary blood flow that induces reperfusion and salvages the dependent myocardium from infarction, but this comes at the price of reperfusion injury (Heusch and Gersh, 2017). Type IV MI is defined as any infarction related to interventional reperfusion (Thygesen et al., 2018) including notably microembolization (Heusch, 2016; Kleinbongard and Heusch, 2022). Thus, the coronary circulation in one form or the other is an integral component of the process of myocardial ischemia, microvascular dysfunction, reperfusion, reperfusion injury, and healing.

The coronary microcirculation is as much a victim of myocardial IRI as the cardiomyocytes. The most extreme form of coronary vascular injury following myocardial I/R is no-reflow as recognized in dogs more than 5 decades ago (Krug et al., 1966; Kloner et al., 1974). Such no-reflow, more specifically microvascular obstruction (MVO) (de Waha et al., 2017) and intramyocardial hemorrhage (Reinstadler et al., 2019), determines the prognosis of patients with reperfused AMI independently of IS (Stone et al., 2016). Because morphologic alterations of the coronary microvasculature are difficult to assess in the absence of reperfusion, it is not clear to what extent MVO is a direct consequence of ischemic injury or is caused by the process of reperfusion. Furthermore, since regions of coronary vascular injury typically occur within the infarcted myocardium, the causal relationship and relative contribution of cardiomyocyte and coronary microvascular injury from I/R is not clear. However, multiple mechanisms contribute to coronary microvascular injury from myocardial I/R: endothelial and interstitial edema (Garcia-Dorado et al., 2012; Fernández-Jiménez et al., 2015; Zhou et al., 2017), impaired vasomotion (Ehring et al., 1995; Kleinbongard et al., 2011), leukocyte adherence (Kupatt et al., 2002), erythrocyte stasis (Driesen et al., 2012), platelet aggregation (Pearson et al., 1990; Folts, 1999), extravascular compression by the interstitial edema (Manciet et al., 1994), and ultimately capillary destruction with consequent intramyocardial hemorrhage (Kloner et al., 1974; Higginson et al., 1982; Bulluck et al., 2016). Clinically, coronary microvascular injury is assessed using various imaging modalities, notably in angiography by decreased thrombolysis in MI (TIMI) flow grade and myocardial blush grade, and in cardiac MRI as edema by T2 weighted mapping and lack of contrast medium in gadolinium-hypercontrasted infarcted myocardium or intramyocardial hemorrhage (Heusch, 2016, 2019a; Hausenloy, Chilian, et al., 2019).

What is fortunate is that coronary microvascular injury is not an all-or-none phenomenon but a process subject to modification and damage limitation (Hausenloy, Chilian, et al., 2019; Heusch, 2019a). In the experimental model, mechanical interventions of ischemic conditioning reduced not only IS but also the area of no-reflow (Skyschally et al., 2017). Ischemic PreC reduced endothelial dysfunction (DeFily and Chilian, 1993; Kaeffer et al., 1996), leukocyte adherence (Kurzelewski et al., 1999), edema formation (Zhao et al., 2003), and MVO (Posa et al., 2010) and improved coronary vasodilator responses to adenosine, nitric oxide (NO), and reactive hyperemia (Gattullo et al., 1999), although not all studies have been consistent in demonstrating such benefit (Bauer et al., 1993). Moreover, delayed ischemic PreC provided endothelial protection and improved coronary vasodilation 24 hours later (Kaeffer et al., 1997; Kim et al., 2007). Ischemic PostC improved endothelial function and vasodilator response to acetylcholine and reduced leukocyte adherence, edema, and no-reflow in dogs and pigs (Zhao et al., 2003; Zhao et al., 2007), but again not all studies were positive (Bodi et al., 2014). RIC reduced IS and area of no-reflow in pigs (Skyschally et al., 2017), again with some studies lacking such effects (Baranyai et al., 2017). Hypothermia in rabbits reduced IS and area of no-reflow, and no-reflow was even reduced when hypothermia was delayed later into reperfusion (Hale et al., 2013). Some drugs, when given at reperfusion, reduced IS and no-reflow [e.g., cyclosporine A (CsA)] (Zalewski et al., 2015) and angiopoietin-like peptide 4 (Galaup et al., 2012). Nitroglycerine can protect both the myocardium and the coronary vasculature but also interferes with RIC (Heusch, 2001; Hauerslev et al., 2018).

The experimental agenda surrounding the reduction of reperfusion injury in the microvasculature has been extensive, but for reasons not clearly understood much has been “lost in clinical translation,” and definitive clinical trials demonstrating benefit are conspicuous by their absence (Heusch, 2017; Heusch and Gersh, 2020). More specifically, there is still debate on the role of coronary microvascular dysfunction as cause or consequence of MI and reperfusion injury (Lerman et al., 2007; Heusch, 2019a). Clinical studies evaluating ischemic PreC’s effect on coronary MVO do not exist. However, several clinical studies notably using MRI looked not only at IS but also at coronary microvascular injury, including edema, MVO, and intramyocardial hemorrhage, in patients with acute STEMI undergoing either ischemic PostC (Thuny et al., 2012; Dwyer et al., 2013; Mewton et al., 2013; Bodi et al., 2014; Eitel et al., 2015; Kim et al., 2015; Traverse et al., 2019), RIC (Crimi et al., 2013; White et al., 2015; Liu et al., 2016), or both in combination (Eitel et al., 2015). The effects on IS and MVO differed and were partly concordantbut partly not, leaving the issue of causality between the 2 manifestations of myocardial IRI open and a challenge for further studies (Heusch, 2019a). The effects of hypothermia on IS and MVO in patients demonstrated modest benefits at best, and the most recent trial actually pointed in the wrong direction in terms of safety signals (Noc et al., 2021). Metoprolol is 1 exception and demonstrated a modest benefit on the reduction of both IS and MVO in patients with AMI (García-Prieto et al., 2017).

In conclusion, the coronary circulation is both a culprit and a victim of myocardial IRI. Cardioprotection notably reduces cardiomyocyte injury, as reflected by IS, but also coronary microvascular injury, as reflected by the area of no-reflow. The targets are clear, but identifying clinical approaches that favorably influence coronary microvascular obstruction and thereby induce cardioprotection have been difficult and remain the “last frontier” of reperfusion therapy (Heusch, 2019a).

1. Classic Pathways

Timewise, there are 3 key steps in the mechanism of cardioprotection: the trigger step, the mediator step, and the end-effector step, which may or may not involve different signaling pathways. These steps can be clearly distinguished only in ischemic PreC but are more difficult to discern in ischemic PostC and RIC (Heusch, 2015; 2020) (Fig. 2). These have been extensively characterized over the past 35 years and are well established, although some controversies still remain (reviewed in Heusch, 2015; Hausenloy et al., 2016). Moreover, given the fact that none of these pathways have resulted in clinically validated cardioprotective drug targets in the last 30 years suggests the possibility that more systematic research approaches will uncover novel, more druggable targets (Varga et al., 2015; Hausenloy et al., 2017; Perrino et al., 2017). Ischemic PreC causes a localized increase in the extracellular concentration of the autacoid mediators adenosine (Liu et al., 1991), bradykinin (Schulz et al., 1998), opioids (Schulz et al., 2001), and sphingosine (Keul et al., 2016), which bind to receptors on the cardiomyocyte plasma membrane and act additively to initiate the trigger pathway. Ligand-receptor signaling leads to the activation of a sequence of cytosolic kinase pathways that ultimately cause opening of mitochondrial ATP-sensitive potassium channels (K_ATP) and allow potassium entry into mitochondria (Liu et al., 1996). Connexin 43 located at the inner mitochondrial membrane forms hemichannels that are also believed to be involved in the passage of potassium into mitochondria and required for cardioprotection by ischemic PreC (Boengler et al., 2005; Heinzel et al., 2005; Miro-Casas et al., 2009; Boengler, Ungefug, Heusch, Leybaert, et al., 2013; Gadicherla et al., 2017; Hirschhäuser et al., 2021). The reperfusion phase of ischemic PreC is crucial for the trigger pathway of cardioprotection, because reoxygenation allows mitochondrial respiration to recommence, which is associated with a small burst of reactive oxygen species (ROS) that activates protein kinase C (PKC) (Liu et al., 1994); the role of PKC in ischemic PreC of larger mammals such as the pig is still contentious (Vahlhaus et al., 1996).

The reperfusion injury salvage kinase (RISK) pathway describes a group of prosurvival kinases that must be activated at the time of reperfusion for ischemic PreC to protect against IRI (Schulman et al., 2002). The relative importance of different signaling pathways appears to vary between species. In rodents, the ischemic PreC signaling pathway is dependent on both the phosphoinositide-3-kinase (PI3K)α/protein kinase B (Akt) 1 and mitogen-activated protein kinase/extracellular signal related kinase (ERK)1/2 signaling pathways (Hausenloy and Yellon, 2004; Kunuthur et al., 2012; Rossello et al., 2017). In pigs and humans, the salvage activating factor enhancement (SAFE) pathway, consisting of gp 130/tumor necrosis factor (TNF) receptor-mediated activation of janus kinase/signal transducers and activators of transcription (STAT) factors, appears to play a more important role (Lecour, 2009; Heusch et al., 2012; Kleinbongard, Skyschally, Heusch., 2017; Hadebe et al., 2018). Evidence suggests that the RISK and/or SAFE pathways are also involved in ischemic PostC and RIC, although the RISK pathway might not be essential to achieve cardioprotection (Skyschally et al., 2009; Inserte et al., 2013). Similarly, ischemic PreC may activate the RISK and SAFE pathways to limit mPTP opening and reduce IS (Davidson et al., 2006; Hausenloy et al., 2004; Lecour, 2009), but it remains unclear whether cardioprotective kinases protect directly via phosphorylation of end-effector proteins, or indirectly via improved mitochondrial respiration, suppression of mitochondrial calcium overload and oxidative stress (Clarke et al., 2008; Heusch et al., 2011; Skyschally et al., 2018). The NOS/NO-cyclic guanosine monophosphate signaling pathway is also necessary for ischemic PreC (Talukder et al., 2010). NO, being a gaseous molecule, can diffuse between organelles and cells to protect mitochondria from I/R by nitrosylating and inhibit mitochondrial complex I, suppressing the production of damaging ROS during early reperfusion (Chouchani et al., 2013; Rassaf et al., 2014).

Since ischemic PostC is applied at reperfusion, the trigger step is obviously different from ischemic PreC and may or may not involve different signaling pathways. By preventing complete reperfusion, ischemic PostC is believed to maintain an acidic myocardial pH, thereby acting at several cell targets involved in cardioprotection (i.e., preventing hypercontraction, calpain-mediated proteolysis, mPTP opening, and gap junction-mediated spread of injury during the first minutes of reflow) (Cohen et al., 2008; Inserte et al., 2011). However, aspects of the signal transduction and end-effector appear to be similar between ischemic PostC and ischemic PreC (e.g., protein kinases and ROS) (Penna et al., 2006; Barsukevich et al., 2015).

The signaling mechanism for RIC necessitates an additional step to enable communication between the triggering pathway in the remote organ and the end-effector pathway in the heart. A wide range of humoral factors have been proposed to mediate this communication, but there is no consensus on the critical molecule/s, and there may be multiple redundant factors (for review see Kleinbongard, Skyschally A, Gent, et al., 2017; Tsibulnikov et al., 2019). The parasympathetic nervous system is also required for transmission of the cardioprotective signal (Donato et al., 2013), meaning that both neural and hormonal signals are required (Lim et al., 2010). The spleen is an important relay organ between the neuronal and humoral signals of RIC (Lieder, Kleinbongard, et al., 2018; Heusch, 2019c). Interestingly, the various forms of ischemic conditioning (ischemic PreC, ischemic PostC, RIC) may all lead ultimately to the same cellular end-effectors to mediate cardioprotection (Heusch, 2015, 2020; Wolfrum et al., 2002). In this respect, the reduction in myocardial IS by RIC also involves activation of the PI3K/Akt pathway (thereby enhancing autophagy) (Gao et al., 2022), activation of STAT3 (Kleinbongard et al., 2018), and adenosine 5′-monophosphate activated protein kinase (Xu et al., 2022).

2. Mitochondria

Mitochondria are at the crossroads of cell death and survival through a plethora of functions that make them not only triggers but also mediators and end-effectors of cardioprotection due to their multifaceted participation in the pathophysiology of IRI, as extensively reviewed elsewhere (Davidson, Ferdinandy, et al., 2019). During ischemia, the interruption in the generation of mitochondrial adenosine triphosphate by oxidative phosphorylation is the triggering mechanism for the profound ionic and biochemical disturbances of cardiomyocytes (see previous discussion), the duration of which determines the fate of the cells (Piper et al., 1998). Upon reperfusion, the abnormal resumption of mitochondrial respiration (leading to an excessive and unregulated ROS production) and mitochondrial matrix calcium accumulation can synergistically exacerbate energy collapse through the activation of irreversible mPTP, a pathologic disruption of the inner membrane that induces massive matrix swelling and culminates in cell death (Halestrap and Richardson, 2015). In support of this, mice in which mitochondrial calcium overload was inhibited by cardiomyocyte-specific deletion of the mitochondrial calcium uniporter were protected against IRI (Luongo et al., 2015).

However, despite the enormous interest aroused by mPTP as a therapeutic target to prevent mitochondrial failure, its molecular entity remains controversial. Either a change in the dimerization of the FoF1-ATP synthase or a structural alteration within the enzyme holocomplex constitute some of the proposed models of the energy-dissipating channel that have received the most experimental support (Giorgio et al., 2013; Alavian et al., 2014; Bonora et al., 2017). Nevertheless, attempts to prevent mPTP by pharmacological inhibition of cyclophilin D (a regulatory protein known to interact with FoF1-ATP synthase; Giorgio et al., 2013) to desensitize mPTP against calcium resulted in inconsistent effects on cardiomyocyte survival during I/R and failed to confer clinical benefit in patients (Lim et al., 2012; Cung et al., 2015; Rahman et al., 2018). Of note, the failure of clinical trials to demonstrate cardioprotection using mPTP inhibitors does not negate its role in human heart but is more likely due to incomplete understanding of required dose/target in humans (Shanmuganathan et al., 2005; see Section IIIa3). In contrast, the attenuation of the complex 1-mediated mitochondrial ROS generation by different interventions, including either pharmacological or genetic inhibition of the reverse electron transport from complex II to complex I reduces mPTP opening and limits IS in mice (Chouchani et al., 2014; Valls-Lacalle et al., 2016; Yin et al., 2021) and in the in vivo pig model (Valls-Lacalle et al., 2018). Malonate—as one example—only reduced infarct size in the isolated mouse heart when administered at reperfusion, whereas an acidic malonate formulation was required to affect infarct size with administration before ischemia (Prag et al., 2022; Schulz and Heusch, 2022). However, malonate turned out not to have additive cardioprotective effects on IS reduction in pigs when combined with RIC (Consegal et al., 2021), despite the fact that conditioning strategies had been previously shown to modulate mPTP susceptibility (Heusch et al., 2011).

In a pig model of IRI with or without ischemic PostC, mitochondrial proteome analysis revealed a dual role for ischemic PostC promoting metabolic reprogramming of the myocardium and a protective response mediated by the voltage-dependent anion channel 2 and DJ-1 in the mitochondria (Gallinat et al., 2022). Cardiac mitochondria are dynamic organelles and organize into differentiated populations. As a general rule, interventions capable of decreasing mitochondrial fission (or increasing mitochondrial fusion) reduce IRI (Hernandez-Resendiz et al., 2020). Hence, genetic or pharmacological inhibition of the fission protein dynamin-related protein 1 mitigated cardiac injury in murine models of I/R, although this treatment failed to protect the heart in the more clinically relevant closed-chest pig model of AMI (Ong et al., 2019). Similarly, the beneficial effects of both aerobic exercise conditioning (Ghahremani et al., 2018) and RIC (Cellier et al., 2016) on IS have been attributed to a better maintenance of the elongated mitochondrial morphology in rat models of in vivo I/R. As for their subcellular location, subsarcolemmal mitochondria have a greater contribution to ROS production (Crochemore et al., 2015) and IRI (Lesnefsky et al., 1997) and are more sensitive toward pharmacological and ischemic conditioning than interfibrillar mitochondria (Holmuhamedov et al., 2012; Sun et al., 2015). Moreover, only subsarcolemmal mitochondria contain connexin 43 at their inner membrane (Boengler, Stahlhofen, et al., 2009), a protein involved in the ischemic PreC cardioprotection (Rodriguez-Sinovas et al., 2006; Ruiz-Meana et al., 2014) that has recently been identified as one of the interactors of the FoF1-ATP synthase (Boengler et al., 2018). Also, STAT 3 activation impacts on mitochondrial function; it increases respiration, ATP formation, and calcium retention capacity and decreases ROS formation in rat and mouse mitochondria of myocardium undergoing IRI with ischemic PreC or PostC (Boengler et al., 2008; Boengler et al., 2010; Heusch et al., 2011; Boengler, Ungefug, Heusch, and Schulz, 2013; Skyschally et al., 2018). Taken together, there are multiple available lines of evidence that link the cardioprotective effect of conditioning strategies with better mitochondrial function and integrity, yet the causality between both phenomena is difficult to establish. In part, a more preserved mitochondrial function and network could simply be the consequence of the otherwise well-known beneficial effects of these maneuvers on cellular ionic homeostasis (Inserte et al., 2014; Hausenloy et al., 2016). Furthermore, the effects on mitochondria can vary depending on the conditioning algorithm, the animal species, and the subtype of mitochondria.

3. Metabolism and Metabolomics

It has long been known that cardiac substrate metabolism is a main determinant of the severity of IRI. This is not surprising considering that I/R is in principle a metabolic pathology, with abruptly altered metabolism and thus energy production during the nonhomeostatic transitions from normoxia to ischemia and from ischemia to reperfusion (Guth et al., 1987). One of the first cardioprotective strategies examined against IRI was a metabolic treatment, employing glucose-insulin-potassium infusions to attenuate electrographic disturbances during MI (Sodi-Pallares et al., 1962). It is now known that almost every specific metabolic substrate pathway can affect cardiac IRI (Zuurbier et al., 2020). However, the complex interactions between these metabolic pathways have likely hindered the development of a singular metabolic treatment providing robust cardioprotection against IRI in the clinical condition. Nevertheless, in terms of metabolic approaches, it seems that activation of glycolysis, glucose, and ketone oxidation and inhibition of fatty acid metabolism and oxygen consumption holds the most promise for protecting the heart against IRI (Zuurbier et al., 2020).

Increases in glucose uptake and glycolysis in rodent hearts are mandatory for protecting the heart during low-flow ischemia (Lochner et al., 1996) and for various cardioprotective interventions such as ischemic PreC (Ji et al., 2013), ischemic PostC (Correa et al., 2008), and nicotinamide adenine dinucleotide (NAD) supplementation (Nadtochiy et al., 2018) to be effective.

The coenzyme NAD⁺ is critical for many biochemical pathways and the cellular stress response, and it is decreased in aging and many pathologies including cardiovascular diseases (Fang et al., 2017). During cardiac I/R, NAD⁺ is acutely decreased in rat heart (Di Lisa et al., 2001), partly due to mPTP opening. NAD⁺ is also used by sirtuins, a group of lysine de-acetylation enzymes controlling metabolism (Anderson et al., 2014). Metabolic overloading such as that present in the metabolic syndrome results in increased acetylation (due to increases in acetyl-coenzyme A) and thereby inhibition of, for example, glucose uptake pathways in rat hearts (Renguet et al., 2017), interfering with cardioprotection. Sirtuins can de-acetylate these pathways and restore cardioprotection. In several studies NAD precursors protected against cardiac IRI (Yamamoto et al., 2014; Nadtochiy et al., 2018; Xiao et al., 2021).

That an activated glucose metabolism is needed for protection is commensurate with the finding that many of the critical signaling molecules (ROS, NO, PKC) triggering and mediating protection (see Fig. 2) are also know activators of glucose metabolism (Tada et al., 2000; Nishino et al., 2004). Metabolomic studies in rat hearts also indicated that protection through PKCε is associated with changes in glucose metabolism (Mayr et al., 2009). It seems that ischemic PreC-activated glucose metabolism (e.g., glycolysis) slows down mitochondrial reactivation following reperfusion (Zuurbier and Ince, 2002), possibly through increased binding of the glycolytic enzyme hexokinase II to mitochondria (Gurel et al., 2009; Nederlof et al., 2017). Glucose and mitochondrially bound hexokinase II are both needed for effective ischemic PreC and reductions in IS and cell death in rodent hearts (Pasdois et al., 2012; Smeele et al., 2011; Sun et al., 2008). Slowing down mitochondrial activity results in decreased oxygen consumption during reperfusion, which has been suggested to contribute to cardioprotection (Burwell et al., 2009). However, the role of mitochondrial function for cardioprotection during reperfusion is somewhat contentious, since STAT3 activation has been shown to mediate cardioprotection by ischemic PostC and RIC through improved mitochondrial function (see previous discussion).

Increased fatty acid uptake and incomplete fatty acid metabolism during ischemia aggravate IRI through the build-up of long-chain acylcarnitines within the mitochondria, resulting in an increase of mitochondrial ROS production (Dambrova et al., 2021). However, although ischemic PreC efficacy is often decreased in conditions of elevated fatty acid metabolism, this is not always observed (Dalgas et al., 2012). Metabolomics studies have suggested that (i) exercise-induced cardioprotection is associated with changes in rat heart ammonia recycling, protein biosynthesis, and pantothenate and coenzyme A biosynthesis (Parry et al., 2018) and (ii) RIC in rats and humans with decreases in plasma ornithine and increases in kynurenine and glycine (Chao de la Barca et al., 2016; Kouassi Nzoughet et al., 2017; Bakhta et al., 2020). Previous work in murine hearts had already indicated the possible important role of alpha-ketoglutarate induced kynurenic acid synthesis in mediating RIC (Olenchock et al., 2016).

Oeing et al. (2021) recently confirmed the importance of glucose metabolism in cardioprotection and ischemic PreC by demonstrating that mechanistic target of rapamycin c1-activated glycolysis at the expense of fatty acid oxidation offers protection of the murine heart against IRI and mediated ischemic PreC protective effects. Lochner et al. (2020) confirmed the mandatory role of glucose in ischemic PreC and that high fatty acid levels prevented ischemic PreC in rat heart.

4. Circulating Cells

Circulating cells can strongly impact on IRI through various mechanisms (for a review, see Davidson, Andreadou, et al., 2019). Among the different circulatory cells, platelets play an important role in myocardial I/R. Platelets, beyond hemostasis and thrombosis, are characterized as versatile cells directly involved in various physiologic and pathophysiological processes (Russo et al., 2017). Several imaging studies have provided evidence that platelets contribute to IRI in in vivo rodent models of cardiac IRI; they are activated early during reperfusion and localized within the ischemic and necrotic areas (von Elverfeldt et al., 2014; Ziegler et al., 2016; Ziegler et al., 2019). Additionally, circulating platelets change their characteristics due to IRI (Eicher et al., 2016; Kaudewitz et al., 2016) as has been shown by proteomic studies in STEMI patients (Ruggeri, 2002).

Platelets also carry and release multiple factors with the potential to reduce IRI (Hjortbak et al., 2021; Kleinbongard et al., 2021), although the role of circulating platelets as signal mediators of cardioprotection is far from being understood. In recent studies, RIC exerted its cardioprotective effect through modulating platelet function by reducing the formation of monocyte-platelet conjugates and thrombus formation (Lanza et al., 2016) and was associated with a reduction in platelet reactivity within the first 48-hour post STEMI (Gorog et al., 2021) (for review see Kleinbongard et al., 2021). More studies are necessary to understand the role of platelets in IRI and their importance for conditioning strategies.

Erythrocyte dysfunction contributes to a reduced NO bioavailability and thereby to increased IS and mortality from IRI in mice (Wischmann et al., 2020); also, erythrocyte stasis contributes to the no-reflow phenomenon (Kyrou et al., 2011).

Neutrophil (polymorphonuclear leukocyte) recruitment to ischemic, and more particularly reperfused, myocardium has been recognized as a pathologic hallmark of AMI for nearly a century. As described earlier, neutrophil adherence and plugging may play a key role in MVO supply and no-reflow. However, a direct causal role of neutrophils in the evolution of myocyte death, at least in the early stages of AMI, has been contentious (Baxter, 2002; Lefer, 2002). There is evidence that effective IS-limiting interventions including ischemic PreC and ischemic PostC result in reduced neutrophil accumulation. One study suggests that neutrophil inhibition is causally related to IS limitation by ischemic PostC (Granfeldt et al., 2012). There is also limited evidence from human RIC studies to suggest that rapid and long-lasting systemic neutrophil inhibition occurs in response to the RIC stimulus (Kharbanda et al., 2001; Shimizu et al., 2010; Saxena et al., 2013). The extent to which these observations are mechanistically important in cardioprotection is unknown.

5. Innate Immunity and the NLRP3 Inflammasome

Neither circulating monocytes nor cardiac-resident macrophages contribute to acute IRI, although they are crucial for longer term infarct and LV remodeling (Bajpai et al., 2019).

During I/R, resident cardiac mast cells degranulate, releasing their proteolytic and damaging contents. Consequently, mast cell stabilizing compounds are cardioprotective (Wang et al., 1996; Rork et al., 2008; Bajpai et al., 2019). However, mast cell degranulation does not appear to contribute to ischemic PreC (Wang et al., 1996).

Necrosis or pyroptosis of cardiac cells during I/R releases their contents into the extracellular milieu, where they are recognized by the innate immune system as damage-associated molecular patterns. Some damage-associated molecular patterns particularly relevant to cardiac I/R injury include proteins of the extracellular matrix, heat shock proteins, S100 proteins, ATP, histones, high-mobility group box 1 (HMGB1), IL-1α, and mitochondrial deoxyribonucleic acid (Vilahur and Badimon, 2014). For example, heat shock protein 60 can induce apoptosis in cardiomyocytes (Kim et al., 2009). Damage-associated molecular patterns are recognized by toll-like receptors, particularly toll-like receptors 2 and 4, and are targets for reducing IRI. Histones released during I/R damage cardiomyocytes by toll-like receptor 4 related mechanism (Shah et al., 2022). Inhibition of toll-like receptor 2 reduces IS in both mouse and pig models (Arslan et al., 2010; Arslan et al., 2012). Knockout of toll-like receptor 3 (Lu et al., 2014) or 4 (Oyama et al., 2004) reduces IS in mice after I/R. Mitochondria are fundamentally involved in innate immunity and sterile inflammation. After exposure to NLRP3 activators, damaged mitochondria accumulate, leading to increased production of oxidized mtDNA fragments. These associate with the NLRP3 inflammasome in the cytosol and are required for its activation (Zhou et al., 2011; Zhong et al., 2018). The complement system forms an important aspect of the innate immune system. Blocking of either the classic, antibody-activated, complement pathway with a C1 esterase inhibitor or of complement factor C5a in the alternative pathway reduces IS in animals subject to I/R (Buerke et al., 1995; van der Pals et al., 2010) (reviewed in Yasuda et al., 1990).

The NLRP3 inflammasome and innate immunity are potential targets for acute cardioprotection. However, Zuurbier et al. showed that NLRP3 is barely expressed in healthy murine hearts, and deletion of NLRP3 had no effect on IS following I/R either in perfused mouse hearts or in vivo (Zuurbier et al., 2012; Jong et al., 2014). In contrast, Sandanger et al. (2013) reported that isolated mouse hearts lacking NLRP3 had smaller IS following global I/R. Generally, it seems that approximately 24 hours reperfusion time is required for priming (expression) of the NLRP3 inflammasome for pyroptosis to make a significant contribution to IS in wildtype hearts (Merkle et al., 2007; Kawaguchi et al., 2011; Sandanger et al., 2013; Jong et al., 2014; Sandanger et al., 2016). In line with this, an NLRP3 inflammasome inhibitor was able to reduce IS even when administered to mice after 1 hour of reperfusion but only when IS was measured 24 hours following infarction (and not after only 3 hours) (Toldo et al., 2016). The selective NLRP3-inflammasome inhibitor MCC950 reduced IS measured 7 days following I/R in pigs (van Hout et al., 2017). A recent study has implicated an NLRP3-independent, oxidative stress-dependent pathway of caspase-11 mediated cleavage of gasdermin D within cardiomyocytes, and release of Il-18 in IRI, and showed that IS was reduced in gasdermin D knockout mice 24 hours after I/R (Shi et al., 2021).

There is some evidence that the NLRP3 inflammasome may be involved in ischemic PreC, although more studies are required to investigate the role of priming. The benefit of ischemic PreC was lost in NLRP3 knockout but not apoptosis-associated speck-like protein containing caspase recruitment domains knockout hearts in the ex vivo Langendorff model (Zuurbier et al., 2012). Pharmacological preconditioning with a toll-like receptor 2 agonist was also lost in NLRP3 knockout hearts (Sandanger et al., 2016). There is limited evidence that RIC might also affect innate inflammation, as recently reviewed (Pearce et al., 2021). Notably, it has been reported that expression of the NLRP3 inflammasome is higher in infiltrating inflammatory cells and murine cardiac fibroblasts (Kawaguchi et al., 2011; Sandanger et al., 2013), so these may be a target for cardioprotection in addition to cardiomyocytes. An important question remains whether comorbidities can prime expression of NLRP3 inflammasome and gasdermin D, which would increase their relevance to the I/R process in diseased hearts.

Indeed, sterile inflammation has been described in other inflammatory conditions such as gout, pseudogout, type 2 diabetes mellitus, metabolic syndrome, atherosclerosis, asbestosis, silicosis, and Alzheimer’s disease (for review see Algoet et al., 2022). There is a growing understanding that these disorders are pathophysiologically linked to and can modulate the course of IRI and its response to treatment. The term “metaflammation” describes a metabolically triggered chronic enhanced systemic inflammatory status that is associated with these conditions (Itoh et al., 2022), and such a chronic inflammatory status is also observed in the elderly population without comorbidities (termed “inflammaging”) (Liberale et al., 2022).

6. Exosomes

Since our last substantial review of this topic, there has been an explosion of interest in cell-derived nanoparticles called exosomes (Prakash et al., 2020). These extracellular vesicles are released from all types of cells and are found in the blood of all species. Although they are derived mainly from erythrocytes and platelets, some are derived from the vasculature and some from cardiomyocytes (Hegyesi et al., 2022), and they play a role in cardioprotection (Sluijter et al., 2018). Ischemic PreC increases the release of exosomes from endothelial cells or from the heart, and these exosomes are cardioprotective via a signaling pathway involving ERK1/2 (Davidson, Riquelme, et al., 2018). The potential for exosomes to be involved in RIC was first suggested in 2014 (Yellon and Davidson, 2014), and at the same time the first evidence for this was provided by the demonstration that exosomes could transfer cardioprotection from 1 isolated heart to another (Giricz et al., 2014). RIC was shown in both humans and rats to increase the number of exosomes in the blood, although in this study no additional protection was seen with exosomes after RIC (Vicencio et al., 2015). Another study measured elevated levels of microRNA (miR)144 in the blood of mice and humans following RIC, which they proposed was circulated via exosomes to the heart to induce cardioprotection in mice (Li et al., 2014). In patients undergoing coronary artery bypass grafting (CABG), prior RIC increased the number of circulating exosomes and notably their miR 20 content along with reduced postoperative troponin release (Frey et al., 2019). It was recently proposed that IPost increases the release of miR 423-3p-containing exosomes from cardiac fibroblasts and that these participate in the cardioprotective effects of ischemic PostC, via the downstream effector RAP2C (member of RAS oncogene family) (Luo et al., 2019). In healthy volunteers undergoing RIC, protection was transferred to isolated rat hearts and mediated by extracellular vesicles and their miR cargo of miR 16-5p, 144-3p and 451a (Lassen, Just, et al., 2021). It remains unclear how miRs are able to act rapidly enough on their target transcripts to affect a rapidly developing AMI. Interestingly, diabetes impairs cardioprotection by exosomes (Davidson, Andreadou, et al., 2018; Wider et al., 2018).

7. Cardiac Transcriptome

Rapid development of ‘omics technologies in the last 2 decades especially with transcriptomics have enabled the measurement of expression of all known coding and noncoding RNAs. Noncoding RNAs exhibit highly organized spatial and temporal expression patterns and are emerging as critical regulators of differentiation, homeostasis, and pathologic states, including in the cardiovascular system (Abbas et al., 2020; Shah et al., 2022). Unbiased bioinformatics evaluation of such data has led to the discovery of novel mechanisms and promising drug targets for cardioprotection (for reviews see Perrino et al., 2017; Parini et al., 2020). It was shown in the early 2000s that ischemic PreC dramatically altered cardiac gene expression pattern at the mRNA level in rats (Onody et al., 2003) (for review see Perrino et al., 2017). Global cardiac miR expression changes are also observed in pig models of ischemic PreC and ischemic PostC (Spannbauer et al., 2019). In 2013, the first evidence was provided that the expression profile of noncoding miR (fine-tune regulators of mRNA expression) in preconditioned and postconditioned hearts are also altered and several miRs expression changes are associated with cardioprotection—these miRs have been termed protectomiRs (Varga et al., 2014). In particular, a mimic of miR 125b has been shown by several groups to play an important role in ischemic PreC in different models (Wang et al., 2014; Bayoumi et al., 2018; Varga et al., 2018). To date, several miRs have been proposed to be involved in cardioprotective signaling, such as miR 1, miR 144, and miR 221. By unbiased molecular network analysis of miR-mRNA interactions, novel gene targets can be explored (for review see Makkos et al., 2021). Other noncoding RNAs such as circular and long-noncoding are also involved in cardioprotective signaling; however, little is known about their function and possible therapeutic relevance (Wu et al., 2017; Cai et al., 2019; Jusic et al., 2020; Lou et al., 2021).

1. Ischemic Preconditioning and Postconditioning

Clinically, the use of ischemic PreC has been largely restricted to patients undergoing cardiac surgery (for review see Buja, 2022). In this setting, early studies demonstrated that intermittent cross-clamping of the aorta reduced myocardial injury (assessed by serum cardiac biomarkers such as creatine kinase-MB or troponin T) (Jenkins et al., 1997), and a meta-analysis found potential benefits with RIC with reduced arrhythmias, less inotrope support requirement, and reduced intensive care unit stay (Walsh et al., 2008). However, given the invasive nature of the ischemic PreC stimulus and the potential risk of cerebral thromboembolism from cross-clamping of an atherosclerotic aorta, ischemic PreC has been largely abandoned in cardiac surgery.

In contrast to ischemic PreC, ischemic PostC could be applied at the time of reperfusion in AMI patients undergoing balloon angioplasty at the time of PPCI (Staat et al., 2005). Although shown to be initially promising, with ischemic PostC reducing myocardial IS and preserving cardiac function in a number of small clinical studies (Staat et al., 2005; Thibault et al., 2008), these were followed by several neutral and even negative studies (Freixa et al., 2012; Heusch, 2012; Tarantini et al., 2012). Unfortunately, the large DANAMI-3 trial, which evaluated the effects of ischemic PostC in 1234 STEMI patients undergoing PPCI, failed to report any beneficial effects on clinical outcomes (Engstrøm et al., 2017). The reasons for this failure are not clear but may relate to the ischemic PostC protocol used in the study with the ischemic PostC protocol being delivered within the stent and/or the low-risk patient population (making the study underpowered). Of note, in a post hoc study patients without thrombectomy had reduced risk of all-cause mortality and hospitalization for HF with ischemic PostC (Nepper-Christensen et al., 2020). Ischemic PostC has also been shown to reduce perioperative myocardial injury in the setting of CABG using intermittent cross-clamping of the aorta once the patient has come off cardiopulmonary bypass (Luo et al., 2008; Candilio and Hausenloy, 2017), but, as for ischemic PreC, the invasiveness of the procedure has limited its application. In a recent multicenter trial, ischemic PostC by 3 cycles of normothermic antegrade blood cardioplegia before release of the aortic clamp did not improve cardiac index (primary endpoint) or reduce troponin T or creatine kinase release but reduced a combined secondary endpoint of intraoperative ventricular fibrillation and postoperative atrial fibrillation and suggested hemodynamic differences in the response to PostC between male and female patients undergoing aortic valve replacement (see Kaljusto et al., 2022 and accompanying editorial Podesser and Kiss, 2022). Both ischemic PreC and ischemic PostC require the cardioprotective intervention to be applied directly to the heart, making the procedure invasive and more challenging to apply in the clinical setting. In this regard, RIC, which allows the cardioprotective stimulus to be applied to an organ or tissue away from the heart, has been intensively investigated as a cardioprotective intervention in the clinical setting.

2. Remote Ischemic Conditioning

The ability to apply the cardioprotective stimulus to the arm or leg by simply inflating and deflating a pneumatic cuff on the upper arm/leg or thigh to induce brief nonlethal episodes of I/R, has greatly facilitated the testing of limb RIC in patients at risk of acute myocardial IRI (Heusch et al., 2015). Single-occasion RIC reduces arterial stiffness and LV remodeling after AMI (Ikonomidis et al., 2021) beyond IS reduction alone, but the effect does not unequivocally translate into a reduction of HF admission in patients (Sloth et al., 2014). Several smaller clinical studies have demonstrated that limb RIC (applying 3 to 4 5-minute cycles of cuff inflation and deflation) prior to CABG surgery reduced perioperative myocardial injury (quantified by serum troponin levels) (Hausenloy et al., 2007; Thielmann et al., 2013), although not all studies have been positive (Rahman et al., 2010). Unfortunately, 3 large, multicenter studies in cardiac surgery patients failed to show any improvement in clinical outcomes with limb RIC applied to cardiac surgery patients (Hong et al., 2014; Hausenloy et al., 2015; Meybohm et al., 2015). The reasons for this failure are not clear but have been attributed to the potential confounding effects of certain comedications such as propofol anesthesia (Kottenberg et al., 2012), nitrates (Candilio et al., 2015), or beta-blockers (Cho et al., 2019) (see Section V). Furthermore, the causes of myocardial injury during CABG are not only due to acute myocardial IRI as coronary embolization, direct handling of the heart, and inflammatory injury associated with cardiopulmonary bypass may be etiological and not amenable to RIC cardioprotection. In contrast, an acute STEMI patient treated by reperfusion using PPCI represents a “purer” setting of acute myocardial IRI, which should be more amenable to the cardioprotective effects of limb RIC.

The first study to demonstrate this in STEMI patients was by Bøtker et al. (2010), who showed applying limb RIC patients in the ambulance on the way to the PPCI center, improved myocardial salvage (assessed by myocardial single-photon-emissions-tomography imaging) but did not reduce myocardial IS. Subsequent studies confirmed the cardioprotective effect of limb RIC administered on arrival at the hospital quantified by cardiac MRI (White et al., 2015) and even at the onset of reperfusion (Crimi et al., 2013), although not all studies have been positive (Verouhis et al., 2016). One large clinical study did demonstrate improved clinical outcomes with less HF hospitalization and cardiac death (Gaspar et al., 2018). In addition, follow-up studies of RIC-treated STEMI patients suggested an improvement in major adverse cardiac events at follow-up (Sloth et al., 2014; Stiermaier et al., 2019). However, despite these promising studies, the large, multicenter CONDI-2/ERIC-PPCI trial of 5401 STEMI patients failed to find any improvement in rates of HF hospitalization and cardiac death at 12 months (Hausenloy, Kharbanda, et al., 2019). The reasons for this failure to translate limb RIC for patient benefit are not clear but may relate to the low-risk population recruited in this trial (Heusch and Gersh, 2020) (see Section IIIa3.).

Most clinical cardioprotective studies with limb RIC have applied 1 stimulus at the time of cardiac surgery or STEMI, but animal studies have suggested that cardioreparative effects of RIC may be induced by repeated daily episodes of limb RIC (Wei et al., 2011). Extended exposure to RIC may add further modulation of myocardial remodeling. Repeated RIC modifies the human inflammatory response and leukocyte adhesion (Shimizu et al., 2010) and improves coronary microcirculation in healthy volunteers and patients with HF (Jones et al., 2014). In contrast to single-occasion RIC, repeated RIC reduces blood pressure (Baffour-Awuah et al., 2021), allowing afterload reduction to modulate myocardial remodeling favorably. Nonetheless, the Daily REmote Ischemic Conditioning Following Acute Myocardial Infarction (DREAM) study demonstrated no effect of 4 weeks of daily RIC-treatment initiated 3 days after PPCI on ventricular function in 73 patients with reduced LV function after the acute coronary event (Vanezis et al., 2018). In pilot studies, repeated RIC as add-on to standard anticongestive treatment in patients with stable chronic HF did not improve LV ejection fraction but decreased circulating NH₂-terminal pro-B-type natriuretic peptide and skeletal muscle function after 28 days of RIC treatment once daily (Pryds et al., 2017), whereas exercise capacity was not different (McDonald et al., 2014). However, prolonged periods of daily limb RIC have been reported to be beneficial in patients with intracranial stenosis at risk of stroke (Meng et al., 2012). Despite the failure to translate the cardioprotective effects of limb RIC into a clinical benefit in cardiac surgery or STEMI patients, it may still have potential in STEMI patients at elevated risk of compromised outcome (Cheskes et al., 2020; Hausenloy et al., 2020) and other settings of acute IRI, such as kidney transplantation (MacAllister et al., 2015).

3. Pharmacological Cardioprotection

A detailed description of comedication administered to treat patients’ comorbidities and its impact on IRI as well as cardioprotective interventions will be discussed extensively in Section V. However, some pharmacological approaches derived from a better understanding of the signaling cascades involved in endogenous cardioprotection have also been evaluated in clinical trials (Table 2).

The most promising of these was CsA, a mPTP inhibitor that had been shown in small and large animal studies to reduce IS (Hausenloy et al., 2002; Argaud et al., 2005; Skyschally et al., 2010), although not all studies had been positive (Karlsson et al., 2010; Lim et al., 2012). While initial phase 2 clinical studies with CsA reported reducing myocardial injury in cardiac surgery patients (Chiari et al., 2014; Hausenloy et al., 2014) and smaller IS in STEMI patients (Piot et al., 2008), 1 study did not show cardioprotection with CsA in STEMI patients (Ghaffari et al., 2013). Unfortunately, 2 large, multicenter clinical studies (CIRCUS and CYCLE) (Cung et al., 2015; Ottani et al., 2016) failed to demonstrate improved clinical outcomes with CsA administered prior to reperfusion in STEMI patients. Why these larger clinical trials failed to confirm the benefit of CsA on either reducing IS or clinical outcomes is unclear but may have been due to a type I error, insufficient dosing, the low-risk population, and the presence of P2Y₁₂ (chemoreceptor for adenosine diphosphate) inhibitors (see Section V).

Other mitochondrial targeting agents such as MTP-131 (which optimizes mitochondrial energetics and attenuates the production of ROS by selectively targeting cardiolipin in the inner mitochondrial membrane) failed to reduce IS in a phase 2 trial in a carefully selected group of anterior STEMI patients (Gibson et al., 2016). The mitochondrial protective agent, TRO40303, (which binds to the translocator protein in the outer mitochondrial membrane) reduced IS in small animals (Schaller et al., 2010) but not in large animals (Hansson et al., 2015) and did not reduce IS when administered to STEMI patients prior to PPCI (Atar et al., 2015).

While some experimental studies demonstrated cardioprotection with intravenous nitrite administered at the onset of reperfusion (Duranski et al., 2005), the National Heart Lung and Blood Institute Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR) Network failed to show IS reduction with nitrite using a multicenter approach in small and large animal I/R models (Lefer and Bolli, 2011; Jones, Tang, et al., 2015; Bolli, 2021). Also, 2 clinical studies failed to demonstrate a significant IS reduction with nitrite administered by either the intravenous (Siddiqi et al., 2014) or intracoronary (Jones, Pellaton, et al., 2015) route in PPCI-treated STEMI patients. The study by Janssens et al. also failed to report any cardioprotective effects with inhaled NO as an adjunct to PPCI in STEMI patients (Janssens et al., 2018), although some benefit was seen in nitrate-naïve patients.

More recently, studies have investigated the cardioprotective effects of targeting interleukin-6 (IL-6), as this cytokine has been shown to contribute to inflammation in coronary artery disease and acute myocardial IRI (Sawa et al., 1998; Interleukin-6 Receptor Mendelian Randomization Analysis Consortium et al., 2012; Ritschel et al., 2013). An initial clinical study in non-ST elevation myocardial infarction patients reported that pretreatment with a single intravenous 60-minute infusion of tocilizumab (an IL-6 receptor antibody) prior to PCI reduced inflammation and PCI-related myocardial injury when compared with placebo (Kleveland et al., 2016). The recently published ASSAIL-MI study in STEMI patients demonstrated that the same treatment regimen initiated during PCI improved myocardial salvage but did not significantly reduce MI size assessed by cardiac MRI performed on day 3 to 7 post-admission, when compared with placebo (Broch et al., 2021).

The importance of NLRP3 inflammasome-driven IL-1β for cardiovascular events was studied in some clinical trials with anakinra (a recombinant IL-1 receptor antibody) in patients with previous MI (VCU-ART and VCUART2; MRC-ILA-Heart Study). These trials were performed in a relatively small number of patients, and the reported results are contentious. The CANTOS trial, which evaluated the long-term effect of canakinumab (a humanized monoclonal IL-1β antibody) in 10061 MI patients, showed a significant 15% reduction in major adverse coronary events (MACE) compared with the placebo group; however, all-cause mortality did not differ between the canakinumab and placebo groups. Recently, the Colchicine Cardiovascular Outcome Trial using low-dose colchicine in 4745 MI patients as well as the subsequently conducted low-dose colchicine 2 trial in 5522 patients with chronic coronary artery disease confirmed a decrease in MACE (including cardiovascular death). Since colchicine can prevent NLRP3 inflammasome assembly, the clinical efficiency of colchicine supports the notion that NLRP3 inflammasome plays a key role in the pathogenesis of atherosclerosis and subsequent atherothrombotic events (for a detailed review see Takahashi, 2022).

1. Improving Preclinical Assessment of Cardioprotective Strategies

One key reason for the failure to translate cardioprotection into the clinical arena has been the lack of rigorous and systematic preclinical testing of novel cardioprotective therapies, the consequence of which has been the premature clinical evaluation of treatments with inconsistent and less than robust cardioprotective effects (Bolli, 2021). Potential strategies for ensuring that only the most robust and reproducible novel cardioprotective therapies are tested in clinical studies include establishing guidelines and criteria for preclinical evaluation of novel cardioprotective therapies and establishing multicenter research networks for testing of novel cardioprotective therapies. The European Union-CARDIOPROTECTION Cooperation in Science and Technology Action CA16225, a pan-European research network of leading experts in experimental and clinical cardioprotection, aims to address some of these issues. It has already published practical guidelines to ensure rigor and reproducibility in preclinical cardioprotection studies (Bøtker, Hausenloy, et al., 2018) and has established the IMproving Preclinical Assessment of Cardioprotective Therapies (IMPACT) criteria for improving the in vivo preclinical evaluation of the efficacy of novel cardioprotective therapies (Lecour et al., 2021). Finally, the EU-CARDIOPROTECTION Cooperation in Science and Technology Action is currently establishing a research network for preclinical multicenter testing of novel cardioprotective therapies. The IMPACT small-animal research network is currently being set up to undertake multicenter evaluation of novel cardioprotective therapies in mice and rat models of acute myocardial IRI, and validation of the research network will be undertaken using ischemic PreC. The CIBERCV (the acronym for Spanish Network-Center for Cardiovascular Biomedical Research) has set up the Cardioprotection Large Animal Platform, a Spanish multicenter network of 5 research centers, to perform experimental pig acute myocardial IRI studies testing the efficacy and reproducibility of promising cardioprotective interventions based on a prespecified design and protocols, centralized randomization, blinding assessment, core laboratory analyses of cardiac MRI imaging, histopathology, and proteomics (Rossello et al., 2019). The network is currently being validated using ischemic PreC. Also, it will be necessary in select preclinical studies to perform a more chronic follow-up and use the same endpoints as used in clinical trials (i.e., mortality over 6 to 12 months and the development of HF) (Heusch, 2018).

2. Multitargeted Approaches to Cardioprotection

One potential strategy for improving the clinical translation is to use a multitarget approach using either single agents (that have more than 1 target) or 2 or more therapies with different targets. The multitargeted approach may be a more effective than a single-targeted approach, especially given the complexity and different proponents of acute myocardial IRI (e.g., cardiomyocytes, endothelial cells, immune cells, platelets, microvasculature) (Davidson, Ferdinandy, et al., 2019). The ongoing PITRI study is testing whether administration of the intravenous P2Y₁₂ platelet inhibitor, cangrelor, prior to PPCI can prevent microvascular obstruction and reduce IS in STEMI patients (Bulluck et al., 2019) (Table 1). Cangrelor offers complete platelet inhibition with 1 to 2 minutes of administration, thereby potentially reducing the risk of MVO in STEMI patients, and has also been reported in small and large animal studies to reduce IS when given at reperfusion (Yang, Cui, et al., 2013; Yang, Liu, et al., 2013a, 2013b) (Table 2). The NACIAM study showed that the addition of an intravenous infusion of N-acetylcysteine on a background of nitroglycerin infusion reduced IS when compared with nitroglycerin infusion (Pasupathy et al., 2017) (Table 2).

The combined effects of limb RIC with ischemic PostC have been tested in a clinical study but were shown to have no additional cardioprotective effects in STEMI patients (Prunier et al., 2014). However, the LIPSIA study did report increased myocardial salvage and improved outcomes (less cardiac death, reinfarction, and new congestive HF at 3.6 years) in patients given both limb RIC and ischemic PostC when compared with control or ischemic PostC alone, although the effects of limb RIC alone were not tested (Eitel et al., 2015; Stiermaier et al., 2019). The ongoing CARIOCA (NCT03155022) study is also testing the combination of limb RIC and ischemic PostC in a large STEMI trial (Table 1). Based on the premise that limb RIC and exenatide had different cardioprotective targets (pig study) (Alburquerque-Béjar et al., 2015), the COMBAT MI study recently compared the IS-limiting effects of combining RIC with exenatide to that of limb RIC or exenatide alone in PPCI-treated STEMI patients. Unfortunately, the combination of RIC and exenatide did not translate into a reduction of IS and, more surprisingly, neither limb RIC nor exenatide alone reduced IS (García Del Blanco et al., 2021) (see Section V) (Table 1).

3. Targeting High-Risk Patients

One potential reason for the failure of limb RIC to improve clinical outcomes in STEMI patients in the CONDI2/ERIC-PPCI trial was the low-risk population recruited: they were optimally treated by PPCI and had relatively short ischemic times (median of 3 hours), and 96% of patients presented in Killip Class I and cardiac mortality (2.7%) was low at 12 months (Heusch and Gersh, 2020). RIC may be more effective in higher risk STEMI patients such as those presenting in HF or cardiogenic shock or those who were still treated by thrombolysis (Bøtker, 2020; Heusch and Gersh, 2020). In this regard, the FIRST study in which RIC was implemented in the clinical setting as part of a pre- and post-implementation study reported potential beneficial effects on MACE in those patients with cardiogenic shock or cardiac arrest (Cheskes et al., 2020). The planned RIC-AFRICA trial (NCT04813159) will evaluate RIC in higher risk STEMI patients treated by thrombolysis due to limited availability of PPCI (Hausenloy et al., 2020), and the RIP-HIGH trial will test the combination of RIC and local ischemic PostC in STEMI patients with heart failure (Killip class≥2) (i.e., those with severe hemodynamic impairment or cardiogenic shock) (NCT 04844931).

In summary, the translation of cardioprotection into the clinical setting for patient benefit has been largely disappointing. On the one hand, there is a wealth of preclinical studies unequivocally demonstrating cardioprotection in a variety of species and experimental models with different endpoints, such as arrhythmias, ventricular dysfunction, IS, and coronary MVO. On the other hand, despite several positive proof-of-concept clinical studies in STEMI patients demonstrating cardioprotection by mechanical and pharmacological approaches, there is now an increasing number of recent phase 2 studies showing no benefits on IS and several large phase 3 studies failing to show benefit on clinical outcomes despite positive phase 2 studies. This discrepancy results from the very different approaches inherent to preclinical research and clinical trials. Preclinical studies aim for novel knowledge and mechanistic understanding and therefore regularly use protocols that maximize the cardioprotective efficacy. In contrast, large clinical trials aim to identify a cure for disease in as many patients as possible and therefore regularly use an all-comer approach that does not consider the need of protection or potential confounders. The disappointment about the lack of translation then reflects the very different mutual expectations and a lack of communication between preclinical researchers and clinicians. The present review aims to improve such communication, with a particular focus on confounders.

On the preclinical side, potential reasons for failure in clinical translation include the lack of rigorous preclinical evaluation of novel cardioprotective therapies. Therefore, strategies for improving the preclinical assessment of novel cardioprotective therapies with the introduction of rigorous criteria that need to be fulfilled before proceeding to clinical studies and the use of multicenter networks of small and large animal research centers to blindly evaluate novel cardioprotective therapies are advocated (Lecour et al., 2021; Kreutzer et al., 2022). On the clinical side, targeting high-risk patients at risk of acute myocardial IRI (Heusch and Gersh, 2020) and consideration of confounders may improve the chances of successfully translating cardioprotection for patient benefit. With respect to our focus on confounders, it is important to note that most published clinical cardioprotection studies in AMI patients have not been suitably powered or specifically designed to test the confounding effects of comorbidities and comedications discussed in this article on the efficacy of cardioprotective therapeutic strategies, despite a significant proportion of recruited patients having these potential confounding factors. In an ideal world, we would propose that only those cardioprotective interventions that have fulfilled stringent criteria and been established in a multicenter network as robust are taken forward from preclinical studies to be tested in a clinical trial, whereas less certain cardioprotective interventions would not even be tested clinically. Conversely, clinical trials for cardioprotection beyond that by reperfusion would be confined to those needing adjunct cardioprotection and not in an all-comer approach; at the least, a prespecified subgroup analysis for STEMI patients with Killip class ≥2 (i.e., those with severe hemodynamic impairment or cardiac shock) would be performed.

1. Aging

2. Sex, IRI, and Cardioprotection

Although ischemic heart disease is a major cause of mortality and morbidity in both males and females, sex differences exist in terms of susceptibility, mechanisms, and outcomes to IRI observed between men and women Confounding factors (comorbidities, comedications) in ischemic heart disease may have sex-specific effects, and mechanisms underlying these differences are multiple and include gonadal hormones (for review see Perrino et al., 2021). Despite sex differences in IRI outcome, which occurs in an age-dependent manner, no clinical studies have yet been able to highlight sex as a confounding factor in the cardioprotective strategy of conditioning (Staat et al., 2005). Although most of the preclinical data exploring the cardioprotective effect of ischemic conditioning have been investigated in healthy young male animals, few studies suggest a sex difference in the cardioprotective response of conditioning against IRI in structurally normal myocardium (for review see Querio et al., 2021). As discussed in Section IVc, notable sex-related differences, however, have been noted in hypertrophied myocardium.

In the preclinical setting, some animal studies suggest that ischemic PreC may be less cardioprotective in females than in males, an effect that is also highly dependent on the age of the animals. In mice, ischemic PreC improved the functional outcome of IRI in both 10- and 18-week-old male mice. In contrast, female mice failed to be protected at an age of 10 weeks (Song et al., 2003; Turcato et al., 2006). In Wistar rats, ischemic PreC successfully reduced IS in 12- and 18-week-old males and females but failed to confer an antiarrhythmic effect in 12-week-old females (Ledvenyiova et al., 2013). Whereas both male and female rats can be preconditioned with endotoxin, the protection in females was only observed with higher doses of endotoxin, thus suggesting that the conditioning threshold may differ between males and females (Pitcher et al., 2005). Similarly, delayed pharmacological PreC with isoflurane protected male but not female rabbits (Wang et al., 2006). It is suggested that the apparent lack of protection with ischemic PreC in young female animals is due to estrogen-mediated better tolerance against IRI compared with males (Song et al., 2003).

In contrast, no sex difference in the cardioprotective effect of ischemic PreC was observed in Lewis rats of mixed age ranging between 10 and 20 weeks (Lieder et al., 2019). Also, in anesthetized Göttinger minipigs, there was no difference in IS, area of coronary MVO, and protection by ischemic PreC between young adult female, castrated male, and male pigs (Kleinbongard, Lieder, et al., 2022a; Kleinbongard, Lieder, et al., 2022b).

Similar findings have been reported with ischemic PostC. Ischemic PostC improved cardiac function and IS in both female and male rat hearts, but the protection differed depending on sex and the severity of the IRI (Crisostomo et al., 2006; Penna et al., 2009). Again, the increased effectiveness of ischemic PostC in males versus females is likely a result of overall reduced IRI (lower IS, less oxidative stress and apoptosis) in females versus males (Ciocci Pardo et al., 2018).

The protective effect of RIC may or may not be sex dependent. Whereas RIC of either 1 or 2 limbs in Lewis rats conferred similar protection in both male and female hearts subjected to I/R (Lieder et al., 2019), plasma isolated from male and female volunteers and perfused into isolated rat hearts subjected to I/R protected the heart in a sex- and age-dependent manner (Heinen et al., 2018). Male but not female plasma collected after RIC protected the isolated rat heart against IRI compared with the non-preconditioned plasma (Heinen et al., 2018).

1. Hypertension

2. Hyperlipidemia

3. Diabetes

5. Atrial Fibrillation

6. Kidney Failure/Uremia

Kidney failure and uremia are important comorbidities for ischemic heart disease (as reviewed earlier; Ferdinandy et al., 2014). Patients with a chronic kidney disease (CKD) have an increased in-hospital mortality after AMI compared with patients with normal renal function (Gansevoort et al., 2013). Experimentally, hearts from animals with CKD (5/6 nephrectomy) (Guo et al., 2018) or uremia (Dikow et al., 2004) had an increased susceptibility to IRI, even when hypertension was treated pharmacologically (Dikow et al., 2004). The mechanisms behind the increase in irreversible injury following I/R in CKD may be related to mitochondrial uncoupling (Taylor et al., 2015) and/or increased endoplasmic reticulum stress (Guo et al., 2018). Uremic rats also exhibited progressive impairment of LV function following MI (Dikow et al., 2010).

Since sex is an independent nonmodifiable risk factor (see previous discussion), more recently the importance of CKD for myocardial IRI and cardioprotective interventions was compared in males and females. While the severity of CKD was similar in males and females following 5/6 nephrectomy, only CKD males developed more severe LV hypertrophy and increased fibrosis. In both sexes, however, ischemic PreC decreased IS in sham and CKD animals. Interestingly, ischemic PreC increased the phospho-STAT3/STAT3 ratio in sham-operated but not CKD animals in both sexes, while no differences in phospho-AKT/AKT and phospho-ERK/ERK ratios existed (Sárközy et al., 2021). Thus, the underlying signaling events might differ between sham (SAFE-pathway-dependent) and CKD animals (SAFE- and RISK-pathway independent). Surprisingly, the effect of kidney failure on RIC has not been studied yet.

In conclusion, although CKD increases myocardial IRI, ischemic PreC still reduces IS in both female and male hearts; protection in males occurs despite the presence of LV hypertrophy and fibrosis. The underlying signaling events might involve endoplasmic reticulum stress as well as mitochondrial function. Other cardioprotective intervention (PostC or RIC) have not yet been studied in CKD and/or uremic animals. There are no data from humans on CKD and cardioprotective intervention available yet. Therefore, further preclinical studies in long-term experimental uremia models, as well as clinical studies, will be necessary to show if mechanical or pharmacological conditioning can still protect the heart in uremic patients.

1. β-Adrenoceptor Antagonists

β-adrenoceptor antagonists form a heterogeneous group of drugs, widely applied as antihypertensive agents since the 1960s and thus, with diuretics, the oldest group of antihypertensive agents in current use. A systematic review has suggested inferiority of first-line hypertension management with β-blockers (mainly atenolol or propranolol) on mortality compared with renin-angiotensin system inhibitors (Wiysonge et al., 2017), although hemodynamic benefits of β-blockers are, at least in part, associated with suppression of the renin-angiotensin cascade. They are also used in the management of patients with established coronary artery disease (e.g., as anti-anginal agents) (Bertero et al., 2021). Although under review, US and European guidelines have advocated the use of particular antagonists in early management of acute coronary syndrome in hemodynamically stable patients (Giannakopoulos and Noble, 2021).

There is a wealth of preclinical data on IS reduction by β-blockers in animal studies of I/R, although, as noted later, none have been successfully and reproducibly translated to humans. Studies in pigs have shown MRI-based evidence for cardioprotection by metoprolol (Ibanez et al., 2007; Ibanez et al., 2011; García-Ruiz et al., 2016; Heusch and Kleinbongard, 2020; Lobo-Gonzalez et al., 2020), and histology-based evidence for cardioprotection by carvedilol (Bril et al., 1992; Feuerstein and Ruffolo, 1995), atenolol, and the short-acting β-blocker landiolol (Park et al., 2011). Carvedilol also reduced the area of no-reflow in pigs following I/R, assessed by myocardial contrast echocardiography (Zhao et al., 2008). There are mixed data suggesting that β3 adrenergic receptor stimulation may be cardioprotective. The β3 adrenergic receptor agonist BRL37344 reduces IS in mice and pigs (Aragon et al., 2011; García-Prieto et al., 2014), but mirabegron, a β3-agonist approved for human use, was not cardioprotective in pigs (Rossello et al., 2018).

Clinical studies conducted in the 1980s provided evidence that β-receptor blockade could reduce IS when given within 4 to 7 hours of symptom onset (Peter et al., 1978; Yusuf et al., 1983; International Collaborative Study Group, 1984; MIAMI Trial Research Group 1985); however, this was in the “pre-reperfusion era” before the use even of thrombolysis. A 2020 patient-pooled meta-analysis of randomized clinical trials testing early intravenous β-blockers in patients undergoing PPCI for STEMI, which included 4 trials and 1150 patients, found no difference in the main outcome of 1-year death or biomarker-based IS (Hoedemaker et al., 2020). Although initial studies reported reduced IS with intravenous metoprolol administered prior to reperfusion (Ibanez et al., 2013; Pizarro et al., 2014; Podlesnikar et al., 2020), the larger EARLY BAMI study of 683 STEMI patients failed to report a reduction in myocardial IS (Garcia-Ruiz et al., 2016; Roolvink et al., 2016; Fabris et al., 2020).

A role of endogenous catecholamines in eliciting ischemic PreC has long been recognized in several species, with contributions from α1-adrenoceptor being initially characterized in rabbit and rat heart (Tsuchida et al., 1994; Mitchell et al., 1995). Transient α1-adrenoceptor stimulation induces pharmacological PreC (Bankwala et al., 1994), and transient β1- or β2-adrenoceptor stimulation also induces pharmacological PreC in rat isolated heart (Salie et al., 2011), with recruitment of similar mechanisms to ischemic PreC. However, the effects of β-blockade on cardioprotective and conditioning responses have been shown in experimental studies to be complex and inconsistent with some but not all studies suggesting a loss of ischemic PreC and ischemic PostC protection, volatile anesthetic PreC, or PostC or RIC in the presence of β-adrenoceptor antagonists (Ferdinandy et al., 2014). There is no obvious or consistent explanation based on the diverse pharmacodynamic profiles of different β-blockers (e.g., lipophilicity, β1 receptor selectivity/cardioselectivity, α receptor antagonism, duration of action, or intrinsic sympathomimetic activity). However, limited evidence from experimental studies suggests that pharmacological PreC or PostC by volatile anesthetic involve recruitment of β-adrenergic signaling (Lange et al., 2009). In more recent clinical studies of conditioning-induced cardioprotection, concomitant β-blockade may be a substantial confounding factor. Cho et al. (2019) examined the effects of limb RIC in healthy human subjects. Plasma dialysate obtained from RIC-treated subjects reduced IS in isolated rat hearts perfused with human RIC dialysate prior to coronary artery occlusion. However, this transfer of protection, likely by some humoral factor in the RIC plasma was abolished if the subjects received RIC in the presence of carvedilol, a β-adrenoreceptor antagonist with ROS-scavenging properties (Feuerstein and Ruffolo, 1995). In a retrospective analysis of a small single-center RCT assessing RIC in patients undergoing CABG surgery, prior use of β-adrenoreceptor antagonists was not found to correlate with troponin I release, a marker of intraoperative IRI (Kleinbongard et al., 2016).

2. ACE Inhibitors and Angiotensin II Receptor Blocker

Inhibitors of the renin-angiotensin system are first-line antihypertensive treatments but are also widely used in the management of established ischemic heart disease and chronic HF. While transient exposure to angiotensin II is known to induce pharmacological PreC via activation of angiotensin II type 1 receptors, and PKC (Liu et al., 1995), several experimental studies have shown ACE inhibitors and angiotensin II receptor blockers (sartans) to be protective in IRI models and to enhance the protective effects of endogenous cardioprotective interventions (ischemic PreC and Ischemic PostC) (Ferdinandy et al., 2014). The mechanisms underlying this beneficial effect are likely to include the potentiation of the production or reduced catabolism of autocrine/paracrine mediators such as bradykinin, prostacyclin, and NO. In pigs with IRI, candesartan pretreatment reduced IS through activation of the angiotensin II type 2 receptor, bradykinin, and prostaglandins, and icatibant or indomethacin abrogated this protection (Jalowy et al., 1998). Sgarra et al. (Sgarra et al., 2014) described differential effects of pharmacological PostC with losartan and irbesartan in a rat isolated heart model of IRI. Losartan given as intermittent pulses during early reperfusion reduced IS whereas continuous losartan perfusion, or intermittent irbesartan treatment, did not. This protective effect was abolished by icatibant (Hoe140), a bradykinin B2 receptor antagonist.

In SHR rats treated with olmesartan for 4 weeks, blood pressure and LV mass were significantly reduced and IS was markedly attenuated after coronary artery occlusion in vivo (Lu, Bi, Chen, and Wang, 2015). In a subsequent study the same group showed that RIC (3 × 5 minute hind limb ischemia during coronary artery occlusion) was absent in SHR but was restored in animals treated with olmesartan for 4 weeks prior to myocardial ischemia (Lu, Bi, and Chen, 2015).

In a model of rapid pacing-induced PostC in rat isolated heart, the IS limiting effect of PostC was abolished in the presence of irbesartan, an angiotensin II type 1 receptor antagonist, suggesting that activation of the angiotensin II type 1 receptor and signaling via the RISK pathway may be involved in this form of conditioning (Babiker et al., 2016). However, both captopril and chymostatin, which inhibit angiotensin II production by ACE-dependent and ACE-independent pathways, respectively, were protective when administered at reperfusion but did not enhance or abolish the effects of superimposed PostC. Thus, the role of locally produced angiotensin II in mediating IRI and conditioning protection are unclear from this study, and the likelihood is that other peptide mediators are modulated by these drugs.

Acute administration of azilsartan during reperfusion was observed to protect isolated rat hearts against IRI, similarly to ischemic PostC. Whereas the effects of ischemic PostC were abrogated in hypercholesterolemic hearts, azilsartan restored the protective effect, likely through upregulation of eNOS activity (Li et al., 2017).

Clinical studies are limited, but given the widespread guideline-directed use of ACE inhibitors and angiotensin II receptor blocker in the management and prevention of multiple cardiovascular diseases, the drugs are frequently present in patients included in clinical cardioprotection trials. The experimental evidence broadly suggests that these drugs can exert independent cardioprotective effects or augment ischemic PreC and PostC responses and could therefore modify responses in trials of conditioning interventions in a variety of settings. Kleinbongard et al. (Kleinbongard et al., 2016), in further analyzing data from their trial of RIC in CABG patients, concluded that neither ACE inhibitors nor angiotensin II receptor blockers were determinants of the major endpoint of protection (plasma troponin I concentration). However, it seems plausible that further analysis of the use of these pleiotropic drugs as potential confounders in larger trials of conditioning interventions, especially in STEMI patients, is warranted.

More recently, neprilysin inhibitors in particular are gaining recognition as a candidate approach for multitarget cardioprotection, given the spectrum of neprilysin substrates that elicit additive or even synergistic cardioprotective signals, including natriuretic peptides, bradykinin, and apelins, among others (Bellis et al., 2020).

3. L-Type Calcium Channel Blockers

L-type calcium channel blockers are a chemically diverse class of agents used in the management of hypertension, certain arrhythmias, and ischemic heart disease. In the context of cardioprotection, extensive experimental evidence points to the IS-limiting potential of all classes of calcium channel blocker when administered prior to the onset of myocardial ischemia, probably by slowing intracellular calcium overload during ischemia. However, there is no benefit if the drugs are administered immediately prior to or during reperfusion. Thus, there is little rationale for their use as adjuncts to reperfusion in STEMI. However, the question of their potential to interfere with conditioning protocols or to confound interpretation of clinical conditioning interventions remains open. There is a paucity of experimental evidence, but nisoldipine did not interfere with ischemic PreC in pig heart (Wallbridge et al., 1996). However, it is plausible that chronic treatment of patients with calcium channel blocker might confer a reduction in susceptibility to IRI, making it difficult to reveal additive benefits of conditioning interventions. Again, further analysis of data from large trials might be helpful in elucidating ideal protocols and patient populations for targeted cardioprotection.

4. Nitrates (and Nitrate Tolerance)

Organic nitrates have been widely used for many decades as one of the main drugs for coronary artery disease treatment. Glyceryl trinitrate (nitroglycerine) is a potent vasodilator that has been used in clinical practice for over a century (Nunez et al., 2005); however, the main constraint of nitrate chronic therapy is the development of rapid tolerance, mainly vascular tolerance, which leads to the loss of clinical efficacy (Csont and Ferdinandy, 2005; Bibli et al., 2019).

Meta-analysis of many experimental studies suggests that IS was limited compared with controls when nitrates were administered through different routes, during ischemia, and/or reperfusion and in different animal species (reviewed in Bice et al., 2016). For example, application of a nitroglycerine patch (designed to deliver 5 mg/d) reduced myocardial IS when applied to mice prior to reperfusion (Yellon et al., 2018). Similarly, low-dose nitroglycerine reduced IS when administered during ischemia both in normal and in animals exhibiting endothelial dysfunction, mainly through the S-nitrosylation and inhibition of cyclophilin D, a component of the mPTP (Bibli et al., 2019). Very recently, administration of a nitrate-functionalized cardiac patch with site-specific delivery of NO directly into the infarcted myocardium demonstrated in a rat model of MI reduced injury at an early stage and suppressed adverse cardiac remodeling, with these results further confirmed in a more clinically relevant porcine model (Zhu et al., 2021).

Clinical trials have provided no consistent evidence of IS limitation associated with nitrate treatment as an adjunct to reperfusion (Bice et al., 2016). However, nitroglycerine showed cardioprotective effects when administered 24 hours before coronary angioplasty compared with patients who received saline (Heusch, 2001; Leesar et al., 2001). This was supported by a study indicating that intracoronary but not intravenous infusion of nitrites reduced IS in STEMI patients with completely occluded arteries (Jones, Pellaton, et al., 2015) and by a recent study indicating that long-term nitrate treatment is cardioprotective, although the mechanism is not fully elucidated (Hauerslev et al., 2018). Additionally, the acute administration of nitrates does not seem to interfere with RIC in patients undergoing CABG surgery under isoflurane anesthesia (Kleinbongard et al., 2013). Interestingly, inhaled NO was able to reduce IS only in a subgroup of nitroglycerine naive STEMI patients (Janssens et al., 2018). This suggests that these patients might be in a nitroglycerine tolerant state that might impair cardioprotection (i.e., an indirect evidence for the hidden cardiotoxic effect of nitroglycerine) (Ferdinandy et al., 2019).

Thus, although tolerance represents a major limitation of the organic nitrates used in the clinic, acute administration and/or site-specific delivery of NO into the myocardium seems to be cardioprotective and may support the translational potential of the use of nitrates as adjunct to reperfusion therapy for IS limitation.

1. Cyclooxygenase Inhibitors

Aspirin may interfere with protection by some forms of ischemic conditioning in experimental studies (Birnbaum et al., 2021). Indomethacin pretreatment abrogates protection from IRI by ACE inhibition and angiotensin II type 1 receptor blockade (Ehring et al., 1994; Jalowy et al., 1998). Thus, cyclooxygenase inhibition can, in principle, interfere with cardioprotection. The cardiac safety of cyclooxygenase 2 inhibitors is still an area of investigation and controversy despite the withdrawal from the market due to increased occurrence of MI (Dubreuil et al., 2018; Abdellatif et al., 2021). Indeed, cyclooxygenase 2 inhibition seems to be cardioprotective in preclinical models; however, its potential hidden cardiotoxic effect has been recently shown in preclinical models of I/R and MI that may hinder their development and indicates safety problems of some cyclooxygenase inhibitors (Brenner et al., 2020).

2. Morphine and Anesthetics

Certain anesthetics are inhibitors of mitochondrial activity (Hanley and Loiselle, 1998; Chen et al., 2018), and some anesthetics are also strong ROS scavengers (Murphy et al., 1992). Cardiac I/R and protection from it are critically dependent on the presence and type of anesthesia (Zaugg et al., 2014). Anesthesia is likely one of the critical factors hampering successful translation in large clinical trials, considering the often large discrepancies between anesthetic regimen in preclinical models (often pentobarbital, ketamine-xylazine) versus the clinical arena (fentanyl, morphine, volatile anesthetics, benzodiazepines, propofol).

The abrogation by propofol of protection by RIC in patients undergoing CABG surgery was first reported by Kottenberg et al. (Kottenberg et al., 2012). The use of propofol may have interfered with cardioprotection by RIC in the larger phase III trials in CABG patients. Also, RIC was beneficial in rats administered pentobarbital and sevoflurane but not in rats receiving propofol (Behmenburg et al., 2018). Subsequently, the effect of anesthesia using sevoflurane or propofol was studied by perfusing plasma dialysates from patients undergoing CABG into isolated rat hearts with I/R. Here, RIC was only protective when no anesthesia was used, whereas both sevoflurane and propofol abolished RIC protection (Cho et al., 2019). Propofol abrogates not only ischemic conditioning but also various pharmacological types of conditioning (Zuurbier et al., 2014; Lucchinetti et al., 2018; Xiao et al., 2021). It thus seems that analgesic and anesthetic agents used in the clinic (opioids, volatile anesthetics, propofol) can interfere with cardioprotective interventions, mandating the incorporation of these agents in preclinical models to improve translation. Such models were recently developed in rats, 1 showing protection by a caspase inhibitor, but not RIC, on a background of heparin, an opioid agonist, and a platelet-inhibitor (He et al., 2020), and another 1 showing protection by a NAD precursor, but not fingolimod, melatonin, or empagliflozin, on a background of fentanyl, benzodiazepine, and a platelet inhibitor (Xiao et al., 2021).

The opioid receptor system has been shown to represent an important candidate for clinical cardioprotection since it beneficially impacts all major determinants of IRI outcome (infarction/apoptosis, arrhythmogenesis, inflammation). A small number of clinical trials have provided evidence of cardiac benefit from morphine or remifentanil in CABG or coronary angioplasty patients (Headrick et al., 2015). Morphine (Stiermaier et al., 2021) and volatile anesthetics can reduce IS following PPCI or CABG procedures (Zaugg et al., 2014) and thus limit the potential for further protective interventions. However, diabetes mellitus mitigates cardioprotective effects of remifentanil PreC in I/R rat heart in association with antiapoptotic pathways of survival (Kim et al., 2010), and hypertrophy (Weil et al., 2006) may influence opioid receptor responses.

1. Antiplatelets

For use of aspirin see Section Vb1.

Clopidrogel, the first P2Y₁₂ inhibitor developed and examined, is now slowly being replaced by the faster-acting prasugrel or ticagrelor. Experimental studies have established that P2Y₁₂ receptor blockers reduce IS during cardiac IRI (Yang, Liu, et al., 2013a, 2013b). Cardioprotective signaling by cangrelor and ticagrelor overlap with the signaling pathways used by conditioning strategies such as ischemic PostC (Yang, Liu, et al., 2013a) and RIC (Cohen and Downey, 2017; He et al., 2020; Hjortbak et al., 2021). However, ischemic PreC remained effective in the presence of the P2Y₁₂ antagonist ticagrelor (Hjortbak et al., 2021). Pharmacological conditioning has been found to remain effective in the presence of P2Y₁₂ agents for the sodium/hydrogen-exchanger inhibitor cariporide (Yang, Cui, et al., 2013), caspase inhibitors (Audia et al., 2018; He et al., 2020), and the NAD⁺ precursor nicotinamide riboside (Xiao et al., 2021) but not for NLRP3 inhibitors (Penna et al., 2020).

In patients undergoing PPCI for STEMI, platelet reactivity in response to dual antiplatelet therapy is a key predictor of the extent of both myocardial and microvascular damage (Massalha et al., 2022). Whether P2Y₁₂ inhibitors indeed possess direct cardioprotective actions against AMI has not been demonstrated in large prospective clinical trials. However, there is some circumstantial evidence from small clinical or large retrospective studies. A recent study showed reduced IS with ticagrelor versus clopidogrel as indirect evidence that P2Y₁₂ agents can reduce IS independent of their platelet inhibitory action (Kim et al., 2017), a finding supported by recent retrospective studies (Hjortbak et al., 2021; Sabbah et al., 2020).

2. Anticoagulants

For decades now, the anticoagulant heparin has been part of the standard of perioperative care during PPCI and cardiac surgical procedures. Experimental studies have established that heparin reduces IS during cardiac IRI (Black et al., 1995; Huang et al., 2017; He et al., 2020). Since 2005, platelet receptor antagonists were added to this standard of care for acute MI patients treated by PCI, and both heparin and P2Y₁₂ antagonists possess cardioprotective actions (Roubille et al., 2012; Kleinbongard et al., 2021). Given their protective potential, both heparin and P2Y₁₂ antagonists should therefore become part of preclinical models testing for cardioprotection, where these agents have been mostly missing (Cohen and Downey, 2017; He et al., 2020).

While oral anticoagulation is obligatory for thromboembolism prophylaxis in AF and for prevention of deep vein thrombosis and pulmonary embolism, current guidelines also recommend oral anticoagulation with the coumarin-derivative warfarin to prevent LV thrombosis in the 3 to 6 months after AMI (Levine et al., 2016; Ibanez et al., 2018; Valgimigli et al., 2018). Increasingly, the direct oral anticoagulants (DOAC) are used off-label in this context (Iqbal et al., 2020). Currently available DOAC include the thrombin inhibitor dabigatran; inhibitors of activated coagulation factor X (FXa) are represented by rivaroxaban, apixaban, edoxaban, and betrixaban. A recently published observational study in patients with AMI who received either warfarin or 1 of rivaroxaban, apixaban, or edoxaban (Jones et al., 2021) showed earlier and greater LV thrombus resolution with the FXa inhibitors compared with warfarin, together with lower bleeding rates but comparable systemic thromboembolic events.

The influence of the activated coagulation system on cardiovascular biology and pathophysiology goes beyond thrombosis. The cardioprotection afforded by antithrombin in murine IRI is independent of its hemostatic effect (Wang et al., 2013). Similarly, the beneficial impact of RIC applied pre- and post-off-pump CABG also appears to be unrelated to perioperative improvement in platelet reactivity to adenosine diphosphate or coagulability status (Kim et al., 2020), pointing to existence of coagulation-independent actions. Thrombin and FXa directly promote endothelial dysfunction, oxidative stress, immune cell activation, cell growth and differentiation, as well as inflammation (Fender et al., 2017; Fender et al., 2020; Ten Cate et al., 2021) through cellular protease-activated receptors and thus need to be considered in the context of IRI and cardioprotection.

Regarding the candidate role of DOAC as cardioprotective agents, no benefit could be noted with dabigatran in a rabbit model of no-reflow after myocardial IRI (Hale and Kloner, 2015). In rodent models of cardiac IRI, application of the FXa inhibitor 1 hour prior to occlusion decreased IS by 21% (Guillou et al., 2020). A PostC benefit of rivaroxaban has also been observed in mice subjected to permanent ligation. Here, the cardioprotective window appeared to persist for 24 hours after ischemia; delaying treatment until day 3 after IRI abolished the observed benefits (Bode et al., 2018). If commenced immediately after surgery or up to 24 hours thereafter, rivaroxaban applied in chow prevents intravascular thrombosis, improved cardiac systolic function, and decreased IS and inflammatory markers to variable extents. Within the infarct zone, levels of TNFα, tissue growth factor β, and both protease-activated receptors 1 and 2 are reportedly suppressed, while noninfarcted areas exhibit lower levels of atrial natriuretic peptide and NH₂-terminal pro-B-type natriuretic peptide, activated ERK, and cardiomyocyte hypertrophy (Bode et al., 2018; Nakanishi et al., 2020). Rivaroxaban also improved cardiac function and survival and suppressed transcription of IL-6 and collagens in a mouse model of secondary IRI prevention. Here, rivaroxaban treatment was commenced after IRI evoked by temporary occlusion and continued over 14 days; a second ischemic event was triggered on day 7 with the application of tissue factor (Goto et al., 2016). Mechanistically, the cardioprotective effects afforded by rivaroxaban could be largely attributed to a blunted signaling though FXa/protease-activated receptors 2 (Bode et al., 2018), and given that apixaban could also blunt post-IRI fibrosis in mice (Shi et al., 2018), it is likely that cardioprotection is a class effect of the FXa blockers rather than a phenomenon specific for rivaroxaban.

A recent study in mice with cardiac IRI elegantly demonstrates the apparent superiority of FXa inhibition versus thrombin inhibition (Gadi et al., 2021). Inhibition of thrombin or FXa was commenced 1 week prior to IRI and reinitiated 2 hours post-surgery and continued for 24 hours or 28 days to examine acute and chronic effects. The dose was adjusted to achieve equivalent anticoagulation. IS was markedly and comparably reduced by both interventions. Remarkable differences between the 2 pharmacological strategies were noted in terms of IRI-triggered inflammation. RNA sequencing analysis showed that approximately 75% of genes aberrantly up- or downregulated by IRI were restored by FXa blockade, while thrombin inhibition reversed only one-third of IRI-regulated genes. The most prominently affected pathways included those related to the NLRP3 inflammasome and fibro-inflammatory stress, with thrombin inhibition tending to increase, while FXa blockade tending to decrease, expression of IL-1β, IL-6, TNFα, and inflammasome components. The authors proposed that the difference might be related to the unique ability of thrombin to induce the activated protein C pathway, which has previously been shown to protect against myocardial IRI (Wildhagen et al., 2014).

Rivaroxaban exerts a direct cytoprotective action in cardiomyocytes subjected to hypoxia/reoxygenation (Guillou et al., 2020). Possible contributing mechanisms include the preservation of mitochondrial function and metabolism through regulation of key mitophagy proteins including mitochondrial dynamin-related protein 1 and Parkin (López-Farré et al., 2014; Zamorano-Leon et al., 2020). Classic cardioprotective cascades such as the RISK and SAFE pathways do not appear to be modulated by FXa blockers; instead, positive regulation of the Wingless and Int-1β-induced PI3K/AKT-activated protein C system may contribute to the cardioprotective benefits of these agents, as was recently reported for edoxaban (Shan et al., 2019). Additional cardioprotection may arise through upregulation of vascular endothelial NOS (Pham et al., 2019) and suppression of angiotensin II-stimulated inflammatory and fibrotic responses in cardiac fibroblasts. Rivaroxaban attenuated angiotensin II-stimulated signaling through nuclear factor kB and mitogen-activated protein kinase/activator protein 1 pathways in mouse cardiac fibroblasts lowered expression of inflammatory proteins and concentration-dependently blunted fibroblast migration and proliferation (Hashikata et al., 2015). Potentially, FXa blockade could help to limit IRI-driven fibrosis and remodeling. In line with this concept, apixaban attenuated fibrosis in mouse hearts subjected to permanent ligation (Shi et al., 2018). The underlying mechanism was shown to depend on inhibition of thrombin formation and suppressed signaling through protease-activated receptors 1/Gq/PKC in cardiac fibroblasts. Data related to the effects of the thrombin inhibitor dabigatran are more limited. At the cellular level, dabigatran counterbalances thrombin-stimulated oxidative stress, inflammatory cytokine expression, and sirtuins-driven autophagy in cardiomyocytes in vitro (Wang, Xu, et al., 2021). More recently, an elegant in silico docking study revealed that dabigatran may be a novel candidate inhibitor of c-jun-N terminal kinase (Zulfiqar et al., 2020).

In patients, data on DOAC and myocardial IRI and protection from it are sparse. The main patient population studied are those with AF. However, in anticoagulated patients with AF, the incidence of AMI is relatively low (Connolly et al., 2009). Thus, one could speculate that oral anticoagulation use goes in hand with a generally low risk of AMI. Early studies examining DOAC added to standard antiplatelet therapy include the Anti-Xa Therapy to Lower Cardiovascular Events in Addition to Standard Therapy in Subjects with Acute Coronary Syndrome 2–Thrombolysis in Myocardial Infarction 51 (ATLAS ACS 2–TIMI 51) trial. Addition of rivaroxaban (2.5 and 5 mg) reported an approximately 9% reduction in subsequent MI in patients with AMI, albeit at the cost of increased major bleeding, but not fatal cerebral bleeds (Mega et al., 2012). The subsequent Cardiovascular Outcomes for People Using Anticoagulation Strategies (COMPASS) trial (Eikelboom et al., 2017), which added very low-dose rivaroxaban to aspirin in patients with chronic coronary and peripheral artery disease, reported a favorable outcome in terms of thrombotic event reduction but no reduction of MI. Standard-dose rivaroxaban alone showed no benefit regarding primary cardiovascular outcomes but increased bleeding rates compared with aspirin alone. The subsequent A Study to Assess the Effectiveness and Safety of Rivaroxaban in Reducing the Risk of Death, Myocardial Infarction, or Stroke in Participants with Heart Failure and Coronary Artery Disease Following an Episode of Decompensated Heart Failure (COMMANDER-HF) trial corroborated that addition of low-dose rivaroxaban on top of standard antiplatelet therapy lowers the rate of ischemic stroke but does not impact beneficially on MACE endpoints including MI (Zannad et al., 2018). Similarly, the Apixaban for Prevention of Acute Ischemic Events 2 (APPRAISE-2) trial, which examined apixaban added to standard antiplatelet therapy in patients with recent AMI and at least 2 additional ischemic risk factors, was terminated early because of increased major bleeding with no evident reduction in cardiovascular events including MI (Alexander et al., 2011).

Thus, appropriate use of triple therapy remains challenging. FXa blockers on top of standard antiplatelet therapy consistently increase bleeding risk, with no counterbenefit in terms of reduced MI (Khan et al., 2018). At least this is the case if substantial coronary artery disease and prior AMI are already evident. It remains to be determined whether DOAC can modulate either the propensity for a first myocardial event to occur, by influencing the underlying coronary artery disease as indicated by some experimental studies (Hara et al., 2015; van Gorp et al., 2021) or, alternatively, whether DOAC impact on the extent of injury and expansion in the aftermath of the infarct (see previous discussion). Thrombin and FXa are also linked to processes pertaining to post-IRI inflammation and remodeling (Raivio et al., 2009; Fender et al., 2019). The thrombin burst that occurs in the context of IRI despite heparinization (Raivio et al., 2006; Raivio et al., 2009) may therefore contribute adversely and in a long-lasting manner to IRI and its sequelae.

1. Sulfonylureas

Preclinical and clinical studies have shown that some sulfonylureas inhibit myocardial protection by conditioning strategies since these drugs have high affinity to myocardial SUR2A/Kir6.2 and smooth muscle SUR2B/Kir6.2 receptors and inhibit the activation of K_ATP channels (Gribble and Ashcroft, 1999). The cardiovascular effect of sulfonylureas in humans is inhibition of the cardioprotective effects of RIC (Loukogeorgakis et al., 2007; Kottenberg et al., 2014). At present, there is no evidence that these effects have clinical consequences. Cross-reactivity between pancreatic and cardiac K_ATP channels varies with the individual sulfonylureas, and in general the later generation sulphonylureas are more specific for the pancreas and therefore bind less to the cardiac K_ATP channels (Gribble and Ashcroft, 1999). The interaction of glimepiride or gliclazide with SUR2 is less than that of glibenclamide, and therefore they do not seem to blunt the cardioprotective effects of ischemic PreC, diazoxide, and nicorandil in isolated rat hearts with I/R (Mocanu et al., 2001; Maddock et al., 2004).

2. Metformin

Pre- and/or post-treatment with metformin protects the heart against IRI and reduces myocardial IS (reviewed in Ye et al., 2011). Metformin PostC reduced IS, attenuated apoptosis, and inhibited myocardial fibrosis, which was largely dependent on the suppression of NLRP3 inflammasome activation in rat hearts and cardiomyocytes (Zhang et al., 2020). Although meta-analyses have supported the cardiovascular safety of metformin in patients with coronary artery disease and chronic HF independent of its glucose-lowering effects (Varjabedian et al., 2018), no acute protection by metformin during CABG was observed (El Messaoudi et al., 2015), questioning the translatability of metformin for protection against acute I/R settings in the clinical situation. Indeed, in contrast to rodent hearts, PostC with high-dose metformin when administered before reperfusion did not reduce myocardial IS or improve LV function in swine (Techiryan et al., 2018), highlighting the importance of rigorously testing therapies in large animal models to facilitate clinical translation of novel cardioprotective therapies.

3. Thiazolidinediones

The effect of thiazolidinediones on IRI is controversial (Riess et al., 2020). Preclinical studies in small animals have shown that these drugs administered either as PreC or PostC agents protect against IRI and limit myocardial IS. Pioglitazone in nondiabetic and diabetic rats reduced IS (Khodeer et al., 2016) and did so, too, in isolated rat hearts when administered prior to I/R (Wynne et al., 2005). Rosiglitazone, however, was associated with enhanced cardiac injury in a similar model (Riess et al., 2020). Rosiglitazone is associated with increased adverse cardiovascular events in diabetic patients (Lincoff et al., 2007).

4. Glucagon-Like Peptide-1 Receptor Agonists

Glucagon-like peptide 1 (GLP-1) receptor agonists exert diverse actions on distinct target tissues, which lead to reduction of blood glucose level and body weight, and they are approved drugs for consideration as monotherapy or in combination with other oral antihyperglycemics (Peng et al., 2016). GLP-1 receptor agonists administered either as PreC or PostC agents limit myocardial IS in small and large animal models (Bose et al., 2005; Sonne et al., 2008; Timmers et al., 2009). A recent meta-analysis indicated that GLP-1 receptor agonists reduced the incidence of MACE and MI in type 2 diabetes patients and attenuated cardiovascular mortality (Sattar et al., 2021 #3585). In rats with I/R, GLP-1 functions as a humoral factor of RIC, involving activation of vagal nerves and M3-muscarinic receptors (Basalay et al., 2016). In the clinical setting, an intravenous infusion of exenatide initiated prior to PPCI reduced myocardial IS in STEMI patients, especially in those patients presenting with short ischemic times from symptom onset (<132 minutes) (Lønborg, Kelbæk, Vejlstrup, Bøtker, Kim, Holmvang, Jørgensen, Helqvist, Saunamäki, Terkelsen, et al., 2012; Lønborg, Vejlstrup, et al., 2012; Woo et al., 2013). Another GLP-1 analog, liraglutide, when administered prior to PPCI and continued for 7 days, improved LV systolic function (Chen et al., 2015). Exenatide activated cardioprotective pathways different from those of RIC and possessed additive effects with RIC on IS reduction in a pig model of I/R (Alburquerque-Béjar et al., 2015). However, in a 2 × 2 factorial follow-up study, neither RIC nor exenatide, nor its combination, reduced IS in STEMI patients when administered as an adjunct to PPCI (García Del Blanco et al., 2021), indicating that, although GLP-1 agonists were promising in preclinical models of MI, they failed in RCTs in humans.

5. Dipeptidyl Peptidase-IV Inhibitors

GLP-1 is enzymatically cleaved and inactivated by dipeptidyl peptidase IV (DPP-IV), leading to the development of DPP-IV inhibitors as potential therapeutics. In rodents and pigs, DPP-IV inhibitors (especially sitagliptin and vildagliptin) limited IS when administered either before or after ischemia. Vildagliptin restored the cardioprotective effects of ischemic PostC on diabetic hearts but did not reduce IS per se (Bayrami et al., 2018). A prospective clinical study assessed the effect of repaglinide and vildagliptin on ischemic PreC in patients with type 2 diabetes and coronary artery disease. Although repaglinide eliminated ischemic PreC, probably due to its effect on the K_ATP channel, vildagliptin did not cause any impairment of ischemic PreC, suggesting a good alternative treatment in these patients (Rahmi et al., 2013). Clinical trials have shown that hospitalization for HF was increased in saxagliptin-treated patients (Scirica et al., 2013), whereas major adverse cardiovascular events were not increased with alogliptin and sitagliptin as compared with placebo (White et al., 2013; Green et al., 2015). A recent Cochrane analysis did not show any beneficial effect of DPP-IV inhibitors on MACE, MI, or cardiovascular mortality (Kanie et al., 2021). In summary, further preclinical studies especially in large animals with diabetes and clinical trials will be warranted to confirm the myocardial protection afforded by DPP-IV inhibitors.

6. Sodium Glucose Cotransporter 2 Inhibitors

Sodium glucose cotransporter 2 (SGLT2) inhibitors are the newest class of antidiabetic drugs. They markedly reduce MACE in large clinical trials in HF patients (Andreadou, Bell, et al., 2020). SGLT2 inhibitors exert cardioprotective effects in animal models of AMI through reduction of IS (Andreadou, Efentakis, et al., 2017; Tanajak et al., 2018; Lim et al., 2019; Sayour et al., 2019; Uthman et al., 2019; Lahnwong et al., 2020; Nikolaou et al., 2021; Seefeldt et al., 2021) and a subsequent attenuation of HF development (Habibi et al., 2017; Yurista et al., 2019; Connelly et al., 2020). Multiple, parallel protective mechanisms of SGLT2 inhibitors have been proposed, such as the attenuation of cardiac and endothelial inflammation or an inhibition of oxidative stress improving cardiac structure and function (Lee et al., 2017; Ye et al., 2017; Andreadou, Bell, et al., 2020). The effect of SGLT2 inhibitors on conditioning mechanisms has not yet been evaluated. Clinical trials examining a potential SGLT2 inhibitory effect on cardiac IRI during cardiac surgery or PPCI procedures are currently missing.