Molecular details, forensic aspects, and treatment possibilities of heart tissue damage from blood flow loss and restoration

AMI is a major global health issue and still represents the single most prevalent cause of death worldwide, despite continuous advances in AMI prevention and treatment [1]. Myocardial infarction (MI) is also a widespread occurrence in forensic medicine, with concerns related to both the differential diagnosis with other cardiovascular conditions and the correct dating of the disease [2,3]. AMI is defined as cardiomyocyte necrosis in a clinical setting consistent with acute myocardial ischemia, as identifiable through a series of clinical (prolonged chest pain), biochemical (elevated cardiac troponin levels), and instrumental (ECG and/or regional wall motion abnormalities) parameters [4]. Ischemia is mainly caused by acute coronary atherothrombotic occlusion or mural thrombus (MI type 1), but it could also be due to other mechanisms reducing coronary flow and/or oxygen supply (MI type 2–5), as recently updated in the fourth universal definition of myocardial infarction, published in 2018 by the Joint Task Force of the European Society of Cardiology, the American College of Cardiology Foundation, the American Heart Association, and the World Heart Federation [5]. This condition of tissue hypoxia triggers specific metabolic (arrest of aerobic metabolism and onset of anaerobic glycolysis with progressive accumulation of catabolites), inflammatory, and oxidant changes, leading to evolving cardiac cell damage—from reversible injury to irreversible death—followed by cardiac repair [6].

The early restoration of the coronary blood flow (i.e., reperfusion therapy) brought by thrombolytic therapy or primary percutaneous coronary intervention (PPCI) is key to save myocardial tissue from ischemic death in patients with STEMI [7], because coronary artery recanalization provides both the oxygen restoration and the availability of inflammatory cells, which can initiate cardiac tissue healing. Nonetheless, it has been known for more than 60 years that reperfusion itself can paradoxically contribute to cardiomyocyte death (through apoptosis, necrosis, necroptosis, and pyroptosis), energy metabolism disorders (mainly due to mitochondrial alterations, linked to oxidative stress and calcium overload), additional inflammatory response, and endothelial cells' death with coronary microvascular dysfunction. These events occur within the first few minutes of reperfusion, leading to an increase in the final IS, which involves residual viable myocytes and causes additional damage, referred to as MIRI [8]. A comprehensive understanding of the pathogenesis and timing of MIRI could lead to the development of new, effective therapeutic strategies to limit the progression to heart failure [9]. In a very recent expert consensus document, published last year from the Canadian Cardiovascular Society, a new classification of atherothrombotic MI with reperfusion therapy is proposed, specifically based on progressive tissue changes in the myocardium due to ischemia/reperfusion injury (IRI), aiming for the development of new tissue-injury-specific therapies for MI [10]. Starting with the CANTOS Trial in 2017, which was the first clinical study able to demonstrate the successful secondary prevention of cardiovascular events by targeting the immune system, especially pro-inflammatory cytokines [11], scientific research intensified the study of the pathobiology of MIRI. Research efforts focused on the role of cardiac oxidative stress and immuno-inflammatory mechanisms, aiming to identify more and more suitable pathways to be targeted by novel therapies, either physical or pharmacological ones, with the final aim being to combine benefits derived from myocardial reperfusion with those from these cardioprotective strategies, with a potentially synergistic action [12]. More importantly, for the forensic pathologist, understanding MIRI may also be key in recognizing that unfavorable outcomes and even death may occur despite successful and early reperfusion, with clear implications for malpractice claims. Finally, the cellular and plasma factors involved in MIRI might be used as immunohistochemical markers of ischemic myocardial damage, therefore representing an additional diagnostic tool for pathologists, allowing them to confirm MIRI as a potential cause of death.

In this article, pathophysiological mechanisms involved in MIRI after AMI are reviewed at the molecular and cellular level, aiming to provide a valid reference for both clinicians and forensic pathologists. In addition, the authors will firstly present a collection of myocardial explicative histological images collected from autopsy cases of AMI death after successful reperfusion, but also potentially MIRI cases after cardiac arrest (and other ischemia situations, without acute infarction), analyzing cardiac expression of inflammatory markers and morphological changes, and secondly a brief focus on the results of recent clinical trials with immunotherapeutic strategies targeting MIRI. The ultimate goal is to provide an up-to-date state-of-the-art overview of the knowledge on the mechanisms underlying MIRI, potential therapeutic interventions, and the forensic implications of this emerging and complex phenomenon.

When oxygen supply is restored with reperfusion after a period of myocardial ischemia, there is an increased production of free radicals from oxygen (reactive oxygen species, ROS) which may also react with nitrogen, producing reactive nitrogen species (RNS). ROS and RNS exhaust ROS scavengers and other antioxidants molecules, resulting in the progressive accumulation of oxidants, which may lead to cell damage and release of inflammatory factors [13,14,15].

Oxygen reduction yields superoxide (O₂⁻) anion, which can determine the hydroxyl (OH) radical formation through two different pathways: one involving spontaneous combination with hydrogen peroxide (H₂O₂), while the other consisting on the Fenton reaction. The RNS group includes nitric oxide (NO), nitrogen dioxide (NO₂), and peroxynitrite (ONOO^–) [14]. During MIRI, sources of ROS include NADPH oxidase, xanthine oxidase, nitric oxide synthase (NOS), and mitochondria [14,16,17]. NADPH oxidase is an enzyme that generates superoxide and consists of a membrane-bound catalytic subunit (NOX) along with several cytosolic regulatory subunits. NOX2 serves as the catalytic subunit of phagocyte NADPH oxidase. Upon activation, the cytosolic components migrate to the transmembrane catalytic protein gp91phox, forming a functional NADPH oxidase complex. Researchers have shown that NADPH oxidase is expressed in cardiomyocytes. NADPH oxidase is a critical source of ROS, which subsequently influences the redox state of the myocardium. However, the most relevant process for ROS generation consists in the production of O₂⁻ anion mediated by the mitochondrial succinate, which activates the REarranged during Transfection (RET) at the level of Complex I [18,19]. O₂⁻ anion is released into the matrix by Complex I, and in the intermembrane space by Complex III [18].

Several cellular and molecular pathways are activated by ROS, leading to cell death, both through apoptosis and pyroptosis. Shen et al. demonstrated the direct linkage between these free radicals and the activation of the nucleotide-binding oligomerization (NOD)-like receptor protein 3 (NLRP3) inflammasome pathway (enhancing both inflammatory response and pyroptosis), after ROS generation by uric acid [20].

Moreover, ROS are the main species responsible for mitochondria dysfunction, and they could also lead to intracellular calcium overload. Indeed, during MIRI, ROS are an important trigger of the opening of the mitochondrial permeability transition pore (mPTP) [16,21], which promotes the escape of Cytocrome C (CytC) in cytosol [22]. As demonstrated by Schriewer et al. [23], mPTP's opening requires ROS presence and can also in turn support ROS production; they showed that another molecule involved in the process of mitochondria-mediated necrosis during ischemia/reperfusion (I/R) was poly (ADP-ribose) polymerase-1 (PARP). PARP can participate in oxidative stress amplification and mediate cell death due to oxidative damage after I/R. During I/R, PARP could participate in mPTP opening through poly ADP-ribosylation of the mitochondrial target, and in turn mPTP opening could enhance PARP activity. The consequentiality of the events is not yet well understood, and these authors proposed PARP inhibition as a possible actionable target to alleviate reperfusion injury even when mPTPs are already opened [23].

One of the effects of the opening of mPTP channels is the release of Ca²⁺ ions. Calcium overload is a typical event that occurs because of reperfusion and contributes to cardiac cells damage; the increased cytosolic concentration of Ca²⁺ ions may lead to apoptosis [24]. Initially during the ischemic phase, the myocardial cells decrease the aerobic glycolysis and Krebs cycle, resulting in a switch to anaerobic metabolism, especially anaerobic glycolysis, which lead to the accumulation of lactate and H⁺ ions, causing a decrease in intracellular pH [25,26]. In this setting of intracellular acidosis, the activation of the Na⁺/H⁺ exchanger (NHE), which extrudes one H⁺ in exchange for one Na⁺ entering the myocyte, results in the cytosolic accumulation of Na⁺. Furthermore, during ischemia, the production of ATP is curtailed, determining the inhibition of both Na⁺/K⁺-ATPase [27], and Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA Ca²⁺-ATPase) [28], which in turn leads to an increase in intracellular Na⁺ (Figure 1).

During reperfusion, the restoration of oxygen levels promotes normalization of extracellular pH and reactivation of mitochondrial respiration, so the NHE continues to support the H⁺ efflux from the cells with a persistent increase in intracellular Na⁺, which in turn activates the Na⁺/Ca²⁺ exchanger (NCX) reverse mode, favoring intracellular Ca²⁺ accumulation [16,28,29,30,31]. Another mechanism that causes an increase in circulating free Ca²⁺ is related to the sarcoplasmic reticulum (SR). The efficient transfer of ATP to sarcoplasmic reticulum (SR) causes Ca²⁺ oscillations, due to accumulation and release of Ca²⁺ by SR. The released Ca²⁺ can be intercepted by adjacent SRs, causing the propagation of calcium waves, and hypercontracture, which can lead to cell death [15,23,28,32].

The increased production of ROS, due to the renewed oxygen supply to tissues, is thus one of the main factors that interferes with proper cellular homeostasis. These highly reactive molecules interact at different levels and with different pathways, causing cellular dysfunction. A clear example of this is the mitochondria, whose damage is, in turn, capable of sustaining ROS production.

In the following sections, histomorphological images are showed illustrating the phenotypic outcomes resulting from pathway alterations related to oxidative stress and calcium overload in MIRI. In particular, Figure 2 depicts a case based on personal observation of a 61-year-old man with chronic hypertension who presented in an emergency room with squeezing retrosternal chest pain and dyspnea. Elevated cardiac enzymes and electrocardiography results suggested acute STEMI since the hospital performed a quick PCI. A control ECG showed nearly complete resolution of the ST-segment elevation. Six days after the treatment, the patient suffered a sudden cardiac arrest due to VF and died.

Inflammasomes are multiprotein complexes involved in the inflammatory response [33], interacting with damage- or pathogen-associated molecular patterns (respectively, DAMPs or PAMPs) [34]. Inflammasomes are a large and heterogenous group of molecules. Regarding their role in MI, the best characterized inflammasome is the nucleotide-binding oligomerization domain (NOD), leuchine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3 (NLRP3), which also plays a role in the inflammation cascade during MIRI [33,34]. The NLRP3 inflammasome is composed of the NLRP3 protein, apoptosis-associated speck-like protein (ASC), and pro-caspase-1. The NLRP3 protein can be in turn divided into three domains: NATCH, a central nucleotide domain mediating oligomerization, a C-terminal LRR domain, and a PYD [34]. A PYD is also present in ASC, combined with the caspase recruitment domain (CARD) [34,35,36].

The expression of the NLRP3 inflammasome can be regulated through Nuclear Factor (NF)-kβ, which is activated by Toll-like receptor 4 (TLR4) once it recognizes tissue damage or a signal of infection (DAMPs or PAMPs). After its activation, NF- kβ in turn also promotes the expression of pro-IL-1β and pro-IL-18, which are then activated by caspase-1, a product of NLRP3 action on pro-caspase-1. IL-1β and IL-18 enhance the inflammatory response by increasing cytokines' production, immune response, and extracellular matrix's turnover [37]. Consistently, in an in vitro model of MIRI, Huang et al. demonstrated the overexpression of the NLRP3 inflammasome by cardiac fibroblasts, with a consequent increase in cellular levels of IL-1β, IL-18, TNF-α, NLRP3, caspase-1, and ASC after hypoxia/reoxygenation (H/R) [38].

Knowledge about NLRP3 has recently widened, with the identification of novel and increasingly complex homeostatic mechanisms interacting with the inflammasome.

Given this increasingly recognized complexity, it comes as no surprise that preclinical studies on the modulation of the inflammasome pathway yielded contrasting results on the attenuation of MIRI. In 2011, Kawaguchi et al. observed that in a mouse model of MIRI, knockout (KO) mice for ASC and caspase-1 showed a less extensive infarcted area and a reduction in cytokines and interleukins' expression compared with the wild-type (WT) mice. Furthermore, the authors provided evidence that ASC and caspase-1 were expressed in the myocardial infarct site [39]. Another study confirmed that the NLRP3 inflammasome is upregulated in the myocardium after MI, and that its deficiency improves myocardial function. Sandanger et al. demonstrated that, in rats and mice models of MIRI, myocardial fibroblasts express TLRs 1–4 and 9 [40]. In support of the involvement of NLRP3 in the inflammatory response following I/R, their study showed that KO mice for NLRP3 maintain contractile function and coronary flow in the heart and have a smaller infarct zone than WT mice, in an ex vivo experiment [40]. However, the same group later surprisingly observed that KO mice for NLRP3 and ASC had a larger infarct size after I/R injury, leading the authors to hypothesize that the NLRP3 inflammasome might contribute to cardioprotection against MIRI, by interacting with the protective reperfusion injury salvage kinase (RISK) pathway [41], which is a combination of the pro-survival kinase signaling cascades phosphatidylinositol-3-OH kinase (PI3K)-Akt and extracellular regulated kinase (ERK)-1/2 [42].

The activation of TLRs by DAMPs and/or PAMPs after myocardial ischemia could enhance the inflammatory response, thus initiating and contributing to MIRI, by activating several molecular pathways other than NLRP3-mediated pathways. Chong et al. detailed the role of TLR4 in MIRI, describing the transmembrane recruitment of the Myeloid differentiation protein (MyD88), and all the distal intracellular signaling leading to inflammation and apoptosis [43]. Many other studies confirmed the central implication of TLR4 signaling in MIRI, focusing on the activation of NF-kβ, and the release of inflammatory factors such as TNF-a, IL-6, and IL-1 [43,44,45,46] (Figure 3).

The involvement of MyD88 in MIRI was demonstrated by several studies in which its inhibition was associated with a decrease in reperfusion damage. In 2020, Miao et al. reported that in a murine model of MIRI, inhibition of MyD88 with TJ-M2010-5 (an experimental drug) resulted in reduced inflammatory response and infarct area [47]; Yang and colleagues also demonstrated that MyD88 inhibition can reduce severe MIRI after heart transplantation in in vivo mice models [48]. MyD88 may interact with other TLRs, in particular TLR9, whose expression is increased during MI [49]. Finally, mitochondrial DNA (mtDNA) may be released in case of significant oxidative stress and activate a similar pathway, resulting in NF-kβ activation [50].

Therefore, there is an astonishing complexity of the inflammatory response during MIRI, due to the multiple cellular and molecular pathways implicated, which may have pleiotropic effects. The NLRP3 inflammasome is seemingly the major activator of the inflammatory response in MIRI, but it might also have a cardioprotective function, complicating the development of therapies targeting this pathway. Further studies will be needed to decipher the homeostatic versus maladaptive role of individual molecules, and to identify novel molecular targets.

In Figure 4, personal observation of a 59-year-old man who presented to the emergency department with acute chest pain is shown. After assessment, he was diagnosed with STEMI. Given the urgency of his condition, he underwent immediate angiography followed by PCI. Initially, after the procedure, he appeared stable, with positive signs of recovery. However, on the second day following PCI, he suddenly became unresponsive and died unexpectedly. An ECG conducted at that time revealed a VF rhythm, indicating a critical arrhythmia associated with his underlying cardiac condition. This severe outcome highlights the complexities and potential complications associated with acute myocardial infarctions and the repercussions of reperfusion therapy.

The term "apoptosis" indicates a highly organized, evolutionary conserved, genetically regulated pathway for maintaining homeostasis in multicellular organisms and was coined by Kerr, Wyllie, and Currie in 1972 [51]. This process refers to a controlled form of cellular death, wherein the cell undergoes distinct structural modifications like contraction and condensation of cytoplasm and nucleus, with the formation of apoptotic bodies [51]. Caspases, a family of cysteine proteases, are responsible for the breakdown and fragmentation of cytoskeletal and nuclear proteins during the progression of apoptosis [52]. Subsequently, macrophages phagocytize apoptotic bodies, which are recognized due to the presence of externalized phosphatidylserine on the outer leaflet of the bilayer envelope, without triggering any inflammation [53]. Previous studies have highlighted that apoptotic cell death is one of the primary forms of cardiomyocyte death during myocardial infarction and MIRI [54,55]. As widely addressed, MIRI induces an elevation in ROS levels, cellular damage, and cardiovascular dysfunction, ultimately resulting in cardiomyocyte apoptosis [56,57]. The induction of apoptosis occurs via three major signaling pathways: the extrinsic pathway, the intrinsic pathway, and the endoplasmic reticulum stress (ERS) pathway [58]. The extrinsic pathway is triggered by the presence of transmembrane death receptors, which are classified as members of the tumor necrosis factor receptor (TNFR) family and possess the "death domain". The transmission of death signals from the cell surface to intracellular pathways is facilitated by the coupling of specific ligands and their corresponding death receptors. Among the various ligand–receptor pairings, the most relevant include the apoptosis-stimulating fragment ligand (FasL) and FasR, TNFα, and TNFR1, as well as TNF-related apoptosis-inducing ligand (TRAIL) and either DR4 or DR5 [58]. Binding of ligands with death receptors results in the recruitment of Fas-associated death domain (FADD) and its subsequent binding to the ligand–receptor complex [59]. FADD activates the pro-caspase-8, leading to the formation of a death-inducing signaling complex (DISC) [60]. This complex then activates caspase-8, which subsequently activates caspase-3 and -7. This process triggers the apoptotic cascade response, ultimately resulting in the execution phase of apoptosis [61,62]. It has been shown that caspase-3 overexpression leads to a higher susceptibility to enhanced myocardial injury following coronary artery ligation, wherein the ischemic tissue is reperfused for a duration of 24 h, resulting in an increase in mortality following MIRI [63]. Additionally, the Fas pathway plays a crucial role in mediating cardiomyocyte apoptosis during MIRI [64], and the onset of reperfusion in mouse models of MIRI is accompanied by an increase in TNF-α and TRAIL levels [65].

The intrinsic pathway is activated by hypoxia, hyperthermia, and low levels of growth factors and is triggered by mitochondria. These stimuli trigger the opening of mPTP and markedly reduce the mitochondrial transmembrane potential, ultimately leading to an increased release in pro-apoptotic proteins, including CytC from the mitochondrial intermembrane space to the cytoplasm [66]. Caspase-9 is activated by CytC, resulting in the production of caspase-3 and -7. The B-cell lymphoma 2 (Bcl-2) family proteins situated in the outer mitochondrial membrane are responsible for regulating the mitochondrial-initiated events mentioned above [67]. By controlling the permeability of the mitochondrial membrane, Bcl-2 family's proteins play a crucial role in regulating the release of CytC. They can be functionally classified as pro-apoptotic and anti-apoptotic proteins. Within the pro-apoptotic group, we find Bad, Bax, Bak, Bid, Bim, Puma, and BNIP3 proteins are included [68]. Interestingly, the extrinsic and intrinsic apoptosis pathways seem to interact: in fact, Fas-mediated apoptosis can lead to mitochondrial damage through the cleavage of Bid by caspase-8 [69]. Overexpression of Bax is observed in the intrinsic apoptosis pathway of ischemic myocardial tissue, and preventing Bax activation has been found to lower apoptosis and enhance MIRI [70]. It was demonstrated that the RISK pathway exerts anti-apoptotic activity during I/R processes by phosphorylating, and therefore inactivating, numerous pro-apoptotic proteins, such as BAD, Bax, BIM, and p53 [71,72,73]. In particular, RISK action on Bax can interfere with its translocation to the mitochondria, thus preventing apoptosis. Remarkably, compelling evidence suggests a correlation between the RISK pathway and the mPTP in rat myocytes. These findings provide evidence that the cardioprotection effect of the PI3K-Akt kinase cascade is achieved through the inhibition of mPTP opening [74].

Finally, the emerging ERS pathway is implicated in a wide range of physiological functions and pathological injuries, encompassing protein folding, intracellular Ca²⁺ storage, oxidative stress, hypoxia, ischemia, and lipid metabolism disorder [75]. ERS plays a crucial role in maintaining cell viability; in fact, not all ERS is harmful. Nevertheless, if it persists for an extended period, it can trigger apoptosis [75]; furthermore, it has been demonstrated that MIRI may be associated with ERS [76]. The presence of unfolded proteins in ERS leads to myocardial injury, which triggers ERS and disrupts the metabolic state of cardiomyocytes, causing greater harm [77]. ERS is amplified during MIRI, and inhibiting ERS has been verified to reduce MIRI [78]. Altered ER oxidation levels result in the abnormal formation of disulfide bonds and the accumulation of peptides. These changes activate intracellular reactions labeled as "the unfolded protein response" (UPR) [79], which leads to the activation of three transmembrane stress sensors: inositol-requiring enzyme 1 (IRE1); activating transcription factor-6 (ATF6) and protein kinase RNA (PKR)-like ER kinase (PERK) [76]. The ERS transcription factor ATF6 initiates a defensive response against MIRI in cardiomyocytes [80,81] (Figure 5).

Necroptosis represents a unique form of cell regulatory necrosis, displaying both necrotic and apoptotic traits [82]. It is widely known that necrosis, in contrast with apoptosis, occurs without proper regulation and is triggered by external factors like hypoxia or inflammation [83]. Necroptosis often involves the upregulation of various pro-inflammatory proteins and compounds, which leads to the rupture of the cell membrane and the subsequent release of cellular contents into the surrounding areas. Therefore, a chain reaction of inflammation and tissue damage occurs. Necroptosis is implicated in a wide range of physiological functions and disease processes, encompassing protein folding, intracellular Ca²⁺ storage, oxidative stress, hypoxia, ischemia, and lipid metabolism disorder [84]. Morphologically, necroptosis primarily presents itself as cellular enlargement and disruption of the cell's plasma membrane. In contrast to apoptosis, necroptosis does not depend on caspase activation and instead relies on distinct signaling pathways. This process is characterized by the release of DAMPs and cytokines, thereby triggering an immune-inflammatory response that exacerbates tissue injury [84]. Necroptosis can arise from various triggers, with TNF-α induced necroptosis being the most prominent. This form of necroptosis is primarily facilitated by receptor interacting protein kinases (RIPK)1 and 3. The interaction between RIPK1 and RIPK3 occurs via the shared RIP homotypic interaction motif (RHIM), resulting in the formation of necrosomes. Subsequently, RIPK1 and RIPK3 undergo phosphorylation and activation [82]. Activated RIPK3 acts on the mixed-lineage kinase domain like protein (MLKL) to phosphorylate, oligomerize, and migrate from the cytoplasm to the cell membrane to form open pores, leading to necrosis [85]. Using a mouse model of MIRI, it has been demonstrated that the levels of RIPK1, RIPK3, and MLKL are increased in the myocardial tissue following reperfusion [86]. Nevertheless, it is crucial to consider the duration of reperfusion and the species-specific vulnerability to cellular death when assessing necroptosis [86]. A study revealed that RIPK3 is also involved in ERS and intracellular Ca²⁺ overload, thereby exacerbating MIRI [87]. The understanding of RIP3-mediated necroptosis and its relationship with apoptosis and other cell death pathways in MIRI remains largely unexplored, with only a few studies addressing this topic. Caspase-8, the pivotal molecule in the process of programmed cell death, can cleave RIPK1, thereby impeding the initiation of necroptosis [82]; as such, caspase-8 represents a connection between apoptosis and necroptosis (Figure 6).

An additional pathway that appears to be involved in myocardial necroptosis is that of phosphoglycerate mutase 5 (PGAM5), which likely acts through mitochondrial fragmentation during MIRI: according to Wang et al., necroptosis can be inhibited through the degradation of PGAM5, which involves the upstream activation of AMP-activated protein kinase (AMPK) and the subsequent stabilization of Kelch-like ECH-associated protein 1 (Keap1) [88]. The molecular mechanisms of necroptosis are still partially unknown; however, in light of the aforementioned pathways and their potential as therapeutic targets in MIRI, it is considered important to further investigate this peculiar form of cell death.

Autophagy is a homeostatic mechanism characterized by a lysosome-dependent degradation [89,90], which is activated by the cells as a response to stress situations, like starvation, or other environment changes [91,92]. It is possible to summarize the well-regulated autophagy process in four principal steps that include the following: (1) the induction of autophagosome formation, (2) the autophagosome formation, (3) the fusion between autophagosome and lysosome, and (4) the degradation of lysosome and its content [92,93,94]. In MIRI, autophagy could be defined as a double edge sword, because it has a cardioprotective effect during ischemia, promoting cell survival and limiting harmful consequences, but its uncontrolled increase during reperfusion becomes detrimental for cardiomyocytes [95,96]. During MIRI, the activation of Beclin 1 is essential to initiate autophagy [89], since this molecular regulator promotes the formation of autophagic lysosomes [92]. In 2007, Matsui et colleagues conducted a preclinical study on mice, concluding that the AMP-activated protein kinase (AMPK) pathway played a regulatory role in autophagy only during myocardial ischemia, and not also in the reperfusion phase, when Beclin-1 is the main molecular autophagic protein [97]. The modulation of autophagy depends on the interaction between Beclin-1 and the Bcl-2 protein, a human apoptotic regulator (anti-apoptotic) which inhibits Beclin-1 when the two molecules are linked, repressing autophagy [98]. However, during MIRI, ROS generation may cause Bcl-2 phosphorylation, promoting the dissolution of the molecular complex with Beclin-1, whose accumulation activates an uncontrolled autophagy [92,99].

On the other hand, during MIRI, the TLR4/NF-kB pathway could downregulate autophagy through the reduced expression of Beclin-1 [100]. Furthermore, another intracellular pathway probably involved in the regulation of cardiomyocyte autophagy during MIRI is the phosphoinositide 3 kinase (PI3K)/Akt/mammalian (or mechanistic) target of the rapamycin (mTOR) pathway, as demonstrated in 2020 by Qiu et al., whose results showed that downregulation of P300/CBP-associated factor (PCAF) inhibited autophagy through activation of PI3K/Akt/mTOR signaling pathway in vitro and in rat models [101]. The same inhibitory role on autophagy by the PI3K/Akt/mTOR pathway was already shown in the study by Jie et al., which was conducted on MIRI models in rats [96]. Another confirmation in the same direction was given by Chen et al., who demonstrated that hyperbaric oxygen treatment could protect against MIRI via the inhibition of inflammation and the modulation of autophagy mediated by upregulation of Akt and mTOR, and decrease in Beclin-1 and tubule-associated protein 1 light chain 3 beta (LC3B), which is another central marker in autophagy [90].

One of the mechanisms that sustains autophagy during MIRI is related to the impaired autophagic efflux that develops due to the interaction of other molecules involved in MIRI with the classical mechanisms that regulate the process under physiological conditions [102]. ROS, whose accumulation during reperfusion has been previously covered, can interact with the expression of LAMP 2 and BECN-1. LAMP 2 is required for phagosome–lysosome fusion, while BECN-1 is able to interfere with this process (Figure 7). The accumulation of ROS causes lower expression of LAMP 2 and overexpression of BECN-1; this causes accumulation of autophagosomes and consequently of damaged cellular components. An impaired autophagy flux was confirmed in other studies which suggested that MIRI may lead to the accumulation of autophagosomes [103]. This accumulation, in turn, enhances inflammation and supports the production of ROS and increased mitochondrial damage [104].

Apoptotic and autophagy markers are highly expressed in myocardial ischemia/reperfusion injury, as shown in Figure 8, referring to a personal observation involving a 59-year-old obese woman with a Body Mass Index (BMI) of 44. While hospitalized and awaiting bariatric surgery, she suddenly experienced chest pain and shortness of breath. A STEMI was diagnosed, and she immediately underwent PCI. Although her symptoms initially improved, six hours later, she suffered a fatal episode of VF that was unresponsive to cardiopulmonary resuscitation.

Pyroptosis is a form of lytic, programmed cell death dependent on caspase-1, firstly observed in Salmonella typhimurium in 2001 [105]. Typically, this process induces formation of pores in the plasma membrane with the release of cell content [106]. This mechanism can be activated through different pathways. The canonical one is dependent on caspase-1, with the essential contribution of the aforementioned inflammasome; the non-canonical one involves caspase-4/5 and 11, activated by lipopolysaccharide (LPS) [107]. Both pathways cause the release of IL-1β and IL-18. GSDMD (Gasdermin D) is the executive protein of pyroptosis, caspase-1 cleaves GSDMD to form N- and C-terminal aminopeptide, and the GSDMD-N can in turn interact with the inner cell membrane; this interaction causes the formation of pores and unregulated leakage of cellular material, which is typical of pyroptosis [106]; GSDMD-C inhibits necrosis [108].

In 2020, Shen et al. demonstrated that in vivo mice subjected to myocardial ischemia and reperfusion had higher levels of NLRP3, ASC, caspase-1, GSDMD, and IL-1β than the sham group; these values were even higher in hyperuricemia mice. Uric acid interacts with the inflammasome, promoting its activation and contributing to ROS accumulation [20]. Later, in 2022, Ye et al. showed that GSDMD participated in MIRI, demonstrating that mice GSDMD-/- subjected to R/I reported a decrease in pro-inflammatory cytokine levels and a reduced infarct zone than WT [109].

In 2023, Zhang et al. studied the effects of cellular FLICE-like inhibitory protein L (cFLIP_L) on the NLRP3/caspase-1/GSDMD (Gasdermin D) pathway and demonstrated its role in attenuating inflammation and modulating pyroptosis. This study showed that overexpression of cFLIP_L decreases the expression of NLRP3 and significantly inhibits the production of the mature caspase-1, resulting in the reduction in IL-1β and IL-18 levels, potentially acting as a natural inhibitor in inflammasome activation [108].

Pyroptosis can also be regulated by calcium overload through calpains. Calpains are calcium-dependent nonlysosomal cysteine proteinases; their activation has been previously demonstrated during MIRI following increased intracellular calcium concentration [110,111]. In 2019, Yue et al. investigated the involvement of calpain in the pyroptotic process using mouse models in vivo and in vitro; the authors showed that calpain can interact with the NLRP3/ASC/caspase-1 axis and participate in the induction of pyroptosis [112].

The markers of pyroptosis are involved in MIRI, in particular calpain 1 and 2. Calpains are proteins that depend on calcium to work and are found in the cells' fluid in an inactive state. They can be activated in various situations and play a crucial role in activating different proteins. These include growth factor receptors, proteins that comprise the cell's structure, proteins that interact with microtubules, and those located in mitochondria. Calpains play a crucial role in regulating the cell cycle, programmed cell death (apoptosis), and cell development [112,113,114].

The process of activating calpains starts when calcium levels inside the cell increase. Changes in calpain's shape enable it to move to the cell membrane, where specific fats help lower the calcium levels required for activation or position calpain near calcium channels, leading to protein activation. Many heart problems are linked to imbalanced calcium levels, particularly during heart damage caused by a lack of blood flow (ischemia) and subsequent restoration of blood flow (reperfusion). Studies of heart tissues have shown that ischemia/reperfusion leads to higher levels of calcium inside the cells. When blood supply to the heart is reduced, sodium and calcium ions accumulate, causing a drop in pH and resulting in tissue acidity. When blood flow returns, there are quick changes in ion movements, which affect how ions are exchanged in the body. The combined harmful effects of ischemia and reperfusion result in excessive calcium accumulation within cells [115,116,117,118].

In Figure 9, we show a personal observation of a 78-year-old man who underwent PPCI for STEMI three months prior to having ankle prosthesis surgery. Forty minutes after the surgery, he experienced the onset of symptoms consistent with STEMI. He was admitted to the coronary care unit, where a coronary angiogram revealed an occluded descending coronary artery. PPCI was performed promptly. However, two hours later, he developed VF and cardiac arrest, which was unresponsive to cardiopulmonary resuscitation.

In summary, pyroptosis is a highly complex process, and the multiple pathways discussed above are involved in MIRI (Figure 10).

The present work confirms that MIRI is a fearful condition that can seriously worsen the outcome of myocardial infarction. Although effective cardioprotective therapies applicable in clinical practice are not available yet, the authors have demonstrated that the goal of understanding the complex and multifactorial pathophysiology of MIRI remains essential, both for clinical and forensic purposes. In fact, only the increasing knowledge of the underlying inflammatory mechanisms and the tissue changes triggered by the multiple molecular/cellular pathways can guarantee the creation of well-designed clinical trials, moving from animal studies, with the final aim to find successful therapeutic targets. Furthermore, the histological and immunohistochemical case series presented from the review authors' institution has an exceptional value because of its exclusivity in the literature and because it underlines the importance for forensic pathologists to be aware of the existence and pathophysiology of MIRI, both to contribute to the tissue characterization of this damage and to recognize the potential deaths related to MIRI, despite early and adequate reperfusion treatment, in malpractice claims.

In conclusion, there is still a great deal of study which pathologists, cardiologists, biologists, and other researchers have to continue on this fundamental topic, focusing on the centrality of the tissue pathology characterization of MIRI.