Molecular Pathways Disturbances during COVID-19 Lead to Cardiomyocyte Necroptosis

1. Background

In December 2019, a local outbreak of a new type of pneumonia was detected in Wuhan, China, which was quickly determined to be caused by a novel coronavirus, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19) [1]. Scientists worldwide quickly started to design and perform studies to recognize this new type of coronavirus. Myocardial injury is the main presentation of COVID-19. Besides, myocarditis is another cause of morbidity among COVID-19 patients. The suggested mechanisms of myocardial injury during COVID-19 include direct viral damage to cardiac cells, viral-induced myocardial fibrosis, systemic inflammation, overstated cytokine reaction due to the activation of type 1 and 2 helper T cells, and hypoxia [2].

Direct viral attack on the myocardium may be the other possible fundamental pathway leading to myocardial damage, in which the noticeable affinity of SARS-CoV-2 with the ACE-2 receptor may lead to direct viral infection in the vascular endothelium and myocardium [3,4,5,6]. SARS-CoV infection of the myocardium is based on the ACE-2 receptors, and the disturbance of ACE-2 causes cardiomyopathy, cardiac dysfunction, and heart failure [7,8]. The association of SARS-CoV and ACE-2 in the myocardium can lead to SARS-related myocardial inflammation and direct damage [9]. The harmful effect of ACE-2 down-regulation may lead the cardioprotective effects of angiotensin to decrease, causing higher TNF-α activation, which as a well-known inflammatory cytokine, develops the inflammatory response in the myocardial damage [2,7,10,11]. Furthermore, TNF-α and JAK/STAT molecular pathway, as other accepted inflammatory pathway, is responsible for several inflammatory processes within the cellular zone [12].

Janus kinase (JAK)/signal transducer and activator for transcription (STAT) pathway have an essential effect in coordinating cell-mediated immune system, particularly cytokine receptors, and may control the modification of T-helper cells. STAT family members are expressed in cardiomyocytes and are the fundamental molecules in myocardial ischemia pathogenesis. Although the association between the renin-angiotensin system (RAS) and STAT clarifies the inhibition of RAS activation, it incites many cardiomyocytes to be saved in myocardial ischemia. Besides, COVID-19’s viral attack and inflammatory cascades may decline during JAK/STAT pathway inhibition over ACE-2 [13,14].

We theorize that COVID-19-related molecular pathways are critical in myocardial damage and necroptosis. SARS-CoV-2-derived molecular pathways lead to myocardial necroptosis due to inflammatory cytokine storm, higher TNF-α activation, and induction of the inflammatory response. This study describes the molecular pathways affecting the myocardium during the infliction of this new type of disease.

2. NFAT Molecular Pathway

Calcium signals result in translocating the nuclear factor of activated T cells (NFAT) from the cytoplasm to the nucleus. Nevertheless, the calcium-activated phosphatase calcineurin, which can be co-transported to the nucleus with NFAT to maintain transcriptionally activity during calcium signaling, regulates this translocation process [15]. NFAT is a key regulatory factor in gene transcription. NFATc1–NFATc4 are four different recognized calcium-related members. Different cell types, such as B lymphocytes, NK cells, mast cells, and neurons, modulate these transcription factors. They can be stimulated by different pathways, for instance, signaling through the B cell receptor (BCR) and other receptors [16,17,18,19,20,21]. NFAT is translocated into the cytoplasm while calcium, NFAT, and calcium-mediated phosphatase calcineurin pathway signal shut down. Different NFAT kinases deal with calcineurin actions and control the nuclear transfer of NFAT. P38/MAPK phosphorylates deal with NFATp.

Furthermore, stimulating this pathway by triggering MKK6 and p38 stabilizes the calcium-induced nuclear increase in NFATp. The activation of the JNK or ERK pathway ineffectively adjusts the nuclear transportation of NFATp. Regularly stimulating p38 leads to down-regulating NFATp-mediated transcription. Hence, the p38 signaling pathway seems to have a key role in controlling NFATp and cellular homeostasis [15].

Although the stimulation process is related to calcium through activating the dephosphorylation of NFAT by the calcium-dependent phosphatase calcineurin, the exact downstream molecular pathway of NFAT is unknown. NFAT functions in T- and B-cell activation and differentiation [22,23]. NFATc1 and NFATc2 knocked-out genes show elevated IgG1 and IgE antibody levels [24]. Likewise, in T-cells, the Tpl-2 kinase accelerates IL-2 expression and stimulates NFAT. Mutant signaling molecules, which inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways, may inhibit the IL-2 promoter stimulation by Tpl-2. Signaling molecules’ stimulation triggers the pathways discussed above. As the NFAT-mediated promoter, IL-2 promoter stimulation depends on Raf1 signals. Consequently, the stimulating NFAT is related to MAPK, and Tpl-2 would be independent of MEK1 and MEK2. Likewise, Tpl-2 also stimulates NF-κB. A combination of calcineurin-dependent and independent pathways controls the Tpl-2-related NFAT stimulation. These dependent and independent pathways are also entirely blocked by Bcl-2 or Bcl-X_L [25]. Nevertheless, Bcl-2 inhibits the apoptosis cascade, which could be induced by several intra/extracellular stimuli. Dissemination of microtubule dynamics kills cells in a Fas/Fas ligand (FasL)-driven situation. The overexpression of Bcl-2 reduces the expression of FasL. This mechanism is based on preventing NFAT by Bcl-2 [26]. Fas/FasL system activates apoptosis in various cell types. Moreover, Bcl-X_L stimulates Fas-related cell death [27].

Besides, SRF can control the expression of MCL-1 as a Bcl family member. NFAT and SRF are the key molecules in controlling cytokine production in lymphocyte lines. NFAT stimulation by the BCR happens through the Ca2+/calcineurin pathway. Conversely, SRF responds to low cellular Ca2+ levels and is less dependent on IP3R expression than NFAT. Ca²⁺-regulated calcineurin is critical in SRF activation combined with diacylglycerol (DAG). DAG is essential for NFAT stimulation. Several DAG effectors, like Ras and Rap1, protein kinase C, and the MEK/ERK pathway, are also needed for SRF and NFAT regulation; however, NFAT is dependent on JNK signals individually [28].

To convert to phosphorylated type, ERK1/2 relies on the activation of MEK1/2 [16]. The partial inhibition in MEK phosphorylation inhibits an increase in c-Fos protein levels, indicating an essential connection between MAPK and c-Fos in MHC-II signaling [16].

3. Necroptosis

Necroptosis is a programmed necrosis that employs various death receptors such as TNF receptor (TNFR) 1, TNFR2, and Fas. It is validated that death receptors possibly induce necroptosis associated with receptor-interacting protein 1 (RIP1) [29]. Moreover, inducing Toll-like receptor (TLR) agonists are known to control caspase-independent necrosis and necroptosis [30,31]. Various genes involved in the TLR pathway are regularly found in necroptotic signaling cascades and lead to necroptosis. So, the TNF-α/TNFR-mediated pathways lead to the necroptosis process. TNF-α, produced by activated macrophages [32], is usually defined as an apoptotic inducer, so, as the first identified agent, it can induce necrosis in tumoral cells [33,34]. Moreover, TNF-α, as a necroptosis inducer, binds to the extracellular part of TNFR1 [35]. This extracellular part of TNFR1, called cysteine-rich domains (CRDs) or pre-ligand assembly domain (PLAD), is essential to develop pre-assembled TNF-α binder receptors [36]. TNF-α binder molecules such as silencer of death domains (SODD) trigger TNFR1 and TNFR2. Subsequently, the downstream signaling cascade forms complex I with TNF-α receptor-associated death domain (TRADD), TNF-α receptor-associated factor 2/5 (TRAF2/5), Fas-associated death domain (FADD), RIP1, and Inhibitor of apoptosis proteins (IAPs), e.g., cIAP1 and cIAP2 [37].

The ubiquitination state of RIP1, as a part of the RIP family homologous N-terminal kinase domain determinant, promotes cell death. TNFR1 involves RIP1, which leads to poly-ubiquitination of TNFR1 by TRAF2/5, cIAP1, and cIAP2 [38,39]. The ubiquitination process of RIP1 activates the IKK complex and NEMO. Therefore, it may up-regulate the NF-κB molecular pathway. RIP1 deubiquitination may inhibit NF-κB molecular pathway and cause a disposition in the cell death pathways program. At this point, cylindromatosis (CYLD), the inhibitor of NF-κB, plays a crucial role in the cell death program [40]. Tumor cells expressing inactive CYLD decrease the incidence of apoptosis [41,42]. Proteasome-dependent degradation of E3 ligases down-regulates NF-κB pathway [43,44]. The ubiquitination of RIP1 is an essential part of the NF-κB molecular pathway activation [45]. Nevertheless, the RIP1 kinase activity [46,47] as the key element in regulating the TNF-induced NF-κB molecular pathway is the ubiquitination status of RIP1, regardless of whether the RIP1 kinase is activated or not. De-ubiquitination of RIP1 is related to complex I and, consequently, to complex II, also called the death-inducing signaling complex (DISC) [48]. The aggregation of TRADD, FADD, RIP1, and caspase-8 forms complex II. The Knock-down of CYLD inhibits TNF-α-induced necroptosis [31], which shows the role of the RIP1 deubiquitination in TNF-α-induced necroptosis [49].

Also, TRAF2, as the other E3 ligase, is demonstrated to be one of the essential molecules in TNF-induced necrosis [50]. This effect is potentially based on TRAF2’s requirement to form complex I. FADD is a critical molecule for TNF-α-induced necroptosis [37], as well as the necroptotic process in several cellular processes which are not based on this cascade [51] (Figure 1). FADD plays a down-regulator role in the necroptosis process for the proliferative phase of the T-cell line [52]. The principal mechanism is based on various roles of FADD, which are explicitly unknown [53,54]. Therefore, although TRADD is essential for the necroptosis process, it may be based on the stimulation type. Complex II might trigger apoptosis and necroptosis as two downstream signaling pathways. While activated, caspase-8 dispossesses RIP1 of its role by splitting dynamic complex II into a pro-apoptosis complex. RIP1 interaction with caspase-8 leads to down-regulating RIP1 and the NF-κB pathway [55] and also shows an inhibitory effect on the necroptosis process [54]. Furthermore, the receptor-interacting protein 3 (RIP3) interface with caspase-8 suppresses RIP3’s capability to increase the caspase-independent cell death rate [56,57].

4. Myocardial Injuries and the Role of Necroptosis

Myocardial infarction is a cause of morbidity worldwide [58]. Despite the significant improvements, current therapies describe the central issue incompletely, derived from acute ischemia or infarction to the chronic type of heart failure. The considerable loss of cardiomyocytes with subsequent remodeling and contractile dysfunction is the leading cause of clinical presentations. Apoptosis, which is related to TNF-mediated death receptors [59] or fas receptor/CD95 [60], is reflected as a well-known molecular cascade of the described events [61]. TNF-α is up-regulated through myocardial infarction (MI) and increases the apoptosis rate [62]. Nevertheless, the overall rate of apoptosis ratio in the infarcted area is approximately 1% in research studies [63,64]. Necroptosis or “programmed necrosis” is suggested as another chief modulator of cell death in myocardial events [63]. This process is closely controlled by individual molecular pathways [65,66,67,68]. Conversely, several studies indicated that the TNF-α-mediated construction of the RIP1 and RIP3 complex is critical in prompting necroptosis [65,66,69]. RIP3 plays a significant role in this process as the regulatory molecule of the RIP1 phosphorylation process [65,69]. An essential function of RIP3-facilitated necroptosis is validated during hepatic viral infections and tissue damage due to inflammatory bowel disease [70]. In the myocardium, inhibition of RIP1 by the small molecule necrostatin-1 causes a decrease in the infarct size [71,72,73], where the efficient consequence of RIP3 on the heart is indistinctly unidentified [74]. RIP3-facilitated necroptosis also expressed in the myocardium, makes a complex with RIP1 in cardiomyocytes, activated by TNF stimulation. However, the RIP1/RIP3 complex inhibition via caspase 8 molecular cascade is suggested as a potential protective mechanism of necroptosis [74,75,76].

Necroptosis and apoptosis are significant severe cardiac pathological situations. While apoptotic signaling pathways are well-defined, the molecular mechanisms inducing cardiomyocyte necroptosis are not yet defined. RIP3 activates the necroptosis cascade by Ca2+-calmodulin-dependent protein kinase (CaMKII) activation. Thus, RIP3-induced triggering of CaMKII causes myocardial necroptosis through molecular events [77].

Permanent cardiac injury causes an emergent contest in dealing with ischemic heart diseases. Molecular chaperone heat shock protein 70 (HSP70) reduces heat-mediated autophagy-related apoptosis. Hence, autophagy may induce the necroptosis process [78].

As discussed above, necroptosis is revealed as a novel cell death model and may be crucial in heart disease. Therefore, specific microRNA (miRNA) and ring finger protein 11 (RNF11)-related cascades presume that oxidative stress, inflammation response, and the mitogen-activated protein kinase (MAPK) pathway may cause necroptosis. Stimulatingly, these molecules play a key role in cardiomyocyte necroptosis occurrence [79].

The discordant stimulation of cell death is strictly associated with the pathogenesis of cardiovascular diseases [80]. Necroptosis is one of the classic pathways for regulating necrosis and depends on the activity of RIP3 kinase. RIP1 substantially relates to RIP3, activating death receptors to produce necrosomes and filamentous amyloid protein complexes [81].

Thus, the mixed lineage kinase (MLKL) domain, by phosphorylation of activated RIP3, oligomerizes and translocates to cellular membranes, cooperating with their capability to preserve homeostasis [82]. The activation of necroptosis organizes a pathophysiological incident in myocardial injury [83,84]. However, whether or not cardiac microvascular endothelium injury is related to necroptosis in myocardial injury is not explicitly defined. PI3K-AKT-eNOS pathway activation up-regulates NO production and decreases necroptosis [85].

Necroptosis is a highly planned mechanism to induce cellular death by caspase activation in order to preserve the homeostasis balance [86]. Expressing the death molecular pathways receptors like Fas and TNF-α stimulates the apoptotic cellular pathway. Nevertheless, it is confirmed that death receptor activation can induce cell death as necrosis in several cell types, while the extrinsic apoptotic pathway is inactivated [87], indicating the existence of regulated necrosis. As defined in necroptosis pathways, RIP1’s serine/threonine kinase action has a key role in cell death. Whereas necrostatin-1, a tryptophan-based molecule, also plays a crucial part as the inhabitant [88,89,90]. This molecule-mediate block of TNF-α induces necrotic cell death by inhibiting RIP1 kinase activity [91,92].

5. COVID-19 and Necroptosis

The COVID-19 virus genome leads to the expression of nonstructural proteins, as an essential requirement for replication, and other proteins related to the receptors of the host cell and interacts with the ACE2 to allow the virus to enter. ACE inhibitors display vasoconstrictive, pro-inflammatory, prooxidative, and prothrombotic effects. SARS-CoV-2 probably controls the inactivation of eukaryotic Initiation Factor 2 (eIF2) by two of the three cellular eIF2 kinases, i.e., protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK). Stimulatingly, PKR up-regulates the autophagy protein p62, related to Nrf2, a regulatory molecule of ACE2, for binding to KEAP1 and further promotes the autophagic degradation of KEAP1, leading to the stimulation of Nrf2 transcriptional activity. Consequently, Nrf2 may reduce the inflammatory process of viral infection. Also, the up-regulation of the Nrf2-transcriptional target heme oxygenase 1 (HO-1, gene name HMOX1) is related to an antiviral process like many viruses like influenza virus and respiratory syncytial virus (RSV). HO-1 can facilitate antiviral reactions by developing a heterodimeric complex. Thus, triggering the Nrf2/HO-1 pathway facilitates SARS-CoV-2 infection [93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141].

Nrf2 is a transcription factor for up-regulating the expressions of many anti-oxidative factors such as SODD and catalase; therefore, it plays a crucial role in maintaining cellular redox homeostasis. However, the level and activity of Nrf2 decrease with age. This statement illustrates the reason for older patients’ morbidity and mortality during COVID-19. Therefore, considering the Nrf2 activators may benefit the elderly who suffer from COVID-19. Well-known nitric oxide (NO), superoxide, and hydroxyl free radicals react with intra/extracellular molecules, cause protein deterioration and cell death, and lead to organ failure. Viral infection induces massive production of free radicals. The pathogenic roles of free radicals in viral infections are profound but overlooked. Free radicals’ damaging role in morbidity and mortality is not declared in several COVID-19 management guidelines. Inflammatory cytokine storms are reported in both COVID-19 and SARS, and the cytokine storm is the most frequently mentioned pathological theory in COVID-19. These inflammatory cytokines are proteins that act as signaling molecules to recruit immune cells to the site of inflammation, induce vascular leakage and exudation, and stimulate the production of free radicals and proteases. For example, IFNɤ, IL-1ẞ, IL-6, and TNF-α can stimulate NO synthesis. IL-2 is highly up-regulated in COVID-19 patients and is known to significantly stimulate producing NO in patients, while NO is the main mediator of IL-2-induced hypotension and vascular leak syndrome. IL-6 is another major inflammatory cytokine up-regulated in COVID-19 patients. IL-6 and TNF-α can provoke generating superoxide in neutrophils, while the production of IL-6 is stimulated by hydrogen peroxide. The inhibition of NO synthesis can decrease the IL-6 generation by more than 50%. The cytotoxicity effect of inflammatory cytokines can be blocked by lipid peroxidation inhibitors [142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157].

Intra/extracellular calcium exchange leads to the activation of calcineurin and its related molecule, NFAT. Intracellular production of NO via calcium-calmodulin-dependent NO synthase (NOS) is a well-determined mechanism in this pathway. Also, NO enhances the calcium-dependent activity of NFAT and promotes the phosphorylation of glycogen synthase kinase-3β (GSK-3β) [158].

Furthermore, vascular endothelial growth factor (VEGF) controls the NOS/NO pathway of endothelial progenitor cells (EPCs) by stimulating the calcineurin/NFAT signaling pathway [159].

NO overproduction acts as RNS, potent oxidants that initiate protein oxidation and peroxidation [160]. Recently, nitration has been displayed to provoke RIP1- and RIP3-related necroptosis cascade, with respiratory complex I [161], which contradicts the well-defined cytoprotective effects of NO, which reduce oxidative stress effects, mitochondrial damage, and tissue infarction [162]. Nitrites also converse protection against ischemia-reperfusion injuries, affecting one of the main intracellular molecules and signal transducers in the myocardium [163].

6. Conclusive Statement

Myocardial injury caused by SARS-CoV-2 infection is a critical event in the pathogenesis of COVID-19. It occurs due to myocardial inflammation caused by ACE-2 and SARS-CoV-2 association, in which ACE-2 down-regulation results in an inflammatory cytokine storm and increases TNF- activation, which results in myocardial damage, necroptosis, and apoptosis. This necroptosis-initiating factor appears to have a role in the pathogenesis of myocardial infarction. TNF-a can cause neutrophils to produce superoxide and induce NO production in the body. The intracellular generation of NO by NOS, dependent on calcium and calmodulin, is a well-established process. NO increases the calcium-dependent activity of the nuclear factor of activated T cells (NFAT).

Additionally, the exchange of intracellular calcium for external calcium activates calcineurin and its related protein, NFAT. Nitration activates the RIP1 necroptosis pathway associated with respiratory complex I. The protective effects of nitrates in the myocardial and activation of the PI3K-AKT-eNOS pathway enhances NO generation and necroptosis decrease in the myocardium.

NO also activates the necrosis molecular pathway by attaching to a portion of the TNFR1 molecule, which activates RIP1, which is a significant participant in cell death during necroptosis. Additionally, NO activates the NF-B, which Tpl-2 also activates. Aside from that, calcineurin regulates Tpl-2-mediated NFAT and is blocked by Bcl-2 or Bcl-XL, respectively. In contrast, the NFAT pathway is dependent on JNK signals; Bcl-2 overexpression produces a decrease in FasL, which triggers apoptosis and inhibits the NFAT pathway, whereas RIP3 activation is responsible for the activation of apoptotic signaling pathways in cardiomyocytes, which are also responsible for the molecular processes of cardiomyocyte necroptosis. Thus, in combination with DAG, which is required for NFAT stimulation, calcium-regulated calcineurin contributes to SRF activation to a limited extent. NFAT depends on signals from DAG effectors such as protein kinase C, Ras, and Rap1 and signals from downstream. Finally, it should be emphasized that inhibiting this intrinsic molecular process can prevent the necroptosis of cardiomyocytes from occurring in some cases.