1. Introduction

Acute myocarditis is commonly associated with viral infections [6,7] including SARS-CoV-2. The focal/diffuse degrees of myocardial inflammation determine the severity of symptoms in patients with myocarditis and the fulminant myocarditis cases predominantly progress with elevated serum cardiac troponins [8,9,10,11,12]. Cardiovascular complications have been described in many COVID-19 patients and myocarditis (from subclinical myocardial injury to fulminant lethal myocarditis) has been proposed to account for a small fraction of cardiac injury among patients infected with SARS-CoV-2 [13,14,15,16,17,18,19,20,21]. (Figure 1).

Figure 1. Schematic representation of myocarditis in patients with COVID-19. Clinical presentation: Most frequently associated with raised troponin blood level, abnormal electrocardiogram (ECG), abnormal echocardiography, late gadolinium enhancement (LGE) involving subepicardial regions, more than 1 abnormality in cardiac magnetic resonance (CMR). Since 2009, a CMR-based diagnosis of myocarditis has been supported by the Lake Louise criteria (LLC), targeting three aspects of myocardial inflammation: oedema, hyperaemia and necrosis and/or fibrosis. Most of the cases of COVID-19-related myocarditis were diagnosed based on CMR findings, with endomyocardial biopsy performed in very few cases because it requires a specific expertise and it is not performed in stable patients. However, endomyocardial biopsy (EMB) remains the gold standard for the diagnosis of myocarditis with identification of SARS-CoV-2 in the myocardium. Beside the identification of myocardial damage, a fundamental step in the diagnostic of myocarditis is the exclusion of obstructive coronary artery disease, especially when the clinical presentation resembles to an acute coronary syndrome. Myocarditis remain uncommon in COVID-19 patients. Since failed human hearts have a higher percentage of ACE2-expressing cardiomyocytes, patients with heart failure are likely to be more susceptible to myocarditis.

Figure 1. Schematic representation of myocarditis in patients with COVID-19. Clinical presentation: Most frequently associated with raised troponin blood level, abnormal electrocardiogram (ECG), abnormal echocardiography, late gadolinium enhancement (LGE) involving subepicardial regions, more than 1 abnormality in cardiac magnetic resonance (CMR). Since 2009, a CMR-based diagnosis of myocarditis has been supported by the Lake Louise criteria (LLC), targeting three aspects of myocardial inflammation: oedema, hyperaemia and necrosis and/or fibrosis. Most of the cases of COVID-19-related myocarditis were diagnosed based on CMR findings, with endomyocardial biopsy performed in very few cases because it requires a specific expertise and it is not performed in stable patients. However, endomyocardial biopsy (EMB) remains the gold standard for the diagnosis of myocarditis with identification of SARS-CoV-2 in the myocardium. Beside the identification of myocardial damage, a fundamental step in the diagnostic of myocarditis is the exclusion of obstructive coronary artery disease, especially when the clinical presentation resembles to an acute coronary syndrome. Myocarditis remain uncommon in COVID-19 patients. Since failed human hearts have a higher percentage of ACE2-expressing cardiomyocytes, patients with heart failure are likely to be more susceptible to myocarditis.

Recently, it has been reported that SARS-CoV-2 infects coronary vessels, inducing pro-atherogenic inflammatory responses that could trigger acute cardiovascular complications and increase the long-term cardiovascular risk [22]. With the rapid rollout of COVID-19 vaccinations, numerous associated and suspected adverse events have been reported nationally and worldwide and their occurrence remains unpredictable. Literature reporting confirmed cases of pericarditis and myocarditis following SARS-CoV-2 mRNA vaccinations [21,23,24,25,26,27,28,29,30,31]. For example, from the 27.3 million Pfizer-BioNTech (BNT162b2) doses administered in Australia to 2 January 2022, there have been 415 reports of likely myocarditis and 735 reports of likely pericarditis and that from the 1.8 million Moderna (mRNA-1273) doses administered in Australia to 2 January 2022, there have been 40 reports of likely myocarditis and 52 reports of likely pericarditis (COVID-19 vaccine weekly safety report - 06-01-2022 | Therapeutic Goods Administration (TGA); accessed on 07.01.2022). However, it is unclear how many of these cases are a direct consequence of the vaccine versus coincidental. An elegant single-centre retrospective analysis of all patients presenting to St Vincent's Hospital, Sydney, Australia with suspected COVID-19 vaccine-related myocarditis (9 suspected cases) and pericarditis (97 suspected cases) was recently published [32]. These authors used the Brighton Collaboration Case Definition of Myocarditis and Pericarditis [12] to categorize patients into groups based on diagnostic certainty and cardiac magnetic resonance imaging findings were reviewed against updated Lake Louise Criteria (LLC, which target three aspects of myocardial inflammation: edema, hyperemia, and necrosis and/or fibrosis) [8,10] for diagnosing patients with suspected myocarditis (Table 1). They confirmed 10 cases of possible or probable myocarditis and pericarditis of which 80% had electrocardiogram abnormalities and one patient had multisystem inflammatory syndrome following vaccination with severely impaired left ventricular ejection fraction.

Table 1. Diagnostic criteria for myocarditis.

Table 1. Diagnostic criteria for myocarditis.

Diagnostic criteria for myocarditis	CDC* criteria	Brighton Collaboration criteria
	Level 1 (confirmed) Symptoms consistent with myocarditis and at least one of: Abnormal histopathology OR Elevated troponin AND abnormal CMR	Level 1 (definitive) Symptoms consistent with myocarditis and at least one of: Abnormal histopathology OR Elevated troponin AND abnormal CMR OR Elevated troponin AND abnormal TTE
	Level 2 (probable)	Level 2 (probable)
	Symptoms consistent with myocarditis and at least one of: Elevated troponin OR Abnormal ECG OR Abnormal TTE OR Abnormal CMR	Symptoms consistent with myocarditis and at least one of: Elevated troponin OR CKMB OR Abnormal ECG OR Abnormal TTE
		Level 3 (possible case)
		Symptoms consistent with myocarditis AND Enlarged heart on CXR OR non-specific ECG abnormalities
CMR diagnostic criteria for myocarditis	Diagnostic target Myocardial edema Myocardial injury Hyperemia Myocardial necrosis	Lake Louise criteria (LLC) T2-weighted imaging, increased Bright signal intensity Increased global early gadolinium enhancement ratio between myocardium and skeletal muscle. At least one focal lesion with non-ischemic regional distribution on late gadolinium enhancement Pericardial effusion; Systolic left ventricular wall motion abnormality

* Abbreviations: .CDC: Center for Disease Control, CMR: Cardiac magnetic resonance imaging; TTE: transthoracic echocardiogram; ECG: electrocardiogram; CKMB: creatine kinase myocardial band; CXR: chest X-ray; LLC: Lake Louise criteria. If two Lake Louise criteria are positive, CMR is considered indicative of active myocardial inflammation. Parametric mapping with CMR permits the routine spatial visualization and quantification of changes in myocardial composition based on changes in T1, T2, and T2*(star) relaxation times and extracellular volume (ECV). The clinical recommendations for CMR mapping can be found in the publication by Messroghli and colleagues [33].

2.3. Shedding light on the manufacture of mRNA-based COVID-19 vaccines

The Pfizer/BioNTech BNT162b2 mRNA vaccine is a lipid nanoparticle (LNP)-encapsulated, nucleoside-modified RNA vaccine (codon-optimized modified spike mRNA), that encodes a SARS-CoV-2 full-length spike protein stabilized in prefusion conformation [42,47].

The production process of the mRNA-based COVID-19 vaccines is quite easy to set up, with a simpler manufacturing process compared to other "more traditional" vaccines, and it theoretically offers several advantages including rapid response and rapid adaptability, enhanced physical stability, and safety. However, this production process involves a number of steps requiring extreme rigor to ensure the presence of intact RNA in the batches [48,49] and an acceptable purity of samples intended for human vaccination.

The manufacturing process of the mRNA-based COVID-19 vaccines includes (Figure 2):

• Isolation of SARS-CoV-2 and the extraction of its RNA genome. Synthesis by reverse transcription (RT) of a double stranded (ds) DNA template of the gene coding for the spike protein. A synthetic DNA sequence encoding the viral spike protein is inserted into a bacterial plasmid (7,824 base pairs for the Pfizer BNT162b2 mRNA vaccine and 6,777 base pairs for the Moderna mRNA 1273 vaccine) that contains a bacterial origin (ori) of replication and a kanamycin resistant (aminoglycoside phosphotransferase Neo/Kan) gene [50]. Notably, the wild type spike sequence (NCBI accession: NC_045512) is found to be 45.3% identical to the BNT162b2 vaccine and the GC content is 37.3% for the wild type and 56.9% for the BNT162b2 vaccine. Eight small ORFs were found to overlap the Pfizer BNT162b2 mRNA vaccines compared to eleven overlapping ORFs in the wild type [51].
• The recombinant bacterial plasmid containing a double stranded DNA copy of the gene coding for the spike protein as well as a DNA-dependent RNA-polymerase promoter and a kanamycin-resistance selection gene are stored at -150°C until use. The, plasmid is then transfected into an Escherichia coli (E. coli) bacteria that has been made competent for DNA uptake (the construct includes missense codons leading to two major changes in the S2 spike protein sequence with K986P and V987P substitution aimed at stabilizing the protein, the proline is a very rigid amino acid forming a bend aimed at improving the stability of the spike protein by preventing the conformational change of the pre-fusion into the post-fusion structure) [52,53].
• E.coli colonies are grown at 37°C for 24 hours on Petri dishes filled with solid medium. During this process the plasmid is transmitted to daughter bacteria of the E.coli colonies when the bacteria divide (bacteria multiply every 20 minutes). To avoid event of plasmid loss its maintenance is enforced by selection with a kanamycin antibiotic added to the growth medium. Bacteria are then grown into flasks filled with medium and then moved into a large fermenter that contains up to 300 liters of a nutrient broth where they are grown for four days. After amplification in bacteria serving as a master cell bank [54], bacteria are chemically broken down and the plasmid DNA is purified from bacterial debris. The products were tested for purity and gene sequence control. Each one liter batch of plasmid DNA is intended to finally produce about one million doses of the vaccine.
• The ring-shaped plasmid is linearized through the action of a restriction enzyme releasing the sequence encoding the synthetic spike. The cell-free in vitro transcription of DNA into RNA is achieved using a T7 RNA polymerase to generate a synthetic mRNA with a 5' cap. The sequence of mRNA is 4100-4300 nucleotides long with a 5'cap [55]. At this stage, the synthetic nucleoside N-methyl-pseudouridine (mψU) is incorporated into the artificial RNA instead of the natural uridine nucleoside to further increase RNA stability, to enhance translation efficiency in host cells, and to remove alternative start codons - avoid overlapping ORFs - and internal ribosome entry sites, thus preventing non-specific recognition by ribosomal complexes [56]. The addition of a 5′ cap structure is a critical part of this production step that has been improved by new technology suitable for large-scale production [57,58]. A poly(A) tail is needed for efficient translation of mRNA vaccines and is also a critical part during manufacture [59].
• In vitro transcription is followed by several steps of mRNA purification, including the removal of DNA and dsRNA, which could lead to an excessive innate immune response by dsRNA sensing [60] and mRNA is filtered and frozen. Analysis of mRNA requires diverse techniques such as RT-qPCR, capillary and gel electrophoresis, high-pressure liquid chromatography (HPLC) and immunoblotting. The Food and Drug Administration (FDA, USA), recommends manufacturers to limit amount of residual DNA in the final product to be below 10 ng/dose and the size of DNA to be below the size of a functional gene [61].
• The thawed mRNA is mixed with water. In a separate process, the oily lipids are mixed with ethanol, and mRNA and lipids (including phospholipids, cholesterol, cationic lipids and polyethylene glycol lipids that are mixed together) are mixed to create lipid nanoparticles [62]. When the lipids come into contact with the mRNA, electric charge pulls them together in a nanosecond. The mRNA is enveloped in several layers of clinically translatable lipid nanoparticles (multilayer liposomes), forming an oily protective vaccine particle. Liposomes or lipid nanoparticles (LNPs) which facilitate the mRNA cytosolic transport, are known to function as adjuvants can also modulate the immune response [63,64]. The newly made vaccine is filtered to remove the ethanol, concentrated and filtered again to remove any impurities, and finally sterilized. Machines inject 0.45 ml of a concentrated vaccine solution into vials, enough for six doses after dilution. The vials are sealed with foil and capped with purple lids and stored at -70°C. After further quality testing, the vials from the same batch are ready to ship.
• Packaged vaccine doses (preserved at low temperature), are shipped and processed for the market. The mRNA-based COVID-19 vaccine is administered by injection into the deltoid muscle leading to capture of mRNA by muscle cells. The lipid nanoparticles protect RNA (a fragile molecule) from RNAse-dependent degradation and facilitate cellular uptake by lipid fusion with lipids of cell membrane. Spike coding RNA is released into target cell cytoplasm.

Figure 2. Schematic representation of the main steps in the production process of mRNA-based COVID-19 vaccine mRNAs: the Pfizer BNT162b2 mRNA vaccine is a lipid nanoparticle-encapsulated, nucleoside-modified RNA vaccine that encodes a SARS-CoV-2 full-length spike protein stabilized in prefusion conformation (see the main text for details).

Figure 2. Schematic representation of the main steps in the production process of mRNA-based COVID-19 vaccine mRNAs: the Pfizer BNT162b2 mRNA vaccine is a lipid nanoparticle-encapsulated, nucleoside-modified RNA vaccine that encodes a SARS-CoV-2 full-length spike protein stabilized in prefusion conformation (see the main text for details).

This vaccine strategy is expected to limit vaccine diffusion near the site of injection and local lymph nodes and is considered safe. However controversial results about the safety of the mRNA-based COVID-19 vaccine have increasingly been reported by different teams [65,66]. There is a concern that the Pfizer BNT162b2 mRNA vaccines are not totally pure and contain traces of plasmid DNA. Furthermore, it was claimed that the robustness of the DNAse I digestion step does not destroy all DNA compounds and that between 10% and 35% of the nucleic acid contained in the vaccines is actually DNA [67]. More recently, Speicher and colleagues [50] reported that all mRNA-based COVID-19 vaccine "exceed the guidelines for residual DNA set by FDA and WHO of 10 ng/dose by 188-509-fold". It was also assumed that the recombinant DNA found in mRNA vaccines can be introduced into the cells at the time of vaccine administration, favored by lipid nanoparticles, just as with the mRNA itself, leading to possible integration [68], an hypothesis that remains the subject of debate [69]. The mRNA vaccines are analyzed using a range of time-consuming and costly methods. Recently Gunter and colleagues [70] described a streamlined method to analyze mRNA vaccines using long read nanopore sequencing (VAX-seq). According to these authors, VAX-seq can comprehensively measure sequence, length, integrity, nucleoside modifications, and purity of mRNA vaccines. Currently, there is also a concern that the vaccine activates the endogenous LINE-1 reverse transcriptase, leading to possible reverse transcription of the mRNA vaccine into DNA with a risk of integration into the host genome [71]. Recently, deep sequencing experiments have provided evidence of heterogeneity in the purity of the mRNA-based COVID-19 vaccine batches and significant traces of DNA [50].

The European Medicines Agency (EMA) guidelines on the quality of clinical aspects associated with DNA vaccines stipulates that DNA should be no more than 0.033% of the total nucleic acids in vaccine doses (Rolling Review Critical Report: https://factreview.gr/wp-content/uploads/2023/07/Rolling-Review-Report-Quality-COVID-19-mRNA-Vaccine-BioNTech.pdf, page 74).

2.4. Biodistribution and persistence of COVID-19 mRNA vaccines

The biodistribution and duration of persistence of the COVID-19 mRNA vaccines may be important for understanding of the adverse side-effects of the vaccine. After injection into the deltoid muscle, the vaccine spike mRNA nanoparticles are expected to be captured and endocytosed by muscle cells. The cellular endocytosis of the mRNA is expected to take about one minute and the translation into spike protein is expected to take about two minutes (as calculated considering a translation speed of 10 amino acids per second and a protein made up of 1273 amino acids) [72,73]. This newly synthesized vaccine spike protein is expected to be delivered at the cell surface in a prefusion conformation, as previously reported by Wrapp and colleagues [47], and to activate an anti-spike immune response including the induction of neutralizing antibodies [74,75,76]. The spike mRNA nanoparticles can also be picked up by the immune cells in the proximal lymph node. Using a cellular model, Fertig and colleagues [77] observed that the endolysosomal compartments of most vaccine-treated cells were enriched with electron dense, multilayered lipid structures as compared to controls, suggesting the successful endocytosis of LNPs, followed by the endosomal-mediated disintegration of LNPs, translation into spike protein and synthesis of spike-like structures clustering on isolated protrusions of the plasma membrane of some cells after 12 hours incubation with the Pfizer/BioNTech BNT162b2 vaccine (10 µg mRNA per 1 × 10⁶ cells). The biodistribution and persistence of mRNA vaccines after intramuscular injection, has been studied in rodents and non-human primates.

These studies indicated that intramuscular injection leads to an initial accumulation of the vaccine spike protein at the injection site within hours of the vaccine injection [77,78]. LNPs are then rapidly transported to proximal lymph nodes by passive draining as well as actively carried by antigen-presenting cells and neutrophils, while the remaining unprocessed LNPs reach systemic circulation [77,79,80]. A study by Krauson and colleagues [81] found that COVID-19 mRNA vaccines elicit antigen-specific germinal center B cell responses only in draining lymph nodes but in mediastinal lymph nodes Using a specific RT-qPCR based assays to detect COVID-19 mRNA vaccine these authors found the presence of spike mRNA in the axillary lymph nodes in the majority of patients who died within 30 days of vaccination. Vaccine was also detected in the myocardium in a subset of patients within 30 days of death. It was reported that a single intramuscular immunization with the Pfizer/BioNTech BNT162b2 vaccine (containing the branched-tail ionizable lipid ALC-0315 that includes a tertiary amine, branched tails and ester linkers biodegradable structure) can activate dendritic cells, monocytes and macrophages in the draining lymph nodes to produce interferons [82]. Another ionizable lipid SM102 (sharing structural features with ALC-0315) used in the Moderna mRNA-1273 vaccine LNPs was found to activate IL-1 Ra cytokine production [83]. It has also been found that exosomes with a spike protein on their surface are induced by the Pfizer/BioNTech BNT162b2 vaccine and circulate prior to the development of specific antibodies. Circulating exosomes expressing spike proteins were found on day 14 after vaccination, followed by anti-spike antibodies being detected 14 days after the second dose [84,85].The beneficial effect of vaccination is usually determined by monitoring the specific anti-spike antibody responses [86], although this represents only part of the immune response against the virus. In a murine animal model, the intramuscular administration of LNPs containing ionizable lipid and mRNA encoding the Spike of SARS-CoV-2, the titres of antibodies against SARS-CoV-2 was increased tenfold with respect to the vaccine encoding for the unadjuvanted antigen [87].

The mRNA-based COVID-19 vaccines, consist of nonreplicating mRNA and are expected to be naturally degraded after translation through intracellular RNAse activities within the cytosol. They should therefore be rapidly eliminated from the injection site. The half-life of this foreign mRNA in the vaccinated subject is unknown, theoretically from a few hours to ten days based on general data for cellular mRNA [88,89], but vaccine-associated synthetic mRNA has been reported to persist in systemic circulation for at least two weeks [77] and full-length or fragments of SARS-CoV-2 spike mRNA vaccine sequences have been found in blood up to four weeks after COVID-19 vaccination [90]. In another study, both the mRNA vaccine and spike protein were found in germinal center in a lymph node up to eight weeks post-injection in some cases [91]. Thus, in contrast to disrupted germinal centers in lymph nodes observed during SARS-CoV-2 infection, the Pfizer/BioNTech BNT162b2 vaccine stimulates robust germinal centers containing vaccine mRNA and vaccine spike antigen.

In a study by Ogata and colleagues [92], 11 of 13 participants exhibited spike antigen in their plasma after a first injection of the Moderna mRNA 1273 COVID-19 vaccine, whereas nucleocapsid concentrations were insignificant in all participants, indicating that the detected spike originated from the vaccination and not from a natural infection. This study suggested that the spike protein leaves the site of the COVID-19 vaccine injection and enters the bloodstream, accumulating in other parts of the body (Figure 3).

Figure 3. Hypothetical model accounting for the possible route taken by vaccine-derived compounds (mRNA coding for the viral spike and/or spike protein) which can be found in the blood circulation and the heart after injection of mRNA-based COVID-19 vaccine in the upper arm (deltoid muscle). Men aged between 18 and 24 are at higher risk of developing myocarditis following mRNA-based COVID-19 vaccination. If undetected, there is a risk of sudden death. When detected, exercises must be prohibited.

Figure 3. Hypothetical model accounting for the possible route taken by vaccine-derived compounds (mRNA coding for the viral spike and/or spike protein) which can be found in the blood circulation and the heart after injection of mRNA-based COVID-19 vaccine in the upper arm (deltoid muscle). Men aged between 18 and 24 are at higher risk of developing myocarditis following mRNA-based COVID-19 vaccination. If undetected, there is a risk of sudden death. When detected, exercises must be prohibited.

This was confirmed by Brogna and colleagues [93] using mass spectrometry examination of biological samples. In vaccinated subjects (n = 20), these authors evidenced the presence of the spike protein translated from the mRNA vaccine (distinguishable from the wild-type protein due to specific amino acid variations introduced to maintain the protein in a prefusion state) in 50% of vaccinated individuals while this was not the case in unvaccinated individual (n = 20).

3. Conclusion

Another question recently addressed by Bozkurt [138] is "why circulating spike protein levels remained elevated despite adequate levels and functionality of anti-spike antibodies ?". This author suggested hypothetical explanations such as vaccine overdose, the possible role of anti-idiotype, and prolonged existence of mRNA. Recently, Japanese radiologists published a study [149] on myocardial 18Fluorine-fluorodeoxyglucose (18F-FDG) uptake on images in 700 asymptomatic vaccinated subjects (The majority of the vaccinated individuals,77.6%, received the Pfizer/BioNTech BNT162b2 vaccine followed by 21% who received the Moderna mRNA-1273 mRNA vaccine. The 700 subjects underwent positron emission tomography (PET)/computed tomography (CT) within a period of 1–180 days after their second vaccination and increased 18F-FDG uptake (a marker of myocardial inflammation of diverse origin including viral myocarditis) was found compared with the unvaccinated group (n = 303). However, this increase was not seen in subjects who underwent imaging more than 180 days after vaccination. These results are corroborated by the data reported in the Krauson's studies [81]. Krauson and colleagues detected spike mRNA vaccine in the axillary lymph nodes and myocardium from recently vaccinated deceased patients. Vaccine was detected in the majority of patients dying within 4 weeks of vaccination, but not in patients dying more than 4 weeks from vaccination.(Figure 4)

Figure 4. Schematic mechanism of action of the COVID-19 mRNA vaccine associated myocarditis. The spike mRNA vaccine was detected in the myocardium from recently vaccinated deceased patients [81]. The LNPs packaging of the COVID-19 mRNA vaccine allows nucleoside-modified RNA vaccine to be stably delivered and enter cardiomyocytes after LNPs had entered the general bloodstream. Once inside a cardiomyocyte, this mRNA forms a complex with initiation factors and the small subunit of the ribosome, where elongation of the spike polypeptide chain starts. The S proteins first assemble to form homotrimers into the cytoplasm and then migrate to the cell surface to protrude with a native-like conformation. Cardiomyocytes expressing the spike protein can possibly be targets for a specific cytotoxic T cells anti-spike immune response and destroyed.

Figure 4. Schematic mechanism of action of the COVID-19 mRNA vaccine associated myocarditis. The spike mRNA vaccine was detected in the myocardium from recently vaccinated deceased patients [81]. The LNPs packaging of the COVID-19 mRNA vaccine allows nucleoside-modified RNA vaccine to be stably delivered and enter cardiomyocytes after LNPs had entered the general bloodstream. Once inside a cardiomyocyte, this mRNA forms a complex with initiation factors and the small subunit of the ribosome, where elongation of the spike polypeptide chain starts. The S proteins first assemble to form homotrimers into the cytoplasm and then migrate to the cell surface to protrude with a native-like conformation. Cardiomyocytes expressing the spike protein can possibly be targets for a specific cytotoxic T cells anti-spike immune response and destroyed.

Whatever the conclusions of this important debate, we now know with certainty that the vaccine spike circulates longer than initially assumed, and that it is directly associated with the increased frequency of myocarditis in young men. Recently Barmada and colleagues [150] investigated a cohort of patients who developed myocarditis and/or pericarditis with elevated troponin and C-reactive protein levels. The patients showed cardiac imaging abnormalities shortly after COVID-19 mRNA vaccination, late gadolinium enhancement, and elevations in circulating interleukins (IL-1 , IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10) and matrix metalloproteases (MMP1, MMP8, MMP9 and TIMP1). Their immune responses analysis indicated expansion of activated cytotoxic T cells, NK cells in the heart and inflammatory and profibrotic monocytes. The precise molecular mechanisms by which the COVID-19 mRNA vaccines lead to myocardial injury and myocarditis remain to be characterized but we can reasonably hypothesize that it is a process which passes through the soluble angiotensin II binding to the transmembrane receptor angiotensin II type I receptor (AT1R; expressed in the arterioles and several organs including heart), and the intracellular factor hypoxia-inducible factor 1 alpha (HIF-1α). (Figure 5)

Figure 5. Potential interaction of COVID-19 mRNA vaccine/spike with the renin-angiotensin system (RAS). A free floating spike can be released from cells expressing the COVID-19 mRNA vaccine, leading to a massive interaction with the cell surface ACE2 receptor. As a result, an increased Ang II level is likely to lead to an overactivation of AT1R signaling which initiates activation of PKC and c-Src that is required for superoxide production by NADPH oxidases (NOX1 and NOX2). NOX2 also stimulates the production of reactive oxygen species (ROS) by mitochondria. The consequence is an inhibition of the hypoxia inducible factor 1 α (HIF-1α) hydroxylation by the prolyl hydroxylase (PHD) pathway and of its polyubiquitinylation by the ubiquitin ligase usually leading to HIF-1 proteosomal degradation (a mechanism contributing to cell homeostasis). After ROS production, HIF-1α translocates to the cell nucleus where it forms heterodimers with the HIF- subunit and binds to the HRE element in the promoter of hypoxia-inducible genes and recruits histone acetyltransferases CREB Binding Protein (CBP)/p300. Thus, it up-regulates ACE1 which contributes to Ang II production and abnormal functioning of the RAS pathway. HIF-1α nuclear translocation also activates the transient receptor potential ankyrin 1 (TRPA1) gene expression which leads to increase in intracellular Ca²⁺ and cell injury as well as expression of several genes involved in the control of cell viability and proliferation among others [132]. In parallel, Ang II triggers an increase in cytoplasmic Ca²⁺ that induces NOX5 to generate H₂O_2. HIF also contributes to the down regulation of the ACE2 gene and activation of ADAM17 which leads to cleavage of the ACE2 protein and the release of soluble ACE2 (sACE2). AT1R signaling activates a number of signaling pathways, such as G protein–mediated (Gq and Gi), Janus kinase/signal transducers and activators of transcription, extracellular signal regulated kinase (ERK), NF-κB, NLRP3 procaspase 1 pathways leading to induction of pro-inflammatory cytokines.

Figure 5. Potential interaction of COVID-19 mRNA vaccine/spike with the renin-angiotensin system (RAS). A free floating spike can be released from cells expressing the COVID-19 mRNA vaccine, leading to a massive interaction with the cell surface ACE2 receptor. As a result, an increased Ang II level is likely to lead to an overactivation of AT1R signaling which initiates activation of PKC and c-Src that is required for superoxide production by NADPH oxidases (NOX1 and NOX2). NOX2 also stimulates the production of reactive oxygen species (ROS) by mitochondria. The consequence is an inhibition of the hypoxia inducible factor 1 α (HIF-1α) hydroxylation by the prolyl hydroxylase (PHD) pathway and of its polyubiquitinylation by the ubiquitin ligase usually leading to HIF-1 proteosomal degradation (a mechanism contributing to cell homeostasis). After ROS production, HIF-1α translocates to the cell nucleus where it forms heterodimers with the HIF- subunit and binds to the HRE element in the promoter of hypoxia-inducible genes and recruits histone acetyltransferases CREB Binding Protein (CBP)/p300. Thus, it up-regulates ACE1 which contributes to Ang II production and abnormal functioning of the RAS pathway. HIF-1α nuclear translocation also activates the transient receptor potential ankyrin 1 (TRPA1) gene expression which leads to increase in intracellular Ca²⁺ and cell injury as well as expression of several genes involved in the control of cell viability and proliferation among others [132]. In parallel, Ang II triggers an increase in cytoplasmic Ca²⁺ that induces NOX5 to generate H₂O_2. HIF also contributes to the down regulation of the ACE2 gene and activation of ADAM17 which leads to cleavage of the ACE2 protein and the release of soluble ACE2 (sACE2). AT1R signaling activates a number of signaling pathways, such as G protein–mediated (Gq and Gi), Janus kinase/signal transducers and activators of transcription, extracellular signal regulated kinase (ERK), NF-κB, NLRP3 procaspase 1 pathways leading to induction of pro-inflammatory cytokines.

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