Can sarcopenia index serve as a predictor of myocarditis from mRNA based COVID-19 vaccine, insights from clustered cases and potential involvement of micro-RNAs in its pathogenesis


 Background: With the current mass international roll out of several vaccines against SARS-Cov-2, several reports of unheeded complications have made headlines. One of which involves myocarditis with the now FDA fully approved vaccine, Pfizer, and others. We hypothesize through this study that a dysregulated micro-RNA response resulting from such type of vaccines can be involved in triggering myocarditis. Methodology: Embase, Medline and the Cochrane Central Register were used to search for specific keywords such as “mRNA COVID-19 vaccines” AND “Myocarditis” for relevant publications up to 1st of September 2021. The systematic review was performed using PRISMA protocolResults:Literature review has identified 26 cases series and reports involving the development of myocarditis from mRNA vaccines, a total of 89 patients were included. Age range was clearly identified in 66 patients. Among those 66 patients, 94% were below 50 years of age, also out of 89 patients, 94% were males. Myocarditis developed, after a median time of 72 hours of the 2nd dose. 90 of cases of myocarditis developed myocarditis after the 2nd dose, the few patients developing myocarditis after the first dose were either predisposed by a history of myocarditis or a history of previous COVID-19 infection. Conclusion:In conclusion, interpretation of the results in view of the suggested hypothesis, reveals that the micro-RNAs implicated in myocarditis in general are as well implicated in the pathogenesis of severe COVID-19, this can explain why patients having a first dose with a history of COVID-19 can develop myocarditis from mRNA vaccines, also the relatively higher likelihood of this complication in males and younger aged individuals can be explained by the upregulation of key myocarditis related miRNAs in those two strata, due to higher muscle mass and suggests performing a sarcopenia index in recipients of the vaccine to correlate it with the likelihood of this complication. This could later set a cut-off of this easy bed-side index to stratify cases a higher risk of this rare complication.

vaccines. Myocarditis is a rare complication, which was more commonly occurring in young adults and adolescents. (3)As mentioned by Bozkurt et al, according to the US centers for disease control and prevention, Myocarditis/Pericarditis rates are =12.6 cases per million doses of second dose mRNA vaccine among individuals 12 to 39 years of age.(4) Interestingly, there is absence of reported cases of Myocarditis with non-mRNA vaccines such as Janssen.
Several mechanisms were suggested as to why the mRNA vaccines speci cally lead to myocarditis. One hypothesis is that mRNA vaccines generate a very high antibody response, like multi-system in ammatory syndrome in children. Molecular mimicry between the protein of SARS-CoV-2 and self-antigens triggering an abnormal immune response has been proposed. (4) In this article we propose a totally different hypothesis related to micro-RNAs. MicroRNAs are short non-coding RNAs that play a crucial role in the regulation of gene expression during cellular processes. It is now established that some of the host-generated miRNAs are known to modulate the antiviral defense during viral infection. Recently, multiple DNA and RNA viruses have been shown to produce miRNAs known as viral miRNAs (v-miRNAs). We hypothesize that mRNA vaccines can either trigger the release of host miRNAs or contain themselves some miRNAs that can trigger this unwanted complication. This suggested hypothesis can have critical diagnostics and therapeutic implications.

Methodology
Embase, Medline and the Cochrane Central Register were used to search for speci c keywords such as "mRNA COVID-19 vaccines" AND "Myocarditis" for relevant publications up to 1st of September 2021. The systematic review was performed using PRISMA protocol

Results
To date, 26 cases' series of myocarditis cases from mRNA vaccines have been reported, comprising a total of 70 cases. Table 1 summarizes the main characteristics of those cases(5)(6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16)(17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) The collected cases series and reports comprised in total 89 cases. A quick analysis of the reported cases reveals that among cases or case series where recipients' ages were reported in details (66 cases out of 89), only 4 cases were above the age of 50 years while the remainder were below 50 years of age ( Figure 1). Among the 89 cases, only 5 were females ( Figure 2). Finally, yet importantly, 90% of patients (80) developed myocarditis after the second dose. 5 out of 9 patients developing myocarditis from the rst dose had history of COVID-19 infection, while 2 had history of myocarditis from another viral illness and the remainder developed it after 7 and 16 days respectively from the rst dose which is longer than the median time of development of myocarditis in other cases (72 hours). (Figure 3) Discussion A)Viruses inducing myocarditis alter host micro-RNAs expression Many studies have identi ed several miRNAs that have a direct relationship with myocarditis. 94 types of miRNAs were studied using gene microarray analysis during viral myocarditis; 27 of which were downregulated and 67 were upregulated during myocarditis in comparison with the controls. One of the miRNAs, called miRNA214 was found to be elevated in plasma during myocarditis. They promote cardiac in ammation by increasing the expression of TNF alpha and IL-6. Their presence in plasma could guide us towards using it as a noninvasive biomarker for diagnosing myocarditis. (28) Many studies are suggesting the therapeutic role of microRNA in myocarditis. For example, the role of microRNA-155 in viral myocarditis caused by Coxsackievirus was explained, and macrophage in ltration was found to be a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, it was found that silencing the microRNA-155 found in cardiac muscles through affecting macrophage polarization and shifting the in ammatory mediators balance, resulted in increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart, which will decrease risks associated with viral myocarditis as cardiomyopathy and heart failure and thus, may be a potential therapeutic target for viral myocarditis. This gives genetic evidence about the role of endogenous miR-155 in modulating macrophage polarization and protecting mice that were experimented on by CVB3-induced VM. (29,30) Another study by Corsten et al revealed that antimiR-155 therapy in VM (Viral Myocarditis) decreased monocytemacrophages numbers and suppressed T-lymphocyte activation, along with inhibition of release of both proand anti-in ammatory cytokines, including TNF-α, Il-6, IL-10, and IFN-γ during the in ammatory phase. Furthermore, suppression of miR-155 reduced body weight loss caused by systemic illness, along with the numbers of circulating leukocytes induced by CVB3 infection. In addition to miRNA-155, miR-146b and miRNA-21 were constantly upregulated during acute in ammation of both human and mouse VM that all contribute to the immune response and in ammatory process (29). miRNA 21 inhibits the pathway of in ammatory NF-κB and antagonizes cardiac myocyte apoptosis in miRNA 21 gene carriers (31). CVB3-upregulated miR-21 can cause heart muscle cell damage by disrupting cardiomyocyte interactions through depressing the levels of components in cell-cell interactions related to the cardiac function. Therefore, during the CVB3 infection, miR-21 expression may lead to the pathogenesis of VMC, and its suppression can reduce host injury(32) It is suggested to prepare miRNA21 mimics for achieving a cardioprotective mechanism. While to antagonize the suppressive effect of proin ammatory miRNAs, some prepared nucleotides were suggested as possible future inhibitors for proin ammatory miRNAs, namely anti-miRNAs oligonucleotides AMOs, antagomirs, locked nucleic acid LNA, antisense nucleotides, peptide nucleic acids and miRNA sponges. (30,33,34) Furthermore, CVB3 reduces the expression of miR-21 which directly inhibits Programmed cell death 4 (PDCD4) resulting in non-stopping apoptosis and worsened myocarditis. CVB3 also cause miR-20b to repress ZFP-148; which is responsible of viral replication, while aberrant miR-20b increases anti-apoptotic proteins B cell lymphoma-2 (Bcl-2) and B-Cell lymphoma-extra large (Bcl-xl) (35). This shows that CVB3 tends to induce miRNAs that balance cardiac cell survival versus apoptosis to create an optimal environment for the virus to replicate. Interestingly, miR-590-5p released extracellularly by CVB3 inhibited pro-apoptotic factors, resulting in prolonged viral replication. Similarly, miR-98 (which can regulate cell apoptosis by interacting with FAS/FASL gene pair) was found to be downregulated in myocarditis patients. Adenosine deaminases acting on RNA bind with Dicer to increase miRNA-222 which inhibits expression of the gene responsible for PTEN (an apoptosis associated protein) during CVB3 induced myocarditis. This shows that non-coding RNAs are important in determining cell fate in enteroviruses infection(36-38).
Another example concerning apoptosis is the miR-29 family's downregulation which has been linked to the pathophysiology of tissue scarring, including heart disease. miR-29b was found to be down-regulated in people and animals with atrial brillation or congestive heart failure, and miR-29b protected against AngII-induced hypertensive cardiac remodeling via inhibiting the TGF/Smad3 signaling pathway. By modifying the extracellular matrix (ECM) and correcting erroneous promoter methylation, it also inhibited cell proliferation and migration. TUG1 taurine up-regulated gene was found to be adversely regulating miR-29b in this study.
Overexpression of miR-29b altered TUG1's effect on cell apoptosis and in ammation, suggesting that miR-29b may have a pro-apoptotic and pro-in ammatory effect in LPS-damaged H9c2 cells (39).
Another example of miRNA being therapeutic is the fact that androgen receptors were found to be involved in increasing cardiac brosis through affecting micro RNA125b (mir-125b) by stimulating more collagen synthesis.
Therefore, stopping the overexpression of ARs, the miR-125b will be inhibited and cardiac in ammation and brosis would be treated. This particularly encouraged treatment with degradation enhancer of AR that limits cardiac brosis in iDCM, thereby providing potentially a therapeutic approach for patients with iDCM.(36) Moreover, after identifying a novel microRNA in mice and humans with myocarditis, they found that the human homologue (hsa-miR-Chr8:96) could be used to distinguish patients with myocarditis from those with myocardial infarction. This was identi ed after inducing autoimmune myocarditis in mice which produced interleukin 17(from TH17 cells) a characteristic feature of myocardial injury in the acute phase of myocarditis.
The microRNA mmu-miR-721 was synthesized by Th17 cells and was present in the plasma of mice with acute autoimmune or viral myocarditis but not in those with acute myocardial infarction 11 . Another example is the role of the miR-221/-222 family that shows that microRNA-221 (miR-221) and miR-222 levels are signi cantly elevated during acute VM caused by Coxsackievirus B3 (CVB3). That is upregulated in VM. The miR-221/-222 cluster was found to have antiviral and in ammatory immune responses to viral infection of the heart. Its inhibition increases viral load, in ammation, and overall cardiac injury upon VM (40).
Exosomes are membrane-bound extracellular vesicles involved in cell signalling 9 . A study by Fan et al, examined the expression of 10 candidate miRNAs in the serum exosome by qRT-PCR in 23 patients with Coxsackievirus B3 (CVB3) viral myocarditis (VM) and 12 controls. Among them, miR-1, -27b, -148a and -133a are heart-associated; miR-146b, 30a, -155, -181d and -125a are immune-associated; miR-21 is brosis associated. The study described 5 miRNAs with disturbed expression in CVB3 VM patients which may be potentially used as biomarkers for VM diagnosis. Not only were they found in serum exosomes, but also, two of these miRNAs were found to have altered expression in cells after CVB3 infection. The miRNAs miR-30a and -181d were signi cantly up-regulated after CVB3 infection. The study further identi ed suppressor of cytokine signaling-3 (SOCS3) as a target for miR-30a and -181d, as predicted by bioinformatics tools and con rmed by dual luciferase assay and Western blot. In addition to this, up-regulated miR-30a and -181d are responsible for the elevated IL-6 levels via modulating SOCS3 expression during CVB3 infection. Furthermore, miRNA inhibitors injection increased mice survival rate after CVB3 infection. Findings of this study suggest that miR-30a and -181d are responsible for the over-activated in ammatory response to CVB3 viral infection of the heart (41) The development of newer modes of treatment of myocarditis was also suggested by a study where RT-qPCR showed miR-141-3p expression to be reduced in mice with experimental autoimmune myocarditis (EAM). In order to test its effect on the pathology of EAM, a group of mice were injected with miR-141-3p agomir. The results showed reduced LVEDd and LVEDs on echocardiography in the non-injected mice while LVEF and LVFS in miR-141-3p agomir injected mice were increased proving its protective effect. Existing literature has proven miRNAs to be involved in mediation of immune in ammation in cardiac diseases 31 . Overexpression of miR-141-3p was found to decrease myocardial in ammation through suppressing STAT4. In this study, the results showed a decrease in miR-141-3p levels with increased STAT4 levels, suggesting that STAT4 is a target gene for miR-141-3p. (42) Lastly, through experimentation on mice it is found that miRNA-98 showed suppression of IL-10 (a strong mediator from B-cells) causing protection and limitation of the severity of myocarditis which may be a new gate for targeted therapy for myocarditis. We can therefore conclude that microRNAs can be used as a diagnostic and therapeutic tool as medicine advances. (43) B)Viral miRNAs interact with host genome, a new miRNA mediated interplay between host and viruses.  C)Why myocarditis from mRNA vaccines seems to decrease with age.
From table 1 we can conclude that most of the cases of myocarditis from mRNA vaccines occurs in individuals aged 50 years or below. Key miRNA involved in induction of myocarditis are expressed in skeletal muscles, an interesting example is miRNA 155, sarcopenia or loss of muscle mass is most pronounced after the age of 50 years, occurring steadily at a rate of 1 to 2% annually. This loss of muscle mass has been correlated to levels of micro RNAs, speci cally microRNA 155, with aging, by Ong and colleagues. They found that miRNA 155 decreases with age. This perfectly matches our hypothesis and might also help in stratifying individuals at higher risk of myocarditis from mRNA vaccines by the sarcopenia index, the higher the index is, the lower the risk of myocarditis from mRNA vaccines. This also could also explain the predominance of male gender in the observed complication, having a higher muscle mass compared to females. (47) D)Diagnostic and therapeutic implications: 1-Predicting mRNA-vaccines related myocarditis.
It is clear from the above that viral RNA can either alter the expression of host miRNA or use cellular machinery to form viral miRNAs. Farr, Rohani, Fani and others have used next generation sequencing to determine micro-RNAs implicated in severe COVID-19. Interestingly, we found that many of the miRNAs implicated in severe COVID-19, have been identi ed as potential targets for viral myocarditis as mentioned earlier (Table3). We summarized in Table 3  The microRNA-132-3p (miR-132) is a regulatory (noncoding) RNA that, in response to cardiomyocyte stress, is upregulated in cardiac tissue. It affects signaling pathways involved in cardiomyocyte growth, autophagy, calcium handling, and contractility. Thus, miR-132 appears as a potentially promising molecular pathophysiological target in HF treatment. CDR132L, a synthetic locked nucleic acid antisense oligonucleotide (ASO) inhibitor with a fully phosphorylated backbone, is a rst-in-class miR-132 inhibitor.
CDR132L treatment resulted in a dose-dependent, sustained miR-132 reduction in plasma. Patients given CDR132L ≥1 mg/kg displayed a median 23.3% NT-proBNP reduction, vs. a 0.9% median increase in the control group. CDR132L treatment induced signi cant QRS narrowing and encouraging positive trends for relevant cardiac brosis biomarkers.CDR132L was safe and well tolerated, without apparent dose-limiting toxicity. A pharmacokinetic/pharmacodynamic dose modelling approach suggested an effective dose level at 1 mg/kg CDR132L.
It was found that not only the direct inhibition of a miRNA was proven useful, but that indirect inhibition can do wonders as well. (51) b-Indirect modulation of miRNAs On the other hand, silencing of DDX17 results in an accumulation of pri-mir26a2 due to its lack of proper processing and cleavage, which in return promotes neurogenesis. In addition to that, accumulated pri-mir26a2 exerted negative feedback on DDX17, which in exchange decreased REST factor's binding to its target promotors favoring further neurogenesis.
Taking the previous two models into consideration, we hypothesize that if we can conduct an inhibitory approach concerning SARS-COV2 viral helicases, we might be able to similarly upregulate certain bene cial myocardial miRNA which will promote reversed myocardial remodeling when it comes to virally induced myocarditis. (53) REST=Repressor Element 1-silencing transcription *Another promising method of indirect inhibition of a miRNA was displayed in the following drug's mechanism of action.
Pirfenidone, an oral, small molecule, anti-brotic agent that acts by inhibiting the activation of cardiac broblasts, and inhibiting the production of peptides, such as transforming growth factor-β. It also works by antisense oligonucleotides directed against cardiotropic long non coding RNA (miRNA , IncRNA), silencing miRNA -21 that promotes cardiac brosis, therefore it manages to diminish broblast proliferation and collagen type 1 production and crosslinking that causes heart brosis.
It is used in idiopathic pulmonary brosis. Results showed great reduction in ECV containing collagen and water from baseline in 52 weeks, where 100 capsules of pirfenidone that were taken (11days ttt t target dose) resulted in mean ECV reduction at 52 weeks of 0.06%, with improvement in 8 out if 10 KCCQ score, including clinical improvements and log NT-proBNP reduction.
The reduction in log NT-proBNP with pirfenidone was suggested to be due to an improvement in left ventricular myocardial stiffness secondary to myocardial brosis regression.
The outcome of decrease in ECV in association with log NT-proBNP reduction, provides further support for its effect in decreasing heart brosis and thus the effectiveness of the drug in treatment of HFpEF, possibly through non-coding miRNA silencing. The drug is effective in the structural wise changes but not the hemodynamic changes occurring in Heart failure. So, RAAS inhibitors should be studied for cardiac hemodynamic effect, synergistically with pirfenidone. Figure 4 summarizes the implications tackled in the article

Conclusion
We can conclude from the above that, miRNA upregulation or downregulation by mRNA vaccines might explain the myocarditis observed from such vaccines. This hypothesis explains why younger individuals are at higher risk of myocarditis, having a larger muscle mass with upregulation of key miRNA such as miRNA 155 being highly expressed in skeletal muscles. Recognition of this might lead to tailoring of multiple tools for risk strati cation of this complication before receiving mRNA vaccines, such as sarcopenia index or testing for some key miRNAs (such as miRNA155). It can also have therapeutic tools by developing anti-sense miRNAs blocking the action of the implicated miRNAs and thus preventing the myocarditis process.    Functions -5p, 5p and -3p ated They are expressed on immune cells and play a role in both innate immunity and myocardial infarction. They regulate the synthesis and release of proinflammatory cytokines in the heart through Toll-like (TRL) receptors and their downstream signaling cascade, which includes the transcription nuclear factor-kappa B (NF-κB). The three were among the most deregulated miRNAs upon SARS-CoV infection.

List Of Abbreviations
-5p ated Significantly up-regulated in severe COVID-19 infection.
One of the miRNAs that are persistently modulated in both human and mouse viral myocarditis. It attributes to myocardial damage by modulating monocyte-macrophage cardiac infiltration and T lymphocyte activation. It is expressed in immune cells and involved in both innate immune response and myocardial infarction or HF. Toll-like (TRL) receptors and their downstream signaling cascade, involving the transcription nuclear factor-kappa B (NF-κB), control the synthesis and secretion of pro-inflammatory cytokines in the heart. 5p ated It inhibits the release of anti-inflammatory cytokines and triggers the release of pro-inflammatory cytokines by targeting PDCD4 (Programmed Cell Death 4), one of the components of the NF-κB pathway. Initially, high levels were linked to serious disease. However, upon further classification into low and high miR concentrations divided by the median concentration, Lower miR-21 levels were associated with more ventilation and vasopressor days, as well as a greater rate of extracorporeal membrane oxygenation (ECMO) and renal replacement therapy.
Despite possible association, further research is necessary due to inconsistent results.
It's expressed on immune cells and plays a role in both innate immunity and myocardial infarction. It regulates the synthesis and release of proinflammatory cytokines in the heart through Toll-like (TRL) receptors and their downstream signaling cascade, which includes the transcription nuclear factor-kappa B (NF-κB).
-3p gulated Through increasing glucose metabolism and inhibiting granulocyte activation, it appears to have cardio-protective and anti-inflammatory properties. As a result, diabetes and CVDs have been linked to a reduction in miR-223-3p levels in tissue and plasma.
-5p, with a-5p ated They are responsible for fine-tuning of macrophage inflammatory response via regulation of NF-κB activity through a unique regulatory network. The activation of NF-κB by a stressor, promotes miR-155-5p production, which amplifies NF-κB activity and allows for a powerful macrophage activation.
MiR-146a-5p transcription is enhanced as the inflammatory response progresses, blocking its targets IRAK1 and TRAF6, and decreasing NF-κB activation.
-3p iR-499ated They were found to be increased in the plasma of individuals with viral cardiomyopathy, and their levels were found to correlate with myocardial damage as measured by Troponin T levels.
They were significantly up-regulated in COVID-19 patients, inducing myocardial damage. Increased levels were linked to elevated procalcitonin and lactate levels. In COVID-19 survivors, upregulation of miR-21, miR155, miR-208a, and miR-499 could be a predictor of chronic myocardial injury and inflammation. Despite, the fact that troponin levels were higher in the Influenza-ARDS group, COVID-19 had higher levels of cardiac specific miR-208a and miR-499. b, miRnd miRated It has been discovered to target ACE2, and in vitro investigations have shown that miR-200c regulation regulates ACE2 expression.
It is involved in CVDs and is upregulated by oxidative stress. This indicates that miR200c-mediated regulation of ACE2 may be essential for SARS-COV-2 entry. a gulated It is associated with macrophage infiltration, cardiac injury, enhanced LV function, and clinical prognosis.    The potential role of micro-RNAs in mRNA-based vaccines Abbreviations: COVID-19: Coronavirus disease 2019, mRNA: messenger Ribonucleic acid, miRNA: Micro Ribonucleic acid