A Single Nucleotide Variant in Ankyrin-2 Inﬂuencing Ventricular Tachycardia in COVID-19 Associated Myocarditis

: Introduction : This paper explores the potential inﬂuence of a single nucleotide variant in the ANK-2 gene on COVID-19 myocarditis-related ventricular tachycardia. Case Description : A 53-year-old female with a history of Crohn’s disease and asthma developed COVID-19. Shortly after infection, she experienced symptoms of chest pressure, palpitations, and shortness of breath, leading to the eventual diagnosis of myocarditis complicated by recurrent ventricular tachycardia. Treatment with mechanistically driven anti-arrhythmic therapy and beta-blockers suppressed this highly symptomatic ventricular tachycardia. Genetic testing to further risk stratify and inﬂuence long term care identiﬁed a single nucleotide variant in the ANK-2 gene, which is known to be associated with arrhythmic risk. Discussion : This case study highlights the use of rationally selected anti-arrhythmic therapy, mexiletine, in the management of ventricular tachycardia associated with COVID-19 myocarditis and the presence of a single nucleotide variant in ANK-2, raising the possibility of its contribution to VT susceptibility and severity. Our patient demonstrated signiﬁcant improvement with administered therapeutics, including the resolution of myocarditis and ventricular tachycardia. The normalization of the QT interval during the resolution phase further supports the potential inﬂuence of the genetic variant in ANK-2 on potassium channel activity.


Introduction
The COVID-19 pandemic persists, as hospitalizations and deaths due to the virus remain prevalent [1].The COVID-19 disease state can vary in clinical intensity in patients, ranging from asymptomatic to cardiac manifestations including myocarditis, which has been associated with mortality [2].COVID-induced myocarditis occurs due to an immunemediated inflammation of the myocardium after infection with SARS-CoV-2.In general, myocarditis due to COVID-19 development can present with no symptoms, or with chest pains, dyspnea, and/or arrhythmias [3].The currently hypothesized mechanisms of these myocardial injuries are related to a dysregulated immune response, renin-angiotensinaldosterone system dysregulation, and/or direct myocardial injury [4].Furthermore, the resultant severity of the clinical manifestation, particularly in COVID-induced fulminant myocarditis, may arise from patient-specific immunogenetic features or genetic variants within myocardial cells that predispose to arrhythmias and cardiomyopathies.
In this report, we present a case of COVID-19 myocarditis-associated ventricular tachycardia in a patient with a single nucleotide variant in ANK-2.Though we believe that her clinical manifestation may likely be attributed to well-established mechanisms of re-entrant VT in myocarditis, we are intrigued by the possibility that her single nucleotide variant in ANK-2, which is known to be associated with arrhythmic risk [5], may have contributed to her disease state.This furthers the well-established understanding that underlying genetic architecture may serve as a basis for driving the severity of disease states.

Clinical History
A 53-year-old Caucasian female with a past medical history significant for Crohn's disease and asthma developed COVID-19 in February of 2021.Approximately three weeks after her COVID-19 diagnosis, she began to experience chest pressure, rapid palpitations, and shortness of breath.At an outside institution, she underwent a standard evaluation, consisting of an EKG, an echocardiogram, and a Holter monitor.Her echo revealed a normal LVEF, no valve concerns, and RVSP, and her Holter revealed salvos of ventricular tachycardia.She underwent cardiac catheterization, which demonstrated normal angiographic coronaries (Figure 1A,B), and subsequently underwent a cardiac MRI (Figure 1C) that revealed diffuse myocardial edema without fibrosis in a pattern diagnostic for myocarditis.At the outside institution, she was started on flecainide 50 mg twice a day.Despite these interventions, she continued to have frequent episodes of highly symptomatic ventricular tachycardia, prompting her referral to our program.Despite her diagnosis, the patient does not take medication to control her Crohn's disease.
Cardiogenetics 2024, 14, FOR PEER REVIEW 2 her clinical manifestation may likely be a ributed to well-established mechanisms of reentrant VT in myocarditis, we are intrigued by the possibility that her single nucleotide variant in ANK-2, which is known to be associated with arrhythmic risk [5], may have contributed to her disease state.This furthers the well-established understanding that underlying genetic architecture may serve as a basis for driving the severity of disease states.

Clinical History
A 53-year-old Caucasian female with a past medical history significant for Crohn's disease and asthma developed COVID-19 in February of 2021.Approximately three weeks after her COVID-19 diagnosis, she began to experience chest pressure, rapid palpitations, and shortness of breath.At an outside institution, she underwent a standard evaluation, consisting of an EKG, an echocardiogram, and a Holter monitor.Her echo revealed a normal LVEF, no valve concerns, and RVSP, and her Holter revealed salvos of ventricular tachycardia.She underwent cardiac catheterization, which demonstrated normal angiographic coronaries (Figure 1A,B), and subsequently underwent a cardiac MRI (Figure 1C) that revealed diffuse myocardial edema without fibrosis in a pa ern diagnostic for myocarditis.At the outside institution, she was started on flecainide 50 mg twice a day.Despite these interventions, she continued to have frequent episodes of highly symptomatic ventricular tachycardia, prompting her referral to our program.Despite her diagnosis, the patient does not take medication to control her Crohn's disease.

Myocarditis and Ventricular Tachycardia (VT)
Myocarditis is an immune-mediated inflammation of the heart muscle that can result in an electrical storm during the acute inflammatory phase, which is a state that can be resistant to anti-arrhythmic therapy.An electrical storm is defined as three or more separate episodes of sustained ventricular tachycardia (VT) within 24 h.Sustained VT with hemodynamic instability calls for prompt cardioversion due to it being a state of poor perfusion.Amiodarone may be used both as a preventative measure and for treatment of recurrent monomorphic VT [6].A recent study showed that polymorphic and irregular ventricular arrhythmias are more common during the active inflammatory phase, whereas monomorphic and regular ventricular arrhythmias are associated with healed myocarditis [7].There are a variety of hypotheses as to what could be triggering viralinduced myocarditis and subsequent ventricular arrhythmia periods.Myocardial fibrosis and secondary hypertrophy could favor a regional slowing of action potentials, resulting in re-entry circuits.Small focal inflammation could also explain electrically sensitive myocardial areas, or abnormal function of myocardial ion channels, all leading to VT [8].

Myocarditis and Ventricular Tachycardia (VT)
Myocarditis is an immune-mediated inflammation of the heart muscle that can result in an electrical storm during the acute inflammatory phase, which is a state that can be resistant to anti-arrhythmic therapy.An electrical storm is defined as three or more separate episodes of sustained ventricular tachycardia (VT) within 24 h.Sustained VT with hemodynamic instability calls for prompt cardioversion due to it being a state of poor perfusion.Amiodarone may be used both as a preventative measure and for treatment of recurrent monomorphic VT [6].A recent study showed that polymorphic and irregular ventricular arrhythmias are more common during the active inflammatory phase, whereas monomorphic and regular ventricular arrhythmias are associated with healed myocarditis [7].There are a variety of hypotheses as to what could be triggering viral-induced myocarditis and subsequent ventricular arrhythmia periods.Myocardial fibrosis and secondary hypertrophy could favor a regional slowing of action potentials, resulting in re-entry circuits.Small focal inflammation could also explain electrically sensitive myocardial areas, or abnormal function of myocardial ion channels, all leading to VT [8].
Upon our review and evaluation of external records, we discontinued flecainide given the presence of myocardial edema/injury on cardiac MRI, and advised a Class IB agent, mexiletine.We rationalized this agent specifically due to our suspicion that her VT originated from depolarized myocarditis associated tissue, resembling a state of injury that can be seen in ischemia.
Mexiletine is a Class IB anti-arrhythmic drug (AAD).Its anti-arrhythmic properties are due to its ability to block fast sodium channels, thereby reducing phase 0 of the cardiac myocyte action potential.Oral mexiletine has proven to be an effective option for the long-term management and prevention of ventricular arrhythmia from various etiologies, including ischemic cardiomyopathy and acute coronary syndrome [9,10].Class IB anti-arrhythmics bind with the highest affinity and avidity to sodium channels in their depolarized state, which elucidates their effectiveness in preventing ventricular arrhythmias associated with ischemia.Specifically, class IB AADs cause prolongation of the effective refractory period (ERP) and, consequently, prolongation of post-repolarization refractoriness in ischemic cells [11].Mexiletine is classified as a Class IB anti-arrhythmic agent, according to the Vaughan-Williams classification system.Class IB drugs, including mexiletine, primarily work by blocking fast sodium channels in cardiomyocytes, preferentially the Nav1.5 in the inactivated state.This reduces the influx of sodium ions into cardiomyocytes during phase 0 of the cardiac myocyte action potential [12].This allows more time for the refractory period and decreases the upstroke velocity, which decreases the action potential duration.The end effect is helping to stabilize the heart rhythm [13].Given the case features, mexiletine offered a logical alternative for the management of ventricular arrhythmias in our patient with ischemic-like tissue who had failed her first anti-arrhythmic.
Along with initiation of mexiletine, we started a low-dose beta blocker for adrenergic attenuation and titrated slowly without any exacerbation of her reactive airway disease.We monitored her progress using mobile cardiac telemetry (MCOT) and were pleased to see complete amelioration of ventricular tachycardia.We initiated Aldactone 25 mg once daily to further manage concomitant hypertension while providing potential anti-fibrotic effects for her at-risk myocardium.Aldactone is a mineralocorticoid receptor (MR) antagonist that impacts fluid, electrolyte, and hemodynamic homeostasis, and plays an important role in tissue remodeling.When mineralocorticoids are hyperactive, there is an increase in tissue inflammation leading to fibrosis.Blockade of these MRs plays a substantial role in limiting fibrotic effects in those with an at-risk myocardium [14].
In her external data, we noted QT prolongation on her index/initial presenting EKG (Figure 2A).Further Kardia and holter monitor data demonstrated salvos of VT (Figure 2B,C).There was no discernible family history of sudden death or known LQTS, and there were no contributions from iatrogenic pharmacologic agents.Though we believed that her ventricular tachycardia was likely secondary to a re-entrant mechanism related to myocarditis-associated injury, her prolonged QT on initial EKG raised suspicion that the severity of her VT was partly driven by underlying genetically mediated arrhythmogenic susceptibility.To probe this further and to guide our long-term management, we pursued clinical genetic testing.

Genetic Testing Results
Sequencing demonstrated a novel single nucleotide variant in ANK2: c.3074G>C p.G1025A.This change results in a glycine to alanine amino acid change, two amino acids with differing properties (Table 1).

Clinical Outcome
After 6 months of mexiletine, b-blocker, and Aldactone therapy, demonstration of myocarditis resolution by repeat cardiac MRI (Figure 3A), and complete absence of any VT by extended Holter monitoring, mexiletine was weaned off (Figure 3B).Interestingly, as shown in Figure 3A, an EKG in this resolution phase demonstrated normalization of her QT interval.We suspect this normalization occurred due to the complete healing of the myocardium, thereby removing this "inducible" factor that appears to have interacted with her ANK-2 susceptibility genotype during acute myocarditis.The patient remains symptom-free with a normal QT on B-blockers.3A), and complete absence of any VT by extended Holter monitoring, mexiletine was weaned off (Figure 3B).Interestingly, as shown in Figure 3A, an EKG in this resolution phase demonstrated normalization of her QT interval.We suspect this normalization occurred due to the complete healing of the myocardium, thereby removing this "inducible" factor that appears to have interacted with her ANK-2 susceptibility genotype during acute myocarditis.The patient remains symptom-free with a normal QT on B-blockers.

Ankyrin-2
The ANK2 gene is located on the long arm of chromosome 4 (4q25-q276) in humans.The gene spans over 500 kb and contains 61 exons that can be alternatively spliced to generate multiple isoforms of the ankyrin-B protein (Figure 4).Ankyrin-B is a relatively large protein, consisting of over 2400 amino acids.It plays an important role in the regulation of the sodium/calcium exchanger in cardiomyocytes [19].

Ankyrin-2
The ANK2 gene is located on the long arm of chromosome 4 (4q25-q276) in humans.The gene spans over 500 kb and contains 61 exons that can be alternatively spliced to generate multiple isoforms of the ankyrin-B protein (Figure 4).Ankyrin-B is a relatively large protein, consisting of over 2400 amino acids.It plays an important role in the regulation of the sodium/calcium exchanger in cardiomyocytes [19].Ankyrin-B plays a critical role in the regulation of ion channels in cardiomyocytes, including those for potassium, sodium, and calcium ions.Ankyrin-B helps to anchor and regulate the activity of potassium channels, including the KCNQ1 and HERG channels.Mutations in the ANK2 gene can disturb the activity of potassium channels, which can cause prolongation of the QT interval and increase the risk of arrhythmias, including LQTS [17].
Ankyrin-B affects cardiac contraction by ensuring that the sodium/calcium ex- Ankyrin-B plays a critical role in the regulation of ion channels in cardiomyocytes, including those for potassium, sodium, and calcium ions.Ankyrin-B helps to anchor and regulate the activity of potassium channels, including the KCNQ1 and HERG channels.Mutations in the ANK2 gene can disturb the activity of potassium channels, which can cause prolongation of the QT interval and increase the risk of arrhythmias, including LQTS [17].
Ankyrin-B affects cardiac contraction by ensuring that the sodium/calcium exchanger, Na/K ATPase, and InsP3 receptors are located in the sarcoplasmic reticulum [20].Mutations in the ANK2 gene can cause mis-localization or altered activity of sodium channels, which can impair electrical signaling and increase the risk of arrhythmias.Ankyrin-B also interacts with several calcium channels, including L-type calcium channels.Changes to L-type calcium channels can decrease heart contractility [15].Some ANK2 variants are associated with increased susceptibility to certain cardiovascular disorders such as LQTS and arrhythmias, while others may have protective effects.For example, the SNP rs180432 on the ANK2 gene is associated with an increased risk of developing ventricular arrhythmia [21].

Discussion: Myocarditis, ANK2, and VT
Our patient's genetic testing revealed a single nucleotide variant in ANK2 c.3074G>C p.G1025A.We suspect that this missense mutation exerted an effect on the activity of the ion channels in her cardiomyocytes during the stress of her myocarditis.The COVID-19induced myocarditis served as a "second hit" compounding the effects of the change in ion channel activity and led to an "inducible" long QT syndrome, which ultimately manifested in her as highly symptomatic VT.To our knowledge, this is the first description of a c.3074G>C p.G1025A nucleotide variant of ANK2 associated with myocarditis-related VT.
Though VT was present in this patient case, myocarditis has been previously associated with various cardiac rhythm irregularities, including both bradyarrhythmias and tachyarrhythmias.These abnormal rhythms may present at any time during the disease course, and all offer a variety of presenting symptoms.Of note, unexplained premature ventricular complexes (PVCs) in children or adults are frequently secondary to underlying myocarditis [22].These ventricular beats may be an asymptomatic finding on an ECG, or may be clinically recognized as palpitations, chest discomfort, and dizziness [23].Novel ECG strategies have been utilized to isolate the origin of PVCs in patients, with the most common sites of origin being the left ventricular outflow tract (LVOT) and the right ventricular outflow tract (RVOT).These strategies revealed that with respect to PVCs that originate specifically from the RVOT, subclinical myocarditis appears to be the underlying mechanism [23].In our case, we unfortunately did not have a PVC captured on a 12 lead EKG during the acute myocarditis phase to aid conclusions regarding a PVC axis, i.e., RVOT basal, LVOT basal, or elsewhere.These known arrhythmogenic properties of myocarditis, paired with the known cause-effect relationship of COVID-19 with cardiac arrythmias [24], further support the hypothesis of a multifactorial etiology responsible for our patient's presentation.
Emerging studies have looked at the genetic susceptibility in humans to COVID-19.Various human leukocyte antigen (HLA) types have been associated with worse or better prognosis in the setting of COVID-19 infection.This is due to the ability of certain HLA types to be more or less efficient at presenting the SARS-CoV-2 peptide [25].In addition, non-classical HLA ligands can be upregulated by the virus and cause an inhibitory effect on the immune system.This leads to a decrease in T cells, NK cells, B cells, and macrophages [26].Other HLA types were also found to have a role in response to the COVID-19 vaccine and may indicate an important finding for vaccine selection [27].Other genetic polymorphisms, some of which are on chromosomes 3 and 9, were found to impact COVID-19 susceptibility as well.These genetic differences allowed the SARS-CoV-2 virus enhanced access to host cells and increased viral production, along with a decreased immune response to the virus [25,28].These genes affect immune regulation, emphasizing the importance of immunogenetics on COVID-19 susceptibility.Interestingly, our subject had an underlying history of established Crohn's disease, and we are intrigued by the pos-sibility that this underlying autoimmune condition may have influenced her susceptibility to myocarditis in the setting of her acute SARS-CoV-2, with her ANK2 SNV influencing the manifestation of her VT.This provides an alluring "multi-hit" hypothesis that should prompt further studies to explore the intersection of the effects of immunogenicity and genetically mediated arrhythmic risk on COVID-19 myocarditis.

Conclusions
These findings further advance the literature surrounding disease phenotypes being influenced by underlying genetic modifications.They also introduce new variants in ANK-2 as a potential source of susceptibility for the development of ventricular tachycardia in patients that are suffering from myocarditis secondary to various etiologies, including infection with SARS-CoV-2.Mutations in ANK2 can disturb the activity of potassium channels, sodium channels, and calcium channels, leading to increased arrhythmia risk.The potential mechanisms behind myocardial injuries and ventricular arrhythmias were explored, including dysregulated immune response, renin-angiotensin-aldosterone system dysregulation, and myocardial injury.This case study suggested that the patient's ventricular tachycardia was likely due to a re-entrant mechanism related to myocarditis-associated injury.The patient's single nucleotide variant in ANK2 (c.3074G>C p.G1025A) was described, and its potential influence on ion channel activity during myocarditis was suggested as a contributing factor to the severity of ventricular tachycardia.The normalization of the QT interval during the resolution phase further supported the potential influence of the genetic variant in ANK2 on potassium channel activity.
This report emphasizes the importance of rationally selected anti-arrhythmic therapy in managing ventricular tachycardia associated with COVID-19 myocarditis, and suggests a potential role for a genetic variant in ion channel genes enhancing the myocarditis disease state to VT susceptibility and severity.We anticipate that this report will lead to further research exploring the genetic and immunogenetic factors contributing to COVID-19 outcomes, and their implications for personalized treatment approaches.

Figure 3 .
Figure 3. (A) Cardiac MRI demonstrating a normal T1/T2 weighted cardiac myocardium without fibrosis or edema.(B) EKG 6 months post-treatment, showing normalization of the QT interval.

Figure 3 .
Figure 3. (A) Cardiac MRI demonstrating a normal T1/T2 weighted cardiac myocardium without fibrosis or edema.(B) EKG 6 months post-treatment, showing normalization of the QT interval.

Figure 4 .
Figure 4.The ANK2 gene contains 61 exons and is responsible for the formation of the large Ankyrin-B protein product.Various mutations have been isolated on the ANK2 gene including R990Q, T1404I, and E1813K.The G1025A mutation, highlighted in yellow, was present in our patient [18].

Figure 4 .
Figure 4.The ANK2 gene contains 61 exons and is responsible for the formation of the large Ankyrin-B protein product.Various mutations have been isolated on the ANK2 gene including R990Q, T1404I, and E1813K.The G1025A mutation, highlighted in yellow, was present in our patient [18].
After 6 months of mexiletine, b-blocker, and Aldactone therapy, demonstration of myocarditis resolution by repeat cardiac MRI (Figure