Buccal cell diagnosis of risk in familial arrhythmia syndromes

Abstract

Described herein are, inter alia, methods for identifying subjects as being at risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or predicting positive response to anti-inflammatory drug therapies, e.g., in subjects who have inflammatory familial cardiac arrhythmias including arrhythmogenic cardiomyopathy (ACM) and Brugada syndrome (BrS), by detecting activated NFκB signaling in buccal cells. The methods can also include selecting and / or administering a treatment for the disease to the subject.

Description

[0001] BUCCAL CELL DIAGNOSIS OF RISK IN FAMILIAL ARRHYTHMIA SYNDROMES

[0002] CLAIM OF PRIORITY

[0003] This application claims priority under 35 USC §119(e) to U.S. Patent Application Serial No. 63 / 461.886, filed on April 25, 2023. The entire contents of the foregoing are hereby incorporated by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government support under Grants No. HL 116906 and HL 148348 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] Described herein are, inter alia, methods for diagnosing increased risk of myocardial injury and arrhythmias by detecting activated NFKB signaling in buccal cells, e.g., in subjects with inflammatory' familial cardiac arrhythmias including arrhythmogenic cardiomyopathy (ACM) and Brugada syndrome (BrS). The methods can also include selecting and / or administering a treatment for the disease to the subj ect.

[0008] BACKGROUND

[0009] Hereditary' or familial arrhythmia syndromes are found in over 50% of all initially unexplained cases of sudden cardiac death in persons under age 40 (Beckman et al., Dtsch Arztebl Int. 201 1 Sep; 108(37): 623-634). Early diagnosis and treatment can reduce the risk of sudden death.

[0010] SUMMARY

[0011] The present inventors have discovered that buccal mucosa cells (non-comified squamous epithelial cells lining the inside of the cheek) from subjects with familial arrhythmia syndromes associated with chronic inflammation, which as shown herein includes arrhythmogenic cardiomyopathy (ACM) and Brugada syndrome (BrS), exhibit changes in NFKB activation that correspond to those seen in cardiac tissues in subjects with myocardial injury, arrhythmias, and sudden cardiac death. The presence of a RelA / p65 nuclear signal indicates NFKB activation and activation of innate immune signaling that is associated with increased risk of myocardial injury and arrhythmias, e.g., in familial arrhythmia syndromes, e.g., ACM and / or BrS. Buccal mucosa cells can be easily and safely obtained, e.g., by simply rubbing the inside of the cheek, e.g., with a swab or scraper; the cells can then be smeared on a slide for subsequent study; larger amounts of cells can also be collected, e.g., using a brush (even a toothbrush) and maintained in tissue culture which allows further analysis in living cells. Thus, the present invention includes methods for determining risk of consequent myocardial inj nrv. arrhythmias, and sudden cardiac death that include obtaining a sample comprising buccal cells, staining the sample for RelA / p65, and detecting the subcellular localization of those proteins. The presence of a RelA / p65 nuclear signal also indicates the presence of or risk of developing a symptomatic familial arrhythmia syndrome, e.g., ACM and / or BrS.

[0012] Thus, provided herein are methods for determining risk, or risk stratification, in subjects, e g., subjects with familial inflammatory’ arrhythmia syndromes including arrhythmogenic cardiomyopathy (ACM) and Brugada syndrome (BrS) Brugada syndrome (BrS). The methods can include obtaining a sample comprising buccal cells from the subject; detecting level and / or localization of RelA / p65 in the subject sample; comparing the level and / or localization of RelA / p65 in the subject sample to a reference level or localization; and optionally determining the presence of risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death and / or predicting positive response to anti-inflammatory drug therapies in the subject when the level in the subject sample is above the reference level, or when the localization differs from the reference localization (i.e., when RelA / p65 is present in the nucleus, at levels above a reference level).

[0013] In some embodiments, the subject may have a heart disease, e.g., a familial inflammatory arrhythmia syndrome, e.g., ACM or BrS, or a disease other than ACM or BrS, in which innate immune signaling in cardiac muscle cells promotes myocardial injury and arrhythmias and in whom anti-inflammatory therapy may be beneficial.

[0014] In some embodiments, the reference level is a level in a reference subject or cohort of reference subjects who do not have a familial inflammatory arrhythmia syndrome, e.g.. does not have BrS or ACM, or a reference subject or a cohort of reference subjects, who do not have myocardial injury, arrhythmias, or sudden cardiac death.

[0015] In some embodiments, the reference localization is localization in a subject who does not have BrS or ACM, or who does not have increased risk of myocardial injury, arrhythmias, or sudden cardiac death, e.g., localization outside the nucleus.

[0016] In some embodiments, the buccal cells were obtained by rubbing the inside of the cheek of the subject with a swab, spatula, or scraper.

[0017] In some embodiments, the methods include applying the buccal cells onto a surface, e.g., a slide, optionally fixing the cells, e.g., with ethanol, and contacting the cells with an antibody or antigen-binding fragment thereof that binds to RelA / p65 protein.

[0018] In some embodiments, the methods include contacting the subject sample with antibodies to RelA / p65.

[0019] In some embodiments, the antibodies are directly or indirectly labeled, and the method comprises detecting the labeled antibodies.

[0020] In some embodiments, the subject is at least 7 years of age, e.g., 8, 9, 10, 11, 12, or 13 years of age or at any age in which it first appears that the subject may have a familial arrhythmia syndrome.

[0021] In some embodiments, the methods further include determining whether a subject identified as having a familial inflammatory arrhythmia syndrome is at risk of myocardial injury, arrhythmias, and sudden cardiac death.

[0022] In some embodiments, the methods include selecting a subject diagnosed with BrS, ACM, or other types of heart disease such as dilated and hypertrophic cardiomyopathies and cardiac sarcoidosis, and determining whether the subject is at risk of myocardial injury, arrhythmias, and sudden cardiac death, using a method described herein for treatment.

[0023] In some embodiments, the methods include administering a treatment to the subj ect.

[0024] For example, in subjects diagnosed with or identified as having BrS and being at risk of developing myocardial injury, arrhythmias, or sudden cardiac death, a treatment comprising one or more of recommending or prescribing or administering an anti-inflammatory treatment, e.g., an NFKB inhibitor or an anti-CD14 antibody or any other small molecule or antibody that interferes with innate immune responses including those that prevent activation of NFKB pathways or prevent nuclear translocation of the pRelA / p65- NFKB complex; recommending or advising the subject to avoid strenuous or intense physical activity or exercise; recommending or prescribing or administering one or more of quinidine, tedisamil, and / or isoproterenol; recommending avoiding administration of agents that exacerbate BrS, including antiarrhythmic agents that block sodium channels; recommending or performing radiofrequency catheter cardiac ablation; or recommending or implanting an implantable cardiac defibrillator (ICD).

[0025] In subjects diagnosed with or identified as having ACM and being at risk of developing myocardial injury, arrhythmias, and sudden cardiac death, the methods can include recommending or prescribing or administering an anti-inflammatory treatment, e.g., an NFKB inhibitor or an anti-CD14 antibody; recommending or advising the subject to avoid strenuous or intense physical activity or exercise; recommending or prescribing or administering one or more Singh Vaughan Williams class II antiarryhthmics (beta blockers) such as propranolol, esmolol, timolol, metoprolol, or atenolol; recommending or prescribing or administering one or more class III anti-arrhythmics (K-channel blockers) such as amiodarone, sotalol, ibutilide, dofetilide, dronedarone or E-4031; recommending or performing cardiac ablation; or recommending or implanting an implantable cardiac defibrillator (ICD).

[0026] In some embodiments, the methods include one or more of recommending or prescribing or administering an anti -infl ammatory treatment, e g., an NF B inhibitor or an anti-CD14 antibody.

[0027] Also provided herein are kits for use in a method described herein. The kits can include antibodies to RelA / p65, and optionally one or more of: instructions for use in a method described herein; a swab, scraper, or spatula for obtaining buccal cells from a subject; reagents for detecting the antibodies; a slide for receiving the buccal cells; and a fixative reagent for fixing the cells.

[0028] In some embodiments, the antibodies are directly or indirectly labeled.

[0029] In some embodiments, the fixative reagent comprises ethanol (e.g., 70% to 90% ethanol), methanol, glutaraldehyde, paraformaldehyde, formaldehyde, and / or saponin or a commercially available spray-on cytology fixative comprising polyglycol in alcoholic solution. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0030] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0031] DESCRIPTION OF DRAWINGS

[0032] Figure 1. Top: Representative immunoperoxidase staining for RelA / p65 in human myocardial samples. Cardiac myocytes show7strong nuclear signal (arrows) in ACM and BrS but not in controls or in the classic ion channelopathies including long QT syndromes types 1 and 2 (LQT 1 and LQT2), and catecholaminergic polymorphic ventricular tachycardia (CPVT). Cytoplasmic RelA / p65 signal is also increased in ACM and BrS samples. Botom: CCR2+ cells are increased in number in hearts of ACM and BrS patients but not in controls or ion channelopathies (note: few er cases were available for CCR2 studies than RelA / p65 staining).

[0033] Figures 2A-E. (A) Examples of RelA / p65 immunoperoxidase staining in myocardial samples from controls and ACM patients. Brown nuclear signal (arrow s) in cardiac myocytes occurred only in ACM samples. (B) Examples of CCR2 immunofl uorescent staining in myocardial samples from controls and ACM patients. Red CCR2-immunoreactive signal (arrows) was seen in small interstitial cells. (C) The number of CCR2+ cells / mm2 in control and ACM patient heart sections; mean ± SEM; * P <0.0001 vs controls; 1-way ANOVA with Tukey multiple comparison test. (D) Examples of RelA / p65 immunofluorescent staining in buccal cells from a control subject and ACM patients with stable disease (pACM16). at initial manifestation of disease (pACM13, or during a ‘hot phase’ of disease deterioration (pACM27). Nuclear RelA signal (white arrows) occurred only in patients undergoing phenoconversion or ‘hot phases’. (E) Immunostaining of buccal mucosa cells obtained from a healthy control patient and from 3 young patients, ages 9 - 17 years, with documented disease-causing ACM variants who had previously shown no clinical evidence of disease. Buccal cells from the 3 ACM patients were obtained at the time of initial presentation of disease including new onset of heart failure, new arrhythmias and pathologic changes on the body surface electrocardiogram and new pathologic changes seen on cardiac imaging studies. Cells were stained with DAPI to show nuclei and with an antibody against RelA / p65. Nuclear signal for RelA / p65 is seen in buccal cells in all 3 ACM patients but not in the control patient.

[0034] Figure 3. NFKB is activated in buccal cells in a patient with Brugada Syndrome. Immunostaining of buccal mucosa cells obtained from a healthy control patient and from a patient with clinically documented Brugada syndrome (BrW). Cells were stained with DAPI (blue) to show nuclei and with an antibody against RelA / p65 (red). Nuclear signal for RelA / p65 is seen in buccal cells of the Brugada patient but not in the control patient.

[0035] DETAILED DESCRIPTION

[0036] Hereditary arrhythmia syndromes are a maj or cause of sudden death in young people. These syndromes include arrhythmogenic cardiomyopathy (ACM) and Brugada syndrome (BrS) and other familial non-ischemic heart muscle diseases including dilated and hypertrophic cardiomyopathies including Naxos disease and Carvajal syndrome. As shown herein, subjects with increased risk of myocardial injury’ and arrhythmias, e.g., in ACM and BrS, have nuclear signal for RelA / p65 in their cardiac myocytes and buccal mucosa cells indicating activation of innate immune signaling. These subjects have elevated levels of NFKB in cardiac tissue that, as shown herein, are replicated in cells of the buccal mucosa. These findings provide evidence of a common pathophysiology that can be used to diagnose, risk-stratify and treat subjects with these diseases, with the goal of reducing the risk of sudden cardiac death.

[0037] As previously described, immunohistochemical analysis of a conventional endomyocardial-biopsy sample for levels and localization of certain marker proteins, e.g., nuclear localization of RelA / p65. has been shown to be characteristic of ACM. However, this still requires cardiac tissue, obtained using an invasive procedure. While it may be reasonable to biopsy the heart of an individual in whom the disease has clinically manifested and who has exhibited signs and symptoms associated with potentially life-threatening arrhythmias, it is not feasible to acquire cardiac tissue samples from family members who may carry a possible disease allele but show no evidence of the disease, or from members of the general public to use as a preventive screening measure.

[0038] The present inventors have discovered that buccal mucosa cells (non-comified squamous epithelial cells lining the inside of the cheek) from subjects with increased risk of developing myocardial injury and arrhythmias or who have recently developed such disease features, e.g.. who have ACM and BrS, exhibit the same changes in localization of RelA / p65 that are seen in heart tissues. Nuclear localization of RelA / p65 indicates that innate immune signaling is activated in the heart, which can be used for risk stratification and identifying patients most likely to benefit from antiinflammatory therapy designed to block innate immune responses in the heart. Buccal mucosa cells can be easily and safely obtained, e.g., by simply rubbing the inside of the cheek with a swab or scraper; the cells can then be smeared on a slide for subsequent study. Thus, the present invention includes methods for the determination of risk of a cardiac event in subjects with inflammatory cardiac arrhythmogenic syndromes, including ACM and BrS, including predicting risk of consequent arrhythmias and sudden cardiac death, that include obtaining a sample comprising buccal cells, staining the sample for RelA / p65 proteins, and detecting the presence, level, and / or localization of those proteins. Nuclear localization of RelA / p65 proteins indicates the presence of or risk of developing an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS.

[0039] Alternatively or in addition, larger amounts of buccal cells can also be collected, e.g., using a brush (even a toothbrush) and maintained in tissue culture which allows further analysis in living cells.

[0040] Arrhythmogenic cardiomyopathy (ACM)

[0041] Arrhythmogenic cardiomyopathy (ACM), also known as arrhythmogenic right ventricular cardiomyopathy in particular (ARVC). is associated with a high frequency of arrhythmias and sudden cardiac death (Marcus et al.. Circulation 1982;65:384-98; Thiene et al., N Engl J Med 1988;318:129-33; Dalal et al.. Circulation 2005;112:3823-32). Mutations in genes encoding desmosomal proteins (including desmoplakin. plakoglobin, plakophilin 2, desmocollin 2, and desmoglein 2) have been identified in approximately 60% of patients with ARVC (te Riele et al.. J Cardiovasc Magn Reson. 2014;16:50). However, genetic analysis remains mainly a research tool, and in every' day practice the determination of risk in subjects with ACM can be challenging. The clinical presentation may be highly variable, and the genetic penetrance is often low. This is especially true in family members of an index patient in whom establishing a determination of risk in subjects with ACM or ARVC requires extensive clinical workup, often leading to equivocal results. Endomyocardial biopsy has not been consistently useful because the structural changes in ARVC tend to spare the subendocardium and do not typically involve the interventricular septum (Thiene et al., 1988, supra) which are the locations in the heart that are typically sampled during conventional endomyocardial biopsy. Thus, the pathological features of ACM / ARVC are often not seen in conventional endomyocardial -biopsy specimens. Moreover, these pathological features tend to be most conspicuous in patients with severe disease and are not well developed in patients with early disease. In the end, the diagnosis usually rests on fulfilling a set of clinical criteria; see, e.g., Marcus et al., Circulation, 2010; 121:1533-1541. Although these criteria are relatively specific, they are not highly sensitive.

[0042] Brugada syndrome (BrS)

[0043] Brugada syndrome (BrS) is an inherited arrhythmogenic disease associated with ST segment elevation in the right precordial leads (VI -V3) and right bundlebranch block.1’4It is estimated to occur in -5 of every 10,000 people and may account for 4 to 12% of all sudden deaths.2It affects both men and women but is 5-8 times more frequent in men.2Given that the frequency of driver mutations should be equally distributed, little is known about why BrS is much more common in men. It may be clinically manifest at any age but most often occurs in young or middle-aged men /

[0044] Coding variants in SCN5A. the gene for the cardiac Na+channel protein Navi.5 (which generates the cardiac Na+current iNa), are found in -20% of BrS cases.6,7Other disease-associated alleles are largely unknown, and little is known about fundamental mechanisms driving disease expression.2A recent GWAS8identified 10 new loci associated with BrS. The gene nearest to one new locus, MAPRE2, encodes the microtubule plus-end binding protein EB2, which plays a key role in organizing microtubules. New functional studies point to a defect in Navi.5 trafficking via microtubules in BrS.8As detailed below, a similar mechanism involving defects in protein trafficking of Cx43 and Navi.5 has been implicated in the pathogenesis of ACM.9

[0045] BrS was originally thought to be an ion channelopathy, but most experts now consider it to be a cardiomyopathy.2Although hearts of some sudden death victims with BrS show only limited structural abnormalities, it has become increasingly clear that a majority of patients exhibit pathologic remodeling, especially of the right ventricular outflow tract.10 11These changes include degenerative changes in cardiac myocytes, fibrosis and occasional fibrofatty replacement.11Structural changes in BrS hearts also include right ventricular dilatation, microaneuiysms seen by angiography and late gadolinium enhancement in the left ventricle.

[0046] Sudden deaths in ACM often occur in athletes engaged in strenuous physical activity,17and it is well established that exercise accelerates disease penetrance and increases risk of adverse outcomes.34The same may be true in BrS. Although data on exercise in BrS are insufficient to warrant definitive clinical guidelines, exercise has been shown to induce ST-segment elevation and monomorphic ventricular arrhythmia in BrS patients.35-39It also amplifies ventricular arrhythmia in BrS patients treated with the Na+channel blocker flecainide.37,40Mechanistic investigation of the contribution of exercise to BrS pathogenesis has focused on the activation-inactivation rates of the mutant Na+channels relative to heart rate in BrS.38A role for aberrant innate immune signaling in BrS cardiac myocytes has not been considered, despite growing evidence from us and others that inflammation promotes arrhythmias in ACM.5

[0047] Methods of Use - Risk Stratification and Treatment

[0048] Included herein are methods for identifying subjects in whom innate immune signaling is activated in the heart, for risk stratification (i.e., determining whether a subject is at increased risk of a cardiac event such as ongoing myocardial injury', arrhythmia, myocardial infarction, or sudden cardiac death, and identifying patients most likely to benefit from anti-inflammatory therapy designed to block innate immune responses in the subject’s heart, e.g., in a subject who has an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS. The methods rely on detection of levels and / or subcellular localization of RelA / p65 in a sample comprising buccal cells. These methods can be practiced on any subject, e.g., a subject who has had a cardiac event (such as ventricular arrhythmia or myocardial infarction, e.g., an event that is otherwise unexplained), or a subject who has had a first, second, or third- degree relative who has had a cardiac event (such as ventricular arrhythmia or myocardial infarction, or sudden cardiac death before 35 or 40 years of age, e.g., an event that is otherwise unexplained). A subject who has no family history or symptoms can also be screened using the methods described herein. In some embodiments, the subject is at least adolescent or post-pubertal, e.g., is at least 10, 11, 12. 13, 14, 15, 16, 17, 18, 19 or 20 years of age. or is at least 7, 8. or 9 years of age. In some embodiments, the subject is a newborn, or is 1, 2, 3, 4, 5 or 6 years of age.

[0049] The methods typically will include obtaining a sample from a subject, and evaluating the subcellular localization and / or level of the RelA / p65 protein in the sample, and comparing the subcellular localization and / or level with one or more references, e.g., a control reference that represents a normal level or subcellular localization of the protein, e.g., a level in an unaffected subject, and / or a disease reference that represents a level and / or subcellular localization of the protein associated with an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, e.g., a level or subcellular localization in a subject having an inflammatory cardiac arrhythmogenic syndrome, e.g., having ACM or BrS.

[0050] As used herein the term “sample”, when referring to the material to be tested for the presence or subcellular localization of a desmosomal protein(s) using a method described herein, includes buccal cells obtained by swabbing or scraping the inside of the cheek of a subject using known methods. In embodiments in which subcellular localization is evaluated, the cells are treated such that they retain their morphology and cytoskeleton substantially intact, though they may be fixed, lysed or permeabilized to permit entrance of antibodies or other labels; suitable methods for fixing the cells include exposing them to one or more of ethanol (e.g., 70% to 90% ethanol), methanol, glutaraldehyde, paraformaldehyde, formaldehyde, and / or saponin or a commercially available spray-on cytology' fixation, e.g., M-FIX spray fixative, comprising polyglycol in alcoholic solution. Other methods can be used, so long as the ability to determine subcellular localization of the cardiac intercalated disk or desmosomal proteins is retained if subcellular localization is to be evaluated.

[0051] An exemplary method for collection of buccal mucosa samples is as follows. A clean cotton-tipped applicator (e.g., a Q-tip™) or wood spatula or a soft cytology brush can be used for collection of the specimen. If the mouth is very’ dry, the applicator or spatula can be moistened (e.g., with saline or sterile water). Usually saliva is sufficient to prevent dehydration. In some cases, the mouth can be rinsed with sterile water before the sample is collected. Material is typically collected from the inside of the cheek by using slight rolling and scraping motions for about 30 seconds on each side. Immediately (e.g., within one minute) after collection, the buccal mucosa sample should be smeared on the center area of each slide (area approximately 2.5 x 2.5 centimeters). Immediately (e.g., within one minute) after smearing the sample on each slide, the slide can be placed in a beaker containing 70% ethyl alcohol, and left in the alcohol for about 1 minute. The slide should not be allowed to dry before being placed into the alcohol. The slide can then be removed and left at room temperature until the alcohol dries (about 10 minutes). Or, the cells smeared on the slide can be sprayed with a commercially available cytology fixative. Slides can be stored, e.g., at room temperature in a box with a tight fitting lid.

[0052] The level or subcellular localization of a protein can be evaluated using appropriate methods known in the art. The methods typically include revealing labels such as fluorescent, chemiluminescent, radioactive, and enzymatic or dye molecules that provide a signal either directly or indirectly. As used herein, the term “label” refers to the coupling (i.e. physical linkage) of a detectable substance, such as a radioactive agent or fluorophore to an antibody or probe, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances include, but are not limited to, the following: radioisotopes (e.g.,3H,14C,3’S,1211,131I), fluorescent labels, (e.g., FITC, rhodamine, lanthanide phosphors, phycoerythrin (PE) or indocyanine (Cy5)), luminescent labels such as luminol; and enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzy matic activity7that can be detected by optical or calorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g.. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualized by electron microscopy. With regard to subcellular localization of the RelA / p65 protein(s), a skilled practitioner can determine whether the desmosomal protein(s) are appropriately localized (e.g., at the cell surface or at cell-cell junctions) or mislocalized (e.g., dispersed within the cytoplasm or greatly reduced in cell surface signal intensity). In some embodiments, a method of detecting the desmosomal protein(s) in situ is used on intact cells such that the presence and subcellular localization of the desmosomal protein(s) can be detected. For example, an antibody that binds to a desmosomal protein is labeled with a detectable substance and the desmosomal protein(s) is localized in tissues and cells based upon the presence of the detectable substance. Known cytochemical methods, e.g., using light, fluorescent, or electron microscopy, can be used to determine the subcellular localization of the desmosomal protein(s). In some embodiments, immunohistochemistry (IHC) can be used, e.g., with either direct or indirect labelling. The sample may also be inspected by fluorescent microscopy when immunofluorescence (IF) is performed, as a variation to IHC. Radiography with radio-emitting labels can also be used. A skilled practitioner, reviewing the results of such a method (e.g., an image of the cells), can determine whether a desmosomal protein is appropriately localized (e.g., at a cell junction / the cell surface) or mislocalized (e.g., dispersed within the cytoplasm, and / or present at such low levels that it is not visualizable). Generally speaking, simple visual examination can be used to see if the signal at the cell surface is normal or greatly reduced. As shown in the figures, this is almost always a very clear distinction that does not require complicated measurements of signal intensity, but is immediately apparent upon visualization.

[0053] In some embodiments, the level of the protein can be determined in addition to or as an alternative to the subcellular localization of the protein.

[0054] In some embodiments, a score is calculated in which the mislocalization of the protein(s) is quantified; see, e.g., the Subcellular Localization Index described in US20150094224.

[0055] In some embodiments, standard electrophoretic and quantitative immunoassay methods can be used for determining levels of proteins, including (but not limited to), Western blot; enzy me linked immunosorbent assay (ELISA); biotin / avidin type assays; protein array detection; radio-immunoassay; immunohistochemistry (IHC); immune-precipitation assay; FACS (fluorescent activated cell sorting); mass spectrometry (Kim (2010) Am J Clin Pathol 134: 157-162; Yasun (2012) Anal Chem 84(14):6008-6015; Brody (2010) Expert Rev Mol Diagn 10(8): 1013-1022; Philips (2014) PLOS One 9(3):e90226; Pfaffe (2011) Clin Chem 57(5): 675-687). For example, an ELISA method may be used to determine the levels of a protein, wherein the wells of a mictrotiter plate are coated with an antibody against which the protein is to be tested. The sample containing or suspected of containing the biological marker is then applied to the w ells. After a sufficient amount of time, during which antibodyantigen complexes would have formed, the plate is washed to remove any unbound moieties, and a detectably labelled molecule is added. Again, after a sufficient period of incubation, the plate is washed to remove any excess, unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. Variations of the ELISA method, such as the competitive ELISA or competition assay, and sandwich ELISA, may also be used, as these are well-known to those skilled in the art.

[0056] Mass spectrometry, and particularly matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) and surface-enhanced laser desorption / ionization mass spectrometry (SELDI-MS), is also useful for the detection of levels of the proteins. (See U.S. Patent No. 5,118,937; 5,045,694; 5,719,060; 6,225,047)

[0057] In some embodiments, where the subcell ular (nuclear) localization and / or level of the RelA / p65 protein is comparable to the subcellular localization and / or level of the protein in the disease reference, and the subject optionally has one or more symptoms associated with an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS or another type of heart disease, then the subject is identified as at risk of (i.e., has an increased risk of experiencing) a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death and / or predicting positive response to anti-inflammatory drug therapies. In some embodiments, the subject has no overt signs or symptoms of an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, but the subcellular localization and / or level of the protein evaluated is comparable to the subcellular localization and / or level of the protein in the disease reference, then the subject is identified as at risk of a cardiac event, ongoing myocardial injury', arrhythmias, and sudden cardiac death and / or predicting positive response to anti-inflammatory drug therapies (i.e., an increased risk as compared to a subject who has normal subcellular localization (e.g., non-nuclear) and / or level of the protein). In some embodiments, once it has been determined that a person is at risk of a cardiac event, ongoing myocardial injury’, arrhythmias, and sudden cardiac death and / or predicting positive response to anti-inflammatory drug therapies, then a treatment, e.g., as known in the art or as described herein, can be administered.

[0058] Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful distribution of the RelA / p65 protein, e.g., a control reference level that represents a normal location of the protein, e.g., a nonnuclear location in an unaffected subject or a subject who is not at risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or a disease reference that represents a distribution of the protein associated w ith a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, e.g., nuclear localization in a subject having an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS.

[0059] Subjects associated with predetermined distribution are typically referred to as reference subjects. For example, in some embodiments, a control reference subject does not have a disorder described herein (e.g., an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS). In some cases it may be desirable that the control subject is a first degree relative of a subject with an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, and in other cases it may be desirable that a control subject is unrelated. In some cases it may be desirable that the control subject has an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS and in other cases it may be desirable that a control subject does not have an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS.

[0060] A disease reference subject is one who has (or has an increased risk of developing) a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, optionally a subject who has an inflammatory cardiac arrhythmogenic syndrome, e.g.. ACM or BrS or another type of heart disease. An increased risk is defined as a risk above the risk of subjects in the general population.

[0061] Thus, in some cases the distribution of the protein in a subject being less than or equal to a reference distribution of the protein is indicative of a clinical status, e.g., indicative of an increased risk of a cardiac event, ongoing myocardial injury', arrhythmias, and sudden cardiac death.

[0062] Antibodies that bind to RelA / p65, or antigen-binding fragments thereof, can be obtained commercially or using methods known in the art.

[0063] RelA / p65

[0064] The RelA / p65 protein is a ubiquitous transcription factor that has been shown to be involved in a number of biological processes. It is held in the cytoplasm as a heterodieric complex with NFkB in an inactive state by specific inhibitors. Upon degradation of the inhibitors, the RelA / p65-NFicB heterodimer translocates to the nucleus and once there it activates transcription of specific genes. NFKB is a complex composed of either an NFKB 1 or NFKB2 protein bound to a REL, RELA, or RELB protein. The most abundant form of NFKB is NFKB1 complexed with the RELA gene product.

[0065] At least four transcript variants encoding different isoforms have been described for the RELA gene. The following table provides GenBank RefSeq Accession numbers for the four major transcripts.

[0066] Variant 1 represents the predominant transcript and encodes the longer isoform 1. Variant 2 uses an alternate in-frame acceptor splice site at one of the coding exons compared to transcript variant 1, encoding a shorter isoform 2 that is missing a 3 aa segment compared to isoform 1. Variant 3 uses an alternate in-frame splice site at the 5' end of the last exon compared to variant 1, encoding isoform 3 that lacks an alternate internal segment compared to isoform 1. Lastly, variant 4 lacks an alternate internal in-frame segment in the last exon compared to variant, encoding isoform 4 that lacks an alternate internal segment compared to isoform 1. Generally speaking, the present methods can include detecting all of the isoforms. Methods of Treatment

[0067] In some embodiments, the methods described herein further include selecting and / or administering a treatment to a subject identified by a method described herein as having or being at risk for an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS. In some embodiments, the methods include one or more of recommending or prescribing or administering an anti-inflammatory treatment, e g., an NFKB inhibitor or an anti-CD14 antibody, or any small molecule or antibody that blocks innate immune signaling via NFkB. NFKB inhibitors are described in detail below. Anti-CD14 antibodies are known in the art and include antibody IC14 (atibuclimab), as well as 18D11 (LSBios), MEM-18 (Bio-Rad antibodies), 4B4F12 (AbCam), TUK4 (Miltenyi). REAL 107 (Miltenyi), and M5E2 (BD Biosciences) antibodies, antigen binding fragments thereof, and humanized and chimeric versions thereof. See, e.g., W02024082008 and W02024082009.

[0068] In subjects identified as being at risk and as having ACM, the methods can include recommending or advising the subject to avoid strenuous or intense physical activity or exercise; recommending or prescribing or administering one or more Singh Vaughan Williams class II antiarryhthmics (beta blockers) such as propranolol, esmolol, timolol, metoprolol, or atenolol; recommending or prescribing or administering one or more class III anti-arrhythmics (K-channel blockers) such as amiodarone, sotalol, ibutilide, dofetilide, dronedarone or E-4031; recommending or performing cardiac ablation; or recommending or implanting an implantable cardiac defibrillator (ICD).

[0069] In subjects identified as being at risk and as having BrS, e.g., a treatment comprising one or more of recommending or prescribing or administering an anti- CD14 antibody (see, e.g., W02024082008 and W02024082009); recommending or advising the subject to avoid strenuous or intense physical activity or exercise; recommending or prescribing or administering one or more of quinidine, tedisamil, and / or isoproterenol; recommending avoiding administration of agents that exacerbate BrS, including antiarrhythmic agents that block sodium channels; recommending or performing radiofrequency catheter cardiac ablation; or recommending or implanting an implantable cardiac defibrillator (ICD).

[0070] The present methods can also be used to stratify subjects in clinical trials, e.g., based on the presence of absence of nuclear RelA / p65. NFKB inhibitors

[0071] Stimulation of cell surface receptors by cytokines. LPS, antigens, etc., activates an IKK (IKB kinase) complex that phosphorylates IKB proteins, and thereby targets them for ubiquitination and degradation. The resulting free NFicB / Rel complexes are further activated by various post-translational modifications (phosphorylation, acetylation and / or glycosylation, reflecting actions of many regulatory enzymes), and translocate to the nucleus where they combine with other transcription factors to regulate gene expression in the immune response.

[0072] Inhibitors of NFKB thus could include the following types (from dow nstream to upstream): DNA binding inhibitors including GYY 4137, p-XSC, CV 3988, and Prostaglandin E2 (PGE2) that inhibit the binding between NFKB / Rel and its target DNA, thus inhibiting any gene expression activated by NFKB; inhibitors of post- translational modifications on NFKB / Rel, e.g. a p65 acetylation inhibitor, including Gallic acid and Anacardic acid that prevents NFKB from activating its target genes; translocation inhibitors including JSH-23, and Rolipram that prevents NFKB / Rel from translocating to the nucleus; IKB degradation inhibitors including BAY 11-7082, MG- 115, MG- 132, Lactacystin, Epoxomicin, Parthenolide, Carfilzomib, and MLN-4924 (Pevonedistat) that prevents ubiquitinated IKB from being degraded, thus maintaining IKB’S suppression of NFKB / Rel functions; IKK inhibitors including TPCA 1, NF-KB Activation Inhibitor VI (BOT-64). BMS 345541, Amlexanox, SC-514 (GK 01140), IMD 0354, and IKK-16 that prevent the phosphorylation of IKB and thus preventing the ubiquitination and degradation of IKB. In some embodiments, NFKB inhibitors are proteasome inhibitors including MG 132, bortezomib, carfilzomib, and ixazomib. In some embodiments, NFKB inhibitors inhibit nuclear translocation inhibitors including dehydroxymethylepoxy quinomicin (DHMEQ), small peptidomimetics, such as SN-50, which encompasses the NLS of p50. In some embodiments, NFKB inhibitors inhibit NFKB’S DNA binding, including sesquiterpene lactone (SL) compounds and decoy oligodeoxynucleotides.

[0073] Person of skills in the art will readily recognize additional types of NFKB inhibitors based on the mechanistic pathways involved, including, e.g., agents that can inhibit protein kinases, protein phosphatases, proteasomes, ubiquitnation, acetylation, methylation, and DNA binding steps have been identified as NFKB inhibitors. (Pires et al., Genes (Basel). 2018 Jan 9;9(1). pii: E24; Gupta, 2010 Oct-Dec; 1799(10- 12):775-87).

[0074] The contents of Pires et al.. Genes (Basel). 2018 Jan 9:9(1) and Gupta et al., Biochim Biophys Acta., 2010 Oct-Dec; 1799(10-12) are hereby incorporated by reference. Table 1 of Gupta et al., Biochim Biophys Acta., 2010 Oct-Dec;1799(10- 12) lists some of the known NFKB inhibitors. Person of skills in the art will understand that the inhibitors may be small molecules, biologies, or other types of agents that block the function of NFKB. In some embodiments, the NFKB inhibitors are antibodies against targets affecting NFKB functions. The antibodies may be blocking antibodies or agonistic antibodies depending on the involvement of the antibody’s target in NFKB functionality. Non-limiting examples of NFKB inhibitors also include 15d-PGJ(2), Calagualine, Conophylline, Evodiamine, Geldanamycin, Perrilyl alcohol, PSK, Rocaglamides, Adenovirus E1A, NS5A (Hep-C virus), Erbin overexpression, Golli BG21, KSR, MAST205, PEDF, Rituximab, TNAP, Betaine, Desloratadine, LY29 and LY30, MOL 294 , Pefabloc. Rhein, SMI and FP, [6]- gingerol, 1 '-Acetoxy chavicol acetate, 20(S)-ProtopanaxatrioL 4-Hydroxynonenal, Acetyl-boswellic acids, Anandamide, Anethole, Apigenin, Artemisia vestital, Baoganning, Betulinic acid, Buddlejasaponin IV, Cacospongionolide B, Calagualine, Cardamomin, Casparol, Cobrotoxin, Cycloepoxy don, Decursin. Dehydroascorbic acid, Dexanabinol. Digitoxin, Diosgenin, Diterpenes, Docosahexaenoic acid. Falcarindol, Flavopiridol, Furonaphthoquinone, Garcinone B, Glycine chloramine, Guggulsterone, Herbimycin A, Honokiol, Hypoestoxide, Indirubin-3'-oxime, Isorhapontigenin, Clarithromycin, Cloricromene, C-K and Rh(2), Cryptotanshinone, Cytochalasin D, Danshenshu, Diterpenoids, Ent-kaurane diterpenoids, Epinastine hydrochloride. Epoxy quinol A, Erythromycin, Evodiamine, Fucoidan, Gallic acid, Ganoderma lucidum, Garcinol, Geranylgeraniol, Ginkgolide B, Glycyrrhizin, Halofuginone, Hematein, Herbal compound 861, Hydroxy ethyl starch, Hydroxyethylpuerarin, Hypericin, Kamebakaurin. Linoleic acid, Lithospermi radix, Macrolide antibiotics, 2-methoxy estradiol, 6-MITC. Oridonin, Plant compound A, Polyozellin, Prenylbisabolane 3, Prostaglandin E2, PSK, Quinic acid, Sanggenon C, Sesamin, Shen-Fu, Silibinin, Sinomenine, Tansinones, Taurine + niacine, TZD MCC- 555, Trichostatin A, Triptolide, Tyrphostin AG-126, Ursolic acid, Withaferin A, Xanthohumol, Xylitol. Yan-gan-wan, Yin-Chen-Hao. Ghrelin, Peptide YY, Rapamycin, Adiponectin, Kahweol, Manumycin A, Monochloramine, N- acetylcysteine, Nitric oxide, Nitrosylcobalamin. Oleandrin, Omega 3 fatty acids, ox- LDL, Panduratin A, PEITC, Petrosaspongiolide M, Phytic acid, Piceatannol, Pinosylvin, Plumbagin, Prostaglandin Al, Quercetin, Rengyolone, Rosmarinic acid, Rottierin, Saikosaponin-d, Sanguinarine, Staurosporine, Sesquiterpene lactones, Scoparone, Silibinin. Silymarin, Sulforaphane, Sulindac, Tetrandine, Theaflavin, Thienopyridine, Tilianin, Ursolic acid. Vesnarinone, Wedel olactone, Withanolides, Xanthoangelol D, Zerumbone, P-carboline, y-mangostin, y-Tocotrienol, 11< I<[3 peptide, NEMO CC2-LZ peptide, Anti-thrombin in, Chorionic gonadotropin, FHIT, HB-EGF, Hepatocyte growth factor, Interferon-a, Interleukin- 10, PAN1, PTEN, SOCS 1, Adenovirus. MC159, MC160, Angiopoietin-1, Antithrombin, -catenin. Bromelain, CaMKK, CD43 overexpression, FLN29 overexpression, FLIP, G-120, Interleukin 4, Transdominant p50, VEGF, ADP ribosylation inhibitor, 7-amino-4- methylcoumarin, Amrinone, Atrovastat, Benfotiamine, Benzamide, Bisphenol A, Caprofen, Carbocisteine. Celecoxib, Gemcitabine, Cinnamaldehyde. 2-methoxy CNA, 2-hydroxy CNA, CDS, CP Compound, Cyanoguanidine, HMP, a- difluoromethylomithine, DTD, Evans Blue, Evodiamine, Fenoldopam, FEX, Fibrates, FK778, Flunixin meglumine, Flurbiprofen, Hydroquinone, IMD-0354, JSH-21, KT- 90, Lovastatin, Mercaptopyrazine, Mevinolin,, Monoethylfumarate, Moxifloxacin, Nicorandil. Nilvadipine, NO-ASA, Panepoxydone, Peptide nucleic acids. Perindopril, PAD, a-PBN, Pioglitazone, Pirfenidone, PNO derivatives, Quinadril, AIDCA derivative, TDZD, TPCA-1, Pyridine derivatives, ACHP, Acrolein, AGROIOO, Amino-pyrimidine, AS602868, Aspirin, Azidothymidine, BAY- 11 -7082, BAY-11- 7083. Benzoimidazole derivative, Benzyl isothiocyanate, BMS-345541, Carboplatin. CDDO-Me, CHS 828, Compound 5, Compound A, Cyclopentenones, CYL-19s, CYL-26z, Diaylpyridine derivative, DPE, Epoxy quinone, Gabexate mesilate, Gleevec, Hydroquinone, Ibuprofen, IQCAD, Indolecarboxamide, Isobutyl nitrite, Jesterone dimer, 15-deoxyspergualine analog, Methotrexate, MLB120, Monochloramine, MX781 (Retinoid antagonist), 4-HPR, Nafamostat mesilate, NSAIDs, PS-1145 (MLN1 145), PQD, Pyridooxazinone derivative, SC-514, Scytonemin, Sodium salicylate, Statins (several), Sulfasalazine, Sulfasalazine analogs, Survanta. Thalidomide, THI 52, YC-1, Lead, Mild hypothermia, Saline (low Na+), 5'- methylthioadenosine, Alachlor, Amentoflavone. Antrodia camphorata, Aucubin, Baicalein, Raxofelast, Ribavirin, Rifamides, Ritonavir, Rosiglitazone, Roxithromycin, DAAS. Serotonin derivative, Simvastatin, SM-7368, T-614, Sulfasalazine, SUN C8079, Tricl osan plus CPC, Tobacoo smoke. Verapamil, Heat (fever-like), Hypercapnic acidosis, Hyperosmolarity, Hypothermia, Alcohol, 4'-DM-6-Mptox, 4- phenylcoumarins, AHUP, Luteolin, Mesuol, Nobiletin, Phomol, Psychosine, Qingkailing, Saucemeol D & E, Shuanghuanglian, Trilinolein, Wortmannin, a- zearalenoL NF-kappaB-repression factor, PIAS3, PTX-B. 17-AAG. TMFC, AQC derivatives, 9-aminoacridine derivatives. Chromene derivatives, D609, Dimethylfumarate, EMDPC, Histidine, Mesalamine, PEITC, Pranlukast, RO31-8220 (PKC, inhibitor), SB203580 (MAPK inhibitor), Tetrathiomolybdate, Tranilast, Troglitazone. Catalposide, Cyclolinteinone, Dihydroarteanniun. Docosahexaenoic acid, Emodin, Ephedrae herba (Mao) extract, EquoL Erbstatin, Ethacrynic acid, Fosfomycin, Genipin, Genistein, Glabridin, Glucosamine sulfate, Isomallotochromanol, Isomallotochromene, Melatonin, Midazolam, Momordin I, Polymyxin B, Prostaglandin. Resiniferatoxin, Thiopental, Tipifamib, TNP-470, Ursodeoxycholic acid, P- PEITC, 8-MSO, -lapachone, Penetratin, VIP, Activated protein C, HSP-70, Interleukin- 13, Intravenous Ig, Murrl gene product, Neurofibromatosis-2 protein, PACAP, SAIF, a-MSH, y-glutamylcysteine synthetase, 1 -Bromopropane, Acetaminophen, Diamide, Dobutamine, Cyclosporin A, Lactacystine. -lactone, APNE, Boronic acid peptide. BTEE, 3.4- dichloroisocoumarin, Deoxyspergualin, DFP, Disulfiram, FK506 (Tacrolimus), Bortezomib, Salinosporamide A, 23-hydroxyursolic acid, Anetholdithiolthione, Apocynin, Arctigenin, Aretemisa p7F, Astaxanthin, Beni dipine, bis-eugenol, BG compounds, BHA, CAPE, Camosol. Carvedilol, Catechol derivatives, Celasterol, Cepharanthine, Chlorogenic acid, Chlorophyllin, Curcumin, DHEA, DHEA sulfate, Dehydroevodiamine, Demethyltraxillagenin, Diethyldithiocarbamate, Diferoxamine, Dihydroisoeugenol, Dihydrolipoic acid, Dilazep, Fenofibric acid, DMDTC, Dimethylsulfoxide, Disulfiram, Ebselen, Edaravone, EGTA, EPC-K1, Epigallocatechin-3-gallate. Ergothioneine. Ethyl pyruvate, Garcinol, y- glutamyl cysteine synthetase, Glutathione, Hematein, Hydroquinone, Hydroquinone, IRFI 042, Iron tetrakis, Isovitexin, Kangen-karyu extract, Ketamine, Lacidipine, Lazaroids, L-cysteine, Lupeol, Magnolol, Maltol, E-73, Ecabet sodium, Gabexate mesilate, Glimepiride, Hypochlorite, Losartin. LY294002, Pervanadate, Phenylarsine oxide, Phenytoin, Rol06-9920, Sabaeksan, U0126 (MEK inhibitor), 15- deoxyspergualin. 2',8"-biapigenin. 5F (from Pteri syeminpinnata), Alginic acid, Apigenin, Astragaloside IV, AT514 (serratamolide), Atorvastatin, Cantharidin, Chiisanoside, Clarithromycin, Eriocalyxin B, Hirsutenone, JM34, KIOM-79, Leptomycin B, Neomycin, Nucling, Oregonin, OXPAPC, Paeoniflorin, Phallacidin, Piperine, Pitavastatin, Rapamycin. Selenomethionine, Shenfu, Sopoongsan, Sphondin, T. poly glycosides, Younggaechulgam-tang, a-pinene. NCPP, PN50, Mangiferin. Melatonin, Mn-SOD, Myricetin, N-acetyl-L-cysteine, Nacyselyn, Naringin, N-ethyl- maleimide, Nitrosoglutathione, NDGA, Ochnaflavone, Orthophenanthroline, Phenylarsine oxide, Pyrithione, Pyrrolinedithiocarbamate, Quercetin, Quinozolines, Rebamipide, Redox factor 1. Resveratrol, Rotenone, Roxithromycin, S-allyl-cysteine, Sauchinone, Sodium 4-Aminosalicylate, Spironolactone, Taxifolin, Tempol, Tepoxaline, tert-butyl hydroquinone, Tetracylic A, Wogonin, xanthohumol, Yakuchinone A, B, a-lipoic acid, a-tocopherol, a-torphryl acetate, a-torphryl succinate, 0-Carotene, Diltiazem, Dioxin, Dipyridamole, Disulfiram, Enalapril. Fluvastatin, Indole-3-carbinol, JSH-23, KL-1156, Leflunomide, Levamisole, Moxifloxacin, Omapatrilat, R-etodolac, Rolipram, SC236 (COX-2 inhibitor), Triflusal, Actinodaphine, Artemisinin, Baicalein, 0-lapachone, Calcitriol, Campthothecin, Capsiate, and Catalposide. See, e.g., Gupta et al., Biochim Biophys Acta., 2010 Oct-Dec;1799(10-12). In some embodiments, the NFKB inhibitor is Andrographolide; Bay 1 1 -7082; Bithionol; Bortezomib; CBL0137 (CBL-0137); Cantharidin; Chromomycin A3; Daunorubicinum; Diethylmaleate; Digitoxin;

[0075] Ectinascidin 743; Emetine; Evodiamine (Isoevodiamine); Fluorosalan; GSK2982772; GSK583; Indole-3-carbinol; JSH-23; Magnolol; Manidipine hydrochloride; Narasin; Lestaurtinib; Omaveloxolone (RTA-408); Ouabain; QNZ (EVP4593); (-)- Parthenolide; Pyrrolidinedithiocarbamate ammonium; Rapamycin; SC75741; Sorafenib tosylate; Sunitinib malate; Tioconazole; Tribromsalan; Triclabendazolum; Triptolide (PG490); or Zafirlukast. In some embodiments, the inhibitor is emetine, fluorosalan, sunitinib malate, bithionol, narasin. tribromsalan. lestaurtinib, ectinascidin 743, chromomycin A3, or bortezomib. See, e.g., Miller et al., Biochem Pharmacol. 2010 May 1; 79(9): 1272-1280. In some embodiments, the NFKB inhibitor is not sodium salicylate. In some embodiments, the NFKB inhibitor is an inhibitor of IKK. IKK comprises two subunits IKKa and IKKp. Each subunit is important for the phosphorylation of IKB. (Mazhar Adli, IKKa and IKKP Each Function to Regulate NF-KB Activation in the TNF-Induced / Canonical Pathway, PLoS One. 2010; 5(2): e9428). In some embodiments, an additional component of IKK is IKKy / NEMO. In some embodiments, the NFKB inhibitor inhibits the function of IKKa; in some embodiments, the NFKB inhibitor inhibits the function of IKKP; and in some embodiments, the NFKB inhibitor inhibits both the function of IKKa and the function of IKKp. In some embodiments, the NFKB inhibitor inhibits IKKy / NEMO. IKK inhibitors can include ATP analogs, allosteric modulators, and agents interfering with the kinase activation loops. (Begalli et. al., Unlocking the NF-KB Conundrum: Embracing Complexity to Achieve Specificity, Biomedicines. 2017 Aug 22;5(3). pii: E50). Examples of ATP analogues include P-carboline, SPC-839, BMS-345541, and SAR-113945. In some embodiments, the NFKB inhibitor is Bay 11-7082. Bay 11- 7082 inhibits both IKKa and the function of IKKp. (Rauert-Wunderlich, The IKK inhibitor Bay 1 1-7082 induces cell death independent from inhibition of activation of NFKB transcription factors, PLoS One. 2013;8(3):e59292). Other known IKK inhibitors include IMD-0354 (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2- hydroxybenzamide), TPCA 1, NF-KB Activation Inhibitor VI (BOT-64), BMS 345541, Amlexanox. SC-514 (GK 01140), IMD 0354, and IKK-16. The contents of Begalli F et. al.. Unlocking the NF-KB Conundrum: Embracing Complexity to Achieve Specificity, Biomedicines. 2017 Aug 22;5(3). pii: E50, and Rauert- Wunderlich et. al., The IKK inhibitor Bay 11-7082 induces cell death independent from inhibition of activation of NFKB transcription factors, PLoS One. 2013;8(3):e59292 are hereby incorporated by reference.

[0076] Various NFKB inhibitors are readily available through public sources. For example, Santa Cruz Biotechnology provides NFKB inhibitors for purchases, including BAY 11-7085. Helenalin, NFkappaB Activation Inhibitor II, JSH-23, QNZ (EVP4593), Andrographolide. etc. (Santa Cruz Biotechnology). Various anti-NFKB antibodies, for example, are available for purchase at Sigma- Aldrich, as well as antibodies against other proteins involved in NFKB functionality, e.g., anti-IKK antibodies available at Sigma-Aldrich.

[0077] See also WO 2019 / 210073. Test / Assay Kits

[0078] Also provided for herein are test or assay kits that can be used in the methods described herein. For example, the kit can include one or more compounds or agents capable of detecting RelA / p65 protein in a sample (e.g., antibodies or antigen-binding fragments thereof, preferably directly or indirectly labeled), and optionally one or more standards or controls. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect RelA / p65 proteins in a method described herein; a swab, scraper, or spatula for obtaining buccal cells from a subject; reagents for detecting the antibodies; a slide or slides for receiving the buccal cells; and a fixative reagent for fixing the cells. Suitable swabs, scrapers, or spatulas are known in the art. including standard cotton swabs and wooden or rubber scrapers, as well as collection systems for buccal cells; see also US 5,738,643; US 20140243706; US 8,317,728; US 8,420,385; US 9,011,358; US20030129738;and US 3163160.

[0079] Methods of Screening

[0080] As described herein, larger amounts of buccal cells can also be collected, e.g., using a brush (even a toothbrush) and maintained in tissue culture (e.g., in the presence of a medium for growth of normal human keratinocytes. e.g., KGM-CD (Lonza) or Stemline™ Keratinocyte Basal Medium (Sigma-Aldrich) optionally supplemented with insulin and / or growth factors, see also Tsao et al., Journal of Cellular Physiology7110(2):219 229 (1982)). These cultured buccal cells can be used, e.g., in drug screens designed to determine if a particular drug is able to reverse the pathological changes seen in buccal mucosa cells in patients with an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS. In these methods, a test compound is applied to the cells and levels and / or localization of RelA / p65 are evaluated. A test compound that is able to restore levels and / or localization of the proteins to normal or near normal is selected as a candidate compound for the treatment of an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS.

[0081] Thus, included herein are methods for screening test compounds, e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds, to identify agents useful in the treatment of an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS. As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3.000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1.500 Da. about 100 to about 1,250 Da, about 100 to about 1.000 Da. about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0082] The test compounds can be, e.g., natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small- Molecular-Weight Compound Libraries , Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czamik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Patent No. 6,503,713, incorporated herein by reference in its entirety.

[0083] Libraries screened using the methods of the present invention can comprise a variety of types of test compounds. A given library can comprise a set of structurally related or unrelated test compounds. In some embodiments, the test compounds are peptide or peptidomimetic molecules. In some embodiments, the test compounds are nucleic acids.

[0084] In some embodiments, the test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g.. using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship. Thus, in some instances, the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds. For example, in one embodiment, a general library’ of small molecules is screened, e.g., using the methods described herein.

[0085] In some embodiments, a test compound is applied to a test sample, e g., a cell or living tissue or organ, e.g., an eye, and one or more effects of the test compound is evaluated. In a cultured or primary' cell for example, the ability of the test compound to normalize (i.e., return to normal or near-normal, at least 60% of normal, e.g., 60%, 70%, 80%, 90%, or 95% of normal) levels and / or localization of the RelA / p65 is evaluated.

[0086] In some embodiments, the test sample is, or is derived from (e.g., a sample taken from) an in vivo model of an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS. For example, an animal model, e.g., a rodent such as a mouse, that expresses a mutation that has been implicated in causing disease in patients with a familial arrhythmogenic syndrome, e.g., ACM or BrS can be used. A number of animal models of ACM are known in the art; see, e.g., McCauley and Wehrens, Dis Model Meeh. 2009 Nov-Dec;2(l l-12):563-70; Pilichou et al.. Circ Cardiovasc Genet. 201 1 Jun;4(3):318-26; Lodder and Rizzo. Front. Physio. 3:221. doi: 10.3389 / fphys.2012.00221 (2012); Delmar and McKenna, Circulation Research. 107:700-714 (2010). Animal models of BrS are described in references 41-45. Cellular models of BrS including hiPSC-CMs with BrS mutations are described in references 42, 46, and 47.

[0087] Alternatively, the test sample can be from a human subj ect who has an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, has a mutation associated with an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, or an unaffected relative, e.g., a first degree relative, thereof. In some embodiments an unaffected relative, e.g., a first degree relative, of a human who has an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS is used as a control; the relative may or may not have a mutation associated with an inflammatory' cardiac arrhythmogenic syndrome, e.g., ACM or BrS. Methods for evaluating each of these effects are known in the art. For example, ability to modulate expression of a protein can be evaluated at the gene or protein level, e.g., using quantitative PCR or immunoassay methods. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999.W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber. Science 2000, 289(5485): 1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect an effect on protein levels. Ability to modulate protein localization and levels can be evaluated, e.g., using imaging methods such as fluorescence microscopy.

[0088] A test compound that has been screened by a method described herein and determined to normalize localization of RelA / p65 can be considered a candidate compound. A candidate compound that has been screened, e.g., in an in vivo model of a disorder, e.g., an animal model with a mutation associated with an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, and determined to have a desirable effect on the disorder, e.g., on one or more symptoms of the disorder, can be considered a candidate therapeutic agent. Candidate therapeutic agents, once screened in a clinical setting, are therapeutic agents. Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and / or derivatized, and formulated with physiologically acceptable excipients to form pharmaceutical compositions.

[0089] Thus, test compounds identified as “hits (e.g., test compounds that normalize localization of RelA / p65 in a first screen can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein. Thus, in one embodiment, the invention includes screening a first library of compounds using a method known in the art and / or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein. Test compounds identified as hits can be considered candidate therapeutic compounds, useful in treating an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS. A variety of techniques useful for determining the structures of “hits” can be used in the methods described herein, e.g., NMR, mass spectrometry, gas chromatography equipped with electron capture detectors, fluorescence and absorption spectroscopy. Thus, the invention also includes compounds identified as “hits” by the methods described herein, and methods for their administration and use in the treatment, prevention, or delay of development or progression of a disorder described herein.

[0090] Test compounds identified as candidate therapeutic compounds can be further screened by administration to an animal model of an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, as known in the art. The animal can be monitored for a change in the disorder, e.g., for an improvement in a parameter of the disorder, e.g., a parameter related to clinical outcome. In some embodiments, the parameter is arrhythmia, e.g., ventricular tachyarrhythmia, and an improvement would be a decrease in frequency or cessation of arrhythmias. In some embodiments, the subject is ahuman, e.g., a human with an inflammatory cardiac arrhythmogenic syndrome, e.g., ACM or BrS, and an implantable cardioverter-defibrillator device, and the parameter is a decrease in activation of the device.

[0091] EXAMPLES

[0092] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0093] Materials and Methods

[0094] The following materials and methods were used in the Examples below.

[0095] Buccal mucosa sampling and preparation of smears: A clean cotton-tipped swab (Q-tip) or a cytology brush was used for collection of the specimen. Material was collected from the inside of the cheek by using slight rolling and scraping motions for about 30 seconds on each side. Immediately after collection, the buccal mucosa material was smeared on the center area of microscope slides (2.5 x 2.5 centimeters), which were subsequently dipped in 70% ethanol for ~1 minute to fix the sample or sprayed with a spray-on cytology fixative product. The slides were allowed to air dry and stored at room temperature before being immunostained. Buccal mucosa culture protocol: The oral cavity was first rinsed thoroughly with antiseptic mouthwash. Buccal mucosa cells were collected with a sterile toothbrush which was lightly scraped on the inside of both cheeks for ~2 minutes. The oral cavity was washed with 15 ml of sterile saline which was collected in a centrifuge tube. The toothbrush itself was washed in an additional 10ml of saline. The same process was repeated one or more times. The resultant cell suspension was centrifuged at 3000g for 5 minutes. The pellet was washed once in sterile PBS. recentrifuged and subsequently re-suspended in 20ml of PBS supplemented with 5% penicillin / streptomycin and 7mM fungizone for 30 minutes. The cells were then washed twice in PBS, reconstituted in KGM™-CD chemically defined keratinocyte growth medium (LONZA) and seeded on matrigel-coated chamber slides, where they were maintained at 37°C, 5% CO2 for up to 7 days. Selected cultures were incubated with SB216763 (5mM) for 24 hours prior to immunostaining.

[0096] Immunohistochemistry: Buccal mucosa smears were immunostained using established protocols.3,8Cells were simultaneously blocked and permeabelized for 45 minutes in 3% normal goat serum, 1% w / v BSA, and 0. 15% Triton X-100 in PBS, followed by an overnight incubation at 4°C with a rabbit monoclonal antibody against Rela / p65 (from Cell Signaling Technology, catalog #8242). Slides were then incubated with Cy 3 -conjugated secondary antibodies (Jackson ImmnunoRes earch) for 2 hours at room temperature and counterstained with DAPI.

[0097] Buccal mucosa cultures were washed in PBS, fixed in 4% paraformaldehyde and, after being washed 3 additional times in PBS, were immunostained as described above. All immunostained preparations were imaged at 40x using a ZEISS inverted confocal microscope.

[0098] Immunoperoxidase:

[0099] Paraffin-embedded myocardial sections (5 pm) were deparaffinized, dehydrated, rehydrated and exposed to 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity. Sections were then incubated first with blocking solution (1 hr) and then with a rabbit polyclonal anti-RelA antibody (LS- B653; LSBiosciences) overnight at 4°C. The following day, sections were incubated with donkey secondary' antibody conjugated to horseradish peroxidase enzy me (1 hr). Peroxidase-conjugated antibodies were detected by the 3,3’-diaminobenzidine (DAB) substrate kit. Heart tissue was counterstained with Mayer's haematoxylin. Bright field images were taken with a Nikon eclipse 80i microscope and recorded with Nikon DS-Fil camera.

[0100] Immunofluores cence :

[0101] Paraffin-embedded myocardial sections (5 pm) were deparaffmized, dehydrated, rehydrated and boiled in citrate buffer (pH=6.0) for 11 mins. They were then first incubated with blocking solution (1 hr) and then with a mouse monoclonal anti-human CCR2 antibody (MAB150-SP; R&D Systems) overnight at 4°C. The following day, sections were incubated with anti-mouse IgG Cy3-labelled secondary antibody and mounted with ProLong Gold. Images w ere recorded using a Nikon AIR confocal microscope.

[0102] Immunocytochemistry:

[0103] Buccal cells were obtained using a soft cytological brush, smeared on glass slides and fixed by M-FIX spray (Merck Millipore). Samples were first incubated with blocking solution (1 hr) and then with a rabbit polyclonal anti-RelA antibody (8242; Cell Signaling Technology) overnight at 4°C. The following day, samples ere incubated with anti-rabbit IgG Cy3-labelled secondary antibody, counterstained with 4',6-diamidino-2-phenylindole (DAPI) and mounted with ProLong Gold. Images were recorded using a Nikon AIR confocal microscope.

[0104] Statistics:

[0105] Prism software (Version 9.2.0 (283); GraphPad Software Inc., San Diego, CA, USA) was used for the statistical analysis of the CCR2+ cell data. The normal distribution of the data was assessed by Kolmogorov-Smirnov test. Normally distributed data was analysed for differences by 1-way analysis of variance (ANOVA) and the Newman-Keuls post-test for multiple comparisons. Data are presented as the mean ± the standard error of the mean (SEM). P-value <0.05 was deemed significant.

[0106] Data were also reported as frequency counts (%) for categorical variables, which were compared betw een groups with Chi-square or Fisher’s exact test using SPSS 21.0 software. p<0.05 was considered significant.

[0107] Example 1. Activation of NFKB in cardiac myocytes and accumulation of CCR2+ inflammatory cells in human familial arrhythmia syndromes

[0108] We have shown in unpublished experimental studies in a mouse model of ACM (Dsg2mubmutmice) that activation of NFkB signaling in cardiac myocytes drives myocardial injury and arrhythmias. The way cardiac myocytes do this is by sending signals that mobilize pro-in fl ammatory “Ml”-like macrophages that express the marker CCR2 (CCR2+ cells) and stimulating them to accumulate in the heart. Once they gain access to the heart, CCR2+ cells cause extensive myocardial injury and arrhythmias. To determine if similar mechanisms occur in patients with familial arrhythmia syndromes, we studied samples of formalin-fixed paraffin-embedded heart tissues. We sought to answer two questions: 1) is NFkB signaling activated in cardiac myocytes in patients with familial sudden death syndromes; and 2) are CCR2+ cells increased in the hearts of patients with familial sudden death syndromes. To answer the first question, we stained sections of human heart samples with an antibody against RelA / p65 and used an immunoperoxidase detection system to look for the presence of signal in cardiac myocyte nuclei (seeing RelA.p65 in a cell nucleus is strong evidence that NFkB signaling has been turned on in that cell). To answer the second question, we stained sections of human heart samples with an antibody against CCR2 and counted the number of positive cells per unit section area. We studied patient heart samples from 36 cases of ACM, 13 cases of BrS, 5 cases of long QT syndrome t pe 1, 5 cases of long QT syndrome type 2, and 11 cases of catecholaminergic polymorphic ventricular tachycardia (CPVT). The latter three conditions are familial arrhythmia syndromes associated with variants in genes that encode ion channels (so-called “ion channelopathies). We compared the results from the patient samples with 19 control heart samples obtained from individuals in the same age-range as the patients and who died from causes unrelated to disease (e g., motor vehicle accidents). We observed strong nuclear signal for RelA / p65 in cardiac myocytes in 34 of 35 cases of ACM and in all 13 cases of BrS but in none of the 19 control cases (representative examples are shown in Figure 1). We also saw no evidence of RelA / p65 signal in cardiac myocytes in subjects with long QT type 1, long QT type 2 or CPVT (examples also shown in Figure 1). These results clearly distinguish ACM and BrS, both cardiomyopathies, from the ion channelopathies and implicate different disease mechanisms. There was a strong correlation between the presence of nuclear signal for RelA / p65 in cardiac myocytes and increased numbers of CCR2+ cells in the heart. As shown in Figure 1, CCR2+ cells were greatly increased in hearts of patients with ACM and BrS compared to controls, but no increase was seen in hearts of patients with familial ion channelopathies. These observations provide compelling evidence that innate immune signaling pathways are activated in cardiac myocytes in ACM and BrS and, further, that this immune response recruits CCR2+ inflammatory cells to the heart where they contribute to disease expression.

[0109] Example 2. Ismate Immune Signaling in Hearts and Buccal Mucosa Cells of Patients with Arrliythmogenic Cardiomyopathy

[0110] Here, we sought to determine if NFKB signaling is activated in cardiac myocytes in patients with ACM and, if so, whether this is associated with accumulation of myocardial CCR2+ cells. We also determined if NFKB signaling is activated in buccal mucosa cells from young individuals who had inherited ACM alleles. Evidence of active NFKB signaling was based on the presence of immunoreactive signal for RelA / p65, the heterodimeric binding partner of NFKB, in nuclei of cardiac myocytes or buccal mucosa cells. Ethical approval for this study was obtained from the UK National Health Service Research Ethics Committee (hearts: 17 / LO / 0747. buccal smears: 17 / LO / 0840). Informed consent was provided by next-of-kin at the time of autopsy and by parents / guardians of children whose buccal cells were sampled.

[0111] Myocardial samples were analyzed from 36 ACM patients and 19 controls. Clinical, diagnostic and genetic information is listed in Table 1. Samples were obtained at autopsy from 29 ACM patients who had died suddenly, from explanted hearts of 6 ACM patients at heart transplantation, and from an endomyocardial biopsy performed in one ACM patient during internal defibrillator placement. Control myocardial samples came age-matched subjects who died from non-cardiac causes and had no cardiovascular disease at autopsy (see Table 1).

[0112] Buccal mucosa cells, obtained as previously reported (2, 3), were analyzed from 28 young individuals (ages 5-17). Clinical, diagnostic and genetic information is listed in Table 2. These subjects all had a family history' of ACM (21 were carriers of ACM disease alleles) and were being followed clinically at Great Ormond Street Hospital. Buccal cells were obtained from: 12 pre-clinical carriers of variants who had never shown clinical disease; 3 at the clinic visit following the first clinical manifestation of disease; 9 with stable disease (without deterioration at the time cells were obtained; termed ‘stable' in Table 2); and 4 sampled during or shortly after clinical exacerbation of disease (‘hot phase’). Buccal smears from 22 children (ages 1-17) without clinical signs or history of cardiomyopathy were used as negative controls.

[0113] Myocardial tissue sections from all ACM patients and controls were analyzed by immunoperoxidase staining using a primary antibody against RelA / p65. When sufficient tissue was available, additional myocardial sections were analyzed by immunofluorescence staining using a primary antibody against CCR2. Buccal mucosa cells were analyzed by immunofluorescence staining using a primary antibody against RelA / p65 and DAPI to independently label nuclei. Details about staining methods and reagents are included in the Methods above.

[0114] Strong immunoperoxidase signal for RelA / p65 was seen in cardiac myocyte nuclei in 34 of 36 ACM patient samples, but in none of the 19 control samples. Representative examples are shown in Figure 2A. The number of cells showing strong immunofluorescent signal for CCR2 was also significantly increased in ACM hearts compared to controls (Figure 2B and 2C). The size and interstitial distribution of CCR2+ cells was consistent with their being macrophages (Figure 2B).

[0115] Buccal cells from the 12 ACM gene carriers with no clinical evidence of disease and the 9 patients with established but quiescent disease showed no nuclear signal for RelA / p65, nor was nuclear signal seen in buccal cells from any of the 22 controls. By contrast, all 7 ACM gene carriers sampled when clinical disease was first manifest or during 'hot phases’ showed strong RelA / p65 signal in buccal cell nuclei. Representative images are shown in the Figures 2D-E. In 2 subjects who showed nuclear RelA / p65 signal during a 'hot phase’, no nuclear signal was seen in previous studies done 7 and 12 months before the ‘hot phase’ (pACM27 and pACM26, respectively). In another patient (pACM13), nuclear signal was lost 20 months after initial disease manifestation, whereas nuclear signal persisted in a repeat sample from a patient (pACM28) who had presented with a dramatic arrhythmia burden 2 months earlier, despite reduced arrhythmias after amiodarone therapy.

[0116] These results indicate that NFKB is activated in cardiac myocytes in patients with ACM. associated with accumulation of pro-inflammatory CCR2+ cells in their hearts. Thus, innate immune signaling in cardiac myocytes may be a major driver of disease in AMC patients, as shown in DSg2mut'mitmice (1). NFKB is also activated in buccal mucosa cells of young ACM gene carriers at the time of disease onset or progression. This finding suggests that ACM is a systemic inflammatory disease, consistent w ith previous studies showing elevated circulating levels of pro- inflammatory cytokines in ACM patients (4). Screening buccal mucosa cells for evidence of immune activation may help identify patients who would benefit from anti-inflammatory therapies. It was not possible or even advisable to obtain heart biopsies from these young patients but the presence of nuclear signal for RelA / p65 in their buccal cells is likely a manifestation of activation of innate immune signaling in their hearts. Lastly, we have analyzed buccal cells in 4 patients with documented BrS. Cells were obtained at the time these individuals had regularly scheduled clinic visits and none had recently experienced any significant adverse disease events. Nevertheless, buccal cells from all 4 patients showed nuclear signal for RelA / p65 (a representative case is shown in Figure 3). These observations show that activation of NFkB signaling in buccal cells occurs in patients with familial arrhythmia syndromes, including ACM and BrS and potentially other heart disease, and this may be considered as a surrogate for activation of NFkB signaling in cardiac myocytes. Accordingly, analyzing buccal cells for nuclear RelA / p65 signal may be a safe, inexpensive and effective way to screen patients and identify those who might benefit from anti-inflammatory therapy designed to target innate immune responses.

[0117] Table 1.

[0118] Study Clinical / postmortem Age at RelA / p65 No data death Sex Genetic data Signal

[0119] Controls: Cause of Death:

[0120] CTR1 murder 37 M NA cytoplasmic

[0121] CTR2 drowning 29 M NA cytoplasmic

[0122] CTR3 electrocution 24 M NA cytoplasmic

[0123] CTR4 drug overdose 20 F NA cytoplasmic

[0124] CTR5 neck compression 40 M NA cytoplasmic

[0125] CTR6 murder 34 M NA cytoplasmic

[0126] CTR7 drug overdose 30 F NA cytoplasmic

[0127] CTR8 suicide 21 M NA cytoplasmic

[0128] CTR9 drug overdose 21 M NA cytoplasmic

[0129] CTR10 drug overdose 21 M NA cytoplasmic

[0130] CTR11 drug overdose 35 M NA cytoplasmic

[0131] CTR12 drug overdose 28 M NA cytoplasmic

[0132] CTR13 drug overdose 26 M NA cytoplasmic

[0133] CTR14 suicide 30 M NA cytoplasmic

[0134] CTR15 head injury 32 M NA cytoplasmic

[0135] CTR16 drowning 19 M NA cytoplasmic

[0136] CTR17 car accident 49 M NA cytoplasmic pulmonary

[0137] CTR18 embolism 29 M NA cytoplasmic

[0138] CTR19 car accident 23 F NA cytoplasmic

[0139] ACM:

[0140] SCD; PM Dx: RV- PKP2; c.2198 2202

[0141] ACM1 dominant ACM 37 M delACACC nuclear

[0142] SCD; PM Dx: RV-

[0143] ACM2 dominant ACM 21 M PKP2; Gin 133X nuclear

[0144] SCD; PM Dx: RV- PKP2;

[0145] ACM3 dominant ACM 20 M c.253_256delGAGT nuclear

[0146] SCD; PM Dx: RV-

[0147] ACM4 dominant ACM 34 M PKP2; Ser688Pro nuclear

[0148] SCD; PM Dx:

[0149] ACM5 biventricular ACM 20 M PKP2; 1216delG cytoplasmic

[0150] SCD; PM Dx:

[0151] ACM6 biventricular ACM 21 M PKP2; 2013delC nuclear

[0152] SCD; PM Dx:

[0153] ACM7 biventricular ACM 26 F DSP; c.4395T>G nuclear

[0154] SCD; PM Dx:

[0155] ACM8 biventricular ACM 28 M DSP; C.5269OT nuclear biopsy at ICD implant; Dx: currently

[0156] ACM9 biventricular ACM 35 F DSP; c.3045del cytoplasmic

[0157] SCD; PM Dx: LV-

[0158] ACM10 dominant ACM 36 F DSP; R1113X nuclear SCD; PM Dx: LV-

[0159] ACM11 dominant ACM 28 F DSP; c.5318del nuclear

[0160] SCD; PM Dx:

[0161] ACM12 biventricular ACM 29 M DSG2; 2036delG nuclear

[0162] SCD; PM Dx:

[0163] ACM13 biventricular ACM 18 M DSC2; G371fsX378 nuclear

[0164] SCD; PM Dx:

[0165] ACM 14 biventricular ACM 34 M DSC2; E896fsX900 nuclear SCD; antemortem JUP; 2157del2,

[0166] ACM15 Dx: Naxos disease 21 M homozygous nuclear

[0167] SCD; PM Dx: PKP2; R735Q &

[0168] ACM16 biventricular ACM 21 M DSP; R270X nuclear

[0169] SCD; PM Dx: DSG2; Mil & Dsg2;

[0170] ACM17 biventricular ACM 32 M I333T nuclear

[0171] SCD; PM Dx: TMEM43;

[0172] ACM18 biventricular ACM 20 M C.1073OT nuclear

[0173] SCD; PM Dx: TMEM43;

[0174] ACM19 biventricular ACM 45 M C.1073OT nuclear SCD; PM Dx: RV- FLNC; c.7552-1

[0175] ACM20 dominant ACM 20 M G>A nuclear SCD; PM Dx: RV-

[0176] ACM21 dominant ACM 36 M FLNC; c.6779A>G nuclear transplant; Dx: biventricular ACM +

[0177] ACM22 HF NA NA PLN; R14del nuclear transplant; Dx: biventricular ACM +

[0178] ACM23 HF NA NA PLN; R14del nuclear transplant; Dx: biventricular ACM +

[0179] ACM24 HF NA NA PLN; R14del nuclear transplant; Dx: biventricular ACM +

[0180] ACM25 HF NA NA PLN; R14del nuclear transplant; Dx: biventricular ACM +

[0181] ACM26 HF NA NA PLN; R14del nuclear

[0182] SCD; antemortem

[0183] ACM27 Dx: ACM 37 M NA nuclear

[0184] SCD; PM Dx:

[0185] ACM28 biventricular ACM 23 F NA nuclear

[0186] SCD; PM Dx:

[0187] ACM29 biventricular ACM 37 M NA nuclear

[0188] SCD; PM Dx: RV-

[0189] ACM30 dominant ACM 41 F NA nuclear

[0190] SCD; PM Dx: RV-dom

[0191] ACM 31 ACM 20 M NA nuclear

[0192] SCD; antemortem

[0193] ACM32 Dx: ACM 40 M NA nuclear

[0194] SCD; PM Dx:

[0195] ACM33 biventricular ACM 24 M NA nuclear

[0196] SCD; PM Dx:

[0197] ACM34 biventricular ACM 43 M NA nuclear

[0198] SCD; PM Dx:

[0199] ACM35 biventricular ACM 33 M NA nuclear transplant; Dx: biventricular ACM + currently no result on gene

[0200] ACM36 HF 47 M panel testing nuclear

[0201] Table 1 Abbreviations:

[0202] ACM arrhythmogenic cardiomyopathy

[0203] PM postmortem Dx diagnosis

[0204] RV right ventricle

[0205] LV left ventricle

[0206] HF heart failure

[0207] NA not available M male

[0208] F female

[0209] PKP2 gene for plakophilin2

[0210] DSP gene for desmoplakin

[0211] JUP gene for junctional plakoglobin DSG2 gene for desmoglein2

[0212] DSC 2 gene for desmocollin2 FLNC gene for filamin C PLN gene for phospholamban

[0213] TMEM43 gene for transmembrane protein 43 SCD sudden cardiac death Table 2.

[0214] Table 2 Abbreviations:

[0215] F female

[0216] M male

[0217] PKP2 gene for plakophilin2

[0218] DSP gene for desmoplakin

[0219] SCN5Agene for sodium voltage-gated channel alpha subunit 5 pACM pediatric arrhythmogenic cardiomyopathy

[0220] NSVT non-sustained ventricular tachycardia SAECG signal averaged electrocardiogram cMRI cardiac magnetic resonance imaging ICD implantable cardioverter defibrillator RV right ventricle

[0221] LV left ventricle

[0222] PVC premature ventricular contraction LGE late gadolinium enhancement

[0223] References Cited

[0224] 1. Brugada P. Brugada J. Right bundle branch block, persistent ST segment elevation and sudden cardiac death: a distinct clinical and electrocardiographic syndrome. A multicenter report. J Am Coll Cardiol 1992; 20: 1391-1396.

[0225] 2. Mizusawa Y, Wilde AAM. Brugada syndrome. Circ Arrhythm Electrophysiol 2012; 5:606-616.

[0226] 3. Aziz HM, Zarzecki MP. Garcia-Zamora S, Kim MS, Bijak P. Tse G. Won H-H, Matusik PT. Pathogenesis and Management of Brugada Syndrome: Recent Advances and Protocol for Umbrella Reviews of Meta Analyses in Major Arrhythmic Events Risk Stratification. J Clin Med Res [Internet]. 2022; 11. Available from: dx.doi.org / 10.3390 / jcml 1071912

[0227] 4. Blok M, Boukens BJ. Mechanisms of Arrhythmias in the Brugada Syndrome. Int J Mol Sci [Internet], 2020;21. Available from: dx.doi.org / 10.3390 / ijms21197051

[0228] 5. Chelko SP, Asimaki A, Lowenthal J, Bueno-Beti C, Bedja D. Scale A, Amat-Codina N, Andersen P, Judge DP, Tung L, Saffitz JE. Therapeutic modulation of the immune response in arrhythmogenic cardiomyopathy. Circulation 2019; 140: 1491-1505.

[0229] 6. Le Scouamec S, et al. Testing the burden of rare variation in arrhythmiasusceptibility genes provides new insights into molecular diagnosis of Brugada syndrome. Hum Mol Genet 2015; 24:2757-2763.

[0230] 7. Bezzina et al. Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death. Nat Genet 2013;45: 1044-1049.

[0231] 8. Bare et al. Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility. Nat Genet 2022;54:232-239.

[0232] 9. Asimaki A, Kapoor S, Plovie E, Arndt AK, Adams E, Liu ZZ, James CA, Calkins H, Churko J. Wu JC, MacRae CA, Kleber AG, Saffitz JE. Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Sci Transl Med 2014; 6(24):240ra74. 10. Catalano O. Antonaci S, Moro G, Mussida M, Frascaroli M. Baldi M, Cobelli F, Baiardi P, Nastoli J. Bloise R, Monteforte N, Napolitano C, Priori SG. Magnetic resonance investigations in Brugada syndrome reveal unexpectedly high rate of structural abnormalities. Eur Heart J 2009; 30:2241-2248.

[0233] 11. Ohkubo K, Watanabe I, Okumura Y, Takagi Y, Ashino S, Kofune M, Sugimura H, Nakai T, Kasamaki Y, Hiryama A. Morimoto S. Right ventricular histological substrate and conduction delay in patients with Brugada syndrome. Int Heart J 2012; 51 :17-23.

[0234] 12. Corrado D, Zorzi A, Cerrone M, Rigato I, Mongillo M, Bauce B, Delmar M. Relationship between arrhythmogenic right ventricular cardiomyopathy and Brugada syndrome: New insights from molecular biology and clinical implications. Circ Arrhythm Electrophysiol 2016; 9(4):e003631.

[0235] 13. Tada H, AiharaN, Ohe T, Yutani C, Hamada S, Miyanuma H, Takamiya M, Kamakura S. Arrhythmogenic right ventricular cardiomyopathy underlies syndrome of right bundle branch block, st-segment elevation, and sudden death. Am J Cardiol 1998; 81 :519-522.

[0236] 14. Morita H, Kusano KF, Miura D, Nagase S, Nakamura K, Morita ST, Ohe T, Zipes DP, Wu J. Fragmented qrs as a marker of conduction abnormality7and a predictor of prognosis of Brugada syndrome. Circulation 2008; 118: 1697-1704.

[0237] 15. Sata PY, Coombs W. Lin X, Nekrasova O. Green KJ, Isom LL, Taffet SM, Delmar M. Interactions between ankyrin-g, phakophilin-2, and connexin43 at the cardiac intercalated disc. Circ Res 2011;109: 193-201.

[0238] 16. Sata PY, Musa H, Coombs W, Guerrero-Sema G, Patino GA, Taffet SM, Isom LL. Delmar M. Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity' in cultured cardiac myocytes. Circ Res 2009; 105:523-526.

[0239] 17. Corrado D, Link M, Calkins H. Arrhythmogenic right ventricular cardiomyopathy. New Eng J Med 2017; 376:61-72.

[0240] 18. Austin KM, Trembley MA, Chandler SF, Sanders SP. Saffitz JE, Abrams DJ, Pu WT. Molecular mechanisms of arrhythmogenic cardiomyopathy. Nat Rev Cardiol [Internet]. 2019; Available from: dx.doi.org / 10.1038 / s41569-019-0200-7

[0241] 19. Chelko SP, Asimaki A. Andersen P, Bedja D, Amat- Alarcon N, DeMazumder D, Jasti R. MacRae CA. Leber R. Kleber AG, Saffitz JE. Judge DP. Central role for GSK3P in the pathogenesis of arrhythmogenic cardiomyopathy. JCI Insight 2016; 1(5). ph: e85923.

[0242] 20. Lazzerini PE, Capecchi PL, Laghi-Pasini F. Systemic inflammation and arrhythmic risk: lessons from rheumatoid arthritis. Eur Heart J 2017; 38: 1717-1727.

[0243] 21. Lazzerini PE, Laghi-Pasani F, Boutjdir M, Capecchi PL. Inflammatory7cytokines and cardiac arrhythmias: the lesson from COVID- 19. Nat Rev Immunol 2022: 22:270-272.

[0244] 22. Misra A, Haudek SB, Knuefermann P, Vallejo JG, Chen ZJ, Michael LH, Sivasubramanian N, Olson EN, Entman ML, Mann DL. Nuclear factor-KB protects the adult cardiac myocyte against ischemia-induced apoptosis in a murine model of acute myocardial infarction. Circulation 2003; 108:3075-3078.

[0245] 23. Boring K, Gosling J, Chensue SW, Kunkel SL, Farese RV Jr, Broxmeyer HE, Charo IF. Impaired monocyte migration and reduced ty pe 1 (Thl ) cytokine responses in C-C chemokine receptor 2 knockout mice. J Clin Invest 1997; 100:2552- 2561.

[0246] 24. Yadav A, Saini V, Arora S. MCI-1: chemoattractant with a role beyond immunity7: a review. Clin Chem Acta 2010; 411 : 1570-1579.

[0247] 25. O’Connor T, Borsig K. Heikenwalder M. CCL2-CCR2 signaling in disease pathogenesis. Endocr Metab Immune Disord Drug Targets 2015; 15: 105-118.

[0248] 26. Braga TT, Correa-Costa M. Silva RC, Cruz MC. Hiyane MI, da Silva JS, Perez KR, Cuccovia IM, Camara NOS. CCR2 contributes to the recruitment of monocytes and leads to kidney inflammation and fibrosis development. Inflammopharmacol 2018; 26:403-411.

[0249] 27. Heymann F, Trautwein C, Tacke F. Monocytes and macrophages as cellular targets in liver fibrosis. Inflamm Allergy Drug Targets 2009; 8:307-318.

[0250] 28. Bajpai G, Bredemeyer A, Li W, Zaitsev K, Koenig AL, Lokshina I, Mohan J, Ivey B, Hsiao HM, Weinheimer C, Kovacs A, Epelman S, Arty omov M, Kreisel D, Lavine KJ. Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial Injury. Circ Res 2019; 124:263-78.

[0251] 29. Fu X, Khalil H, Kanisicak O, Boyer JG, Vagnozzi RJ, Maliken BD, Sargent MA, Prasad V, Valiente- Alandi I, Blaxall BC, Molkentin JD. Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart. J Clin Invest. 2018; 128:2127-43.

[0252] 30. Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, Rivera-Feliciano J, Jiang D, von Gise A, Ikeda S, Chien KR, Pu WT. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 2008;454: 109-113.

[0253] 31. Bonny A. Tonet J, Marquez MF, De Sisti A, Temfemo A, Himbert C, Guiffaf F, Larrazet F, Ditah I, Frank F, Hidden-Lucet F. Fontaine G. C-reactive protein levels in the Brugada syndrome. Cardiol Res and Pract 2011; article ID 341521.

[0254] 32. Li A, Tung R, Shivkumar K, Bradfield JS. Brugada syndrome - malignant phenotype associated with acute cardiac inflammation? Heart Rhythm Case Reports 2017;3:384-388.

[0255] 33. Pieroni M, Notarstefano P, Oliva A, Campuzano O, Santangeli P, Coll M, Nesti M, Carnevali A, Fraticelli A, Iglesias A, Grassi S, Brugada R, Bolognese L. Electroanatomic and pathologic right ventricular outflow tract abnormalities in patients with Brugada syndrome. J Am Coll Cardiol 2018; 72:2747- 2757.

[0256] 34. Rojas A, Calkins H. Present understanding of the relationship between exercise and arrhythmogenic right ventricular dysplasia / cardiomyopathy. Trends Cardiovas Med 2015; 25: 181-183.

[0257] 35. Esperer HD, Hoos O. Hottenrott K. Syncope due to Brugada syndrome in a young athlete. Br J Sports Med 2007; 41 : 180-181 .

[0258] 36. Grimster A, Segal OR, Behr ER. Type I Brugada electrocardiogram pattern during the recovery phase of exercise testing. Europace 2008; 10:897-898.

[0259] 37. Ranger S. Talajic M, Lemerry R. Roy D. Nattel S. Amplification of flecainide-induced ventricular conduction slowing by exercise. A potentially significant clinical consequence of use-dependent sodium channel blockade. Circulation 1989 79: 1000-1006.

[0260] 38. Veldkamp MW, Viswanathan PC, Bezzina C, Baartscheer A, Wilde AA, Balser JR. Two distinct congenital arrhythmias evoked by a mutidysfunctional Na(+) channel. Circ Res 2000; 86:E91-97.

[0261] 39. Boersma LV, Jaarsma W, Jessurun ER, Van Hemel NH, Wever EF. Brugada syndrome: a case report of monomorphic ventricular tachycardia. Pacing Clin Electrophysiol 2001 ;24: 112-115. 40. Falk RH. Flecainide-induced ventricular tachycardia and fibrillation in patients treated for atrial fibrillation. Ann Intern Med 1989; 111: 107-111.

[0262] 41. Sendfeld F, Selga E, Scomik FS, Perez GJ, Mills NL, Brugada R. Experimental models of Brugada syndrome. Int J Mol Sci 2019; 20:2123-2124.

[0263] 42. Li Y, Lang S, Akin I, Zhou X, El-Battra\\y I. Brugada Syndrome: Different Experimental Models and the Role of Human Cardiomyocytes From Induced Pluripotent Stem Cells. J Am Heart Assoc 2022; ! 1 : e024410.

[0264] 43. Man JCK, Mohan RA, van den Boogaard M, Hilvering CRE, Jenkins C, Wakker V, Bianchi V, de Laat W, Barnett P, Boukens BJ, Christoffels VM. An enhahncer cluster controls gene activity and topology of the SCN5A-SCN10A locus in vivo. Nat Commun 2019; 10:4943.

[0265] 44. Papadatos GA, Wallerstein PMR, Head CEG, Ratcliff R, Brady PA, Benndorf K, Saumarez RC, Trezise AEO, Huang CLH, Vanderberg JI, Colledge WH, Grace AA. Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a. Proc Natl Acad Sci USA 2002; 99:6210- 6215.

[0266] 45. Remme CA, Verkerk AO, Nuyens D, van Ginneken ACG, van Brunschot S, Belterman CNW, Wilders R, van Roon MA, Tan HL, Wilde AA, Carmeliet P, de Bakker JMT, Veldkamp MW, Bezzina CR. Overlap sy ndrome of cardiac sodium channel disease in mice carrying the equivalent mutation of human SCN5A- 1795insD. Circulation 2006; 114:2584-2594.

[0267] 46. Nijak A, Saenen J, Labro AJ, Schepers D, Loeys BL, Alaerts M. iPSC- Cardiomyocyte Models of Brugada Syndrome- Achievements, Challenges and Future Perspectives. Int J Mol Sci [Internet]. 2021;22. Available from: dx. doi.org / 10.3390 / ijms22062825

[0268] 47. Ma D, Liu Z, Loh LJ, Zhao Y, Li G, Liew R, Islam O, Wu J, Chung YY, Teo WS, Ching CK, Tan BY, Chong D. Ho KL, Lim P. Yong RYY, Panama BK, Kaplan AD, Bett GCL, Ware J, Bezzina CR, Verkerk AO, Cook SA, Rasmusson RL, Wei H. Identification of an / \a-dependent and / to-mediated proarrhythmic mechanism in cardiomyocytes derived from pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient. Sci Rep 2018; 8:11246.

[0269] 48. Wang G, McCain ML, Yang L, He A, Pasqualini FD, Agarwal A, Yuan H, Jiang D, Zhang D, Zang L. Geva J, Roberts AE. Ma Q, Ding K. Chen K, Wang DZ, Li K, Wang K, Wanders RJA, Kulik W, Vaz FM, Laflamme MA. Murry CE, Chien KR, Kelley RL Church GM, Parker KK. Pu WT. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on- chip technologies. Nat Med 2 014; 20:616-623.

[0270] 49. Park SJ, Zhang D, Qi Y, Li Y, Lee KY, BesseridesVJ, Wang P, Xia S, Kim SL, Liu X, Lu F, Pasqualini FD, Campbell PH. Geva J, Roberts AE, Kleber AG, Abrams DJ, Pu WT, Parker KK. Insights into the pathogenesis of catecholaminergic polymorphic ventricular tachycardia from engineered human heart issue. Circulation 2019; 140-390-404.

[0271] 50. Bezzerides VJ, Cabaellero A, Wang S, Ai Y, Hylind RJ. Lu F, Helms- Waldron DA, Chambers KD, Zhang D, Abrams DJ. Pu WT. Gene therapy for catecholaminergic polymorphic ventricular tachycardia by inhibition of Ca2+ / calmodulin-dependent kinase II. Circulation 2019; 140:405-419.

[0272] 51. Liu X, Wang S, Guo X, Li Y, Ogurlu R, Lu F, Prondzynski M, de la Sema Buzon S, Ma Q, Zhang D, Wang G, Cotton J, Guo Y, Xiao L. Milan DJ, Xu Y. Schlame M, Bezzerides VJ, Pu WT. Increased reactive oxygen species-mediated Ca2+ / calmodulin-dependent kinase II activation contributes to calcium handling abnormalities and impaired contraction in Barth syndrome. Circulation 2021; 143: 1894-1911.

[0273] 52. Wang G, Yand L, Grishin D. Rios X, Ye LY, Hu Y. Li K. Zhang D, Church GM, Pu WT: Efficient, footprint-free human iPSC genome editing by consolidation of Cas9 / CRISPR and piggyBac technologies. Nat Protoc 2017; 12:88- 103.

[0274] 53. Zhang JZ, Belbachir N, Zhang T, Liu Y, Shrestha R, Wu JC. Effects of Cryopreservation on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Assessing Drug Safety Response Profiles. Stem Cell Reports 2021;16: 168-181.

[0275] 54. Young D, Popovic ZB, Jones WK, Gupta S. Blockade of NF-kappaB using IkappaB alpha dominant- negative mice ameliorates cardiac hypertrophy in myotrophin-overexpressed transgenic mice. J Mol Biol 2008;381 :559-568.

[0276] 55. Breckwoldt K, Letuffe-Breniere D, Mannhardt I, Schulze T, Ulmer B, Wemer T, Benzin A, Klampe B, Reinsch MC, Laufer S, Shibamiya A, Prondzy nski M, Mearini G, Schade D, Fuchs S, Neuber C, Kramer E, Saleem U, Schulze ML, Rodriguez ML. Eschenhagen T, Hansen A. Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat Protoc 2017; 12: 1177-1197.

[0277] OTHER EMBODIMENTS

[0278] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method comprising: obtaining a sample comprising buccal cells from the subject; and detecting one or both of the level or localization of RelA / p65 in the subject sample.

2. A method of determining risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or predicting positive response to antiinflammatory drug therapies, in a subject, optionally a subject who has an inflammatory cardiac arrhythmogenic syndrome, optionally arrhythmogenic cardiomyopathy (ACM) or Brugada Syndrome, the method comprising: obtaining a sample comprising buccal cells from the subject; detecting localization of RelA / p65 protein in the subject sample; comparing the localization of the RelA / p65 protein in the subject sample to a reference localization; and identifying the subject as being at risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or predicting positive response to antiinflammatory drug therapies when the localization differs from the reference localization (optionally when nuclear localization is present).

3. The method of claim 2, where the reference level is a level in a subject who is not at risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or not likely to have a positive response to anti-inflammatory drug therapies, or who does not have an inflammatory cardiac arrhythmogenic syndrome, optionally does not have ACM or BrS.

4. The method of claim 2, wherein the reference localization is localization in a subject who does not have an inflammatory cardiac arrhythmogenic syndrome, optionally does not have ACM or BrS, optionally localization outside the nucleus.

5. The method of claim 1 or 2, wherein the buccal cells were obtained by rubbing the inside of the cheek of the subject with a swab, spatula, or scraper.

6. The method of claim 1 or 2, comprising applying the buccal cells onto a surface; optionally fixing the cells; and contacting the cells with an antibody or antigenbinding fragment thereof that binds to a desmosomal or gap junction protein.

7. The method of claim 2, wherein the inflammatory cardiac arrhythmogenic syndrome is BrS.

8. The method of claim 2, wherein the inflammatory cardiac arrhythmogenic syndrome is ACM.

9. The method of claim 1 or 2, comprising contacting the subject sample with antibodies to RelA / p65.

10. The method of claim 9, wherein the antibodies are directly or indirectly labeled, and the method comprises detecting the labeled antibodies.

11. The method of claim 1 or 2, wherein the subject is at least 7 years of age.

12. The method of claims 2-11, further comprising one or more of recommending or prescribing or administering an anti-inflammatory agent, optionally an NFKB inhibitor, to a subject identified as being at risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or predicting positive response to anti-inflammatory drug therapies.

13. The method of claim 13, further comprising selecting a subject who has ACM and is identified as being at risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or predicting positive response to anti-inflammatory drug therapies, for treatment.

14. The method of claim 14, further comprising administering the treatment to the identified subject.

15. The method of claim 14 or 15, wherein the treatment comprises one or more of recommending or advising the subject to avoid strenuous or intense physical activity or exercise; recommending or prescribing or administering one or more SinghVaughan Williams class TI antiarryhthmics (beta blockers) such as propranolol, esmolol, timolol, metoprolol, or atenolol; recommending or prescribing or administering one or more class III anti-arrhythmics (K-channel blockers) such as amiodarone, sotalol, ibutilide, dofetilide, dronedarone or E-4031; recommending or performing cardiac ablation; or recommending or implanting an implantable cardiac defibrillator (ICD).

16. The method of claim 13, further comprising selecting a subject who has BrS and is identified as being at risk of a cardiac event, ongoing myocardial injury, arrhythmias, and sudden cardiac death, and / or predicting positive response to anti-inflammatory drug therapies, for treatment.

17. The method of claim 16, further comprising administering the treatment to the identified subject.

18. The method of claim 14 or 15, wherein the treatment comprises one or more of: recommending or advising the subject to avoid strenuous or intense physical activity or exercise; recommending or prescribing or administering one or more of quinidine, tedisamil, and / or isoproterenol; recommending avoiding administration of agents that exacerbate BrS, including anti arrhythmic agents that block sodium channels; recommending or performing radiofrequency catheter cardiac ablation; or recommending or implanting an implantable cardiac defibrillator (ICD).

19. A kit comprising antibodies to RelA / p65, and and one, two, or more of a swab, scraper, or spatula for obtaining buccal cells from a subject; reagents for detecting the antibodies; a slide for receiving the buccal cells; and a fixative reagent for fixing the cells.

20. The kit of claim 20, wherein the antibodies are directly or indirectly labeled.

21. The kit of claim 20, wherein the fixative reagent comprises one or more of ethanol, methanol, glutaraldehyde, paraformaldehyde, formaldehyde, and / or saponin, or polyglycol in alcoholic solution.

Images