Background SARS-CoV-2-mediated COVID-19 may cause sudden cardiac death (SCD). Factors contributing to this increased risk of potentially fatal arrhythmias include thrombosis, exaggerated immune response, and treatment with QT-prolonging drugs. However, the intrinsic arrhythmic potential of direct SARS-CoV-2 infection of the heart remains unknown. Objective To assess the cellular and electrophysiological effects of direct SARS-CoV-2 infection of the heart using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods hiPSC-CMs were transfected with recombinant SARS-CoV-2 spike protein (CoV-2 S) or CoV-2 S fused to a modified Emerald fluorescence protein (CoV-2 S-mEm). Cell morphology was visualized using immunofluorescence microscopy. Action potential duration (APD) and cellular arrhythmias were measured by whole cell patch-clamp. Calcium handling was assessed using the Fluo-4 Ca 2+ indicator. Results Transfection of hiPSC-CMs with CoV-2 S-mEm produced multinucleated giant cells (syncytia) displaying increased cellular capacitance (75±7 pF, n = 10 vs. 26±3 pF, n = 10; P <0.0001) consistent with increased cell size. The APD90 was prolonged significantly from 419±26 ms (n = 10) in untransfected hiPSC-CMs to 590±67 ms (n = 10; P <0.05) in CoV-2 S-mEm-transfected hiPSC-CMs. CoV-2 S-induced syncytia displayed delayed afterdepolarizations, erratic beating frequency, and calcium handling abnormalities including calcium sparks, large “tsunami”-like waves, and increased calcium transient amplitude. After furin protease inhibitor treatment or mutating the CoV-2 S furin cleavage site, cell-cell fusion was no longer evident and Ca 2+ handling returned to normal. Conclusion The SARS-CoV-2 spike protein can directly perturb both the cardiomyocyte’s repolarization reserve and intracellular calcium handling that may confer the intrinsic, mechanistic substrate for the increased risk of SCD observed during this COVID-19 pandemic.

Triadin knockout syndrome (TKOS) is a malignant arrhythmia disorder caused by recessive null variants in TRDN-encoded cardiac triadin. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two unrelated TKOS patients and an unrelated control. CRISPR-Cas9 gene editing was used to insert homozygous TRDN-p.D18fs∗13 into a control line to generate a TKOS model (TRDN−/−). Western blot confirmed total knockout of triadin in patient-specific and TRDN−/− iPSC-CMs. iPSC-CMs from both patients revealed a prolonged action potential duration (APD) at 90% repolarization, and this was normalized by protein replacement of triadin. APD prolongation was confirmed in TRDN−/− iPSC-CMs. TRDN−/− iPSC-CMs revealed that loss of triadin underlies decreased expression and co-localization of key calcium handling proteins, slow and decreased calcium release from the sarcoplasmic reticulum, and slow inactivation of the L-type calcium channel leading to frequent cellular arrhythmias, including early and delayed afterdepolarizations and APD alternans.

SARS-CoV-2-mediated COVID-19 can affect the heart and cause sudden cardiac death (SCD). Multiple factors contribute to this increased risk of potentially fatal arrhythmias including thrombosis, exaggerated immune response, and treatment with QT-prolonging drugs. However, the intrinsic arrhythmic potential of direct SARS-CoV-2 infection of the heart is unknown.

Catecholaminergic polymorphic ventricular tachycardia is characterized by stress-induced ventricular tachycardia often leading to syncope and sudden death. Pathogenic variants in the RYR2-encoded ryanodine receptor 2 (RyR2) underlie CPVT1 and cluster into 4 distinct regions with cluster-I spanning from amino acids 77-466.

HomeCirculation: Genomic and Precision MedicineVol. 13, No. 6Prevalence and Phenotypic Correlations of Calmodulinopathy-Causative CALM1-3 Variants Detected in a Multicenter Molecular Autopsy Cohort of Sudden Unexplained Death Victims Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBPrevalence and Phenotypic Correlations of Calmodulinopathy-Causative CALM1-3 Variants Detected in a Multicenter Molecular Autopsy Cohort of Sudden Unexplained Death Victims Daniel J. Clemens, BS, Belinda Gray, BSc(Med), MBBS, PhD, Richard D. Bagnall, PhD, David J. Tester, BS, John R. Giudicessi, MD, PhD, Joseph J. Maleszewski, MD, Lia Crotti, MD, PhD, Peter J. Schwartz, MD, Emma Matthews, MRCP, Christopher Semsarian, MBBS, PhD, MPH, Elijah R. Behr, MD, PhD, MPH and Michael J. Ackerman, MD, PhD Daniel J. ClemensDaniel J. Clemens https://orcid.org/0000-0003-2102-5306 Windland Smith Rice Sudden Death Genomics Laboratory, Department of Molecular Pharmacology & Experimental Therapeutics (D.J.C., D.J.T., M.J.A.), Mayo Clinic, Rochester, MN. , Belinda GrayBelinda Gray https://orcid.org/0000-0002-9966-0329 Molecular & Clinical Sciences Research Institute, St George's University of London (B.G., E.R.B.). Cardiology Clinical Academic Group, St George's University Hospitals' National Health Service (NHS) Foundation Trust, London, United Kingdom (B.G., E.R.B.). Sydney Medical School Faculty of Medicine & Health (B.G., C.S.) Department of Cardiology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia (B.G., R.D.B., C.S.). , Richard D. BagnallRichard D. Bagnall https://orcid.org/0000-0002-2476-9864 Agnes Ginges Centre for Molecular Cardiology at Centenary Institute, The University of Sydney (R.D.B., C.S.). Department of Cardiology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia (B.G., R.D.B., C.S.). , David J. TesterDavid J. Tester Windland Smith Rice Sudden Death Genomics Laboratory, Department of Molecular Pharmacology & Experimental Therapeutics (D.J.C., D.J.T., M.J.A.), Mayo Clinic, Rochester, MN. Division of Heart Rhythm Services, Department of Cardiovascular Medicine (D.J.T., J.R.G., J.J.M., M.J.A.), Mayo Clinic, Rochester, MN. , John R. GiudicessiJohn R. Giudicessi https://orcid.org/0000-0001-5871-0265 Division of Heart Rhythm Services, Department of Cardiovascular Medicine (D.J.T., J.R.G., J.J.M., M.J.A.), Mayo Clinic, Rochester, MN. , Joseph J. MaleszewskiJoseph J. Maleszewski https://orcid.org/0000-0003-3239-6460 Division of Heart Rhythm Services, Department of Cardiovascular Medicine (D.J.T., J.R.G., J.J.M., M.J.A.), Mayo Clinic, Rochester, MN. Department of Laboratory Medicine & Pathology (J.J.M.), Mayo Clinic, Rochester, MN. , Lia CrottiLia Crotti https://orcid.org/0000-0001-8739-6527 Istituto Auxologico Italiano, IRCCS, Center for Cardiac Arrhythmias of Genetic Origin & Laboratory of Cardiovascular Genetics (L.C., P.J.S.). Istituto Auxologico Italiano, IRCCS Department of Cardiovascular, Neural & Metabolic Sciences, San Luca Hospital (L.C.). Department of Medicine & Surgery University of Milano-Bicocca, Milan, Italy (L.C.). , Peter J. SchwartzPeter J. Schwartz https://orcid.org/0000-0003-0367-1048 Istituto Auxologico Italiano, IRCCS, Center for Cardiac Arrhythmias of Genetic Origin & Laboratory of Cardiovascular Genetics (L.C., P.J.S.). , Emma MatthewsEmma Matthews https://orcid.org/0000-0002-3810-306X Queen Square Centre for Neuromuscular Diseases, National Hospital for Neurology and Neurosurgery & University College London Institute of Neurology, London, United Kingdom (E.M.). , Christopher SemsarianChristopher Semsarian https://orcid.org/0000-0001-6441-274X Sydney Medical School Faculty of Medicine & Health (B.G., C.S.) Agnes Ginges Centre for Molecular Cardiology at Centenary Institute, The University of Sydney (R.D.B., C.S.). Department of Cardiology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia (B.G., R.D.B., C.S.). , Elijah R. BehrElijah R. Behr https://orcid.org/0000-0002-8731-2853 Molecular & Clinical Sciences Research Institute, St George's University of London (B.G., E.R.B.). Cardiology Clinical Academic Group, St George's University Hospitals' National Health Service (NHS) Foundation Trust, London, United Kingdom (B.G., E.R.B.). and Michael J. AckermanMichael J. Ackerman Correspondence to: Michael J. Ackerman, MD, PhD, Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory, Guggenheim 501, Mayo Clinic, 200 First St SW, Rochester, MN 55905. Email E-mail Address: [email protected] https://orcid.org/0000-0002-8011-3333 Windland Smith Rice Sudden Death Genomics Laboratory, Department of Molecular Pharmacology & Experimental Therapeutics (D.J.C., D.J.T., M.J.A.), Mayo Clinic, Rochester, MN. Division of Heart Rhythm Services, Department of Cardiovascular Medicine (D.J.T., J.R.G., J.J.M., M.J.A.), Mayo Clinic, Rochester, MN. Division of Pediatric Cardiology, Department of Pediatric & Adolescent Medicine (M.J.A.), Mayo Clinic, Rochester, MN. Originally published15 Nov 2020https://doi.org/10.1161/CIRCGEN.120.003032Circulation: Genomic and Precision Medicine. 2020;13:e003032Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: November 15, 2020: Ahead of Print Sudden unexplained death is a profoundly tragic event for families and their communities. These cases can be categorized into 2 main groups including sudden infant death syndrome (SIDS, <1year of age) and sudden unexplained death in the young (SUDY, 1–35 years). In the United States alone, SIDS accounts for ≈3000 sudden deaths each year, whereas SUDY occurs in up to 5000 individuals annually.1 Approximately 10% of SIDS and 25% of SUDY may be caused by pathogenic variants in cardiac channelopathy-susceptibility genes.2Three different calmodulin genes, CALM1 (chr14q21), CALM2 (chr2p21), and CALM3 (chr19q13), each of which has a distinct genomic locus and unique nucleotide sequence, all encode for an identical 149 amino acid CaM (calmodulin).3 Since 2012, pathogenic variants in CALM1-3-encoded CaM, ≈90% of which are de novo, have been implicated as the underlying cause of multiple arrhythmic phenotypes including long-QT syndrome (CaM-LQTS), catecholaminergic polymorphic ventricular tachycardia (CaM-CPVT), and idiopathic ventricular fibrillation (CaM-IVF), collectively termed the calmodulinopathies.4Data from the recently published International Calmodulinopathy Registry found that 68% of 74 calmodulinopathic patients have suffered at least one major arrhythmic event at an average onset age of 4 years, and 27% experienced sudden cardiac death.4 However, despite its malignant and potentially lethal phenotype, the prevalence of calmodulinopathic variants in cases of SIDS and SUDY remains unknown.Here, we determined the spectrum and prevalence of CALM1-3 pathogenic variants in a large multicenter cohort of 599 SIDS and 258 SUDY cases contributed from 3 international medical centers in the United States, United Kingdom, and Australia. To prevent the reidentification of individuals included in this study, individual patient data will not be made available to other researchers. This study complies with the Declaration of Helsinki; locally appointed ethics committees including Mayo Clinic's Institutional Review Board have approved the research protocol.Of the SIDS cases, 362 (60%) were male, and 237 (40%) were female. The median age at death was 2 months (interquartile range, 1–4 months), and 349 (58%) of these cases were white. In the SUDY cohort, 176 (68%) cases were male, and 82 (32%) were female. The median age at death was 21 years (interquartile range, 16–29 years) with 88% dying between the ages of 1 to 35 years and 196 (76%) were white.Postmortem genomic DNA, derived from each decedent, underwent either whole exome or targeted gene panel sequencing followed by a gene-specific analysis of CALM1, CALM2, and CALM3, using Ingenuity Variant Analysis software. Only rare (minor allele frequency≤0.005% in gnomAD) nonsynonymous variants with a call quality score of ≥20 and a read depth of ≥10 were considered. Identified variants were classified according to the American College of Medical Genetics guidelines.Overall, we identified a pathogenic CALM1-3 variant in 3 out of 857 sudden unexplained death cases (0.035%). Interestingly, none of these variants were present in our SIDS cohort (0/599, 0%), but all 3 were identified in cases of SUDY (3/258, 1.2%; P=0.027; Figure [A]). The yield of pathogenic CALM 1-3 variants was significantly higher in SUDY cases dying between the ages of 1 and 10 years (3/32, 9.4%) compared with those older than 10 years at age of death (0/226, 0%; P=0.002; Figure [A]). In comparison, ≈7% of our SIDS cohort, and 26% of our SUDY cohort hosted a rare nonsynonymous variant in one of the four major channelopathy genes (KCNQ1, KCNH2, SCN5A, or RYR2).Download figureDownload PowerPointFigure. Yield and location of CALM variants identified in sudden infant death syndrome (SIDS) and sudden unexplained death in the young (SUDY).A, A table showing the yield of CALM variants in the SIDS and SUDY cohorts and in SUDY cases between the ages of 1 to 10 y and those >10 y of age. B, A schematic of the CaM (calmodulin) protein showing the N and C domains, each with 2 EF hands (EF-I through EF-IV) with calcium (red) bound. Blue circles represent WT residues, and white circles represent variants identified in our SUDY cohort. Combined Annotation Dependent Depletion (CADD) score >20 is considered an in silico threshold for possible pathogenicity. CPVT indicates catecholaminergic polymorphic ventricular tachycardia; LQTS, long-QT syndrome; and WT, wild type.A CaM-p.Asn54Ile variant (CALM1, c.161A>T) was identified in a 9-year-old female who died suddenly following extreme emotion. This variant is located in the inter-EF-hand I-II linker domain and is known to cause CaM-CPVT (Figure [B]).4 The second variant, CaM-p.Phe90Leu (CALM2, c.268T>C), which resides in the inter-EF-hand II-III linker domain was found in a 5-year-old male who experienced sudden death during physical exertion. Although this variant has not been identified before in CALM2, CALM1-p.Phe90Leu has been associated previously with CaM-IVF (Figure [B]).4 The CaM-p.Asn98Ser variant (CALM2, c.293A>G) was identified in a 2-year-old male who died suddenly while engaging in toddler play. This variant is located in the EF-hand III domain and has been associated with both LQTS and CPVT in CALM1- and CALM2-encoded calmodulin (Figure [B]).4 However, patients with the p.Asn98Ser variant typically do not express an overt LQTS phenotype.5Although CaM-LQTS patients exhibit a more malignant phenotype and have a higher rate of sudden cardiac death than other calmodulinopathy phenotypes, it is not surprising that the CALM variants identified in our SUDY cases have been associated with CaM-CPVT or CaM-IVF and not CaM-LQTS. Typically, CaM-LQTS manifests with severe and readily detectable clinical features (QTc >550 ms, bradycardia, 2:1 AV block, T-wave alternans) often occurring during infancy and is, therefore, likely detected before the occurrence of sudden death. Thus, the absence of CaM-LQTS variants in SIDS and SUDY may be explained by their high penetrance and marked expressivity. In contrast, CaM-CPVT and CaM-IVF patients do not display the same readily detectable clinical feature and may elude detection until a sentinel event of sudden cardiac death after the first year of life. Additionally, channelopathies are responsible for a greater percentage of SUDY than SIDS and, therefore, larger number of cases may be needed to identify CALM-related SIDS cases.Although pathogenic variants in CALM1-3 do not contribute meaningfully to SIDS, about 1% of SUDY overall stems from pathogenic CALM variants. Additionally, CALM variants may account for up to 10% of the sudden unexplained death cases occurring during childhood. Therefore, the CALM1, CALM2, and CALM3 genes should be included in postmorterm genetic testing (aka, the molecular autopsy), especially in children who have died between the ages of 1 to 10 years.Nonstandard Abbreviations and AcronymsCaMcalmodulinCPVTcatecholaminergic polymorphic ventricular tachycardiaIVFidiopathic ventricular fibrillationLQTSlong-QT syndromeSIDSsudden infant death syndromeSources of FundingThis work was supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. Dr Giudicessi thanks the Mayo Clinic Clinician-Investigator Training Program and Department of Cardiovascular Medicine for fostering an outstanding environment for physician-scientist training. Dr Semsarian is the recipient of a National Health and Medical Research Council Practitioner Fellowship (no. 1154992). B. Gray is the recipient of a National Health and Medical Research Council Early Career Fellowship (no. 1122330). E. Matthews is the recipient of a Wellcome Clinical Research Career Development Fellowship (no. 209583/Z/17/Z).DisclosuresDr Ackerman is a consultant for Abbott, ARMGO Pharma, Audentes Therapeutics, Biotronik, Boston Scientific, Daiichi Sankyo, Invitae, LQT Therapeutics, Medtronic, MyoKardia, and UpToDate. Dr Ackerman and Mayo Clinic are involved in an equity/royalty relationship with AliveCor, Blue Ox Health Corporation, and Stemonix. These relationships are all modest, and none of these entities has contributed to this study in any manner. The other authors report no conflicts.FootnotesThis manuscript was sent to Ruth McPherson, Guest Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page 717.Correspondence to: Michael J. Ackerman, MD, PhD, Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory, Guggenheim 501, Mayo Clinic, 200 First St SW, Rochester, MN 55905. Email ackerman.[email protected]eduReferences1. Kung HC, Hoyert DL, Xu J, Murphy SL. Deaths: final data for 2005.Natl Vital Stat Rep. 2008; 56:1–120.MedlineGoogle Scholar2. Semsarian C, Ingles J, Wilde AA. Sudden cardiac death in the young: the molecular autopsy and a practical approach to surviving relatives.Eur Heart J. 2015; 36:1290–1296. doi: 10.1093/eurheartj/ehv063CrossrefMedlineGoogle Scholar3. Fischer R, Koller M, Flura M, Mathews S, Strehler-Page M, Krebs J, Penniston J, Carafoli E, Strehler E. Multiple divergent mRNAs code for a single human calmodulin.J Biol Chem. 1998; 263:17055–17062.CrossrefGoogle Scholar4. Crotti L, Spazzolini C, Tester DJ, Ghidoni A, Baruteau AE, Beckmann BM, Behr ER, Bennett JS, Bezzina CR, Bhuiyan ZA, et al.. Calmodulin mutations and life-threatening cardiac arrhythmias: insights from the International Calmodulinopathy Registry.Eur Heart J. 2019; 40:2964–2975. doi: 10.1093/eurheartj/ehz311CrossrefMedlineGoogle Scholar5. Makita N, Yagihara N, Crotti L, Johnson CN, Beckmann BM, Roh MS, Shigemizu D, Lichtner P, Ishikawa T, Aiba T, et al.. Novel calmodulin mutations associated with congenital arrhythmia susceptibility.Circ Cardiovasc Genet. 2014; 7:466–474. doi: 10.1161/CIRCGENETICS.113.000459LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails December 2020Vol 13, Issue 6Article InformationMetrics Download: 336 © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCGEN.120.003032PMID: 33191766 Originally publishedNovember 15, 2020 Keywordsgeneticspediatricssudden infant deathcalmodulindeathPDF download Advertisement SubjectsArrhythmiasGeneticsPediatricsSudden Cardiac Death

Background: Triadin knockout syndrome (TKOS) is a potentially lethal arrhythmia disorder caused by recessively inherited null variants in TRDN -encoded cardiac triadin. Despite its malignant phenotype, the prevalence of TKOS in sudden infant death syndrome and sudden unexplained death in the young is unknown. Methods: Exome sequencing was performed on 599 sudden infant death syndrome and 258 sudden unexplained death in the young cases. Allele frequencies of all TRDN null variants identified in the cardiac-specific isoform of TRDN in the Genome Aggregation Database were used to determine the estimated prevalence and ethnic distribution of TKOS. Results: No triadin null individuals were identified in 599 sudden infant death syndrome and 258 sudden unexplained death in the young exomes. Using the Genome Aggregation Database, we estimate the overall prevalence of TKOS to be ≈1:22.7 million individuals. However, TKOS prevalence is 5.5-fold higher in those of African descent (≈1:4.1 million). Conclusions: TKOS is an exceedingly rare clinical entity that does not contribute meaningfully to either sudden infant death syndrome or sudden unexplained death in the young. However, despite its rarity and absence in large sudden death cohorts, TKOS remains a malignant and potentially lethal disorder which requires further research to better care for these patients.

BACKGROUND Triadin knockout syndrome (TKOS) is a rare arrhythmia syndrome caused by recessive null variants in TRDN-encoded cardiac triadin 1. TKOS has presented frequently with cardiac arrest in childhood. OBJECTIVE The purpose of this study was to elucidate the underlying genetic mechanism of disease in a genetically elusive patient displaying a characteristic TKOS phenotype. METHODS Genome sequencing and a TRDN gene-specific trio analysis were completed on the patient. RNA and protein isolated from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes were used to determine the effects of the identified variants using reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS Genome sequencing revealed compound heterozygous putative splice-error variants (maternal c.22129A.G and paternal c.48411189G.A). The novel paternally derived c.48411189G.A variant is located within 24 base pairs of a predicted alternative exon 6 (exon 6a), which resides within the intron between canonical exons 5 and 6. We determined that this previously unrecognized exon 6a produces a short TRDN transcript and potentially a novel protein isoform in the normal human heart. The c.48411189G.A variant not only results in abnormal splicing of the exon 6a-containing transcript leading to a frameshift mutation but also results in the abolishment of the 8-exon cardiac triadin 1 transcript. CONCLUSION Here, we present evidence for a novel alternative exon 6a-containing TRDN transcript in the normal heart. The novel deep intronic TRDN variant identified in a patient with TKOS leads to splicing error of a newly recognized exon 6a and loss of triadin. Considering that both TRDN variants in this patient were missed after commercial testing, these results highlight the importance of using genome sequencing when identifying patients with TKOS.

Viruses spread between hosts through particles, but within hosts, viral genomes can spread from cell to cell through fusion, evading antiviral defenses and obviating costly infectious virion production1-3. Billions of electromechanically coupled cardiomyocytes (CMs) make myocardium inherently vulnerable to pathological electromechanical short circuits caused by intercellular viral spread 4-6. Beyond respiratory illness, COVID-19 affects the heart7 and cardiac injury and arrhythmias are serious public health concerns8-12. By studying myocardium of a young woman who died suddenly, diagnosed postmortem with COVID-19, we discovered highly focal myocardial SARS-CoV-2 infection spreading from one CM to another through intercellular junctions identified by highly concentrated sarcolemmal t-tubule viral spike glycoprotein. SARS-CoV-2 permissively infected beating human induced pluripotent stem cell (hiPSC)-CMs building multinucleated cardiomyotubes (CMTs) through cell type-specific fusion driven by proteolytically-activated spike glycoprotein. Recombinant spike glycoprotein, co-localizing to sarcolemma and sarcoplasmic reticulum, produced multinucleated CMTs with pathological structure, electrophysiology and Ca2+ excitation-contraction coupling. Blocking cleavage, a peptide-based protease inhibitor neutralized SARS-CoV-2 spike glycoprotein pathogenicity. We conclude that SARS-CoV-2 spike glycoprotein, efficiently primed, activated and strategically poised during biosynthesis, can exploit the CM’s inherent membranous connectivities to drive heart damage directly, uncoupling clinically common myocardial injury from lymphocytic myocarditis, often suspected but rarely confirmed in COVID-19.

The functional maturation status of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has a notable impact upon their use in pharmacological studies, disease modeling, and therapeutic applications. Non-cardiomyocytes (non-CMs) produced in the differentiation process have previously been identified as having an extrinsic influence upon hiPSC-CM development, yet the underlying mechanisms are not fully understood. Herein, we aimed to modulate electrophysiological properties of hiPSC-CMs within co-cultures containing varied proportions of non-CMs and investigate the nature of interactions between these different cell types. Therefore, we sorted cardiac differentiations on day 10 and subsequently replated the cells at ratios of 7:3, 1:1, 3:7, and 1:9 non-CMs to CMs. After a month of co-culture, we evaluated electrophysiological properties through the genetically encoded voltage indicator ArcLight. We ultimately identified that co-cultures with approximately 70%–90% CM purity demonstrated the highest action potential (AP) amplitude and maximum upstroke velocity by day 40 of differentiation, indicative of enhanced electrophysiological maturation, as well as more ventricular-like AP morphologies. Notably, these findings were distinct from those observed for co-cultures of hiPSC-CMs and dermal fibroblasts. We determined that the co-culture phenotypes could not be attributed to paracrine effects of non-CMs due to the inability of conditioned media to recapitulate the observed effects. This led to the further observation of a distinctive expression pattern of connexin 43 (Cx43) at cell-cell interfaces between both CMs and non-CMs. Depletion of Cx43 by short hairpin RNA (shRNA) specifically in the non-CM population within a co-culture environment was able to recapitulate electrophysiological phenotypes of a purer hiPSC-CM population. Collectively, our data demonstrate that abundant non-CM content exerts a significant negative influence upon the electrophysiological maturation of hiPSC-CMs through Cx43-mediated cell-cell-contacts, and thus should be considered regarding the future production of purpose-built hiPSC-CM systems.

BACKGROUND:Triadin knockout syndrome (TKOS) is a rare, inherited arrhythmia syndrome caused by recessive null mutations in TRDN-encoded cardiac triadin. Based previously on 5 triadin null patients, TKOS has been characterized by extensive T-wave inversions, transient QT prolongation, and severe disease expression of exercise-induced cardiac arrest in early childhood refractory to conventional therapy.METHODS:We have established the International Triadin Knockout Syndrome Registry to include patients who have genetically proven homozygous/compound heterozygous TRDN null mutations. Clinical/genetic data were collected using an online survey generated through REDCap.RESULTS:Currently, the International Triadin Knockout Syndrome Registry includes 21 patients (11 males, average age of 18 years) from 16 families. Twenty patients (95%) presented with either cardiac arrest (15, 71%) or syncope (5, 24%) at an average age of 3 years. Mild skeletal myopathy/proximal muscle weakness was noted in 6 (29%) patients. Of the 19 surviving patients, 16 (84%) exhibit T-wave inversions, and 10 (53%) have transient QT prolongation > 480 ms. Eight of 9 patients had ventricular ectopy on exercise stress testing. Thirteen (68%) patients have received implantable defibrillators. Despite various treatment strategies, 14 (74%) patients have had recurrent breakthrough cardiac events.CONCLUSION:TKOS is a potentially lethal disease characterized by T-wave inversions in the precordial leads, transient QT prolongation in some, and recurrent ventricular arrhythmias at a young age despite aggressive treatment. Patients displaying this phenotype should undergo TRDN genetic testing as TKOS may be a cause for otherwise unexplained cardiac arrest in young children. As gene therapy advances, enrollment into the International Triadin Knockout Syndrome Registry is encouraged to better understand TKOS and to ready a well-characterized cohort for future TRDN gene therapy trials.

Introduction: Loss-of-function (LOF) variants in the KCNH2 -encoded Kv11.1 potassium channel cause type 2 long QT syndrome (LQT2). To date, hundreds of KCNH2 missense variants (MVs) have been published as “disease-causative”. However, it has been suggested that 10% of rare LQTS variants in the literature may be "false positives”. Objective: To determine previously published KCNH2 variants that are likely false positives and warrant demotion to at least likely benign status. Methods: A list of all case-derived LQT2-associated MVs from six large published compendia was compiled. The frequency of each MV within the Genome Aggregation Database (gnomAD, n = 141,352 individuals) was recorded. Eight in silico variant assessment tools were used to grade each MV. Variants (n=8) absent in gnomAD but predicted “benign” by all 8 tools were considered potential false positives, despite being ultra-rare, and were characterized functionally using whole-cell patch clamp. Results: Overall, 339 KCNH2 MVs were identified among LQTS cases. Of these, 13 (4%) were seen at a minor allele frequency (MAF) > 0.0004 in gnomAD. This MAF cut-off is derived from the frequency of R176W-KCNH2, a well-established albeit weakly penetrant (i.e. < 20%) LQT2-causative MV. Variants seen at MAF > R176W-KCNH2 are unlikely to be LQT2-contributing. However, 253 MVs (75%) were absent in gnomAD. Of these, 8 (4%) MVs (I96V, G187S, A203T, P241L, H254Q, G314S, P935S, and P963T) were predicted by all 8 in silico tools to be benign and had never been characterized functionally. Patch clamp studies did not demonstrate any LOF perturbation for these 8 MVs. Conclusion: This study offers compelling evidence for the demotion from LQT2-causative status to likely benign status for 21 (6%) of the 339 previously published LQT2 MVs based either on i) gnomAD MAF unacceptably high for a 1:8000 disorder (LQT2) for 13 MVs or ii) ultra-rarity (never seen in gnomAD) but in silico variant assessment tools predicting benign impact of the amino acid substitution and in vitro functional validation studies devoid of LOF for 8 additional MVs. This meticulous “pruning” exercise must be conducted for all published variants previously implicated as the monogenic cause for LQTS in particular and all genetic heart diseases in general.

Introduction: Loss-of-function (LOF) and gain-of-function (GOF) pathogenic variants in the KCNH2 -encoded Kv11.1 potassium channel cause type 2 long QT syndrome(LQT2) and type 1 short QT syndrome (SQT1), respectively. Given the prevalence of KCNH2- mediated heart disease (1 in 8000 for LQT2,1 in 100,000 for SQT1) and the reduced penetrance/variable expressivity seen in LQTS and SQTS, large databases such as the Genome Aggregation Database (gnomAD, n=141,352 individuals) might harbor subjects with unidentified LQT2/SQT1-causative mutations. Objective: To determine which “ultra-rare” KCNH2 missense variants (MVs) in gnomAD are most likely to be LQT2- or SQT1-causative. Methods: A list of gnomAD-derived MVs in KCNH2 was compiled. The frequency of each MV in gnomAD was assessed. Each MV was analyzed by 8 in silico pathogenicity prediction tools. MVs that were seen once in gnomAD and predicted damaging by all 8 tools were considered possible LQT2/SQT1-causative MVs worthy of functional validation via whole-cell patch clamp. Results: Overall, 233/474 (49%) of gnomAD KCNH2 MVs were seen in just 1 of >140,000 subjects. Of these, 2 (0.4%) MVs (S320W, S379Y) were predicted damaging by 8 in silico tools. Both S320W and S379Y localize to the N-terminus of the Kv11.1 potassium channel. There was no significant change in peak or tail current density between heterozygously expressed KCNH2-S320W (n=13, p=NS) and KCNH2-WT (n=13). However, heterozygous KCNH2-S379Y (n=12) produced a marked increase in peak current density across the range -30 mV to +20 mV and shifted the V 1/2 of activation by -17 mV (p < 0.001). Also, tail current density of S379Y channels significantly increased across the range -20 mV to +20 mV and the V 1/2 of deactivation shifted by -26 mV (p < 0.001). Conclusion: With this stringent requirement of ultra-rarity, universal prediction of damaging impact, and functionally abnormal electrophysiological properties, one SQT1-causative pathogenic variant in gnomAD was identified consistent with the predicted frequency of SQTS. The search continues among the remaining 32 ultra-rare MVs in KCNH2 where at least 75% of the in silico tools predicted deleterious impact of the MV to find the 15-20 subjects in gnomAD’s population who ought to have an LQT2-causative MV.

BACKGROUND Mutations in the KCNQ1-encoded Kv7.1 potassium channel cause long QT syndrome (LQTS) type 1 (LQT1). It has been suggested that similar to 10%-20% of rare LQTS case-derived variants in the literature may have been published erroneously as LQT1-causative mutations and may be "false positives." OBJECTIVE The purpose of this study was to determine which previously published KCNQ1 case variants are likely false positives. METHODS A list of all published, case-derived KCNQ1 missense variants (MVs) was compiled. The occurrence of each MV within the Genome Aggregation Database (gnomAD) was assessed. Eight in silico tools were used to predict each variant's pathogenicity. Case-derived variants that were either (1) too frequently found in gnomAD or (2) absent in gnomAD but predicted to be pathogenic by <= 2 tools were considered potential false positives. Three of these variants were characterized functionally using whole-cell patch clamp technique. RESULTS Overall, there were 244 KCNQ1 case-derived MVs. Of these, 29 (12%) were seen in >= 10 individuals in gnomAD and are demotable. However, 157 of 244 MVs (64%) were absent in gnomAD. Of these, 7 (4%) were predicted to be pathogenic by <= 2 tools, 3 of which we characterized functionally. There was no significant difference in current density between heterozygous KCNQ1-F127L, -P477L, or -L619M variant-containing channels compared to KCNQ1-WT. CONCLUSION This study offers preliminary evidence for the demotion of 32 (13%) previously published LQT1 MVs. Of these, 29 were demoted because of their frequent sighting in gnomAD. Additionally, in silico analysis and in vitro functional studies have facilitated the demotion of 3 ultra-rare MVs (F127L, P477L, L619M).