Ackerman, Michael

Background: Calcium release channel deficiency syndrome (CRCDS) is caused by biogenic or biophysical loss-of-function (LOF) pathogenic variants in the RYR2 -encoded ryanodine receptor (RyR2), a key intracellular Ca 2+ release channel. Previously, we identified a novel homozygous duplication involving the promoter and exons 1-4 of RYR2 , leading to 80% RyR2 protein loss and exertion-related sudden death in the young. Here, we generated a RYR2 knockout (RYR2-KO) induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model to explore intracellular and electrophysiological compensatory mechanisms that overcome this extreme loss of RyR2 protein. Methods: Using CRISPR/Cas9 gene editing, a homozygous c.163delT variant (p.S55Pfs*46) was inserted into a normal iPSC line (isogenic control) to create a homozygous RYR2-KO iPSC line. After re-engineering the lines into ventricular-like cardiomyocytes (iPSC-CMs), intracellular Ca 2+ handling was assessed by Fluo-4 AM cell imaging (0.5 Hz stimulation). Electrical remodeling in the L-type Ca 2+ current (I CaL ) was assessed using the whole-cell patch clamp technique. Results: Significant differences were observed in Ca 2+ transient parameters between RYR2-KO and isogenic control iPSC-CMs. Biogenic RyR2 loss significantly reduced Ca 2+ transient peak amplitude (CTA: 0.16 ± 0.01 ΔF/F0, p<0.0001) and upstroke velocity (CTV: 0.42 ± 0.06 (ΔF/F0)/t, p<0.0001), and prolonged Ca 2+ transient duration (CTD 90 : 1.59 ± 0.03 s, p=0.01) as compared to isogenic control (CTA: 0.48 ± 0.04 ΔF/F0; CTV: 1.30 ± 0.15 (ΔF/F0)/t; CTD 90 : 1.46 ± 0.05 s). Additionally, RyR2 loss abolished SR Ca 2+ leak (0.9 ± 0.9 % versus 35.1 ± 15.5 %, p=0.03). Lastly, a significant reduction in I CaL density was observed in RYR2-KO iPSC-CMs as compared to isogenic control iPSC-CMs (at 0 mV, RYR2-KO: -8.54 ± 1.60 pA/pF, control: -17.45 ± 3.69 pA/pF, p=0.0004). These data indicate that I CaL reduction may contribute to the reduced Ca 2+ transient peak. Conclusions: Complete loss of RyR2 in iPSC-CMs profoundly disrupts intracellular Ca 2+ handling, abolishes Ca 2+ sparks frequency, and secondarily down-regulates the sarcolemmal L-type Ca 2+ channel.

Background: Long QT syndrome (LQTS) is a potentially lethal cardiac channelopathy that stems primarily from pathogenic variants in KCNQ1 (LQT1), KCNH2 (LQT2), or SCN5A (LQT3) and is characterized by prolongation of the cardiac action potential duration (APD) at the cellular level. Despite current therapeutic options, there is a pressing need for novel, precision medicine-based, effective pharmacological therapies for LQTS, particularly for LQT2 and LQT3. Goal: To develop a high-throughput drug screening platform to identify novel therapeutics for LQTS using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods: iPSC-CMs were made for patients with LQT2 (p.G604S) or LQT3 (p.P1332L). Voltage dye assays were performed using a kinetic image cytometer (KIC) in a 96 well format. The APD at 90% repolarization (APD90) was measured in treated/untreated iPSC-CMs. Cyteseer image analysis software was used for optical APD analysis. SCN5A-P1332L iPSC-CMs were treated with 10 uM mexiletine (MEX) as a positive control. 114 compounds from a commercially available library of FDA-approved drugs were tested in triplicate to assess their APD shortening efficacy. Whole-cell patch clamp was used to confirm the lead compound’s effect in KCNH2-G604S iPSC-CMs. Results: MEX significantly shortened the APD90 of SCN5A-P1332L iPSC-CMs by 13% (669 ± 5 to 586 ± 10 ms; p<0.0001; n=50). Of the 114 tested compounds, 6 shortened the APD90 of SCN5A-P1332L iPSC-CMs by greater than 20%. The lead compound shortened the APD90 of SCN5A-P1332L iPSC-CMs by 32% (571 ± 2 ms; n=314, to 387 ± 4 ms; n=80; p<0.0001). All 6 candidate compounds also shortened the APD90 of KCNH2-G604S iPSC-CMs by greater than 20% in KIC screening assays. Whole-cell patch clamp confirmed APD shortening by the lead compound in KCNH2-G604S iPSC-CMs (759 ± 35 ms; n=13, to 599 ± 89 ms; n=9; p<0.03). Conclusion: The high-throughput KIC screen confirmed mexiletine’s APD shortening effects in SCN5A-P1332L iPSC-CMs, validating the assay's potential for efficient, large-scale drug screening. In a pilot screen, we identified 6 novel LQTS drug candidates which shortened the APD90 in SCN5A-P1332L (LQT3) and KCNH2-G604S (LQT2) iPSC-CMs more than mexiletine. The lead compound’s APD shortening effects in KCNH2-G604S iPSC-CMs were confirmed with whole cell patch clamp. The KIC platform offers a robust method for identifying novel QT shortening compounds by enabling reproducible APD measurements in a high-throughput format.

Abstract Background and Aims Type 1 short QT syndrome (SQT1) is a genetic channelopathy caused by gain-of-function variants in KCNH2, resulting in shortened cardiac repolarization and QT intervals, which predispose patients to ventricular arrhythmias and sudden cardiac death. This study aimed to investigate the therapeutic efficacy of KCNH2-specific suppression-and-replacement (KCNH2-SupRep) gene therapy in a transgenic rabbit model of SQT1. Methods KCNH2-SupRep was developed by combining a KCNH2-shRNA with its corresponding shRNA-immune KCNH2-cDNA into an AAV9 vector, delivered directly into the aortic root (1 × 1010 vg/kg). Therapeutic efficacy was evaluated in vivo by electrocardiogram, ex vivo by optical mapping, and at cellular levels by patch-clamp, calcium imaging, and qPCR in ventricular cardiomyocytes (VCMs). Results In vivo, KCNH2-SupRep normalized the heart rate-corrected QT interval (QTc) in SQT1 rabbits, without affecting repolarization heterogeneity. Ex vivo, KCNH2-SupRep corrected the action potential duration (APD90) and resolved the increased apicobasal APD90 heterogeneity observed in untreated (UT)-SQT1 hearts, supporting an antiarrhythmic effect, which was further validated by reduced re-entry formation in silico. At cellular levels, KCNH2-SupRep prolonged APD90 in VCMs from SupRep-SQT1 rabbits closer to wildtype levels compared with UT- and sham-SQT1. Additionally, KCNH2-SupRep restored the cellular surrogate of the electro-mechanical window and normalized IKr in nearly 50% of VCMs, in line with a 50%–60% suppression of the mutant KCNH2 transcript. Conclusions This proof-of-concept study is the first to demonstrate the efficacy of gene therapy for SQT1 in a medium-sized animal model. KCNH2-SupRep gene therapy successfully corrected the pathologic phenotype in vivo, ex vivo, and at cellular levels in transgenic SQT1 rabbits.

Background: Many medications, electrolyte perturbations, and several diseases can prolong the QTc beyond its 99 th percentile value resulting in acquired QT prolongation (AQTP). In contrast, approximately 1 in 2,000 people have congenital long QT syndrome (LQTS) hallmarked by pathological QT prolongation. Recently, we developed an AI-ECG model to distinguish these two groups, however this model does not elucidate which ECG segments distinguish LQTS from AQTP. Methods: Using the cohort from our previous study, all patients with LQTS evaluated in the Mayo Clinic Genetic Heart Rhythm Clinic and controls were included. Next, every patient/control with ≥1 ECG above age- and sex-specific 99 th percentile for QTc was selected and matched at 1:100 ratio. Subsequently, 5 ML algorithms (logistic regression, Naïve Bayes, random forest, Gradient boosting and XGBoost) were trained using 52 features representing segments of the 12-lead ECG (e.g. wave peak times, amplitudes, areas under wave, duration; 52x12 features). Training and testing sets were spilt at 80:20 ratio and 5-fold cross validation was performed to prevent overfitting. Following analysis, the top 100 features were re-analyzed to refine results. Results: Among >1,600 patients with LQTS, 690 had ≥ 1 ECG with a QTc above the established threshold compared to 28,186 controls. Following age- and sex-matching and splitting, 22,410 (training), and 5,776 (testing) ECGs were used. Of the 5 ML algorithms, XGBoost demonstrated best performance (AUC 0.913, accuracy 0.836, sensitivity 0.857, specificity 0.835) to distinguish LQTS from AQTP. Following filtering for the top 100 features, performance of the algorithm remained high with AUC of 0.912 (accuracy 0.84, sensitivity 0.841, specificity 0.84). The features with the greatest impact on classification output included time from T wave onset to T wave peak (leads V1, V4, V4), QRS interval duration (leads V1, V3, V4, aVR), and amplitude at end of ST segment (aVF). Conclusions: Distinguishing monogenetically driven LQTS from multi-factorial AQTP is critical in clinical practice for proper management, mitigation of offending QT-prolonging factors, and treatment. Building on our previously developed AI-ECG-LQTS ‘mutation detector’, this model identifies some of the elements within the ‘black box’ that distinguish an ECG and QTc stemming from a patient with LQTS compared to a similar prolonged QTc value that is arising due to non-genetic, acquired QT-prolonging factors.

Background: We identified a novel, ultra-rare missense variant (p.R534S) in DENND3 as a potential disease-susceptibility variant in a multi-generational pedigree of familial idiopathic ventricular fibrillation (IVF). DENND3 is a guanine nucleotide exchange factor and activator for small GTPase Rab proteins involved in endosome and ion channel membrane trafficking. However, the role of DENND3 in the heart and its potential involvement in cardiac arrhythmias are unknown. Objective: To functionally characterize DENND3-p.R534S and determine its impact on channel trafficking. Methods: Heterologous co-expression studies in TSA201 cells were performed with DENND3-WT or DENND3-p.R534S and ion channels: KCNQ1 (Kv7.1, IKs), KCNH2 (Kv11.1, IKr), SCN5A (Nav1.5, INa), or CACNA1C (Cav1.2, ICa) as well as Rab proteins (Rab4, Rab5, Rab6, Rab7, Rab11, Rab12). Patient-specific DENND3-p.R534S iPSC-derived cardiomyocytes (iPSC-CMs) and isogenic controls (IC) were created. Localization and expression of ion channel and Rab proteins was assessed using immunofluorescence (IF) by super resolution AiryScan microscopy. GTPase-Glo assays measured Rab protein activity. Electrophysiology studies using patch-clamp and FluoVolt were performed on iPSC-CM models. Results: IF imaging showed significant increases in membrane expression of KCNQ1 (11714±157to 21929±378, p<0.05), KCNH2 (11378±160 to 31965±544, p<0.05), , SCN5A (10339±8 to 11225±23, p<0.05), and CACNA1C (11879±83 to 13749±138, p<0.05) in DENND3-p.R534S compared to DENND3-WT expressing TSA201 cells. Rab5 distribution was altered significantly by the DENND3-p.R534S variant, with increased dispersion away from the nucleus (0.23±0.01 vs. 0.17±0.01, p<0.05) and increased proximity to the plasma membrane (0.16±0.02 vs. 0.23±0.01, p<0.05). Additionally, Rab5 activity was enhanced significantly (57% increase) by p.R534S versus WT (p<0.05). iPSC-CMs revealed similar increases in channel membrane accumulation and irregular arrhythmic activity including frequent action potential duration alternans and afterdepolarizations in 78% of p.R534S iPSC-CMs versus none in IC cells (p<0.05). Conclusion: DENND3 is a novel ion channel trafficking protein in the heart. The DENND3-p.R534S variant disrupts cardiac ion channel trafficking. These findings highlight DENND3’s role in cardiac ion channel distribution, cellular function, and potential association with arrhythmogenic risk.

Background: Cardiac channelopathies are a heterogenous group of genetic heart diseases (GHDs) that affect cardiac ion channel function. The most prevalent among these are long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) which predispose patients to potentially life threatening ventricular arrhythmias. Accurate and timely diagnosis is important for the management and risk stratification of the index case and identification of potentially at-risk relatives. Unfortunately, despite rising awareness of these GHDs, critical warning signs are overlooked. Objective: To describe the phenotypes of patients who present with a delayed or missed diagnosis of LQTS or CPVT. Methods: EMR data was reviewed for all patients evaluated and treated at Mayo Clinic between 2000-2023 who arrived with a mis- or delayed diagnosis of LQTS or CPVT. Data was abstracted for patient demographics, clinical characteristics, family history of SCD, and cardiac and genetic test results. Results: Overall, 47/2119 (2%) patients [33 (70%) female, mean age at correct diagnosis 26±18 years] diagnosed with LQTS (30; 2% of LQTS cohort) or CPVT (17; 7% of CPVT cohort) arrived with an outside misdiagnosis (37, 79%) or underdiagnosis (10, 21%). The most common misdiagnosis was epilepsy (20; 43%) or vasovagal syncope (14; 30%) and 3 CPVT patients (6%) were misdiagnosed with LQTS. Of the 10 underdiagnosed patients, 4 (40%) had QT prolongation, 4 (40%) family history of SCD, 3 (30%) bilateral deafness, and 1 (10%) fetal bradycardia and syndactyly. Overall, 40/47 (85%) were symptomatic (syncope: 29 [73%], seizures: 21 [53%], SCA: 10 [25%]) prior to diagnosis. Of note, 34/40 (85%) had symptoms with exertion, 5 (13%) at rest, and trigger was unknown in 1 (2%). Of the 7 asymptomatic patients (15%), 4 (57%) had QT prolongation and 4 (57%) had family history of SCD. In total, 20/47 patients (43%) had positive family history of SCD under 45 years of age. Overall, patients experienced a median delay in diagnosis of 8 years during which a median of 4 events (range: 1-150) occurred. As a result, an additional 29 family members of these patients received a delay in their diagnosis. Conclusions: Recognizing the signs and symptoms of patients with GHDs is important to reduce the ongoing burden of misdiagnosis or delayed diagnosis in LQTS and CPVT. The most important factors for suspicion of an inherited channelopathy are symptoms during exertion and family history of SCD.

Abstract 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.

Background: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. Methods: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. Results: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance ( P <5×10 −8 ) near NOS1AP , KCNQ1 , and KLF12 , and 1 missense variant in KCNE1 (p.Asp85Asn) at the suggestive threshold ( P <10 −6 ). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation ( h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (r g =0.40; P =3.2×10 −3 ). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls ( P <10−13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive ( P <0.005). Conclusions: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.

Abstract Aims Brugada syndrome (BrS) is characterized by a unique electrocardiogram (ECG) pattern and life-threatening arrhythmias. However, the Type 1 Brugada ECG pattern is often transient, and a genetic cause is only identified in &lt;25% of patients. We sought to identify an additional biomarker for this rare condition. As myocardial inflammation may be present in BrS, we evaluated whether myocardial autoantibodies can be detected in these patients. Methods and results For antibody (Ab) discovery, normal human ventricular myocardial proteins were solubilized and separated by isoelectric focusing (IEF) and molecular weight on two-dimensional (2D) gels and used to discover Abs by plating with sera from patients with BrS and control subjects. Target proteins were identified by mass spectrometry (MS). Brugada syndrome subjects were defined based on a consensus clinical scoring system. We assessed discovery and validation cohorts by 2D gels, western blots, and ELISA. We performed immunohistochemistry on myocardium from BrS subjects (vs. control). All (3/3) 2D gels exposed to sera from BrS patients demonstrated specific Abs to four proteins, confirmed by MS to be α-cardiac actin, α-skeletal actin, keratin, and connexin-43, vs. 0/8 control subjects. All (18/18) BrS subjects from our validation cohorts demonstrated the same Abs, confirmed by western blots, vs. 0/24 additional controls. ELISA optical densities for all Abs were elevated in all BrS subjects compared to controls. In myocardium obtained from BrS subjects, each protein, as well as SCN5A, demonstrated abnormal protein expression in aggregates. Conclusion A biomarker profile of autoantibodies against four cardiac proteins, namely α-cardiac actin, α-skeletal actin, keratin, and connexin-43, can be identified from sera of BrS patients and is highly sensitive and specific, irrespective of genetic cause for BrS. The four involved proteins, along with the SCN5A-encoded Nav1.5 alpha subunit are expressed abnormally in the myocardium of patients with BrS.