Optical Mapping Research Articles

Genome assembly of a diversity panel of Chenopodium quinoa

Quinoa (Chenopodium quinoa) is an important crop for the future challenges of food and nutrient security. Deep characterization of quinoa diversity is needed to support the agronomic improvement and adaptation of quinoa as its worldwide cultivation expands. In this study, we report the construction of chromosome-scale genome assemblies of eight quinoa accessions covering the range of phenotypic and genetic diversity of both lowland and highland quinoas. The assemblies were produced from a combination of PacBio HiFi reads and Bionano Saphyr optical maps, with total assembly sizes averaging 1.28 Gb with a mean N50 of 71.1 Mb. Between 43,733 and 48,564 gene models were predicted for the eight new quinoa genomes, and on average, 66% of each quinoa genome was classified as repetitive sequences. Alignment between the eight genome assemblies allowed the identification of structural rearrangements including inversions, translocations, and duplications. These eight novel quinoa genome assemblies provide a resource for association genetics, comparative genomics, and pan-genome analyses for the discovery of genetic components and variations underlying agriculturally important traits.

Diagnostic and Prognostic/Therapeutic Significance of Comprehensive Analysis of Bone and Soft Tissue Tumors Using Optical Genome Mapping and Next-generation Sequencing.

Detecting somatic structural variants (SVs), copy number variants (CNVs), and mutations in bone and soft tissue tumors is essential for accurately diagnosing, treating, and prognosticating outcomes. Optical genome mapping (OGM) holds promise to yield useful data on SVs and CNVs but requires fresh or snap-frozen tissue. This study aimed to evaluate the clinical utility of data from OGM compared to current standard-of-care cytogenetic testing. We evaluated 60 consecutive specimens from bone and soft tissue tumors using OGM and karyotyping, FISH, gene fusion assays, and deep next-generation sequencing (NGS). OGM accurately identified diagnostic SVs/CNVs previously detected by karyotyping and FISH (specificity=100%). OGM identified diagnostic and pathogenic SVs/CNVs (∼23% of cases) undetected by karyotyping (cryptic/submicroscopic). OGM allowed detection and further characterization of complex structural rearrangements including chromoanagenesis (27% of cases) and complex 3-6-way translocations (15% of cases). In addition to identifying 321 SVs and CNVs among cases with chromoanagenesis events, OGM identified approximately 9 SVs and 12 CNVs per sample. A combination of OGM and deep NGS data identified diagnostic and pathogenic SVs, CNVs, and mutations in ∼98% of cases. Our cohort contained the most extensive collection of bone and soft tissue tumors profiled by OGM. OGM had excellent concordance with standard-of-care cytogenetic testing, detecting and assigning high-resolution genome-wide genomic abnormalities with higher sensitivity than routine testing. This is the first and largest study to provide insights into the clinical utility of combined OGM and deep sequencing for the pathologic diagnosis and potential prognostication of bone and soft tissue tumors in routine clinical practice.

Optical genome mapping technology and its applications in genetic disease diagnosis

Optical genome mapping (OGM) is an emerging technology for the detection of genetic diseases based on physical mapping, which can detect numerical chromosomal abnormalities, copy number variation (CNV) and structural variation (SV) on a genome-wide scale. In recent years, a number of studies have proved that OGM, as a new generation of cytogenomic technique, has higher resolution and stronger ability to discover genomic variants compared with conventional genetic techniques. This article has systematically reviewed the principles, characteristics, advantages and limitations of OGM technology, and its applications in the diagnosis of genetic disorders.

Enhanced resolution of optical genome mapping utilizing telomere-to-telomere reference in genetic disorders.

Reference genomes serve as a baseline criterion for comparison of personal genomes to deduce clinical variants. The widely used reference genome, GRCh38, contains stretches of gaps and unresolved bases particularly in complex regions which could obscure variant discovery. In contrast, the gapless telomere-to-telomere CHM13 (T2T-CHM13) reference genome can be used to assess difficult regions of the genome. Optical genome mapping (OGM), an imaging technique for structural variation identification has improved resolution compared to traditional cytogenetic methods. Our study showcases the utility of the T2T-CHM13 reference genome for enhanced structural variant (SV) detection in complex regions. We illustrate this through two clinical cases, where improved alignment with T2T-CHM13 led to significantly higher confidence scores for critical SVs. We demonstrate improved clinical diagnostic outcomes with the updated T2T-CHM13 reference and advocate its adoption.

Retrospective study on the utility of optical genome mapping as a follow-up method in genetic diagnostics

BackgroundCurrent standard-of-care (SOC) methods for genetic testing are capable of resolving deletions and sequence variants, but they mostly fail to provide information on the breakpoints of duplications and balanced structural...

Multiscale Elasticity of Epoxy Networks by Rheology and Brillouin Light Spectroscopy.

The response of soft materials to an imposed oscillatory stress is typically frequency dependent, with the most utilized frequency range falling in the range of 10-2-102 rad/s. In contrast to most conventional contact techniques for measuring material elasticity, like tensile or shear rheology and atomic force microscopy, or invasive techniques using probes, such as microrheology, Brillouin light spectroscopy (BLS) offers an optical, noncontact, label-free, submicron resolution and three-dimensional (3D) mapping approach to access the mechanical moduli at GHz frequencies. Currently, the correlation between the experimental viscoelastic (at lower frequencies) and elastic (at higher frequencies) moduli has fundamental and practical relevance, but remains unclear. We utilize a series of solvent-free epoxy polymer networks with variable cross-link density as models to compare the storage modulus, G', (in the MPa range) obtained from shear rheology and the longitudinal modulus, M', (in the GPa range) extracted from BLS. Our results show that G' exhibits a much stronger increase with increasing cross-link density than M' (by a factor of about 3.5). This finding is discussed in the context of the phantom network model for G' and Wood's inverse rule of mixtures for M'. The epoxy polymer network displays an unexpectedly fast hypersonic dispersion compared to its uncross-linked precursor. These results testify the importance of obtaining reliable information about the elasticity of networks and will hopefully trigger further investigations in the direction of bridging the elasticity of soft materials at different scales.

Resveratrol alleviates heart failure by activating foxo3a to counteract oxidative stress and apoptosis

Resveratrol has been extensively studied for its multifaceted health benefits. Nonetheless, the pharmacological mechanisms of resveratrol for heart failure remain elusive, especially the cardioprotective effects. To address this knowledge gap, we performed high-throughput drug screening using zebrafish and discovered that resveratrol significantly alleviated heart failure, including rescuing abnormalities in heart rate, blood flow, cardiac output, and nppb overexpression. Mechanically, calcium optical mapping revealed that resveratrol diminished the prolongation of calcium duration at 90 % repolarization (CaD90). Membrane potential assay demonstrated that resveratrol alleviated mitochondrial damage, subsequently relieved the excessive accumulation of reactive oxygen species (ROS). Tunel staining further showed that resveratrol inhibited cardiomyocyte apoptosis both in zebrafish and human AC16 cell. Given the close relationship between the Forkhead Box O (foxo) family and oxidative stress and apoptosis, we used qPCR and noted that resveratrol could regulate the heart failure-induced expressions of foxo1b and foxo3a to normal levels. Furthermore, we conducted in situ hybridization to confirm the effective down-regulation patterns of foxo3a after resveratrol treatment. To investigate whether resveratrol’s effects are mediated via foxo3a, we used gardenoside to inhibit foxo3a expression, noting that resveratrol’s cardioprotective effects were reduced with foxo3a inhibition. Overall, our study underscores the molecular mechanisms by which resveratrol confers cardioprotection and provides a reference for heart failure therapeutic approaches.

De novo establishment of an ex vivo culture for living myocardial slices applying a microphysiological system – MPSlms

Abstract Cardiovascular disease is a global health burden. To develop novel treatment options complex in vitro model systems are needed that resemble the pathophysiological situation ex vivo. Nevertheless, current pre-clinical in vitro models for pharmacological research are limited in complexity. Basic cell culture models lack cardiac tissue architecture and intercellular communication, limiting their translational capability. Force measurement methods on ex vivo cultured living myocardial slices (LMS) have been described for contraction analysis studies after compound treatment. Here, we combined LMS with a microphysiological system (MPS) to develop MPSlms as heart-on-chip approach that enables advanced nutrition circulation and integrates electrical pacing (MPSpacer) of the ex vivo cardiac tissue. To optimize the LMS technique, we designed a novel isometric tissue holder (ITH) and miniaturized the LMS format, allowing for extended condition testing and thus refinement of animal experiments. The contractile performance of cardiomyocytes was quantified by applying optical mapping of movement detection, which revealed precise and local variations in contraction within one LMS.

Optical genome mapping identified deletions, inversions, and insertions in hemophilia

Optical genome mapping identified deletions, inversions, and insertions in hemophilia

Full characterization of unresolved structural variation through long-read sequencing and optical genome mapping

Structural variants (SVs) are important contributors to human disease. Their characterization remains however difficult due to their size and association with repetitive regions. Long-read sequencing (LRS) and optical genome mapping (OGM) can aid as their molecules span multiple kilobases and capture SVs in full. In this study, we selected six individuals who presented with unresolved SVs. We applied LRS onto all individuals and OGM to a subset of three complex cases. LRS detected and fully resolved the interrogated SV in all samples. This enabled a precise molecular diagnosis in two individuals. Overall, LRS identified 100% of the junctions at single-basepair level, providing valuable insights into their formation mechanisms without need for additional data sources. Application of OGM added straightforward variant phasing, aiding in the unravelment of complex rearrangements. These results highlight the potential of LRS and OGM as follow-up molecular tests for complete SV characterization. We show that they can assess clinically relevant structural variation at unprecedented resolution. Additionally, they detect (complex) cryptic rearrangements missed by conventional methods. This ultimately leads to an increased diagnostic yield, emphasizing their added benefit in a diagnostic setting. To aid their rapid adoption, we provide detailed laboratory and bioinformatics workflows in this manuscript.

Analysis of complex excitation patterns using Feynman-like diagrams

Many extended chemical and biological systems self-organise into complex patterns that drive the medium behaviour in a non-linear fashion. An important class of such systems are excitable media, including neural and cardiac tissues. In extended excitable media, wave breaks can form rotating patterns and turbulence. However, the onset, sustaining and elimination of such complex patterns is currently incompletely understood. The classical theory of phase singularities in excitable media was recently challenged, as extended lines of conduction block were identified as phase discontinuities. Here, we provide a theoretical framework that captures the rich dynamics in excitable systems in terms of three quasiparticles: heads, tails, and pivots. We propose to call these quasiparticles ‘cardions’. In simulations and experiments, we show that these basic building blocks combine into at least four different bound states. By representing their interactions similarly to Feynman diagrams in physics, the creation and annihilation of vortex pairs are shown to be sequences of dynamical creation, annihilation, and recombination of the identified quasiparticles. We draw such diagrams for numerical simulations, as well as optical voltage mapping experiments performed on cultured human atrial myocytes (hiAMs). Our results provide a new, unified language for a more detailed theory, analysis, and mechanistic insights of dynamical transitions in excitation patterns.

Effects and mechanisms of dexmedetomidine preconditioning on isoproterenol-induced ventricular arrhythmias

Dexmedetomidine (DEX) is commonly used in clinical practice because of its sedative, analgesic, antisympathetic, hemodynamic stabilization and antianxiety effects. Previous clinical studies have demonstrated that DEX plays a role in both the prevention and treatment of perioperative arrhythmias. However, the precise mechanisms underlying the effects of DEX remain unclear. Furthermore, few studies have examined the effect of DEX on cardiac electrophysiology. ECG recording was performed in vivo and ex vivo on C57 mice. Simultaneous recording of membrane voltage (Vm) and [Ca2+]i changes was achieved with dual-dye optical mapping, in which voltage- and Ca2+-sensitive dyes are employed. Simultaneous programmed electrical stimulation was used to pacing and induce arrhythmias. Simulating catecholamine-induced arrhythmias with isoprotereno (ISO) and preconditioning with DEX to investigate the antiarrhythmic effects of DEX. Our findings demonstrated that ISO increased the incidence of ventricular tachycardia or ventricular fibrillation in mice during rapid pacing stimulation. DEX preconditioning reduced the incidence of ISO-induced ventricular arrhythmias. Optical mapping with simultaneous recordings of dual dyes (Vm dye and intracellular Ca2+ dye) revealed that DEX pretreatment attenuated the ISO-induced shortening of action potential duration (APD), calcium transient duration (CaTD), and time-to-peak (TTP) of calcium transients, as well as the ISO-induced increase in repolarization heterogeneity. DEX also slowed the conduction velocity. More importantly, DEX preconditioning significantly reduced the calcium transient alternans ratio at 80-ms, 70-ms, and 60-ms pacing cycles. These findings suggest that DEX preconditioning can reduce the incidence of ventricular arrhythmias induced by acute stress simulated by ISO. Prolongation of action potential duration and calcium transient duration and the maintenance of intracellular calcium homeostasis may be the electrophysiological mechanisms involved.

Dual calcium-voltage optical mapping of regional voltage and calcium signals in intact murine RyR2-R2474S hearts

Abnormal regional variations in electrical and calcium homeostasis properties have been implicated in catecholaminergic polymorphic ventricular tachycardias (CPVT) attributable to abnormal RyR2-mediated store Ca2+ release, but their underlying mechanism have not been well explored in intact hearts. MethodsWe performed in vivo and ex vivo studies including high throughput mapping of Ca2+ transients (CaT) and transmembrane voltage (Vm) in murine wild-type (WT) and heterozygous RyR2-R2474S/+ hearts, before and during isoprenaline (ISO) challenge. ResultsISO-challenged RyR2-R2474S/+ showed increased incidence of arrhythmia accompanied by abnormal Ca2+ transients compared to WT. CaT duration (CaTD) in the LV apex amongst regions studied both before and during ISO challenge in both WT and RyR2-R2474S/+ ventricles. RyR2-R2474S/+ ventricles showed prolonged CaTD, both before and during isoprenaline (ISO) challenge. Conversely, action potential durations (APD) were the same in WT and RyR2-R2474S/+ ventricles and identically reduced by ISO challenge. RyR2-R2474S/+ showed Vm-CaT latencies at time to half decay, but not rise time to peak, which were significantly prolonged compared to WT in all ventricular regions examined with ISO challenge. Following burst pacing, ventricular localized concordant alternans in CaT and APD were readily observed in RyR2-R2474S/+ but not in WT mice. Such CaT and APD alternans occurred mostly semiannually in specific regions of the ventricular pre-occurrence of VT. ConclusionThe pro-arrhythmic RyR2-R2474S/+ phenotype in intact hearts thus directly parallels delayed regional CaT recovery properties and alteration of Vm-CaT latencies. Studies suggest that discordant localized calcium alternans are mechanistically responsible for action potential duration alternans and occurrence of VT in RyR2-R2474S/+ mice.

Insulin mitigates acute ischemia induced atrial fibrillation and sinoatrial node dysfunction ex vivo.

Acute atrial ischemia is a well-known cause of postoperative atrial fibrillation (POAF). However, mechanisms through which ischemia contributes to the development of POAF are not well understood. In this study, ex vivo Langendorff perfusion was used to induce acute ischemia and reperfusion in the heart in order to mimic POAF. Inducibility of atrial fibrillation (AF) was evaluated using programmed electrical stimulation and confirmed with open-atrium optical mapping. Compared to the control group without ischemia, 25 minutes of ischemia substantially increased the incidence of AF. The right atrium was more susceptible to AF than the left atrium. Administering insulin for 30 minutes before ischemia and during reperfusion with 25 minutes of ischemia greatly reduced the vulnerability to AF. However, insulin treatment during reperfusion only did not show substantial benefits against AF. Optical mapping studies showed that insulin mitigates ischemia-induced abnormal electrophysiology, including shortened action potential duration and effective refractory period, slowed conduction velocity, increased conduction heterogeneity, and altered calcium transients. In conclusion, insulin reduced the risk of acute ischemia/reperfusion-induced AF via improving the electrophysiology and calcium handling of atrial cardiomyocytes, which provides a potential therapy for POAF.

Adipocyte-Mediated Electrophysiological Remodeling of PKP-2 Mutant Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder responsible for nearly a quarter of sports-related sudden cardiac deaths. ACM cases caused by mutations in desmosome proteins lead to right ventricular enlargement, the loss of cardiomyocytes, and fibrofatty tissue replacement, disrupting electrical and mechanical stability. It is currently unknown how paracrine factors secreted by infiltrating fatty tissues affect ACM cardiomyocyte electrophysiology. A normal and a PKP2 mutant (c.971_972InsT) ACM hiPSC line were cultivated and differentiated into cardiomyocytes (CMs). Adipocytes were differentiated from human adipose stem cells, and adipocyte conditioned medium (AdCM) was collected. Optical mapping and phenotypic analyses were conducted on human iPSC-cardiomyocytes (hiPSC-CMs) cultured in cardiac maintenance medium (CMM) and either with AdCM or specific cytokines. Significant differences were observed in voltage parameters such as the action potential duration (APD80, APD30), conduction velocity (CV), and CV heterogeneity. When cultured in AdCM relative to CMM, the APD80 increased and the CV decreased significantly in both groups; however, the magnitudes of changes often differed significantly between 1 and 7 days of cultivation. Cytokine exposure (IL-6, IL-8, MCP-1, CFD) affected the APD and CV in both the normal and PKP2 mutant hiPSC-CMs, with opposite effects. NF-kB signaling was also found to differ between the normal and PKP2 mutant hiPSC-CMs in response to AdCM and IL-6. Our study shows that hiPSC-CMs from normal and mPKP2 ACM lines exhibit distinct molecular and functional responses to paracrine factors, with differences in RNA expression and electrophysiology. These different responses to paracrine factors may contribute to arrhythmogenic propensity.

Multiple paralogues and recombination mechanisms contribute to the high incidence of 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder. Why the incidence of 22q11.2DS is much greater than that of other genomic disorders remains unknown. Short read sequencing cannot resolve the complex segmental duplicon structure to provide direct confirmation of the hypothesis that the rearrangements are caused by nonallelic homologous recombination between the low copy repeats on Chromosome 22 (LCR22s). To enable haplotype-specific assembly and rearrangement mapping in LCR22 regions, we combined fiber-FISH optical mapping with whole genome (ultra-)long read sequencing or rearrangement-specific long-range PCR on 24 duos (22q11.2DS patient and parent-of-origin) comprising several different LCR22-mediated rearrangements. Unexpectedly, we demonstrate that not only different paralogous segmental duplicon but also palindromic AT-rich repeats (PATRR) are driving 22q11.2 rearrangements. In addition, we show the existence of two different inversion polymorphisms preceding rearrangement, and somatic mosaicism. The existence of different recombination sites and mechanisms in paralogues and PATRRs which are copy number expanding in the human population are a likely explanation for the high 22q11.2DS incidence.

Abstract 4132820: S-nitrosylation of cardiac Cx43 hemichannels at Cys271 promotes arrhythmogenicity and myocardial injury upon cardiac stress in Duchenne Muscular Dystrophy

Connexin-43 (Cx43) plays a critical role in the propagation of action potentials among cardiomyocytes and proper cardiac contractility. In healthy cardiomyocytes, Cx43 is located at the intercalated disk; however, Cx43 remodeling is observed in cardiac pathologies and is linked with arrhythmogenesis and sudden cardiac death. Utilizing a mouse model of Duchenne muscular dystrophy (DMD mdx ), we have previously demonstrated that cardiac Cx43 is laterally localized, forming undocked hemichannels that activate via S-nitrosylation in response to isoproterenol-evoked cardiac stress. This activation leads to the disruption of cardiac membrane permeability, triggered activity, and deadly arrhythmogenic behaviors. To establish the direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we developed a specific knock-in mouse line in which the single Cx43 site for S-nitrosylation, cysteine 271 (Cys271), was substituted with a serine (C271S +/- ). Here, we developed a DMD mdx :C271S +/- line (4–6 months old), exhibiting reduced levels of S-nitrosylated Cx43 after crossing DMDmdx mice and C271S +/- mouse lines to assess the effect of β-adrenergic stimulation-induced cardiac stress and heart dysfunction. We show that cardiac Cx43 remodeling was not prevented in DMD mdx :C271S +/- , similar to what was shown in DMD mdx mice via immunofluorescence analysis. In addition, DMD mdx mice displayed an increased number of deadly arrhythmogenic events, increased Ca 2+ signaling, and prolonged action potentials in Langendorff-perfused whole hearts via optical mapping, compared to wild-type and DMD mdx :C271S +/- mice. Similarly, isoproterenol treatment evoked severe myocardial injury, increased levels of plasmatic cardiac troponin I (cTnI), and 40% mortality in DMD mdx mice. Notably, DMD mdx :C271S +/- mice, similar to DMD mdx mice treated with the Cx43 hemichannel blocker Gap19, exhibited cardioprotection compared to the cardiac dysfunction observed in DMD mdx mice. Therefore, these findings strongly suggest that S-nitrosylation of Cx43 proteins at site Cys271 represents a fundamental NO-mediated mechanism involved in the induction of arrhythmias and myocardial injury in DMD mdx after β-adrenergic stress.

Abstract 4144530: IL-37 promotes resolution of Right Heart Disease-induced inflammation and atrial fibrillation.

INTRODUCTION: Atrial Fibrillation (AF) is the most common cardiac arrhythmia. Hypertension, obesity, diabetes or right heart disease (RHD) are important risk factors for AF. Inflammation stands as a common denominator among most AF risk factors. Studies have shown that patients with AF present high levels of circulating IL-18. Our group recently described that inflammation-resolution may prevent AF in RHD, however, the mechanism remains unclear. To inhibit IL-18-induced inflammation, IL37, a specific antagonist of IL-18-receptor was shown to attenuate cardiac fibrosis in a mice model of myocardial infarction, but little is known about its efficacy to prevent AF. HYPOTHESIS: IL-37 curbs RHD-induced atrial inflammation and AF vulnerability. METHODS: To induce right-sided cardiac hypertrophy and dilation, pulmonary artery banding (PAB) was performed in male and female Wistar rats (250-300g). Sham animals did not receive the ligation. Animals were randomized into four groups: Sham and PAB treated or not with IL37 (100 ng/ml/kg). Electrophysiological studies and echocardiography were performed in vivo before sacrifices at day (D)0, D7, D14 and D21. To analyze the atrial conduction, right atrial (RA) optical mapping was performed on Langendorff-perfused hearts. Histology, western blot, and qPCR were performed to study the expression of proteins and genes involved in atrial inflammation, fibrosis, and electrical remodeling. RESULTS: Twenty-one days after surgery, PAB rats were more vulnerable to AF and developed severe right-sided hypertrophy and dilation compared to Sham. Reliable to clinical evidence, female rats might be more resistant to AF than males. AF was associated with increased RA levels of inflammatory biomarkers including IL18, IL1β, and IL6. IL37 treatment was associated with decreased atrial expression of inflammatory markers. CONCLUSION: IL37 is a potential new therapeutic strategy to reduce atrial inflammation and prevent AF vulnerability in RHD.

Abstract 4138278: A Titin Missense Variant Causes Atrial Fibrillation

Background: Rare loss-of-function variants in TTN , the gene encoding the sarcomeric protein titin, have been causally associated with atrial fibrillation (AF). Missense TTN variants ( TTN mv) are generally regarded as benign in relation to dilated cardiomyopathy, but their role in AF is unknown. Hypothesis: TTN mv are associated with clinical outcomes and AF pathogenesis. Aims: We aimed to determine if TTN mv are associated with AF/heart failure (HF) hospitalizations and investigate if a rare missense variant ( TTN -T32756I) caused AF using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). Methods: Whole exome sequencing was performed in 131 adults at an urban academic healthcare system who identified as non-Hispanic Black (NHB) or Hispanic/Latinx (HL). Multivariable Cox proportional hazard models were used to assess cumulative risk of AF/HF hospitalization. Isogenic TTN -T32756I iPSC-aCM mutants were generated using the CRISPR-Cas9 technique. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping and patch-clamping to assess the electrophysiological (EP) properties. Results: We identified 137 TTN mvs in 76/131 (58.0%) of subjects, which were most commonly in the TTN A-band. 64 AF-related and 110 HF-related hospitalizations occurred after a median follow-up time of 4.16 years. Hospitalization risk was higher in TTN mv carriers (HR 1.81, Fig. 1A ). We also identified three patients with early-onset AF who shared a rare missense variant T32756I. TTN-T32756I -iPSC-aCMs displayed aberrant contractility ( Fig. 1B-D ), shortened action potential duration ( Fig. 1G-H ), increased activity of cardiac K + channel ( Fig. 1I ), and dysregulated Ca 2+ homeostasis ( Fig. 1J ) without compromising sarcomeric integrity ( Fig. 1E-F ). The titin-binding protein, Four-and-a-Half Lim domains 2 (FHL2), also has increased binding with KCNQ1 and its modulatory subunit KNCE1 in TTN-T32756I -iPSC-aCMs, which may enhance the slow delayed rectifier K + current ( I ks ). Suppression of FHL2 normalized I ks , supporting FHL2 as a mediator of the increased I ks ( Fig. 1K) . Conclusion: In a single-center multi-ethnic AF cohort, TTN mv were associated with increased AF/HF hospitalization, and the T32756I- TTN mv causes ion channel remodeling and an AF-like EP phenotype. These findings establish a mechanistic link between TTN mv, K + ion channels, and sarcomeric regulatory proteins that may represent a novel therapeutic target.

Abstract 4129195: Mechanisms of Healthy Aging in the Prevention of Atrial Fibrillation

Introduction: Atrial Fibrillation (AF) is the most frequent cardiac arrhythmia. AF can lead to severe complications such as myocardial infarction, strokes, and sudden death. With 70% of AF patients aged 65 and older, aging stands as the major risk factor for AF. Our group have recently contributed to reveal that old Wistar rats (20 months) are more susceptible to AF than younger rats (5 months). Interestingly, Lou/c/jall (LOU) rats, a model of healthy aging derived from Wistar rats, can live up to ~35 months old (equivalent to 100 human years). LOU rats have been identified as resistant to age-related diseases such as obesity, hypertension, and neurodegenerative disorders, which are major risk factors for AF. However, the susceptibility of LOU rats to AF have never been reported. Hypothesis: LOU rats may carry specific molecular adaptations conferring resistance to age-related cardiac remodeling and low AF vulnerability. Objectives: Characterize the vulnerability of very old LOU rats (32 months) to AF compared to old Wistar rats (22 months). Determine the atrial inflammatory profile of LOU rats and identify the molecular adaptations that may explain their resistance to AF Methods: Male and Female rats were divided into five groups: LOU and Wistar rats of 5 months (young), 20 months (old), and 32 months (very old: LOU/c rats only). AF inducibility was assessed in vivo using transesophageal electrophysiological study and cardiac structure and function were analyzed by in vivo echocardiography. Atrial electrical conduction was examined using ex vivo optical mapping. Histological analyses were performed to assess atrial fibrosis. Additionally, immunoblot analysis, qPCR, and RNA sequencing were employed to investigate the levels of biomarkers involved in atrial inflammation and fibrosis. Result: Old Wistar rats were more vulnerable to AF than young rats. Old and very old LOU were significantly more resistant to AF than old Wistar animals. LOU rats’ low susceptibility to AF was accompanied with absence of cardiac structural and functional remodeling and low levels of atrial fibrosis compared to old Wistar. Additionally, advanced age did not affect LOU rats’ atrial electrical conduction properties as compared to old Wistar rats. Conclusion: Promotion of healthy aging conditions mimicking LOU rats’ resistance to cardiac remodeling and reduction of atrial fibrosis may contribute to prevent AF in subjects with pathological aging.