Diane Fatkin

BACKGROUND: Cardiomyopathy guidelines recommend routine screening of at-risk relatives with dilated cardiomyopathy (DCM). Titin-truncating variants are the most prevalent cause. However, the diagnostic yield of screening is low. Risk-based stratification could optimize screening intervals and resource allocation. This study aims to develop a safe, evidence-based gene-specific longitudinal screening algorithm for DCM development. METHODS: We included likely pathogenic/pathogenic titin-truncating variant relatives from 7 centers who underwent cardiac screening. Relatives were stratified based on their baseline clinical phenotype: genotype-positive/phenotype-negative (no left ventricular dysfunction and dilatation) or partial DCM (ie, fulfilling only 1 criterion). DCM development predictors were identified based on follow-up data. Risk profiles were established and used to develop a multistate model to create a longitudinal screening algorithm to safely and effectively monitor titin-truncating variant relatives. RESULTS: Among 413 relatives, follow-up data were available in 301 relatives (median follow-up 5.7 years), of whom 24.6% developed left ventricular dysfunction or dilatation and 17.3% developed DCM. In total, 16 relatives (5.3%) experienced a major adverse cardiac event after DCM diagnosis. Based on the identified risk factors, age ≥30 years, male sex, and partial DCM, 3 distinct profiles were established: (1) relatives with partial DCM, (2) females ≥30 years and males, and (3) female relatives <30 years. A screening algorithm was developed, recommending intervals of 1, 3, and 5 years, optimizing the balance between safety and effectiveness. CONCLUSIONS: An evidence-based genotype-specific longitudinal screening algorithm that integrates age, sex, and echocardiographic measurements may improve patient care and improve efficiency of clinical resource allocation.

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20’s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20 ^+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20 ^+/+ ). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20- centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20 ^+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

Rare coding genetic variants may exert large effects on risk of common disease, yet their contribution to disease architecture and their utility in gene prioritization remain limited by inadequate sample sizes. Here, we performed a massive-scale rare variant association study (RVAS), analyzing over 1.1 million sequenced participants among which 130,000 had atrial fibrillation (AF). Through a multi-mask burden testing approach, we identified 15 genes significantly associated with AF through rare large-effect variation. Integrative analyses revealed strong convergence between genes implicated by rare and common variation, and highlighted instances where RVAS data may aid in GWAS prioritization. Nevertheless, several RVAS genes were not among GWAS loci ( FAM189A2 , ACTC1 , FNIP1 , FBN1 ), or were not nominated through contemporary GWAS prioritization ( KDM5B , ZFP36L2 ). Finally, we observed that ultra-rare protein-disrupting variants - concentrated in a small number of large-effect size genes - explained at least 2% of AF susceptibility across European and African ancestry groups. These findings refine the genetic architecture of AF, while highlighting the value and cost of RVAS for genomic discovery in common disease.

Background: Truncating TTN variants (TTNtv) are the main genetic cause of dilated cardiomyopathy (DCM) and are independently associated with atrial fibrillation (AF). In DCM, AF usually arises from left atrial (LA) remodeling attributable to left ventricular systolic dysfunction. Whether TTNtv are linked to primary LA dysfunction independent of left ventricular systolic dysfunction remains unclear. Methods: This retrospective, multicenter study evaluated atrial function by strain echocardiography in TTNtv carriers across the left ventricular ejection fraction (LVEF) spectrum and its relationship with AF. Individuals with TTNtv and LVEF ≥50% (TTNtv+/DCM–) were compared with matched healthy controls, and those with LVEF P P P P P =0.200). Conclusions: LA myopathy, detected by strain imaging, appears early in TTNtv+/DCM– individuals and may represent an early phenotypic marker independent of left ventricular systolic dysfunction. When LVEF declines below 50%, atrial dysfunction worsens in parallel. AF was more frequent in TTNtv carriers, supporting further research on LA strain for AF risk stratification within this population.

BACKGROUND: Sinus bradycardia is a well-recognized physiological adaptation in endurance athletes, primarily attributed to sinus node remodeling or increased vagal modulation. Although genetic influences on resting heart rate (HR) have been observed, the genetic contribution to athletic bradycardia has not been elucidated. METHODS: We phenotyped current and former elite endurance athletes in the Pro@Heart cohort study using multimodal cardiac imaging, cardiopulmonary exercise testing, and Holter monitoring. Genetic susceptibility to bradycardia was assessed using a validated HR-associated polygenic risk score (HR-PRS), in which lower scores are associated with a lower HR, and compared with healthy nonathletic controls. Clinical and genetic features of bradycardic endurance athletes with minimum HR ≤40 bpm on a Holter monitor (bradycardic athletes [BAs]) were compared with non-BAs). A healthy cohort of nonathletes from the ASPREE study (Aspirin in Reducing Events in the Elderly) were used for genetic comparisons. RESULTS: Among 465 endurance athletes (median age, 23 [18–49] years, 75% men), 175 (38%) had a minimum HR on a Holter monitor ≤40 bpm, of whom 7 (2% of total) had a HR ≤30bpm. Pauses ≥2 s were observed in 115 (25%) athletes, of whom 12 (3% of total) had pauses ≥3 s. Mobitz I second-degree atrioventricular block was observed in 15 (3% of total) athletes. BAs were younger and fitter and exhibited greater athletic cardiac remodeling than non-BAs. Mean HR-PRS was significantly lower in all athletes compared with ASPREE nonathletes ( P P =0.006). When the distribution of HR-PRS within our athletic cohort was considered, athletes with scores in the bottom quartile had a lower minimum HR (median HR, 41 [35–45] bpm versus 45 [40–49] bpm, P P P= 0.004). Neither bradycardia nor pauses were associated with increased risk of adverse outcomes over 5.5 years. CONCLUSIONS: Resting bradycardia (HR ≤40 bpm) and pauses of 2 to 3 s are present in a significant proportion of endurance athletes and are well tolerated. Our data suggest that both fitness and genetic variation contribute to sinus node function in endurance athletes. Intriguingly, HR-PRS differed between athletes and nonathletes, raising the possibility that genetics may be a determinant of athleticism.

BACKGROUND: Truncating variants in the TTN gene ( TTN tv), encoding the giant sarcomeric protein titin, cause a range of human cardiac and skeletal muscle disorders of varying penetrance and severity. The effects of variant location on clinical manifestations are incompletely understood. METHODS: We generated 6 zebrafish lines carrying truncating ttn.2 variants in the Z-disk, I-band, A-band, and M-band titin regions. Expression of titin transcripts and protein levels was evaluated using quantitative polymerase chain reaction and proteomics. Phenotype analysis was performed during embryonic development and in adult hearts. RESULTS: Homozygous embryos from all lines except the C-terminal line, e232, showed a significant reduction of Z-disk and I-band ttn.2 transcripts, but A-band and M-band transcript levels were reduced only in lines with truncations distal to the cronos promoter. These homozygous embryos uniformly died by 7 to 10 days postfertilization with marked impairment of cardiac morphology and function. Skeletal muscle motility and sarcomere organization were more disrupted in mutants with truncations distal to the cronos promoter compared with those proximal. In contrast, homozygous e232 embryos, which lacked only the titin kinase and M-band regions, had relatively preserved cardiac function with incorporation of truncated Ttn.2/Cronos protein and normal sarcomere assembly, but selective degradation of fast skeletal muscle sarcomeres. All heterozygous embryos were phenotypically indistinguishable from wild type. High-frequency echocardiography in adult heterozygous fish showed reduced ventricular contraction under resting conditions in A-band mutants. Heterozygous Z-disk and I-band mutants had no significant baseline impairment but were unable to augment ventricular contraction in response to acute adrenaline exposure, indicating a lack of cardiac reserve. CONCLUSIONS: Our data suggest that cardiac and skeletal muscle dysfunction associated with truncating ttn.2 variants is influenced by age, variant location, and the amount of functional titin protein. The distinctive phenotype associated with distal C-terminal truncations may reflect different requirements for C-terminal titin for maintenance of fast, slow, and cardiac muscle sarcomeres.

Hypertrophic cardiomyopathy (HCM), a genetic heart disease defined by unexplained cardiac wall thickening, is a leading cause of sudden death worldwide. However, the three-dimensional organization of cardiac tissue underlying left ventricular hypertrophy remains poorly understood. We developed CaMVIA-3D, a deep-learning volumetric imaging and analysis pipeline to characterize cardiac microarchitecture. Analysis of tissues from HCM hearts revealed genotype-specific differences in cardiomyocyte volume, morphology, and extracellular volume, with pathogenic variants exhibiting greater concentric cellular hypertrophy and disarray and variant-negative cases showing predominant fibrosis. Longitudinal profiling of a pig HCM model revealed early-onset fibrosis preceding cardiomyocyte hypertrophy. Integrating transcriptomic and morphologic changes, we identified genes associated with cellular and extracellular remodeling. These findings define genotype-specific microstructural differences in HCM, offering insights to improve diagnostics and targeted therapies.

SUMMARY: Zebrafish and medaka are powerful cardiovascular models, yet cellular and molecular investigations of adult heart cells have been constrained by suboptimal dissociation and characterization methods. To overcome these barriers, we developed a physiological-temperature workflow that generates high-yield, viable single-cell suspensions for FACS, imaging, and low-input molecular profiling. Using transgenic fluorescent reporters, we consistently isolate ∼6,000 cardiomyocytes per adult zebrafish ventricle and ∼12,000 from medaka, preserving cellular, structural, and molecular integrity. Single-cell morphometrics revealed cardiomyocyte heterogeneity and demonstrated that swimming exercise induces both hypertrophy and hyperplasia, while ventricular injury triggers expansion of regenerative gata4 ⁺ cardiomyocytes. We integrated proteomics and RNA-seq from FACS-purified cells to construct cell-type-specific proteo-transcriptomic atlases. Functional enrichment, transcription factor, and network analyses identified protein hubs and regulatory circuits defining cardiomyocyte and endothelial cell identity. Our platform delivers cell-type-resolved molecular datasets of adult teleost cardiac cells, establishing a systems-level resource for heart regeneration research, cardiovascular disease modeling, and drug discovery. Teaser: A robust workflow for isolation, FACS, imaging, and multi-omics profiling of cardiomyocytes and cardiac endothelial cells in fish. HIGHLIGHTS: Temperature-optimized dissociation and standardized FACS enable high-yield isolation of viable cardiac cells. Single-cell imaging uncovers morphological heterogeneity in adult ventricular cardiomyocytes. Sustained exercise induces both hypertrophy and hyperplasia of zebrafish cardiomyocytes. Proteo-transcriptomics defines core molecular programs and interaction networks in cardiomyocytes and endothelial cells.