Rate- and contractility-modulating drugs, such as adrenergic agonists and antagonists, are widely used in the treatment of cardiovascular conditions. Preclinical assessment of new modulators of rate, inotropy and metabolism can be aided by high-throughput (HT) methods for chronic measurements, coupled with scalable human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we evaluate the utility of long-term optical (label-free) measurements of pericellular oxygen in a HT format (96-well plates) for the assessment of the effectiveness of adrenergic drugs in hiPSC-CMs. Quantitative oxygen consumption metrics were derived and correlated to measurements performed in the same samples using all-optical electrophysiology. Adrenergic agonists significantly increased oxygen consumption rate (OCR), best seen in the kinetics of initial depletion of pericellular oxygen, i.e. time to reach 5%. Adrenergic antagonists decreased OCR, best quantified using steady-state values for pericellular oxygen after at least 5 h. OCR-based drug type identification correlated well with the acute spontaneous rate measurements in the same samples. Direct rate modulation with chronic optogenetic pacing sped up OCR in hiPSC-CMs. Blebbistatin, an excitation-contraction uncoupler, significantly reduced OCR. Computational modeling helped interpret our results by capturing the effects of pacing rate, adrenergic stimulation, and blebbistatin on oxygen consumption, thereby highlighting the key contribution of inotropy and mechanical contraction to OCR in hiPSC-CMs. We conclude that HT label-free optical oxygen measurements and the comprehensive in silico hiPSC-CM models, constrained by such measurements, represent valuable human-based approaches for non-invasive assessment of rate- and metabolism-modulating drugs in preclinical studies.

BACKGROUND:Caveolae are nanoscale, plasma membrane invaginations that compartmentalize ion channels and transporters, including those involved in sinoatrial node (SAN) activity. However, role of caveolae in cardiac pacemaking remains unknown. OBJECTIVES:This study sought to determine the role of caveolae in SAN pacemaking and sinus node dysfunction (SND). METHODS:In vivo electrocardiography, ex vivo optical mapping, in vitro Ca2+ imaging, immunofluorescent and electron microscopy were performed in wild-type, cardiac-specific Cav3 knockout and 8-week post-myocardial infarction heart failure mice. Mouse and human donor SAN tissues were used for biochemical protein copurification studies. A novel 3-dimensional single SAN cell mathematical model was used to determine the impact of protein localization on SAN pacemaking. RESULTS:In both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1, and Na+-Ca2+ exchanger (NCX1) proteins within discrete pacemaker signalosomes via direct association with Cav3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local sarcoplasmic reticulum Ca2+ release events, which diffuse across ∼15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (If) activation curve, and a 40% reduction of NCX1 function, along with ∼2.3-times widening of the sarcolemma-sarcoplasmic reticulum distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extracaveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by local sarcoplasmic reticulum Ca2+ release events and trigger action potentials. Heart failure remodeling mirrored caveolae disruption leading to NCX1-local sarcoplasmic reticulum Ca2+ release event uncoupling and SND. CONCLUSIONS:SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, demonstrating a new dimension of SAN remodeling and revealing a novel therapeutic target.

Atrial fibrillation (AF), the most common sustained arrhythmia, is both cause and consequence of atrial remodeling, with atrial fibrosis playing a key role in AF maintenance, progression, and treatment response. AF prevalence rises with age, as sex hormone (estrogen, E2, and testosterone, TS) levels decline in both sexes, and aging-associated extracellular matrix (ECM) remodeling parallels these hormonal transitions during menopause and andropause. Furthermore, extensive experimental evidence supports the protective effect of E2 and TS against fibrotic remodeling. However, the mechanistic basis of sex hormone-dependent antifibrotic effects remains unclear. To identify potential underlying mechanisms, we extended our computational model of atrial-enriched fibroblast (Fb) by incorporating E2 and TS pathways. We validated predictions against a broad set of independent experimental data, demonstrating 81% concordance in cardiac Fbs and 100% in atrial Fbs under two AF-relevant profibrotic stimuli: angiotensin-II (AngII) and transforming growth factor-β (TGFβ). E2 and TS significantly attenuated profibrotic remodeling triggered by both AngII and TGFβ. E2 exerted protection by suppressing Smad3 and upstream regulators of Ca2+ signaling, reactive oxygen species (ROS) formation, and Jun N-terminal kinase (JNK). TS showed limited protection against TGFβ-induced fibrogenesis, but significantly blunted AngII-induced fibrotic responses mainly through Smad3. These analyses identified hormone-specific regulatory nodes through which E2 and TS mitigate atrial fibrogenesis, offering mechanistic insight into how loss of sex hormone-mediated protection may contribute to age- and sex-dependent atrial remodeling. Our findings provide a quantitative framework for exploring sex hormone-mediated regulation of atrial remodeling and highlight potential therapeutic targets for antifibrotic AF treatment.NEW & NOTEWORTHY We developed a sex-informed computational model of atrial Fb signaling and used it to identify the mechanistic basis of sex hormone-dependent antifibrotic effects. TS acted primarily through Smad3, whereas ROS-JNK signaling emerged as a dominant mediator of E2-dependent antifibrotic protection, largely through coordinated feedback regulation. These findings offer hormone-specific regulatory nodes that may serve as a potential target for durable antifibrotic therapy in AF across sex and age.

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is a major contributor to stroke, heart failure, and mortality worldwide. Although AF affects both men and women at a similar rate, accumulating experimental and clinical evidence indicates that its underlying mechanisms, disease progression, and treatment responses differ by sex. However, current antiarrhythmic drug development and clinical management of AF remains largely sex neutral, likely contributing to limited efficacy and increased adverse effects. To address this gap, we developed a computational drug-screening pipeline based on experimentally constrained, sex-specific human atrial cardiomyocyte models to predict and evaluate sex-specific pharmacological strategies for AF. The pipeline integrates multivariable regression with mechanistic modeling to systematically test multi-target combinations of ion channel inhibitors and Ca2+ handling modulators and identify interventions that reduce arrhythmia vulnerability by restoring sex-specific electrophysiological and Ca2+ handling properties toward normal sinus rhythm (nSR). Application of this approach revealed a greater number of successful inhibitory drug combinations in males than in females. In males, optimal recovery to nSR primarily required inhibition of Na+ and K+ channels to prolong repolarization and refractoriness, increase Ca2+ transient amplitude (CaTAmp), and reduce susceptibility to action potential duration (APD) alternans. In females, modulation of Ca2+-related pathways was additionally required to suppress delayed afterdepolarizations (DADs). Forward single-cell simulations confirmed the predictions of the drug-analysis pipeline, demonstrating recovery of APD, CaTAmp, and arrhythmia vulnerability indices without introducing instabilities. Importantly, extension of these interventions to two-dimensional atrial tissue simulations demonstrated that sex-specific drug strategies reduce vulnerability to triggered activity, while suppression of reentry was most effective when combined with partial recovery of cell-cell coupling. Our results establish a multiscale computational pipeline for identifying sex-informed, multi-target antiarrhythmic therapies, amenable to experimental validation and translation to the clinic.

Atrial fibrillation (AF), the most common arrhythmia, is characterized by irregular electrical activity which contributes to electrophysiological and Ca 2+ cycling remodeling alongside extensive structural modifications, including atrial fibrosis. Fibrosis is marked by excessive extracellular matrix (ECM) deposition impacting AF maintenance and treatment outcomes. While fibrosis is a hallmark of AF in both sexes, sex-specific differences have been observed in ECM protein expression: Young females exhibit lower ECM levels than males, but these levels increase with age in females, likely due to estrogen (E2) loss during menopause. However, the mechanistic basis for E2 protective role against AF fibrogenesis remains unclear. To identify and quantitatively interrogate the mechanisms underlying E2 modulatory role in fibrogenesis during AF, we expanded our previously developed atrial fibroblasts (Fb) model by integrating E2-dependent signaling pathways based on experimental evidence in Fb-related cells. By adjusting E2 initial levels and maximal E2 receptor activation, we developed sex-specific models representing males, premenopausal females, and postmenopausal females. Our findings indicate that ECM levels significantly increase under high profibrotic signals—such as Angiotensin II (AngII) and Transforming Growth Factor β (TGFβ)— in postmenopausal female and male models, but remain unchanged in premenopausal females, aligning with experimental data. Mechanistically, we show that E2 mitigates AngII- and TGFβ-induced ECM upregulation, particularly by the suppression of Plasminogen Activator Inhibitor-1 (PAI1) consistent with experimental data from postmenopausal females. Specifically, E2 counteracts AngII-induced PAI1 by activating Protein Kinase A and inhibiting AngII Receptor Type-1 and Reactive Oxygen Species. Our novel model identifies E2 protective mechanisms against ECM production in Fbs and highlights key regulatory nodes as potential therapeutic targets. Future extensions will incorporate progesterone and testosterone to further refine sex-specific pathways in AF fibrogenesis. Ultimately, this work provides a foundation for developing targeted anti-AF therapies that prevent or reverse atrial fibrosis.

Atrial fibrillation (AF), the most common cardiac arrhythmia, shows marked sex differences in clinical presentation, treatment response and outcomes. Although prevalence is similar, women often experience more severe symptoms, higher rates of adverse drug effects and reduced treatment efficacy. To investigate the underlying sex-specific AF mechanisms, we developed and validated male and female human atrial cardiomyocyte models that integrate sex-based differences in electrophysiology and calcium (Ca2+) handling under normal sinus rhythm (nSR) and chronic AF (cAF) conditions. Although the model parameterizations and assumptions (based on limited human data) may not capture the full spectrum of clinical variability, the models reproduced key reported sex-dependent differences in human atrial cardiomyocyte action potential (AP) and Ca2+ transient (CaT) dynamics. Simulations revealed that both sexes exhibited shortened effective refractory periods and wavelengths in cAF vs. nSR. Females were more prone to delayed afterdepolarizations (DADs), whereas males were more susceptible to AP duration (APD) and CaT amplitude (CaTAmp) alternans. Population-based modelling identified distinct parameter associations with arrhythmia mechanisms: DAD vulnerability was associated with enhanced ryanodine receptor Ca2+ sensitivity in females, and alternans in males correlated with reduced L-type Ca2+ current maximal conductance. Pharmacological simulations revealed sex-specific responses to antiarrhythmic therapies. In males, multiple drug combinations restored APD at 90% repolarization (APD90), CaTAmp and reduced alternans susceptibility, whereas females responded to only one combination improving APD90 and CaTAmp but with minimal impact on DAD risk. These findings underscore the need for sex-specific therapeutic strategies and support use of computational modelling in guiding precision medicine against AF. KEY POINTS: Atrial fibrillation (AF) is a common heart rhythm disorder that presents differently in males and females, but how the underlying mechanisms differ in males and females is not fully understood. We developed and validated computer models of male and female human atrial cardiomyocytes that incorporate known sex differences in ion channels and calcium handling under normal sinus rhythm and AF conditions. Under normal rhythm, males and females showed distinct electrical activity, which became less pronounced in AF. In AF, both sexes showed reduced effective refractory period and wavelength and depressed calcium transients. Males were more susceptible to electrical alternans, whereas females showed a greater tendency for calcium-driven delayed afterdepolarizations. Simulated drug treatments showed greater benefit in male models, particularly with combinations targeting multiple potassium channels, whereas female models showed limited response. These results highlight the need for sex-specific approaches to treating AF and may help guide future drug development.

Females exhibit longer QT intervals and a higher risk of long QT syndrome (LQTS) associated arrhythmogenesis compared with males. Although several studies suggest these sex disparities result from the effect of sex hormones on cardiac ion channels, the underlying mechanisms remain incompletely understood. This research investigates the arrhythmogenic effects, sex-specific risk, and mechanisms associated with LQTS linked to either to loss-of-function of the rapidly activating delayed rectifier K+ current (IKr), or gain-of-function of the L-type Ca2+ current (ICaL). We primarily used the Tomek-Rodriguez (ToR-ORd) model of human ventricular cardiomyocytes and incorporated sex-specific parameterizations based on previous studies. The O'Hara-Rudy and Grandi-Bers models were used to demonstrate model-independence of the findings. We used a populations-of-models approach to assess early afterdepolarization (EAD) susceptibility in control and LQTS male and female groups. All female models had consistently longer action potentials and were more prone to EADs than male models. In the ToR-ORd model, IKr loss-of-function led to EADs in 65.8% of females versus 22.8% of males. ICaL gain-of-function led to EADs in 66.2% of females but only 3.6% of males. Using logistic regression analysis, we identified key ionic predictors of EAD susceptibility, with maximal conductance of the L-type Ca2+ current (GCaL) and maximal transport rate of the Na+/Ca2+ exchanger (GNCX) consistently emerging as positively and maximal conductance of the rapidly activating delayed rectifier K+ current (GKr) as negatively associated to EADs across both sexes and LQTS types. Notably, higher GNCX but lower GKr in female versus male cardiomyocytes could explain heightened female EAD risk. Our studies explore the ionic traits that favor (or confer resilience against) EADs with potential implications for personalized treatments. NEW & NOTEWORTHY We explored sex disparities in long QT syndrome (LQTS) using sex-specific human ventricular cardiomyocyte models. We showed that females exhibit greater susceptibility to early afterdepolarizations (EADs) than males, and identified key ionic predictors of EAD risk, including increases in the voltage-gated L-type Ca2+ current and electrogenic Na+/Ca2+ exchanger, and downregulation of the rapidly activating delayed rectifier K+ current. These findings offer new insights into sex-specific mechanisms underlying arrhythmogenesis in LQTS, with potential implications for personalized treatments.