Biology subjects

Bers, D. M.

Publications and source records attributed to Bers, D. M..

10 recordsLinked to original sources

bioRxiv2026

Dynamic visualization of physiological CaMKII activity using sensitive FRET biosensors

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key mediator of complex physiological processes throughout the body, from the brain to the reproductive system, where CaMKII translates spatiotemporally dynamic calcium elevations into specific biological functions. Directly visualizing CaMKII activity dynamics in living cells using genetically encoded fluorescent biosensors can thus provide crucial insights into the molecular regulation of health and disease. Yet the ability to sensitively and specifically monitor endogenous CaMKII activity in physiologically relevant contexts is limited by the lack of sensors that can achieve robust, quantitative visualization of CaMKII responses. Here, we leveraged a recent serine/threonine kinome-wide substrate atlas to rationally engineer a powerful suite of Forster resonance energy transfer (FRET)-based CaMKII kinase activity reporters with high specificity, sensitivity, and signal-to-noise ratio. Using these biosensors, we were able to sensitively and robustly visualize endogenous CaMKII activity dynamics in both cultured cell lines and primary cells, including cardiomyocytes, oocytes, and neurons. We further utilized 2pFLIM imaging of organotypic hippocampal slices to quantitatively track LTP-induced CaMKII activity within single dendritic spines, highlighting a major advance in the study of physiological CaMKII signaling.

bioengineering↗

bioRxiv2025

Role of Dynamical Instability in QT Interval Variability and Early Afterdepolarization Propensity

Beat-to-beat variability of the QT interval (QTV) is a well-established marker of cardiac health, with increased QTV (> 5 ms) often associated with a higher risk of arrhythmias. However, the underlying mechanisms contributing to this phenomenon remain poorly understood. Recently, we showed that cardiac instability is a major cause of QTV. Early afterdepolarizations (EADs) are abnormal electrical oscillations that occur during the plateau phase of the cardiac action potential (AP), often arising when the membrane potential becomes unstable. In this study, we use a physiologically detailed computational model of rabbit ventricular myocytes with stochastic ion channel gating to investigate the relationship between QTV and EAD propensity. We found that increased AP duration (APD) variability, which serves as a surrogate for QTV on the ECG at the single-cell level, can arise even in the absence of apparent EADs, driven by intrinsic dynamical instability. As the cellular state approaches the threshold for EAD generation, small perturbations in membrane voltage are amplified, leading to increased APD variability. The phase-plane analysis in the voltage-calcium channel inactivation space demonstrates that proximity to the EAD-generating basin of attraction strongly influences repolarization variability, establishing a mechanistic link between QTV and EAD propensity. Furthermore, we observed that QTV increases at longer pacing cycle lengths (PCLs), distinguishing it from alternans-associated APD variability, which increases at shorter PCLs. These findings suggest that increased QTV may serve as an early indicator of arrhythmic risk before the manifestation of EADs, potentially offering a critical window for preventive intervention. Our results provide novel insights into the fundamental mechanisms underlying QTV and its potential role in arrhythmia prediction. Significance statementThis study investigates the relationship between beat-to-beat variability of the QT interval (QTV) and the propensity for early afterdepolarizations (EADs), abnormal electrical oscillations linked to life-threatening arrhythmias. Using a computational model, we show that increased QTV can precede apparent EADs, driven by inherent dynamical instability of cardiac cells. As cells approach an arrhythmia-prone state, small membrane voltage fluctuations are amplified, increasing repolarization variability and thus QTV. Furthermore, QTV increases with slower heart rates, distinguishing it from alternans, another type of instability arising at faster rates. Thus, increased QTV may serve as an early warning signal for arrhythmia risk, potentially enabling preventative interventions. Our results provide novel insights into the fundamental mechanisms underlying QTV and its potential role in arrhythmia prediction.

biophysics↗

bioRxiv2025

De novo Design of a Peptide Modulator to Reverse Sodium Channel Dysfunction Linked to Cardiac Arrhythmias and Epilepsy

Ion channels orchestrate electrical signaling in excitable cells. In nature, ion channel function is customized by modulatory proteins that have evolved to fulfill distinct physiological needs. Yet, engineering synthetic modulators that precisely tune ion channel function is challenging. One example involves the voltage-gated sodium (NaV) channel that initiates the action potential, and whose dysfunction amplifies late/persistent sodium current (INaL), a commonality that underlies various human diseases including cardiac arrhythmias and epilepsy. Here, using a computational protein design platform, we engineered a de novo peptide modulator, ELIXIR, that binds NaV channels with submicromolar affinity. Functional analysis revealed an unexpected selectivity in inhibiting pathogenic INaL and confirmed its effectiveness in reversing NaV dysfunction linked to both cardiac arrhythmias and epilepsy in cellular and murine models. These findings exemplify the efficacy of de novo protein design for engineering synthetic ion channel modulators and sets the stage for rational design of future therapeutic approaches.

biophysics↗

bioRxiv2025

T-World: A highly general computational model of a human ventricular myocyte

Cardiovascular disease is the leading cause of death, demanding new tools to improve mechanistic understanding and overcome limitations of stem cell and animal-based research. We introduce T-World, a highly general virtual model of human ventricular cardiomyocyte suitable for multiscale studies. T-World shows comprehensive agreement with human physiology, from electrical activation to contraction, and is the first to replicate all key cellular mechanisms driving life-threatening arrhythmias. Extensively validated on unseen data, it demonstrates strong predictivity across applications and scales. Using T-World we revealed a likely sex-specific arrhythmia risk in females related to restitution properties, identified arrhythmia drivers in type 2 diabetes, and describe unexpected pro-arrhythmic role of NaV1.8 in heart failure. T-World demonstrates strong performance in predicting drug-induced arrhythmia risk and opens new opportunities for predicting and explaining drug efficacy, demonstrated by unpicking effects of mexiletine in Long QT syndrome 2. T-World is available as open-source code and an online app.

cell biology↗

bioRxiv2025

Time-dependent Mitochondrial Remodeling in Experimental Atrial Fibrillation and Potential Therapeutic Relevance

BACKGROUNDChanges in mitochondria have been implicated in atrial fibrillation (AF), but their manifestations and significance are poorly understood. Here, we studied changes in mitochondrial morphology and function during AF and assessed the effect of a mitochondrial-targeted intervention in a large animal model. METHODS AND RESULTSAtrial cardiomyocytes (ACMs) were isolated from dogs in electrically-driven AF for periods of 24 hours to 3 weeks and from humans with/without longstanding persistent AF. Mitochondrial Ca2+-concentration ([Ca2+]Mito), reactive oxygen species (mtROS) production, membrane potential ({Delta}{Psi}m), permeability transition-pore (mPTP) opening and flavin adenine dinucleotide (FAD) were measured via confocal microscopy; nicotine adenine dinucleotide (NADH) under ultraviolet light. mtROS-production increased within 24 hours and superoxide-dismutase type-2 was significantly reduced from 3-day AF. [Ca2+]Mito and mPTP-opening frequency/duration increased progressively during AF. Mitochondrial depolarization was detectable 24 hours after AF-onset. NADH increased by 15% at 24-hour AF, concomitant with increased pyruvate-dehydrogenase expression, then gradually decreased. Mitochondria enlarged and elongated at 24-hour and 3-day AF, followed by progressive fragmentation, rupture and shrinkage. Mitochondrial fusion protein-1 (MFN1) was reduced from 3-day to 3-week AF and phosphorylated dynamin-related protein-1 (p-DRP1ser-616) increased after 1 week of canine AF and in human AF. Addition of the mitochondrial antioxidant MitoTempo attenuated action-potential shortening and L-type Ca2+-current (ICaL)-downregulation in canine and human AF ACMs in vitro. Administration of the orally-active mitochondrial-targeted ubiquinone mitoquinone to dogs during 3-week AF prevented mitochondrial Ca2+-overload, mtROS-overproduction, structural damage and abnormalities in {Delta}{Psi}m and respiration. Functionally, mitoquinone reduced AF-induced Ca2+-current downregulation, action-potential abbreviation, contractile dysfunction and fibrosis, preventing AF-substrate development and AF-sustainability. CONCLUSIONSMitochondria show a series of changes during AF, with early hyperfunction and enhanced ROS-generation, followed by progressive damage and dysfunction. Mitochondrial-targeted therapy prevents mitochondrial dysfunction and attenuates adverse AF-related remodeling, positioning mitochondrial protection as a potential novel therapeutic target in AF.

pathology↗

bioRxiv2023

Nitric Oxide modulates spontaneous Ca2+ release and ventricular arrhythmias during β adrenergic signalling through S-nitrosylation of Calcium/Calmodulin dependent kinase II

RationaleNitric oxide (NO) has been identified as a signalling molecule generated during {beta}-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKII{delta}) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence. ObjectiveWe test the role of S-nitrosylation of CaMKII{delta} at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during {beta}-AR stimulation. Methods and ResultsWe measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKII{delta} expression (CaMKII{delta}-KO) or with deletion of the S-nitrosylation site on CaMKII{delta} at Cys-273 or Cys-290 (CaMKII{delta}-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 M), sodium nitroprusside (SNP; 200 M) and/or {beta}-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKII{delta}-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKII{delta}-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKII{delta}-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKII{delta}-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKII{delta}-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKII{delta}-C273S but was apparent in CaMKII{delta}-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKII{delta}-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events. ConclusionsWe conclude that prior S-nitrosylation of CaMKII{delta} at Cys-273 can limit subsequent {beta}-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain {beta}-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

physiology↗

bioRxiv2023

Kinetics and Mapping of Ca-driven Calmodulin conformations on Skeletal and Cardiac Muscle Ryanodine Receptors

Calmodulin (CaM) transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during relaxation and contraction in cardiac and skeletal muscle. However, the structural dynamics by which CaM modulates the SR Ca2+ release channel (ryanodine receptor, RyR) at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime detection of FRET between RyR-bound FKBP and CaM, we resolved different structural states of CaM and Ca-driven shifts in the conformation of CaM bound to RyR. We found that CaM becomes more compact in contracting high-Ca2+ vs. relaxing low-Ca2+. Skeletal and cardiac RyR isoforms show different CaM-RyR conformations, and binding and structural kinetics reflect functional roles. Furthermore, our FRET methods provide insight representative of physiological CaM-RyR structure, in synergy and comparison with cryo-EM models, which result from more disrupted samples. This technology will drive future studies focusing on pathologic CaM-RyR interactions, and on RyR dynamics with other important modulators.

biophysics↗

bioRxiv2023

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide, (DPc10) detects leaky RyR2 in cardiomyocytes. Conversely, calmodulin (CaM) inhibits RyR2 leak. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. Here we used FRET to understand the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with analysis of fluorescence-lifetime-detected FRET indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, this effect being larger in micromolar vs. nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

biophysics↗

bioRxiv2020

Quantitative cross-species translators of cardiac myocyte electrophysiology: model training, experimental validation, and applications

Animal experimentation is key in the evaluation of cardiac efficacy and safety of novel therapeutic compounds. However, inter-species differences in the mechanisms regulating excitation-contraction coupling can limit the translation of experimental findings from animal models to human physiology, and undermine the assessment of drugs efficacy and safety. Here, we built a suite of translators for quantitatively mapping electrophysiological responses in ventricular myocytes across species. We trained these statistical operators using a broad dataset obtained by simulating populations of our biophysically detailed computational models of action potential and Ca2+ transient in mouse, rabbit, and human. We then tested our translators against experimental data describing the response to stimuli, such as ion channel block, change in beating rate, and {beta}-adrenergic challenge. We demonstrate that this approach is well suited to predicting the effects of perturbations across different species or experimental conditions, and suggest its integration into mechanistic studies and drug development pipelines.

systems biology↗

bioRxiv2019

Alpha-Actinin-1 promotes activity of the L-type Ca2+ Channel CaV1.2

The L-type Ca2+ channel CaV1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of -actinin to the IQ motif of CaV1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1646-1664) bound to -actinin-1 and to apo-calmodulin (apoCaM). The CaV1.2 K1647A and Y1649A mutations, which impair -actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not -actinin-1 binding, decreased single channel open probability, gating charge movement, and its coupling to channel opening. Thus, -actinin recruits CaV1.2 to defined surface regions and simultaneously boosts its open probability so that CaV1.2 is mostly active when appropriately localized.

molecular biology↗