A computational model predicts sex-specific responses to calcium channel blockers in mammalian mesenteric vascular smooth muscle.

The function of the smooth muscle cells lining the walls of mammalian systemic arteries and arterioles is to regulate the diameter of the vessels to control blood flow and blood pressure. Here, we describe an in silico model, which we call the 'Hernandez-Hernandez model', of electrical and Ca2+ signaling in arterial myocytes based on new experimental data indicating sex-specific differences in male and female arterial myocytes from murine resistance arteries. The model suggests the fundamental ionic mechanisms underlying membrane potential and intracellular Ca2+ signaling during the development of myogenic tone in arterial blood vessels. Although experimental data suggest that KV1.5 channel currents have similar amplitudes, kinetics, and voltage dependencies in male and female myocytes, simulations suggest that the KV1.5 current is the dominant current regulating membrane potential in male myocytes. In female cells, which have larger KV2.1 channel expression and longer time constants for activation than male myocytes, predictions from simulated female myocytes suggest that KV2.1 plays a primary role in the control of membrane potential. Over the physiological range of membrane potentials, the gating of a small number of voltage-gated K+ channels and L-type Ca2+ channels are predicted to drive sex-specific differences in intracellular Ca2+ and excitability. We also show that in an idealized computational model of a vessel, female arterial smooth muscle exhibits heightened sensitivity to commonly used Ca2+ channel blockers compared to male. In summary, we present a new model framework to investigate the potential sex-specific impact of antihypertensive drugs.

High blood pressure is a major risk factor for heart disease, which is one of the leading causes of death worldwide. While drugs are available to control blood pressure, male and female patients can respond differently to treatment. However, the biological mechanisms behind this sex difference are not fully understood. Blood pressure is controlled by cells lining the artery walls called smooth muscle cells which alter the width of blood vessels. On the surface of smooth muscle cells are potassium and calcium channels which control the cell's electrical activity. When calcium ions enter the cell via calcium channels, this generates an electrical signal that causes the smooth muscle to contract and narrow the blood vessel. Potassium ions then flood out of the cell via potassium channels to dampen the rise in electrical activity, causing the muscle to relax and widen the artery. There are various sub-types of potassium and calcium channels in smooth muscle cells. Here, Hernandez-Hernandez et al. set out to find how these channels differ between male and female mice, and whether these sex differences could alter the response to blood pressure medication. The team developed a computational model of a smooth muscle cell, incorporating data from laboratory experiments measuring differences in cells isolated from the arteries of male and female mice. The model predicted that the sub-types of potassium and calcium channels in smooth muscle cells varied between males and females, and how the channels impacted electrical activity also differed. For instance, the potassium channel Kv2.1 was found to have a greater role in controlling electrical activity in female mice, and this sex difference impacted blood vessel contraction. The model also predicted that female mice were more sensitive than males to calcium channel blockers, a drug commonly prescribed to treat high blood pressure. The findings by Hernandez-Hernandez et al. provide new insights into the biological mechanisms underlying sex differences in response to blood pressure medication. They also demonstrate how computational models can be used to predict the effects of drugs on different individuals. In the future, these predictions may help researchers to identify better, more personalized treatments for blood pressure.

Harnessing AlphaFold to reveal state secrets: Prediction of hERG closed and inactivated states.

To design safe, selective, and effective new therapies, there must be a deep understanding of the structure and function of the drug target. One of the most difficult problems to solve has been resolution of discrete conformational states of transmembrane ion channel proteins. An example is KV11.1 (hERG), comprising the primary cardiac repolarizing current, IKr. hERG is a notorious drug anti-target against which all promising drugs are screened to determine potential for arrhythmia. Drug interactions with the hERG inactivated state are linked to elevated arrhythmia risk, and drugs may become trapped during channel closure. However, the structural details of multiple conformational states have remained elusive. Here, we guided AlphaFold2 to predict plausible hERG inactivated and closed conformations, obtaining results consistent with myriad available experimental data. Drug docking simulations demonstrated hERG state-specific drug interactions aligning well with experimental results, revealing that most drugs bind more effectively in the inactivated state and are trapped in the closed state. Molecular dynamics simulations demonstrated ion conduction that aligned with earlier studies. Finally, we identified key molecular determinants of state transitions by analyzing interaction networks across closed, open, and inactivated states in agreement with earlier mutagenesis studies. Here, we demonstrate a readily generalizable application of AlphaFold2 as a novel method to predict discrete protein conformations and novel linkages from structure to function.

A computational model predicts sex-specific responses to calcium channel blockers in mammalian mesenteric vascular smooth muscle.

The function of the smooth muscle cells lining the walls of mammalian systemic arteries and arterioles is to regulate the diameter of the vessels to control blood flow and blood pressure. Here, we describe an in-silico model, which we call the "Hernandez-Hernandez model", of electrical and Ca2+ signaling in arterial myocytes based on new experimental data indicating sex-specific differences in male and female arterial myocytes from murine resistance arteries. The model suggests the fundamental ionic mechanisms underlying membrane potential and intracellular Ca2+ signaling during the development of myogenic tone in arterial blood vessels. Although experimental data suggest that KV1.5 channel currents have similar amplitudes, kinetics, and voltage dependencies in male and female myocytes, simulations suggest that the KV1.5 current is the dominant current regulating membrane potential in male myocytes. In female cells, which have larger KV2.1 channel expression and longer time constants for activation than male myocytes, predictions from simulated female myocytes suggest that KV2.1 plays a primary role in the control of membrane potential. Over the physiological range of membrane potentials, the gating of a small number of voltage-gated K+ channels and L-type Ca2+ channels are predicted to drive sex-specific differences in intracellular Ca2+ and excitability. We also show that in an idealized computational model of a vessel, female arterial smooth muscle exhibits heightened sensitivity to commonly used Ca2+ channel blockers compared to male. In summary, we present a new model framework to investigate the potential sex-specific impact of anti-hypertensive drugs.

Toward Digital Twin Technology for Precision Pharmacology.

The authors demonstrate the feasibility of technological innovation for personalized medicine in the context of drug-induced arrhythmia. The authors use atomistic-scale structural models to predict rates of drug interaction with ion channels and make predictions of their effects in digital twins of induced pluripotent stem cell-derived cardiac myocytes. The authors construct a simplified multilayer, 1-dimensional ring model with sufficient path length to enable the prediction of arrhythmogenic dispersion of repolarization. Finally, the authors validate the computational pipeline prediction of drug effects with data and quantify drug-induced propensity to repolarization abnormalities in cardiac tissue. The technology is high throughput, computationally efficient, and low cost toward personalized pharmacologic prediction.

Elucidating molecular mechanisms of protoxin-II state-specific binding to the human NaV1.7 channel.

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells, and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula venom peptide protoxin-II (PTx2) has high selectivity for hNaV1.7 and is a valuable scaffold for designing novel therapeutics to treat pain. Here, we used computational modeling to study the molecular mechanisms of the state-dependent binding of PTx2 to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to the deactivated VSD II and activated VSD IV. These state-specific interactions are mediated primarily by PTx2's residues R22, K26, K27, K28, and W30 with VSD and the surrounding membrane lipids. Our work revealed important protein-protein and protein-lipid contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.

Structural modeling of hERG channel-drug interactions using Rosetta.

The human ether-a-go-go-related gene (hERG) not only encodes a potassium-selective voltage-gated ion channel essential for normal electrical activity in the heart but is also a major drug anti-target. Genetic hERG mutations and blockage of the channel pore by drugs can cause long QT syndrome, which predisposes individuals to potentially deadly arrhythmias. However, not all hERG-blocking drugs are proarrhythmic, and their differential affinities to discrete channel conformational states have been suggested to contribute to arrhythmogenicity. We used Rosetta electron density refinement and homology modeling to build structural models of open-state hERG channel wild-type and mutant variants (Y652A, F656A, and Y652A/F656 A) and a closed-state wild-type channel based on cryo-electron microscopy structures of hERG and EAG1 channels. These models were used as protein targets for molecular docking of charged and neutral forms of amiodarone, nifekalant, dofetilide, d/l-sotalol, flecainide, and moxifloxacin. We selected these drugs based on their different arrhythmogenic potentials and abilities to facilitate hERG current. Our docking studies and clustering provided atomistic structural insights into state-dependent drug-channel interactions that play a key role in differentiating safe and harmful hERG blockers and can explain hERG channel facilitation through drug interactions with its open-state hydrophobic pockets.

The formation of KV2.1 macro-clusters is required for sex-specific differences in L-type CaV1.2 clustering and function in arterial myocytes.

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

A multiscale predictive digital twin for neurocardiac modulation.

Cardiac function is tightly regulated by the autonomic nervous system (ANS). Activation of the sympathetic nervous system increases cardiac output by increasing heart rate and stroke volume, while parasympathetic nerve stimulation instantly slows heart rate. Importantly, imbalance in autonomic control of the heart has been implicated in the development of arrhythmias and heart failure. Understanding of the mechanisms and effects of autonomic stimulation is a major challenge because synapses in different regions of the heart result in multiple changes to heart function. For example, nerve synapses on the sinoatrial node (SAN) impact pacemaking, while synapses on contractile cells alter contraction and arrhythmia vulnerability. Here, we present a multiscale neurocardiac modelling and simulator tool that predicts the effect of efferent stimulation of the sympathetic and parasympathetic branches of the ANS on the cardiac SAN and ventricular myocardium. The model includes a layered representation of the ANS and reproduces firing properties measured experimentally. Model parameters are derived from experiments and atomistic simulations. The model is a first prototype of a digital twin that is applied to make predictions across all system scales, from subcellular signalling to pacemaker frequency to tissue level responses. We predict conditions under which autonomic imbalance induces proarrhythmia and can be modified to prevent or inhibit arrhythmia. In summary, the multiscale model constitutes a predictive digital twin framework to test and guide high-throughput prediction of novel neuromodulatory therapy. KEY POINTS: A multi-layered model representation of the autonomic nervous system that includes sympathetic and parasympathetic branches, each with sparse random intralayer connectivity, synaptic dynamics and conductance based integrate-and-fire neurons generates firing patterns in close agreement with experiment. A key feature of the neurocardiac computational model is the connection between the autonomic nervous system and both pacemaker and contractile cells, where modification to pacemaker frequency drives initiation of electrical signals in the contractile cells. We utilized atomic-scale molecular dynamics simulations to predict the association and dissociation rates of noradrenaline with the ß-adrenergic receptor. Multiscale predictions demonstrate how autonomic imbalance may increase proclivity to arrhythmias or be used to terminate arrhythmias. The model serves as a first step towards a digital twin for predicting neuromodulation to prevent or reduce disease.

Rate-dependent effects of state-specific sodium channel blockers in cardiac tissue: Insights from idealized models.

A common side effect of pharmaceutical drugs is an increased propensity for cardiac arrhythmias. Many drugs bind to cardiac ion-channels in a state-specific manner, which alters the ionic conductances in complicated ways, making it difficult to identify the mechanisms underlying pro-arrhythmic drug effects. To better understand the fundamental mechanisms underlying the diverse effects of state-dependent sodium (Na+) channel blockers on cellular excitability, we consider two canonical motifs of drug-ion-channel interactions and compare the effects of Na+ channel blockers on the rate-dependence of peak upstroke velocity, conduction velocity, and vulnerable window size. In the literature, both motifs are referred to as "guarded receptor," but here we distinguish between state-specific binding that does not alter channel gating (referred to here as "guarded receptor") and state-specific binding that blocks certain gating transitions ("gate immobilization"). For each drug binding motif, we consider drugs that bind to the inactivated state and drugs that bind to the non-inactivated state of the Na+ channel. Exploiting the idealized nature of the canonical binding motifs, we identify the fundamental mechanisms underlying the effects on excitability of the various binding interactions. Specifically, we derive the voltage-dependence of the drug binding time constants and the equilibrium fractions of channels bound to drug, and we then derive a formula that incorporates these time constants and equilibrium fractions to elucidate the fundamental mechanisms. In the case of charged drug, we find that drugs that bind to inactivated channels exhibit greater rate-dependence than drugs that bind to non-inactivated channels. For neutral drugs, the effects of guarded receptor interactions are rate-independent, and we describe a novel mechanism for reverse rate-dependence resulting from neutral drug binding to non-inactivated channels via the gate immobilization motif.

The formation of K V 2.1 macro-clusters is required for sex-specific differences in L-type Ca V 1.2 clustering and function in arterial myocytes.

In arterial myocytes, the canonical function of voltage-gated Ca V 1.2 and K V 2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, K V 2.1 also plays a sex-specific role by promoting the clustering and activity of Ca V 1.2 channels. However, the impact of K V 2.1 protein organization on Ca V 1.2 function remains poorly understood. We discovered that K V 2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of K V 2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the K V 2.1 clustering site (K V 2.1 S590A ) eliminated K V 2.1 macro-clustering and sex-specific differences in Ca V 1.2 cluster size and activity. We propose that the degree of K V 2.1 clustering tunes Ca V 1.2 channel function in a sex-specific manner in arterial myocytes.

The formation of KV2.1 macro-clusters is required for sex-specific differences in L-type CaV1.2 clustering and function in arterial myocytes.

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

Elucidating Molecular Mechanisms of Protoxin-2 State-specific Binding to the Human NaV1.7 Channel.

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula venom peptide protoxin-2 (PTx2) has high selectivity for hNaV1.7 and serves as a valuable scaffold to design novel therapeutics to treat pain. Here, we used computational modeling to study the molecular mechanisms of the state-dependent binding of PTx2 to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to the deactivated VSD II and activated VSD IV. These state-specific interactions are mediated primarily by PTx2's residues R22, K26, K27, K28, and W30 with VSD as well as the surrounding membrane lipids. Our work revealed important protein-protein and protein-lipid contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.

Elucidation of a dynamic interplay between a beta-2 adrenergic receptor, its agonist, and stimulatory G protein.

G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (ß2AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (Gs). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to ß2AR by Gs protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from ß2AR and the conformational interconversions of Gs between closed and open conformations in the NE(+)-ß2AR-Gs ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter Gs α subunit (Gsα) conformational transitions. Our simulations showed that the interdomain movement and the stacking of Gsα α1 and α5 helices are significant for increasing the distance between the Gsα and ß2AR, which may indicate a partial dissociation of Gsα The distance increase commences when Gsα is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of ß2AR interacting with Gsα, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation.

Structural modeling of the hERG potassium channel and associated drug interactions.

The voltage-gated potassium channel, KV11.1, encoded by the human Ether-à-go-go-Related Gene (hERG), is expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. Gating of the hERG channel is characterized by rapid, voltage-dependent, C-type inactivation, which blocks ion conduction and is suggested to involve constriction of the selectivity filter. Mutations S620T and S641A/T within the selectivity filter region of hERG have been shown to alter the voltage dependence of channel inactivation. Because hERG channel blockade is implicated in drug-induced arrhythmias associated with both the open and inactivated states, we used Rosetta to simulate the effects of hERG S620T and S641A/T mutations to elucidate conformational changes associated with hERG channel inactivation and differences in drug binding between the two states. Rosetta modeling of the S641A fast-inactivating mutation revealed a lateral shift of the F627 side chain in the selectivity filter into the central channel axis along the ion conduction pathway and the formation of four lateral fenestrations in the pore. Rosetta modeling of the non-inactivating mutations S620T and S641T suggested a potential molecular mechanism preventing F627 side chain from shifting into the ion conduction pathway during the proposed inactivation process. Furthermore, we used Rosetta docking to explore the binding mechanism of highly selective and potent hERG blockers - dofetilide, terfenadine, and E4031. Our structural modeling correlates well with much, but not all, existing experimental evidence involving interactions of hERG blockers with key residues in hERG pore and reveals potential molecular mechanisms of ligand interactions with hERG in an inactivated state.

Pore opening, not voltage sensor movement, underpins the voltage-dependence of facilitation by a hERG blocker.

A drug that blocks the cardiac myocyte voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene (hERG) carries a potential risk of long QT syndrome and life-threatening cardiac arrhythmia, including Torsade de Points Interestingly, certain hERG blockers can also facilitate hERG activation to increase hERG currents, which may reduce proarrhythmic potential. However, the molecular mechanism involved in the facilitation effect of hERG blockers remains unclear. The hallmark feature of the facilitation effect by hERG blockers is that a depolarizing preconditioning pulse shifts voltage-dependence of hERG activation to more negative voltages. Here we utilize a D540K hERG mutant to study the mechanism of the facilitation effect. D540K hERG is activated by not only depolarization but also hyperpolarization. This unusual gating property enables tests of the mechanism by which voltage induces facilitation of hERG by blockers. With D540K hERG, we find that nifekalant, a hERG blocker and Class III antiarrhythmic agent, blocks and facilitates not only current activation by depolarization but also current activation by hyperpolarization, suggesting a shared gating process upon depolarization and hyperpolarization. Moreover, in response to hyperpolarizing conditioning pulses, nifekalant facilitates D540K hERG currents but not wild-type currents. Our results indicate that induction of facilitation is coupled to pore opening, not voltage per se We propose that gated access to the hERG central cavity underlies the voltage-dependence of induction of facilitation. This study identifies hERG channel pore gate opening as the conformational change facilitated by nifekalant, a clinically important antiarrhythmic agent. Significance Statement Nifekalant is a clinically important antiarrhythmic agent and a hERG blocker which can also facilitate voltage-dependent activation of hERG channels after a preconditioning pulse. Here we show that the mechanism of action of the preconditioning pulse is to open a conductance gate to enable drug access to a facilitation site. Moreover, we find that facilitation increases hERG currents by altering pore dynamics, rather than acting through voltage sensors.

Research Opportunities in Autonomic Neural Mechanisms of Cardiopulmonary Regulation: A Report From the National Heart, Lung, and Blood Institute and the National Institutes of Health Office of the Director Workshop.

This virtual workshop was convened by the National Heart, Lung, and Blood Institute, in partnership with the Office of Strategic Coordination of the Office of the National Institutes of Health Director, and held September 2 to 3, 2020. The intent was to assemble a multidisciplinary group of experts in basic, translational, and clinical research in neuroscience and cardiopulmonary disorders to identify knowledge gaps, guide future research efforts, and foster multidisciplinary collaborations pertaining to autonomic neural mechanisms of cardiopulmonary regulation. The group critically evaluated the current state of knowledge of the roles that the autonomic nervous system plays in regulation of cardiopulmonary function in health and in pathophysiology of arrhythmias, heart failure, sleep and circadian dysfunction, and breathing disorders. Opportunities to leverage the Common Fund's SPARC (Stimulating Peripheral Activity to Relieve Conditions) program were characterized as related to nonpharmacologic neuromodulation and device-based therapies. Common themes discussed include knowledge gaps, research priorities, and approaches to develop novel predictive markers of autonomic dysfunction. Approaches to precisely target neural pathophysiological mechanisms to herald new therapies for arrhythmias, heart failure, sleep and circadian rhythm physiology, and breathing disorders were also detailed.

Biological noise is a key determinant of the reproducibility and adaptability of cardiac pacemaking and EC coupling.

Each heartbeat begins with the generation of an action potential in pacemaking cells in the sinoatrial node. This signal triggers contraction of cardiac muscle through a process termed excitation-contraction (EC) coupling. EC coupling is initiated in dyadic structures of cardiac myocytes, where ryanodine receptors in the junctional sarcoplasmic reticulum come into close apposition with clusters of CaV1.2 channels in invaginations of the sarcolemma. Cooperative activation of CaV1.2 channels within these clusters causes a local increase in intracellular Ca2+ that activates the juxtaposed ryanodine receptors. A salient feature of healthy cardiac function is the reliable and precise beat-to-beat pacemaking and amplitude of Ca2+ transients during EC coupling. In this review, we discuss recent discoveries suggesting that the exquisite reproducibility of this system emerges, paradoxically, from high variability at subcellular, cellular, and network levels. This variability is attributable to stochastic fluctuations in ion channel trafficking, clustering, and gating, as well as dyadic structure, which increase intracellular Ca2+ variance during EC coupling. Although the effects of these large, local fluctuations in function and organization are sometimes negligible at the macroscopic level owing to spatial-temporal summation within and across cells in the tissue, recent work suggests that the "noisiness" of these intracellular Ca2+ events may either enhance or counterintuitively reduce variability in a context-dependent manner. Indeed, these noisy events may represent distinct regulatory features in the tuning of cardiac contractility. Collectively, these observations support the importance of incorporating experimentally determined values of Ca2+ variance in all EC coupling models. The high reproducibility of cardiac contraction is a paradoxical outcome of high Ca2+ signaling variability at subcellular, cellular, and network levels caused by stochastic fluctuations in multiple processes in time and space. This underlying stochasticity, which counterintuitively manifests as reliable, consistent Ca2+ transients during EC coupling, also allows for rapid changes in cardiac rhythmicity and contractility in health and disease.

Mechanisms of cAMP compartmentation in cardiac myocytes: experimental and computational approaches to understanding.

The small diffusible second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is found in virtually every cell in our bodies, where it mediates responses to a variety of different G protein coupled receptors (GPCRs). In the heart, cAMP plays a critical role in regulating many different aspects of cardiac myocyte function, including gene transcription, cell metabolism, and excitation-contraction coupling. Yet, not all GPCRs that stimulate cAMP production elicit the same responses. Subcellular compartmentation of cAMP is essential to explain how different receptors can utilize the same diffusible second messenger to elicit unique functional responses. However, the mechanisms contributing to this behaviour and its significance in producing physiological and pathological responses are incompletely understood. Mathematical modelling has played an essential role in gaining insight into these questions. This review discusses what we currently know about cAMP compartmentation in cardiac myocytes and questions that are yet to be answered.