Cardiac Dyad Research Articles

473 Application of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling of Ankyrin-2 p.R990Q variant-induced ventricular arrhythmia and personalized medicine

OBJECTIVES/GOALS: The cytoskeletal protein α²II spectrin interacts with actin and ankyrin-2 in cardiomyocytes which is essential to orchestrate ion channels and membrane proteins in the cardiac dyad. Our goal is to understand molecular mechanism causing severe ventricular arrhythmias due to spectrin dysfunction and explore novel therapies to treat such conditions. METHODS/STUDY POPULATION: We previously published a case of a 36-year-old woman with an ankyrin-2 p.R990Q (ANK2) variant, presented with severe ventricular arrhythmias and sudden cardiac arrest, caused by a novel mutation in the ankyrin-B gene (c.2969G>A) that disrupts the interaction of ankyrin-B/βII spectrin. To model the condition, we will use human induced pluripotent stem cell (DF 19-9-7T, WiCell)-derived ventricular cardiomyocytes (iPSC-CMs) having ANK2 variant, engineered using CRISPR/Cas9 method (Synthego Corp.). We will validate the differentiation of iPSCs into ventricular lineage and characterize the ANK2 ventricular phenotype. Next, we will express light-gated cation channel Channelrhodopsin (ChR2) in the ANK2 iPSC-CMs and investigate the potential role of optogenetics in treating such severe arrhythmias. RESULTS/ANTICIPATED RESULTS: Immunostaining shows 87.339% of iPSC-CMs, treated with All-trans retinoic acid (RA) (1 uM) on days 7 and 12 [RA 7,12], and 23.84% of those, treated on days 3 and 5 [RA 3,5], expressed MLC-2V (p<0.001). Calcium reuptake (τ) is 0.5914 s in RA 7, 12 while 0.2247s in RA 3, 5 (p<0.001). APD90 and APD50 of RA 7, 12 are 2- and 5-fold higher than RA 3, 5, showing distinct ventricular and atrial phenotypes. Protein expression of βII-spectrin and ankyrin-2 and their co-localizations were reduced in the ANK2 phenotype compared to the healthy phenotype. We found prolongation of Ca2+ waves and τ with blue light on iPSC-CMs, expressing ChR2. We anticipate that such prolongation of calcium transients would prevent aberrant calcium spikes, rescue Ca2+/calpain-induced βII-spectrin loss and provide electrical stability. DISCUSSION/SIGNIFICANCE: Animal models cannot accurately recapitulate human cardiac electrophysiology. The proposed human iPSC-CM-based ANK2 model would provide better mechanistic insights of severe ventricular arrhythmias. Also, the proposed optogenetic cardioversion has the potential to provide safe, targeted and painless cardioversion to manage arrhythmias.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling cardiac arrhythmias: strengths, challenges and potential solutions.

Ion channels and cytoskeletal proteins in the cardiac dyad play a critical role in maintaining excitation-contraction (E-C) coupling and provide cardiac homeostasis. Functional changes in these dyad proteins, whether induced by genetic, epigenetic, metabolic, therapeutic, or environmental factors, can disrupt normal cardiac electrophysiology, leading to abnormal E-C coupling and arrhythmias. Animal models and heterologous cell cultures provide platforms to elucidate the pathogenesis of arrhythmias for basic cardiac research; however, these traditional systems do not truly reflect human cardiac electro-pathophysiology. Notably, patients with the same genetic variants of inherited channelopathies (ICC) often exhibit incomplete penetrance and variable expressivity which underscores the need to establish patient-specific disease models to comprehend the mechanistic pathways of arrhythmias and determine personalized therapies. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) inherit the genetic background of the patient and reflect the electrophysiological characteristics of the native cardiomyocytes. Thus, iPSC-CMs provide an innovative and translational pivotal platform in cardiac disease modeling and therapeutic screening. In this review, we will examine how patient-specific iPSC-CMs historically evolved to model arrhythmia syndromes in a dish, and their utility in understanding the role of specific ion channels and their functional characteristics in causing arrhythmias. We will also examine how CRISPR/Cas9 have enabled the establishment of patient-independent and variant-induced iPSC-CMs-based arrhythmia models. Next, we will examine the limitations of using human iPSC-CMs with respect to in vitro arrhythmia modeling that stems from variations in iPSCs or toxicity due to gene editing on iPSC or iPSC-CMs and explore how such hurdles are being addressed. Importantly, we will also discuss how novel 3D iPSC-CM models can better capture in vitro characteristics and how all-optical platforms provide non-invasive and high- throughput electrophysiological data that is useful for stratification of emerging arrhythmogenic variants and drug discovery. Finally, we will examine strategies to improve iPSC-CM maturity, including powerful gene editing and optogenetic tools that can introduce/modify specific ion channels in iPSC-CMs and tailor cellular and functional characteristics. We anticipate that an elegant synergy of iPSCs, novel gene editing, 3D- culture models, and all-optical platforms will offer a high-throughput template to faithfully recapitulate in vitro arrhythmogenic events necessary for personalized arrhythmia monitoring and drug screening process.

Abstract P2067: Loss Of Cardiomyocyte β2 Spectrin Promotes Cardiac Dyad Dysfunction And Accelerated Heart Failure

Background: The cardiac dyad is composed of the transverse-tubules, junctional sarcoplasmic reticulum (jSR), and its associated calcium (Ca 2+ ) release units, which are integral for cardiac excitation-contraction (E-C). Proper E-C coupling requires Ca 2+ influx via L-type Ca 2+ channels (LTCC) which triggers Ca 2+ release from adjacent ryanodine receptor 2 (RyR2) receptors located in jSR. Loss of the βII-spectrin scaffolding protein alters RyR2 localization; however, its contribution to cardiac dyad organization and E-C coupling remains unclear. Hypothesis: We hypothesized that βII-spectrin is required for cardiac dyad organization and proper cardiomyocyte calcium signaling. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control (βII-flox) mice were generated and hearts were harvested for immunoblotting, qRT-PCR, and transmission electron microscopy (TEM). Cardiomyocytes were isolated and stained using proximity ligation assay (PLA) and visualized by confocal microscopy. To assess heart response to pressure overload, control and βII-cKO mice were subjected to transaortic constriction surgery followed by treatment with or without, diltiazem and monitored by echocardiography, and electrocardiography. Results: Cardiac dyad structure and components are significantly altered in βII-cKO hearts. Ultrastructural analysis of βII-cKO hearts showed fragmented cardiac dyads, dilated jSR and reduced jSR contact with T-tubules. Quantitative RT-PCR and immunoblotting analysis revealed altered expression of dyad mRNAs and proteins. Preliminary PLA demonstrated reduced density of LTCC-RyR2 complexes. Furthermore, pressure-induced overload of βII-cKO hearts promoted accelerated heart failure (HF), which can be rescued by treatment with diltiazem. Conclusions: We demonstrate that βII-spectrin is required for cardiac dyad integrity, and that βII-spectrin-mediated HF can be attenuated by diltiazem.

Cardiomyocyte Maturation During Regeneration

The interplay between calcium signaling and sarcomeres plays a crucial role in directing cardiomyocyte maturation during cardiac regeneration. During cardiac regeneration, various processes occur to restore damaged or lost cardiac tissue, including the proliferation and differentiation of precursor cells into functional cardiomyocytes. These newly formed cardiomyocytes undergo a maturation process to acquire the structural and functional characteristics of adult cardiomyocytes. Nguyen and colleagues found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat-containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. They found that its function was conserved in mammalian cardiomyocytes. Their study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes. Science. 2023 May 19;380(6646):758-764. doi: 10.1126/science.abo6718.

A cardiac dyad component controls cardiomyocytes maturation in regenerative hearts.

A cardiac dyad component controls cardiomyocytes maturation in regenerative hearts.

Interplay between calcium and sarcomeres directs cardiomyocyte maturation during regeneration

Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat–containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.

Cardiac signaling cloud regulates intracellular [Na+], [Ca2+] and adrenergic signaling in atrial myocytes.

An important but often overlooked "silent" partner of atrial myocyte function is intracellular Na+ concentration ([Na+]i). We report here on four surprising and inter-related discoveries concerning [Na+]i in atrial myocytes. (1). Using highly sensitive, calibrated fluorescence measurements we report that the heart rate-dependent [Na+]i is significantly lower (∼40%) in atrial myocytes than the benchmark values reported for ventricular myocytes. (2). Na+/K+ ATPase (NKA), the main cytosolic Na+ extrusion pump, has much higher protein expression levels (3 X higher) in atrial tissue and NKA current densities (2 X higher) than expected in atrial myocytes. (3). NKA is exquisitely sensitive to ß-adrenergic activation in atrial tissue due to a huge phosphorylation capacity of its regulatory protein phospholemman (PLM). This enhances the high baseline Na+ extrusion rate in atrial myocytes during adrenergic stress. (4). Two-color super-resolution microscopy (dSTORM) reveals a novel ion channel nanodomain in atrial and ventricular myocytes that is formed by the co-clustering of NKA (NKAα1) with the L-type Ca2+ channel (Cav1.2) at the cardiac dyad. In atrial myocytes, each signaling nanodomain consists of large NKAα1 and Cav1.2 "superclusters". Functional studies further reveal that the regulatory proteins associated with NKAα1 and Cav1.2 locally regulate channel function in a proximity-dependent "signaling cloud" that is in direct apposition to ryanodine receptor (RyR2) clusters. Taken together these atrial Na+ signaling features provide robust signaling with an increased margin of safety for a system with known arrhythmogenic vulnerability. Finally, the novel cardiac NKAα1-Cav1.2 signaling cloud localizes the Na+ pump to the cardiac dyad, where it contributes to the regulation of the subsarcolemmal [Na+]i and Ca2+ concentration.

Abstract 13200: Loss of Cardiomyocyte β2 Spectrin Promotes Cardiac Dyad Dysfunction and Accelerated Heart Failure

Background: The cardiac dyad and associated calcium (Ca 2+ ) release units (CRUs) are integral for cardiac excitation-contraction (E-C) coupling. The cardiac dyad is a multicomponent complex composed of the transverse-tubules, junctional sarcoplasmic reticulum (jSR), and CRU-associated proteins. Proper E-C coupling requires Ca 2+ influx via long-lasting-L-type Ca 2+ channels (Cav1.2), which triggers Ca 2+ release from adjacent ryanodine receptor 2 (RyR2) located in jSR. βII-spectrin loss alters RyR2 localization, but its contribution to cardiac dyad organization and E-C coupling remains unclear. Hypothesis: We hypothesized that βII-spectrin is required for cardiac dyad organization and proper cardiomyocyte calcium signaling. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control mice (βII-flox) were generated and harvested for immunoblotting, qRT-PCR, and imaging by transmission electron microscopy (TEM). Mice hearts were also interrogated by echocardiography following transaortic constriction. Results: Cardiac dyad structure and CRU components are significantly altered in βII-cKO hearts. Ultrastructural analysis of βII-cKOhearts by TEM showed fragmented cardiac dyads, dilated jSR and reduced jSR contact with T-tubules. Quantitative RT-PCR showed increased expression of calsequestrin 1 and 2, decreased phospholambam, Ncx and Cacna1c (major Cav1.2 subunit), while Ryr2 , Jph2 , Serca2a are unchanged. Protein expression of NCX was decreased, while expression of CAV1.2, RYR2, CASQ2, and SERCA2A remained unchanged. Furthermore, pressure-induced overload of βII-cKO hearts promoted accelerated heart failure (HF). Conclusions: This is the first demonstration that the βII-spectrin cytoskeletal protein is critically important for maintaining the structural integrity of the cardiac dyad. βII-spectrin loss and the resulting alterations in CRU structure, genes, and protein composition promoting accelerated HF.

Evolution of the cardiac dyad.

Cardiac dyads are the site of communication between the sarcoplasmic reticulum (SR) and infoldings of the sarcolemma called transverse-tubules (TT). During heart excitation–contraction coupling, Ca2+-influx through L-type Ca2+ channels in the TT is amplified by release of Ca2+-from the SR via type 2 ryanodine receptors, activating the contractile apparatus. Key proteins involved in cardiac dyad function are bridging integrator 1 (BIN1), junctophilin 2 and caveolin 3. The work presented here aims to reconstruct the evolutionary history of the cardiac dyad, by surveying the scientific literature for ultrastructural evidence of these junctions across all animal taxa; phylogenetically reconstructing the evolutionary history of BIN1; and by comparing peptide motifs involved in TT formation by this protein across metazoans. Key findings are that cardiac dyads have been identified in mammals, arthropods and molluscs, but not in other animals. Vertebrate BIN1 does not group with members of this protein family from other taxa, suggesting that invertebrate BINs are paralogues rather orthologues of this gene. Comparisons of BIN1 peptide sequences of mammals with those of other vertebrates reveals novel features that might contribute to TT and dyad formation. The analyses presented here suggest that the cardiac dyad evolved independently several times during metazoan evolution: an unexpected observation given the diversity of heart structure and function between different animal taxa.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

CMYA5 establishes cardiac dyad architecture and positioning

Cardiac excitation-contraction coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse tubules and junctional sarcoplasmic reticulum. Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac development, homeostasis, and disease. Here, we use proximity proteomics in intact, living hearts to identify proteins enriched near dyads. Among these proteins is CMYA5, an under-studied striated muscle protein that co-localizes with Z-lines, junctional sarcoplasmic reticulum proteins, and transverse tubules in mature cardiomyocytes. During cardiac development, CMYA5 positioning adjacent to Z-lines precedes junctional sarcoplasmic reticulum positioning or transverse tubule formation. CMYA5 ablation disrupts dyad architecture, dyad positioning at Z-lines, and junctional sarcoplasmic reticulum Ca2+ release, leading to cardiac dysfunction and inability to tolerate pressure overload. These data provide mechanistic insights into cardiomyopathy pathogenesis by demonstrating that CMYA5 anchors junctional sarcoplasmic reticulum to Z-lines, establishes dyad architecture, and regulates dyad Ca2+ release.

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after β-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

CaV channels reject signaling from a second CaM in eliciting Ca2+-dependent feedback regulation

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.

The architecture and function of cardiac dyads.

Cardiac excitation-contraction (EC) coupling, which links plasma membrane depolarization to activation of cardiomyocyte contraction, occurs at dyads, the nanoscopic microdomains formed by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). In a dyadic junction, EC coupling occurs through Ca2+-induced Ca2+ release. Membrane depolarization opens voltage-gated L-type Ca2+ channels (LTCCs) in the T-tubule. The resulting influx of extracellular Ca2+ into the dyadic cleft opens Ca2+ release channels known as ryanodine receptors (RYRs) in the jSR, leading to the rapid increase in cytosolic Ca2+ that triggers sarcomere contraction. The efficacy of LTCC-RYR communication greatly affects a myriad of downstream intracellular signaling events, and it is controlled by many factors, including T-tubule and jSR structure, spatial distribution of ion channels, and regulatory proteins that closely regulate the activities of channels within dyads. Alterations in dyad architecture and/or channel activity are seen in many types of heart disease. This review will focus on the current knowledge regarding cardiac dyad structure and function, their alterations in heart failure, and new approaches to study the composition and function of dyads.

Presentation of Early Heart Failure Phenotypes in βII-Spectrin Conditional Knockout Mice

Background βII spectrin is an actin associated protein which regulates the localization of cytoskeletal and plasma membrane protein complexes necessary for normal cellular function. In cardiac myocytes βII spectrin recruits ankyrin-B to the cardiac dyad and modulates the functions of Na+/Ca2+ exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Disruption of the βII spectrin/ankyrin-B complex due to genetic mutation results in the onset of fatal arrhythmias. This was associated to the dysregulation of Ca2+ channels via ryanodine receptor 2 which forms the cardiac dyad. We hypothesize that loss of spectrin leads to acceleration of heart failure (HF), and plays a critical role in the SMAD pathway in the heart, and spectrin based arrhythmias are caused by abnormalities with the cardiac dyad. Methods Transgenic βII conditional knockout (cKO) mice were regenerated with a cleaner genetic profile (C57BL/6J) via Cre/LoxP system. Cardiac function of flox and cKO confirmed mice is assessed every 4 weeks via transthoracic echocardiogram (TTE) and electrocardiogram. We used the following antibodies: βII-spectrin (lab created), Ankryin B (lab created), βI (Novos), Nav (Abcam), Cav1.2 (Abcam), RyR receptor 2 (Abcam), Girk1 (alomone), Girk4 (alomone). A 2-tailed Student's t-test was performed on all data obtained from immunoblots. A p value of Results βII spectrin cKO mice display HF phenotypes as early as 4 weeks, verified by TTE. The observed ejection fraction is notably lower in the βII spectrin cKO mice compared to control (Flox) mice (LVEF 46.2% vs 57.8%, p=0.046). Expression of ankyrin-B was lower in the conditional knockout mice cardio myocytes relative to flox control (55.6 % vs 76.5%, n=3, p=0.38). Consistent with our previous work, expression of Ryanodine receptor 2 is significantly decreased in cKO relative to flox control (0.78 % vs 2.02, n=3, p=0.036). Conclusions The regeneration of cardiac specific βII-spectrin knockout mice model using the genetically homogenous C57BL/6J mice has been successful and confirmed to have decreased βII spectrin expression. Of note, these cKO mice do not require an external stressor for the development of heart failure phenotypes and exhibit this phenotype quite early on. Future studies we will explore other spectrin based regulatory pathways (SMADs) and assess the role of spectrins in organizing the cardiac dyad.

Na+ microdomains and sparks: Role in cardiac excitation-contraction coupling and arrhythmias in ankyrin-B deficiency

Cardiac sodium (Na+) potassium ATPase (NaK) pumps, neuronal sodium channels (INa), and sodium calcium (Ca2+) exchangers (NCX1) may co-localize to form a Na+ microdomain. It remains controversial as to whether neuronal INa contributes to local Na+ accumulation, resulting in reversal of nearby NCX1 and influx of Ca2+ into the cell. Therefore, there has been great interest in the possible roles of a Na+ microdomain in cardiac Ca2+-induced Ca2+ release (CICR). In addition, the important role of co-localization of NaK and NCX1 in regulating localized Na+ and Ca2+ levels and CICR in ankyrin-B deficient (ankyrin-B+/−) cardiomyocytes has been examined in many recent studies. Altered Na+ dynamics may contribute to the appearance of arrhythmias, but the mechanisms underlying this relationship remain unclear. In order to investigate this, we present a mechanistic canine cardiomyocyte model which reproduces independent local dyadic junctional SR (JSR) Ca2+ release events underlying cell-wide excitation-contraction coupling, as well as a three-dimensional super-resolution model of the Ca2+ spark that describes local Na+ dynamics as governed by NaK pumps, neuronal INa, and NCX1. The model predicts the existence of Na+ sparks, which are generated by NCX1 and exhibit significantly slower dynamics as compared to Ca2+ sparks. Moreover, whole-cell simulations indicate that neuronal INa in the cardiac dyad plays a key role during the systolic phase. Rapid inward neuronal INa can elevate dyadic [Na+] to 35–40 mM, which drives reverse-mode NCX1 transport, and therefore promotes Ca2+ entry into the dyad, enhancing the trigger for JSR Ca2+ release. The specific role of decreased co-localization of NaK and NCX1 in ankyrin-B+/− cardiomyocytes was examined. Model results demonstrate that a reduction in the local NCX1- and NaK-mediated regulation of dyadic [Ca2+] and [Na+] results in an increase in Ca2+ spark activity during isoproterenol stimulation, which in turn stochastically activates NCX1 in the dyad. This alteration in NCX1/NaK co-localization interrupts the balance between NCX1 and NaK currents in a way that leads to enhanced depolarizing inward current during the action potential plateau, which ultimately leads to a higher probability of L-type Ca2+ channel reopening and arrhythmogenic early-afterdepolarizations.

Cardiac T-Tubule Microanatomy and Function.

Unique to striated muscle cells, transverse tubules (t-tubules) are membrane organelles that consist of sarcolemma penetrating into the myocyte interior, forming a highly branched and interconnected network. Mature t-tubule networks are found in mammalian ventricular cardiomyocytes, with the transverse components of t-tubules occurring near sarcomeric z-discs. Cardiac t-tubules contain membrane microdomains enriched with ion channels and signaling molecules. The microdomains serve as key signaling hubs in regulation of cardiomyocyte function. Dyad microdomains formed at the junctional contact between t-tubule membrane and neighboring sarcoplasmic reticulum are critical in calcium signaling and excitation-contraction coupling necessary for beat-to-beat heart contraction. In this review, we provide an overview of the current knowledge in gross morphology and structure, membrane and protein composition, and function of the cardiac t-tubule network. We also review in detail current knowledge on the formation of functional membrane subdomains within t-tubules, with a particular focus on the cardiac dyad microdomain. Lastly, we discuss the dynamic nature of t-tubules including membrane turnover, trafficking of transmembrane proteins, and the life cycles of membrane subdomains such as the cardiac BIN1-microdomain, as well as t-tubule remodeling and alteration in diseased hearts. Understanding cardiac t-tubule biology in normal and failing hearts is providing novel diagnostic and therapeutic opportunities to better treat patients with failing hearts.

Dysfunction in the βII spectrin-dependent cytoskeleton underlies human arrhythmia.

The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin-deficient mice. Our findings identify βII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

Modeling CaMKII-mediated regulation of L-type Ca(2+) channels and ryanodine receptors in the heart.

Excitation-contraction coupling (ECC) in the cardiac myocyte is mediated by a number of highly integrated mechanisms of intracellular Ca2+ transport. Voltage- and Ca2+-dependent L-type Ca2+ channels (LCCs) allow for Ca2+ entry into the myocyte, which then binds to nearby ryanodine receptors (RyRs) and triggers Ca2+ release from the sarcoplasmic reticulum in a process known as Ca2+-induced Ca2+ release. The highly coordinated Ca2+-mediated interaction between LCCs and RyRs is further regulated by the cardiac isoform of the Ca2+/calmodulin-dependent protein kinase (CaMKII). Because CaMKII targets and modulates the function of many ECC proteins, elucidation of its role in ECC and integrative cellular function is challenging and much insight has been gained through the use of detailed computational models. Multiscale models that can both reconstruct the detailed nature of local signaling events within the cardiac dyad and predict their functional consequences at the level of the whole cell have played an important role in advancing our understanding of CaMKII function in ECC. Here, we review experimentally based models of CaMKII function with a focus on LCC and RyR regulation, and the mechanistic insights that have been gained through their application.

Control of Sarcoplasmic Reticulum Ca2+ Release by Stochastic RyR Gating within a 3D Model of the Cardiac Dyad and Importance of Induction Decay for CICR Termination

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca2+ spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca2+, Mg2+, and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca2+ sparks, Ca2+ blinks, and Ca2+ spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca2+ dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca2+ dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca2+, as well as the time course of local Ca2+ gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca2+ spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca2+ spark initiation and subsequent Ca2+ spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca2+ in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca2+ spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca2+ flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca2+ sensitivities, Ca2+ spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Termination of Calcium Sparks: An Emergent Property from Stochastic RyR Gating and Dyad Geometry

Many cells depend on ‘calcium-induced calcium release' (CICR), an inherently regenerative process due to the Ca2+-dependent gating of ryanodine receptors (RyRs) in the SR. Ca2+ sparks, as seen in muscle, reflect the concerted gating of groups of RyRs in specialized signalling domains in the junctions between the SR and surface membrane. However, the mechanism(s) responsible for the termination of regenerative CICR during the evolution of Ca2+ sparks remain uncertain. Rat cardiac RyR gating was recorded at physiological Ca2+, Mg2+ and ATP levels and incorporated into a 3D spatial model of the cardiac dyad which reproduced the time-course of Ca2+ sparks, Ca2+ blinks and Ca2+ spark restitution. Model CICR termination was robust, relatively insensitive to the number dyadic RyRs and automatic. This emergent behavior arose from the rapid development and dissolution of nanoscopic Ca2+ gradients within the dyad. These simulations show that CICR does not require intrinsic inactivation mechanisms for stability and cessation of regeneration arises from local control at the molecular scale via a process we call ‘induction decay'.