Axial Tubules Research Articles

Different effects of cardiomyocyte contractile activity on transverse and axial tubular system luminal content dynamics

BackgroundEfficient excitation-contraction coupling of mammalian ventricular cardiomyocytes depends on the transverse-axial tubular system (TATS), a network of surface membrane invaginations. TATS enables tight coupling of sarcolemmal and sarcoplasmic reticulum membranes, which is essential for rapid Ca2+-induced Ca2+ release, and uniform contraction upon electrical stimulation. The majority of TATS in healthy ventricular cardiomyocytes is composed of transverse tubules (TT, ∼90 % of TATS in rabbit). The remainder consists of mostly axial tubules (AT), which are less abundant and less well studied. In disease, however, the relative abundance of TT and AT changes. The mechanisms and relevance of this change are not known, and understanding them requires a more targeted effort to study the dynamics of AT structure and function.While TATS content is continuous with the interstitial space, it is contained within a domain of restricted diffusion. We have previously shown that TT are cyclically squeezed during stretch and contraction. This can contribute to TT content mixing and accelerates luminal content exchange with the environment. Here, we explore the effects of cardiomyocyte stretch and contraction on AT. MethodsTATS structure and diffusion dynamics were studied using 3D electron tomography of rabbit left ventricular cardiomyocytes, preserved at rest or during contraction, and ventricular tissue preserved at rest or during stretch, as well as live-cell TATS content exchange measurements. ResultsWe show (i) that cardiomyocyte contraction is associated with an increase in the apparent speed of diffusion of TT content that scales with beating rate and degree of cell shortening. In contrast, (ii) AT develop membrane folds and constrictions during contraction, (iii) with no effect of contraction on luminal exchange dynamics, while (iv) cardiomyocyte stretch is associated with AT straightening and AT and TT ‘squeezing’ that (v) supports an acceleration of the apparent speed of diffusion in AT and TT. Finally, (vi) we present a simple computational model outlining the potential relevance of AT in healthy and diseased cells. ConclusionsOur results indicate that TT and AT are differently affected by the cardiac contractile cycle, and suggest that AT may play a role in ensuring TATS network content homogeneity in diseased cardiomyocytes. Further research is needed to explore the interplay of structural and functional remodelling of different TATS components in failing myocardium.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

PO-01-209 THE MECHANISMS OF IMPAIRED CALCIUM HANDLING UNDERLYING THE INCREASED SUSCEPTIBILITY TO ATRIAL FIBRILLATION IN METABOLIC SYNDROME

Atrial fibrillation (AF) is becoming increasingly common partly due to the prevalence of metabolic syndrome (MetS). Altered Ca2+-handling proteins on the atrial cellular membrane as well as across the sarcoplasmic reticulum (SR) network and axial tubules due to this condition can precipitate AF through elevation of Ca2+ in junctional clefts, triggering Ca2+ sparks. Through the stochastic recruitment of these sparks, a Ca2+ wave can manifest to activate neighbouring ryanodine receptors (RyRs), stimulating Na+/Ca2+ exchangers (INaCa) into potentially triggering arrhythmogenic spontaneous action potentials (SAPs). However, the mechanisms by which MetS can disrupt Ca2+ handling and trigger SAPs is not well understood. To investigate the mechanisms of impaired calcium handling underlying the increased susceptibility to AF in MetS. We utilized a 3D model of subcellular Ca2+ signalling by Zhang et al. 2022, which compromises of a network of Ca2+-release units (CRUs) and ion channel and exchanger formulations from the Grandi et al. common pool model. The Zhang et al. model far more accurately captures the processes by which Ca2+ sparks can occur and terminate through the inclusion of crucial membrane ion channels/transporters within the extensive network of axial tubules interwoven across the CRUs (Fig A). Based on experimental studies, MetS meditated functional change in Ca2+-handling proteins was replicated by increasing the conductivity of SERCA and RyR and reducing ICaL and INaCa. To evaluate the contribution of each individual remodeled Ca2+-handling component to the membrane potential and Ca2+ sparks, a series of simulations was conducted where we first incorporated each individual ionic remodeling into the control model, then from the MetS model, we reversed each remodeled Ca2+-handling component separately. We have demonstrated that SAPs were attributed to a decrease in INaCa and an increase in SERCA. For the former, this also corresponded to a much greater incidence of Ca2+ sparks compared to other cases. However, for the latter, increasing SERCA surprisingly had a negligible impact on the genesis of these sparks (Fig B). This suggests that an alternate pathway can potentially exist by which upregulated SERCA function can induce SAPs. Increased Ca2+ spark incidence due to functional changes in Ca2+-handling proteins is not the sole mechanism to SAP genesis. Our study therefore highlights that MetS-mediated AF can occur through different pathways.

Dysferlin facilitates the de novo biogenesis of tubular membranes in hypertrophic remodelling

Abstract Introduction Ferlins are transmembrane proteins with multiple C2 domains that may mediate Ca2+ dependent membrane fusion and repair events. In ventricles, Dysferlin expression is dominant, and patient mutations have been associated with cardiomyopathies. Hence, we hypothesized that Dysferlin is essential for the functional integrity of the tubular endomembrane system in ventricular myocytes (VM), and may facilitate the cellular hypertrophic remodelling in pressure-overload. Results In 30 weeks old Dysferlin knockout (KO) mice, echocardiography revealed dilative cardiomyopathy with reduced left-ventricular ejection fractions (difference between means KO vs. wildtype (WT) −13.45±2.17%, p<0.001, n=16/20 mice). Asking for the subcellular relevance of Dysferlin, we investigated VM nanodomains by immunolabelling in STimulated Emission Depletion (STED) nanoscopy and visualized punctual Dysferlin clusters decorating the T-tubule system of mouse and human VM. Precisely, Dysferlin clusters localized to junctions of the tubular system and the sarcoplasmic reticulum (SR) in nanometric proximity to RyR2 Ca2+ release units. Complexome profiling (LC-MS/MS) also suggested interactions of Dysferlin with RyR2 and the SR membrane-tethering protein Junctophilin-2 (JP2) in high molecular weight complexes. In JP2-overexpressing mice, live-cell STED membrane imaging captured polyadic junctional membrane structures in VM, specifically surrounded by dense Dysferlin clusters, which proposes Dysferlin as stabilizing protein of tubule-SR junctions. Further, we studied the ultrastructural remodelling of the tubular system in a mouse model of cardiac hypertrophy and heart failure induced by transaortic constriction (TAC). Interestingly, mortality four weeks post TAC was not increased in KO mice. However, echocardiography uncovered that Dysf-KO prevents left-ventricular hypertrophy in response to pressure-overload, which was confirmed by single cell measurements (VM area: WT 3559±120 μm2 vs. KO 2671±87 μm2, n=163/158 VM, p<0.001). Thus, we assumed Dysferlin to mediate tubular network proliferation during hypertrophic remodelling. Indeed, WT VM showed an augmented Dysferlin expression post TAC (158±0,125%, p=0,005, n=7/7 mice), and immunofluorescence STED nanoscopy detected an increased Dysferlin network complexity. In detail, we visualized highly abundant Dysferlin clusters decorating newly shaped axial tubules, which were quantified by component-specific tubule network analysis. Conclusion Nanoscale imaging proposes Dysferlin as stabilizer of tubule-SR junctions, thereby promoting functional excitation-contraction coupling, and demonstrates the crucial importance of Dysferlin in pressure-overload induced hypertrophic remodelling, facilitating the de novo biogenesis of axial tubule membranes and tubule-SR junctions necessary for VM growth. Thus, Dysferlin may emerge as potential novel therapeutic rationale to control subcellular membrane remodelling in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Multiscale Bioimaging Cluster of Excellence: From Molecular Machines to Networks of Excitable Cells;German Research Foundation (DFG) - Collaborative Research Centre 1002 (SFB 1002): Modulatory Units in Heart Failure

A junctional cAMP compartment regulates rapid Ca2+ signaling in atrial myocytes

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3′,5′-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas β-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

Nanoscale Study of Calcium Handling Remodeling in Right Ventricular Cardiomyocytes Following Pulmonary Hypertension.

Pulmonary hypertension is a complex disorder characterized by pulmonary vascular remodeling and right ventricular hypertrophy, leading to right heart failure. The mechanisms underlying this process are not well understood. We hypothesize that the structural remodeling occurring in the cardiomyocytes of the right ventricle affects the cytosolic Ca2+ handling leading to arrhythmias. After 12 days of monocrotaline-induced pulmonary hypertension in rats, epicardial mapping showed electrical remodeling in both ventricles. In myocytes isolated from the hypertensive rats, a combination of high-speed camera and confocal line-scan documented a prolongation of Ca2+ transients along with a higher local Ca2+-release activity. These Ca2+ transients were less synchronous than in controls, likely due to disorganized transverse-axial tubular system. In fact, following pulmonary hypertension, hypertrophied right ventricular myocytes showed significantly reduced number of transverse tubules and increased number of axial tubules; however, Stimulation Emission Depletion microscopy demonstrated that the colocalization of L-type Ca2+ channels and RyR2 (ryanodine receptor 2) remained unchanged. Finally, Stimulation Emission Depletion microscopy and super-resolution scanning patch-clamp analysis uncovered a decrease in the density of active L-type Ca2+ channels in right ventricular myocytes with an elevated open probability of the T-tubule anchored channels. This may represent a general mechanism of how nanoscale structural changes at the early stage of pulmonary hypertension impact on the development of the end stage failing phenotype in the right ventricle.

Dysferlin links excitation-contraction coupling to structure and maintenance of the cardiac transverse-axial tubule system.

The multi-C2 domain protein dysferlin localizes to the T-Tubule system of skeletal and heart muscles. In skeletal muscle, dysferlin is known to play a role in membrane repair and in T-tubule biogenesis and maintenance. Dysferlin deficiency manifests as muscular dystrophy of proximal and distal muscles. Cardiomyopathies have been also reported, and some dysferlinopathy mouse models develop cardiac dysfunction under stress. Generally, the role and functional relevance of dysferlin in the heart is not clear. The aim of this study was to analyse the effect of dysferlin deficiency on the transverse-axial tubule system (TATS) structure and on Ca2+ homeostasis in the heart. We studied dysferlin localization in rat and mouse cardiomyocytes by immunofluorescence microscopy. In dysferlin-deficient ventricular mouse cardiomyocytes, we analysed the TATS by live staining and assessed Ca2+ handling by patch-clamp experiments and measurement of Ca2+ transients and Ca2+ sparks. We found increasing co-localization of dysferlin with the L-type Ca2+-channel during TATS development and show that dysferlin deficiency leads to pathological loss of transversal and increase in longitudinal elements (axialization). We detected reduced L-type Ca2+-current (ICa,L) in cardiomyocytes from dysferlin-deficient mice and increased frequency of spontaneous sarcoplasmic reticulum Ca2+ release events resulting in pro-arrhythmic contractions. Moreover, cardiomyocytes from dysferlin-deficient mice showed an impaired response to β-adrenergic receptor stimulation. Dysferlin is required for TATS biogenesis and maintenance in the heart by controlling the ratio of transversal and axial membrane elements. Absence of dysferlin leads to defects in Ca2+ homeostasis which may contribute to contractile heart dysfunction in dysferlinopathy patients.

Influence of the tubular network on the characteristics of calcium transients in cardiac myocytes.

Transverse and axial tubules (TATS) are an essential ingredient of the excitation-contraction machinery that allow the effective coupling of L-type Calcium Channels (LCC) and ryanodine receptors (RyR2). They form a regular network in ventricular cells, while their presence in atrial myocytes is variable regionally and among animal species We have studied the effect of variations in the TAT network using a bidomain computational model of an atrial myocyte with variable density of tubules. At each z-line the t-tubule length is obtained from an exponential distribution, with a given mean penetration length. This gives rise to a distribution of t-tubules in the cell that is characterized by the fractional area (F.A.) occupied by the t-tubules. To obtain consistent results, we average over different realizations of the same mean penetration length. To this, in some simulations we add the effect of a network of axial tubules. Then we study global properties of calcium signaling, as well as regional heterogeneities and local properties of sparks and RyR2 openings. In agreement with recent experiments in detubulated ventricular and atrial cells, we find that detubulation reduces the calcium transient and synchronization in release. However, it does not affect sarcoplasmic reticulum (SR) load, so the decrease in SR calcium release is due to regional differences in Ca2+ release, that is restricted to the cell periphery in detubulated cells. Despite the decrease in release, the release gain is larger in detubulated cells, due to recruitment of orphaned RyR2s, i.e, those that are not confronting a cluster of LCCs. This probably provides a safeguard mechanism, allowing physiological values to be maintained upon small changes in the t-tubule density. Finally, we do not find any relevant change in spark properties between tubulated and detubulated cells, suggesting that the differences found in experiments could be due to differential properties of the RyR2s in the membrane and in the t-tubules, not incorporated in the present model. This work will help understand the effect of detubulation, that has been shown to occur in disease conditions such as heart failure (HF) in ventricular cells, or atrial fibrillation (AF) in atrial cells.

P1230Impact of regulated junctophilin-2 clustering at axial tubule junctions on atrial excitation-contraction coupling and therapeutic implications

Abstract Objectives Atrial dysfunction is highly prevalent and known to significantly aggravate heart failure. While rapid excitation-contraction (EC) coupling depends on axial tubule junctions in atrial myocytes (AMs), the mechanisms leading to atrial loss-of-function remain unclear. Junctophilin-2 (JP2), a tail-anchored protein of the sarcoplasmic reticulum, stabilizes the integrity of ventricular Ca2+ release units, which is disrupted in ventricular myocytes by reduced JP2 expression or proteolysis. Here we aim to characterize the abundance and subcellular localisation of JP2 in AMs, to assess the impact of decreased JP2 expression on atrial remodelling, and to investigate the potential to correct JP2 expression and atrial dysfunction. Results We identified 5-fold lower JP2 levels in atrial compared to ventricular tissue in mouse and human hearts by SDS-PAGE. Surprisingly, in AMs, this resulted in subcellular expression of large JP2 clusters at axial tubule junctions together with highly phosphorylated ryanodine receptor (RyR2) channels visualized by STED superresolution microscopy. Importantly, left atrial hypertrophy induced by aortic pressure overload led to an additional strong decrease in JP2 expression compared to sham control, disrupted junctional RyR2 clustering and EC-coupling. This loss-of-function mechanism was confirmed by conditional shRNA-mediated JP2 knockdown. Quantitative image analysis after atrial JP2 knockdown showed a 50% decrease in area overlap between RyR2 and JP2 in AMs (JP2 knockdown 0.03±0.003 μm2 vs. control 0.06±0.004 μm2, p<0.001), and a ∼2-fold increased Ca2+ spark frequency, consistent with decreased left atrial fractional shortening (JP2 knockdown 12.9±0.8% vs. control 16.5±0.9%, p<0.01). Whereas atrial-ventricular dysfunction due to aortic pressure overload resulted in 40% mortality, additional JP2 knockdown exacerbated mortality to 100% (n: 10 control vs. 9 JP2 knockdown mice). In contrast, transgenic JP2 overexpressor mice showed greatly improved atrial contractility without mortality after induced aortic pressure overload (n: 21 control vs. 16 JP2 overexpressor mice). JP2-OE not only augmented atrial RyR2-clustering, but induced the de-novo biogenesis of large poly-adic junctional membrane complexes, which were resolved by STED microscopy via high-resolution cholesterol-based membrane staining in live AMs and electron tomography. Conclusions Nanoscale imaging identifies a new subcellular mechanism of significantly limited atrial JP2 protein expression in large clusters at axial tubule junctions. In atrial hypertrophy, JP2 is further decreased with junctional RyR2 cluster disruption leading to impaired Ca2+ release and decreased contractility. Importantly, JP2 overexpression effectively protected from atrial dysfunction, providing a novel therapeutic rationale for atrial cardiomyopathies.

Effects of Varying Transverse and Axial Tubules in a Three-Dimensional Model of Calcium Signaling in the Human Atrial Myocyte

T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in ventricular myocytes. Atrial myocytes are generally thought to possess sparse irregular transverse-tubular (TT) components, as opposed to the highly dense and regular ventricular TT system. Axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum that include ryanodine receptor (RyR) clusters, characterized by rapid activation of Ca2+-induced Ca2+ release, have also been identified in atrial myocytes. AT and TT remodeling and changes in distribution, composition, and phosphorylation status of Ca2+ release units are thought to underlie Ca2+ abnormalities in atrial fibrillation (AF), the most common cardiac arrhythmia. Here, we performed a computational analysis to investigate how changes in the tubular network affect human atrial electrophysiology. We modified our well-established three-dimensional model of Ca2+ signaling in the rabbit ventricular myocyte to develop an analogous model in the human atrium. We also coupled the Ca2+ signaling model to our well-established model of membrane electrophysiology in the human atrial myocyte. We systematically varied TT and AT density and RyR distribution and assessed the effect on Ca2+ spark and wave properties. When TT density is low, as shown in isolated atrial myocytes, the model recapitulates the typical U-shaped Ca2+ wave seen experimentally in transverse confocal line-scan images. Introduction of ATs resulted in W-shaped Ca2+ waves, reflecting more synchronous Ca2+ release. The frequency and amplitude of Ca2+ release events were affected by both density of TT and AT, as well as RyR distribution. Our newly developed three-dimensional model of the human atrial myocyte will be employed to investigate whether and how AF-induced cellular electrical and structural remodeling effects collectively contribute to the membrane potential and Ca2+ abnormalities seen in AF.

Axial Tubule Junctions Activate Atrial Ca2+ Release Across Species.

Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling.Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca2+ signaling proteins across species from mouse to human.Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca2+ current was similar in human and mouse AMs, the intracellular Ca2+ transient showed quantitative differences. Importantly, local intracellular Ca2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections.Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial “super-hub” Ca2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.

The Subcellular Distribution of Ryanodine Receptors and L-Type Ca2+ Channels Modulates Ca2+-Transient Properties and Spontaneous Ca2+-Release Events in Atrial Cardiomyocytes.

Spontaneous Ca2+-release events (SCaEs) from the sarcoplasmic reticulum play crucial roles in the initiation of cardiac arrhythmias by promoting triggered activity. However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs.

Heterogeneity of transverse-axial tubule system in mouse atria: Remodeling in atrial-specific Na+–Ca2 + exchanger knockout mice

Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a characteristic “V”-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the “V”-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na–Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a “V”-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm.

Axial tubule junctions control rapid calcium signaling in atria.

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon "super-hubs" thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias.

Atrial-specific pathways for control of intracellular signaling and myocyte function.

Atrial fibrillation (AF) is a cardiac arrhythmia that arises from electrical and contractile dysfunction in the atria. Atrial function is regulated by a variety of intracellular signaling networks that facilitate rapid communication and coordinate responses of atrial myocytes. In this issue of the JCI, Brandenburg and colleagues describe the identification and characterization of "super-hub" signaling nodes located on atrial axial tubules that regulate atrial contraction. Together, the results of this study provide important insight into the regulation of atrial contraction and describe potential therapeutic targets to be explored in future studies.

Sarcoplasmic reticulum is an intermediary of mitochondrial and myofibrillar growth at the intercalated disc.

In cardiomyocytes columns of intermyofibrillar mitochondria run up to the intercalated disc (ID); half are collinear with those in the neighbouring cell, suggesting coordinated addition of sarcomeres and mitochondria both within and between cells during cardiomyocyte growth. Recent evidence for an association between sarcoplasmic reticulum (SR) and mitochondria indicates that the SR may be an intermediary in this coordinated behaviour. For this reason we have investigated the arrangement of SR and t tubules with respect to mitochondria and myofibrils, particularly at the ID. In the body of the cardiomyocyte the mitochondrial columns are frequently intersected by transverse tubules. In addition, we find that a majority of axial tubules are sandwiched between mitochondria and myofibril. No tubules are found at the ID. SR coats mitochondrial columns and fibrils throughout their length and reaches towards the peaks of the ID membrane where it attaches in the form of junctional (j)SR. These peripheral ID couplings are often situated between mitochondria and ID membrane, suggesting an SR connection between the two. In dilated cardiomyopathy (DCM) the mitochondria are somewhat disordered and clumped. In a mouse model for DCM, the muscle LIM protein KO, we find that there is a lack of mitochondria near the ID, suggesting the uncoupling of the myofibril/mitochondria organisation during growth. SR still coats the fibrils and reaches the ID folds in a jSR coupling. Unlike in control tissue, however, loops and long fingers of ID membrane penetrate into the proximal sarcomere suggesting a possible intermediary state in cardiomyocyte growth.

Super-Resolution Localization and Distribution of Proteins within the Mammalian Couplon

We have used 3D dSTORM to characterize the distribution and localization of the ryanodine receptor (RyR), the L-type calcium channel (Cav1.2) and the sodium-calcium exchanger (NCX) within the couplon of the rat cardiomyocyte. The images have a resolution of 20nm in X and Y and 50 nm in Z, and cover areas of up to 1200 µm2 in XY with depths of up to 700nm in Z. In the case of RyR, hundreds of individual clusters could be identified and characterized. The clusters varied greatly in both size and structure; their internal structure showing little evidence of the checkerboard arrangement that has been thought to be predominant. RyR clusters were identified both in the transverse and axial tubules and in some cases could be seen to be separated by a gap typical of the width of a t-tubule. In general, the clusters of Cav1.2 were smaller and far denser than their RyR counterparts, with their centers appearing to be tightly packed. NCX was much more widely distributed than either RyR or Cav1.2 and formed a dense carpet along the cell surface with little clustering or identifiable detail. While small isolated clusters of NCX were present in the tubular system, there were other regions where the labeling was more widespread and the clusters were poorly defined.

Axial Tubules of Rat Ventricular Myocytes Form Multiple Junctions with the Sarcoplasmic Reticulum

Ryanodine receptors (RyRs) are located primarily on the junctional sarcoplasmic reticulum (SR), adjacent to the transverse tubules and on the cell surface near the Z-lines, but some RyRs are on junctional SR adjacent to axial tubules. Neither the size of the axial junctions nor the numbers of RyRs that they contain have been determined. RyRs may also be located on the corbular SR and on the free or network SR. Because determining and quantifying the distribution of RyRs is critical for both understanding and modeling calcium dynamics, we investigated the distribution of RyRs in healthy adult rat ventricular myocytes, using electron microscopy, electron tomography, and immunofluorescence. We found RyRs in only three regions: in couplons on the surface and on transverse tubules, both of which are near the Z-line, and in junctions on most of the axial tubules—axial junctions. The axial junctions averaged 510 nm in length, but they occasionally spanned an entire sarcomere. Numerical analysis showed that they contain as much as 19% of a cell's RyRs. Tomographic analysis confirmed the axial junction's architecture, which is indistinguishable from junctions on transverse tubules or on the surface, and revealed a complexly structured tubule whose lumen was only 26 nm at its narrowest point. RyRs on axial junctions colocalize with Cav1.2, suggesting that they play a role in excitation-contraction coupling.

Functional groups of ryanodine receptors in rat ventricular cells

Ryanodine receptors (RyR2s) are ion channels in the sarcoplasmic reticulum (SR) that are responsible for Ca2+ release in rat ventricular myocytes. Localization of RyR2s is therefore crucial for our understanding of contraction and other Ca2+-dependent intracellular processes. Recent results (e.g. circular waves and Ca2+ sparks in perinuclear area) raised questions about the classical views of RyR2 distribution and organization within ventricular cells. A Ca2+ spark is a fluorescent signal reflecting the activation of a small group of RyR2s. Frequency and spatio-temporal characteristics of Ca2+ sparks depend on the state of cytoplasmic and intraluminal macromolecular complexes regulating cardiac RyR2 function. We employed electron microscopy, confocal imaging of spontaneous Ca2+ sparks and immunofluorescence to visualize the distribution of RyR2s in ventricular myocytes and to evaluate the local involvement of the macromolecular complexes in regulation of functional activity of the RyR2 group. An electron microscopy study revealed that the axial tubules of the transverse-axial tubular system probably do not have junctions with the network SR (nSR). The nSR was found to be wrapped around intermyofibrillar mitochondria and contained structures similar to feet of the junctional cleft. Treatment of ventricular myocytes with antibodies against RyR2 showed that in addition to the junctional SR, a small number of RyR2s can be localized at the middle of the sarcomere and in the zone of perinuclear mitochondria. Recordings of spontaneous Ca2+ sparks showed the existence of functional groups of RyR2s in these intracellular compartments. We found that within the sarcomere about 20% of Ca2+ sparks were not colocalized with the zone of the junctional or corbular SR (Z-line zone). The spatio-temporal characteristics of sparks found in the Z-line and A-band zones were very similar, whereas sparks from the zone of the perinuclear mitochondria were about 25% longer. Analysis of the initiation sites of Ca2+ sparks within the same junctional SR cluster suggested that 18-25 RyR2s are in the functional group producing a spark. Because of the similarity of the spatio-temporal characteristics of sarcomeric sparks and ultrastructural characteristics of nSR, we suggest that the functional groups of RyR2s in the middle of the sarcomere are macromolecular complexes of approximately 20 RyR2s with regulatory proteins. Our data allowed us to conclude that a significant number of functional RyR2s is located in the middle of the sarcomere and in the zone of perinuclear mitochondria. These RyR2s could contribute to excitation-contraction coupling, mitochondrial and nuclear signalling, and Ca2+-dependent gene regulation, but their existence raises many additional questions.

Voltage-gated K+Channel, Kv4.2, Localizes Predominantly to the Transverse-axial Tubular System of the Rat Myocyte

Kv4.2 subunit, a member of K+channel gene family, is considered to play a major role in the formation of depolarization-activated transient outward K+current channels in the mammalian heart. We investigated the subcellular localization of Kv4.2 subunit in the rat heart by immunofluorescence and immunoelectron microscopy. In atrial cells, Kv4.2 immunofluorescent staining was intensely observed in the peripheral sarcolemma and the intercalated disks, but seldom found in transverse tubules, which are rare or absent in atrial cells. In ventricular cells, the labeling of Kv4.2 immunofluorescent staining was found throughout the entire cell membrane, and the staining was stronger in the transverse-axial tubular system than in the peripheral sarcolemma. Correlative immunoconfocal and immunoelectron microscopy using FluoroNanogold confirmed that Kv4.2 distributed in the transverse-axial tubular system including the longitudinally oriented axial tubules. Immunogold electron microscopy of ultrathin cryosections revealed that Kv4.2 was distributed on the plasma membranes of the T-tubules. The extensive distribution of Kv4.2 on the entire cell membrane of myocytes would provide rat myocardial cells with a large capability for the transport of K+ions through the channels in the repolarization phase.