Transverse-axial Tubular System 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.

A new approach to the determination of tubular membrane capacitance: passive membrane electrical properties under reduced electrical conductivity of the extracellular solution

The transverse-axial tubular system (tubular system) of cardiomyocytes plays a key role in excitation–contraction coupling. To determine the area of the tubular membrane in relation to the area of the surface membrane, indirect measurements through the determination of membrane capacitances are currently used in addition to microscopic methods. Unlike existing electrophysiological methods based on an irreversible procedure (osmotic shock), the proposed new approach uses a reversible short-term intermittent increase in the electrical resistance of the extracellular medium. The resulting increase in the lumen resistance of the tubular system makes it possible to determine separate capacitances of the tubular and surface membranes. Based on the analysis of the time course of the capacitive current, computational relations were derived to quantify the elements of the electrical equivalent circuit of the measured cardiomyocyte including both capacitances. The exposition to isotonic low-conductivity sucrose solution is reversible which is the main advantage of the proposed approach allowing repetitive measurements on the same cell under control and sucrose solutions. Experiments on rat ventricular cardiomyocytes (n = 20) resulted in the surface and tubular capacitance values implying the fraction of tubular capacitance/area of 0.327 ± 0.018. We conclude that the newly proposed method provides results comparable to the data obtained by the currently used detubulation method and, in addition, by being reversible, allows repeated evaluation of surface and tubular membrane parameters on the same cell.

Fraction of the T-Tubular Membrane as an Important Parameter in Cardiac Cellular Electrophysiology: A New Way of Estimation.

The transverse-axial tubular system (t-tubules) plays an essential role in excitation-contraction coupling in cardiomyocytes. Its remodelling is associated with various cardiac diseases. Numerous attempts were made to analyse characteristics essential for proper understanding of the t-tubules and their impact on cardiac cell function in health and disease. The currently available methodical approaches related to the fraction of the t-tubular membrane area produce diverse data. The widely used detubulation techniques cause irreversible cell impairment, thus, distinct cell samples have to be used for estimation of t-tubular parameters in untreated and detubulated cells. Our proposed alternative method is reversible and allows repetitive estimation of the fraction of t-tubular membrane (f t) in cardiomyocytes using short-term perfusion of the measured cell with a low-conductive isotonic sucrose solution. It results in a substantial increase in the electrical resistance of t-tubular lumen, thus, electrically separating the surface and t-tubular membranes. Using the whole-cell patch-clamp measurement and the new approach in enzymatically isolated rat atrial and ventricular myocytes, a set of data was measured and evaluated. The analysis of the electrical equivalent circuit resulted in the establishment of criteria for excluding measurements in which perfusion with a low conductivity solution did not affect the entire cell surface. As expected, the final average f t in ventricular myocytes (0.337 ± 0.017) was significantly higher than that in atrial myocytes (0.144 ± 0.015). The parameter f t could be estimated repetitively in a particular cell (0.345 ± 0.021 and 0.347 ± 0.023 in ventricular myocytes during the first and second sucrose perfusion, respectively). The new method is fast, simple, and leaves the measured cell intact. It can be applied in the course of experiments for which it is useful to estimate both the surface and t-tubular capacitance/area in a particular cell.

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.

Homozygous G650del nexilin variant causes cardiomyopathy in mice.

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948–1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn’s function in tubular system organization and normal cardiac function.

Accounting for cardiac t-tubule increase with age and myocyte volume to improve measurements of its membrane area and ionic current densities

In-silico models of cardiac myocytes allow simulating experiments in numbers on series of myocytes as well as on large populations of myocytes assembled in 3D structures. The simulated myocyte populations should have realistic values and statistical dispersions of biophysical parameters such as myocyte dimensions and volume and areas of the peripheral membrane and transverse-axial tubular system (TATS). Dependencies among these variables also have to be taken into account. In this work, we propose a quantitative representation of the changes in the fraction of membrane area in the TATS that integrates published dependencies with body weight (age) and size of rat ventricular cardiac myocytes while respecting the above constraints. Imposing a constant total membrane area-to-volume ratio appears to account for the increase of this fraction with myocyte size (i.e.: volume) within a given age group. The agreement of our results with published data was discussed and reasons for discrepancies were analysed. On the basis of our framework, strategies are proposed for minimizing the influence of non-random dispersion related to myocyte volume on measurements of the area of TATS and surface membrane compartments and of ionic current densities. The next step will be to quantitatively compare these strategies by evaluating the impact of myocyte morphological parameters and their dependencies, sample size, biases and errors, on the output of experiments.

Abstract 418: Caveolar Disruption of L-type Calcium Channel and Ryanodine Receptor Facilitates Atrial Ectopy and Arrhythmogenesis in Heart Failure Mice

Atrial fibrillation (AF) often occurs during heart failure (HF). Ectopic foci that trigger AF, are linked to discrete atrial regions that experience the highest remodeling and clinically used for AF ablation; however, mechanisms of their arrhythmogenic propensity remain elusive. We employed in vivo ECG telemetry, in vitro optical mapping and confocal imaging of Ca 2+ transients (CaT) from myocytes isolated from the right atrial appendage (RAA) and inter-caval region (ICR) of wild type (WT, n=10), caveolin-3 knockout (KO, n=6) and 8-weeks post-myocardial infarction HF (n=8) mice. HF and KO mice showed an increased susceptibility to pacing-induced AF and enhanced ectopy originated exclusively from ICR. Optical mapping in isolated atria showed prolongation of CaT rise up time (CaT-RT) in HF ICR, which suggested a remodeled coupling between L-type Ca 2+ channels (LTCCs) and ryanodine receptors (RyRs) in this specific region. In WT mice, RAA consists of structured myocytes with a prominent transverse-axial tubular system (TATS) while ICR myocytes don’t have TATS. In RAA, CaT-RT depends on LTCCs in TATS triggering RyR, while in ICR, all the LTCCs are localized in surface caveolae where they can activate subsarcolemmal RyRs and lead to a slow diffusion of Ca 2+ inside the cell interior. Downregulation of caveolae was observed specifically in HF ICR. To mimic this, we used cav3-KO mice. Triggered activities were observed in myocytes isolated from HF and KO ICR, which presumably underlie the ectopic activities in tissue level. These myocytes presented significantly unsynchronized sarcoplasmic reticulum (SR) Ca 2+ releases (synchronization index: 10.8±0.9 in WT vs 38.3±4.1 in HF vs 21.5±2.1 in KO, p <0.01 for HF and KO vs WT respectively) especially at the subsarcolemmal space that prolongs CaT-RT (62.2±4.1 ms in WT vs 122.5±12.8 ms in KO, p <0.01). In addition, failing ICR myocytes showed a higher occurrence and size of spontaneous Ca 2+ sparks which were linked to CaMKII activity and associated phosphorylation of RyR. Our findings demonstrate that in HF, caveolar disruption creates “hot spots” for arrhythmogenic ectopic activity emanated from discrete vulnerable regions of the right atrium which are associated with desynchronized SR Ca 2+ release and elevated fibrosis.

Nexilin Is Necessary for Maintaining the Transverse-Axial Tubular System in Adult Cardiomyocytes.

NEXN (nexilin) is a protein of the junctional membrane complex required for development of cardiac T-tubules. Global and cardiomyocyte-specific loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy and premature death. Therefore, little is known as to the role of NEXN in adult cardiomyocytes. Transverse-axial tubular system remodeling are well-known features in heart failure. Although NEXN is required during development for T-tubule formation, its role, if any, in mature T-tubules remains to be addressed. Nexn inducible adult cardiomyocyte-specific KO mice were generated. Comprehensive morphological and functional analyses were performed. Heart samples (n>3) were analyzed by molecular, biochemical, and electron microscopy analyses. Isolated single adult cardiomyocytes were analyzed by confocal microscopy, and myocyte shortening/re-lengthening and Ca2+ transient studies were conducted. Inducible cardiomyocyte-specific loss of Nexn in adult mice resulted in a dilated cardiomyopathy with reduced cardiac function (13% reduction in percentage fractional shortening; P<0.05). In vivo and in vitro analyses of adult mouse heart samples revealed that NEXN was essential for optimal contraction and calcium handling and was required for maintenance of T-tubule network organization (transverse tubular component in Nexn inducible adult cardiomyocyte-specific KO mice reduced by 40% with respect to controls, P<0.05). Results here reported reveal NEXN to be a pivotal component of adult junctional membrane complexes required for maintenance of transverse-axial tubular architecture. These results demonstrate that NEXN plays an essential role in the adult cardiomyocyte and give further understanding of pathological mechanisms responsible for cardiomyopathy in patients carrying mutations in the NEXN gene.

Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

Measuring Electrical Conductivity of the Cardiac T-Tubular System

Cardiomyocytes are characterized by a complex network of membrane invagination (the transverse axial tubular system, TATS) that propagates the action potential to the cell core guarantying a synchronous and uniform cell contraction. Pathological settings are commonly associated with structural remodelling of TATS that may cause failures of action potential propagation at single T-tubular level. Here, we investigate whether structural alterations occurring in TATS are linked to overall changes in its electrical conductivity which in turn hinder the regular propagation of action potentials across the network. Exploiting the formal analogy between diffusion and electrical conductivity we linked the conductivity with the diffusion properties of TATS. Fluorescence recovery after photo-bleaching (FRAP) microscopy is used to probe the diffusion properties of the tubular system in isolated cardiomyocytes: the fluorescent dextran inside TATS lumen is photo-bleached and the diffusion of unbleached dextran from extracellular space to TATS is monitored. We designed a mathematical model that correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the dextran. Then, taken advantage of the analogy between diffusion and conductivity, the apparent diffusion is used to assess TATS conductivity. This value is used to evaluate the efficiency of the passive spread of voltage changes along TATS. To probe how TATS conductivity is influenced by geometrical features, we used a model of acutely detubulated cells. We found that the overall reduction of TATS halves tubular conductivity. Then we tested our method in a pathological setting characterized by structural alteration, i.e. the heart failure rat model. We found that the tubular conductivity is not changed as compared to control. Actually, this result should not be surprising since heart failure is characterized by compound alterations that potentially compensate each other in their individual effect on global conductivity.

Electrical defects of the transverse-axial tubular system in cardiac diseases.

Electrical excitability is an essential feature of cardiomyocytes and the homogenous propagation of the action potential is guaranteed by a complex network of membrane invaginations called the transverse‐axial tubular system (TATS). TATS structural remodelling is a hallmark of cardiac diseases and we demonstrated that this can be accompanied by electrical defects at single T‐tubular level. Using a random‐access multi‐photon (RAMP) microscope, we found that pathological T‐tubules can fail to conduct action potentials, which delays local Ca2+ release. Although the underlying causes for T‐tubular electrical failure are still unknown, our findings suggest that they are likely to be related to local ultrastructural alterations. Here, we first review the experimental approach that allowed us to observe and dissect the consequences of TATS electrical dysfunction and then propose two different strategies to unveil the reasons for T‐tubular electrical failures. The first strategy consists in a correlative approach, in which the failing T‐tubule identified with the RAMP microscope is then imaged with electron microscopy. The second approach exploits the diffusion of molecules within TATS to gain insights into the local TATS structure, even without a thorough reconstruction of the tubular network. Although challenging, the local electrical failure occurring at single T‐tubules is a fundamental question that needs to be addressed and could provide novel insights in cardiac pathophysiology.

168-03: Axial membrane tubules in atrial myocytes initiate rapid intracellular calcium signals through a new “super-hub” mechanism

Invaginated sarcolemmal membrane structures in cardiac myocytes form transverse tubules (TTs). The TTs form electrical and chemical signaling conduits from the myocyte exterior to regions deep in the cell such as the junctional sarcoplasmic reticulum (jSR) critical in excitation-contraction (EC) coupling. Local control of jSR Ca2+ release in ventricular myocytes (VM) depends critically on this organization. In contrast, atrial myocytes (AM) normally have few if any TTs (although the exact abundance has been controversial). In atrial myocytes, the normally sparse distribution of TTs suggests that a critical role in EC coupling remains unlikely. Here, we identify a structurally distinct and mechanistically novel role for even sparsely set TTs in atrial EC coupling. Using novel membrane-preserving live cell techniques, we identify relatively abundant axial membrane tubules (ATs). These ATs are deep intracellular branches of the relatively rare TTs in 100% of AM investigated (n = 29). Using live cell STimulated Emission Depletion (STED) superresolution microscopy, we show that atrial AT structures are characteristically different from TTs. ATs represent both the major component of the cell-wide tubule network and the largest tubules: AT circumference in AM 921 ± 24 nm vs. VM 630 ± 26 nm; p < 0.001 (physiological live cell conditions). These dimensions indicate unprecedentedly large AT surfaces with a positive membrane curvature at the AM center suggesting that ATs could function as a central-axial Ca2+ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined AT and intracellular Ca2+ transient (CaT) imaging. High-amplitude CaTs originated directly from ATs creating highly localized central-axial Ca2+ signals. Importantly, local CaT onset measurements showed that atrial CaTs occurred significantly earlier at AT compared to surface membrane locations (p < 0.05). To explain the observed CaT behaviors, we imaged in situ the Ser-2808 phospho-epitope of ryanodine receptor (RyR2) Ca2+ release channels necessary for PKA phosphorylation. RyR2 channel clusters at AT-hubs showed increased PKA phosphorylation in contrast to the low phosphorylation state of non-junctional RyR2 clusters in unstimulated AM. Sarcomere shortening experiments confirmed significantly larger and faster AM compared with VM axial contractions (p < 0.05); this effect was significantly augmented by isoproterenol (1 µm) through non-junctional cluster recruitment. Consistent with the proposed AT super-hub model, genetic ablation of RyR2 PKA phosphorylation in Ser-2808-Ala knockin mice significantly attenuated atrial sarcomere shortening and in vivo atrial but not ventricular contractile function. Our data suggest a fundamentally new atrial EC-coupling model where AT structures in atrial myocytes function as large signaling super-hubs that couple central-axial Ca2+ signaling to rapid contractile activation. This supports a critical role of the transverse-axial tubular system in cell-specific atrial EC-coupling mechanisms (i.e. faster contractions than in ventricles) and questions previous models of atrial EC-coupling based on slow intracellular Ca2+ diffusion. Genetic ablation of RyR2 cluster phosphorylation unmasks a previously unknown role of atrial PKA regulation, which may explain atrial function in health versus decreased contractility occuring in atrial fibrillation and heart failure.

Diffusion Properties of Cardiac T-Tubular System

Action potentials (APs), via the transverse axial tubular system (TATS), synchronously trigger uniform Ca2+ release throughout the cardiomyocyte. In cardiomyocytes from diseased hearts we previously documented the presence of TATS elements which failed to propagate AP (Sacconi et al. PNAS 2012). Using an imaging method to simultaneously assess TATS electrical activity and local Ca2+ release we recently demonstrated that failing tubular elements show a slower local Ca2+ transient compared with regions with electrically coupled elements (Crocini et al. PNAS 2014). The causes of AP propagation failure remained, however, unsolved. TATS structural remodelling that is generally associated with pathological settings may increase the tubular electrical resistance, augmenting the probability that the depolarization wavefront fails in reaching the AP threshold at the tubular membrane. TATS structural changes can modify the diffusion properties of T-tubule lumen. Here, we used fluorescence recovery after photo-bleaching (FRAP) microscopy to probe the diffusion properties of TATS lumen in cardiomyocytes. T-tubules of isolated cardiomyocytes from rodent models of cardiac diseases are labelled using fluorescent dextran that diffuses from extracellular space to TATS lumen. The fluorescent dextran present inside TATS lumen is photo-bleached and the diffusion of unbleached dextran from extracellular space to TATS is monitored using confocal imaging. Analysing the recovery of the fluorescence signal at pixel-by-pixel level, a diffusional map of the tubular system is obtained. Diffusion maps performed at different dextran molecular weight are acquired to probe structural features of the TATS lumen. Finally, diffusion maps in different pathological models are reconstructed to find potential mechanisms for the presence of AP failing TATS elements.

Bachmann’s Bundle

Bachmann’s bundle (BB), also known as the interatrial bundle, is well recognized as a muscular bundle comprising of parallel aligned myocardial strands connecting the right and left atrial walls and is considered to be the main pathway of interatrial conduction.1 Disruption of the bundle’s structure causes interatrial conduction block (IAB),2 which is associated with development of various atrial tachyarrhythmias3,4 and with electromechanical dysfunction of the left atrium.5 Technological progress providing sophisticated mapping and imaging techniques in the past decade has increased our knowledge of specific anatomic structures and their role in development of both atrial brady- and tachyarrhythmias. This review outlines the current knowledge of the relation between anatomic and electrophysiological properties of BB and its possible role in initiation and perpetuation of atrial fibrillation (AF). In 1963, Thomas N. James described 3 pathways connecting the sinus node to the atrioventricular node (AVN), namely the anterior, medial, and posterior internodal pathways.6 Whether these conduction pathways were because of the presence of specialized conduction tissue or because of the anisotropic orientation of the muscle fibers remains controversial. Nevertheless, James described the anterior pathway as leaving the sinus node in anterior direction and giving off a secondary branch at the level of the superior vena cava to form BB.1 BB stretches subepicardially across the interatrial groove (septal raphe). It is at the interatrial groove that the BB can be identified as a discrete bundle (Figures 1 and 2) separated by fatty tissues from the infolded right atrial wall that is the limbus of the oval fossa. Notably, the bundle is not surrounded by a fibrous tissue sheath. Instead, the bundle is comprised of strands of atrial myocardium that are similarly aligned in parallel fashion. Its rightward and leftward extensions bifurcate to pass to either …

The transverse-axial tubular system of cardiomyocytes.

A characteristic histological feature of striated muscle cells is the presence of deep invaginations of the plasma membrane (sarcolemma), most commonly referred to as T-tubules or the transverse-axial tubular system (TATS). TATS mediates the rapid spread of the electrical signal (action potential) to the cell core triggering Ca(2+) release from the sarcoplasmic reticulum, ultimately inducing myofilament contraction (excitation-contraction coupling). T-tubules, first described in vertebrate skeletal muscle cells, have also been recognized for a long time in mammalian cardiac ventricular myocytes, with a structure and a function that in recent years have been shown to be far more complex and pivotal for cardiac function than initially thought. Renewed interest in T-tubule function stems from the loss and disorganization of T-tubules found in a number of pathological conditions including human heart failure (HF) and dilated and hypertrophic cardiomyopathies, as well as in animal models of HF, chronic ischemia and atrial fibrillation. Disease-related remodeling of the TATS leads to asynchronous and inhomogeneous Ca(2+)-release, due to the presence of orphan ryanodine receptors that have lost their coupling with the dihydropyridine receptors and are either not activated or activated with a delay. Here, we review the physiology of the TATS, focusing first on the relationship between function and structure, and then describing T-tubular remodeling and its reversal in disease settings and following effective therapeutic approaches.

Superresolution Imaging—Caveolae, Caveolins, Mitochondria, and Function in Heart

Our ability to investigate questions improves as methods advance. Such advances may permit some issues to be resolved while other questions develop a new complexion and engender new and/or refined questions. Here experiments that combine superresolution optical imaging (or stochastic optical reconstruction microscopy, STORM (1,2)) and electron microscopy (EM) tomography are presented in a study on cardiac structures by Wong et al. (3). The focus is primarily on the spatial organization of the cardiac transverse tubular (TT) system, also called the transverse-axial tubular system (TATS). While the transverse character of the TT/TATS system clearly dominates in healthy mammalian adult ventricular myocytes, heart failure leads to disorganization and dedifferentiation of this tubular system (4). In addition, the organization of caveolin-3 and the ryanodine receptors (RyR2 or the Ca2+ release channel in the junctional sarcoplasmic reticulum) are presented and discussed. The work seeks to link dynamic cellular structures and nanoscopic organization to cellular function. This work is also a very nice demonstration of the value of combining superresolution optical microscopy (STORM, PALM, and STED—see below (1,2,5–7)) and EM tomography (8,9). The name of the protein caveolin comes from the name of structures with which they are reported to be associated: the caveolae (10). The caveolae are omega-shaped invaginations of the surface membrane (10,11) of cells with localized function and organization (12). In cardiac and skeletal muscle cells, the caveolae are thought to be associated with both the external sarcolemmal membrane and the membranes of the TATS. In some cases, caveolae are associated with mitochondria (13) and in adipocytes with lipid metabolism (14). It is thus often thought that the spatial organization of the caveolae as well as the specific array of proteins and lipids in the very small omega-shaped structure underlie caveolar function. There is, however, a distinction of the concept between caveolin-rich membranes and caveolae as presented in the work by Wong et al. (3). The authors show that the majority of the surface membrane (external SL and TATS membranes) containing caveolin-3 does not include caveolar invaginations. Although there are TT dilations in the region of the junctional sarcoplasmic reticulum, they are not caveolae. This suggests that some quantitative reexamination of a number of hypotheses may be in order. Perhaps the exciting recent findings by Makarewich et al. (15), for example, would be recast if caveolae were not present. Nevertheless, the findings of Wong et al. (3) serve mainly to draw attention to a critical question: What exactly do the caveolins do? In addition, the work demonstrates both the power and utility of superresolution optical imaging combined with EM tomography.

Imaging T-tubules: dynamic membrane structures for deep functions

This editorial refers to ‘Emerging mechanisms of T-tubule remodelling in heart failure’ by A. Guo et al ., pp. 204–215, this issue and ‘Ultrastructural uncoupling between T-tubules and sarcoplasmic reticulum in human heart failure’ by H.-B. Zhang, et al ., pp. 269–276, this issue. Extensive intracellular contacts of the endo/sarcoplasmic reticulum (ER/SR) with transverse tubules (TTs) are fundamental for intracellular Ca2+ cycling in cardiac muscle. For this, TTs extend from the cytoplasmic surface deep into the cell, setting up a complex three-dimensional (3D) membrane network, referred to as ‘transverse axial tubular system’ (TATS) ( Figure 1 A ). The TATS extends over several orders of magnitude in size, e.g. from local TT diameters <100 nm to cell-wide continuous networks expanding over 100 μm, in order to provide thousands of subcellular TT contacts with the ER organelle via junctional SR (jSR) contacts in a typical ventricular myocyte (VM). Not surprisingly, approaches to study the TT membrane structures and properties of the TATS network encompass different imaging and physiological methods. However, until recently ultrastructural TT resolution vs. continuous representation of TATS regions had to be traded against each other. Just imagine representative EM studies (smallest field of view, possible resolution ≤1 nm) when compared with confocal fluorescence microscopy of histological samples (field of view of ∼150 × 150 µm at ∼250 nm resolution using ×63 objectives). Meanwhile this methodological gap is addressed by recent super-resolution fluorescence microscopy approaches (see below).1 Despite apparent gaps between imaging resolution and the nanometric nature of subcellular membrane compartments, conventional fluorescence microscopy is essential to characterize TATS membranes in living samples. In particular, functional imaging studies of isolated cells and two-photon imaging of intact heart tissue have related typical TATS architectures to subcellular functions like Ca2+ release events (sparks) in resting cells (for diastole) or Ca2+ transients …

Spatiotemporal Relationship between Ca2+ Release and Action Potential in Cardiomyocytes Probed by Random Access Microscopy

Action potential, via the transverse axial tubular system (TATS), synchronously triggers uniform Ca2+ release throughout the cardiomyocyte. Cardiac diseases associated with TATS structural remodeling preclude a uniform Ca2+ release across the myocyte, contributing to contractile dysfunction. A simultaneous recording of intracellular local Ca2+ release and action potential in tubular network can be useful to unravel the link between TATS abnormality and dysfunctional EC coupling. Here we combine the advantage of an ultrafast random access multi-photon (RAMP) microscope with a double staining approach to optically record AP in several TATS elements and, simultaneously, the corresponding local Ca2+ transient. Isolated rat cardiomyocytes were labeled with a novel voltage sensitive dye (VSD) and a calcium indicator. RAMP microscope rapidly scans between lines drawn across the TATS of the cardiomyocyte to perform a multiplexed measurement of the two fluorescence signals. Although the calcium and voltage indicators can be excited at the same wavelength, the large Stokes shift of the VSD emission allows us to use spectral unmixing to resolve the voltage and calcium responses. In healthy cardiomyocytes, we found uniform AP propagation within the TATS and homogeneous Ca2+ release throughout the whole cell. The capability of our technique in probing spatiotemporal relationship between Ca2+ and electrical activity was then explored in a model of acute detubulation in which failure to conduct AP in disconnected TATS may cause local delay of Ca2+ transient rise leading to non-homogenous Ca2+ release. The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement 284464.

Action potential propagation in transverse-axial tubular system is impaired in heart failure

The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.

Electrical Behavior At the Transverse-Axial Tubular Membrane of Hypertrophic Cardiomyocytes in Situ

Cardiac hypertrophy is associated with restructuring of the transverse-axial tubular (TAT) system. It is unknown whether such changes are accompanied by altered electrical behavior at the transverse-axial tubules. Here, we used confocal microscopy and the voltage-sensitive dye ANNINE-6plus to visualize the TAT network and to resolve action potentials at the surface and TAT membrane of in situ left ventricular cardiomyocytes within normal and hypertrophic mouse hearts. Transgenic (TG) hearts overexpressing Gαq and non-transgenic (NTG) hearts were perfused in Langendorff-mode during image acquisition. Cardiomyocytes in hypertrophic TG hearts exhibited patchy loss and gain, respectively, of transverse and axial elements. Action potentials were recorded at the TAT membrane within two adjacent cardiomyocytes (denoted T1 and T2) and at the end-to-end junctions (E) between them. Action potential onset occurred synchronously within the TAT network of NTG and TG cardiomyocytes. Whereas the repolarization time course was similar between the three recording sites in NTG or TG hearts (Figure A), overall repolarization was prolonged in TG cardiomyocytes (Figure B). Thus, electrical coupling between the surface and TAT membrane was maintained despite pronounced TAT membrane restructuring in this model of cardiac hypertrophy.View Large Image | View Hi-Res Image | Download PowerPoint Slide