T-tubules Research Articles

Early transverse tubule development begins in utero in the sheep heart.

The ventricular cardiomyocytes of adult mammals contain invaginations of the plasma membrane known as transverse (t)-tubules. These regular structures are essential for the synchronisation of excitation-contraction (EC) coupling throughout the cell, which is a vital process for cardiac function. T-tubules form a close association with the sarcoplasmic reticulum (SR) to form junctions, where several key proteins involved in EC coupling are localised, including the SR calcium release channels-the ryanodine receptors (RyR). The lipophilic SR protein junctophilin-2 (JPH2) has been implicated in the development of both the junctions and t-tubules. Several studies have identified that t-tubules develop only postnatally in rodents, while historical electron microscopy data indicate that this is not the case in larger mammals, including humans. We have performed, to our knowledge, the first fluorescent, target-specific study to characterise t-tubule development in the large mammalian fetal heart, focussing on the sheep. T-tubules were present in fetal sheep hearts from 114days gestation (with term being 145days), with occurrence progressively increasing with gestational age, and further maturation after birth. This was accompanied by an increasing intracellular localisation of JPH2, which progressively increased its association with RyR within the cardiomyocytes as they undergo hypertrophy. These findings indicate that large mammalian hearts exhibit a significantly different temporal pattern of development compared to that of the rodent. Our findings have potential implications for human cardiac development, including the future investigation of congenital heart disease.

Phosphatidylinositol 3-kinase inhibition restores Ca2+ release defects and prolongs survival in myotubularin-deficient mice

Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation-contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear. During normal skeletal muscle EC coupling, transverse (t) tubule depolarization triggers sarcoplasmic reticulum (SR) Ca2+ release through ryanodine receptor channels gated by conformational coupling with the t-tubule voltage-sensing dihydropyridine receptors. We report that MTM1 deficiency is associated with a 60% depression of global SR Ca2+ release over the full range of voltage sensitivity of EC coupling. SR Ca2+ release in the diseased fibers is also slower than in normal fibers, or delayed following voltage activation, consistent with the contribution of Ca2+-gated ryanodine receptors to EC coupling. In addition, we found that SR Ca2+ release is spatially heterogeneous within myotubularin-deficient muscle fibers, with focally defective areas recapitulating the global alterations. Importantly, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity rescues the Ca2+ release defects in isolated muscle fibers and increases the lifespan and mobility of XLMTM mice, providing proof of concept for the use of PtdIns 3-kinase inhibitors in myotubular myopathy and suggesting that unbalanced PtdIns 3-kinase activity plays a critical role in the pathological process.

Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology

During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striated muscle utilizes alternative splicing to generate specific isoforms required for function of adult tissue. We deliver morpholinos into flexor digitorum brevis muscles in adult mice to redirect splicing of four trafficking genes to the fetal isoforms. The splicing switch results in multiple structural and functional defects, including transverse tubule (T-tubule) disruption and dihydropyridine receptor alpha (DHPR) and Ryr1 mislocalization, impairing excitation-contraction coupling, calcium handling, and force generation. The results demonstrate a previously unrecognized role for trafficking functions in adult muscle tissue homeostasis and a specific requirement for the adult splice variants.

Dyssynchronous calcium removal in heart failure-induced atrial remodeling.

We tested the hypothesis that in atrial myocytes from a rabbit left ventricular heart failure (HF) model, spatial inhomogeneity and temporal dyssynchrony of Ca removal during excitation-contraction coupling together with increased Na/Ca exchange (NCX) activity generate a substrate for proarrhythmic Ca release. Ca removal occurs via Ca reuptake into the sarcoplasmic reticulum and extrusion via NCX exclusively in the cell periphery since rabbit atrial myocytes lack transverse tubules. Ca removal kinetics were assessed by the time constant τ of decay of local peripheral subsarcolemmal (SS) and central (CT) action potential (AP)-induced Ca transients (CaTs) recorded in confocal line scan mode (using Fluo-4). Spatial and temporal dyssynchrony of Ca removal was quantified by CV TAU, defined as the standard deviation of local τ along the transverse cell axis divided by mean τ. In normal cells CT CaT decline was slower compared with the SS domain, while in HF cells decline was accelerated, became equal in SS and CT regions, and a significant increase of CV TAU indicated an increased Ca removal dyssynchrony. In HF atrial cells NCX upregulation was accompanied by an overall higher incidence of spontaneous Ca waves and a higher propensity of arrhythmogenic Ca waves, defined as waves that triggered APs due to NCX-mediated membrane depolarization. NCX inhibition normalized CV TAU in HF atrial cells and decreased the propensity of Ca waves. In summary, HF atrial myocytes show accelerated but dyssynchronous diastolic Ca removal and altered sarcoplasmic reticulum Ca-ATPase (SERCA) and NCX activity that result in increased susceptibility to arrhythmia.

Regional distribution of T-tubule density in left and right atria in dogs

The peculiarities of transverse tubule (T-tubule) morphology and distribution in the atrium-and how they contribute to excitation-contraction coupling-are just beginning to be understood. The objectives of this study were to determine T-tubule density in the intact, live right and left atria in a large animal and to determine intraregional differences in T-tubule organization within each atrium. Using confocal microscopy, T-tubules were imaged in both atria in intact, Langendorf-perfused normal dog hearts loaded with di-4-ANEPPS. T-tubules were imaged in large populations of myocytes from the endocardial surface of each atrium. Computerized data analysis was performed using a new MatLab (Mathworks, Natick, MA) routine, AutoTT. There was a large percentage of myocytes that had no T-tubules in both atria with a higher percentage in the right atrium (25.1%) than in the left atrium (12.5%) (P < .02). The density of transverse and longitudinal T-tubule elements was low in cells that did contain T-tubules, but there were no significant differences in density between the left atrial appendage, the pulmonary vein-posterior left atrium, the right atrial appendage, and the right atrial free wall. In contrast, there were significant differences in sarcomere spacing and cell width between different regions of the atria. There is a sparse T-tubule network in atrial myocytes throughout both dog atria, with significant numbers of myocytes in both atria-the right atrium more so than the left atrium-having no T-tubules at all. These regional differences in T-tubule distribution, along with differences in cell width and sarcomere spacing, may have implications for the emergence of substrate for atrial fibrillation.

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.

Recent advances in understanding cardiac contractility in health and disease.

The aim of this review is to provide the reader with a synopsis of some of the emerging ideas and experimental findings in cardiac physiology and pathophysiology that were published in 2015. To provide context for the non-specialist, a brief summary of cardiac contraction and calcium (Ca) regulation in the heart in health and disease is provided. Thereafter, some recently published articles are introduced that indicate the current thinking on (1) the Ca regulatory pathways modulated by Ca/calmodulin-dependent protein kinase II, (2) the potential influences of nitrosylation by nitric oxide or S-nitrosated proteins, (3) newly observed effects of reactive oxygen species (ROS) on contraction and Ca regulation following myocardial infarction and a possible link with changes in mitochondrial Ca, and (4) the effects of some of these signaling pathways on late Na current and pro-arrhythmic afterdepolarizations as well as the effects of transverse tubule disturbances.

Excitation contraction uncoupling by high intracellular [Ca2+] in frog skeletal muscle: a voltage clamp study.

Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]i intracellular solution, permeabilization of the plasma membrane by a Ca2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2±1.24 to 9.9±0.94 nC/μF meanwhile the Ca2+ release flux was reduced from 13.5+0.7 to 2.2±0.3μM/ms (n=33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40% of the uncoupled cells we observed that the [Ca2+]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.

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.

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.

The T-System Provides a Dynamic Ca2+-Buffer in Human Skeletal Muscle Fibres

The tubular (t-) of skeletal muscle fibres is known to be able to vacuolate in response to osmotic stress, stretch and fatigue. We trapped rhod-5N in the t-system of skinned fibres to image changes in t-system structure and [Ca2+] in human muscle fibres isolated from biopsies on a confocal microscope. Imaging showed variation in numbers and volume of vacuoles, from none to virtually totally obscuring the transverse tubules. To observe Ca2+ movements between the t-system and cytoplasm, Ca2+sup was rapidly released from the sarcoplasmic reticulum and this was followed by introduction of 28-1342nM [Ca2+]. Once formed, vacuoles persisted through Ca2+ load and release cycles. While Ca2+ release depleted transverse tubules via SOCE, vacuoles took up Ca2+ which slowly diffused out. However, if vacuoles were exposed to high [Ca2+]cyto or Ca2+ release from a heavily loaded SR, chronically activated SOCE did not deplete Ca2+ from vacuoles. Many minutes of bathing in a solution containing no Ca2+ was required for vacuoles to begin to decrease in size. Upon application of 5mM Ca2+ and ionophore, vacuoles could show a many-fold increase in volume and thus Ca2+-holding capacity. Subsequent application of 0 Ca2+ caused vacuoles to deform in seconds. A further addition of 5 mM Ca2+ saw the transverse tubules reappear by filling with Ca2+. We conclude that vacuoles are able to take up and hold mM Ca2+ and can deform as Ca2+ is lost. This suggests that vacuoles can dynamically buffer Ca2+ in skeletal muscle fibres.

Sheet-Like Remodeling of the T-System of Ventricular Cardiomyocytes in Heart Failure

The transverse tubular system (t-system) is a membrane specialization of striated myocytes crucial for excitation-contraction coupling. Various studies demonstrated remodeling of t-system in heart disease in species including rodent, canine and human. Commonly, disease was associated with heterogeneous t-system depletion. Here, we test the hypothesis that t-system remodeling underlies inefficient excitation-contraction coupling in human heart failure.We investigated the t-system in left-ventricular myocardium samples from 5 patients undergoing cardiac surgery. The tissues were labeled for ryanodine receptors (RyRs), and the extracellular space including the t-system. We acquired 3D images with a Leica SP8 confocal microscope using a 63x oil immersion lens. After image deconvolution, we segmented individual cardiomyocytes. We reconstructed the t-system and quantified the RyR intensity and distance of RyR clusters to the t-system. Furthermore, isolated myocytes were labeled with di-8-ANEPPS and Fluo-4 to acquire 2D image sequences of calcium transients using a Zeiss Live 5 Duo confocal microscope.Our studies revealed a novel type of t-system remodeling, characterized by flat sheets rather than tubular structures. Three-dimensional reconstructions indicated that sheet cavities were connected with the extracellular space. Principal component analysis showed that sheets were aligned with the longitudinal myocyte axis. One sample did not show sheets and served as control. Comparing 5 cells with sheets to 5 control cells, we found significantly increased distances between RyR clusters and the t-system (0.82±0.12µm and 0.5±0.06µm, respectively) and a significantly higher fraction of RyRs present >1µm from the sarcolemma (49±6.9% and 21±5.5%, respectively). Cardiomyocytes with sheets exhibited spatial heterogeneity and asynchrony of calcium transients.Our studies present a novel phenotype of remodeled t-system, with a strikingly increased number of non-junctional RyR clusters. We suggest that the associated spatially heterogeneous and asynchronous calcium release contributes to reduced contractility in heart failure.

Axial Membrane Tubules in Atrial Cardiomyocytes Confine Ultrarapid Intracellular Calcium Signals through a New Super-Hub Mechanism

Endocytic membrane structures called transverse tubules (TTs) are thought to represent the main mechanism for contacts with the junctional sarcoplasmic reticulum (jSR) and intracellular Ca2+ release in ventricular cardiomyocytes (VCM). In contrast, in atrial cardiomyocytes (ACM) we identify previously not described large axial membrane tubules (ATs) at the cell core. Using optimized ACM isolation and live cell superresolution microscopy, we show that AT membrane structures represent both the major (ie. >90% of endocytic tubule structures) and largest tubule component (mean AT circumference 921±24 nm). Due to the unusually large AT membrane surface with a positive curvature exposed to the cytoplasm, we hypothesized that the endocytic AT membrane surface functions as central-axial Ca2+ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined imaging of AT membranes with fast intracellular Ca2+ imaging. Indeed, high-amplitude Ca2+ transients originated in a highly localized fashion rapidly from the AT-hubs. Importantly, Ca2+ transients originating from AT-hubs occured significantly earlier compared to cellular surface locations (p<0.05). This suggests a new EC-coupling model where AT-hubs control rapid centrifugal Ca2+ signals. Sarcomere shortening confirmed significantly faster ACM versus VCM EC-coupling (p<0.05). Furthermore, in situ phospho-epitope imaging correlated ryanodine receptor (RyR2) channel phosphorylation with AT-hub function. Only junctional RyR2 clusters at AT-hubs showed increased phosphorylation compared to non-junctional clusters. Genetic ablation of RyR2-specific phosphoepitopes by Ala-knockin impaired ACM contractility, consistent with loss-of-function and disruption of AT-hub-dependent signaling. Hence, our data support a fundamentally new ACM model of EC-coupling, where AT-membranes function as signaling super-hubs that control high-amplitude ultrarapid Ca2+ signals at the cell core. Genetic ablation of RyR2 cluster phosphorylation further suggests that the super-hub model is relevant to explain atrial function in health and decreased contractility typical for atrial disease forms.

Effects of Beta-Adrenergic Stimulation on Rat Failing Cardiomyocytes

Beta-adrenoceptors start the most powerful signaling pathway for the sympathetic control of the cardiac function. Human and animal models of heart failure (HF), one of the most important causes of mortality worldwide, show alterations of the sympathetic system. Number and function of beta-adrenoceptors, namely the beta-1 subtype, are impaired in HF, representing a major (mal)adaptive feature of myocardial remodeling. Moreover, HF is characterized by structural and functional remodeling of T-tubules (TT). We previously observed failure of action potential conduction in about 6% of HF TTs accompanied by profound alterations of local Ca2+ transient (Crocini et al. PNAS 2014). Here, we employ an ultrafast random access multi-photon (RAMP) microscope to assess the effect of beta-adrenoceptor activation on Ca2+ release in electrically coupled and uncoupled tubular elements. We find that HF cells exhibit a significantly higher Ca2+ sparks frequency compared to controls that are not affected by beta-receptor activation (10-7 M isoproterenol). Control cardiomyocytes treated with isoproterenol show instead a more than 3-fold increase of Ca2+ sparks frequency. To better examine this difference, we assess beta-adrenergic signaling role on Ca2+ release. We observe an accelerated Ca2+ rise exclusively in the vicinity of electrically coupled TT, while Ca2+ rise close to failing elements is unchanged in beta-adrenergic stimulated cells. This finding indicates a patchy response of HF cells to beta-adrenergic stimulation. On the contrary, isoproterenol reduces the beat-to-beat variability of Ca2+ release time to peak at every membrane site of HF cardiomyocytes. Simultaneous recording of action potential and Ca2+ transient allows us to probe the effect of beta-adrenergic stimulation at subcellular level, disclosing specific local effects that may help understanding the changes of beta-adrenergic signaling in HF.

Relationship between Multi-Scale Cardiomyocyte Organization and Function in Trabeculae of the Failing Human Heart

Invaginations of the plasma membrane within cardiomyocytes form the transverse tubule network (t-tubules), and at “dyadic junctions” these come into close association with regions of the sarcoplasmic reticulum (SR). In addition to many of the key excitation-contraction (EC) coupling proteins, junctions contain junctophilin-2 (JPH2), which is implicated in both the formation and maintenance of junctions. In human heart failure there is a loss of cardiac function which is generally associated with impairment of EC coupling processes, as well as changes to t-tubule structure and protein distribution. The aim of this study was to investigate the link between structural re-organization of cardiomyocytes and the loss of contractile function in trabeculae from failing human hearts.We obtained trabeculae from explanted human hearts in end-stage heart failure and performed force measurements followed by immuno-labelling to determine correlations between protein distribution and functional performance in the failing human heart. Cardiac trabeculae were processed along with samples taken from the free wall to investigate the variability of structure and protein distribution, including t-tubules, JPH2 and ryanodine receptors (the SR calcium release channel) using confocal imaging. Findings from these experiments revealed that there is a high degree of variability of trabecular structure and function, both within and between hearts. Trabeculae exhibiting poor contractile function often had low cardiomyocyte content and remaining myocytes showed disorganization of t-tubules and EC coupling proteins.These findings demonstrate a clear link between trabecula structure and function in the diseased human heart and support the idea that cardiomyocyte organization at cell and tissue levels is correlated with cardiac function. Furthermore, while trabeculae are useful for studying cardiac function, trabecula and cardiomyocyte structure must be considered when interpreting functional results.

Ryanodine Receptor Sensitivity Governs the Stability and Synchrony of Local Calcium Release during Cardiac Excitation-Contraction Coupling

Synchronous coordination of calcium-induced calcium release (CICR) across thousands of calcium release units (CRUs) within the cardiomyocyte is critical for normal excitation-contraction coupling (ECC). Here we utilize a novel “local control” mathematical model of ECC, designed to simulate a mouse cardiomyocyte, to investigate physiological and pathophysiological calcium (Ca2+) signaling. The model contains 20,000 independent CRUs each composed of 6 stochastically gated L-type Ca2+ channels (LCCs) in the transverse tubule membrane that are positioned across a local “dyadic subspace” from a junctional sarcoplasmic reticulum (JSR) compartment containing a cluster of 50 stochastically gated ryanodine receptors (RyR2s). Action potential depolarization of the sarcolemmal membrane activates LCC openings which elevate subspace [Ca2+] ([Ca2+]ds) across each of the cell's 20,000 CRUs. This rise in [Ca2+]ds promotes RyR2 opening to initiate the fundamental element of triggered Ca2+ release, the Ca2+ spark. The mechanistic design and true SR Ca2+ pump/leak balance displayed by our model allows us to quantify ECC fidelity and Ca2+ spark fidelity (the probability that a LCC opening or RyR2 opening induces a Ca2+ spark, respectively). We study the effect of excess SR Ca2+ leak in the context of “catecholaminergic polymorphic ventricular tachycardia (CPVT)” caused by the R33Q mutation in calsequestrin (CASQ2). CPVT is simulated by increasing RyR2 sensitivity to [Ca2+]i and reducing JSR Ca2+ buffering capacity to levels identified by prior studies of the CASQ2-R33Q “knock-in” mice. Our model effectively reproduces both normal and arrhythmogenic CPVT RyR2 [Ca2+]i sensitivity, local Ca2+ spark activity, and global Ca2+ signals. Under CPVT conditions enhanced RyR2 open probability is critical for the development of “unstable”, quiescent Ca2+ sparks that fail to terminate robustly as well as asynchronous systolic Ca2+ spark activity and increased diastolic SR Ca2+ leak.

Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation.

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.

Evidence of iguanodontian dinosaurs from the Lower Cretaceous of Tunisia

The fossil record of ornithischian dinosaurs from Africa is particularly scarce and limited to a few historic localities. In this study we describe new ornithischian remains from the Albian deposits of southern Tunisia (Tataouine Governorate), represented by isolated teeth of large-bodied iguanodontians. Teeth display a wide, diamond-shaped crown with a primary ridge dividing the occlusal surface in two unequal parts and two or more secondary ridges. Hook-like denticles are present on both mesial and distal crown margins and do not display mammillae. In overall morphology, specimens are comparable to those of many Early Cretaceous basal hadrosauriforms, including isolated ornithopodan teeth from comparably-aged levels of Niger. Transversal sections of the crowns permitted identification of dental tissues, which include a thick enamel, and well developed longitudinal and transverse giant tubules. Their relative extents appear to be related to the size, thus developmental age, of the tooth. Teeth are representative of the Oum ed Diab Member, a unit characterized by coastal deposits accumulated under arid to xeric climatic conditions and dominated by fish, crocodilians, and hydraulically transported rebbachisaurid and spinosaurid remains. Sedimentological data and preservation bias strongly support selective taphonomic causes for the fossil distribution of ornithischians in southern Tunisia questioning the purported geographic and paleoecologic distribution of isolated Saharan dinosaurs.

Detachment of surface membrane invagination systems by cationic amphiphilic drugs.

Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulin inhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs also caused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of human platelets. Anionic amphiphiles, U73343 (a less electrophilic U73122 analogue) and a range of kinase inhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cell membrane where they bind to anionic lipids, especially PI(4,5)P2. We therefore propose that surface detachment of membrane invaginations results from an ability of CADs to interfere with PI(4,5)P2 interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a large class of pharmaceutical compounds.

Paul Fatt. 13 January 1924 — 28 September 2014

Paul Fatt made discoveries that are fundamental to our understanding of synaptic transmission in the nervous system. He grew up in the USA and saw service in World War II, but came to London in 1948 as a research student supported by the GI Bill. His seminal work with Bernard Katz at University College London (UCL), John Eccles in Canberra, and Bernard Ginsborg at UCL was carried out during an intense period between 1950 and 1960. His work with Katz demonstrated for the first time that neurotransmitter is released in small packets, or ‘quanta’. His work with Eccles (and Katz) provided an understanding of the mechanism underlying synaptic inhibition, and his work with Ginsborg identified voltage-gated calcium currents for the first time. Furthermore, in the early 1960s his electrical measurements of the muscle transverse tubule system contributed to the early models of excitation–contraction coupling in muscle. The final period of his research career was spent working on phototransduction in the visual system.