Transverse Tubule System Research Articles

Matriglycan maintains t-tubule structural integrity in cardiac muscle

Maintaining the structure of cardiac membranes and membrane organelles is essential for heart function. A critical cardiac membrane organelle is the transverse tubule system (called the t-tubule system) which is an invagination of the surface membrane. A unique structural characteristic of the cardiac muscle t-tubule system is the extension of the extracellular matrix (ECM) from the surface membrane into the t-tubule lumen. However, the importance of the ECM extending into the cardiac t-tubule lumen is not well understood. Dystroglycan (DG) is an ECM receptor in the surface membrane of many cells, and it is also expressed in t-tubules in cardiac muscle. Extensive posttranslational processing and O-glycosylation are required for DG to bind ECM proteins and the binding is mediated by a glycan structure known as matriglycan. Genetic disruption resulting in defective O-glycosylation of DG results in muscular dystrophy with cardiorespiratory pathophysiology. Here, we show that DG is essential for maintaining cardiac t-tubule structural integrity. Mice with defects in O-glycosylation of DG developed normal t-tubules but were susceptible to stress-induced t-tubule loss or severing that contributed to cardiac dysfunction and disease progression. Finally, we observed similar stress-induced cardiac t-tubule disruption in a cohort of mice that solely lacked matriglycan. Collectively, our data indicate that DG in t-tubules anchors the luminal ECM to the t-tubule membrane via the polysaccharide matriglycan, which is critical to transmitting structural strength of the ECM to the t-tubules and provides resistance to mechanical stress, ultimately preventing disruptions in cardiac t-tubule integrity.

Ligand-grafted nanoparticles as tools to study L-type calcium channel and β-adrenergic receptor function on surface and T-tubule membranes

The cell membrane of adult ventricular myocytes (AVMs) is characterized by many invaginations, constituting the transverse tubule (TT) system. TT membrane (TTM) is continuous with the outer surface membrane (OSM) to allow the rapid electrical coupling during the action potential and represents about 30% of the total cell membrane. Most membrane proteins, such as L-type Ca 2+ channels (LTCC) and β-adrenergic receptors (β- AR) are located in both OSM and TTM, but at different densities. However, how the location of these proteins in OSM vs. TTM impacts on their function remains poorly understood, mainly because of the impossibility to selectively activate or inhibit a protein in one compartment without acting on its counterpart in the other compartment. For example, an important function of the TT system is to provide proximity between LTCCs in TTM and ryanodine receptor type 2 in the sarcoplasmic reticulum membrane. However, the role of their counterparts located in OSM is unknown. The goal of our study is to meet this challenge using an innovative nanotechnology approach. We designed nanoparticles (NPs) with immobilized ligands on their surface in order to prevent their access to TTM. The first step was to graft ligands on 5kDa PEG (polyethylene glycol) to favour ligand flexibility. PEG- and NPs-ligand affinity were determined by radioligand binding assays. PEG- and NPs-ligand potency were evaluated by FRET experiments. Confocal microscopy has been used to visualize the membrane distribution of the fluorescent PEGs and NPs after incubation of AVMs. PEG-ligand affinity and potency are lower than affinity and potency of free ligand. The confocal microscopy assay shows that neither NPs nor PEGs penetrate in the TT network. PEG-ligands seem to act on proteins located on the surface membrane. After this validation, functional experiments will be realized to discriminate the respective role of β-AR and LTCC subpopulations.

A continuum-mechanical skeletal muscle model including actin-titin interaction predicts stable contractions on the descending limb of the force-length relation.

Contractions on the descending limb of the total (active + passive) muscle force—length relationship (i. e. when muscle stiffness is negative) are expected to lead to vast half-sarcomere—length inhomogeneities. This is however not observed in experiments—vast half-sarcomere—length inhomogeneities can be absent in myofibrils contracting in this range, and initial inhomogeneities can even decrease. Here we show that the absence of half-sarcomere—length inhomogeneities can be predicted when considering interactions of the semi-active protein titin with the actin filaments. Including a model of actin—titin interactions within a multi-scale continuum-mechanical model, we demonstrate that stability, accurate forces and nearly homogeneous half-sarcomere lengths can be obtained on the descending limb of the static total force—length relation. This could be a key to durable functioning of the muscle because large local stretches, that might harm, for example, the transverse-tubule system, are avoided.

A novel mechanism of tandem activation of ryanodine receptors by cytosolic and SR luminal Ca2+ during excitation-contraction coupling in atrial myocytes.

In atrial myocytes excitation-contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca2+ release starts in the cell periphery and propagates towards the cell centre by Ca2+ -induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store. The cytosolic Ca2+ sensitivity of the ryanodine receptor (RyRs) Ca2+ release channel is low and it is unclear how Ca2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra-SR calcium revealed a transient elevation of store Ca2+ that we termed 'Ca2+ sensitization signal'. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca2+ through a 'fire-diffuse-uptake-fire' (or FDUF) mechanism: Ca2+ uptake by SR Ca2+ pumps at the propagation front elevates Ca2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca2+ activation threshold. In atrial myocytes Ca2+ release during excitation-contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca2+ store into the peripheral subsarcolemmnal junctional (j-SR) and the much more abundant central non-junctional (nj-SR) SR. Action potential (AP)-induced Ca2+ entry activates Ca2+ -induced Ca2+ release (CICR) from j-SR ryanodine receptor (RyR) Ca2+ release channels. Peripheral elevation of [Ca2+ ]i initiates CICR from nj-SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca2+ ]i ; with the fluorescent Ca2+ indicator rhod-2) and intra-SR ([Ca2+ ]SR ; fluo-5N) Ca2+ in rabbit atrial myocytes revealed that Ca2+ release from j-SR resulted in a cytosolic Ca2+ transient of higher amplitude compared to release from nj-SR; however, the degree of depletion of j-SR [Ca2+ ]SR was smaller than nj-SR [Ca2+ ]SR . Similarly, Ca2+ signals from individual release sites of the j-SR showed a larger cytosolic amplitude (Ca2+ sparks) but smaller depletion (Ca2+ blinks) than release from nj-SR. During AP-induced Ca2+ release the rise of [Ca2+ ]i detected at individual release sites of the nj-SR preceded the depletion of [Ca2+ ]SR , and during this latency period a transient elevation of [Ca2+ ]SR occurred. We propose that Ca2+ release from nj-SR is activated by cytosolic and luminal Ca2+ (tandem RyR activation) via a novel 'fire-diffuse-uptake-fire' (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca2+ uptake by sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) at the propagation front elevates local [Ca2+ ]SR , leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR.

Membrane Cholesterol in Skeletal Muscle: A Novel Player in Excitation-Contraction Coupling and Insulin Resistance.

Membrane cholesterol is critical for signaling processes in a variety of tissues. We will address here current evidence supporting an emerging role of cholesterol on excitation-contraction coupling and glucose transport in skeletal muscle. We have centered our review on the transverse tubule system, a complex network of narrow plasma membrane invaginations that propagate membrane depolarization into the fiber interior and allow nutrient delivery into the fibers. We will discuss current evidence showing that transverse tubule membranes have remarkably high cholesterol levels and we will address how modifications of cholesterol content influence excitation-contraction coupling. In addition, we will discuss how membrane cholesterol levels affect glucose transport by modulating the insertion into the membrane of the main insulin-sensitive glucose transporter GLUT4. Finally, we will address how the increased membrane cholesterol levels displayed by obese animals, which also present insulin resistance, affect these two particular skeletal muscle functions.

Dyssynchronous CA Removal in Atrial Cardiac Myocytes

In heart failure atrial remodeling (AR) leads to impaired contractility, relaxation and atrial fibrillation and contributes to negative clinical outcomes. In myocytes cytosolic Ca-removal after systolic release is primarily achieved by SERCA-pump and Na/Ca-exchange (NCX) activity, allowing atrial relaxation. In atrial myocytes that lack a transverse-tubule system, NCX is located exclusively in the cell periphery where it is exposed to high concentrations of Ca during the cardiac cycle. Previously we found that the relative contribution of NCX to Ca-removal was increased in AR and was associated with a higher propensity of arrhythmogenic Ca waves, i.e. Ca waves triggering action potentials (APs). We hypothesize that dyssynchrony of Ca-removal globally (whole-cell) and locally (peripheral subsarcolemmal (SS) and central (CT) domains) is responsible for impaired relaxation and increased arrhythmogenicity. We used confocal linescan-imaging and Ca sensitive dyes to investigate Ca-removal in remodeled atrial myocytes from a rabbit left ventricular volume-pressure overload systolic HF model. In control atrial myocytes Ca-removal (assessed by the time constant of decay (TAU) of local SS and CT AP-induced Ca transients) is faster in the cell center (CT region) compared to the SS domain (342 ± 32 vs. 393 ± 31 ms, n = 10 cells). In AR myocytes however, CT TAU was not different from SS TAU (196 ± 11 vs. 190 + 14 ms, n = 10 cells). In addition, in AR cells local dyssynchrony (defined as the standard deviation of TAU divided by mean TAU) in the CT domain was significantly increased compared to control (0.11 ± 0.03 vs. 0.04 ± 0.01, n = 10 AR and CTRL cells). However despite the increased dyssynchrony, CT Ca-removal was 50% and SS Ca-removal 56% faster in AR myocytes as compared to control (n = 10 AR and CTRL cells). In summary, AR myocytes show accelerated but dyssynchronous diastolic Ca-removal which may result in impaired relaxation and increased susceptibility to rhythm disorders.

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.

Abstract 16781: Dyssynchronous Ca Removal in Atrial Cardiac Myocytes From Failing Hearts

In heart failure atrial remodeling (AR) leads to impaired contractility and relaxation as well as, atrial fibrillation and contributes to negative clinical outcomes. In myocytes cytosolic Ca removal after systolic release is primarily achieved by SERCA pump and Na/Ca exchange (NCX) activity, allowing atrial relaxation. In atrial myocytes that lack a transverse tubule system, NCX is located exclusively in the cell periphery where it is exposed to high concentrations of Ca during the cardiac cycle. Previously we found that the relative contribution of NCX to Ca removal was increased in AR and was associated with a higher propensity of arrhythmogenic Ca waves, i.e. Ca waves that triggered action potentials (APs). We hypothesize that dyssynchrony of Ca removal globally (whole cell) and locally (peripheral subsarcolemmal (SS) and central (CT) domains) is responsible for impaired relaxation and increased arrhythmogenicity. We used confocal line scan imaging and Ca sensitive dyes (Fluo-4/AM) to investigate Ca removal in remodeled atrial myocytes from a rabbit left ventricular volume-pressure overload systolic heart failure model. In control atrial myocytes Ca removal (assessed by the time constant of decay (TAU) of local SS and CT AP-induced Ca transients) is faster in the cell center (CT region) compared to the SS domain (342±32 vs. 393±31 ms, n=10 cells). In AR myocytes however, CT TAU was not different from SS TAU (196±11 vs. 190+14 ms, n=10 cells). In addition, in AR cells local dyssynchrony (defined as the standard deviation of TAU divided by mean TAU) in the CT domain was significantly increased compared to control (0.11±0.03 vs. 0.04±0.01, n=10 AR and CTRL cells). However despite the increased dyssynchrony, CT Ca removal was 50% and SS Ca removal 56% faster in AR myocytes as compared to control cells (n=10 AR and CTRL cells). In summary, AR myocytes show accelerated but dyssynchronous diastolic Ca removal that may result in impaired relaxation and increased susceptibility to rhythm disorders.

MG29/SYPL2 Contributes to Dysregulation of Lipid Composition and Store Operated Ca2+ Entry in Aging Skeletal Muscle

We proposed over one decade ago that age-related muscle weakness and not the loss of muscle size is the principal contributor to disability. In searching for cellular and molecular explanations for this weakness that exceeds the loss of muscle mass, we found that elements of contractile force generation in skeletal muscle are dependent on store-operated Ca2+ entry (SOCE) and that this capacity is lost in aging skeletal muscle. This altered SOCE is directly associated with reduced content of mitsugumin 29 (MG29), a synaptophysin family protein (SYPL2) containing a MARVEL domain involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the formation of the transverse-tubule system and efficient SOCE, creating a link between reduced MG29 expression in aged muscle and the development of muscle dysfunction in sarcopenia as our previous data demonstrate that aspects of aged skeletal muscle are present in young mg29-/- muscle. Here we test how the loss of MG29 affects the phospholipid composition of skeletal muscle fibers and if producing similar changes in muscle fibers can influence SOCE activity. Lipidomic analysis of young mg29-/- skeletal muscle showed decreased cholesterol content and altered levels of various phospholipid species compared to young control muscle. To simulate the reduced cholesterol content observed in mg29-/- muscle we partially extracted it from mature C2C12 myotubes with methyl-β-cyclodextrin (MCD) and then measured SOCE in these myotubes. Reduced SOCE was observed following MCD extraction, similar to the limited SOCE we previously observed in young mg29-/- and wild-type aged muscle fibers. Further studies will determine if mutation of conserved residues in the MARVEL domain of MG29 modulate SOCE function in, and the lipid composition of, skeletal muscle cells.

Automatic quantitative analysis of t-tubule organization in cardiac myocytes using ImageJ.

The transverse tubule system in mammalian striated muscle is highly organized and contributes to optimal and homogeneous contraction. Diverse pathologies such as heart failure and atrial fibrillation include disorganization of t-tubules and contractile dysfunction. Few tools are available for the quantification of the organization of the t-tubule system. We developed a plugin for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. This plugin (TTorg) analyzes raw confocal microscopy images. Analysis options include the whole image, specific regions of the image (cropping), and z-axis analysis of the same image. Batch analysis of a series of images with identical criteria is also one of the options. There is no need to either reorientate any specimen to the horizontal or to do a thresholding of the image to perform analysis. TTorg includes a synthetic "myocyte-like" image generator to test the plugin's efficiency in the user's own experimental conditions. This plugin was validated on synthetic images for different simulated cell characteristics and acquisition parameters. TTorg was able to detect significant differences between the organization of the t-tubule systems in experimental data of mouse ventricular myocytes isolated from wild-type and dystrophin-deficient mice. TTorg is freely distributed, and its source code is available. It provides a reliable, easy-to-use, automatic, and unbiased measurement of t-tubule organization in a wide variety of experimental conditions.

Methyl‐β‐cyclodextrin increases GLUT4‐mediated glucose transport in skeletal muscle fibers from insulin resistant mice

Insulin stimulates glucose uptake in skeletal muscle fibers by promoting the translocation of Glut4 glucose transporters to the transverse tubule (TT) system. Cholesterol is highly enriched in TT, raising the possibility that modifying TT cholesterol content may affect insulin‐induced glucose transport. By feeding mice a high fat diet (HFD), we increased TT cholesterol content and modified insulin‐induced glucose uptake. Male C57BL/6J mice were fed a normal or a HFD for 8 weeks. Cholesterol levels in triads from HFD‐fed mice were 30% higher than in controls whereas insulin‐stimulated glucose uptake in fibers from HFD‐fed mice was lower than in controls. In FDB muscle, pre‐incubation with low doses of Methyl‐β‐cyclodextrin (MβCD), reported to extract membrane cholesterol, decreased Akt phosphorylation without altering the activity of AMPK, and increased both basal and insulin‐induced glucose uptake in muscle fibers from controls or HFD‐fed mice. MβCD restored insulin sensitivity in HFD‐fed mice, even after Akt or CaMKII inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, inhibited the effect of MβCD. Our findings suggest that TT cholesterol content has a critical role in GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may be a strategy to improve insulin resistance. Supported by ACT1111 and FONDECYT 3110105.

Membrane Receptor Neighborhoods

A provocative new study in the current issue of Circulation Research by Ibarra et al1 suggests that insulin-like growth factor 1 receptors (IGF-1R) exist in plasma membrane invaginations that come in very close proximity to (or even invade) the nuclear membrane in cardiac myocytes to selectively raise local nuclear [Ca2+]. This article raises an intriguing and novel mechanism by which plasma membrane receptors may have preferential local access to nuclear signaling, even in ventricular myocytes which are large cells with central nuclei. Article, see p 236 The IGF-1R is a tyrosine kinase growth factor receptor that connects with many downstream signaling cascades.2 The 2 best-known pathways are Ras-Raf-mitogen-activated protein kinase and phosphatidylinositide 3 kinase-protein kinase B (Akt). However, Ibarra et al1 focus on a less well-studied pathway in which IGF-1 induces Gi-dependent phospholipase C activation3 and 1,4,5-inositol trisphosphate (InsP3) that can release Ca2+ from intracellular InsP3 receptor (InsP3R) stores.4 They show that in cardiac myocytes IGF-1 triggers nuclear Ca2+ transients that precede cytosolic Ca2+ transients, and that blocking the nuclear Ca2+ transient prevents the cytosolic Ca2+ transient, but not vice versa. Their data are consistent with the idea that a substantial fraction of plasma membrane IGF-R1 are in patches of plasma membrane invaginations that are extremely close to the nucleus, potentially as extensions of the transverse tubule (T-tubule) system. Further, they infer that these IGF-1R are Gi-coupled and that upon activation they produce phospholipase C and InsP3-dependent nuclear Ca2+ transients that contributes to Ca-dependent regulation of gene transcription (Figure B). …

Arrhythmogenic coupling between the Na+–Ca2+ exchanger and inositol 1,4,5-triphosphate receptor in rat pulmonary vein cardiomyocytes

Atrial fibrillation, the most common sustained arrhythmia, is believed to be triggered by ectopic electrical activity originating in the myocardial sleeves surrounding the pulmonary veins (PVs). It has been reported that myocardial sleeves have the potential to generate automaticity in response to norepinephrine. This study investigated the cellular mechanisms underlying norepinephrine-induced automaticity in PV cardiomyocytes isolated from rats. Application of 10 μM norepinephrine to PV cardiomyocytes induced repetitive and transient increases in intracellular Ca(2+) concentrations. The Ca(2+) transient was accompanied by depolarization, and induced automatic rhythmic action potentials at approximately 4Hz in perforated patch clamp preparations in 27% of myocytes were observed. When the recording mode was switched from current-clamp to voltage-clamp mode during the continuous presence of automaticity, an oscillatory current was observed. The oscillatory current was always inward, irrespective of the membrane potential, indicating that the current was derived mainly from the Na(+)-Ca(2+) exchanger (NCX). The norepinephrine-induced automaticity was suppressed by blocking either the β(1)- or α(1)-adrenoceptor. Additionally, this automaticity was blocked by inhibitors of phospholipase C and the inositol 1,4,5-triphosphate receptor (IP(3)R) but not by a protein kinase C inhibitor. We observed that the transverse-tubule system was enriched in cardiomyocytes in the PV, in contrast to those of the atrium, and that the NCX and IP(3)R were co-localized along transverse tubules. These findings suggest that a functional coupling between the NCX and IP(3)R causes arrhythmic excitability of the PV during the presence of combined β(1)- and α(1)-adrenoceptor stimulation.

Lactic acid restores skeletal muscle force in an in vitro fatigue model: are voltage-gated chloride channels involved?

High interstitial K(+) concentration ([K(+)]) has been reported to impede normal propagation of electrical impulses along the muscle cell membrane (sarcolemma) and then also into the transverse tubule system; this is one considered underlying mechanism associated with the development of muscle fatigue. Interestingly, the extracellular buildup of lactic acid, once considered an additional cause for muscle fatigue, was recently shown to have force-restoring effects in such conditions. Specifically, it was proposed that elevated lactic acid (and intracellular acidosis) may lead to inhibition of voltage-gated chloride channels, thereby reestablishing better excitability of the muscle cell sarcolemma. In the present study, using an in vitro muscle contractile experimental setup to study functionally viable rectus abdominis muscle preparations obtained from normal swine, we examined the effects of 20 mM lactic acid and 512 μM 9-anthracenecarboxylic acid (9-AC; a voltage-gated chloride channel blocker) on the force recovery of K(+)-depressed (10 mM K(+)) twitch forces. We observed a similar muscle contractile restoration after both treatments. Interestingly, at elevated [K(+)], myotonia (i.e., hyperexcitability or afterdepolarizations), usually present in skeletal muscle with inherent or induced chloride channel dysfunctions, was not observed in the presence of either lactic acid or 9-AC. In part, these data confirm previous studies showing a force-restoring effect of lactic acid in high-[K(+)] conditions. In addition, we observed similar restorative effects of lactic acid and 9-AC, implicating a beneficial mechanism via voltage-gated chloride channel modulation.

3D Imaging of Nanoscopic Membrane Systems Regulating Cardiac Excitation-Contraction Coupling: Multi-Color Optical Super-Resolution and Serial Block-Face Scanning Electron Microscopy

Cardiac excitation contraction (E-C) coupling is controlled by ionic fluxes in couplons, nano-scale junctional structures between sarcolemma and sarcoplasmic reticulum (SR) membranes. The 3-dimensional (3D) geometry of cardiac couplons and their relationship to the transverse-tubule system (t-system) is complex with little 3D data available. Using super-resolution microscopy, we successfully imaged couplons in normal adult mouse ventricular myocytes with a resolution of about 30nm. A fluorescence microscope equipped with an insertable cylindrical lens and an EMCCD camera enabled multi-color 3-dimensional dSTORM imaging and recorded fluorophore “blinking”: rapid transitions between a photon-emitting “on”-state and laser-induced “dark”-state. This data was analyzed with custom-written software to localize individual fluorophores and render super-resolution images. We used ryanodine receptor (RyR) to identify couplons and the sarcolemmal protein Caveolin-3 (CAV3) to visualize the t-system. Separately, we applied an advanced 3D electron microscopic technology, Serial Block-face Scanning Electron microscopy (SBFSEM), to cardiac muscle for the first time to image, segment, and annotate couplons and the t-system in mouse cardiac myocytes. Expectedly, the majority of RyR clusters aggregated near the t-system. The individual morphology and size of couplons visualized with dSTORM and SBFSEM were both highly variable, in agreement with our recent publications. Furthermore, both microscopies strongly suggested a significant enlargement in diameter of the t-system at loci where couplons accumulated. This enlargement may contribute to minimizing ion accumulation or depletion. Alternatively, it could reflect a geometrical requirement for forming larger junctional complexes. Collectively, the study revealed the strength of integrating new advanced 3D light and electron microscopies. Knowledge of nanoscale anatomy may be critical both to mechanistic understanding of E-C coupling and as a resource to generate realistic computational models that can simulate E-C coupling and other muscle biology.

A shortcut to a skeletal muscle DHPR knock‐in?

A shortcut to a skeletal muscle DHPR knock‐in?

T-Tubule Remodeling During Transition From Hypertrophy to Heart Failure

The transverse tubule (T-tubule) system is the ultrastructural substrate for excitation-contraction coupling in ventricular myocytes; T-tubule disorganization and loss are linked to decreased contractility in end stage heart failure (HF). We sought to examine (1) whether pathological T-tubule remodeling occurs early in compensated hypertrophy and, if so, how it evolves during the transition from hypertrophy to HF; and (2) the role of junctophilin-2 in T-tubule remodeling. We investigated T-tubule remodeling in relation to ventricular function during HF progression using state-of-the-art confocal imaging of T-tubules in intact hearts, using a thoracic aortic banding rat HF model. We developed a quantitative T-tubule power (TT(power)) index to represent the integrity of T-tubule structure. We found that discrete local loss and global reorganization of the T-tubule system (leftward shift of TT(power) histogram) started early in compensated hypertrophy in left ventricular (LV) myocytes, before LV dysfunction, as detected by echocardiography. With progression from compensated hypertrophy to early and late HF, T-tubule remodeling spread from the LV to the right ventricle, and TT(power) histograms of both ventricles gradually shifted leftward. The mean LV TT(power) showed a strong correlation with ejection fraction and heart weight to body weight ratio. Over the progression to HF, we observed a gradual reduction in the expression of a junctophilin protein (JP-2) implicated in the formation of T-tubule/sarcoplasmic reticulum junctions. Furthermore, we found that JP-2 knockdown by gene silencing reduced T-tubule structure integrity in cultured adult ventricular myocytes. T-tubule remodeling in response to thoracic aortic banding stress begins before echocardiographically detectable LV dysfunction and progresses over the development of overt structural heart disease. LV T-tubule remodeling is closely associated with the severity of cardiac hypertrophy and predicts LV function. Thus, T-tubule remodeling may constitute a key mechanism underlying the transition from compensated hypertrophy to HF.

Effect of supplemental vitamin D3 concentration on concentrations of calcium, phosphorus, and magnesium relative to protein in subcellular components of the longissimus and the distribution of calcium within longissimus muscle of beef steers.

The effect of supplementing diets with various levels of vitamin D3 to provide 0, 0.5, 1, and 5 million IU/(steer x d) for 8 d before slaughter on the mineral content and localization of Ca in LM and muscle fragments was studied during the postmortem aging process. Twelve feedlot steers of three biological types were given access to the four levels of vitamin D for 8 d before slaughter. Differential centrifugation techniques were used to determine the concentrations of minerals relative to protein in different muscle fragments on d 3 and 21 postmortem. Electron microscopy visualization of bound Ca indicated that vitamin D3 mobilized Ca from the sarcoplasmic reticulum and transverse tubule system into the myofibrils. Bound Ca was concentrated near the Z-line at the A-band/I-band juncture within the sarcomere. Supplementing steers with 1 and 5 million IU/(steer x d) of vitamin D3 increased (P < 0.05) Ca, P, and Mg concentrations per unit of protein in the cytosol. Soluble cytosolic Ca concentrations were greater (P < 0.05) on d 21 than on d 3 postmortem only when steers were supplemented with 5 million IU/d. Concentrations of Ca, P, and Mg in isolated tissues were increased (P < 0.05) in nuclei and myofibrilar proteins by supplementing steers with 1 and 5 million IU/ (steer x d) of vitamin D3. All supplemental vitamin D3 treatments also increased (P < 0.001) Mg concentrations in the cytosol, regardless of aging treatment, and increased Mg concentrations (P < 0.04) within the mitochondria at d 3 postmortem. Thus, supplementation of feedlot steers with vitamin D3 at levels of 0.5 to 5 million IU/(steer x d) increased Ca concentrations within respiring muscle, resulting in increased bound tissue Ca concentrations. When the respiring muscle was converted to meat, the increased bound tissue Ca resulting from vitamin D3 treatment released Ca concentrations into the cytosol during aging (P < 0.05). Results of this study indicate that vitamin D3 supplementation increased total cytosolic Ca, P, and Mg concentrations in meat.

T-tubule localization of the inward-rectifier K(+) channel in mouse ventricular myocytes: a role in K(+) accumulation.

1. The properties of the slow inward 'tail currents' (I(tail)) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K(+) currents in these myocytes were followed by a slowly decaying inward I(tail) on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K(+) currents, I(K1), produced by steps to potentials negative to the holding potential, were not followed by I(tail). 2. For depolarizations of equal duration, the magnitude of I(tail) increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of I(tail) was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased. 3. Removal of external Na(+) and Ca(2+) had no significant effect on the magnitude or time course of I(tail). BaCl(2) (0.25 mM), which had no effect on the magnitude of outward currents, abolished I(tail) and I(K1) simultaneously. 4. Accordingly, I(tail) in mouse ventricular myocytes probably results from K(+) accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K(+) into the t-tubules during outward currents produced by depolarization shifts the K(+) Nernst potential (E(K)) from its 'resting' value (close to -80 mV) to more depolarized potentials. This suggests that I(tail) is produced by I(K1) in the t-tubules and is inward because of the transiently elevated K(+) concentration and depolarized value of E(K) in the t-tubules. 5. Additional evidence for the localization of I(K1) channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes.

RyR3 Amplifies RyR1-mediated Ca2+-induced Ca2+ Release in Neonatal Mammalian Skeletal Muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.