Transverse-axial Tubular System Research Articles

TRPC3 channels colocalize with Na+/Ca2+exchanger and Na+pump in axial component of transverse-axial tubular system of rat ventricle

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na(+)-K(+)-ATPase (NKA) pump and the Na(+)/Ca(2+) exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca(2+)-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.

The functional role of cardiac T-tubules explored in a model of rat ventricular myocytes

The morphology of the cardiac transverse-axial tubular system (TATS) has been known for decades, but its function has received little attention. To explore the possible role of this system in the physiological modulation of electrical and contractile activity, we have developed a mathematical model of rat ventricular cardiomyocytes in which the TATS is described as a single compartment. The geometrical characteristics of the TATS, the biophysical characteristics of ion transporters and their distribution between surface and tubular membranes were based on available experimental data. Biophysically realistic values of mean access resistance to the tubular lumen and time constants for ion exchange with the bulk extracellular solution were included. The fraction of membrane in the TATS was set to 56%. The action potentials initiated in current-clamp mode are accompanied by transient K+ accumulation and transient Ca2+ depletion in the TATS lumen. The amplitude of these changes relative to external ion concentrations was studied at steady-state stimulation frequencies of 1-5 Hz. Ca2+ depletion increased from 7 to 13.1% with stimulation frequency, while K+ accumulation decreased from 4.1 to 2.7%. These ionic changes (particularly Ca2+ depletion) implicated significant decrease of intracellular Ca2+ load at frequencies natural for rat heart.

Aqueous Diffusion Pathways as a Part of the Ventricular Cell Ultrastructure

The physical organization of the ventricular myocyte includes barriers for the movement of objects of varying dimensions ranging from ions to solid particles. There are two kinds of diffusion in the cell: lateral (in membranes) and aqueous. Here we examine the size constraints of aqueous diffusion pathways and discuss their impact on cellular physiology. Calibrated gold nanoparticles were used to probe the accessibility of the entire transverse-axial tubular system (TATS), the sarcoplasm, and intracellular structures. The TATS tubules, although up to 300nm in diameter, permitted only particles ≤11nm to enter. When calibrated nanoparticles were added to permeabilized ventricular cells, particles ≤11nm were found in the sarcoplasm. The distribution of nanoparticles in the cells allowed us to conclude that 1), the TATS and the sarcoplasm are accessible only for particles ≤11nm; 2), the gaps between T-tubules and junctional sarcoplasmic reticulum (jSR), jSR and mitochondria, and intermitochondrial contacts are inaccessible for particles with physical size >3nm; 3), the mitochondrial voltage-dependent anion channel and the nuclear pore complex in ventricular cells could not be penetrated by particles ≥6nm; and 4), there is a difference in size clearance between transversal and longitudinal sarcoplasmic diffusional pathways.

Role of the transverse-axial tubule system in generating calcium sparks and calcium transients in rat atrial myocytes.

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse-axial tubular system (TATS). To investigate the possible role of the TATS in excitation-contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di-8-ANEPPS) simultaneously with Ca2+ transients and/or Ca2+ sparks (fluo-4). In confocal transverse linescan images of field-stimulated cells, whole-cell Ca2+ transients had two morphologies: 'U-shaped' transients and irregular or 'W-shaped' transients with a varying number of points of origin of the Ca2+ transient. About half (54 %, n =289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 +/- 2.8 microm) than cells without TATS (11.7 +/- 2.0 microm) and were more common in the left atrium (n = 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca2+ sparks and sarcolemmal structures showed that cells without TATS had U-shaped transients that started at the cell periphery, and cells with TATS had W-shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) 'spontaneous' (non-depolarized) Ca2+ sparks occurred within 1 microm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 microm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca2+ transient and for spontaneous Ca2+ sparks.

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

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

A treadmill exercise regimen for identifying cardiovascular phenotypes in transgenic mice.

Cardiovascular stress in response to treadmill exercise is frequently used to detect cardiac abnormalities that are not readily apparent at rest. Herein we describe a treadmill exercise protocol for mice that allows for quantitation of the performance of an animal and the ability to gather metabolic information in a nonrestraining manner using telemetry implant devices. Transgenic (TG) mice overexpressing ventricular myosin regulatory light chain (MLC2v) were subjected to a 5-wk exercise regimen. The TG mice had significant decreases in their capacity for exercise at relatively high treadmill speeds compared with their nontransgenic (NTG) littermates. There was no indication of a hypertrophic response occurring in TG or NTG animals in response to the exercise protocol, and exercise had no effect on MLC2v phosphorylation. Ultrastructural examination of TG atria showed overtly normal myofibrillar organization but a proliferation of the transverse-axial tubular system. This exercise protocol should prove useful in detecting subtle phenotypes that occur in mice as a result of genetic manipulation of the cardiac compartment.

Transverse-axial tubular system in guinea pig ventricular cardiomyocyte: 3D reconstruction, quantification and its possible role in K + accumulation-depletion phenomenon in single cells

K + accumulation-depletion (AD) phenomena were found in single guinea pig ventricular myocytes using the patch-clamp method in whole cell configuration. We suggest that the cardiomyocyte transverse-axial tubular system (TATS) lumen is the restricted extracellular space where the K + AD could take place. A three-dimensional (3D) reconstruction of the TATS in a cardiomyocyte segment from serial ultrafine sections was made by three-dimensional isosurface rendering and quantitative data were obtained from the image processing. This original approach of the TATS intricated network gave a new vision of this membrane system; moreover, quantitative data about the tubular membrane importance (52.6% of the total plasma membrane) and its surface area versus the tubular volume fraction ( S TATS V TATS = 13.5 μ m 2 /μ m 3 ) would fit in the electrophysiological results. The hypothesis whereby this ‘extracellular’ compartment could play, in single cells, a role as important as that of narrow clefts in the whole heart is discussed.

Transverse‐axial tubular system in guinea pig ventricular cardiomyocyte: 3D reconstruction, quantification and its possible role in K+ accumulation‐depletion phenomenon in single cells

K+ accumulation-depletion (AD) phenomena were found in single guinea pig ventricular myocytes using the patch-clamp method in whole cell configuration. We suggest that the cardiomyocyte transverse-axial tubular system (TATS) lumen is the restricted extracellular space where the K+ AD could take place. A three-dimensional (3D) reconstruction of the TATS in a cardiomyocyte segment from serial ultrafine sections was made by three-dimensional isosurface rendering and quantitative data were obtained from the image processing. This original approach of the TATS intricated network gave a new vision of this membrane system; moreover, quantitative data about the tubular membrane importance (52.6% of the total plasma membrane) and its surface area versus the tubular volume fraction (STATS/VTATS = 13.5 micron 2/micron 3) would fit in the electrophysiological results. The hypothesis whereby this 'extracellular' compartment could play, in single cells, a role as important as that of narrow clefts in the whole heart is discussed.

Ultra-high-resolution scanning electron microscopic studies on the sarcoplasmic reticulum and mitochondria of the rat intrafusal muscle fibers. Part II: The extracapsular region.

The three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum (SR) and mitochondria in the extracapsular region of the intrafusal muscle fibers of the rat lumbrical muscle was observed by ultra-high-resolution scanning electron microscopy after removal of the cytoplasmic matrix by the osmium-DMSO-osmium procedure. Nuclear chain fibers possessed thick to moderately sized column-forming mitochondria with occasional I-band branches. The transverse tubules (T-tubules) running at the level of the A-band side of the A-I junction were sandwiched between two large terminal cisternae for most of their length, forming triads. The sarcotubules arising from the terminal cisternae formed single-layered networks at the A-band level, and well developed, double-layered, three-dimensional networks at the I-band level. Nuclear bag1 fibers possessed slender column-forming mitochondria devoid of I-band branches. The T-tubules were located at the level of the A-I junction. Both the T- and the axial-tubules were occasionally coupled with small terminal cisternae, forming dyads or triads. At the I-band level, the SR was well developed and formed single layered networks, but at the A-band level only a few longitudinally arranged sarcotubules and axial tubules were observed. Nuclear bag2 fibers contained medium to large diameter column-forming mitochondria without I-band branches. The T-tubules ran at the level of the A-band adjacent to the A-I junction, and were coupled at some intervals with terminal cisternae of various lengths. Swollen, large terminal cisternae were frequently seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultra-High-Resolution Scanning Electron Microscopic Studies on the Sarcoplasmic Reticulum and Mitochondria of the Rat Intrafusal Muscle Fibers. Part I. The Capsular Sleeve Region

The three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum (SR) and mitochondria in the capsular sleeve region of the intrafusal muscle fibers of the rat lumbrical muscle was observed by ultra-high-resolution scanning electron microscopy after removal of the cytoplasmic matrix by the osmium-DMSO-osmium procedure. Nuclear chain fibers were seen to have slender I-band limited mitochondria, and thick column-forming mitochondria which occasionally exhibited I-band branches. The transverse tubules (T-tubules) ran at the level of the A-band side of the A-I junction and were sandwiched between two large terminal cisternae for most of their length, forming triads. The sarcotubules arising from the terminal cisternae formed single-layer networks at the A-band level, and well developed, double-layer, three-dimensional networks at the I-band level. Occasionally, tetrads and pentads were also seen. Nuclear bag2 fibers possessed column-forming mitochondria of medium size, practically devoid of I-band branches. The T-tubules ran at the level of A-band adjacent to the A-I junction and were coupled with terminal cisternae of various lengths at intervals. As a rule, the SR formed single-layered networks at the level of the A-band and double-layered networks at the I-band level, and was less developed than in chain fibers. Nuclear bag1 fibers contained slender column-forming mitochondria devoid of I-band branches. The T-tubules were located at the level of the A-I junction. Both the T- and the axial-tubules were occasionally coupled with small terminal cisternae, forming dyads or triads. At the I-band level, the SR was well developed and formed single layer networks, but at the A-band level only a few longitudinally arranged sarcotubules and axial tubules were recognized.

High‐resolution scanning electron microscopic studies on the three‐dimensional structure of the transverse‐axial tubular system, sarcoplasmic reticulum and intercalated disc of the rat myocardium

The three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum (SR), and intercalated disc of the rat left ventricle was examined by high-resolution scanning electron microscopy after removal of the cytoplasmic matrices by the osmium-DMSO-osmium procedure. In the intermyofibrillar space, the transverse tubules (T-tubules) are accompanied by longitudinally oriented axial tubules and together form a transverse-axial system. The junctional SR is usually small but occasionally medium or large in size and couples with the T- or with the axial tubules. On the surface of the junctional SR facing the T- or the axial tubule, tiny junctional processes are seen. One or two sarcotubules, the so-called Z-tubules, frequently run parallel to the T-tubule. The sarcotubules derived from the junctional SR or from the Z-tubule run longitudinally or obliquely and form polygonal meshes around the myofibrils. On the surface of the SR at the H-band level, small fenestrations of 12-40 nm in diameter, and tiny hollows 8-20 nm in diameter are seen. Bulbous swellings of the SR, the corbular SR, are preferentially seen near the Z-band. The large and flat SR, known as the cisternal SR, intercalates among the SR meshes. In the subsarcolemmal space, the sarcotubules form a multilayered network (peripheral SR). The cisternal SR is frequently intercalated in these meshes and closely associated with the inner surface of the sarcolemma. The intercalated disc appears as a prominently undulated membrane demarcating the border between two adjacent heart muscle cells, and occasionally small projections 60-90 nm in diameter and 200-600 nm in length display on its surface.

The atrial myocardial cells of mouse heart: A structural and stereological study

Structural and stereological studies of mouse atrial myocardial cells, carried out in the same fashion as our previous investigations on mouse ventricle, demonstrate an extremely well-developed sarcoplasmic reticulum (SR) in atrial cells. The volume fraction ( V v) of the SR exceeds 12% in mouse atrial cells; perimyofibrillar network SR constitutes the major portion. We have confirmed the findings of Bossen et al. (1981, Tissue Cell 13, 71–77) of a difference between atria in terms of coupling density, the right atrium having a significantly lower incidence of interior junctional SR than the left. The SR of mouse atrium comprises a rich variety of specialized segments, including the IJSR, peripheral junctional SR, corbular SR, cisternal SR (including regions similar to fenestrated collars of striated skeletal muscle SR), as well as a peculiar form of extended junctional SR (EJSR). Although less frequent in occurrence than corbular SR, the EJSR seems closely related, since it occurs in multiple clusters at or near the Z-line regions, contains internal granular densities, and bears surface-connected structures resembling junctional processes. Seen in thin sections, mouse atrial EJSR elements are more complex than corbular SR, being larger in diameter and frequently circular in profile. Thick-section and serial-section analyses reveal that bodies of EJSR are in fact hollow spheroids. The transverse-axial tubular system of mouse atrium is rather poorly developed in comparison to its ventricular counterpart. The Golgi apparatus and associated specific atrial granules are prominent cell components. “Focal ellipsoidal deposits” (FEDs) previously described by Page and co-workers (1986, Amer. J. Physiol.) are consistently located adjacent to the Golgi region, but immunocytochemical staining for two different segments of atrial natriuretic peptide reveals no specific reaction in FEDs, whereas the SAGS are densely labeled for both antibodies.

Ultrastructure of the myocardium of the least shrew, Cryptotis parva say

The heart of the least shrew, Cryptotis parva Say, is an extremely active organ, capable of achieving rates of 800-1,200 beats/minute. The general features of myocardial cell ultrastructure in this insectivore are much like those of other small mammals; no single striking feature of fine structure is present to which the physiological properties of this heart might necessarily be attributed. Still there exist in these myocardial cells a number of atypical properties. These include 1) mitochondria having a wide variety of sizes and internal configurations 2) a pleiomorphic, highly ramified, small-diameter transverse-axial tubular system (TATS) 3) numerous "labyrinths," which are proliferated components of the TATS, and 4) myofibril-free regions, located both in juxtanuclear and other myoplasmic levels and populated by a concentration of TATS elements and fibrillar structures. Features (2) and (3) are also characteristic of another fast-beating heart, that of the mouse. The sinoatrial and atrioventricular nodal regions, as well as a Purkinje system, have been identified in the least shrew heart, along with sparsely distributed atrial cells whose myofibrils contain proliferated Z-band material. A feature frequently encountered in atrial working muscle cells is the occurrence of close appositions between gap junctions and tubules of sarcoplasmic reticulum; such appositions are also present in other regions of the shrew heart, as are complexes composed of gap junctions and mitochondria.

Membrane systems of guinea pig myocardium: Ultrastructure and morphometric studies

The structure and quantitative contribution of membrane systems (transverse-axial tubular system [TATS] and sarcoplasmic reticulum [SR]) have been investigated in the heart of the adult guinea pig. Although previous quantitative studies have been made of guinea pig myocardium, this is the first such study that has utilized tissue in which membrane system elements were clearly identified by selective staining (in this case by the osmium-ferrocyanide [OsFeCN] postfixation method). Both membrane systems are highly developed in ventricular cells, but a TATS is essentially absent from atrial myocytes. The ventricular TATS consists principally of large-bore elements which may be oriented transversely, axially, or obliquely, making numerous anastomoses with one another to form a highly interconnected system of extracellular spaces that penetrate to all myoplasmic depths of the ventricular cell. The cell coat that lines the lumina of these tubules is structured, containing fibrillar structures that run along the length of the tubule. The volume fraction (VV) of the ventricular TATS is low (2.5-3.2%), in consideration of the qualitative prominence of the TATS in these cells. The relative total population of sarcoplasmic reticulum is higher in the atria (VV of 10-11%) than in the ventricles (VV of ca. 8%). In all guinea pig myocytes, several major structural divisions of SR can be discerned, which include network SR, junctional SR, corbular SR, and cisternal SR. Junctional SR (J-SR) in the atrial cells is limited almost exclusively to peripheral saccules of junctional SR (PJSR), whereas both interior J-SR and PJSR are present in the ventricle. Two distinct morphological types of PJSR appear in atrial cells, including both flattened and distended saccules, the latter resembling PJSR of lower vertebrate heart. Spheroidal bodies of SR with opaque contents (corbular SR) are prominent at or near Z-line levels of the sarcomeres of atrial and ventricular cells. Cisternal SR is likely a subset of network SR, but some examples appear related to rough endoplasmic reticulum. An overall impression obtained from this study is that guinea pig atria are composed of structurally primitive cells, whereas the ventricular cardiac muscle cells are more highly developed entities.

The sarcoplasmic reticulum of mouse heart: Its divisions, configurations, and distribution

The sarcoplasmic reticulum (SR) is a prominent, highly ramified component of mouse myocardial cells. The use of ferrocyanide-reduced osmium tetroxide (OsFeCN) as a postfixative solution facilitates appreciation of both its extent and three-dimensional architecture. We have found that the individual volume fractions (Vv) of myofibrils, mitochondria, and SR are similar in cells of the right and left ventricular walls.Vv(total SR) is approximately 7%, a value considerably larger than previously reported. We attribute this disparity in large part to the recognition factor which comes into play with OsFeCN-treated tissue. Previous observations pertaining to the stereology of myocardial SR have likely substantially underestimated both volume fraction and surface density of this membrane system, since none to this point has utilized specific staining such as that conferred by the OsFeCN regimen. Our stereological measurements of different depths of the ventricular cell indicate that although considerable differences are found between SR configuration at peripheral and deep cell levels, no significant difference exists between the volume fractions of either the total SR or its individual constituents. Two different stereologic regimens gave close agreement on volume fractions of the various SR segments; the majority (approximately 92%) of the total SR is network SR, whereas the remainder is composed of the various categories of junctional SR (peripheral, apposed to the surface sarcolemma;interior, complexed with the transverse-axial tubular system;corbular, existing free of sarcolemmal contact). In the adult mouse, interior junctional SR greatly preponderates the other types of junctional SR; corbular SR is qualitively assessed to be a far more common component of atrial cells than of ventricular cardiomyocytes.

The transverse-axial tubular system (TATS) of mouse myocardium: its morphology in the developing and adult animal.

Invaginations of the sarcolemma that generate the transverse-axial tubular system (TATS) of the ventricular myocardial cells have begun to develop in the mouse by the time of birth. The formation of the TATS appears to be derived from the repetitive generation of caveolae, which forms "beaded tubules". Beaded tubules are retained in the adult, in which they frequently present a spiraled topography. Development of the TATS progresses so rapidly that complex systems are already present in the cardiac muscle cells of young mice; by 10-14 days of age, the ultrastructure is essentially identical to that of the adult. The mouse myocardial TATS is composed of anastomosed elements that are directed transversely and axially (longitudinally). Many tubules have an oblique orientation, however, and most elements of the TATS are highly pleiomorphic. In this respect the TATS of the mouse heart is relatively primitive in appearance in comparison with the more ordered TATS latticeworks typical of the ventricular cells of other mammals. Stereological analysis of the mouse TATS indicates that the volume fraction (VV) and surface density (SV) are considerably greater than previously reported (3.24% and 0.5028 micron-1, respectively). The most complex ramifications of the TATS are embodied in the subsarcolemmal caveolar system and the deeper tubulovesicular "labyrinths", both of which can be found in early postnatal and adult ventricular cells. In atrial cells, TATS development is initiated several days later than in the ventricular cells. The TATS of adult atrial myocardial cells is less prominent than the ventricular TATS and consists largely of axial elements; the incidence of the TATS, furthermore, is more pronounced in the left than in the right atrium.

An orderly lattice of axial tubules which interconnect adjacent transverse tubules in guinea-pig ventricular myocardium

Electron microscopy of guineapig heart muscle (ventricular) shows that large-diameter axial tubules are present in between the myofibrils. There is free communication between the lumina of these axial tubules and the transverse tubules. This transverse-axial tubular system forms an interconnected orderly network of “intracellular” tubules lined with basement membrane and open to the bulk interstitial fluid. Micropinocytotic-like vesicles are found along the axial tubules as well as the transverse tubules. Electron-opaque diffusion tracers, such as horseradish peroxidase (diameter of about 55 Å) and colloidal thorium dioxide (diameter of 60 Å) readily enter into these tubules. This tubular lattice occupies a large fraction of the cell volume (> 7.7%), and this fraction increases in hypertonic solutions due to swelling of the transverse-axial system. A network of closed or small SR sarcotubules into which tracer does not penetrate are also present surrounding the myofibrils, and often this system is in close proximity to the transverse-axial system. The transverse-axial tubular system is absent in the guinea-pig atrial muscle, but these cells do contain a closed SR sarcotubular system. Thus, in the ventricular cells the myofibrils and mitochondria have close access to a space that is continuous with the bulk interstitial fluid and which may be of similar cationic composition; such an arrangement should facilitate excitation-contraction coupling.