Modelling the cardiac transverse-axial tubular system

The transverse-axial tubular system (TATS) of cardiac ventricular myocytes is a complex network of tubules that arises as invaginations of the surface membrane; it appears to form a specialised region of cell membrane that is particularly important for excitation–contraction coupling. However, much remains unknown about the structure and role of the TATS. In this brief review we use experimental data and computer modelling to address the following key questions: (i) What fraction of the cell membrane is within the TATS? (ii) Is the composition of the TATS membrane the same as the surface membrane? (iii) How good is electrical coupling between the surface and TATS membranes? (iv) What fraction of each current is within the TATS? (v) How important is the complex structure of the TATS network? (vi) What is the effect of current inhomogeneity on lumenal ion concentrations? (vii) Does the TATS contribute to the functional changes observed in heart failure? Although there are many areas in which experimental evidence is lacking, computer models provide a method to assess and predict the possible function of the TATS; such models suggest that although the surface and TATS membranes are electrically well coupled, concentration of ion flux pathways within the TATS, coupled to restricted diffusion, may result in the ionic composition in the TATS lumen being different from that in the bulk extracellular space, and varying with activity and in pathological conditions.     

A model of the guinea-pig ventricular cardiac myocyte incorporating a transverse-axial tubular system

A model of the guinea-pig cardiac ventricular myocyte has been developed that includes a representation of the transverse–axial tubular system (TATS), including heterogeneous distribution of ion flux pathways between the surface and tubular membranes. The model reproduces frequency-dependent changes of action potential shape and intracellular ion concentrations and can replicate experimental data showing ion diffusion between the tubular lumen and external solution in guinea-pig myocytes. The model is stable at rest and during activity and returns to rested state after perturbation. Theoretical analysis and model simulations show that, due to tight electrical coupling, tubular and surface membranes behave as a homogeneous whole during voltage and current clamp (maximum difference 0.9 mV at peak tubular I_Na of −38 nA). However, during action potentials, restricted diffusion and ionic currents in TATS cause depletion of tubular Ca²⁺ and accumulation of tubular K⁺ (up to −19.8% and +3.4%, respectively, of bulk extracellular values, at 6 Hz). These changes, in turn, decrease ion fluxes across the TATS membrane and decrease sarcoplasmic reticulum (SR) Ca²⁺ load. Thus, the TATS plays a potentially important role in modulating the function of guinea-pig ventricular myocyte in physiological conditions.     

Immunogold labeling study of the distribution of GLUT-1 and GLUT-4 in cardiac tissue following stimulation by insulin or ischemia

Whereas glucose transporter 1 (GLUT-1) is thought to be responsible for basal glucose uptake in cardiac myocytes, little is known about its relative distribution between the different plasma membranes and cell types in the heart. GLUT-4 translocates to the myocyte surface to increase glucose uptake in response to a number of stimuli. The mechanisms underlying ischemia- and insulin-mediated GLUT-4 translocation are known to be different, raising the possibility that the intracellular destinations of GLUT-4 following these stimuli also differ. Using immunogold labeling, we describe the cellular localization of these two transporters and investigate whether insulin and ischemia induce differential translocation of GLUT-4 to different cardiac membranes. Immunogold labeling of GLUT-1 and GLUT-4 was performed on left ventricular sections from isolated hearts following 30 min of either insulin, ischemia, or control perfusion. In control tissue, GLUT-1 was predominantly (76%) localized in the capillary endothelial cells, with only 24% of total cardiac GLUT-1 present in myocytes. GLUT-4 was found predominantly in myocytes, distributed between sarcolemmal and T tubule membranes (1.84 +/- 0.49 and 1.54 +/- 0.33 golds/microm, respectively) and intracellular vesicles (127 +/- 18 golds/microm(2)). Insulin increased T tubule membrane GLUT-4 content (2.8 +/- 0.4 golds/microm, P < 0.05) but had less effect on sarcolemmal GLUT-4 (1.72 +/- 0.53 golds/microm). Ischemia induced greater GLUT-4 translocation to both membrane types (4.25 +/- 0.84 and 4.01 +/- 0.27 golds/microm, respectively P < 0.05). The localization of GLUT-1 suggests a significant role in transporting glucose across the capillary wall before myocyte uptake via GLUT-1 and GLUT-4. We demonstrate independent spatial translocation of GLUT-4 under insulin or ischemic stimulation and propose independent roles for T-tubular and sarcolemmal GLUT-4.     

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

Summary— 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 μm²/μm³ 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.     

Store-operated Ca2+ entry uncoupled with ryanodine receptor and junctional membrane complex in heart muscle cells

Store-operated Ca2+ entry (SOCE) is the Ca2+ influx that is activated on depletion of intracellular Ca2+ stores. Although SOCE is found in a variety of cell types, its activation mechanism and molecular identity remain to be clarified. Current experimental results suggest that SOCE channels are activated by direct coupling with Ca2+ release channels on depleted stores. Here we report SOCE in cardiac myocytes, that was prominently sensitive to Zn2+ but resistant to inhibitors for voltage-dependent Ca2+ channels and Na+/Ca2+ exchangers. The SOCE activity may be developmentally regulated, because the SOCE was easily detected during embryonic and neonatal stages but not in mature myocytes from adult hearts. In cardiac myocytes, ryanodine receptor type 2 (RyR-2) is thought to be the sole Ca2+ release channel on the intracellular store, and junctophilin type 2 (JP-2) contributes to formation of the junctional complex between the cell surface and store membranes. Using the knockout mice, we also examined possible involvement of the Ca2+ release channel and junctional membrane complex in cardiac SOCE. Apparently normal SOCE activities were retained in mutant myocytes lacking RyR-2 or JP-2, suggesting that neither the Ca2+ release channel nor junctional membrane complex is involved in activation of cardiac SOCE.     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Structural heterogeneity of the basement membrane in the rat proximal tubule

Summary The ultrastructure of the basement membrane of the rat proximal tubule was observed by transmission electron microscopy after the use of a cold dehydration technique. The basement membrane of the P1 segment is thick and possesses several structural specializations that are rare in other basement membranes; these include intraepithelial ridges, dense bars, and basement membrane vesicles. The intraepithelial ridges are found in the intercellular spaces between interdigitating processes of the proximal tubule cells. The ridges and the interdigitating processes run circumferentially around the tubule. The dense bars are frequently found in the intraepithelial ridges. They are especially prominent on the concave side of the tubular bends and to a lesser extent near sites where intracellular actin filaments anchor onto the basal cell membranes. The basement membrane vesicles are bounded by unit membranes; they are variable in both their electron density and their size. They are usually found in association with dense bars, and the grade of their accumulation is positively correlated with the development of the dense bars. These three specializations have no topographical relationship with the interstitial structures, such as fibrobalasts and collagen fibrils. The specializations are best developed on the concave side of tubular bends where the circumferential stresses caused by the intraluminal hydraulic pressure are presumably the largest; we therefore propose that they are an adaptation to, or a manifestation of, the increased wall stress in the proximal tubule.     

A quantitative model of the cardiac ventricular cell incorporating the transverse-axial tubular system

The role of the transverse-axial tubular system (TATS) in electrical activity of cardiac cells has not been investigated quantitatively. In this study a mathematical model including the TATS and differential distribution of ionic transfer mechanisms in peripheral and tubular membranes was described. A model of ventricular cardiac cell described by Jafri et al. (1998) was adopted and slightly modified to describe ionic currents and Ca2+ handling. Changes of concentrations in the lumen of the TATS were computed from the total of transmembrane ionic fluxes and ionic exchanges with the pericellular medium. Long-term stability of the model was attained at rest and under regular stimulation. Depletion of Ca2+ by 12.8% and accumulation of K+ by 4.7% occurred in the TATS-lumen at physiological conditions and at a stimulation frequency of 1 Hz. The changes were transient and subsided on repolarization within 800 ms (Ca2+) and 300 ms (K+). Nevertheless, the course of action potentials remained virtually unaltered. Simulations of voltage clamp experiments demonstrated that variations in tubular ionic concentrations were detectable as modulation of the recorded membrane currents.     

Effect of input resistance voltage-dependency on DC estimate of membrane capacitance in cardiac myocytes

The measure of membrane capacitance (C(m)) in cardiac myocytes is of primary importance as an index of their size in physiological and pathological conditions, and for the understanding of their excitability. Although a plethora of very accurate methods has been developed to access C(m) value in single cells, cardiac electrophysiologists still use, in the majority of laboratories, classical direct current techniques as they have been established in the early days of cardiac cellular electrophysiology. These techniques are based on the assumption that cardiac membrane resistance (R(m)) is constant, or changes negligibly, in a narrow potential range around resting potential. Using patch-clamp whole-cell recordings, both in current-clamp and voltage-clamp conditions, and numerical simulations, we document here the voltage-dependency of R(m), up to -45% of its resting value for 10-mV hyperpolarization, in resting rat ventricular myocytes. We show how this dependency makes classical protocols to misestimate C(m) in a voltage-dependent manner (up to 20% errors), which can dramatically affect C(m)-based calculations on cell size and on intracellular ion dynamics. We develop a simple mechanistic model to fit experimental data and obtain voltage-independent estimates of C(m), and we show that accurate estimates can also be extrapolated from the classical approach.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

In situ localization of cholesterol in skeletal muscle by use of a monoclonal antibody.

A common perception is that cholesterol, the major structural lipid found in mammalian membranes, is localized nearly exclusively to the plasma membrane of living cells and that it is found in much smaller quantities in internal membranes. This perception is based almost exclusively on cell fractionation studies, in which density gradient centrifugation is used for purification of discrete subcellular membrane fractions. Here we describe a monoclonal antibody, MAb 2C5-6, previously reported to detect purified cholesterol in synthetic membranes (Swartz GM Jr, Gentry MK, Amende LM, Blanchette-Mackie EJ, and Alving CR. Proc Natl Acad Sci USA 85: 1902-1906, 1988), that is capable of detecting cholesterol in situ in the membranes of skeletal muscle sections. Localization of cholesterol, the dihydropyridine receptor of the T tubule, and the Ca(2+)-ATPase of the sarcoplasmic reticulum (SERCA2) by means of double and triple immunostaining protocols clearly demonstrates that cholesterol is primarily localized to the sarcoplasmic reticulum membranes of skeletal muscle rather than the sarcolemmal or T tubule membranes. The availability of this reagent and its ability to spatially localize cholesterol in situ may provide a greater understanding of the relationship between membrane cholesterol content and transmembrane signaling in skeletal muscle.     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β₁-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.     

Surface:volume relationship in cardiac myocytes studied with confocal microscopy and membrane capacitance measurements: species-dependence and developmental effects.

The quantitative analysis of the contribution of ion fluxes through membrane channels to changes of intracellular ion concentrations would benefit from the exact knowledge of the cell volume. It would allow direct correlation of ionic current measurements with simultaneous measurements of ion concentrations in individual cells. Because of various limitations of conventional light microscopy a simple method for accurate cell volume determination is lacking. We have combined the optical sectioning capabilities of fluorescence laser scanning confocal microscopy and the whole-cell patch-clamp technique to study the correlation between cell volume and membrane capacitance. Single cardiac myocytes loaded with the fluorescent dye calcein were optically sectioned to produce a series of confocal images. The volume of cardiac myocytes of three different mammalian species was determined by three-dimensional volume rendering of the confocal images. The calculated cell volumes were 30.4 +/- 7.3 pl (mean +/- SD) in rabbits (n = 28), 30.9 +/- 9.0 pl in ferrets (n = 23), and 34.4 +/- 7.0 pl in rats (n = 21), respectively. There was a positive linear correlation between membrane capacitance and cell volume in each animal species. The capacitance-volume ratios were significantly different among species (4.58 +/- 0.45 pF/pl in rabbit, 5.39 +/- 0.57 pF/pl in ferret, and 8.44 +/- 1.35 pF/pl in rat). Furthermore, the capacitance-volume ratio was dependent on the developmental stage (8.88 +/- 1.14 pF/pl in 6-month-old rats versus 6.76 +/- 0.62 pF/pl in 3-month-old rats). The data suggest that the ratio of surface area:volume of cardiac myocytes undergoes significant developmental changes and differs among mammalian species. We further established that the easily measurable parameters of cell membrane capacitance or the product of cell length and width provide reliable but species-dependent estimates for the volume of individual cells.     

Shaping up for action

The Malpighian tubule is the main organ for excretion and osmoregulation in most insects. During a short period of embryonic development the tubules of Drosophila are shaped, undergo differentiation and become precisely positioned in the body cavity, so they become fully functional at the time of larval hatching a few hours later. In this review I explore three developmental events on the path to physiological maturation. First, I examine the molecular and cellular mechanisms that generate organ shape, focusing on the process of cell intercalation that drives tubule elongation, the roles of the cytoskeleton, the extracellular matrix and how intercalation is coordinated at the tissue level. Second, I look at the genetic networks that control the physiological differentiation of tubule cells and consider how distinctive physiological domains in the tubule are patterned. Finally, I explore how the organ is positioned within the body cavity and consider the relationship between organ position and function.     

Isolation and characterization of single myocardial cells from the perch, Perca fluviatilis

In applying the enzymatic cell isolation technique to the fish heart about 40% of the dispersed myocytes maintained their spindle-shaped morphology, and about half of them tolerated physiological concentration of Ca2+ and excluded the vital dye, Evans blue. The length of spindle-shaped myocytes was on average 133 +/- 3 micron and the maximum width was 4.2 +/- 0.1 micron. The mean length of the sarcomeres was 2.1 +/- 0.1 micron. The sizes of the myocytes did not vary significantly with the weights of the fish. Electron microscopic examinations showed typical fish myocardial cell structure; absence of transverse tubule system, a sparse network of sarcoplasmic reticulum and from a few up to eight or more myofibrils. The cells were mononuclear. Most of the Ca2+-tolerant myocytes were quiescent, but the contraction in them could be induced by electric field stimulation. Both the spontaneous and electrically triggered contractions were of twitch type. The slowly propagating contraction waves, so-called phasic contractions common in isolated mammalian cardiac myocytes, could not be seen at all.     

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.     

Novel determinant of PKC-epsilon anchoring at cardiac Z-lines

The Z-line represents a critical link between the transverse tubule network and cytoskeleton of cardiac cells with a role in anchoring structural proteins, ion channels, and signaling molecules. Protein kinase C-epsilon (PKC-epsilon) regulates cardiac excitability, cardioprotection, and growth, possibly as a consequence of translocation to the Z-line/T tubule region. To investigate the mechanism of PKC-epsilon translocation, fragments of its NH2-terminal 144-amino acid variable domain, epsilonV1, were fused with green fluorescent protein and evaluated by quantitative Fourier image analysis of decorated myocytes. Deletion of 23 amino acids from the NH2-terminus of epsilonV1, including an EAVSLKPT motif important for binding to a receptor for activated C kinase (RACK2), reduced but did not abolish Z-line binding. Further deletions of up to 84 amino acids from the NH2-terminus of epsilonV1 also did not prevent Z-line decoration. However, deletions of residues 85-144 from the COOH-terminus strongly reduced Z-line binding. COOH-terminal deletions caused 2.5-fold greater loss of binding energy (deltadeltaG) than did NH2-terminal deletions. Synthetic peptides derived from these regions modulated epsilonV1 binding and cardiac myocyte function, but also revealed considerable heterogeneity within populations of adult cardiac myocytes. The COOH-terminal subdomain important for Z-line anchoring maps to a surface in the epsilonV1 crystal structure that complements the eight-amino acid RACK2 binding site and two previously identified membrane docking motifs. PKC-epsilon anchoring at the cardiac Z-line/T tubule appears to rely on multiple points of contact probably involving protein-lipid and protein-protein interactions.     

In vitro studies on adult cardiac myocytes: attachment and biosynthesis of collagen type IV and laminin

The interactions between adult rat cardiac myocytes and the basement membrane components collagen type IV and laminin were investigated in attachment experiments and biosynthesis studies and by immunofluorescence staining. Adult myocytes attached equally well to native collagen type IV and laminin but did not attach to collagen type IV solubilized with pepsin (P-CIV) or to collagen type I. However, when laminin was used to coat P-CIV, attachment was enhanced. Affinity-purified antibodies against laminin inhibited the attachment of myocytes to dishes coated with native collagen type IV, indicating that cell surface-bound laminin mediated attachment of the cells to this substrate. Immunofluorescence staining of freshly isolated myocytes, using antibodies against laminin or collagen type IV, revealed the presence of laminin but not of collagen type IV on the surface of freshly isolated cells, indicating that during the isolation procedure collagen IV was removed from the cell surface. Metabolic labeling followed by immunoprecipitation demonstrated synthesis of both laminin and collagen type IV in cardiac myocytes as they progressed into culture over a 14-day period. This synthesis was accompanied by the deposition of the collagen type IV and laminin into distinctly different patterns as revealed by immunofluorescence staining. As the cells progressed into culture, newly synthesized laminin formed a network radiating from the center of the reorganizing cell into the pseudopods. The laminin was redistributed and remodeled with time in culture to form a dense layer beneath the cell. Collagen type IV was also synthesized with time in culture, but the pattern was a much finer network as opposed to the denser pattern of laminin staining. These studies demonstrate that adult cardiac myocytes synthesize and remodel the basement membrane as they adapt to the culture environment.     

Abnormal features in skeletal muscle from mice lacking mitsugumin29

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.