Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca²⁺ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.     

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.     

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 300 nm in diameter, permitted only particles 3 nm; 3), the mitochondrial voltage-dependent anion channel and the nuclear pore complex in ventricular cells could not be penetrated by particles >/=6 nm; and 4), there is a difference in size clearance between transversal and longitudinal sarcoplasmic diffusional pathways.     

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 (Vv) 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.     

THE ULTRASTRUCTURE OF THE CAT MYOCARDIUM : II. Atrial Muscle

The ultrastructure of the cells specialized for contraction in the atrium and ventricle of young adult cats are compared. The cells specialized for conduction are not included. In addition to possessing distinctive atrial granules, the cells of the atrium are smaller in diameter (5–6 µ) than ventricular cells (10–12 µ) and have strikingly fewer T tubules. These latter differences are discussed in terms of their possible significance for the rate of conduction of the action potential. It is suggested that the very small number of T tubules in atrial cells may compensate for the small cell diameter, and thus permit rapid conduction of the action potential across the surface of the atrium. Coated dense vesicles found in association with the sarcoplasmic reticulum at the level of the Z line in ventricular muscle are more evident in atrial cells. In the virtual absence of T tubules in atrial cells, the sub-sarcolemmal cisternae of the sarcoplasmic reticulum are almost exclusively at the cell periphery. The ends of the cells and their processes in ventricular muscle are rectilinear with the interdigitated portions of the intercalated discs oriented transversely, whereas those of the atrium are often oblique to the myofilament axis. This difference may be related to the lower mechanical tension on atrial cells.     

Ultrastructure of the heart of the sturgeon Acipenser stellatus (Chondrostei)

Summary The ultrastructure of atrial and ventricular myocardial cells from Acipenser stellatus is described. The cells of the atrium are more loosely connected than those of the ventricle. Cell contact is by simple intercalated discs and by desmosomes. The cells are flattened, with peripheral myofibrils and a central region of mitochondria and the nucleus. The sarcoplasmic reticulum consists of subsarcolemmal tubules, that frequently extend towards the central mitochondria. Dyads are small and positioned at any sarcomeric level. No T-tubules are present. Specific granules are restricted to the atrial cell, and are sometimes present within the SR tubules.     

Structures located at the levels of the Z bands in mouse ventricular myocardial cells

Within ventricular myocardial cells of the mouse, the myoplasmic regions located immediately adjacent to the Z lines of the sarcomeres contain a variety of structures. These include: (1) transversely oriented 10 nm (‘intermediate’) filaments that apparently contribute to the cytoskeleton of the myocardial cell; (2) the majority of the transverse elements of the T-axial tubular system; (3) specialized segments of the sarcoplasmic reticulum (SR) that are closely apposed to the sarcolemma or T-axial tubules (junctional SR); (4) ‘extended junctional SR’ (‘corbular SR’) that exists free of association with the cell membrane; (5) ‘Z tubules’ of SR that are intimately apposed to the Z line substance; and (6) leptofibrils. In addition, fasciae adherentes supplant Z lines where myofibrils insert into the transverse borders (intercalated discs) of the cells. The concentration of these myocardial components at the level of the Z lines suggests that a particular specialization of structural and physiological activities exists in the Z-level regions of the myoplasm. In particular, it appears that the combination of intermediate filaments, T tubules, and Z-level SR elements forms a series of parallel planar bodies that extend across each myocardial cell to impart transverse rigidity. The movement and compartmentation of calcium ion (Ca²⁺) would seem especially active near the Z lines of the myofibrils, in view of the preferential location there of Ca²⁺-sequestering myocardial structures such as T tubules, junctional SR, extended junctional SR and Z tubules.     

Secretion of atrial natriuretic peptide (ANP) from fish atrial and ventricular myocytes in tissue culture

Primary cultures of atrial and ventricular myocytes (approx. 1 x 10(5) cells/culture) were prepared from adult teleost fish Gila atraria and maintained for 10 days. Immunoreactive atrial natriuretic peptide (ir-ANP) from fish atrial and ventricular cells was 3.9 and 2.8 ng/culture respectively, values not significantly different. Atriocytes from rat and mouse secreted comparable amounts of ANP which were not significantly different from atrial fish cultures (5.2 and 4.3 ng/culture). In contrast, their ventricular myocytes secreted only small quantities of ANP (0.8 and 0.3 ng/culture). When analyzed by reversed-phase HPLC, the media of both fish atrial and ventricular myocytes contained a peptide which exhibited properties similar to authentic human ANP (Ser 99-Tyr 126), suggesting a significant degree of sequence homology between fish and mammalian ANP. Fish ventricular cells, unlike normal mammalian ventricular cells, secrete substantial quantities of immunoreactive-ANP.     

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.     

Loss of the plateau of the cardiac action potential in hypertonic solutions

The effect of hypertonicity on the electrical properties of vertebrate myocardial cells was studied in ventricular muscle fibers of guinea pig, cat, frog, and chicken. The latter two species do not have a T-tubule system, whereas the former two do. In hypertonic solutions (2 x isotonic) produced by addition of sucrose or excess of NaCl, cell diameter decreased and there was a slight hyperpolarization and decrease in action potential overshoot. In guinea pig and cat, the hypertonic solution caused a decrease in input resistance and the plateau of the action potential to disappear in some of the cells; contractions of the entire ventricle also became depressed. These effects were reversed by returning the muscle fibers to isotonic solution. Addition of 5 mM SrCl₂ to the hypertonic solution also caused the plateau component and contraction to reappear. In frog and chick cells, loss of the plateau component and contraction never occurred in hypertonic solution, and input resistance increased. Urea and glycerol hyperosmolarity (2 x) caused no loss of the plateau component or contraction. If the frog and chicken ventricular, and guinea pig atrial myocardial cells (all of which lack T tubules) were to serve as an adequate control for possible effects of hypertonicity on the surface membrane and on contractile proteins, then the results suggest that swelling of the T tubules of mammalian myocardial cells leads to loss of the plateau component.     

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

Rationale

Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.

Methods and Results

In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.

Conclusion

Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.     

Oracle, a novel PDZ-LIM domain protein expressed in heart and skeletal muscle

In order to identify novel genes enriched in adult heart, we performed a subtractive hybridization for genes expressed in mouse heart but not in skeletal muscle. We identified two alternative splicing variants of a novel PDZ-LIM domain protein, which we named Oracle. Both variants contain a PDZ domain at the amino-terminus and three LIM domains at the carboxy-terminus. Highest homology of Oracle was found with the human and rat enigma proteins in the PDZ domain (62 and 61%, respectively) and in the LIM domains (60 and 69%, respectively). By Northern hybridization analysis, we showed that expression is highest in adult mouse heart, low in skeletal muscle and undetectable in other adult mouse tissues. In situ hybridization in mouse embryos confirmed and extended these data by showing high expression of Oracle mRNA in atrial and ventricular myocardial cells from E8.5. From E9.5 low expression of Oracle mRNA was detectable in myotomes. These data suggest a role for Oracle in the early development and function of heart and skeletal muscle.     

Transverse tubules are a common feature in large mammalian atrial myocytes including human

Transverse (t) tubules are surface membrane invaginations that are present in all mammalian cardiac ventricular cells. The apposition of L-type Ca(2+) channels on t tubules with the sarcoplasmic reticulum (SR) constitutes a "calcium release unit" and allows close coupling of excitation to the rise in systolic Ca(2+). T tubules are virtually absent in the atria of small mammals, and therefore Ca(2+) release from the SR occurs initially at the periphery of the cell and then propagates into the interior. Recent work has, however, shown the occurrence of t tubules in atrial myocytes from sheep. As in the ventricle, Ca(2+) release in these cells occurs simultaneously in central and peripheral regions. T tubules in both the atria and the ventricle are lost in disease, contributing to cellular dysfunction. The aim of this study was to determine if the occurrence of t tubules in the atrium is restricted to sheep or is a more general property of larger mammals including humans. In atrial tissue sections from human, horse, cow, and sheep, membranes were labeled using wheat germ agglutinin. As previously shown in sheep, extensive t-tubule networks were present in horse, cow, and human atrial myocytes. Analysis shows half the volume of the cell lies within 0.64 ± 0.03, 0.77 ± 0.03, 0.84 ± 0.03, and 1.56 ± 0.19 μm of t-tubule membrane in horse, cow, sheep, and human atrial myocytes, respectively. The presence of t tubules in the human atria may play an important role in determining the spatio-temporal properties of the systolic Ca(2+) transient and how this is perturbed in disease.     

Expression of TASK-1, a pH-sensitive twin-pore domain K(+) channel,in rat myocytes

We have investigated the expression of TASK-1, a pH-sensitive, twin-pore domain K(+) channel in the rat heart. A mammalian cell line of Chinese hamster ovary cells (CHO), transfected with a plasmid containing mouse TASK-1, demonstrated the specificity of the anti-TASK-1 antibody. TASK-1 expression in cardiac tissue was initially demonstrated by Western blot and then localized by immunofluorescence. In single rat ventricular myocytes, strong staining of the TASK-1 protein was located at the intercalated disks and across the cell in a striated pattern, corresponding to the transverse axial tubular network (T tubules). In contrast, single rat atrial myocytes were stained at the intercalated disks with a weak punctate, striated pattern corresponding to underdeveloped T tubules. Also, formamide was used to induce the detubulation of ventricular myocytes, which enabled confirmation that TASK-1 protein expression occurs in T tubules. Consistent with this, RT-PCR revealed the expression of TASK-1 mRNA in total RNA from both the ventricles and atria. In this study, we conclusively demonstrated that TASK-1 protein and mRNA were expressed in rat atrial and ventricular tissue. The extensive distribution of TASK-1 shown to exist within myocyte membranes may provide a potential future target for antiarrhythmic drugs.     

Atrial natriuretic peptide levels during development of chronic heart failure after myocardial infarction in rats

Serum levels of atrial natriuretic peptide (ANP) are elevated in chronic heart failure presumably due to dilatation of the left atrium resulting from increases in intracardiac pressures. To define the time course of changes in serum ANP levels and to determine the relationship to left ventricular end-diastolic pressure, rats were subjected to coronary artery ligation to produce myocardial infarction and left ventricular failure. Atrial natriuretic peptide levels were measured weekly for four weeks thereafter. In rats with myocardial infarction and elevation of left ventricular end-diastolic pressure there was no change in ANP levels at 7 and 14 days. However, at day 21 and 28, ANP levels were elevated more than 3 fold. There was a correlation between ANP levels and left ventricular end-diastolic pressures. There was no correlation between ANP levels and right atrial pressures or serum sodium concentrations. We conclude that the chronic elevation of left ventricular end-diastolic pressure is required to produce an increase in ANP after myocardial infarction which results in chronic heart failure.     

Increasing myocardial contraction and blood pressure in C57BL/6 mice during early postnatal development

Knowledge of the developmental changes of cardiovascular parameters in the genetic background of a mouse strain is important for understanding phenotypic changes in transgenic or knockout mouse models for heart disease. We studied arterial blood pressure and myocardial contractility in mice of the common background strain C57BL/6, aged 21 days [postnatal day 21 (P21)] to 580 days. Heart rate increased during maturation from 396 beats/min at P21 to 551 beats/min at postnatal day 50 (P50), and mean arterial blood pressure increased in parallel from 86 to 110 mmHg and remained constant afterward. Echocardiographically determined left ventricular myocardial wall dimensions (R = 0.79, P < 0.0001) and left ventricular mass calculated using the area-length algorithm correlated strongly with histomorphometrical measurements (R = 0.93, P < 0.001). Sarcomere shortening records from isolated ventricular myocytes used as a measure for myocardial contractility revealed a negative shortening-frequency relation under a pacing frequency of 2 Hz and a positive relation above 2 Hz. Shortening amplitudes recorded from P21 myocytes were smaller, and the shortening-frequency relation was less steep than in adult myocytes. A stimulation pause was followed by a negative "staircase" at pacing frequency of < or =6 Hz and a positive staircase at > or =6 Hz. P21 myocytes developed positive staircases at 8 and 10 Hz, and adult myocytes also developed them at 6 Hz. Blood pressure increase during maturation until P50 may originate from increasing single cardiomyocyte contractility.     

Freeze-fracture studies of the sinoatrial and atrioventricular nodes of the caprine heart,with special reference to the nexus

Summary The caprine sinoatrial node (SAN) and atrioventricular node (AVN) were studied by freeze-fracture techniques, and their nexus or gap junction structure were compared with that of ordinary atrial and ventricular muscle cells. The general features of the nexus in both the SAN and AVN were essentially identical. Approximately two-thirds of the nexuses observed in the nodal cells consisted of typical macular arrangements of nexal particles, and the remaining third, of atypical configurations of either circular arrangements or linear arrays of particles in continuity with the macular nexuses. Such atypical nexuses were never observed in the ordinary adult myocardial cells. Quantitative analysis revealed that all of the nexuses in the nodal cells measured, were less than 0.1 m², whereas the majority of the nexuses in ordinary myocardial cells (64% in the atrium and 76% in the ventricle) were larger than 0.1 m². No significant differences in diameter and center-to-center distance of nexal particle were found between the nodal cells and ordinary myocardial cells.     

Relation of fast and slow skeletal, and atrial and ventricular myosin in various mammals

Myosin was isolated from adult mouse, rat, rabbit and cat atrial and ventricular myocardium and fast and slow skeletal muscles and examined by measuring Ca2+-ATPase activity and by electrophoretic fractionation of chymotryptic peptides and MLCs. The myosin from mouse atrial and ventricular myocardium were very similar. The properties of cat soleus muscle myosin and ventricular myocardium were also very similar (ATPase activity and electrophoretic pattern of chymotryptic peptides of myosin). The electrophoretic pattern of MLCs, however, was distinct when comparing mouse and feline muscles. These observations are consistent with the idea that atrial and ventricular alpha MHCs are closely related and that beta MHCs from ventricular myocardium and slow skeletal muscle fibres are also closely related.     

Immunohistochemical analysis of the adaptation of adult guinea-pig cardiomyocytes in long-term cultures and in cocultures with cardiac neurons: A novel model for studies of myocardial function

In this study, we used laser confocal scanning microscopy and immunofluorescent markers to describe the establishment of long-term cultures of adult guinea-pig cardiomyocytes and their cocultures with adult intrinsic cardiac neurons. We have also investigated the effect of plating density on the adaptation of the myocytes in culture. Providing that the preparation of freshly isolated cardiomyocytes consists mostly (> 80%) of rod-shaped, Ca-tolerant, and quiescent cells and these are plated under optimal conditions and density (105/cm2), these myocytes have the following characteristics: (1) they remain elongated with regular ultrastructural characteristics and quiescent for several days; (2) within 10-14 days, they reestablish their intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layers; (3) they retain their regular myofibrilar striation all through the adaptation to culture conditions without any sign of dedifferentiation or redifferentiation; (4) these characteristics are lost when the cells are plated at too low (< 104/cm2) or too high (2 × 105/cm2) a density and they exhibit signs of dedifferentiation; (5) the adult ventricular myocytes appear to retain their ability to express atrial natriuretic peptide (ANP), as indicated by immunoreactivity to anti-ANP antibody; (6) this activity seems to be directly related to the surface area of the myocytes in contact with the substrate (i.e. to the stretch of the myocytes); (7) the intrinsic cardiac neurons grow intricate networks of neurites, which form a free-ending type of contact with the cocultured myocytes.abstract typed in here; if it is more than one paragraph use Long-term cultures of adult guinea-pig ventricular myocytes, alone or in their cocultures with cardiac neurons in which both are fully active functionally, provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial function under acute or chronic effects of various intrinsic and/or extrinsic factors, including neuroregulation.