Response of rat myocardial mast cells to experimental ischemia.

The effects of ischemia on mast cells in the rat heart were studied. After 5 hours morphological signs of mediator release: granule dissolution, were observed. After 4 weeks the number of mast cells was significantly increased in comparison with nonischemic. Furthermore, 4 weeks after ischemia there was an increased number of regenerating mast cells. An increase in the number of mast cells and morphological signs of granule synthesis in the ventricular or septal muscle of the sham operated controls indicates that the surgical procedure itself can affect the mast cells of the heart generally. Morphological signs of granule synthesis were observed within mast cells of all experimental groups, and the figure was highly increased after 4 weeks of ischemia.

Calcium in myocardial tissue and isolated mitochondria from a teleost.

A method is presented for isolating cardiac mitochondria from bony-fish. Calcium levels in ventricular whole tissue and isolated mitochondria of Gadus virens L. are determined by atomic absorption flame spectroscopy, and were found to be about 8 and 16 nmolCa/mg prot., respectively. In conclusion, the calcium concentration within the myocardial mitochondria in this species may be nearly three times higher than at the outside, and probably these structures serve as a "calcium sink". The results are compared with those previously reported for mammals.

A correlative transmission and scanning electron microscopic study of the pigeon myocardial cell.

A comparative study of the pigeon ventricular myocardial cell has been performed by transmission electron microscopy (TEM) and by scanning electron microscopy (SEM). Three-dimensional access to the cell interior was obtained by cryo-fracturing paraffin-embedded tissue immersed in liquid nitrogen. The TEM studies revealed parallelly arranged myofibils separated by rows of mitochondria. The sarcoplasmic reticulum is represented by a well-developed network of tubules which, at the Z- and H-band level of the sarcomere, expands to form belt-like cisternae. The cisternae at the Z-band level lie in close proximity to both myofilaments and mitochondria. Transverse tubules are absent and thus only peripheral couplings are present. SEM observations of the fractured tissue revealed the spatial relationship between the different cell organelles, the most important of these being the parallel myofibrils and the mitochondria. The conspicuous ridges transversing the myofibril at the Z-band level consist mainly of expanded Z-bands, but overlying SR-tubules also contribute to these ridges. Traces of the SR can sometimes be seen covering the myofibrils. The close proximity between the SR and the mitochondria was also confirmed in the SEM. Preparation and examination of SEM prepared tissue in the TEM confirmed that no essential damage or reorganization of cell organelles had taken place during tHE SEM procedure. On the other hand some shrinkage of the tissue, which was probably caused by critical point drying, was noticed.

The T-tubule system in the myocardia of the sand rat and mouse as demonstrated by horseradish peroxidase.

The transverse tubule (T-tubule) system in papillary muscles of the sand rat and the mouse were studied with the aid of a diffusion tracer (horseradish peroxidase). The T-tubule system in the sand rat showed a typical mammalian pattern with sarcolemmal tubules invaginating at the Z-band level of the sarcomere. These tubules follow a transverse direction in the cell with frequent longitudinal side-branches which connect tubules at different Z-band levels. In the mouse myocardium, the T-tubules also start as lateral invaginations from the sarcolemma at the Z-band levels. In the cell interior, however, the tubules ramify and brake up into a complicated system of spirally running tubules. These spirals, of relative small diameter (400A--700A), frequently expand and form lobulated cisternae at the Z-band levels.

Cardiac hypothermia evaluated by ultrastructural studies in man.

Transmural left ventricular biopsies from 13 patients undergoing cardiopulmonary bypass were studied. The hypertrophic myocardium was protected by deep hypothermia (15 degrees C.) during ischemic arrest for a maximal period of 96 minutes. Biopsies were taken at the start of bypass, at the end of aortic cross-clamping, and after 20 minutes of reperfusion. The structure of the mitochondria remained normal, whereas cellular alterations in the form of widened intercalated discs, interstitial and intracellular edema, myelin figures, and slight myofibrillar lysis were observed in all stages. As no clear correlation between these structural injuries and aortic cross-clamping time was seen, they are a result of cardiac hypertrophy rather than intraoperative ischemia.

Cryofracture of paraffin-embedded heart muscle cells.

A comparative study of internal cellular structures of the sheep ventricular myocardium has been conducted by scanning electron microscopy (SEM) and by transmission electron microscopy (TEM). Access to the cell interior for three-dimensional studies was obtained by cryofracturing paraffin-embedded tissue frozen in liquid nitrogen. For accurate localization of structures of special interest thick paraffin sections were examined in the light microscope (LM). Based on the information gained, it was possible to fracture the block in a desired plane. The fracturing was carried out by a light blow to a precooled scalpel held against the surface of the block, which was immersed in liquid nitrogen. After thawing and deparaffinizing at room temperature in several baths of xylene, the tissue pieces were critical point dried using CO2. As xylene was found to be miscible with CO2, it also served as an intermediate fluid. This method resulted in good preservation fo the myofibrils, mitochondria, sarcoplasmic reticulum and transverse tubules (T-tubules), which was confirmed by TEM studies of conventionally prepared tissue and of tissue originally prepared for SEM.

Substructure of inner mitochondrial membranes of myocardial cells as shown by cryo-ultramicrotomy.

The substructure of the inner mitochondrial membranes has been studied by cryo-ultramicrotomy under conditions during which denaturation of proteins by treatment with chemical solutes has been totally avoided. In such preparations, the inner membrane has a substructure consisting of globular subunits. These subunits have an average diameter of ca. 20 A-ca. 62 A and are fairly regularly spaced. Intracristal space is absent in the unstained, freeze-dried preparations, whereas a space of ca 40 A is seen in preparations lightly treated by OsO4-vapour. It is concluded that the subunits of the inner mitochondrial membranes probably consist either of single protein molecules or of complexes of protein molecules.

Preservation of shock-frozen myocardial tissue as shown by cryo-ultramicrotomy and freeze-fracture studies.

Myocardial tissue was quickly quench-frozen by letting the specimen fall either into liquid nitrogen (LN2) or into LN2-slush, or, by drill-propelling the specimen into LN2 at a speed of 1.5 m/s. The preservation of the tissues was studied in ultrathin, dry-cut and freeze-dried cryo-sections. Shock-freezing by propelling into LN2 yields extensive areas of well preserved tissue without hole damage. Quenching into LN2-slush shows variable results, while a maximum of hole damage is obtained by letting the specimen fall into LN2. Replicas of freeze-fractured tissue which had been quench-frozen by the same methods confirmed these observations. By the drill-propelling, areas 2000-5000 micrometer2 wide reveal good preservation without visible ice crystals. Such areas occur within a superficial band of tissue to a depth of c. 20 micrometer. Even at 35 micrometer and 50 micrometer depth good preservation may be registered at the periphery of the replicas. Description is given of modifications in accessories and methods in order to make the LKB CryoKit more suitable as a routine instrument. These modifications cover replacement of the knife coolant container by a coolant brass container in open connection with three copper tubes, replacement of one of the LKB Dewar flasks by a 25 litre Dewar equipped with a Balzers filling device, and replacement of the LKB plastic sleeve fixed to the back of the specimen coolant container, by two perspex-glass discs mounted in the slit between the Ultrotome base and the cryo-chamber. In addition, modified constructions are presented of the grid-carrier, the press-assembly as well as of the container for freeze-drying and warming up of the frozen sections.

Primary 9 + 0 cilia in the embryonic and the adult human heart.

Primary 9 + 0 cilia have been found in the embryonic and the adult human heart. Proximally the cilia show the typical 9 + 0 filament arrangement. Rearrangement of the filaments occur in their distal regions. The cilia are usually found in deep invaginations of the cell membrane, but can also be found in a superficial position. Close connections are frequently found between cilia and thin cytoplasmic extensions from neighbouring cells.

Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis.

Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N2-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600 A-900 A in diameter in the normal myocardium of the sand rat and the mouse and from 250 A-400 A in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.

Ultrastructure of abnormal membrane inclusions in nuclei of human myocardial cells.

The ultrastructure of the nuclear envelope was studied in cardiac muscle cells of 12 patients undergoing cardiopulmonary bypass surgery. Two main types of membrane inclusions, globular and tubular, were observed in the nuclei. The globular type was found in 6 patients. The globular inclusions were about 0-5mu to 1-5mu in diameter and lined by two unit membranes equal in structure to that of the nuclear envelope. The lining was probably always in continuity with the nuclear membranes. Such inclusions contained a granular matrix in which no cytoplasmic organelles could be seen, with the exception of a limited number of ribosomes. The tubular type of nuclear inclusion was observed in the cardiac tissue of 3 patients. These tubules ran in straight or slightly bent segments of about 0-2 mu to 1-6 mu length, interrupted by bendings at obtuse or right angles. The diameter of their lumina measured about 300 A to 800 A. Such tubules were nearly always found in areas of the nucleoplasm rich in heterochromatin. Nuclear pore-like structures were occasionally found in the tubular wall. The tubular wall consisted of a single unit membrane shown to be in continuity with the inner membrane of the nuclear envelope. Since these nuclear aberrances have been observed at all of the stages examined, the possibility is considered that they may represent a specific nuclear response to the process of cardiac hypertrophy.

Ultrastructural studies on the growth of filaments and sarcomeres in mechanically overloaded human hearts.

The ultrastructure of myocardial cells was studied in four patients with left ventricular myocardial hypertrophy due to aortic and/or mitral valvular disease. Signs of cellular lysis and synthesis were present in juxtanuclear, interfibrillar, and subsarcolemmal areas. Subsarcolemmal areas contained a granular matrix with foci of polyribosomes, mitochondria, well preserved membranes, a proliferating sarcoplasmic reticulum, and thick and thin filaments and Z-band formations at different stages of development. Z-band substance and thin filaments appeared in the cytoplasm in close proximity to free polyribosomes and endoplasmic membranes and were seemingly not associated with the sarcolemma. These findings were interpreted as evidence for a proteosynthetic activity leading to the formation of new myofibrils and sarcomeres during the chronic stage of hypertrophy. Comparative studies showed that this process has much in common with the genesis of contractile cardiac units in the mammalian embryo. Anomalous Z-band accumulations were studied in normal myocardial cells of various vertebrates and in cells of the overloaded human heart. No evidence of a proteosynthetic activity was detected in the expanded Z-bands.

Ultrastructure of mitochondria-containining nuclei in human myocardial cells.

The ultrastructure of myocardial cell nuclei was examined in a group of 12 patients undergoing cardiopulmonary bypass surgery. In one patient (aged 60 years) with rheumatic heart disease, myocardial cells were observed which contained mitochondria with well preserved membranes within their nuclei. Such intranuclear mitochondria were observed with equal frequency in all of the three stages examined, i.e. at the start and end of aortic crossclamping and after 20 min of reperfusion. Approximately 2-3% of the total number of nuclei studied in this patient contained mitochondria. A partial disintegration of the membranes of the affected nuclei was sometimes seen. These findings are discussed in relation to the hypothesis of a de novo synthesis of mitochondria inside the nucleus, and also, in relation to the "trapping theory" of mitochondria through the nuclear envelope.

Granule containing cells and fibres in the sinus venosus of elasmobranchs.

The concentrations of catecholamines in the heart chambers of elasmobranchs were measured by the fluorimetric method of Bertler et al. (1958). Noradrenaline (NA) can be detected in all the chambers, but the sinus venosus is by far the richest in NA. This can either be due to the presence of storage sites for this amine in the sinus wall, or to a transport of amine to the sinus venosus from the anterior chromaffin bodies. The sinus wall contains large numbers of "granule containing cells" and axon-like processes, both with numerous dense-core vesicles of about 1800 A diameter. The dense-core vesicles contain a uranophilic matrix indicating the presence of protein, phospholipids and/or nucleic acid. The reactions failed to demonstrate amine, which may be due to a loss of amine by diffusion, to a relatively low intravesicular amine concentration, or, to the absence of amines in these granule-containing cells and processes. Heavy accumulations of granule-containing processes occur in the subendothelial area. The endothelium contains fenestrae and pores through which granule-containing fibres protrude into the venous cavity. Granule-containing cells are innervated by presumed cholinergic nerve endings. It is suggested that the granule-containing cells and fibres belong to the neurosecretory system with a cholinergic input, releasing the contents of the dense-core vesicles into the blood stream at the level of the venous cavity.

Troponin bound calcium in electron micrographs of myocardial cells as demonstrated by the potassium-antimonate technique.

Following the K-antimonate reaction in atrial myocardial tissue, a pattern of evenly spaced cross striations of antimonate precipitates is demonstrated along the myofilaments. This spacing, found in both turtle and mouse atria, has a periodicity of about 400 A. In order to test the shifts of the antimonate reaction product in the tissue, a comparison is made between the localization of the antimonate precipitate as seen in viz. thin plastic sections and in cryo-ultra sections being dry-cut at -90 degree C from N2 frozen tissue. Preliminary results suggest only minor distributional differences in the sarcomeric pattern. On the basis of these tests, and, on the basis of previous studies by means of X-ray microanalysis, it is suggested that the periodic pattern of evenly spaced precipitates, reflects the localization of troponin bound calcium along the thin filaments during contraction.

A comparative study in the transmission electron microscope and scanning electron microscope of intracellular structures in sheep heart muscle cells.

The internal cellular structures of the sheep ventricular myocardium have been comparatively studied in the transmission electron microscope (TEM) and in the scanning electron microscope (SEM). For TEM studies the tissue was prepared according to standard methods. Thick sections (10 mum) of paraffin embedded material were, after they had been deparaffinized in toluene, critical point dried, coated with gold and examined in the SEM. The comparative TEM and SEM investigations revealed very good correspondence, and it is evident that the described preparation procedure for SEM has preserved the fine structures of myofibrils, mitochondria, T-Tubules and sarcoplasmic reticulum in an excellent life-like pattern. Of special interest was the three-dimensional demonstration of triads and circumferentially arranged T-tubules.

Identification of nerve fibres in the scanning electron microscope.

The use of silver as a "stain" for nerve fibres in scanning electron microscopy (SEM) preparations has been investigated. Samples of cardiac tissues were treated according to the Bielschovsky silver impregnation method. Following embedding in paraffin, successive sections were selected for light microscopy and for SEM studies, respectively. The silver impregnated fibres, when examined in SEM preparations, appeared nearly white against a greyish background of cardiac tissue. They were therefore easy to localize even at a low magnification. These nerve fibres were identified in the same tissue, but different blocks by means of transmission electron microscopy (TEM) studies and by fluorescence microscopy.