Thorvald Sætersdal

Ultrastructure and innervation of the teleostean atrium.

Abstract Since the vagus nerve is regarded as the only pathway of effector innervation to the heart in teleosts, the teleostean atrium was chosen as a model of a purely parasympathetic innervated cardiac tissue. The atrial muscle cells contained a well developed sarcoplasmic reticulum, peripheral couplings and large aggregations of atrial granules, but no transverse tubules. Short nexuses were common. The endothelium consisted of two different types of cells. One type contained small, dense granules within bodies of moderate electron density. The granules were stained by a modification of the chromaffin reaction, indicating the presence of catecholamines. The atrium was heavily innervated by myelinated and unmyelinated axons of large diameter. The latter were surrounded by one or more loose turns of Schwann cell expansions. Silver stained structures were mostly found to have a neural origin. Axons ran beneath the endothelium, between the muscle cells, and, ensheated by endothelial cells, in fibres between the trabecules. Vesiculated, cholinergic nerve processes made close contacts with the surface of the muscle cells, which showed a certain degree of postsynaptic specialization. Vesiculated axons were found in close relation to the catecholamine-storing endothelial cells. It is suggested that these fibres may cause a release of catecholamines from the moderately dense bodies, capable of affecting the contractility of the myocardium.

Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy

SummaryWe have investigated the associations between β-tubulin and mitochondria in freshly isolated cardiac myocytes from the rat. Beta-tubulin was identified by using monoclonal antibodies for immunofluorescence and high resolution immunogold electron microscopy. In addition, conventional transmission and scanning electron microscopic studies were performed. After chemical stabilization in a formaldehyde solution, the myocytes were shock-frozen at −150°C, cryosectioned at −70°C and subsequently processed for immunohistochemical and immunocytochemical microscopy. A characteristic of the rod shaped myocytes is the presence of a dense network of microtubules in the cytoplasm displaying a pattern of strong anti-β-tubulin reaction. The complexity of this network however varies considerably among the myocytes reflecting microtubule dynamic instability. Further, our findings demonstrate that the β-tubulin label in rod cells is confined to the perinuclear and interfibrillar spaces and, therefore, is largely colocalized with the cytoplasmic organelles. In myocytes undergoing severe contracture the distribution of β-tubulin is entirely restricted to the outer mitochondrial-containing domain. This implies that, in a cell model with marked segregation of the contractile filaments and organelles, mitochondria are codistributed with microtubules in the total absence of desmin intermediate filaments. Moreover, our immunogold preparations demonstrate anti-β-tubulin labelling in the outer mitochondrial membrane as well as of fibres in close apposition to this membrane. These results indicate the presence of a specific β-tubulin binding to the outer mitochondrial membrane that probably also involves microtubule based translocators and/or MAPs.

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

SummaryPrimary 9+0 cilia have been found in the embryonic and the adult human heart. Proximally the cilia show the typical (+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.

The calcium paradox phenomenon: a flow rate and volume response study of calcium-free perfusion.

A dose-response study concerning the importance of the flow rate (0.5 to 12 ml/min) and volume (2.5 to 60 ml) of calcium-free coronary perfusion (duration 5 min) in the induction of a calcium paradox on reperfusion (duration 15 min) with calcium-containing medium has been performed in the isolated rat heart (37 degrees C). On the basis of enzymatic, physiological, and metabolic assessments three different levels of tissue injury were identified: a minimal paradox at 1.0 ml/min or 5 ml, a subtotal paradox at 2 ml/min or 10 ml and a total paradox at 9 ml/min or 45 ml. Ultrastructural examination revealed that cellular injury following calcium repletion was always severe, and that an increase in the flow rate and volume of calcium-free perfusion increased the number of severely injured cells. During calcium-free perfusion the external lamina largely remained intact over the surface coat of the sarcolemma, but variable degrees of separation of intercalated discs were observed. It is concluded that the calcium paradox model of myocardial injury presents a rather sharp threshold related to the flow rate or volume of calcium-free coronary perfusion and that on trespassing this threshold there is a narrow zone characterized by a decreasing number of viable cells. Furthermore, the study indicates that a separation of the external lamina from the surface coat of the sarcolemma is not a prerequisite for the induction of a calcium paradox, and that cell injury may occur in the presence of intact intercalated discs.

Effects of isoproterenol on the dense core and perigranular membrane of atrial specific granules

SummaryFollowing subcutaneous injections of isoproterenol hydrochloride (ISO), atrial cells present a large number of partly degranulated or completely clear “specific granules” enclosed by an intact membrane. Such profiles were never encountered in normal controls and might suggest ISO-induced release of a secretory product. Permeability of perigranular membrane was tested using the extracellular macromolecular tracer horseradish peroxidase (HRP). Reaction product was entirely absent within granules of atrial cells in which the sarcolemma was made permeable to HRP molecules by the ISO injections. This seemed to be the case even in heavily labelled cells in which the peroxidase had penetrated the mitochondrial membranes. In atrial cells impermeable to the tracer, the specific granules closely apposed to the sarcolemma were always HRP-negative. The release mechanism of a possible secretory substance from the specific granules is discussed.

Regional appearance of atrial natriuretic peptide in the ventricles of infarcted rat hearts

SummaryThe appearance of atrial natriuretic peptide (ANP) in the ventricular myocardium was investigated in rat hearts subjected to severe left ventricular infarction. The left coronary artery was ligated for 1, 2, 3, 4 and 6 days and for 3 weeks, and the tissue was prepared for microscopic examination of immunoreactive ANP and for electron microscopy. In the normal and sham-operated hearts, and in hearts subjected to 1 day of coronary ligation, ANP immunoreactivity was restricted to a few ventricular myocytes of the conduction system. Following 2–3 days of coronary ligation, ANP immunoreactivity was detected in the viable myocardium of the lateral border of the infarct and in a few layers of viable cardiac myocytes located in the subendocardial areas of the ischemic left free ventricular wall. Further, during the following days and after 3 weeks of coronary ligation, a gradient of specific labeling was commonly seen across the lateral border area of the infarct. Thus, the strongest immunoreactivities were present in the cardiac myocytes located adjacent to the non-contracting myocardium. Electron microscopic examination of the immunoreactive cardiac myocytes confirmed the presence of electron-dense specific granules within these cells. The present findings suggest that the increased regional production of ANP within the ventricular myocardium is induced by increased mechanical stretch of the cardiac myocytes, and that this might contribute to the increased release of ANP in myocardial infarction.

Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against α-ANF and β-tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.

Ultrastructure of the pigeon papillary muscle with special reference to the sarcoplasmic reticulum

Abstract The contractile material of the pigeon papillary muscle cell, constitutes one single mass of myofilaments which contains sarcoplasmic tubules and parallel rows of mitochondria and lipid droplets. These rows do not demarcate separate myofibrillar subunits as in the skeletal muscle. The sarcoplasmic reticulum represents a well developed network of tubules of different sizes. Transversely orientated and dilated tubules are present both at the Z line-I band and H-line levels. The tubules at the Z line-I band levels, form belt-like cisternae penetrating the myocardial cell in close proximity to myofilaments and mitochondria. It is suggested that these cisternae, and possibly also the longitudinal parts of the sarcoplasmic reticulum, may function as intracellular storage sites for calcium. The structure and the functional significance of the peripheral couplings is discussed. Transverse tubules and, accordingly, internal couplings are not present in the tissue studied. Zebra bodies are commonly seen, interpolated between the sarcolemma and the contractile material of the cell. The observations indicate that their main function is contractile movements of actin-like myofilaments. It is suggested that these contractions may be likely to exert an adjusting and regulatory effect on the movements of the cellular surface during cardiac activity.

Regional activation of the immediate-early response gene c-fos in infarcted rat hearts

Regional infarction of the left ventricle is followed by hypertrophy of the viable myocardium. This compensatory growth of cardiac myocytes requires induction of gene transcription and synthesis of proteins. In this study, we examined the expression of the immediate‐early response gene c‐fos following ligation of the left coronary artery in rat hearts. RNase protection assay demonstrated a rapid increase in the c‐fos mRNA level in the ventricular myocardium. After two days of infarction, the c‐fos expression was attenuated and was comparable to that observed in sham‐operated control hearts. In situ tissue distribution of Fos protein‐like immunoreactivity revealed the appearance of positively stained cells adjacent to the lateral border of the ischaemic myocardium, in the left ventricular subendocardial areas, in the papillary muscles of the left ventricle, in the proximity of great transmural vessels, and focally in the normo‐perfused subepicardial myocardium. Double staining using antibodies recognizing the Fos protein and α‐actinin, confirmed that the accumulation of nuclear Fos protein‐like immunoreactivity was mainly seen in the cardiac myocytes. However, double staining of the Fos protein and Hoechst DNA labelling showed that detectable immunoreactivity occurred only in a limited proportion of the total nuclei present in these myocardial regions. Moreover, the regions showing c‐fos activation correspond to the areas in which the appearance of subsequent growth responses are most pronounced following myocardial infarction. The present results therefore indicate that an early and regional c‐fos activation is taking place in viable cardiac myocytes following left coronary artery ligation, and that c‐fos is a possible regulating factor of sequential events leading to altered pattern of gene expression and protein synthesis in the hypertrophying heart.

The application of various electron microscopic techniques for ultrastructural characterization of the human papillary heart muscle cell in biopsy material

Various electron microscopical techniques have been applied to biopsy material obtained from patients suffering from mitral stenosis in order to characterize the subcellular organization of the hypertrophied papillary muscle. Small pieces of the same sample were processed for correlative transmission - (TEM) and scanning -(SEM) electron microscopical studies. TEM was carried out on conventionally fixed tissue with or withouten bloc staining with a Cu-Pb citrate solution, and on freeze fracture replicas, while cryofractured material was studied by SEM. Stereo electron micrographs of the Cu-Pb impregnated tissue and of the cryofractured material were especially useful for studying the spatial distribution and relationship between various cell organelles. The myofilaments of the hypertrophied cells were arranged in a normal hexagonal pattern. Regions with irregular orientation of the myofibrils were occasionally seen. Accumulations of interfilamentous glycogen particles adjacent to the Z-bands were characteristic patterns of the contracted muscle cells. The extensive nexuses frequently observed in the subsarcolemmal regions may reflect functional alterations of the intercommunication between hypertrophied cells. The T-tubules were relatively few and irregularly distributed, and the complexity of the sarcotubular system (SR) revealed regional variations. Excellent visualization of the interior couplings between the SR and the T-tubules was achieved by studying thick sections of Cu-Pb impregnated tissue in the TEM. The dense staining of the various intracellular membranes when compared with the almost unstained external membranes including the free cell surface, intercalated disc and T-system, strongly indicates differences in chemical and functional properties of the two membrane systems.En bloc staining resulted also in contrasted glycogen as well as components of the nucleolus and the heterochromatin. The biochemical basis for the selective staining remains obscure; it may be a result of binding of heavy metal ions to carboxyl groups of specific proteins, and/or it may represent deposits of lead phosphate.