Histochemical investigations of the accumulation of lipids in heart papillary muscle

Synopsis The lipid composition and fatty change of the papillary muscle, arterioles and chordae tendineae of the left cardiac ventricle of sixty-two patients who had died of various diseases in hospital were analysed qualitatively by histochemical techniques and thin-layer chromatography.The histochemical techniques revealed considerable differences in the fatty change of the myocardium, arterioles and chordae tendineae. The myocardial change seems to be due solely to accumulation of triglycerides. The fatty change in the chordae tendineae appears to be the result mainly of accumulation of cholesterol esters and, to some extent, of free fatty acids. The arteriolar fatty change arises mainly from accumulation of cholesterol esters and some triglycerides. The myocardial and arteriolar fatty change is greatest at the tip of the papillary muscle.The results of thin-layer chromatography showed that the lipid composition of the papillary muscle was qualitatively highly constant. The triglycerides and free fatty acid content varied.The osmium tetroxide--naphthylamine technique, which indicates phospholipids, stained the cardiac and arteriolar muscle fibres uniformly. No focal staining, suggestive of fatty change, was noted.All three tissue sites studied reacted with Baker's acid Haematein test. Staining was focal and clearly associated with fatty change. The results observed suggested that Baker's acid Haematein test is not specific for phospholipids, but it may stain neutral fats and cholesterol esters whenever large amounts of these are present in a tissue.     

Neuropeptide Y modulates the action of vasodilator agents in guinea-pig epicardial coronary arteries

Recently, we have demonstrated that guinea-pig epicardial coronary arteries are supplied by numerous nerve fibres containing neuropeptide Y (NPY) immunoreactivity. However, examination of vasomotor responses revealed that NPY did not elicit a contractile response in these arteries. In contrast, acetylcholine (ACh), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) all relaxed precontracted arteries. In the present study, we have used histochemical, immunohistochemical and in vitro pharmacological techniques, in order to further investigate the possible role of NPY in guinea-pig epicardial coronary arteries. A double-immunofluorescence staining technique revealed that CGRP and substance P were co-localized in nerve fibres distinct from those displaying NPY immunoreactivity. Furthermore, using a method combining immunofluorescence and histochemical techniques, we observed that putative cholinergic nerve fibres (identified by their acetylcholinesterase content) and NPY-immunoreactive nerve fibres are two different nerve populations. An in vitro pharmacological method demonstrated that NPY markedly inhibited the relaxant responses mediated by ACh, VIP, substance P and isoprenaline but had no effect on CGRP. These results suggest that NPY-containing nerves associated with guinea-pig epicardial coronary arteries may be predominantly involved in modulating the action of vasodilator agents.     

Skeletal-muscle sarcolemma from normal and dystrophic mice. Isolation, characterization and lipid composition.

1. Mouse skeletal-muscle sarcolemma was isolated, and the preparations obtained from normal mouse muscle and from muscle of mice with hereditary muscular dystrophy were characterized with respect to appearance under the optical and electron microscopes, distribution of marker enzymes, histochemical properties and biochemical composition. 2. The sarcolemmal membranes from normal and dystrophic muscle were subjected to detailed lipied analysis. Total lipid content was shown to increase in sarcolemma from dystrophic mice as a result of a large increase in neutral lipid and a smaller increase in total phospholipids. Further analysis of the neutral-lipid fraction showed that total acylglycerols increased 6-fold, non-esterified fatty acid 4-fold and cholesterol esters 2-fold, whereas the amount of free cholesterol remained unchanged in sarcolemma from dystrophic muscle. Significant increases were found in lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in dystrophic-muscle sarcolemma; however, the relative composition of the phospholipid fraction remained essentially the same as in the normal case. 3. The overall result of alterations in lipid composition of the sarcolemma in mouse muscular dystrophy was an increase in neutral lipid compared with total phospholipid, and a 4-fold decrease in the relative amount of free cholesterol in the membrane. The possible impact of these changes on membrane function is discussed.     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)     

Molecular forms and localization of acetylcholinesterase and nonspecific cholinesterase in regenerating skeletal muscles

Molecular forms and histochemical localization of acetylcholinesterase and nonspecific cholinesterase were analysed in muscle regenerates obtained from rat EDL and soleus muscles after ischaemic-toxic degeneration and irreversible inhibition of preexistent enzymes. Regenerating myotubes and myofibres produce the 16S AChE form in the absence of innervation. The 10S AChE form prevails over 4S form with maturation into striated fibres. Although the patterns of AChE molecular forms in normal EDL and soleus muscles differ significantly no such differences were observed in noninnervated regenerates from both muscles. Two types of focal accumulation of AChE appear on the sarcolemma of regenerating muscles: first, in places of former motor endplates and, second, in extrajunctional regions. The 4S form of nonspecific cholinesterase is prevailing in regenerating myotubes whereas its asymmetric forms or focal accumulations could not be identified reliably. The satellite cells which survive after muscle degeneration probably originate from some type of late myoblasts and transmit the information concerning the ability to synthesize the asymmetric AChE forms and to focally accumulate AChE to regenerating muscle cells. Synaptic basal lamina from former motor endplates may locally induce AChE accumulations in regenerating muscles.Special Issue Dedicated to Dr. Abel Lajtha.     

A review of clinical trials in dietary interventions to decrease the incidence of coronary artery disease

Of the associations between dietary elements and coronary artery disease (CAD), the greatest body of evidence deals with the beneficial effect of reducing the dietary intake of saturated fatty acids and cholesterol. Furthermore, it is well established, on the basis of convincing evidence, that reduction in serum total cholesterol results in reduction in coronary morbidity and mortality, as well as in regression of other atherosclerotic manifestations.In fact, dietary intervention studies revealed that it is possible to reduce the incidence of coronary death and nonfatal myocardial infarction, as well as manifestations of atherosclerosis in cerebral and peripheral arteries, by reducing dietary intake of saturated fat and cholesterol. In two recently reported dietary interventions the incidence of coronary events, especially coronary mortality, and total mortality were reduced by increased intake of n-3 long-chain polyunsaturated fatty acids and by a modification of the diet toward a Mediterranean-type diet (rich in α-linolenic acid. In addition to those findings, the potential efficacy of the dietary newcomers phytostanol and phytosterol esters on reducing coronary incidence is discussed in the present review.     

Effects of phorbol 12-myristate 13-acetate on triglyceride and cholesteryl ester synthesis in cultured coronary smooth muscle cells and macrophages

In cultured pig coronary smooth muscle cells phorbol 12-myristate 13-acetate (PMA) stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and the incorporation of [2-3H]glycerol into triglycerides 6.4- and 4.5-fold, respectively. The maximal effects occurred after 3 h of treatment and there was a return to basal values after 72 h. In the presence of 400 microM oleic acid, PMA stimulated the conversion of [4-14C]cholesterol into cholesteryl esters and that of [2-3H]glycerol into triglycerides 5.3- and 2.3-fold, respectively. The stimulatory effects were more sustained (still significant after 72 h) and their maxima were delayed (peaks after 24 h). PMA was also found to increase 2-fold the amount of triglyceride that accumulated in the cells in the presence of oleic acid after 24 h. In macrophages IC-21, the effects of PMA were observed only in the presence of oleic acid. They consisted of a 1.9-fold stimulation in the conversion of [4-14C]cholesterol into cholesteryl esters after 72 h and of a 1.7-fold stimulation in the incorporation of [2-3H]glycerol into triglycerides after 24 h. PMA also increased the amount of triglyceride that accumulated in the cells 1.9-fold after 72 h. It is concluded that PMA, and possibly growth factors, may promote lipid storage in smooth muscle cells and that fatty acids favor long lasting effects of PMA in smooth muscle cells and are necessary for any effect of PMA in macrophages.     

Disorders of myocardial contraction and cardiomyocyte ultrastructure after emotional stress

By means of physiological, electron microscopical and histochemical methods, changes in intensity of mechanisms participating in calcium transport and the cations localization in cardiomyocytes have been studied, the changes resulted from an emotional-painful stress. After the stress, the contractile function of the rat isolated heart becomes more dependent on calcium concentration in the perfusate. As demonstrate combined electron microscopical and histochemical methods and, first of all, determination of calcium antimonate in the cardiomyocytes, together with increased cardiac function, an elevated amount of calcium localized in sarcoplasm of cardiomyocytes is observed, especially in the subsarcolemmic area. The data obtained demonstrate that the membrane mechanisms of calcium transport are injured and calcium content in the sarcoplasm is increased; that can play a definite role in the development of focal lesions in the myocardium at a stress.     

"Red" fibres of pigeon pectoralis major muscle are "type II red".

On the basis of the histochemical activity of succinic dehydrogenase, only two fibre-types are distinguished in pigeon pectoralis major muscle. These are narrow "Red" and broad "White". The histochemical activity of myofibrillar ATPase was studied in these two distinct fibre-types. Both fibre-types showed high activity for the ATPase. "Red" fibres of pigeon pectoralis were not alkali-labile, at incubation pH 9.4, as were the "Type I" fibres of both avian and mammalian muscles. Again unlike "Type I" fibres, the "Red" fibres of pigeon pectoralis lacked the characteristic activation of acid-preincubated ATPase reaction. Pigeon pectoralis "Red" fibres are known to possess some characteristics of fast-twitch fibres (e.g. high fat, considerable phosphorylase, fibrillenstruktur myofibrillar arrangement, focal "en plaque" pattern of nerve endings). It is emphasized, therefore, that the pigeon pectoralis "Red" fibres are not equivalent to "Type I or slow-twitch", muscle fibres, but they are possibly "fast-twitch fatigue resistent or Type II Red" muscle fibres.     

The effect of low density lipoproteins, cholesterol, and 25-hydroxycholesterol on apolipoprotein B gene expression in HepG2 cells.

The purpose of the present study was to examine the effects of exogenous cholesterol on the apolipoprotein (Apo) B gene expression in HepG2 cells. Pure cholesterol had no significant effect on either the cellular content of cholesteryl esters or the net accumulation of neutral lipids and ApoB in the culture medium. By contrast, addition of 25-hydroxycholesterol increased the net accumulation of cholesteryl esters in cells and medium by 2-3-fold and decreased that of unesterified cholesterol by 50% in both compartments. A 33% reduction in the cellular content of triglycerides was commensurate with a 40% increase in their accumulation in the medium. A significant 3-fold increase in the net accumulation of ApoB in the medium was predominantly due to enhanced secretion of newly synthesized ApoB as established by pulse-chase studies. The stimulation in ApoB secretion was accompanied by a 55% increase in cellular ApoB mRNA. Under these experimental conditions, the low density lipoprotein receptor activity was decreased by only 12-20%. Addition of progesterone prevented the effects of 25-hydroxycholesterol. The changes in the concentration of neutral lipids and ApoB were reflected in the composition of secreted "low-density" lipoproteins. These particles had increased percentage contents of cholesteryl esters and ApoB and a decreased percentage content of unesterified cholesterol in comparison with lipoproteins produced by control cells. The rate of ApoB production was not correlated with the triglyceride mass in the cells but was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. With the exception of unchanged cellular unesterified cholesterol and ApoB mRNA levels, plasma low density lipoprotein had similar, although less pronounced, effects on the production of neutral lipids and ApoB. These results demonstrate that in HepG2 cells the synthesis and secretion of ApoB and cholesteryl esters are tightly coupled and that 25-hydroxycholesterol increased the concentration of ApoB-containing lipoproteins primarily by stimulating their production rather than reducing their catabolism.     

Identification of [I]endothelin binding sites in human coronary tissue

In vitro receptor autoradiography has been used to study the distribution of [¹²⁵I]endothelin binding sites in human coronary tissue from patients undergoing cardiac transplantation. Dense binding of [¹²⁵I]endothelin was associated with the smooth muscle of epicardial coronary arteries as well as to perivascular regions of these vessels. Binding was also associated with the ventricular myocardium. There was an increased binding of [¹²⁵I]endothelin to atheromatous tissue, both coronary arteries and vein graft.

The [¹²⁵I]endothelin binding sites identified using in vitro autoradiography are likely to be functionally relevant since endothelin causes a concentration-dependent contraction of segments of human epicardial coronary arteries in vitro and also has positive inotropic activity on isolated human cardiomyocytes.

The presence of specific binding sites for [¹²⁵I]endothelin on coronary tissue and the increased binding in atheromatous tissue suggest that endothelin is a peptide which may play a role in the maintenance of vascular tone and/or the pathogenesis of ischaemic heart disease.     

Effects of saturated and unsaturated fats given with and without dietary cholesterol on hepatic cholesterol synthesis and hepatic lipid metabolism

Hepatic cholesterol synthesis was studied in rats after consuming diets of varying neutral lipid and cholesterol content. Cholesterol synthesis was evaluated by measuring 3-hydroxy-3-methylglutaryl-CoA reductase and by determining the rate of 3H-labeled sterol production from [3H]mevalonate. Results were correlated with sterol balance data and hepatic lipid content. Hepatic cholesterol synthesis was relatively great when cholesterol was excluded from the diet. The source of neutral dietary lipids, saturated vs. unsaturated, produced no change in hepatic sterol synthesis. Values for fecal sterol outputs and hepatic cholesterol levels were also similar in rats consuming either saturated or unsaturated fats. When 1% cholesterol was added to the diet, hepatic cholesterol synthesis was suppressed but the degree of suppression was greater in rats consuming unsaturated vs. saturated fats. This was associated with greater accumulation of cholesterol in livers from rats consuming unsaturates and a reduction in fecal neutral sterol output in this group as opposed to results from rats on saturated fats. Cholesterol consumption also altered the fatty acid composition of hepatic phospholipids producing decreases in the percentages of essential polyunsaturated fatty acids. It is concluded that dietary cholesterol alters cholesterol and fatty acid metabolism in the liver and that this effect is enhanced by dietary unsaturated fats.     

Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: production of very low density lipoproteins and bile acids

Primary cultures of rabbit hepatocytes were used to investigate the effect of purified (B-100 free) chylomicron remnants (CR) on lipid and bile acid metabolism. ApoB-100-containing lipoproteins were removed from the CR-enriched plasma fraction by affinity column chromatography on Sepharose 4B conjugated with anti-apoB-100 monoclonal antibodies. CR were shown to stimulate the accumulation of neutral lipids in hepatocytes in a dose-response manner. After 24-hour preincubation of rabbit hepatocytes with 50 micrograms protein/ml CR the cellular neutral lipid content increased: 1.9-4-fold for triglycerides, 1.5-3.7-fold for free cholesterol and 1.5-2.5-fold for esterified cholesterol. This accumulation was accompanied by a decreasing incorporation of [14C] acetate into cholesterol (80-90%) and triglycerides (70-80%). At the same time the incorporation of [14]oleate into triglycerides increased by 50-65%. The inhibited biosynthesis of fatty acids might account for this effect. No effect of CR on cholesterol esterification by [14C]oleate was observed. CR increased the amount of triglycerides and free cholesterol secreted in very low density lipoproteins (VLDL). The secretion of taurocholic acid was decreased. These data confirm our hypothesis that dietary cholesterol is preferentially secreted by hepatocytes within VLDL but is not accumulated as cholesterol esters or oxidized to bile acids.     

Intracellular cholesterol accumulation is accompanied by enhanced proliferative activity of human aortic intimal cells

Cholesterol accumulation in smooth muscle cells of unaffected human aortic intimal tissue occurred in the following conditions: (1) incubation of cells with atherogenic blood serum from patients with coronary heart disease (CHD), (2) cultivation of cells in the presence of insoluble associates containing low density lipoprotein (LDL). Preincubation of cells with blood serum from CHD patients resulted in a 2-5-fold increase in intracellular cholesterol and in 1.5-3-fold increase in cellular [3H]thymidine uptake. Blood serum collected from healthy donors had no significant effect on cultivated smooth muscle cells. When intimal cells were preincubated with insoluble associates containing LDL and components of fibrous extracellular matrix, the level of intracellular cholesterol increased from 2-4 times and uptake of [3H]thymidine increased 1.5-2.5-fold. Thus, a strong correlation was found between [3H]thymidine incorporation and intracellular cholesterol accumulation. The current study suggests that intracellular lipid accumulation may stimulate the proliferative activity of human aortic intimal cells from uninvolved tissue.     

Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells

We describe herein the effects of Marek's disease herpesvirus (MDV) on cholesterol and cholesteryl ester metabolism in cultured chicken arterial smooth muscle cells. Infection of arterial smooth muscle cells from specific pathogen-free chickens with MDV, but not a virus control, herpesvirus of turkeys led to a 7-10-fold increase in the accumulation of free and esterified cholesterol and a 2-fold increase in phospholipids. The cellular lipid changes observed in the MDV-infected arterial smooth muscle cells resulted, in part, from the following: decreased low-density lipoprotein-cholesteryl ester hydrolysis due to decreased lysosomal (acid) cholesteryl ester hydrolytic activity; increased de novo synthesis of cholesterol; decreased excretion of free cholesterol; and, both increased cholesteryl ester synthetic activity and decreased cytoplasmic (neutral) cholesteryl ester hydrolytic activity which resulted in increased incorporation of oleic acid into cholesteryl ester. Other changes noted in the MDV-infected cells as compared to uninfected cells included a 2-fold increase in both total protein synthesis and lysosomal and microsomal marker enzyme activities. These alterations in lipid and protein metabolism in MDV-infected arterial smooth muscle cells may explain in part our in vivo findings that herpesvirus (MDV) infection of specific pathogen-free chickens fed a normocholesterolemic diet will induce arterial thickening and lipid accumulation resembling human atherosclerosis.     

Histochemical reactions of myocardial proteases during open heart surgery

Histochemical analysis of some lysosomal and sarcoplasmic proteolytic enzymes was assayed in human myocardial biopsies taken from 26 cardiopathic patients subjected to open heart operations, under extracorporeal circulation and protection with cardioplegic solution and hypothermia. The investigated myocardial proteases were: cathepsin B, cysteine aminopeptidase, acid gelatinases, trypsin-like endopeptidase, chymotrypsin-like endopeptidase and neutral gelatinases. The effects of surgical interventions appreciated by comparing the myocardium fragments harvested before, and at various intervals after aorta clamping (6-90 minutes) revealed disorders in the activity and compartmentalization of all the investigated proteases, whose histochemical reactions increased between 10 and 20 minutes after aorta clamping and manifested a lowering tendency with sarcoplasmic diffusion and extracellular release at longer periods than 20 minutes. The early activation of the neutral proteases and their sarcolemmal expression even before 10 minutes after aorta clamping, suggested the involvement of the nonlysosomal proteases in the first proteolytic events implied in the molecular membrane damage of the myocardial fibre. Sequential proteolytic cascades of abnormal neutral and acid proteases were emphasized as possible mediators and effectors of molecular and subcellular damages suffered by the myocardial fibers during the open heart operations, even under cardioplegic and hypothermic protection.     

Lipid storage myopathy: successful treatment with propranolol.

Lipid storage myopathies are a rare but serious cause of muscle weakness characterised by the accumulation of abnormal amounts of neutral fat in type 1 fibres. A case is reported in which the patient presented with weakness of the proximal limb muscles and greatly increased activities of creatine kinase and lactate dehydrogenase. After two years lipid myopathy was diagnosed when electron microscopy confirmed the presence of large numbers of lipid particles within muscle fibres. Twelve years after the initial presentation propranolol (40 mg thrice daily) was started. Strength gradually improved and enzyme activities returned to normal. The improvement in the patient''s condition was almost certainly due to the propranolol, although the mode of action of the drug remains unknown.     

So-called muscular "bridges" and their interrelationship with blood vessels]

Fifty hearts of corpses of humans of different age groups were studied by means of macro-microscopical anatomical dissection of muscle fibres and cardiac blood vessels. The muscle bridges were found over the coronary arteries in all preparations of the heart. They were most frequently met on the anterior interventricular branch of the left coronary artery. The topography of muscle bridges is described and the miocardium muscle bundles participating in their formation are revealed. The character of the course of fibres in the muscle bridge is related to the course of the fibres of the muscle bundle forming it.     

Quantitative succinate-dehydrogenase histochemistry. III. Variations in histochemical succinatedehydrogenase activity in different cross-sections of the same muscle fibre.

The variation in histochemical SDH-activity at different levels in the same muscle fibre was determined in muscle fibre cross-sections both by visual classification and quantitative determination of the formazan-deposits. This work resulted in a confirmation of the earlier micro-biochemical studies of Spamer AND Pette (1977, 1979) and Lowrey et al. (1978) that the activity of enzymes of the citric acid cycle is not homogeneously distributed in a muscle fibre over its entire length. In addition it is shown that the observed variations in histochemical SDH-activity strongly interfere with the visual muscle fibre typing. Some of the possible causes for these variations in histochemical SDH-activity (section-thickness, presence of the motor-endplate) and the implications of these findings for the relation between histochemical characteristics and functional properties of the muscle fibres are briefly discussed.     

Various aspects of lipid metabolism in the rat testicle after sulpiride treatment]

Testicular lipids were extracted from 20 rats which had received 40 mg sulpiride per kg per day for 20 days as well as from 20 controls. The extracts were weighed and chromatographed on thin layer in order to analyse them for the main fatty constituants : phospholipids and neutral fats. In addition total cholesterol, triglycerides and lipid phosphorus were determined. The total testicular lipid content and the phospholipids were unaltered. The distribution of phospholipids wasn't influenced either. Among the neutral fats the precursors of steroid hormones such as cholesterol and its esters didn't show any modification. The triglycerides on the other hand suffered a significant decrease which was partly compensated in situ by the increase of free fatty acids. Since plasma electrophoresis didn't reveal a parallel increase of the complex albumin--free fatty acids, the hypothesis of an in situ lipolysis is proposed. But it is not possible yet to attribute to sulpiride the role of a direct activation on a testicular triglyceride lipase nor that of an indirect activator by way of prolactine for example.