Biomedical subjects

H Dalen

Publications and source records attributed to H Dalen.

At least 37 records · Page 2Linked to original sources

PubMed1996

Suppression of intimal hyperplasia in experimental vein grafts by oral l-arginine supplementation and single ex vivo immersion in deferoxamine manganese.

PURPOSE: Vein grafts undergo morphologic and functional changes after insertion into the arterial circulation with the development of intimal hyperplasia, as well as significant alterations in endothelial and smooth muscle cell physiologic responses. METHODS: Forty New Zealand white rabbits underwent jugular vein interposition grafting of the common carotid artery. Ten animals were controls, 10 animals received 2.25% L-arginine supplementation in their drinking water (200 ml/day; 2 gm/kg) 7 days before surgery and continued thereafter until harvest, in 10 animals the veins were immersed in deferoxamine manganese (DFMn; 10(-3) mol/L in heparinized Ringer's lactate for 15 minutes) before implantation, and 10 received both L-arginine supplementation and either histologic (n=6) or isometric tension studies (n=4). The function of the vein grafts was compared with that of jugular veins. RESULTS: Treatment with DFMn, l-arginine, amd DFMn L-arginine produce increases in mean intimal thickness of 39% (51 +/ -7 microm; p<0.05), 51% (41 +/- 7 microm; p<0.05), and 65% (29 +/- 6 microm; p< 0.01), respectively, compared with control vein grafts (83 +/- 12 microm). Compared with the control group, the intimal ratio ([intima]/[intima + media]) decreased by 16% (difference not significant), 8% (difference not significant), and 47% (p<0.01) in the DFMn-, L-arginine- and and DFMn/L-arginine-treated vein grafts, respectively. Jugular veins relaxed to acetylcholine (53% +/- 12% maximal relaxation), whereas control vein grafts did not relax. In contrast, vein grafts from each of the experimental groups relaxed to acetylcholine with maximal relaxations of 26% +/- 7% (p<0.05 compared with the jugular vein), 22% +/- 8% (p<0.05), and 44% +/- 14% (difference not significant) in the DFMn, L-arginine, DFMn/L-arginine groups, respectively. Neither DFMn nor l-arginine had a significant effect on the alterations in smooth muscle contractility that occur in control vein grafts. CONCLUSION: This study demonstrates that an agent that modulates free radical production combined with a precursor of nitric oxide formation will lead to a significant decrease in the formation of intimal hyperplasia in arterial vein grafts with the preservation of endothelial-derived relaxation.

Administration, Oral↗

PubMed1996

Characterisation of angiotensin II receptor mediated responses and inhibition of intimal hyperplasia in experimental vein grafts by the specific angiotensin II receptor inhibitor, L158,809.

OBJECTIVES: This study characterises pharmacologically the angiotensin II receptor in experimental vein grafts and examines the effect of the angiotensin II receptor (type 1) antagonist (L158,809) on the formation of vein graft intimal hyperplasia in vivo, as well as the in vitro physiological response to angiotensin II of vein grafts after chronic oral L158,809 treatment. MATERIALS: Thirty New Zealand White rabbits had a right carotid interposition bypass graft using the external jugular vein and were killed on the 28th postoperative day. DESIGN: To characterise the angiotensin II receptors, concentration response curves to angiotensin II were obtained in vitro in the presence or absence of L158,809. To determine the effect of L158,809 on the development of intimal hyperplasia, 10 animals received chronic oral therapy with L158,809 (10 mg/kg/day; begun 5 days before surgery and continued until harvest) and 10 animals received vehicle only as controls. These grafts were harvested either for histology (n = 6 per group) or for in vitro isometric tension studies to angiotensin II. RESULTS: The monophasic contractile response to angiotensin II in the untreated vein grafts could be inhibited in a concentration dependent manner by L158,809 with first order kinetics. Chronic oral treatment with L158,809 produced a 48% decrease in intimal thickness from 82 +/- 1 micron (mean +/- S.E.M.) in the controls to 43 +/- 7 microns in the treated vein grafts (p = 0.002). There was also a significant decrease (45%) in the medial thickness between the control (76 +/- 6 microns) and L158,809 treated (42 +/- 6 microns) vein grafts (p = 0.007). The responses to angiotensin II were abolished in the vein grafts by chronic L158,809 therapy. CONCLUSIONS: This study suggests that vein graft angiotensin II responses are mediated through a type 1 receptor and that chronic inhibition with L158,809, significantly reduces intimal hyperplasia and medial hypertrophy in experimental vein grafts and concomitantly abolishes the in vitro responses to angiotensin II. Therefore, angiotensin II acting through AT1 receptors mediates a significant part of the intimal hyperplastic response in vein grafts.

Angiotensin II↗

PubMed1995

Control of accelerated vein graft atheroma with the nitric oxide precursor: L-arginine.

Hyperlipidemia contributes to the development of intimal hyperplasia and subsequent accelerated atherosclerosis in vein bypass grafts. This study examines the effect of dietary supplementation with L-arginine on the development of intimal hyperplasia and the vasomotor function of vein grafts in hypercholesterolemic animals. Thirty male New Zealand White rabbits had a right carotid vein bypass graft and were sacrificed at 28 days postoperatively. Twenty animals received a 1% cholesterol diet for 4 weeks prior to surgery and this diet was continued until harvest. Of these, 10 also received L-arginine (2.25%, 2 g/kg, p.o.) 7 days preoperatively and thereafter until harvest. The last 10 animals were controls. Vein grafts were harvested either for morphology or for in vitro isometric tension studies. Cumulative dose-response curves to norepinephrine, serotonin, and bradykinin were recorded, and following norepinephrine precontraction, relaxation to acetylcholine and sodium nitroprusside were determined. After in situ pressure fixation, intimal thicknesses of the vein grafts were measured by videomorphometry. The addition of L-arginine doubled the serum arginine concentrations. Intimal hyperplasia of both groups of hypercholesterolemic vein grafts contained foam cells and lipid-laden endothelial and smooth muscle cells. There was a 24% reduction in the intimal thickness of vein graft intimal hyperplasia in the L-arginine group compared to that in the hypercholesterolemia group (P < 0.05). All hypercholesterolemic vein grafts were two-fold thicker than in the control group. L-arginine supplementation resulted in the preservation of acetylcholine-mediated relaxation but did not change hypercholesterolemia-induced contractile agonist supersensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Exposure of cells to nonlethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization.

The cytotoxicity of hydrogen peroxide is, at least partly, mediated by the induction of intralysosomal iron-catalyzed oxidative reactions with damage to lysosomal membranes and leakage of destructive contents. We hypothesize that minor such leakage may be nonlethal, and the ensuing cellular degeneration repairable. Consequently, we investigated, using a model system of cultured J-774 cells, the effects of hydrogen peroxide in moderate concentrations on cellular viability, lysosomal membrane integrity, morphology, and ATP and reduced glutathione concentrations. These parameters were initially estimated directly after a 30 min exposure to a bolus dose of hydrogen peroxide in phosphate buffered saline at 37 degrees C, and then again following subsequent recovery periods of different lengths under ordinary culture conditions. All cells survived an exposure to 250 microM hydrogen peroxide for 30 min, whereas 350 and 500 microM exposure was lethal to a small fraction of cells. The oxidative stress caused early, time- and dose-dependent, partial relocalization of the lysosomotropic weak base acridine orange from the lysosomal compartment to the cytosol. This phenomenon is known to parallel leakage of damaging lysosomal contents such as hydrolytic enzymes. There were also signs of cellular damage in the form of surface blebbing and increased autophagocytosis, more marked with the higher doses of hydrogen peroxide. Also found was a rapid depletion of ATP and GSH. These alterations were all reversible, as long as cells were exposed to nonlethal amounts of hydrogen peroxide. Based on these and previous findings, we suggest that lysosomes are less stable organelles than has hitherto been assumed. Restricted lysosomal leakage might be a common event, for example, during sublethal oxidative stress, causing reversible, degenerative alterations, which are repaired by autophagocytosis.

Acridine Orange↗

PubMed1995

The influence of the combined presence of diabetes mellitus and hypercholesterolaemia on the function and morphology of experimental vein grafts.

OBJECTIVES: Diabetes and hypercholesterolaemia are known risk factors for the development of atherosclerosis and are considered to influence the development of vein graft intimal hyperplasia. This study examines the combined effect of diabetes for 12 weeks (alloxan-induced) and hypercholesterolaemia for 8 weeks (1% cholesterol diet) on the formation of intimal hyperplasia and the vasomotor function of vein grafts. MATERIALS AND DESIGN: Thirty-two New Zealand White rabbits underwent a carotid vein bypass graft. Eight were controls, eight were diabetic, eight were hypercholesterolaemic and eight had both diabetes and hypercholesterolaemia. All vein grafts were harvested at 4 weeks postoperatively for morphology (n = 4) or contractility studies (n = 4). RESULTS: Compared to controls, both diabetes and hypercholesterolaemia increased intimal thickness by 20% and 63% respectively; medial thicknesses of these vein grafts were unchanged compared to control. In contrast, diabetes with hypercholesterolaemia dramatically increased intimal and medial thickness (1.8 fold and 1.6 fold respectively, compared to control). Smooth muscle cell contractility was enhanced in both the diabetic and hypercholesterolaemic groups. The presence of diabetes with hypercholesterolaemia did not further alter the enhanced smooth muscle cell contractile responses. CONCLUSIONS: This study suggests that the combination of both the atherogenic risk factors, hypercholesterolaemia and diabetes, significantly augments the formation of intimal hyperplasia in experimental vein grafts.

Animals↗

PubMed1994

Systemic hypertension and hypercholesterolemia in vein grafts: effects on the function and morphology of experimental vein grafts.

Hypertension and hypercholesterolemia are known risk factors for the development of atherosclerosis and are considered to influence the development of vein graft intimal hyperplasia. This study examines the effect of hypertension (two-kidney, one-clip model for 8 weeks) and hypercholesterolemia (1% cholesterol diet for 8 weeks) individually and in combination on the formation of intimal hyperplasia and the vasomotor function of vein grafts. Forty New Zealand White rabbits underwent a carotid vein bypass graft. Ten were controls, 10 were hypertensive, 10 were hypercholesterolemic, and 10 had both hypertension and hypercholesterolemia. Hypertension and/or hypercholesterolemia were present for 4 weeks prior to and for 4 weeks after surgery. All vein grafts were harvested at 4 weeks postoperatively for histology (n = 6) or contractility studies (n = 4). Compared to controls, hypercholesterolemia increased intimal but not medial thicknesses of the vein grafts and enhanced smooth muscle cell contractility. Hypertension did not increase vein graft intimal or medial thicknesses but did augment vein graft contractility compared to controls. Hypertension with hypercholesterolemia increased intimal and medial thicknesses and enhanced vasoreactivity in vein grafts. The results show that hypertension influences the vasoreactivity but not the development of intimal hyperplasia in vein grafts. When hypertension is combined with hypercholesterolemia, there is both an increase in the medial thickness and an attenuation of vasomotor function compared to hypercholesterolemia alone, although there is no further increase in intimal hyperplasia formation. Therefore, this study suggests that the combination of both atherogenic risk factors does not act synergistically in promoting either the formation of intimal hyperplasia or vasomotor dysfunction in vein grafts.

Animals↗

PubMed1994

The functional and morphological consequences of balloon catheter injury in veins.

Endovascular procedures are now used to treat occlusive arterial and venous diseases. However, after arterial catheter injury, intimal hyperplasia develops and there is altered endothelial and smooth muscle cell vasoreactivity of the vessel segment. This study examines the morphology and vasoreactivity of veins which have undergone balloon catheter injury. Under direct vision, 22 jugular veins of 11 New Zealand White rabbits were catheterized with a Fogarty balloon catheter (4Fr, three passes, 0.75 ml H2O inflation). Ten uninstrumented jugular veins from 5 additional animals were used as controls. Immediately after balloon injury, 10 jugular veins were harvested. Scanning electron microscopy of these vessels confirmed complete endothelial denudation and no in vitro endothelium-dependent relaxation was observed. After 28 days, 12 veins were harvested. There were no occlusions. Histologically, the 28-day balloon-injured veins had an almost confluent endothelium. However, there was medial hypertrophy but no development of intimal hyperplasia. There were no significant changes in the contractile responses of the balloon-injured veins after 28 days to norepinephrine. Bradykinin and histamine responses were markedly attenuated. Following bradykinin precontraction, balloon-injured veins showed diminished acetylcholine sensitivity with a reduced maximal response, and a small decrease in serotonin sensitivity with a markedly decreased maximal response. In contrast, there was an increased sensitivity in response to sodium nitroprusside without a change in maximal response in precontracted balloon-injured veins. This study shows that after 28 days venous balloon injury does not induce the development of intimal hyperplasia but does reduce overall endothelial dependent relaxation (acetylcholine- and serotonin-mediated) with increased sensitivity to endothelial independent relaxation (sodium nitroprusside-mediated). These findings would suggest that when compared to arterial balloon injury, venous balloon injury does not carry the same histological and vasoconstrictive sequelae but does demonstrate similarly impaired endothelial cell function.

Animals↗

PubMed1994

Diabetes mellitus and experimental vein graft structure and function.

PURPOSE: Diabetes mellitus is a known risk factor for accelerated atherosclerosis and for postangioplasty restenosis. METHODS: This study examines the effect of chronic, uncontrolled, alloxan-induced diabetes on the structure and vasomotor function of vein bypass grafts in 20 male New Zealand white rabbits with diabetes and in 10 controls. After 8 weeks of diabetes, a common carotid vein bypass graft was performed. Four weeks after operation, vein grafts and contralateral jugular veins were harvested. RESULTS: Diabetes induced a twofold increase in the vein graft intimal thickness compared with control. There was no change in medial thickness. Electron microscopy of the vein grafts in diabetes revealed intercellular gaps in the endothelium lining and abnormal endothelial cell junctions compared with controls. Diabetes significantly increased the maximal contractions generated in vein grafts to all contractile agonists tested without any change in sensitivity. CONCLUSIONS: This study demonstrates that diabetes alters endothelial cell structure and increases the development of intimal hyperplasia with increased maximal contractility in vein grafts and therefore suggests that the vein grafts in diabetes are more susceptible to early stenosis.

Animals↗

PubMed1994

Control of the structural and functional consequences of vein graft intimal hyperplasia with a 21-aminosteroid--U74389G.

Following angioplasty and vein bypass grafting, there is endothelial cell injury, infiltration of leukocytes and smooth muscle cell (SMC) proliferation leading to intimal hyperplasia which may result in stenosis and can lead to eventual occlusion. This study examines the effect of the 21-aminosteroid U74389G (Upjohn Company), on the formation of vein graft intimal hyperplasia in vivo and on SMC DNA synthesis and proliferation in vitro. Twenty New Zealand White rabbits had a right carotid interposition bypass graft using the ipsilateral external jugular vein. Ten animals received chronic oral therapy with U74389G (25 mg/kg/day; begun 5 days before surgery and continued until harvest) and 10 control animals received vehicle only. All animals were sacrificed on the 28th postoperative day. Vein grafts were harvested either for histology/videomorphometry (n = 6 per group) or for in vitro isometric tension studies (n = 4; four 5 mm rings per graft). The incorporation of [3H]thymidine into the cellular DNA of serum-stimulated rabbit aortic SMC (passage 6th to 12th) was assessed in the presence of increasing concentrations of U74389G (10(-9) to 10(-4) M). The effect of U74389G on in vitro cell proliferation was also assessed. Treatment with U74389G produced a 44% decrease in overall mean intimal thickness from 82 +/- 1 microM (mean +/- S.E.M.) in the controls to 57 +/- 10 microM in the U74389G treated vein grafts (p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Effect of sodium butyrate treatment on the granule morphology, histamine level and elemental content of the bone marrow-derived mast cell.

Mast cells derived from the bone marrow of BALB/mice (BMMC) were cultured and their growth ceased with sodium butyrate. Sodium butyrate treatment (1mM, 4 days) caused maturation of the granules, an increased histamine content from approx. 1 pg/cell to 4 pg/cell. X-ray microanalysis revealed that maturation of the granules was accompanied by the increase in relative weight percent of sodium, phosphorus and sulphur, with concomitant decrease in chloride. The sulphur to potassium ratio increased three-fold in butyrate-treated mast cells. The existence of a different elemental composition during mast cell maturation may provide additional parameter for rapid discrimination of mast cell subpopulations.

Animals↗

PubMed1994

Postoperative reduction of high serum cholesterol concentrations and experimental vein bypass grafts. Effect on the development of intimal hyperplasia and abnormal vasomotor function.

Hypercholesterolemia is an important contributor to the development of intimal hyperplasia and superimposed accelerated atherosclerosis in vein bypass grafts. This study examines the effect of dietary modification of serum cholesterol on the development of intimal hyperplasia and vasomotor function of vein grafts. Thirty male New Zealand White rabbits had a right carotid vein bypass graft and were put to death 28 days after the operation. Twenty animals received a 1% cholesterol diet for 4 weeks before the operation. In 10 animals this diet was continued until harvest (hypercholesterolemia group). In another 10 animals the diet was changed to standard rabbit chow on the day of the surgical procedure and continued until harvest (cholesterol reduction group). The last 10 animals were control subjects. Vein grafts were harvested either for histologic study or for in vitro isometric tension studies. Cumulative dose response curves to norepinephrine, serotonin, bradykinin, and endothelin-1 were determined. After in situ pressure fixation, intimal thicknesses of the vein grafts were measured by videomorphometry. The change in diet produced a 74% reduction in serum cholesterol concentration within 28 days. There was a 26% reduction in the intimal thickness of vein graft intimal hyperplasia and the macroscopic disappearance of atheromatous lesions from the graft wall, which are always observed in vein grafts from the hypercholesterolemia group. Cholesterol reduction did not change hypercholesterolemia-induced agonist supersensitivity. Therefore, cholesterol reduction slows the formation of intimal hyperplasia in vein grafts but does not prevent the persistence of the hypercholesterolemia-associated smooth muscle phenotype.

Animals↗

PubMed1994

Reduction of experimental vein graft intimal hyperplasia and preservation of nitric oxide-mediated relaxation by the nitric oxide precursor L-arginine.

BACKGROUND: Previous studies in animals and human beings have shown that vein bypass grafts exhibit diminished endothelium-dependent relaxation and the development of intimal hyperplasia. This study examines the effect of L-arginine on experimental vein graft endothelial cell function and the development of intimal hyperplasia. METHODS: Common carotid vein bypass grafts were performed in 24 New Zealand White rabbits: 12 were controls and 12 received L-arginine (2.25%) orally 7 days before operation and thereafter until harvest 28 days after operation. Intimal and medial dimensions were determined by planimetry on pressure-fixed vessels. Relaxation to acetylcholine, serotonin, calcium ionophore (A23187), and sodium nitroprusside was performed on precontracted vessel rings. RESULTS: Arginine-treated vein grafts showed a 47% reduction in mean intimal thickness (p < 0.001) compared with controls. By scanning and transmission electron microscopy, all vein grafts showed a confluent endothelium. In contrast to control grafts, which do not relax to acetylcholine and serotonin, arginine-treated vein grafts relaxed in response to both agonists. There was a significant increase (p < 0.05) in the maximal relaxation to calcium ionophore (A23187) in arginine-treated vein grafts compared with control grafts. Non-endothelium-dependent responses to sodium nitroprusside were equivalent in all vein grafts. CONCLUSIONS: This study shows that oral L-arginine supplementation significantly reduces intimal hyperplasia and preserves nitric oxide-mediated relaxation in experimental vein grafts, suggesting a role for nitric oxide in the regulation of the cellular events that lead to intimal hyperplasia.

Animals↗

PubMed1993

The integrity of experimental vein graft endothelium--implications on the etiology of early graft failure.

INTRODUCTION: the vascular endothelium serves as a functional barrier between the circulating blood and the vessel wall. It is an essential element for the maintenance of vascular homeostasis and is implicated in the pathogenesis of vascular disease. Reversed vein bypass grafting is considered to be a devastating procedure for the endothelial cell layer of the graft during the first 7 days. At this time, smooth muscle cell proliferation, the forerunner of intimal hyperplasia, begins. Loss of endothelial cell integrity is cited as an important factor in this smooth muscle cell response. The integrity of the vein graft endothelial lining after grafting was examined in this study. METHODS: reversed vein bypass grafting of the common carotid artery using external jugular vein was performed in 24 New Zealand white rabbits. All grafts were pressure fixed (80 mmHg) in situ, at 0 and 10 min, 6 h and 1, 3, 5, 7 and 14 days postoperatively. The endothelial cell layer was examined by light microscopy (LM), scanning (SEM) and transmission electron microscopy (TEM) and immunohistochemistry (Factor VIII) using standard histological procedures. RESULTS: the endothelium was observed by SEM and confirmed by both LM, Factor VIII and TEM in all specimens. It covered almost the entire surface examined. At 0 and 10 min, endothelial cells were present and displayed minimal evidence of injury. At 6 h and 1 day, numerous red cells, polymorphonucleocytes (PMNs), platelets and fibrin were adherent to the luminal surface. Blood cells were also seen beneath the endothelium. At day 3, the adherent fibrin and cellular elements were reduced with most of the endothelial lining intact. Within 10 min, TEM demonstrated that these cells were stretched, very thin with few microvesicles and a blurred cytoplasm, which would indicate viability but a degree of cellular injury. By day 1, the endothelial cells were lifted from their underlying structures by subendothelial oedema and an infiltrate predominantly of PMNs. By day 5, the blood cells and fibrin which were adherent to the endothelium had been dispersed and the subendothelial infiltrate was to a large extent replaced by disintegrated PMNs. On days 7 and 14, a viable confluent endothelial cell layer was present and a degree of intimal hyperplasia was noted. The endothelial cells appeared to have enlarged nucleoli and cytoplasms filled with a considerable quantity of rough endoplasmic reticulum. CONCLUSION: the endothelium of reversed vein grafts is preserved at the time of implantation and at all time intervals studied in this model. These findings do not support the assumption that endothelial denudation is a prerequisite for intimal hyperplasia. Endothelial cell dysfunction and morphological changes are maximal within the first 3 days after grafting but appear to recover by the 5th postoperative day. The gross preservation of the endothelial cell layer implies that therapeutic approaches, to mitigate endothelial cell injury and its consequences, should be focused on the preoperative period and the first 5 days following implantation.

Animals↗

PubMed1993

Regression of intimal hyperplasia with restoration of endothelium-dependent relaxing factor-mediated relaxation in experimental vein grafts.

BACKGROUND: The reversibility of the morphologic and functional alterations that occur in veins transplanted into the arterial circulation was examined in this study. METHODS: Common carotid vein bypass grafts (VG) were performed in 20 male New Zealand White rabbits. Ten VG and jugular veins (CV) were harvested after 14 days, and ten VG were reimplanted as venovenous bypass grafts (REV) and harvested after an additional 14 days. Vessels were taken for structural or isometric tension studies to norepinephrine, serotonin, and bradykinin and to acetylcholine and sodium nitroprusside after precontraction. RESULTS: There was a decrease in the thickness of the intima (p = 0.02) and the media (p = 0.002) in REV compared with VG. In REV, sensitivity to norepinephrine decreased (p = 0.0007) with a reduced maximal tension to norepinephrine (p = 0.02) and to serotonin (p = 0.0001). Bradykinin sensitivity increased in REV (p = 0.003 vs VG) and was greater than in CV. Only the precontracted CV and REV relaxed to acetylcholine. All tissues relaxed to sodium nitroprusside. CONCLUSIONS: This study suggests that intimal hyperplasia can be reversed with restoration of endothelium-dependent relaxing factor-mediated relaxation but that only a partial regression of the contractile abnormalities can be achieved.

Animals↗

PubMed1992

Fibronectin and laminin in transverse tubules of cardiac myocytes studied by laser confocal microscopy and immunocytochemistry.

The distribution of two non-collagenous glycoproteins of high molecular weight, fibronectin (FN) and laminin (LMN), was investigated in myocardial cells from the ventricle of rats, and from biopsies collected from the auricle of patients undergoing a coronary bypass operation. In order to elucidate the expression of FN and LMN across cells, non-invasive serial sectioning has been carried out by laser scanning confocal microscopy of frozen, immunostained tissue sections. In addition, immunoelectron microscopy was used to study the distribution of these antigens at higher magnifications. These studies show that FN is part of the basement membrane of the surface sarcolemma of both ventricular and atrial cells, in addition to being an abundant protein of the extracellular matrix (ECM). Along transverse tubular(TT)-membranes, FN was only detected in tubules exceeding 200 nm in diameter. Even here, the intensity of labelling varied greatly and was generally low. By contrast, a heavy investment of LMN was organized in the basal lamina along the surface sarcolemma and along ramifications of the entire TT-system in ventricular heart muscle cells. In this way, the network of TT-membrane systems of working heart muscle cells provides a supply of LMN to all depths of the myocardial fibre. In human atrial muscle cells, a regular TT-system appears to be absent. Instead occasional, deep sarcolemmal invaginations occur with diameters of 300-500 nm, the surfaces of which are also invested with LMN. The significance of the present findings has been discussed, with special reference to LMN as a possible component of a series of proteins involved in transmembrane communication between the ECM and the sarcoplasm.

Animals↗

PubMed1992

Cryopreservation evaluated with mitochondrial and Z line ultrastructure in striated muscle.

Single, intact, frog skeletal muscle fibres and whole frog hearts were quick-frozen on a polished, liquid-He-cooled copper block and examined in the electron microscope after freeze-substitution and freeze-fracture. In both kinds of striated muscle, collapse of the peripheral and intracristal membrane spaces in mitochondria was found to increase with increasing distance from the point of first impact (PFI) of the muscle cells on the cold copper block. The changes correlated with a previously described gradient of Z line and A band cryodamage occurring with distance from the PFI. The findings in thin sections from freeze-substituted preparations were confirmed by freeze-fracture preparations. It is concluded that, since the mitochondrial membrane changes are concurrent with, and follow the same spatial distribution of, other manifest cryoartefacts, the cryoartefactual nature of the mitochondrial changes must be excluded before functional significance is attributed to them. The collapse of mitochondrial membrane spaces as a sensitive indicator of quality of cryopreservation may apply to non-muscle cells as well.

Animals↗

PubMed1992

Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.

A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.

Animals↗

PubMed1991

To excite a heart: a bird's view.

Ultrastructural investigations of avian cardiac muscle, including ratite hearts, have provided great insights into the mechanisms as to how excitation leads to contraction in the heart. The geometry of the conduction fibers of ratite hearts confirms earlier observations on birds showing that the geometry of the conduction system and its component cells is adapted to hearts of different sizes and rates of contraction so as to maintain a differential in conduction velocities between the conduction system and the working fibers. The study of the ratite conduction fibers bears out the idea of an inverse relationship between the size of the gap junctions and the input resistance of cardiac cells. The anomalous extended junctional SR typical of all avian hearts, proscribes the notion of direct contact transduction into calcium release for contraction of an excitatory signal propagating at the cell surface. Couplings appear well suited to maintain direct, if transitory, connections to the extracellular space in addition to harboring channels for intracellular calcium release.

Actin Cytoskeleton↗