Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster

Previous studies have shown that cardiac Na+ -K+ ATPase activity in the UM-X7.1 hamster strain is decreased at an early stage of genetic cardiomyopathy and remains depressed; however, the mechanism for this decrease is unknown. The objective of the present study was to assess whether changes in the expression of cardiac Na+-K+ ATPase subunits in control and UM-X7.1 cardiomyopathic hamsters are associated with alterations in the enzyme activity. Accordingly, we examined sarcolemmal Na+-K+ ATPase activity as well as protein content and mRNA levels for the alpha1, alpha2, alpha3 and beta1-subunit of the Na+-K+ ATPase in 250-day-old UM-X7.1 and age-matched, control Syrian hamsters; this age corresponds to the severe stage of heart failure in the UM-X7.1 hamster. Na+-K+ ATPase activity in UM-X7.1 hearts was decreased compared to controls (9.0 +/- 0.8 versus 5.6 +/- 0.8 micromol Pi/mg protein/hr). Western blot analysis revealed that the protein content of Na+-K+ ATPase alpha1- and beta1-subunits were increased to 164 +/- 27% and 146 +/- 22% in UM-X7.1 hearts respectively, whereas that of the alpha2- and alpha3-subunits were decreased to 82 +/- 5% and 69 +/- 11% of control values. The results of Northern blot analysis for mRNA levels were consistent with the protein levels; mRNA levels for the alpha1- and beta1-subunits in UM-X7.1 hearts were elevated to 165 +/- 14% and 151 +/- 10%, but the alpha2-subunit was decreased to 60 +/- 8% of the control value. We were unable to detect mRNA for the alpha3-subunit in either UM-X7. 1 or control hearts. These data suggest that the marked depression of Na+-K+ ATPase activity in UM-X7.1 cardiomyopathic hearts may be due to changes in the expression of subunits for this enzyme.     

Altered expression of connexin43 contributes to the arrhythmogenic substrate during the development of heart failure in cardiomyopathic hamster

Heart failure is known to predispose to life-threatening ventricular tachyarrhythmias even before compromising the systemic circulation, but the underlying mechanism is not well understood. The aim of this study was to clarify the connexin43 (Cx43) gap junction remodeling and its potential role in the pathogenesis of arrhythmias during the development of heart failure. We investigated stage-dependent changes in Cx43 expression in UM-X7.1 cardiomyopathic hamster hearts and associated alterations in the electrophysiological properties using a high-resolution optical mapping system. UM-X7.1 hamsters developed left ventricular (LV) hypertrophy by ages 6 approximately 10 wk and showed a moderate reduction in LV contractility at age 20 wk. Appreciable interstitial fibrosis was recognized at these stages. LV mRNA and protein levels of Cx43 in UM-X7.1 were unaffected at age 10 wk but significantly reduced at 20 wk. The expression level of Ser255-phosphorylated Cx43 in UM-X7.1 at age 20 wk was significantly greater than that in control golden hamsters at the same age. In UM-X7.1 at age 10 wk, almost normal LV conduction was preserved, whereas the dispersion of action potential duration was significantly increased. UM-X7.1 at age 20 wk showed significant reduction of cardiac space constant, significant decrease in conduction velocity, marked distortion of activation fronts, and pronounced increase in action potential duration dispersion. Programmed stimulation resulted in sustained ventricular tachycardia or fibrillation in UM-X7.1. LV activation during polymorphic ventricular tachycardia was characterized by multiple phase singularities or wavebreaks. During the development of heart failure in the cardiomyopathic hamster, alterations of Cx43 expression and phosphorylation in concert with interstitial fibrosis may create serious arrhythmogenic substrate through an inhibition of cell-to-cell coupling.     

Pathological changes of myocardial cytoskeleton in cardiomyopathic hamster

Immunocytochemical investigation was performed on the cytoskeletal proteins in cardiac tissue of the cardiomyopathic hamster. Male cardiomyopathic UM-X7.1 hamsters at 180 days of age (n=8) and age- and sex-matched normal BIO-RB hamsters (n=8) were used in this study. Immunofluorescence microscopy using monoclonal antibodies against desmin, -actinin, titin, and vincullin was employed. The heart weight to body weight ratio was significantly increased in the heart of cardiomyopathic hamster compared with that of normal hamster. In cardiomyopathic hamster, the left ventricular cavity was markedly dilated. Light microscopically, hypertrophy and atrophy of myocytes and myocardial fibrosis were prominently observed in cardiomyopathic myocardium. Immunocytochemically, desmin, -actinin and titin showed the cross striations along the myofibers in normal myocardium. In contrast, in cardiomyopathic myocardium, desmin was irregularly distributed in myocytes and the amount of desmin was increased. Loss of cross striations of -actinin and titin were frequently observed. Immunofluorescence against vinculin was not significantly altered. We conclude that the alterations of cytoskeletal proteins in myocardial cells may relate to decreased myocardial function in cardiomyopathic hamster failing heart.     

Kinetic changes of ethanolamine base exchange activity and increase of viscosity in sarcolemmal membranes of hamster heart during development of cardiomyopathy

The activity of the phospholipid base exchange enzyme specific for ethanolamine has been measured in cardiac sarcolemmal membrane preparations from Syrian golden and UM-X7.1 cardiomyopathic hamsters. In Syrian golden hamsters, the Km of the enzyme for ethanolamine does not change with age, whereas it almost doubles in membranes from cardiomyopathic animals, from the 30th to the 150th day of age. During the same period, the membrane cholesterol content increases by 68% in cardiomyopathic hamsters, whereas it does not change significantly in the Syrian golden hamster strain. As a consequence, in the adult animal, the cholesterol to phospholipid ratio and the viscosity of sarcolemmal membranes are higher in UM-X7.1 strain than in Syrian golden hamsters. A cause consequence relationship between the enzymatic changes and the compositional modifications in the sarcolemma occurring in UM-X7.1 hamsters during the development of cardiomyopahhy is proposed. (Mol Cell Biochem 116: 89–93,1992)     

ETB receptor dependent alteration in aortic responses to ET-1 in the cardiomyopathic hamster

The aim of this study was to verify whether an alteration in the aortic endothelin-1 (ET-1) response takes place in UM-X7.1 cardiomyopathic hamsters. Our results showed that ET-1 (10(-12) - 10(-5) mol/L) induces dose-dependent sustained increases in tension in the intact and endothelium denuded aortas from both normal and cardiomyopathic hamsters. The EC50 values of ET-1 of both intact and endothelium denuded aortas of normal hamsters were similar (2.2 x 10(-9) mol/L and 1.8 x 10(-9) mol/L, respectively). However, in cardiomyopathic hamsters, the EC50 of ET-1 in intact aortas was higher (1.5 x 10(-8) mol/L) than that of the endothelium denuded preparations (2.7 x 10(-9) mol/L). The EC50 of ET-1 in normal and cardiomyopathic hamster denuded aortas were similar. However, the EC50 of ET-1 in intact aortas of cardiomyopathic hamster was higher (1.5 x 10(-8) mol/L) than that of normal hamsters (2.2 x 10(-9) mol/L). Pre-treatment with the ETA receptor antagonist ABT-627 (10(-5)mol/L) of intact and endothelium denuded aortas from both normal and cardiomyopathic hamsters significantly prevented ET-1 (10(-7) mol/L) from inducing an increase in tension. Pre-treatment with the ETB receptor antagonist A-192621 (10(-5) mol/L) had no effect on the ET-1-induced increase in tension in endothelium denuded aortas of both normal and cardiomyopathic hamsters, as well as in intact preparations of normal animals. However, blockade of the ETB receptors in intact aortas of cardiomyopathic hamsters significantly (p < 0.001) potentiated the ET-1-induced increase in tension. In summary, an attenuation of the contraction response to ET-1 was found in UM-X7.1 cardiomyopathic hamsters when compared with normal age-matched hamsters. This alteration of the ET-1 effect in the aortas of cardiomyopathic hamsters seems to be dependent on the presence of the endothelium and could be due, in part, to an increase in the contribution of endothelial ETB receptors to relaxation, which in turn acts as a physiological depressor of ET-1 vasoconstriction. Our results suggest that an increase in the endothelium ETB receptor density may play a role in the development of hypotension in UM-X7.1 cardiomyopathic hamsters.     

Prevention of hereditary cardiomyopathy in the hamster by verapamil and other agents.

Verapamil, Prenylamine and, to a lesser extent, Cinnarizine were highly efficient in preventing the development or reducing the severity of heart lesions in cardiomyopathic hamster of the UM-X7.1 line. The calcium antagonistic compounds did not protect against the skeletal muscle changes already present at the time when treatment was initiated. The cardiac lesions were also significantly diminished in frequency and severity by a low calcium diet and in apparent contradiction, by parenteral administration of calcium gluconate. The relation of these electrolytes and creatine phosphokinase is not yet fully understood but suggests that a primary defect in muscles cell membranes may be responsible for hereditary cardiomyopathy in hamsters.     

Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

Fourier transform infrared evaluation of microscopic scarring in the cardiomyopathic heart: effect of chronic AT1 suppression

Our primary aim was to investigate the use of Fourier transform infrared (FTIR) spectromicroscopy as an accurate assay of cardiac extracellular matrix remodeling. Abnormal rearrangement or remodeling of the cardiac extracellular matrix is known to contribute to cardiac dysfunction. The microscopic multifocal necrosis and scarring are modulated by chronic AT(1) receptor blockade in experimental cardiomyopathy; thus, we also wished to rationalize the spectromicroscopic differences among control, untreated cardiomyopathic (CMP), and losartan-treated cardiomyopathic (LOS) hearts according to the pathogenesis of experimental cardiomyopathy. Male UM-X7.1 cardiomyopathic Syrian hamsters at early and late (65 and 200 days) stages of cardiomyopathy were subjected to 4-week losartan (15 mg/kg/day continuous infusion) treatment. Focal collagen microdomain distribution was confirmed spectroscopically by observation of the collagen IR fingerprint in the 1000-1800 cm(-1) region. Synchrotron FTIR spectromicroscopic map data were obtained from control (F1-beta strain) hamsters, nontreated cardiomyopathic, and losartan-treated CMP animals and imaged with mapping software, according to intensity of collagen fingerprint. Compared to controls, untreated late-stage CMP myocardium was characterized by elevated levels of fibrillar collagens and this was partially normalized with a 4-week losartan treatment. FTIR spectromicroscopy revealed that elevated collagen expression in focal microdomains is present in late-stage cardiomyopathy, and 4-week AT(1) blockade is associated with attenuation of collagen absorption in these lesions.     

Age-related changes of the ultrastructure in the cardiomyopathic hamster (UM-X7.1 Syrian hamster) parathyroid gland

We qualitatively and quantitatively investigated parathyroid glands of the UM-X7.1 cardiomyopathic hamster at 1, 2, 6 and 12 months of age to compare them with those of the normal hamster. We found that at 1 month of age in the UM-X7.1 hamster, the Golgi apparatus, lipid droplets and secretory granules decreased. There were no significant differences between the UM-X7.1 hamster and the control hamster at 2 months of age. At 6 months of age, the Golgi apparatus, rER and the secretory granules significantly increased in the UM-X7.1 hamster. At 12 months of age, the Golgi apparatus and lysosomes increased, while the secretory granules decreased. Ultrastructurally, we consider that in the UM-X7.1 hamster, the synthesis and release of the parathyroid at 6 months of age may be activated by an excessive amount of circulating catecholamine, and the functional activity of the parathyroid glands at 12 months of age may be depressed by the increased plasma calcium level. These findings suggest that the activities of the synthesis and release of the parathyroid hormone were the highest at 6 months of age in the UM-X7.1 hamster.     

Polysome structure in sea urchin eggs and embryos: an electron microscopic analysis

Primary cultures of cardiac myocytes from newborn normal and genetically cardiomyopathic (strain UM-X7.1) hamsters were analyzed by electron microscopy and immunofluorescent staining for myosin, actin, tropomyosin, and alpha-actinin. Antibody staining of these contractile proteins demonstrates that both normal and cardiomyopathic (CM) myocytes contain prominent myofibrils after 3 days in culture, although the CM myofibrils are disarrayed and not aligned as those in normal cells. The disarray becomes even more pronounced in CM cells after 5 days in culture. The immunofluorescent staining patterns of individual myofibrils in normal and CM cells were similar for myosin, actin, and tropomyosin. However, alpha-actinin staining reveals that the CM myofibrils have abnormally wide and irregularly shaped Z bands. Electron microscopy confirms the irregular Z-band appearance as well as the myofibril disarray. Thus, CM cardiomyocytes clearly show an aberrant pattern of myofibril structure and organization in culture.     

Nuclear colocalization of cellular and viral myc proteins with HSP70 in myc-overexpressing cells.

The c-myc oncogene and its viral counterpart v-myc encode phosphoproteins which have been located within cell nuclei, excluding nucleoli. We have expressed the c-myc gene under the simian virus 40 early promoter and studied the distribution of its protein product in transient expression assays in COS, HeLa, and 293 cells. We found three distinct patterns of c-myc immunofluorescence in the transfected cells: one-third of the c-myc-positive cells displayed a diffuse nuclear distribution, and in two-thirds of the cells the c-myc fluorescence was accumulated either in small amorphous or in large multilobed phase-dense nuclear structures. Unexpectedly, these structures also stained for the HSP70 heat shock protein in both heat-shocked and untreated cells. Our results indicate that both transient and stable overexpression of either the c-myc or v-myc protein induces translocation of the endogenous HSP70 protein from the cytoplasm to the nucleus, where it becomes sequestered in structures containing the myc protein. Interestingly, the closely related N-myc protein does not stimulate substantial nuclear expression of the HSP70 protein. Studies with chimeric myc proteins revealed that polypeptide sequences encoded by the second exon of c-myc are involved in colocalization with HSP70.     

Altered coronary and cardiac adrenergic response in the failing hamster heart: role of cyclooxygenase derivatives

Although the influence of the adrenergic system has been studied in the presence of heart failure, controversies still exist. Since cyclooxygenase derivatives appear to modulate coronary and cardiac adaptation in the failing heart, we hypothesized that cyclooxygenase derivatives may participate in the altered adrenergic responses in this situation. Isolated hearts from cardiomyopathic (UM-X7.1 subline) and normal hamsters, aged > 240 days, were utilized. Coronary and cardiac response to alpha1-, beta1-, and beta2-adrenergic stimulations was observed before and after pretreatment with indomethacin, a cyclooxygenase inhibitor. Reduction of coronary flow elicited by alpha1-adrenergic stimulation was unchanged in the presence of heart failure, while beta1- and beta2-induced vasodilatations were reduced. Inotropic response to alpha1 and beta1 stimulations were also reduced in failing hearts, while beta2-adrenergic action was unchanged. Pretreatment with indomethacin exacerbated coronary flow reduction observed with alpha1 stimulation in failing hearts only. Beta2-induced coronary vasodilatation and inotropic response to alpha1 and beta2 stimulations were impaired similarly in the presence of indomethacin in normal and failing hearts. The results suggest a complex interaction between adrenergic and cyclooxygenase activation.     

Effects of induced hyperthyroidism in normal and cardiomyopathic hamsters.

Thyroid hormones (TH) enhance cardiac function and reverse gene changes typical of pathological hypertrophy. However, reports in humans, but not animals, indicate that excess TH can cause heart failure. Also, the effects of TH on normal and cardiomyopathic hearts are likely to be different. The goal of this study was to characterize the effects of prolonged hyperthyroidism on cardiac function, chamber and cellular remodeling, and protein expression in both normal and cardiomyopathic hearts. Hyperthyroidism was induced in 3-mo-old normal BIO F1B and dilated cardiomyopathic BIO TO2 hamsters. After TH treatment for 10 days and 2 mo, hemodynamics, echos, myocyte length, histology, and protein expression were assessed. After 10 days and 2 mo, there were no differences between TO2-treated (Tx) and TO2-untreated (Untx) hamsters in chamber diameters or left ventricular function. After 2 mo of treatment, however, F1B-Tx showed evidence of dilated heart failure vs. F1B-Untx. Chamber diameters were increased, and ejection fraction and positive and negative changes in pressure over time were reduced. In F1B-Tx and TO2-Tx hamsters, beta-myosin isoform expression was reduced, whereas alpha-myosin increased significantly in F1B-Tx only. In TO2-Tx hamsters, the percent of viable myocardium was increased, and percent fibronecrosis was reduced vs. TO2-Untx. Myocyte length increased with TH treatment in both hamster strains. We conclude that 1) excess TH can induce heart failure in normal animals as observed in humans, 2) reversal of myosin heavy chain expression does not necessarily improve heart function, and 3) excess TH altered cellular remodeling but did not adversely affect chamber function or dimensions in TO2 hamsters.     

Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an important role in maintaining myofibril alignment in cardiac cells.     

A metabolic approach to the treatment of dilated cardiomyopathy in BIO T0-2 cardiomyopathic Syrian hamsters

Mechanisms underlying dilated cardiomyopathy (DCM) are poorly understood and effective therapy is still unavailable. The aim of this study was to examine the heart ultrastructure and dynamic of BIO T0-2 cardiomyopathic hamsters, an animal model of DCM, and to study in these animals, the effects of a co-formulation (HS12607) of propionyl-L-carnitine, coenzyme Q(10) and omega-3 fatty acids on cardiac mechanical parameters. Sarcomere length, Frank-Starling mechanism and force-frequency relations were studied on isolated ventricular papillary muscle from age-matched BIO F1B normal Syrian hamsters, BIO T0-2 control and BIO T0-2 HS12607-treated cardiomyopathic Syrian hamsters. At the optimum length to maximum active force, electron microscopy of left ventricular papillary muscle revealed that seven out of ten muscles studied showed shorter sarcomeres (1.20 +/- 0.29 microm), and the remaining three showed longer sarcomeres (2.80 +/- 0.13 microm), compared to those of normal hamsters (2.05 +/- 0.06 microm, n = 10). Severe alterations of the Frank-Starling mechanism, force-frequency relations and derivative parameters of contractile waves were also observed in vitro in the BIO T0-2 control hamsters. Long-term (8 weeks) treatment with HS12607 prevented alterations in sarcomere length in the BIO T0-2 cardiomyopathic hamsters; the Frank-Starling mechanism and force-frequency relations were also significantly (P < 0.05) improved in these hamsters. Therefore results of the present study strongly suggest the need for clinical studies on metabolic therapeutic intervention in the effort to stop the progression of dilated cardiomyopathy.     

Expression of the preproenkephalin A gene in the heart of cardiomyopathic hamsters.

The expression of the preproenkephalin A gene (Enk gene), which codes for the precursor of enkephalins, was investigated in the heart of hamsters with a hereditary cardiomyopathy at four different stages of the disease: the prenecrotic stage (30 days), the necrotic stage (60 days), the hypertrophic stage (120 days), and the final stage (200 days). In control atria and ventricles, the relative abundance of the Enk mRNA, as assessed by Northern blot analysis, did not change upon ageing. In the ventricles of cardiomyopathic hamsters, however, it increased about two- to three-fold at the necrotic stage, but was unaltered at the other time points studied; whereas in the atria, it progressively decreased to reach about half that of control hamsters at the final stage. Enkephalin levels, as measured by radioimmunoassay, decreased at 60 days in both the atria and ventricles of control hamsters, and also in the atria of cardiomyopathic hamsters, and remained stable thereafter, corresponding to one-third to one-half of those at 30 days. However, in the ventricles of cardiomyopathic hamsters, the peptide level decreased only slightly, the consequence being that at 60, 120, and 200 days, it was about two- to three-fold that of control hamsters. The lack of correlation between peptide levels and the relative abundance of the Enk mRNA suggests that translational and (or) post-translational mechanisms are important in the control of the expression of the Enk gene in the heart of hamsters.     

Studies on adenylate cyclase-cyclic AMP system of the myopathic hamster (UM-X7.1) skeletal and cardiac muscles.

Cyclic AMP content, adenylate cyclase (EC 4.6.1.1) activity and phosphodiesterase I (EC 3.1.4.1) activity of the hind leg skeletal muscle and cardiac muscle in 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters were examined. In 60-day-old myopathic animals, cardiac cyclic AMP levels were higher and phosphodiesterase I activity was lower, without any changes in the basal adenylate cyclase activity, whereas in 150-day-old myopathic hamsters, cardiac cyclic AMP and basal adenylate cyclase activity were lower, without any changes in the homogenate phosphodiesterase I activity. On the other hand, basal adenylate cyclase and phosphodiesterase I activities in the skeletal muscle homogenate from 60- and 150-day-old myopathic animals were not different from the normal values but the skeletal muscle cyclic AMP levels were significantly less in 60-day-old myopathic hamsters only. The plasma cyclic AMP levels in 60-day-old myopathic hamsters, unlike 150-day-old myopathic animals, were higher than the normal. Although these results reveal differences in myopathic cardiac and skeletal muscles, it is concluded that changes in adenylate cyclase-cyclic AMP system in myopathy are dependent upon the degree of disease.     

The role of osteopontin in the development of granulomatous lesions in lung

Osteopontin (OPN) has been shown to be expressed by cells in granulomas of various origins, but whether it plays a functional role in granuloma formation is not known. Here we used a cardiomyopathic hamster (TO2) model, to test the hypothesis that OPN contributes functionally to granuloma development. We immunized cardiomyopathic and normal hamsters by subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, and assessed various tissues for both OPN RNA expression and granuloma formation. Cardiomyopathic hamsters expressed OPN, and formed granulomatous lesions, in heart tissue in both immunized and untreated animals. In addition, immunization induced expression of OPN in lung and lymph nodes of cardiomyopathic (but not normal) hamsters, and also induced granuloma formation in these organs. To test whether OPN expression could play a functional role in inducing granulomas, we produced an adenoviral vector containing the murine OPN gene, and introduced this vector intratracheally into the lungs of normal hamsters. The OPN-containing vector, but not the control vector, induced pulmonary granuloma formation. These studies provided direct in vivo evidence that OPN can contribute functionally to the formation of granulomatous lesions, and suggest that OPN expression may be a common factor involved in formation of granulomas of various origin.     

Isolation,long-term culture,and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells

Summary A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture. This study was supported by a grant from the American Heart Association of Michigan, National Institutes of Health grant HL-25482, and by an Oakland University Biomedical Research Support Grant.