Normal Hamster Hearts Research Articles

Accommodation after lung xenografting from hamster to rat1

Long-term xenograft survival can be achieved in hamster hearts transplanted into rats treated with cobra venom factor (CVF) and cyclosporine A (CsA). This phenomenon of "accommodation" is associated with expression of protective genes such as bcl-2, bcl-X(L), and heme-oxygenase-1. We examined whether accommodation could be induced in hamster-to-rat lung xenografts and whether the pattern of protective genes is similar to cardiac xenografts. We used hamster-to-rat cardiac and lung xenotransplantation models. Cardiac xenotransplants were treated with CVF+CsA and compared with untreated controls. Lung xenotransplants were treated with either CVF+CsA or FK506 and cyclophosphamide (Cp) and compared with untreated controls. All recipients were killed by 21 days after transplantation. We examined graft survival and protein expression of protective genes, and we performed histologic and immunohistologic analyses. Rejection occurred rapidly in untreated rats. CVF+CsA or FK506+Cp treatment significantly influenced graft survival. Eight of 12 CVF+CsA-treated heart transplants survived 21 days. Seven of 16 CVF+CsA-treated lung grafts and five of 12 FK506+Cp-treated lung xenografts survived 21 days. We observed significant protein expression of bcl-2, bcl-X(L), and heme-oxygenase-1 in cardiac xenografts treated with CVF+CsA at 2, 14, and 21 days after transplantation, compared with normal hamster hearts. We also observed significant expression of these proteins in lung xenografts treated with either CVF+CsA or FK506+Cp at 21 days after transplantation, compared with normal lungs. Accommodation may be a general phenomenon for all organs, mediated through protective genes. Induction of accommodation does not require disruption of the complement system.

Ectopic expression of an embryonic skeletal myosin heavy chain in human fetal and Syrian hamster hearts.

The mammalian heart is known to contain only two isoformic myosin heavy chain (MHC) genes, alpha and beta. A previously uncharacterized MHC gene was isolated in Syrian hamster hearts (McCully et al., JMol Biol 1991). We identified the novel MHC gene as a hamster embryonic skeletal MHC gene based on the developmental stage- and tissue-specific expression pattern: the restricted expression ofmRNA to striated muscles was highest in embryonic skeletal muscle and was developmentally down-regulated. We confirmed that the embryonic skeletal MHC gene exhibited higher expression in cardiomyopathic than in normal hamster hearts, and was up-regulated during the development of cardiomyopathy. The sporadic expression was highly localized in the endocardium. The present study identified that a very small number of undifferentiated myogenic cells existed in adult hamster endocardium. Moreover, using RT-PCR, a homologue of embryonic skeletal MHC mRNA was also expressed in human embryonic, but not adult ventricles. Our data provide a new insight into the regulatory mechanisms of MHCs in the cardiomyopathic hamster heart.

Age- and myopathy-dependent changes in connexins of normal and cardiomyopathic syrian hamster ventricular myocardium

The conduction of cardiac action potentials depends on the flow of excitation through gap junctions, which are hexameric protein associations of connexins (Cxs). The major Cx reported in the heart is Cx43, although some Cx40 and Cx45 are also present. There is some evidence for altered Cx content in heart failure. In heart failure, conduction is depressed and slowed conduction may contribute to arrhythmogenesis and (or) the maintenance of arrhythmia. Cx content and distribution were determined in ventricular tissues from normal and cardiomyopathic Syrian hamsters, an animal model of heart failure which has reproducible age-specific cardiomyopathy resulting in heart failure and age-matched controls in three groups: young (3-5 weeks), adult (13-18 weeks), and old (>45 weeks). Frozen, unfixed sections of ventricular tissues were immunofluorescently stained using antibodies against Cx43, Cx40, and Cx45. Cx43 was the predominant Cx detected in all samples. In normal hamsters, Cx43 was localized predominantly at the intercalated disc region, while in myopathic myocytes, it was scattered. In Western blots, Cx43 content of normal hamster hearts was highest in the adult hearts compared with young and old hamster hearts. In contrast, Cx43 content was significantly lower in adult cardiomyopathic hamster hearts compared with all other groups. The alterations of content and distribution of gap junction Cx43 may contribute to diminished conduction, pump function, and arrhythmogenesis in heart failure.

Delineation of genomic deletion in cardiomyopathic hamster

Cardiomyopathic hamster is a representative animal model for autosomal recessive cardiomyopathy. We have previously shown that the transcript of δ-sarcoglycan is missing in the heart of cardiomyopathic hamster due to genomic deletion. Here we define the normal genomic region deleted in cardiomyopathic hamster, which spans about 30 kb interval and includes the two first exons of the δ-sarcoglycan gene. RNA blot analysis using genomic DNA fragments covering the entire deletion as probes failed to detect any transcript other than δ-sarcoglycan in normal hamster heart, suggesting that δ-sarcoglycan is the only transcript defective in the heart of cardiomyopathic hamster.

ANTIGEN PAINTING TO A XENOGRAFTED HEART'S CONTENT: TH2 CYTOKINE-INDUCED CHANGES IN PROTEOGLYCAN EXPRESSION RESULT IN ENDOTHELIAL COATING BY HOST MHC CLASS I AND REGULATORS OF COMPLEMENT ACTIVATION.

33 Historically, definitions of xenograft accommodation were so vague that therapeutic approaches and mechanisms responsible for long-term survival, despite the presence of normal host antibody and complement (C) responses, were unknown. Recently, we have shown that brief decomplementing with cobra venom factor (CVF) plus daily cyclosporin (CsA) result in permanent survival of >75% of hamster->rat cardiac xenografts. Evaluation of accommodated xenografts showed antibody and C deposition and a Th2 cytokine response, in conjunction with infiltration by macrophages, NK and T cells; we now provide further insights into the mechanisms of accommodation in this model. Analysis with monoclonal antibodies (mAbs) of cardiac xenografts in which accommodation was induced by 1-2 doses of CVF plus daily CsA showed high levels of endothelial expression of hamster-specific heparan sulphate and CD44(hyaluronic acid receptor). Accordingly, baby hamster kidney cells (BHK) cells were cultured in the presence of rat Th1 (IL-2, IFN-γ) or Th2 (IL-4, IL-10) cytokines and evaluated (flow cytometry, ELISA) for changes in proteoglycan expression. Th2 but not Th1 cytokines induced dramatic increases in proteoglycan and CD44 production. Increased proteoglycan expression in vitro led to markedly enhanced binding of rat MHC class I and Crry (a rat complement regulatory protein with properties shared by both CD46 and CD55), as assessed using rat-specific mAbs. Moreover, the latter binding was associated with increased resistance to complement- or NK cell-mediated lysis in vitro. Consistent with these data, immunohistologic assessment of accommodated hamster->rat cardiac xenografts vs. rejecting or normal hamster hearts showed dense vascular labeling for hamster proteoglycans and CD44, plus endothelial staining with mAbs to rat MHC class I and Crry molecules throughout follow-up (day 4 - day 100). Together, these studies show that rat Th2 cytokines can induce increased expression of proteogylcans by hamster cells in vitro and in vivo, and such expression of highly charged molecules facilitates the coating or“antigen painting” of vascular surfaces by host molecules. i.e. Local binding of Crry appears to make hamster cells more resistant to attack by rat C, the concentration of MHC class I dampens rat NK cell-mediated lysis, and CD44 expression may contribute to selective T cell recirculation through the xenograft. These findings indicate mechanisms for xenograft (and potentially allograft) accommodation which are testable in large animal models, and suggest means to promote long-term survival with minimal host toxicity.

T-type Ca2+ channels are abnormal in genetically determined cardiomyopathic hamster hearts.

Although there is substantial evidence of abnormal Ca2+ homeostasis in heart cells of the cardiomyopathic Syrian hamster (Bio 14.6 strain), the mechanism by which these myocytes become Ca(2+)-overloaded is not known. To elucidate the role of voltage-sensitive Ca2+ channels in the pathogenesis of myopathy, whole-cell Ca2+ currents were measured in myopathic and normal control cardiac myocytes. These studies demonstrate the presence of two voltage-sensitive Ca2+ channel types in ventricular myocytes isolated from 200- to 300-day-old cardiomyopathic and age-matched normal hamsters. The two Ca2+ channel types were identified by their unitary conductance properties and pharmacologic sensitivities. Both L-type and T-type Ca2+ channels were present in cardiomyopathic and normal cells. Current density through L-type Ca2+ channels was the same in cardiomyopathic and normal control myocytes. However, the mean current density of T-type Ca2+ channels in cardiomyopathic cells was significantly higher than in normal cells (myopathic, 12.3 +/- 1.8 pA/pF; normal, 5.8 +/- 1.1 pA/pF; n = 8; P < .01). The T-type Ca2+ current in cardiomyopathic myocytes was activated and inactivated at more negative potentials than in cells from normal hamster hearts. These findings demonstrate no abnormality of the dihydropyridine-sensitive voltage-dependent L-type Ca2+ channel. In contrast, the observed abnormalities in T-type Ca2+ channel function in cardiomyopathic hamster myocytes suggest that this alteration may be related to the pathogenesis of Ca2+ overload and the arrhythmias in this genetically determined form of cardiomyopathy.

Enhanced alpha 1-adrenergic responsiveness in cardiomyopathic hamster cardiac myocytes. Relation to the expression of pertussis toxin-sensitive G protein and alpha 1-adrenergic receptors.

The pathogenesis of the myopathy occurring in the heart of the cardiomyopathic strain of the Syrian hamster is not well understood but is believed to be associated with abnormal calcium handling by myopathic cells. The purpose of this study was to determine whether the cardiomyopathy occurring in strain BIO 14.6 animals is associated with an enhanced alpha 1-adrenergic receptor-mediated rise in cytosolic calcium, whether a pertussis toxin-sensitive G protein is involved in coupling the alpha 1-adrenergic receptor to changes in intracellular calcium and whether enhanced alpha 1 responsiveness is associated with an increase in the level of expression of the alpha 1-adrenergic receptor or in the pertussis toxin-sensitive G protein or proteins. To test the hypothesis that the cardiomyopathic state is associated with a greater alpha 1-receptor-mediated rise in cytosolic calcium, we studied the effect of phenylephrine (in the presence of propranolol) on time-averaged cytosolic calcium concentration ([Ca2+]i) in isolated cardiac myocytes from cardiomyopathic and age-matched control hamsters. Phenylephrine caused a greater increase both in time-averaged [Ca2+]i (an increase of 48 +/- 8% versus 12 +/- 3%, p less than 0.01) and in contractility (+181 +/- 22% versus +35 +/- 9%, p less than 0.01) in cardiomyopathic than in normal cardiac myocytes. Exposure to pertussis toxin (200 ng/ml for 3 hours) attenuated the alpha 1-adrenergic receptor-mediated increase in contractility and time-averaged [Ca2+]i in both cardiomyopathic and normal cells. The level of pertussis toxin-sensitive G protein, as determined by pertussis toxin-mediated [32P]ADP-ribosylation, was 1.6-fold higher in cardiomyopathic versus normal hamster hearts. The density of alpha 1-adrenergic receptors, as measured by the antagonist radioligand [3H]prazosin and the affinity of the receptor for agonist and antagonist were similar in myopathic and normal heart membranes. Thus, in cardiac myocytes from hamsters, the alpha 1-adrenergic receptor-mediated effects on [Ca2+]i and contractility appear to be mediated by a pertussis toxin-sensitive G protein or proteins. In myocytes from cardiomyopathic hamsters, these alpha 1-adrenergic effects were increased in magnitude, as was the level of pertussis toxin-sensitive G protein, but there was no measurable alteration in the density or ligand binding properties of alpha 1-adrenergic receptors.

Angiotensins and the failing heart. Enhanced positive inotropic response to angiotensin I in cardiomyopathic hamster heart in the presence of captopril.

We examined the hypothesis that the positive inotropic effect of angiotensin I (Ang I) may be retained in the presence of angiotensin converting enzyme inhibitors so that it may have a direct beneficial effect on the heart. Accordingly, isolated perfused hearts (Langendorff preparation) of 300-day-old cardiomyopathic hamsters (a model of spontaneous cardiomyopathy) and age-matched normal hamsters (controls) were infused with Ang I in the presence of captopril; propranolol was added to the perfusing medium to block catecholamine-mediated effects of angiotensins on the heart. Left ventricular developed pressure and the rate of increase in left ventricular developed pressure increased significantly (p less than 0.001) in both the cardiomyopathic and the normal hamster heart despite concomitant reduction in myocardial flow rate favoring a direct inotropic effect of Ang I in both normal and myopathic hearts; these changes were significantly higher by almost threefold in the cardiomyopathic than in the normal hamsters (p less than 0.01) and were blocked by the angiotensin II (Ang II) antagonist [Sar1,Thr8]Ang II. Comparing dose-left ventricular contractility response curves for Ang I and Ang II, ED50 for responses was identical in both normal and myopathic hearts, whereas peak responses to Ang II were double those to Ang I in normal hearts but were almost identical in the myopathic hearts. Binding of [125I]Ang II in six cardiomyopathic and four normal hamster hearts was of high affinity, but there was no evidence for Ang I-saturable high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural localization of calcium in the myocardium of cardiomyopathic Syrian hamsters

Cardiomyopathy of the Syrian hamster is characterized by myocardial calcium overload and focal myocardial necrosis. The cause of the myocardial calcium overload is not yet fully understood. In this study, the ultrastructural localization of calcium was determined in normal hamster hearts and in non-necrotic and necrotic myocardium of cardiomyopathic hamsters (strain BIO 82.62). In many cells from the non-necrotic myocardium of the cardiomyopathic hamsters the calcium deposits, visible as 20 nm particles, were confined to the inner leaflet of the plasma membrane, the T-tubules and the intercalated disks. This corresponds to the calcium distribution found in normal hamsters and other mammalian species. A number of morphologically normal cells, however, displayed an increased amount of calcium precipitate in the mitochondria as well as at the sarcolemma indicating that, in the cardiomyopathic hamster, focal calcium overload is detectable cytochemically in cells which otherwise do not show gross abnormalities. In cells showing morphological signs of myolytic degeneration a marked redistribution of calcium precipitate took place. Sarcolemma became devoid of calcium deposits whereas an enormous amount of clustered precipitate occurred in largely swollen mitochondria. These data are in support of a relationship between impaired ion homeostasis and degeneration events in cardiomyopathy. Furthermore, there appears to be a clear parallelism in calcium redistribution between hypoxic and cardiomyopathic myocardium.

Energy levels at systole vs. diastole in normal hamster hearts vs. myopathic hamster hearts.

The following studies were carried out to examine energy metabolites and cardiac performance of the failing heart (hereditary cardiomyopathy) of the Syrian hamster (strain UM-X7.1) perfused either by normal or stress conditions, and to determine whether cyclical changes in energy-related metabolites occurred in the glucose-perfused hearts of both normal and heart failure animals. Hamster hearts from 250-day-old animals with moderate heart failure were removed and perfused either as nonworking hearts (Langendorff method, an afterload pressure of 90 mm Hg and 2.5 mM calcium in the perfusate) or as working hearts with stress conditions [an afterload of 110 mm Hg, high calcium concentrations in the perfusate (3.5 mM), and 10(-8) M isoproterenol]. Mechanical parameters (developed pressure and max dP/dt) and measurements of oxygen consumption indicated that both contractility and oxygen consumption had fallen 50% in myopathic hearts, compared with those of normal hamsters perfused with either of the two conditions. By means of a specially designed stimulator-triggered freeze clamp, hearts were terminated at systole and diastole, and tissue content of ATP, ADP, AMP, adenosine, phosphocreatine, creatine, pyruvate, lactate, and inorganic phosphate were analyzed. A 50% reduction in cardiac performance of the cardiomyopathic hamster hearts was associated with a corresponding reduction in systolic ATP, adenosine, and phosphocreatine values, while inorganic phosphate and lactate increased. With glucose as the sole substrate, the high energy phosphates, ATP and phosphocreatine, reached maximum values during diastole and minimum values during systole.(ABSTRACT TRUNCATED AT 250 WORDS)

An effect of ouabain on isolated heart tissue from cardiomyopathic Syrian hamsters

The effect of ouabain on respiration of mechanically chopped cardiac tissue from normal and cardiomyopathic hamster was measured. When compared to a “ouabain-sensitive” species, i.e. the guinea pig, the respiration of normal hamster heart was not markedly altered by ouabain (10 −8 m or 10 −6 m). However, the cardiomyopathic hamster strain, BIO-40.54, exhibited significant respiratory stimulation in response to 10 −8 m-ouabain when compared to the control hamsters. A decreased association of [H 3]-ouabain with hamster heart particulate subcellular fractions was noted with reference to similar fractions from guinea pig heart. A similar pattern was observed in the subcellular fractions from the cardiomyopathic hamster but with a trend toward increased recovery of [H 3]-ouabain in the particulate fractions. An increased sensitivity of the cardiomyopathic hamsters to ouabain is suggested.