Inducible ATP1B1 Upregulates Antiviral Innate Immune Responses by the Ubiquitination of TRAF3 and TRAF6.

The antiviral innate immune responses are crucial steps during host defense and must be strictly regulated, but the molecular mechanisms of control remain unclear. In this study, we report increased expression of human ATPase Na+/K+ transporting subunit ß 1(ATP1B1) after DNA and RNA virus infections. We found that the expression of ATP1B1 can inhibit viral replication and increase the levels of IFNs, IFN-stimulated genes, and inflammatory cytokines. Knockdown of ATP1B1 by specific short hairpin RNA had the opposite effects. Upon viral infection, ATP1B1 was induced, interacted with TRAF3 and TRAF6, and potentiated the ubiquitination of these proteins, leading to increased phosphorylation of downstream molecules, including TGF-ß-activated kinase 1 (TAK1) and TANK-binding kinase 1 (TBK1). These results reveal a previously unrecognized role of ATP1B1 in antiviral innate immunity and suggest a novel mechanism for the induction of IFNs and proinflammatory cytokines during viral infection.

Activation of Na+-K+-ATPase with DRm217 attenuates oxidative stress-induced myocardial cell injury via closing Na+-K+-ATPase/Src/Ros amplifier.

Reduced Na+-K+-ATPase activity has close relationship with cardiomyocyte death. Reactive oxygen species (ROS) also plays an important role in cardiac cell damage. It has been proved that Na+-K+-ATPase and ROS form a feed-forward amplifier. The aim of this study was to explore whether DRm217, a proved Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could disrupt Na+-K+-ATPase/ROS amplifier and protect cardiac cells from ROS-induced injury. We found that DRm217 protected myocardial cells against hydrogen peroxide (H2O2)-induced cardiac cell injury and mitochondrial dysfunction. DRm217 also alleviated the effect of H2O2 on inhibition of Na+-K+-ATPase activity, Na+-K+-ATPase cell surface expression, and Src phosphorylation. H2O2-treatment increased intracellular ROS, mitochondrial ROS and induced intracellular Ca2+, mitochondrial Ca2+ overload. DRm217 closed Na+-K+-ATPase/ROS amplifier, alleviated Ca2+ accumulation and finally inhibited ROS and mitochondrial ROS generation. These novel results may help us to understand the important role of the Na+-K+-ATPase in oxidative stress and oxidative stress-related disease.

Anticancer and Immunogenic Properties of Cardiac Glycosides.

Cardiac glycosides (CGs) are natural compounds widely used in the treatment of several cardiac conditions and more recently have been recognized as potential antitumor compounds. They are known to be ligands for Na/K-ATPase, which is a promising drug target in cancer. More recently, in addition to their antitumor effects, it has been suggested that CGs activate tumor-specific immune responses. This review summarizes the anticancer aspects of CGs as new strategies for immunotherapy and drug repositioning (new horizons for old players), and the possible new targets for CGs in cancer cells.

Activation of Na+/K+-ATPase attenuates high glucose-induced H9c2 cell apoptosis via suppressing ROS accumulation and MAPKs activities by DRm217.

Hyperglycemia is one of the major factors responsible for the myocardial apoptosis and dysfunction in diabetes. Many studies have proved that there is a close relationship between decreased Na+/K+-ATPase activity and diabetic cardiomyopathy. However, the effect of directly activated Na+/K+-ATPase on high glucose-induced myocardial injury is still unknown. Here we found that DRm217, a Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could prevent high glucose-induced H9c2 cell injury, reactive oxygen species (ROS) release, and mitochondrial dysfunction. High glucose-treatment decreased Na+/K+-ATPase activity and increased intracellular Ca2+ level, whereas DRm217 increased Na+/K+-ATPase activity and alleviated Ca2+ overload. Inhibition of Ca2+ overload or closing sodium calcium exchanger (NCX channel) could reverse high glucose-induced ROS increasing and cell injury. In addition, DRm217 could significantly attenuate high glucose-induced p38, JNK and ERK1/2 phosphorylation, which were involved in high glucose-induced cell injury and ROS accumulation. Our findings suggest that DRm217 may protect against the deleterious effects of high glucose in the heart. Prevention of high glucose-induced myocardial cell injury by specific Na+/K+-ATPase activator may be an attractive therapeutic option.

Acid-sensing ion channels are involved in epithelial Na+ uptake in the rainbow trout Oncorhynchus mykiss.

A role for acid-sensing ion channels (ASICs) to serve as epithelial channels for Na(+) uptake by the gill of freshwater rainbow trout was investigated. We found that the ASIC inhibitors 4',6-diamidino-2-phenylindole and diminazene decreased Na(+) uptake in adult rainbow trout in a dose-dependent manner, with IC50 values of 0.12 and 0.96 µM, respectively. Furthermore, we cloned the trout ASIC1 and ASIC4 homologs and demonstrated that they are expressed differentially in the tissues of the rainbow trout, including gills and isolated mitochondrion-rich cells. Immunohistochemical analysis using custom-made anti-zASIC4.2 antibody and the Na(+)-K(+)-ATPase (α5-subunit) antibody demonstrated that the trout ASIC localizes to Na(+)/K(+)-ATPase-rich cells in the gill. Moreover, three-dimensional rendering of confocal micrographs demonstrated that ASIC is found in the apical region of mitochondrion-rich cells. We present a revised model whereby ASIC4 is proposed as one mechanism for Na(+) uptake from dilute freshwater in the gill of rainbow trout.

Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

BACKGROUND: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats. METHODS: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 µg/kg) or saline 20 minutes before LPS (200 µg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured. RESULTS: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1ß, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain. CONCLUSION: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

Na(+), K(+)-ATPase subunit composition in a human chondrocyte cell line; evidence for the presence of α1, α3, ß1, ß2 and ß3 isoforms.

Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na(+), K(+)-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons). Studying the distribution of Na(+), K(+)-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na(+), K(+)-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na(+), K(+)-ATPase in a human chondrocyte cell line (C-20/A4) using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, ß1 and ß2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na(+), K(+)-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na(+), K(+)-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na(+), K(+)-ATPase expression and regulation in chondrocytes.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia).

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule.

Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues.

(Na+ + K+)-ATPase correlated with a major group of intramembrane particles in freeze-fracture replicas of cultured chick myotubes.

Immunofluorescence microscopy with a fluorescein-labeled monoclonal antibody was used to map the distribution of sodium- and potassium-ion stimulated ATPase [( Na,K]-ATPase) on the surface of tissue-cultured chick skeletal muscle. At this level of resolution it appeared that the (Na,K)-ATPase molecules were distributed nearly uniformly over the plasma membrane. These molecules could be cross-linked by use of the monoclonal antibody followed by a second antibody directed against the monoclonal antibody; the resulting fluorescent pattern was a set of small dots (patches) on the muscle surface. This pattern was stable over several hours, and there was little evidence of interiorization or of coalescence of the patches. Myotubes labeled with immunofluorescence were fixed in glutaraldehyde, cryoprotected with glycerin, then fractured and replicated by standard methods. Replicas of the immunofluorescence-labeled myotubes revealed clusters of intramembrane particles (IMP) only when the immunofluorescent images indicated a patching of the (Na,K)-ATPase molecules. Double antibody cross-linking of antigenic sites on myotubes with each of three other monoclonal antibodies to plasma membrane antigens likewise resulted in patched patterns of immunofluorescence, but in none of these cases were clusters of intramembrane particles found in freeze-fracture replicas. In each case it was shown that the (Na,K)-ATPase molecules were not patched. Other control experiments showed that patching of (Na,K)-ATPase molecules did not cause co-patching of one of the other plasma membrane proteins defined by a monoclonal antibody and did not cause detectable co-clustering of acetylcholine receptors. Detailed mapping showed that there was a one-to-one correspondence between immunofluorescent patches related to the (Na,K)-ATPase and clusters of IMP in a freeze-fracture replica of the same cell. We conclude that the intramembrane particles patched by double antibody cross-linkage of the (Na,K)-ATPase are caused by (Na,K)-ATPase molecules in the fracture plane. Quantification of the IMP indicated that the (Na,K)-ATPase-related particles account for up to 50% of particles evident in the replicas, or up to about 400 particles/micrometers2 of plasma membrane. Particles related to the (Na,K)-ATPase were similar to the average particle size and were as heterodisperse in size as the total population of IMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunolocalization of Na+/K+-ATPase and Na+/K+/2Cl- cotransporter in the tubular epithelia of sea snake salt glands.

The sublingual salt gland is the primary site of salt excretion in sea snakes; however, little is known about the mechanisms mediating ion excretion. Na(+)/K(+)-ATPase (NKA) and Na(+)/K(+)/2Cl(-) cotransporter (NKCC) are two proteins known to regulate membrane potential and drive salt secretion in most vertebrate secretory cells. We hypothesized that NKA and NKCC would localize to the basolateral membranes of the principal cells comprising the tubular epithelia of sea snake salt glands. Although there is evidence of NKA activity in salt glands from several species of sea snake, the localization of NKA and NKCC and other potential ion transporters remains unstudied. Using histology and immunohistochemistry, we localized NKA and NKCC in salt glands from three species of laticaudine sea snake: Laticauda semifasciata, L. laticaudata, and L. colubrina. Antibody specificity was confirmed using Western blots. The compound tubular glands of all three species were found to be composed of serous secretory epithelia, and NKA and NKCC were abundant in the basolateral membranes. These results are consistent with the morphology of secretory epithelia found in the rectal salt glands of marine elasmobranchs, the nasal glands of marine birds and the gills of teleost fishes, suggesting a similar function in regulating ion secretion.

A mutation of the Drosophila sodium pump alpha subunit gene results in bang-sensitive paralysis.

A bang-sensitive enhancer trap line was isolated in a behavioral screen. The flies show a weak bang-sensitive paralysis, recovering after about 7 s. The P element insert is localized at 93B1-2 on the salivary chromosomes, the site of the (Na+,K+)ATPase alpha subunit gene. Molecular characterization demonstrates that the transposon is inserted into the first intron of this gene. This insertion leads to normal-sized transcripts, but reduced levels of expression. This change is also reflected in lower amounts of a normal-sized alpha subunit protein. Mutant flies show a much greater sensitivity to ouabain, likewise indicating, on a functional level, a reduction in Na+ pump activity. Furthermore, the bang-sensitive behavior can also be mimicked by injecting sublethal doses of ouabain into wild-type flies. The molecular and functional evidence indicates that the insertion has produced a hypomorphic mutation of the (Na+,K+)ATPase alpha subunit gene, opening the way to future studies of the regulation of the Na+ pump.

A novel role for MNTB neuron dendrites in regulating action potential amplitude and cell excitability during repetitive firing.

Principal cells of the medial nucleus of the trapezoid body (MNTB) are simple round neurons that receive a large excitatory synapse (the calyx of Held) and many small inhibitory synapses on the soma. Strangely, these neurons also possess one or two short tufted dendrites, whose function is unknown. Here we assess the role of these MNTB cell dendrites using patch-clamp recordings, imaging and immunohistochemistry techniques. Using outside-out patches and immunohistochemistry, we demonstrate the presence of dendritic Na+ channels. Current-clamp recordings show that tetrodotoxin applied onto dendrites impairs action potential (AP) firing. Using Na+ imaging, we show that the dendrite may serve to maintain AP amplitudes during high-frequency firing, as Na+ clearance indendritic compartments is faster than axonal compartments. Prolonged high-frequency firing can diminish Na+ gradients in the axon while the dendritic gradient remains closer to resting conditions; therefore, the dendrite can provide additional inward current during prolonged firing. Using electron microscopy, we demonstrate that there are small excitatory synaptic boutons on dendrites. Multi-compartment MNTB cell simulations show that, with an active dendrite, dendritic excitatory postsynaptic currents (EPSCs) elicit delayed APs compared with calyceal EPSCs. Together with high- and low-threshold voltage-gated K+ currents, we suggest that the function of the MNTB dendrite is to improve high-fidelity firing, and our modelling results indicate that an active dendrite could contribute to a 'dual' firing mode for MNTB cells (an instantaneous response to calyceal inputs and a delayed response to non-calyceal dendritic excitatory postsynaptic potentials).

Immunization with Na+/K+ ATPase DR peptide prevents bone loss in an ovariectomized rat osteoporosis model.

Osteoporosis is characterized by decreased bone strength and microarchitectural deterioration of bone tissue leading to an increase in bone fracture. Here we report a new agent named DR peptide, a conserved sequence of Na+/K+ ATPase (NKA), can be used to prevent osteoporosis. Our results showed that immunization with DR peptide conjunct with Keyhole limpet hemocyanin (DR-KLH) significantly strengthened trabecular bone and improved bone mineral density of femur and the 5th lumbar in the ovariectomized (OVX) rats when compared with those in OVX rats immunized with KLH alone. To study the underlying mechanisms, anti-DR antibody (DR-Ab) and IgG were purified from the serum of rats immunized with DR-KLH or KLH alone in OVX rats. We found that DR-Ab had no significant effects on the proliferation of osteoclasts precursors, but it alleviated the inhibitory effect of H2O2 on the expression of osteogenic marker genes, Runx2, alkaline phosphatase and bone sialoprotein. DR-Ab also reversed the loss of collagen and the reduced bone nodule formation caused by H2O2 treatment. Knockdown of NKA with siRNA transfection reduced osteoblast differentiation and rendered the osteoblasts more vulnerable to oxidative challenge. Immunostaining analysis confirmed that NKA expression was reduced in osteoblasts in the femur of OVX rats, but this effect was attenuated by immunization with DR-KLH. Therefore, the protective effects of DR-Ab on osteoblasts might be associated with preservation of NKA level. Taken together, our findings reveal a novel mechanism for the development of osteoporosis and DR peptide and DR-Ab are potential approaches for the treatment of osteoporosis.

Ligation of Na, K ATPase ß3 subunit on monocytes by a specific monoclonal antibody mediates T cell hypofunction.

T cells play a crucial role in orchestrating body immune responses. T cell hyperfunction, however, leads to inflammation and induction of autoimmune diseases. Understanding of T cell regulation mechanisms and successful modulation of T cell responses is beneficial in treatment of disease associated to T cell hyperresponsiveness. Our previous study indicated that monoclonal antibody (mAb) P-3E10, a mAb to Na, K ATPase ß3 subunit, inhibited anti-CD3-induced PBMC proliferation. In the current study, we further investigated the mechanism of mAb P-3E10 in the induction of T cell hypofunction. We demonstrated that mAb P-3E10 decreased T cell proliferation and Th1, Th2 and Th17 cytokine production. Monocytes were the cells playing a key role in mediation of mAb P-3E10 induced T cell hypofunction. The inhibition of T cell activation by mAb P-3E10 required cell contact between monocytes and T cells. The mAb P-3E10 induced the down-expression level of MHC class II and CD86 and increased IL-6, IL-10 and TNF-α production of monocytes. We concluded that ligation of the Na, K ATPase ß3 subunit on monocytes by mAb P-3E10 arbitrated T cell hypofunction. This mAb might be a promising novel immunotherapeutic antibody for the treatment of hyperresponsive T cell associated diseases.

Na+, K+-ATPase Activating Antibody Displays in vitro and in vivo Beneficial Effects in the Pilocarpine Model of Epilepsy.

Na+, K+-ATPase is an important regulator of brain excitability. Accordingly, compelling evidence indicates that impairment of Na+, K+-ATPase activity contributes to seizure activity in epileptic mice and human with epilepsy. In addition, this enzyme is crucial for plasma membrane transport of water, glucose and several chemical mediators, including glutamate, the major excitatory transmitter in the mammalian brain. Since glucose hypometabolism and increased glutamate levels occur in clinical and experimental epilepsy, we aimed the present study to investigate whether activation of Na+, K+-ATPase activity with specific antibody (DRRSAb) would improve glucose uptake and glutamate release in pilocarpine-treated mice. We found decreased uptake of the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-il)amino]-2-desoxi-d-glucose (2-NBDG) in cerebral slices from pilocarpine-treated animals. Interestingly, decreased 2-NBDG uptake was not detected in DRRSAb-treated slices, suggesting a protective effect of the Na+, K+-ATPase activator. Moreover, DRRSAb prevented the increase in glutamate levels in the incubation media of slices from pilocarpine-treated mice. In addition, in vivo intrahippocampal injection of DRRSAb restored crossing activity of pilocarpine-treated mice in the open-field test. Overall, the present data further support the hypothesis that activation of the Na+, K+-ATPase is a promising therapeutic strategy for epilepsy.

Cerebral Malaria Causes Enduring Behavioral and Molecular Changes in Mice Brain Without Causing Gross Histopathological Damage.

Malaria, parasitic disease considered a major health public problem, is caused by Plasmodium protozoan genus and transmitted by the bite of infected female Anopheles mosquito genus. Cerebral malaria (CM) is the most severe presentation of malaria, caused by P. falciparum and responsible for high mortality and enduring development of cognitive deficits which may persist even after cure and cessation of therapy. In the present study we evaluated selected behavioral, neurochemical and neuropathologic parameters after rescue from experimental cerebral malaria caused by P. berghei ANKA in C57BL/6 mice. Behavioral tests showed impaired nest building activity as well as increased marble burying, indicating that natural behavior of mice remains altered even after cure of infection. Regarding the neurochemical data, we found decreased α2/α3 Na+,K+-ATPase activity and increased immunoreactivity of phosphorylated Na+,K+-ATPase at Ser943 in cerebral cortex after CM. In addition, [3H]-Flunitrazepam binding assays revealed a decrease of benzodiazepine/GABAA receptor binding sites in infected animals. Moreover, in hippocampus, dot blot analysis revealed increased levels of protein carbonyls, suggesting occurrence of oxidative damage to proteins. Interestingly, no changes in the neuropathological markers Fluoro-Jade C, Timm staining or IBA-1 were detected. Altogether, present data indicate that behavioral and neurochemical alterations persist even after parasitemia clearance and CM recovery, which agrees with available clinical findings. Some of the molecular mechanisms reported in the present study may underlie the behavioral changes and increased seizure susceptibility that persist after recovery from CM and may help in the future development of therapeutic strategies for CM sequelae.

Evidence that the H1-H2 domain of alpha1 subunit of (Na++K+)-ATPase participates in the regulation of cardiac contraction.

(Na++K+)-ATPase (NKA) plays an important role in ion homeostasis and regulates cardiac contraction. To understand the molecular basis of its cardiac regulatory functions, we investigated whether the primary structure of the H1-H2 domain in alpha-1 (alpha1) subunit of the enzyme plays a role in myocardial contractile regulation. Here we show that site-specific binding to this 1 H1-H2 domain with a targeted antibody (SSA78) markedly augments intracellular Ca2+ transients and contraction of rat ventricular cardiomyocytes without inactivating NKA. In vivo SSA78 infusion in mice results in a positive inotropic effect with enhanced contractile function yet no change in relaxation, indicating a direct cardiac effect linked to the H1-H2 domain. Competitive immunofluorescent staining and flow cytometry reveal that SSA78 binding is antagonized by ouabain, supporting the interaction of SSA78 at one of the glycoside-effecter sites. These new findings suggest that the H1-H2 domain of 1 subunit of NKA is a critical determinant of enzyme biologic activity, which couples to enhanced myocyte calcium transient and inotropic action.

Antigen-specific effects of autoantibodies against sarcolemmal Na-K-ATPase pump in immunized cardiomyopathic rabbits.

OBJECTIVES: We examine antigen-specific actions of autoantibodies directed against sarcolemmal Na-K-ATPase. BACKGROUND: Autoantibodies against some receptors or pumps were detected in patients with dilated cardiomyopathy. Although immunoglobulin adsorption therapy improved cardiac function in such patients, direct pathogenic effects of autoantibodies remain to be proven. METHODS: Japanese white rabbits were immunized once a month with purified Na-K-ATPase (NKA rabbits, n=10) or a synthetic peptide corresponding to the second extracellular loop of beta1-adrenergic receptors (beta rabbits, n=10), respectively. Control rabbits (n=10) received vehicle in the same manner. RESULTS: At 6 months, cardiac hypertrophy along with increased left ventricular end-diastolic pressure was observed in both NKA and beta rabbits, and inhibitory G protein level increased in both NKA and beta rabbits. Histological findings showed similar myocyte hypertrophy and interstitial fibrosis in both rabbits. Enzymatic activities of Na-K-ATPase were lower in NKA rabbits than in other groups. Immunoblotting showed that alpha3-isoform of Na-K-ATPase was selectively reduced in myocardium from NKA rabbits. CONCLUSIONS: Our present findings suggested that isoform-specific alterations of myocardial Na-K-ATPase activity were induced by immunizing rabbits. This was not secondary change due to cardiac hypertrophy. Thus, autoantibodies against sarcolemmal Na-K-ATPase have antigen-specific effect on the heart in vivo.

Clinical implications of anti-cardiac immunity in dilated cardiomyopathy.

Criteria of organ-specific autoimmunity are fulfilled in a subset of patients with myocarditis/dilated cardiomyopathy (DCM). In particular, circulating heart-reactive autoantibodies are found in such patients and symptom-free relatives. These autoantibodies are directed against multiple antigens, some of which are expressed in the heart (organ-specific), others in heart and some skeletal muscle fibres (partially heart-specific) or in heart and skeletal muscle (muscle-specific). Distinct autoantibodies have different frequency in disease and normal controls. Different techniques detect one or more antibodies, thus they cannot be used interchangeably for screening. It is unknown whether the same patients produce more antibodies or different patient groups develop autoimmunity to distinct antigens. IgG antibodies, shown to be cardiac- and disease-specific for myocarditis/DCM, can be used as autoimmune markers for relatives at risk as well as for identifying patients in whom immunosuppression may be beneficial. Some autoantibodies may also have a functional role, but further work is needed.