Hypokalaemia induces Ca²âº overload and Ca²âº waves in ventricular myocytes by reducing Na⁺,K⁺-ATPase α2 activity.

KEY POINTS: Hypokalaemia is a risk factor for development of ventricular arrhythmias. In rat ventricular myocytes, low extracellular K(+) (corresponding to clinical moderate hypokalaemia) increased Ca(2+) wave probability, Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) load and induced SR Ca(2+) leak. Low extracellular K(+) reduced Na(+),K(+)-ATPase (NKA) activity and hyperpolarized the resting membrane potential in ventricular myocytes. Both experimental data and modelling indicate that reduced NKA activity and subsequent Na(+) accumulation sensed by the Na(+), Ca(2+) exchanger (NCX) lead to increased Ca(2+) transient amplitude despite concomitant hyperpolarization of the resting membrane potential. Low extracellular K(+) induced Ca(2+) overload by lowering NKA α2 activity. Triggered ventricular arrhythmias in patients with hypokalaemia may therefore be attributed to reduced NCX forward mode activity linked to an effect on the NKA α2 isoform. ABSTRACT: Hypokalaemia is a risk factor for development of ventricular arrhythmias. The aim of this study was to determine the cellular mechanisms leading to triggering of arrhythmias in ventricular myocytes exposed to low Ko. Low Ko, corresponding to moderate hypokalaemia, increased Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) load, SR Ca(2+) leak and Ca(2+) wave probability in field stimulated rat ventricular myocytes. The mechanisms leading to Ca(2+) overload were examined. Low Ko reduced Na(+),K(+)-ATPase (NKA) currents, increased cytosolic Na(+) concentration and increased the Na(+) level sensed by the Na(+), Ca(2+) exchanger (NCX). Low Ko also hyperpolarized the resting membrane potential (RMP) without significant alterations in action potential duration. Experiments in voltage clamped and field stimulated ventricular myocytes, along with mathematical modelling, suggested that low Ko increases the Ca(2+) transient amplitude by reducing NKA activity despite hyperpolarization of the RMP. Selective inhibition of the NKA α2 isoform by low dose ouabain abolished the ability of low Ko to reduce NKA currents, to increase Na(+) levels sensed by NCX and to increase the Ca(2+) transient amplitude. We conclude that low Ko, within the range of moderate hypokalaemia, increases Ca(2+) levels in ventricular myocytes by reducing the pumping rate of the NKA α2 isoform with subsequent Na(+) accumulation sensed by the NCX. These data highlight reduced NKA α2 -mediated control of NCX activity as a possible mechanism underlying triggered ventricular arrhythmias in patients with hypokalaemia.

SERCA2 activity is involved in the CNP-mediated functional responses in failing rat myocardium.

BACKGROUND AND PURPOSES: Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca(2+) ATPase2 (SERCA2) activity is essential for these responses. EXPERIMENTAL APPROACH: Contractility, cGMP levels, Ca(2+) transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction. KEY RESULTS: CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser(16) and troponin I (TnI) Ser(23/24) phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca(2+) transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca(2+) channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene. CONCLUSIONS AND IMPLICATIONS: We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.

A morphological characterization of Borrelia anserina.

The morphology and ultrastructure of two strains of Borrelia anserina were investigated by electron microscopy of negatively stained and ultrathin sectioned cells. One was a cultivable strain originally isolated in the USA and the other was originally isolated in Nigeria and maintained in chickens. The cells were regularly helical, 9-21 microns long and 0.22-0.26 microns wide with a helix wavelength of about 1.7 microns. The cells were surrounded by a surface layer and appeared to divide by binary fission. The structure of the cells from each of the two strains was identical except that those of the USA strain possessed seven flagella inserted at each end and those from the Nigerian strain had eight.

Isolation of a new serovar of the genus Leptonema in the family Leptospiraceae.

A leptospira-like spirochete was isolated from a lymphocyte culture from a HIV-I and HTLV-I/II-positive patient. On the basis of serological, biological and morphological characteristics of the cells of the isolated strain (strain Lisboa), we conclude that it is a member of the genus Leptonema.

Morphological and ultrastructural studies of Mycoplasma flocculare and Mycoplasma hyopneumoniae in vitro.

Cells of Mycoplasma flocculare were found to vary in size and shape, especially in the later phases of growth, whereas those of Mycoplasma hyopneumoniae were fairly uniform irrespective of growth phases. Filamentous cells were present in cultures of M flocculare in the stationary and declining phases, but were never found in cultures of M hyopneumoniae. The filamentous and bizarre forms observed when mycoplasmas were suspended in phosphotungstic acid probably result from the action of the hypotonic solution. The surface of all cells was covered by a fuzzy coat consisting of fine hairs or bristles. An electron-lucent region was usually seen in cells negatively stained after centrifugation, but was only occasionally seen in cells negatively stained directly from the medium. Intracytoplasmic membranes were present in sectioned cells. No attachment organelle was found in cells of either species.

Taxonomy of Borrelia spp.

An increasing number of phenotypically heterogeneous borrelia isolates have been obtained by culture. Genetic analysis of some of them suggest that a reorganisation in delineation of the different species within the genus Borrelia may be necessary. Specifically increasing numbers of variants phenotypically different from the American type strain of B. burgdorferi have been described. In this workshop experts have discussed phenotypic and genotypic characters for (a) differentiation of B. burgdorferi strains from other borrelia and from treponemes, (b) characterization of B. burgdorferi isolates at the subspecies level. Ultrastructure was discussed by K. Hovind-Hougen, antigen structure by B. Wilske and genotypic characters by G. Baranton, A. G. Barbour, R. C. Johnson and V. Preac-Mursic. J. F. Anderson discussed differences between strains isolated from a wide range of vectors and hosts in North America. The outcome of the workshop was that B. burgdorferi may comprise different genomic species which however share common epitopes recognized by certain monoclonal antibodies. At the subspecies level a broad heterogeneity was demonstrated using methods as restriction endonuclease analysis, hybridization with whole B. burgdorferi-DNA or specific probes as well as plasmid analysis. A serotyping system based on monoclonal antibody reactivity against the outer surface protein OspA was proposed. A comparison of the different typing methods is impaired by use of different strains for analysis. In the future a broad variety of phenotypically different and defined strains need to be analysed for genotypic clusters and later on reexamined for phenotypic characters. To accomplish this close cooperation of different research groups is necessary.

Morphological heterogeneity among spirochetes isolated from cases of swine dysentery.

Pathogenic and non-pathogenic spirochetes isolated from the intestines of pigs were examined by electron microscopy using the negative staining and ultrathin sectioning techniques. Morphological differences were observed among cells of different strains. The cells differed in length as well as in width and in the number of flagella inserted at each end. In addition, the cells from different strains also varied in their resistance to the action of the detergents, Teepol and sodium deoxycholate. Three of the strains studied contained weakly haemolytic spirochetes, two of which differed markedly in their morphology from the cells of the other strains. These spirochetes had fewer flagella inserted at each end than those from other isolates and showed a distinct lattice-like substructure covering the ends of the cells. The spirochetes examined were found to be morphologically more similar to those of the genus Borrelia than to those of the genus Treponema but were clearly different from the cells of both of these genera. The taxonomic implications of the observations are discussed in brief.

Cultivation and characterization of spirochetes from cerebrospinal fluid of patients with Lyme borreliosis.

Attempts were made to culture spirochetes from cerebrospinal fluid samples of 105 patients suspected of having Lyme borreliosis with neurological complications. At the final evaluation, only 38 patients fulfilled the criteria of neuroborreliosis. Spirochetes were cultured from cerebrospinal fluid samples of four of these patients. All four patients had pleocytosis in their cerebrospinal fluid and a history of neurological symptoms of only 4 to 10 days in duration. Two of them had no detectable antibodies against any of the isolated spirochetes in their cerebrospinal fluid, both when tested with an enzyme-linked immunosorbent assay and when tested by immunoblotting. An antibody reaction against the homologous isolate that was distinctly stronger than that against the heterologous isolates was found in the serum and cerebrospinal fluid samples from one patient. The cells of the isolates were morphologically similar and showed a very similar protein pattern when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells of all isolates reacted with the monoclonal antibodies H5332 and H9724, which also react with Borrelia burgdorferi B31, the type strain. One isolate lost a major protein of 23 kilodaltons after subcultivation for 4 months. We conclude that isolation of spirochetes from cerebrospinal fluid might prove successful in clinically selected cases of Lyme borreliosis.

First isolation of Borrelia burgdorferi, the agent of Lyme borreliosis, from Ixodes ricinus (Acari: Ixodidae) in Berlin (West).

In 1984, two human cases of tick-borne Lyme borreliosis with considerable neurologic involvement were reported in Berlin (West). The diagnosis of Lyme borreliosis was serologically confirmed. The ticks which had transmitted the Borrelia were from Berlin (West). In the autumn of 1985, 156 ticks were collected in forests of Berlin (West) for the cultural detection of spirochetes by using BSK II medium. Three strains of spirochetes were isolated (from a pooled sample of two nymphs, and samples of one nymph and one female tick, respectively). These isolates were identified as Borrelia burgdorferi by means of SDS-PAGE, Western blot (using monoclonal antibody H 5332), microscopic agglutination test and electron microscopy. Investigations with the electron microscope showed that cells of two isolates (strains 2/B45 and 3/B56) had 8 flagella inserted at each end. The cells of the third isolate (strain 1/B29) had 9 flagella inserted at each end. This type had not been observed before.

Ultrastructural differences among spirochetes isolated from patients with Lyme disease and related disorders, and from Ixodes ricinus.

Previous studies on cells of strains B31 isolated in the U.S.A. from Ixodes dammini and strain G25 isolated in Sweden from Ixodes ricinus, showed that their ultrastructure was similar, but not identical. For this reason the studies were extended to spirochetes isolated directly from patients with Lyme disease and related disorders. Included in the present study were three strains isolated from skin, blood and spinal fluid, respectively, from patients with Lyme disease, two strains from patients with erythema chronicum migrans and one strain from a patient with acrodermatitis chronica atrophicans. Three additional strains isolated in Sweden from Ixodes ricinus were also studied. All spirochetes were examined after negative straining with 1% ammonium molybdate. The cells of each individual strain were identical except for one strain isolated from a tick. This isolate was found to consist of two morphologically different spirochetes. Comparison of morphological features of cells from various isolates revealed certain differences. The cells of the different strains could be divided into at least four groups for which cell size and shape as well as number of flagella varied. By morphological criteria, all cells were found to belong to the genus Borrelia.

Newly recognized Leptospira species ("Leptospira inadai" serovar lyme) isolated from human skin.

Leptospira strain 10, which represents a new Leptospira species, was isolated from a skin biopsy of a patient with Lyme disease. Although pathogenic for laboratory animals, the organism was not considered to have a significant role in the patient's illness.

Infectious typhlitis in chickens caused by spirochetes.

A weakly haemolytic spirochete was detected with an unabsorbed fluorescent antiserum to Treponema hyodysenteriae in smears and cultures of scrapings of caecal mucosa of laying hens with diarrhoea. Two groups of experimental chickens were fed a pure culture of this spirochete or homogenated intestinal contents of affected birds. Both groups showed clinical signs of disease such as increased water content of faecal material and slight retardation of growth. A non-specific typhlitis which histologically resembled milder forms of swine dysentery was seen in the birds from which spirochetes were isolated. The isolate obtained differed in cultural, biochemical, anti-genic and morphological characteristics from T. hyodysenteriae. The pathological significance of intestinal spirochetes and their possible epidemiological relation to swine dysentery are discussed.

Intestinal spirochetosis of the vermiform appendix.

A series of 681 surgically removed appendices were examined for spirochetes. Five hundred seventy-four appendices were removed because of suspected acute appendicitis; the rest were removed per occasionem. One hundred six of the former were histologically normal, whereas 421 showed acute appendicitis. The remaining 47 specimens showed a variety of other pathological conditions, for example, tumors and diverticula. Spirochetes were found in 13 (12.3%) of the appendices removed from patients clinically suspected to suffer from acute appendicitis, but whose appendices were otherwise histologically normal (pseudoappendicitis). Only 3 patients (0.7%) with histologically confirmed acute appendicitis (p less than 0.05) did show spirochetes in their appendices. Of the 107 patients who had their appendices removed per occasionem, 2 patients (1.9%) had spirochetosis (p less than 0.05). The ultrastructure of the spirochetes obtained from appendices with spirochetosis was studied by means of negative staining and ultrathin sectioning. The morphology of these spirochetes was very similar to that of Brachyspira aalborgi, a spirochete recently isolated from rectal biopsy specimens obtained from patients with colorectal spirochetosis.

Ultrastructure of spirochetes isolated from Ixodes ricinus and Ixodes dammini.

Two strains of Ixodes spirochetes, one isolated in the United States (B31) and the other in Sweden (G25), were examined by electron microscopy. Cells of strain G25 were 11-25 micron long with a wavelength of 2.1-2.4 micron and an amplitude of 0.4 micron. Eleven flagella were inserted subterminally at each end of the cell. Cells of strain B31 were similar but had eleven or seven flagella. Cytoplasmic tubules were not seen in cells of either strain. Although not identical, both strains showed ultrastructural details characteristic of the genus Borrelia.

Morphological changes upon subculturing of freshly isolated strains of Leptospira interrogans serovar hardjo.

The morphology of freshly isolated strains of Leptospira interrogans serovar hardjo was studied by electron microscopy and was compared to that of established laboratory strains of the same serovar and to some other serovars within the Hebdomadis serogroup. Changes in the morphology of freshly isolated strains of serovar hardjo were observed when the isolates were subcultured in the laboratory. Cells of strains subcultured more than 20 times were longer and had longer wavelengths, and electron lucent inclusions were observed less frequently than in cells of strains subcultured less than 10 times. Upon hamster passage, cells subcultured 40 and 50 times became more similar to cells of low passage number than to those of the inoculate. The results are discussed and the importance of environmental factors for the growth of leptospires is emphasized.

Colorectal spirochetosis: clinical significance of the infestation.

Mucosal biopsy specimens from 300 consecutive patients with symptoms requiring sigmoidoscopy were examined for spirochetosis by light and electron microscopy. Colorectal spirochetosis was detected in 15 of the patients (5%). Apart from the spirochetal infestation, the mucosa appeared normal with no inflammatory reaction. The spirochetes were eliminated upon treatment with neomycin and bacitracin, but the symptoms of the patients remained unchanged. It is concluded that in the present material, colorectal spirochetosis was without clinical significance.

Intestinal spirochetosis: morphological characterization and cultivation of the spirochete Brachyspira aalborgi gen. nov., sp. nov.

The ultrastructure of spirochetes obtained from rectal biopsies of patients with intestinal spirochetosis was studied by means of negative staining and ultrathin sectioning. The cells were sigmoidal with tapered ends, 2 to 6 microns long, with a wavelength of 2 microns. Four flagella were inserted at each end of the cells. The maximal cell width was about 0.2 microns. The spirochetes were cultured on tryptose soy blood agar plates. They were anaerobic and grew, although very slowly, at 37 to 38.5 degrees C in an atmosphere of 5% CO2-95% H2. Two types of colonies could be distinguished. The growth characteristics and the morphology of the isolated spirochetes differ from those of previously isolated spirochetal strains. Consequently, it is proposed that the present strains constitute a new genus, Brachyspira, of the family Treponemataceae. The type species is Brachyspira aalborgi, the type strain of which is 513A (NCTC 11492).

Leptospira parva sp.npv.: some morphological and biological characters.

Two lines of a spirochete were isolated as contaminants in a culture of a leptospire and from a bottle of uninoculated medium. Studies on morphological and biological characters led to the conclusion that these lines represent a new species of the genes Leptospira, Leptospira parva sp. nov. The morphology of the cells was rather similar to that of previously examined leptospires, but the cells were shorter and had a shorter wavelength (more tightly wound). Furthermore, the surface layer of cells of L. parva formed numerous small blebs with no apparent substructure when detached during preparation for negative staining, while the surface layer of previously studied leptospires formed cross-striated tubules under similar conditions. L. parva shows biological characteristics of both parasitic and saprophytic leptospires. The deoxyribonucleic acid base composition of cells of this organism differed from that found in leptospires, as well as from that found in cells of Leptonema illini.

Isolation of a heat-stable antigen from Treponema Reiter, using an immunoadsorbent with antibodies from syphilitic patients.

It was attempted to isolate antigens from Treponema Reiter, relevant to syphilis serology, by immunoadsorption with patients' antibodies coupled to CNBr--Sepharose 4B. One antigen was desorbed by 2 M KSCN in 0.05 M Tris--barbital buffer, pH 8.6. The recovery was 3% and 7% in two experiments. A small amount of human antibodies in the isolate was removed on an immunoadsorbent column with insolubilized rabbit antibodies against normal human serum proteins. The antigen thus obtained was immunologically pure when analysed by crossed immunoelectrophoresis. By electron microscopy of immunoprecipitates and by tandem crossed immunoelectrophoresis it was shown that the antigen differed from the flagellar antigen of T. Reiter, but was identical to antigen d previously described in T. Reiter. Antigen d could also be isolated from supernatants of T. Reiter cultures. The d antigen was not denatured at pH 2.8, by 8 M urea or by 3 M KSCN, and it resisted heating to 100 degrees C for 30 min. No protein could be detected in a concentrated preparation, and the antigen might be a polysaccharide. Antigen d is probably present in the sorbent used in the fluorescence treponemal antibody absorption (FTA-ABS) test and may constitute the active substance of this reagent.