Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada1
Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA2
Author for correspondence: Guangming Zhong. Tel: +1 210 567 1169. Fax: +1 210 567 0293. e-mail: Zhongg{at}UTHSCSA.edu
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ABSTRACT |
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Keywords: myocarditis, myocytes, chlamydial infection, apoptosis
Abbreviations: i.f.u., inclusion-forming unit(s); LDH, lactate dehydrogenase
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INTRODUCTION |
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Despite the apparent differences in tissue tropism between the chlamydial species, all three major species have been shown to be associated with or to induce myocarditis (Bachmaier et al., 1999 ; Blessing et al., 2000
; Diaz & Collazos, 1997
; Fan et al., 1999
; Gnarpe et al., 1997
; Schinkel et al., 2000
). Infection with C. psittaci has been demonstrated to not only cause endocarditis in animals but is also infrequently associated with myocarditis in humans working with infected animals (Schinkel et al., 2000
). Epidemiological investigations have presented evidence for a role of C. pneumoniae infection in myocarditis and sudden unexpected cardiac death. Not only are C. pneumoniae-specific antibody levels increased in patients with inflammatory heart muscle diseases (Gnarpe et al., 1997
), but C. pneumoniae antigens have also been detected in endomyocardial biopsy samples (Wesslen et al., 1992
). A C. trachomatis murine strain was demonstrated to cause myocarditis in mice (Bachmaier et al., 1999
; Fan et al., 1999
). An autoimmune hypothesis was proposed as the potential mechanism of the Chlamydia-induced myocarditis (Bachmaier et al., 1999
; Diaz & Collazos, 1997
). However, chlamydial organisms were detected in the cardiac tissues of infected subjects, suggesting that the organisms may be able to invade the heart tissues. Thus, it is important to determine whether Chlamydia can actually infect myocytes and cause direct damage to the cardiac tissues. In the present study, we have used rat neonatal myocytes as the model cells to address these questions. We found that C. trachomatis (LGV) and C. pneumoniae (AR39) successfully infected myocytes and produced infectious progeny in myocytes. The chlamydial infection induced lactate dehydrogenase (LDH) release, a clinical indicator of myocarditis, and superoxide production, a known agent that can damage cardiac tissues. The Chlamydia-induced myocyte damage is accompanied with a reduced ATP level. However, no apparent nuclear apoptosis was detected in the infected myocytes, despite the obvious cellular damage.
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METHODS |
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Chlamydial organisms and infection.
The C. trachomatis LGV (serovar L2) and C. pneumoniae AR39 strains were obtained from Washington Research Foundation, Seattle, USA, and were propagated and purified as described previously (Hu et al., 1999 ; Zhong et al., 2001
). Aliquots of the organisms were frozen at -80 °C before being used for infection. For AR39 infection, the organisms were diluted in DMEM without FBS and inoculated onto cell monolayers in a volume of 200 µl per well at an m.o.i. of 2, or as indicated in individual experiments. After incubation for 2 h at 37 °C, the inoculating solution was removed and 1 ml of DMEM with 10% FBS was added to each well. For L2 infections, the organisms were directly diluted in 10% FBS/DMEM, and 1 ml of the organism-containing solution was added to each well. The organisms were allowed to grow for various periods of time, as indicated in individual experiments. No cycloheximide was added to any of the myocyte infection cultures. However, 2 µg cycloheximide ml-1 was kept in the culture medium used for titrating the chlamydial organisms in HeLa cells. For determining the burst size of the myocyte-born chlamydial inclusions, lysates harvested from the infected myocytes were serially diluted in PBS and inoculated onto HeLa cell monolayers grown on coverslips. Forty-eight (for L2-infected myocyte lysate sample) or 72 (for AR39-infected myocyte lysate sample) hours post-infection, the coverslips were fixed for immunofluorescence staining as described below and the number of chlamydial inclusions (also designated as inclusion-forming units, i.f.u.) per view was counted for 10 random views from duplicate coverslips. The total number of i.f.u. generated from a given myocyte lysate sample was calculated based on the lysate volume, dilution factors and number of views per coverslip. Finally, the burst size of the inclusions grown in myocytes was determined by dividing the total number of i.f.u. obtained from the titration of the myocyte lysates on HeLa cells by the number of i.f.u. used to initially infect myocytes.
Immunofluorescence staining.
The myocyte samples (with or without infection) grown on coverslips in 24-well plates were fixed with a 2% paraformaldehyde/PBS solution for 30 min at room temperature, followed by permeabilization with 0·5% saponin for 30 min at room temperature. After blocking with 1% milk/PBS for 1 h at room temperature, a mouse anti-chlamydial LPS mAb (G. Zhong et al., unpublished data) was used to stain chlamydial antigens. The first antibody staining was visualized with a goat-anti-mouse IgG conjugated with Cy3 (red; Jackson Immunologicals). A DNA dye, Hoechst (blue; Sigma), was used to simultaneously reveal host-cell nuclei. Images were acquired individually for each staining using a SesiCam Imaging System (The Cooke Corporation, NY, USA) connected to a Nikon diaphot inverted microscope and the single colour images were merged in-frame into the dual colour image using the software SIMPLEPCI. However, for publication purposes, all colour images were flattened into black and white pictures. As a result, only the Cy3 staining (red) of chlamydial inclusions was shown as white on a black background.
LDH release assay.
LDH activity was measured spectrophotometrically as described by Bergmeyer & Bernt (1974) . Aliquots of the culture supernatants from each well were collected and centrifuged for 5 min at 500 g. The supernatant was saved in a sterile microcentrifuge tube and stored at -80 °C. All solutions used for this assay were made fresh on the day of the assay. The substrate for the LDH reaction was made by mixing 10% (v/v) NADH and 10% (v/v) pyruvate in Tris/KCl buffer and equilibrated to 25 °C in a water bath prior to use. Quadruplicate 20 µl aliquots of the supernatants (containing LDH) were added to quartz cuvettes containing 800 µl of the substrate solution in a Cary Win UV spectrometer. The change in absorbance at 340 nm is directly proportional to LDH activity in the supernatant samples.
Detection of ATP levels.
Rat cardiomyocytes with or without chlamydial infection were rinsed twice with cold PBS, and then treated with lysis buffer (0·1 M potassium phosphate, 1% Triton X-100, 1 mM DTT, 2 mM EDTA, pH 7·8) for 15 min at 65 °C. The cell lysates were collected and frozen at -80 °C. The ATP levels in the lysates were determined using a standard firefly luciferaseluciferrin bioluminescent assay Kit (Sigma) on a Beckman LS 6500 multi-purpose scintillation counter with single photo monitor. The results were expressed as ATP concentration (nM). Each measurement was carried out in duplicate and two experiments were performed.
Assessment of oxidative stress.
The intracellular oxidative stress in cardiomyocytes was assessed by monitoring the oxidation of 2,7-dichlorofluorescein diacetate (H2DCF) to highly fluorescent dichlorofluorescein (Swift & Sarvazyan, 2000 ). Briefly, a 10 mM stock solution of H2DCF (Sigma) was freshly dissolved in ethanol and kept at -20 °C. The stock solution was diluted to 10 µM with PBS (containing both Ca2+ and Mg2+) prior to the experiment. Rat neonate myocytes were washed twice with warm Ca2+,Mg2+-containing PBS and incubated with 10 µM H2DCF (0·5 ml per well of a 24-well plate) at room temperature in the dark. After 20 min incubation, cells were washed twice with PBS to remove the extracellular probe, and image analysis (488 nm excitation wavelength, 515 nm emission wavelength) was conducted immediately using an inverted Nikon microscope with an Axon Integrated Imaging System (Axon Instruments). All images were collected at room temperature. Mean fluorescence intensity of individual cells was calculated by Axon Imaging Workbench Software and the images were presented in grey scale so that the degree of the intracellular fluorescence brightness is directly proportional to the level of oxidative species in individual cells.
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RESULTS |
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DISCUSSION |
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During epithelial infection of respiratory and urogenital tracts, the Chlamydia-induced diseases are largely due to persistent infection. It is not known whether C. trachomatis or C. pneumoniae can establish persistence in heart muscle cells. Nevertheless, either the direct damage caused by productive infection or the inflammatory response provoked by persistent infection in the heart can be equally fatal to the infected hosts. It is unlikely that heart muscle cells are the natural target cells for chlamydial organisms, since transmission to new hosts from this tissue is difficult. However, once chlamydial organisms are accidentally carried to the heart of the host, chlamydial growth inside the myocytes may cause damage to the heart tissue. This hypothesis is supported by our current finding that chlamydial infection in myocytes induced the production of reactive oxygen species (Fig. 3), agents known to cause cell damage, and triggered release of LDH (Fig. 2
), an indicator of cell membrane permeability alteration.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 July 2002;
revised 20 August 2002;
accepted 29 August 2002.
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