Abstract
ObjectivesAnthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca2+ properties.MethodsMurine cardiomyocyte contractile function and intracellular Ca2+ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca2+ decay rate. Stress signaling and Ca2+ regulatory proteins were assessed using Western blot analysis.ResultsIn vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca2+ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca2+ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca2+ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca2+ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.ConclusionsOur findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca2+ through a NADPH oxidase-dependent mechanism.
Highlights
Bacillus anthracis (B. anthracis), a spore-forming, Gram-positive bacterium, has been widely known as the causative agent of anthrax [1]
lethal factor (LF) is a Zinc-metalloprotease which cleaves the NH2terminal of mitogen-activated protein kinase kinases (MEKs) resulting in inactivation of the kinases
Given the notion that proteins affording protection against ROS may play an important role in cell survival to lethal toxin infection as evidenced by proteomic studies [18], we examined superoxide anion production and involvement of the main superoxide anion generating enzyme NADPH oxidase in the heart following lethal toxin exposure
Summary
Bacillus anthracis (B. anthracis), a spore-forming, Gram-positive bacterium, has been widely known as the causative agent of anthrax [1]. Accidental or intentional exposure of B. anthracis spores by oral, cutaneous or pulmonary routes often triggers infection resulting in a high mortality rate associated with severe hypotension [2,3,4]. Treatment remedies such as administration of antibiotics, vaccination or antibody to the toxin have shown some promises in the clinical management of B. anthracis infection [1,5]. Expression of the stress signaling molecules MEK1/2, p38 MAP kinase, ERK and JNK as well as the essential Ca2+ regulatory proteins were evaluated in an effort to better understand the mechanism of action behind lethal toxin-induced cardiomyocyte mechanical and intracellular Ca2+ handling responses, if any
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