Background
The 2001 anthrax bioterrorism in the United States has drawn the interest of the scientific community in understanding the pathophysiology of anthrax infection. Anthrax is a pathological condition caused by a spore-forming, Gram-positive bacterium
Bacillus anthracis. Infection by inhalation of
B. anthracis spores can result in a mortality rate up to 96% [
1‐
3]. Major routes of infection have been confirmed through inhalation of, skin contact with or ingestion of
Bacillus anthracis spores. Anthrax toxin is the major virulence factor of
Bacillus anthracis, containing three polypeptides, namely: edema factor (EF), lethal factor (LF) and protective antigen (PA). LF is a zinc metalloprotease which specifically cleaves the NH
2-terminal of mitogen-activated protein kinase kinases resulting in inactivation of the kinases. EF is a calmodulin-dependent adenylyl cyclase which promotes intracellular cAMP accumulation and associated cellular responses [
4‐
7]. PA binds the cellular receptors tumor endothelial marker 8 and capillary morphogenesis protein 2 [
8,
9]. The combination of LF and the receptor binding PA yields the lethal toxin [
10]. Once bound to the receptor and proteolytically activated, PA forms a heptamer to deliver EF and/or LF to the cytoplasm following receptor-mediated endocytosis. Following anthrax exposure, patients usually develop refractory hypotension unresponsive to antibiotics, fluid, pressor and respiratory support [
11]. Anthrax lethal toxin was found to decrease the heart rate, left ventricular ejection fraction and mean arterial pressure [
12,
13]. In addition, anthrax lethal toxin has been reported to directly compromise myocardial function [
14‐
17]. However, the underlying mechanisms behind lethal toxin-induced unfavorable cardiac effects remain elusive.
Accumulation of reactive oxygen species (ROS) has been known to trigger cellular injury, including oxidation of DNA and lipids, mitochondrial damage and dysregulated autophagy [
18,
19]. Evidence suggests that anthrax lethal toxin initiates ROS accumulation; in particular, generation of superoxide and other ROS in macrophages and neutrophils [
14,
20,
21]. We previously reported that anthrax lethal toxin stimulates myocardial superoxide generation and thus impairs cardiac contractility [
14]. To this end, our present study was designed to examine the effect of the antioxidant enzyme catalase on lethal toxin-induced cardiac contractile anomalies and the underlying mechanism. Catalase is an antioxidant enzyme converting hydrogen peroxide (H
2O
2) produced from highly reactive superoxide (O
2
-) by superoxide dismutase to water and oxygen molecules. Given that autophagy has been implicated in anthrax infection [
14], essential protein markers for autophagy, including microtubule- associated protein light chain 3 (LC3), Beclin-1, autophagy related gene-7 (Atg-7) and green fluorescent protein-tagged LC3 puncta (GFP-LC3), were monitored in myocardial tissues or H9C2 myoblasts with or without lethal toxin challenge. Given the pivotal role of ubiquitin-proteasome system (UPS) in maintaining the protein synthesis and degradation parallel to the autophagic quality control mechanism [
22], proteasome function was assessed using chymotrypsin-like and caspase-like activities.
Discussion
The salient findings of our study are that anthrax lethal toxin exposure elicits enhanced ROS accumulation, myocardial contractile dysfunction, impaired intracellular Ca2+ handling, decreased mitochondrial membrane potential, enhanced ubiquitination and overt autophagy. Intriguingly, cardiac catalase overexpression abrogated or attenuated anthrax lethal toxin-induced cardiac contractile and intracellular Ca2+ anomalies. The catalase-offered beneficial effects against lethal toxin were closely associated with the alleviation of lethal toxin-induced accumulation of O2
- and ROS, loss of mitochondrial membrane potential, increase in ubiquitination, decrease in proteasome activity and induction of autophagy, depicting a possible role autophagy and mitochondrial integrity in catalase-offered protection against lethal toxin. Our data further revealed that autophagy induction mitigated catalase-offered a cardioprotective effect, consistent with the finding of obliteration of lethal toxin-induced cardiomyocyte contractile anomalies. These findings collectively support a causal role of autophagy in lethal toxin-induced cardiac injury and catalase enzyme-offered cardioprotection.
Ample experimental evidence has revealed hemodynamic and cardiac anomalies following anthrax exposure [
1,
13‐
15,
17,
33,
34]. Data from our current study revealed that anthrax lethal toxin inhibits cardiomyocyte contractile function and intracellular Ca
2+ handling including depressed peak shortening amplitude and maximal velocity of shortening/re-lengthening, prolonged duration of re-lengthening as well as reduced electrically-stimulated intracellular Ca
2+ rise (ΔFFI), and delayed intracellular Ca
2+ clearance. These results are consistent with our previous report [
14]. Intriguingly, lethal toxin-induced anomalies in cardiac contractile and intracellular Ca
2+ properties were significantly attenuated or mitigated by overexpression of the antioxidant catalase. These findings denote a possible role of intracellular Ca
2+ homeostasis in lethal toxin- and/or catalase-elicited mechanical responses. Our observations of unchanged intracellular Ca
2+ regulatory proteins, including SERCA2a, Na
+-Ca
2+ exchanger and phospholamban, were consistent with our earlier report [
14], suggesting possible involvement of certain post-translational modification process in these intracellular Ca
2+ regulatory proteins en route to altered intracellular Ca
2+ homeostasis. In our hands, catalase overexpression alleviated lethal toxin-induced oxidative stress (O
2
- and ROS) and autophagy, favoring a possible role of oxidative modification and autophagy regulation in intracellular Ca
2+ homeostasis in our experimental setting.
Autophagy is a tightly regulated cellular process through which mammalian cells degrade and recycle protein aggregates and organelles [
18]. Under physiological conditions, autophagy helps to maintain the amino acid pool during starvation, and prevents neurodegeneration, aging and tumor development through clearance of intracellular microbes [
18,
35,
36]. Impaired autophagy is often associated with cardiac diseases due to poor autophagic removal of damaged cellular components [
35]. Autophagy can be activated by pathophysiological stress stimuli such as, hypoxia, energy depletion and ER stress as well as bacterial, viral and parasite infections [
35,
36]. Although up-regulated autophagy serves to offset cardiac hypertrophy via protein degradation [
37,
38], excessive autophagy usually compromises cardiomyocyte survival and subsequently ventricular function. Enhanced autophagy is observed in failing hearts and a wide array of cardiovascular diseases resulting in cardiac death and impaired cardiac performance [
39‐
41]. Data from our study revealed that lethal toxin exposure induced overt myocardial autophagy, the effect of which was mitigated by catalase overexpression and NAC. More importantly, our data revealed that induction of autophagy with rapamycin nullified the cardioprotective benefit of catalase overexpression against lethal toxin whereas autophagy inhibition using 3-MA mimicked catalase-elicited beneficial effect. These data convincingly support a causality relationship of autophagy in lethal toxin- and catalase enzyme-induced cardiac mechanical responses. The fact that NAC ablated lethal toxin-induced up-regulated in autophagy (Figure
8A-D) further depicts a permissive role of oxidative stress in lethal toxin-induced induction of autophagy. ROS has been shown to activate starvation-induced autophagy, antibacterial autophagy and autophagic cell death through distinct mechanisms, depending on cell types and stimulation conditions [
42]. It is unclear at this time why NAC treatment itself lowered Beclin-1 levels, which may be related to intercellular redox state-governed autophagy initiation.
Mitochondrion plays a pivotal role in energy generation, intracellular Ca
2+ homeostasis and ROS production [
43,
44]. In addition to its primary function for energy generation to meet the high demand of the beating heart, mitochondria also regulates cell death in response to a wide variety of stress signals, such as oxidative stress, infection and DNA damage [
45]. Anthrax lethal toxin stimulates ROS production in macrophages and cardiomyocytes [
14]. It was also shown that mitochondrial impairment is a critical event leading to macrophage cytolysis during anthrax toxin exposure [
46]. This is also supported by our early finding of the involvement of NADPH oxidase, a mitochondrial-based O
2
--generating enzyme, in lethal toxin-induced cardiac anomalies [
14]. Data from our study revealed that anthrax lethal toxin exposure stimulates O
2
- and ROS generation and reduces mitochondrial membrane potential. Interestingly, lethal toxin-induced changes in O
2
-, ROS and mitochondrial membrane potential were attenuated or ablated by catalase overexpression, suggesting a likely role of mitochondria in catalase-offered protection against lethal toxin exposure. Nonetheless, it is surprising for the catalase enzyme to attenuate the catalase-resistant O
2
- generation. Although the precise mechanism responsible for such unexpected inhibition is still elusive at this time, a couple of scenarios may be considered. It is possible that catalase enzyme may rapidly clear the H
2O
2 produced by SOD, thus favoring a rightward shift for superoxide dismutation reaction to facilitate O
2
- removal. In addition, the appearance of O
2
- in myocytes could be induced by the external H
2O
2 [
47]. It is plausible to speculate that catalase-induced partial attenuation of O
2
- production may be associated with the reduction of H
2O
2 levels. Our data also suggested that damaged mitochondria in response to a lethal toxin challenge may be related to induction of autophagy. Although efficient removal of dysfunctional mitochondria by way of autophagy is critical for the maintenance of cell homeostasis, excessive autophagy induction, (in particular, mitophagy), may trigger loss of ATP production and mitochondrial membrane potential leading to mitochondrial injury [
48,
49]. Further study is warranted to examine the putative mechanisms whereby alterations in the autophagic removal of damaged mitochondria intervene in the process of lethal toxin toxicity.
Ubiquitin proteasome system (UPS) plays an essential role in regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, protein quality control DNA repair, transcription and immune response [
50‐
52]. UPS degrades both mis-folded and damaged proteins by covalently attaching ubiquitin to target proteins followed by proteasomal degradation of these proteins. Nonetheless, proteasome functional insufficiency (PFI) often develops under pathophysiological conditions as a result of impaired proteasome activity or insufficient activity to cope with the increased demand. Proteasome functional insufficiency may lead to decreased degradation of mis-folded proteins, resulting in accumulation of protein aggregates to further inhibit proteasomal activities and deteriorate cellular stress. Increased protein ubiquitination and aberrant protein aggregation are typical signs of PFI, which is commonly seen in cardiac proteinopathy, myocardial ischemia-reperfusion injury, idiopathic dilated cardiomyopathy and hypertrophic cardiomyopathy [
53‐
55]. PFI may compromise cardiac function through impairing protein quality control [
56]. Data from our study revealed a significant increase in ubiquitination accompanied by impaired proteasome activity in response to lethal toxin challenge. Our data also revealed a reminiscent inhibitory effect of H
2O
2 on proteasomal activity, the effect of which was reversed by the antioxidant NAC. One likely explanation for the decreased proteasome activity following lethal toxin exposure could be due to the inactivation of 26S proteasome subunits by oxidative modification [
57]. We and others have reported overt O
2
- accumulation in neutrophils, macrophages and myocardium following lethal toxin exposure [
14,
20,
21]. Inhibition of 26S proteasome could be caused by oxidative products, such as protein aggregates, oxidized lipids or by oxidative modification (4-hydroxy-nonenalyation) of several proteasome subunits [
35,
42,
52]. Moreover, oxidative stress can induce the dissociation of the 20S core particle from the 19S regulatory particle of the 26S proteasome, which results in loss of the activities of the 26S proteasome and thus results in accumulation of ubiquitinated proteins [
22]. Cardiac-specific catalase overexpression effectively mitigates lethal toxin-induced impairment of ubiquitin proteasome function (although not in ubiquitin level), suggesting a possible role of UPS in the antioxidant-offered beneficial effect against lethal toxin.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MRK participated in the study design, carried out in vivo studies, acquired contractile and Western blot data, performed in vitro experiments, performed statistical analysis and drafted the manuscript. XY participated in Western blot experiments. AEF participated in the design, coordination and manuscript writing. JR participated in the study design, performed statistical analysis and took part in manuscript writing. All authors have read and approved the final version of the manuscript.