Cardiac-specific catalase overexpression rescues anthrax lethal toxin-induced cardiac contractile dysfunction: role of oxidative stress and autophagy

Abstract

Background: Lethal and edema toxins secreted by Bacillus anthracis during anthrax infection were found to incite serious cardiovascular complications. However, the underlying mechanisms in anthrax lethal toxin-induced cardiac anomalies remain unknown. This study was designed to evaluate the impact of antioxidant enzyme catalase in anthrax lethal toxin-induced cardiomyocyte contractile dysfunction.

Methods: Wild type (WT) and cardiac-specific catalase overexpression mice were challenged with lethal toxin (2 μg/g, intraperotineally (i.p.)). Cardiomyocyte contractile and intracellular Ca(2+) properties were assessed 18 h later using an IonOptix edge-detection system. Proteasome function was assessed using chymotrypsin-like and caspase-like activities. GFP-LC3 puncta and Western blot analysis were used to evaluate autophagy and protein ubiquitination.

Results: Lethal toxin exposure suppressed cardiomyocyte contractile function (suppressed peak shortening, maximal velocity of shortening/re-lengthening, prolonged duration of shortening/re-lengthening, and impaired intracellular Ca(2+) handling), the effects of which were alleviated by catalase. In addition, lethal toxin triggered autophagy, mitochondrial and ubiquitin-proteasome defects, the effects of which were mitigated by catalase. Pretreatment of cardiomyocytes from catalase mice with the autophagy inducer rapamycin significantly attenuated or ablated catalase-offered protection against lethal toxin-induced cardiomyocyte dysfunction. On the other hand, the autophagy inhibitor 3-MA ablated or significantly attenuated lethal toxin-induced cardiomyocyte contractile anomalies.

Conclusions: Our results suggest that catalase is protective against anthrax lethal toxin-induced cardiomyocyte contractile and intracellular Ca(2+) anomalies, possibly through regulation of autophagy and mitochondrial function.

Figure 1

Effect of LeTx on survival rate and cell shortening in cardiomyocytes from WT and catalase mice. A: Survival rate (%); B: Peak shortening (PS, normalized to resting cell length); C: Maximum velocity of shortening (+ dL/dt); D: Maximum velocity of relengthening (-dL/dt); E: time-to-peak shortening (TPS); and F: time-to-90% re-lengthening (TR₉₀). For panel A; n = 8 mice per group. For panel B-F data were presented as Mean ± SEM, n = 100 to 150 cells from two to three mice per group, *P <0.05 vs. WT group, # P <0.05 vs. WT-LeTx group.

Figure 2

Effect of LeTx on intracellular Ca²⁺ transients in cardiomyocytes from WT and catalase mice. A: Resting fura-2 fluorescence intensity (FFI); B: electrically stimulated rise in FFI (ΔFFI); C: Single exponential intracellular Ca²⁺ transient decay rate; and D: Bi-exponential intracellular Ca²⁺ transient decay rate. Mean ± SEM, n = 60 to 75 cells from two to three mice per group, *P <0.05 vs. WT group, #P <0.05 vs. WT-LeTx group.

Figure 3

Effect of catalase (CAT) overexpression on LeTx-induced generation of O₂^- and ROS. A: Representative images depicting DHE fluorescence in myocardium from WT and catalase transgenic mice treated with or without lethal toxin. B: Pooled data of O₂^- production; C: Representative images depicting DCF fluorescence in myocardium from WT and catalase transgenic mice; and D: ROS levels in isolated cardiomyocytes from WT and catalase transgenic mice treated with or without lethal toxin. Mean ± SEM, n = 3 mice (panel B) or 6 isolations (panel D) per group, *P <0.05 vs. WT group, #P <0.05 vs. WT-LeTx group.

Figure 4

Effect of catalase (CAT) overexpression on LeTx-induced cardiomyocyte mitochondrial damage. A: Representative fluorescent images of JC-1 in control and LeTx exposure conditions. CCCP (10 μM) was used as a positive control; B: Summarized JC-1 ratio representing mitochondrial membrane potential in response to LeTx and CCCP exposure; and C: Cardiomyocyte mitochondrial membrane potential over time. Mean ± SEM, n = 6 isolations per group, *P <0.05 vs. WT group, #P <0.05 vs. WT-LeTx group.

Figure 5

Effect of catalase (CAT) overexpression and antioxidant on LeTx-induced changes in ubiquitin-proteasome activity. Expression and activity of the ubiquitin-proteasome system were determined in myocardium from WT and catalase transgenic mice treated with or without lethal toxin. For positive control, a cohort of isolated cardiomyocytes from WT mice were incubated with H₂O₂ (250 μM) for 2 h in the presence or absence of the antioxidant NAC (500 μM). A: Representative gel blots in triplicates depicting ubiquitination; B: Ubiquitin expression; C: Chymotrypsin-like activity; and D: Caspase-like activity. Mean ± SEM, n = 6 mice or isolations per group, *P <0.05 vs. WT group, #P <0.05 vs. WT-LeTx group, ψ P <0.05 vs. WT-H₂O₂ group.

Figure 6

Effect of LeTx on autophagosome formation. A: LC3-II in H9C2 cells treated with different concentrations of lethal toxin (25 to 200 ng/ml); B: LC3-II in H9C2 cells treated with 100 ng/ml lethal toxin for different durations of times (1 to 9 h). Inserts depicting expression LC3-I/II and GAPDH (loading control); C: Representative fluorescent microscopic images of GFP-LC3-II (DAPI's blue staining depicts nucleus; white arrows points autophagosomes); D: Quantitation of number of autophagosome per cell. Mean ± SEM, n = 3 independent cultures, *P <0.05 vs. Control.

Figure 7

Effect of catalase (CAT) overexpression on LeTx-induced autophagy. A: Representative gel blot depicting expression of Beclin-1, Atg-7, LC3-I/II and GAPDH (loading control); Triplicates in the gel blot represents samples from three mice of each treatment group. B: Beclin-1; C: Atg-7; and D: LC3-II. Mean ± SEM, n = 6 mice per group, *P <0.05 vs. WT group, #P <0.05 vs. WT-LeTx group.

Figure 8

Effect of ROS inhibition on lethal toxin-induced autophagy. A-D: Isolated cardiomyocytes were incubated with LeTx (100 ng/ml) for 3 h in the absence or presence of the NAC (500 μM). A: Representative gel blots depicting expression of Beclin-1, Atg-7, LC-3 and GAPDH (used as loading control); B: Beclin-1; C: Atg-7; D: LC3-II. Each lane in a treatment group on the gel represents sample from independent experiment. Mean ± SEM, n = 4 independent isolations per group, *P <0.05 vs. control group, # P <0.05 vs. LeTx group. Effect of autophagy induction or inhibition on lethal toxin induced cardiac contractile dysfunction. E-J: Isolated cardiomyocytes from WT and CAT mice were incubated with lethal toxin (100 ng/ml) for 3 h in presence or absence of the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) or autophagy inducer rapamycin (5 μM) respectively. E: Peak shortening (PS, normalized to resting cell length); F: Maximum velocity of shortening (+ dL/dt); G: Maximum velocity of relengthening (- dL/dt); H: time-to-90% re-lengthening (TR₉₀). Mean ± SEM, n = 60 to 75 cells from three mice per group, *P <0.05 vs. WT group, # P <0.05 vs. WT-LeTx group.