RETRACTED: Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced myocardial contractile dysfunction: Role of autophagy

Abstract

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. After an institutional investigation into the work of Dr. Jun Ren, University of Wyoming subsequently conducted an examination of other selected publications of Dr. Ren's under the direction of the HHS Office of Research Integrity. Based on the findings of this examination, the University of Wyoming recommended this article be retracted due to concerns regarding data irregularities inconsistent with published conclusions. Specifically, University of Wyoming found evidence of data irregularities and image reuse in Figure 2 that significantly affect the results and conclusions reported in the manuscript.

Fig. 1

A: Effect of catalase (CAT) overexpression on animal survival following endotoxemic shock using LPS. Mice (n = 15 and 21 for FVB and CAT groups, respectively) were given intraperitoneal injection of LPS (30 mg/kg). Animals were monitored for lethality every 6 hours for up to 3 days. Log-rank test was used to determine the median survival time (FVB: 47.8 hr vs. CAT: 65.9 hr, p < 0.05 between the two groups); B: H&E staining of left ventricular myocardium from FVB and CAT transgenic mice treated with or without LPS (6 mg/kg, i.p.) or saline for 4 hrs. Original magnification 400×; LPS challenge triggers inflammatory leukocyte infiltration, the effect of which is abolished by CAT overexpression; C: TNF-α protein expression. Inset: Representative gel blots depicting expression of TNF-α and α-Tubulin (used as loading control) using specific antibodies; D: TNF-α mRNA expression; E: Liver protein carbonyl levels; and F: Liver caspase-3 activity. Mean ± SEM, n = 4–5 mice per group (panels C–F), * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig. 2

Echocardiographic properties of FVB and catalase (CAT) transgenic mice injected with LPS (6 mg/kg, i.p.) or saline for 4 hrs. A: Representative M-mode images; B: Heart rate (bpm: beat per minute); C: LV posterior wall thickness in diastole (LVPWd); D: LV end systolic diameter (LVESD); E: LV end diastolic diameter (LVEDD); and F: Fractional shortening (%). Mean ± SEM, n = 5–7 mice per group, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig. 3

Cardiomyocyte contractile properties in FVB and catalase (CAT) transgenic mice treated with LPS (6 mg/kg, i.p.) or saline for 4 hrs. A: Resting cell length; B: Peak shortening (normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (−dL/dt); E: Time-to-peak shortening (TPS); and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 71 – 72 cells from 3 mice per group; * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig. 4

Intracellular Ca²⁺ properties in cardiomyocytes from FVB and catalase (CAT) transgenic mice treated with LPS (6 mg/kg, i.p.) or saline for 4 hrs. A: Resting fura-2 fluorescence intensity (FFI); B: Electrically-stimulated rise in FFI (ΔFFI); C: Single exponential intracellular Ca²⁺ decay rate; and D: Bi-exponential intracellular Ca²⁺ decay rate. Mean ± SEM, n = 43–49 cells from 3 mice per group, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig. 5

Western blot analysis of autophagy markers in myocardium from FVB and catalase (CAT) transgenic mice treated with LPS (6 mg/kg, i.p.) or saline for 4 hrs. A: Representative gel blots depicting expression of LC-3I/II, Atg-5 and Atg-7 and Beclin-1 (α-tubulin used as loading control) using specific antibodies; B: LC3-II/LC3-I ratio; C: Atg-5; D: Atg-7 and E: Beclin-1; Mean ± SEM, n = 4–5, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig. 6

Effect of LPS (6 mg/kg, i.p., 4 hrs) or saline treatment on myocardial apoptosis in FVB and catalase (CAT) transgenic mice. A: Representative TUNEL assay images; All nuclei were stained with DAPI (blue) in the left column. TUNEL-positive nuclei were visualized with fluorescein (green) in the right column; B: Quantification of TUNEL assay from 15 fields (3 mice per group); C: Caspase-3 activity: D: Representative gel blots depicting expression of cleaved caspase-3, Bax, Bcl-2 and α-tubulin (loading control) using specific antibodies; and E: Pooled data of cleaved caspase-3, Bax and Bcl-2 (normalized to α-tubulin). Mean ± SEM, n = 4 – 6 samples per group (Panel B–D), * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig. 7

Ultrastructural changes, ROS/O₂⁻ production and protein carbonyl formation in FVB and catalase (CAT) transgenic mice treated with LPS (6 mg/kg, i.p.,) or saline for 4 hrs. A: Transmission electron microscopic micrographs of left ventricular tissues FVB-LPS hearts present myofibril disorientation and loss (asterisks), mitochondrial degeneration and autophagosome-like vacuoles (arrows). CAT-LPS hearts reveal largely normal myofibril structure and mitochondria assembly with the exception of some mild disruption cristae density (arrowheads); B: Generation of ROS and O₂⁻ with staining myocardial sections using 5-(6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (DCF, green fluorescence, left column) and dihydroethidium (DHE, red fluorescence, right column); C: Pooled DCF fluorescence intensity; D: Pooled DHE fluorescence intensity; and E: Protein carbonyl formation. Mean ± SEM, n = 10 – 12 fields (panel C–D) or 5 hearts (panel E), * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group;

Fig. 8

Western blot analysis of JNK, p38, Akt and PGC-1α in myocardium from FVB and catalase (CAT) transgenic mice treated with LPS (6 mg/kg, i.p.) or saline for 4 hrs. A: Representative gel blots depicting expression of JNK, p38, Akt, PGC-1α and α-tubulin (used as loading control) using specific antibodies; B: pJNK-to-JNK ratio; C: pp38-to-p38 ratio; D: pAkt-to-Akt ratio; and E: PGC-1α expression; Mean ± SEM, n = 4–5 mice per group, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig.9

Effect of the autophagy inhibitor 3-MA and antioxidant NAC on lipopolysaccharide (LPS)-induced cardiomyocyte contractile defects. Freshly isolated cardiomyocytes from normal FVB mice were incubated with LPS (100 μM) in the presence or absence of 3-MA (10 μM) or NAC (500 μM) for 1 hr. A: Resting cell length; B: Peak shortening (normalized to resting cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (− dL/dt); E: Time-to-peak shortening (TPS) and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 45–50 cells from 3 mice per group, * p < 0.05 vs. Control group, # p < 0.05 vs. LPS group.