Cardiac overexpression of metallothionein rescues cardiac contractile dysfunction and endoplasmic reticulum stress but not autophagy in sepsis

Abstract

Sepsis is characterized by systematic inflammation where oxidative damage plays a key role in organ failure. This study was designed to examine the impact of the antioxidant metallothionein (MT) on lipopolysaccharide (LPS)-induced cardiac contractile and intracellular Ca(2+) dysfunction, oxidative stress, endoplasmic reticulum (ER) stress and autophagy. Mechanical and intracellular Ca(2+) properties were examined in hearts from FVB and cardiac-specific MT overexpression mice treated with LPS. Oxidative stress, activation of mitogen-activated protein kinase pathways (ERK, JNK and p38), ER stress, autophagy and inflammatory markers iNOS and TNFalpha were evaluated. Our data revealed enlarged end systolic diameter, decreased fractional shortening, myocyte peak shortening and maximal velocity of shortening/relengthening as well as prolonged duration of relengthening in LPS-treated FVB mice associated with reduced intracellular Ca(2+) release and decay. LPS treatment promoted oxidative stress (reduced glutathione/glutathione disulfide ratio and ROS generation). Western blot analysis revealed greater iNOS and TNFalpha, activation of ERK, JNK and p38, upregulation of ER stress markers GRP78, Gadd153, PERK and IRE1alpha, as well as the autophagy markers Beclin-1, LCB3 and Atg7 in LPS-treated mouse hearts without any change in total ERK, JNK and p38. Interestingly, these LPS-induced changes in echocardiographic, cardiomyocyte mechanical and intracellular Ca(2+) properties, ROS, stress signaling and ER stress (but not autophagy, iNOS and TNFalpha) were ablated by MT. Antioxidant N-acetylcysteine and the ER stress inhibitor tauroursodeoxycholic acid reversed LPS-elicited depression in cardiomyocyte contractile function. LPS activated AMPK and its downstream signaling ACC in conjunction with an elevated AMP/ATP ratio, which was unaffected by MT. Taken together, our data favor a beneficial effect of MT in the management of cardiac dysfunction in sepsis.

Fig. 1

Median lethal dose (LD₅₀) and lysosomal membrane stability in FVB and MT mice treated with or without LPS. A: Various dosages of LPS-induced mortality in mice over a period of 72 hrs. LD₅₀ was calculated using the probit analysis method; B: Lysosomal membrane stability evaluated by the lysosomal enzyme β-glucuronidase activity in FVB and MT mice treated with or without LPS (4 mg/kg, i.p.) for 4 hrs. Mean ± SEM, n = 30 mice in each strain for panel A; n = 6–7 per group for panel B, p value provided in panel A exhibited significant difference between the two.

Fig. 2

Cardiomyocyte contractile properties in FVB and MT transgenic mice treated with or without LPS (4 and 40 mg/kg, i.p.). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (− dL/dt); E: Time-to-PS (TPS); and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 85–87 cells from 4 mice per group, * p < 0.05 vs. FVB group; # p < 0.05 vs. respective FVB-LPS group.

Fig. 3

Intracellular Ca²⁺ properties in cardiomyocytes from FVB and MT mice treated with or without LPS (4 and 40 mg/kg, i.p.). A: Resting intracellular Ca²⁺; B: Peak intracellular Ca²⁺; C; Electrically-stimulated rise (peak – resting) in intracellular Ca²⁺; and D: Intracellular Ca²⁺ decay rate. Mean ± SEM, n = 58–60 cells from 4 mice per group, * p < 0.05 vs. FVB group; # p <0.05 vs. respective FVB-LPS group.

Fig. 4

ROS production and oxidative stress in cardiomyocytes from FVB and MT transgenic mice treated with or without LPS (4 mg/kg, i.p.). A: ROS production measured by DCF fluorescence; B: Glutathione (GSH) and oxidized glutathione (GSSG) levels; and C: GSH/GSSG ratio. Mean ± SEM, n = 5 – 6 mice per group, * p < 0.05 vs. FVB; # p < 0.05 vs. FVB-LPS group.

Fig. 5

Western blot analysis exhibiting phosphorylation of ERK, JNK and p38 in myocardium from FVB and MT transgenic mice treated with or without LPS (4 mg/kg. i.p.). A: Representative gel blots depicting total and phosphorylated ERK, JNK and p38 proteins using specific antibodies; B: pERK-to-ERK ratio; C: pJNK-to-JNK ratio; and D: pp38-to-p38 ratio. Mean ± SEM, n = 4–6 samples per group, * p < 0.05 vs. FVB group, # p < 0.05 vs. FVB-LPS group.

Fig. 6

Effect of MPA kinase inhibition on LPS-induced cardiomyocyte contractile dysfunction. FVB (control) cardiomyocytes were incubated with LPS (4 µg/ml) at 37°C for 2 hrs in the absence or presence of the ERK inhibitor U0126 (1 µM), the JNK inhibitor SP600125 (1 µM) or the p38 MAPK inhibitor SB203580 (1 µM) prior to functional assessment. A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (− dL/dt); E: Time-to-PS (TPS); and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 52–54 cells from 3 mice per group, * p < 0.05 vs. control, # p < 0.05 vs. LPS group.

Fig. 7

Western blot analysis displaying expression of the ER stress markers GRP78, Gadd153, PERK, eIF2α and IRE1α in myocardium from FVB and MT transgenic mice treated with or without LPS (4 mg/kg, i.p.). A: Representative gel blots depicting GRP78, Gadd153, PERK, eIF2α and IRE1α proteins using specific antibodies; B: Pooled data of GRP78, Gadd153, PERK, eIF2α and IRE1α proteins (normalized to GAPDH). Mean ± SEM, n = 3–6 samples per group, * p < 0.05 vs. FVB, # p < 0.05 vs. FVB-LPS group.

Fig. 8

Effect of inhibition of ER stress and oxidative stress on LPS-induced cardiomyocyte contractile dysfunction. FVB (control) cardiomyocytes were incubated with LPS (4 µg/ml) at 37°C for 2 hrs in the absence or presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 500 µM) or the antioxidant N-acetylcysteine (NAC, 500 µM) prior to mechanical assessment. A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (− dL/dt); E: Time-to-PS (TPS); and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 49–52 cells from 3 mice per group, * p < 0.05 vs. control, # p < 0.05 vs. LPS group.

Fig. 9

Expression of total and phosphorylated AMPK and ACC proteins as well as the AMP-to-ATP ratio in myocardium from FVB and MT transgenic mice treated with or without LPS (4 mg/kg, i.p.). A: pAMPK-to-AMPK ratio; B: pACC-to-ACC ratio; and C: AMP-to-ATP ratio. Mean ± SEM, n = 3–5 per group, * p < 0.05 vs. FVB, #p < 0.05 vs. FVB-LPS group.

Fig. 10

Protein expression of the autophagy markers Beclin-1, LC3B and Atg7 as well as the proinflammatory markers iNOS and TNFα in myocardium from FVB and MT mice treated with or without LPS (4 mg/kg, i.p.). A: Representative gel blots depicting Beclin-1, LC3B, Atg7, iNOS, TNFα and GAPDH (loading control) proteins using specific antibodies; B: Beclin 1; C: LC3B; D: Atg7; E: iNOS; and F: TNFα. Mean ± SEM, n = 3–4 per group, * p < 0.05 vs. FVB group.