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, 47 (3), 382-90

Overexpression of Hsp20 Prevents Endotoxin-Induced Myocardial Dysfunction and Apoptosis via Inhibition of NF-kappaB Activation

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Overexpression of Hsp20 Prevents Endotoxin-Induced Myocardial Dysfunction and Apoptosis via Inhibition of NF-kappaB Activation

Xiaohong Wang et al. J Mol Cell Cardiol.

Abstract

The occurrence of cardiovascular dysfunction in sepsis is associated with a significantly increased mortality rate of 70% to 90% compared with 20% in septic patients without cardiovascular impairment. Thus, rectification or blockade of myocardial depressant factors should partly ameliorate sepsis progression. Heat shock protein 20 (Hsp20) has been shown to enhance myocardial contractile function and protect against doxorubicin-induced cardiotoxicity. To investigate the possible role of Hsp20 in sepsis-mediated cardiac injury, we first examined the expression profiles of five major Hsps in response to lipopolysaccharide (LPS) challenge, and observed that only the expression of Hsp20 was downregulated in LPS-treated myocardium, suggesting that this decrease might be one of the mechanisms contributing to LPS-induced cardiovascular defects. Further studies using loss-of-function and gain-of-function approaches in adult rat cardiomyocytes verified that reduced Hsp20 levels were indeed correlated with the impaired contractile function. In fact, overexpression of Hsp20 significantly enhanced cardiomyocyte contractility upon LPS treatment. Moreover, after administration of LPS (25 microg/g) in vivo, Hsp20 transgenic mice (10-fold overexpression) displayed: 1) an improvement in myocardial function; 2) reduced the degree of cardiac apoptosis; and 3) decreased NF-kappaB activity, accompanied with reduced myocardial cytokines IL-1beta and TNF-alpha production, compared to the LPS-treated non-transgenic littermate controls. Thus, the increases in Hsp20 levels can protect against LPS-induced cardiac apoptosis and dysfunction, associated with inhibition of NF-kappaB activity, suggesting that Hsp20 may be a new therapeutic agent for the treatment of sepsis.

Figures

Figure 1
Figure 1
Time course of major Hsps’ expression in the mouse heart after i.p. administration of LPS(25 μg/g). (A) At the indicated intervals, mouse hearts were excised and homogenized to assess major Hsps’ expression by Western blot analysis. (B) The quantitative results from the Western-blots. α-actin was used as an internal control (n = 4 hearts for each time point, *P < 0.05 versus control 0 h ).
Figure 2
Figure 2
Effect of decreased Hsp20 expression on cardiaomyocyte contractility.(A) Photomicrographs of Ad.Hsp20-AS-infected or Ad.GFP-infected adult rat cardiomyocytes (500 MOI)) were taken after infection for 48 h. (B) Expression of Hsp20 in infected adult rat cardiomyocytes. The endogenous Hsp20 protein was down-regulated in Ad.Hsp20-AS-infected myocytes by 40% compared with Ad.GFP-infected myocytes. α-Actin was used as a loading control. (C) Representative traces of cardiomyocyte mechanics in Ad.GFP-infected and Ad.Hsp20-AS-infected adult rat cardiomyocytes. (D–F) Decreased myocyte percent fractional shortening (FS%; B) and decreased maximal rates of contraction and relaxation (±dL/dt; E and F) in Ad.Hsp20-AS-infected myocytes compared with Ad.GFP-infected cells. n = 5 hearts for each group and 15–25 myocytes/heart. Values are means ± SD. *P < 0.05.
Figure 3
Figure 3
Effect of increased Hsp20 expression on cardiaomyocyte contractility and viability after LPS treatment. (A) Expression of Hsp20 in infected adult rat cardiomyocytes. The total Hsp20 protein was up-regulated in Ad.Hsp20- infected myocytes by 2.5 fold compared with Ad.GFP-infected myocytes. α-Actin was used as a loading control. (B) Representative traces of cardiomyocyte mechanics in Ad.GFP-infected and Ad.Hsp20-infected adult rat cardiomyocytes with or without LPS treatment (1 μg/ml). (C–E) LPS treatment resulted in a marked decrease in FS% (C) and maximal rates of contraction and relaxation (±dL/dt; D and E) in Ad.GFP-infected myocytes, whereas these decreases were greatly attenuated in Ad.Hsp20-infected myocytes (C–E). LPS-induced cell death (F) and apoptosis (G) were significantly reduced in Ad.Hsp20-infected cardiomyocytes. n = 6 hearts for each group and 15–25 myocytes/heart for contractility measurement. For assessment of cell viability and apoptosis, similar results were observed in three additional, independent experiments. Values are means ± SD. *P < 0.001, # P < 0.05 vs. vehicle.
Figure 4
Figure 4
Overexpression of Hsp20 in vivo enhances cardiac function (A and B) and improves LPS-induced myocardial dysfunction. (A) 10-fold overexpression of Hsp20 protein was observed in Hsp20-hearts. LPS treatment (25μg/g) for 6 h resulted in reduced levels of Hsp20 in Non-transgenic (NTG) hearts, whereas no significant alteration was observed in Hsp20-hearts. In addition, Hsp20 overexpression did not alter the levels of Hsp70 and Hsp60 in the heart under either control conditions or LPS treatment for 6 h._ (B) Representative M-mode echocardiograms are shown in NTG mice and Hsp20 mice with vehicle or LPS treatment for 6 h. (C) Left ventricular ejection fraction (LVEF) and (D) left ventricular end diastolic diameters (LVEDD) were dramatically depressed in NTG-hearts treated with LPS. By contrast, LPS-induced cardiac depression was significantly attenuated in Hsp20-hearts (C and D). *P < 0.001, # P < 0.05 verse vehicle. n=6 for each group.
Figure 5
Figure 5
Myocardial TNF-α (A) and IL-1β (B) protein production after LPS challenge. NTG and Hsp20-TG mice were administered an injection of LPS i.p. (25μg/g). Values represent mean ± SD from 3 different animals at each time point (*P<0.05 vs NTG).
Figure 6
Figure 6
Cardiac-specific expression of Hsp20 inhibits NF-κB activation. (A) Representative autoradiographs of an EMSA for NF-κB, and (B) Image analysis of activity of NF-κB. Results are representative of 3 separate time course experiments. *P < 0.05 vs. NTG mice. C) Representative Western blot analysis of phosphorylated and total IκBα in NTG and Hsp20-TG mice with or without LPS treatment (25μg/g) for 30 min. α-Actin was used as a loading control. D) Image analysis of phosphorylated and total IκBα determined by densitometry. Fold increase was calculated vs. respective NTG value (vehicle) set to 1.0. *P < 0.001, # P < 0.05 vs. vehicle mice, n=4 per group.
Figure 6
Figure 6
Cardiac-specific expression of Hsp20 inhibits NF-κB activation. (A) Representative autoradiographs of an EMSA for NF-κB, and (B) Image analysis of activity of NF-κB. Results are representative of 3 separate time course experiments. *P < 0.05 vs. NTG mice. C) Representative Western blot analysis of phosphorylated and total IκBα in NTG and Hsp20-TG mice with or without LPS treatment (25μg/g) for 30 min. α-Actin was used as a loading control. D) Image analysis of phosphorylated and total IκBα determined by densitometry. Fold increase was calculated vs. respective NTG value (vehicle) set to 1.0. *P < 0.001, # P < 0.05 vs. vehicle mice, n=4 per group.
Figure 7
Figure 7
Cardiac-specific expression of Hsp20 suppresses LPS-induced myocardial apoptosis and caspase-3 activity. (A) Triple-staining with anti–α-sarcomeric actin antibody (red), DAPI (blue), and TUNEL (green, see arrow) to determine apoptosis in the myocardium of LPS-treated Hsp20 mice compared with LPS-treated NTG mice. (B) LPS-treated Hsp20 TG hearts exhibited significantly reduced number of TUNEL-positive (green fluorescence) nuclei (n=5, with 3 sections per heart). *P<0.05, vs LPS-treated NTG hearts. (C) The extent of apoptosis was further assessed using an ELISA kit, which measures DNA fragmentation. *P<0.05 vs LPS-treated NTG hearts (n=6). (D) Serum LDH levels were significantly reduced in Hsp20-TG mice after LPS treatment for 6h, compared with those of NTG mice (n=5, *P<0.05). (E) Caspase-3 activity measurement in the myocardium of LPS-treated NTG and Hsp20-TG mice. Fold change was calculated vs. NTG value set to 100%. * P < 0.05 vs. LPS-treated NTG mice, n=6 per group.
Figure 8
Figure 8
Proposed scheme for protection of Hsp20 against LPS-triggered myocardial dysfunction and apoptosis. Overexpression of Hsp20 suppresses NF-κB activation and reduces caspase-3 activity triggered by LPS stimulation, leading to decreased production of myocardial proinflammatory cytokines including TNF-α and IL-1β, and inhibition of cardiac apoptosis. Consequently, LPS-induced myocardial dysfunction is attenuated in Hsp20-mice.

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