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Proteasome inhibition attenuates coxsackievirus-induced myocardial damage in mice - PubMed

Proteasome inhibition attenuates coxsackievirus-induced myocardial damage in mice

Guang Gao et al. Am J Physiol Heart Circ Physiol. 2008 Jul.

Abstract

Coxsackievirus B3 (CVB3) is one of the most prevalent pathogens of viral myocarditis, which may persist chronically and progress to dilated cardiomyopathy. We previously demonstrated a critical role of the ubiquitin-proteasome system (UPS) in the regulation of coxsackievirus replication in mouse cardiomyocytes. In the present study, we extend our interest to an in vivo animal model to examine the regulation and role of the UPS in CVB3-induced murine myocarditis. Male myocarditis-susceptible A/J mice at age 4-5 wk were randomized to four groups: sham infection + vehicle (n = 10), sham infection + proteasome inhibitor (n = 10), virus + vehicle (n = 20), and virus + proteasome inhibitor (n = 20). Proteasome inhibitor was administered subcutaneously once a day for 3 days. Mice were killed on day 9 after infection, and infected hearts were harvested for Western blot analysis, plaque assay, immunostaining, and histological examination. We showed that CVB3 infection led to an accumulation of ubiquitin conjugates at 9 days after infection. Protein levels of ubiquitin-activating enzyme E1A/E1B, ubiquitin-conjugating enzyme UBCH7, as well as deubiquitinating enzyme UCHL1 were markedly increased in CVB3-infected mice compared with sham infection. However, there was no significant alteration in proteasome activities at 9 days after infection. Immunohistochemical staining revealed that increased expression of E1A/E1B was mainly localized to virus-damaged cells. Finally, we showed that application of a proteasome inhibitor significantly reduced CVB3-induced myocardial damage. This observation reveals a novel mechanism of coxsackieviral pathogenesis, and suggests that the UPS may be an attractive therapeutic target against coxsackievirus-induced myocarditis.

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Figures

Fig. 1.
Fig. 1.

Coxsackievirus B3 (CVB3) infection leads to an accumulation of protein-ubiquitin conjugates in mouse heart. A/J mice were infected with CVB3 [105 plaque-forming units (PFU) of Nancy stain] or phosphate-buffered saline (PBS; sham infection). At 9 days (d) after infection (pi) mice were killed and heart tissue was harvested. A: Western blot was performed to detect the ubiquitinated [(Ub)n] proteins with an anti-ubiquitin antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was probed as a protein loading control. Protein levels of protein-ubiquitin conjugates [molecular mass (MM) from 82.2 to ∼230 kDa] were quantitated by densitometric analysis with the NIH ImageJ program and normalized to GAPDH expression. Data are means ± SE (sham-infected group: n = 3; CVB3 group: n = 3), and significance was determined by Student's t-test. *P < 0.05 vs. sham infection. B: heart homogenates were prepared, and proteasome activity was measured with the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (SLLVY-AMC). Results are expressed as the amount of AMC formed by the enzymatic cleavage of substrate (means ± SE of 3 independent measurements from each animal; sham-infected group: n = 8; CVB3 group: n = 9). NS, no significant difference vs. sham infection.

Fig. 2.
Fig. 2.

Expression of ubiquitinating enzymes is upregulated in CVB3-infected mouse heart. A/J mice were infected with CVB3 or sham infected with PBS as described in Fig. 1. Nine days after infection, hearts were collected. A: Western blot was performed with anti-E1A/E1B, anti-UbcH7, anti-E6-AP, and anti-GAPDH (loading control) antibodies. Levels of expression were quantitated by densitometric analysis with the NIH ImageJ 1.37 program and normalized to GAPDH expression. Data are means ± SE (sham-infected group: n = 7; CVB3 group: n = 6). *P < 0.05 vs. sham infection; NS, no significant difference vs. sham infection. B: immunohistochemical staining for ubiquitin-activating enzyme E1A/E1B (red) was carried out as described in

materials and methods

. Nuclei were counterstained with hematoxylin (blue). Scale bar, 50 μm.

Fig. 3.
Fig. 3.

Expression of deubiquitinating enzyme is increased in CVB3-infected mouse heart. A/J mice were infected and hearts collected as described in Figs. 1 and 2. Anti-ubiquitin COOH-terminal hydrolase L1 (UCHL1) antibody was used for immunoblotting of deubiquitinating enzyme UCHL1. Protein expression was quantitated and analyzed as described in Fig. 2. Data are means ± SE (sham-infected group: n = 7; CVB3 group: n = 6). *P < 0.05 vs. sham infection.

Fig. 4.
Fig. 4.

Proteasome inhibitor MLN353 inhibits CVB3 replication in mouse cardiomyocytes. HL-1 cells were preincubated with various concentrations of MLN353 (as described in

materials and methods

) for 30 min and then infected with CVB3 (multiplicity of infection = 100) for 1 h. A: 7 h after infection, cell lysates were collected and immunoblotted with anti-VP1 and anti-β-actin (as loading control) antibodies. Data are representative of 3 independent experiments. B: 18 h after infection, medium was collected from CVB3-infected cells and virus titer was determined by plaque assay. Values are means ± SE of 3 independent experiments. *P < 0.001 vs. vehicle-treated cells.

Fig. 5.
Fig. 5.

MLN353 treatment reduces proteasome activity in mouse heart. A: Kaplan-Meier plot of survival curves of vehicle- and MLN353-treated mice 9 days after CVB3 infection. The numbers in parentheses at 9 days after infection are the numbers of surviving mice over the numbers of total experimental mice. P > 0.05 vs. vehicle-treated mice by log-rank test. B: A/J mice were sham infected with PBS or CVB3 infected and treated with vehicle or MLN353. At 9 days after infection, heart homogenates were prepared and proteasome activity was measured as described in Fig. 1. Results are means ± SE of 3 independent measurements from each animal (vehicle group: n = 9; MLN353 group: n = 11). *P < 0.05 vs. vehicle-treated mice at 9 days after infection.

Fig. 6.
Fig. 6.

Effect of proteasome inhibition on CVB3 viral titer in mice. A/J mice were CVB3 infected with vehicle or MLN353 treatment. Heart tissues were collected at 9 days after infection, and heart homogenates were used for plaque assay (means ± SE; vehicle group: n = 8, MLN353 group: n = 11). NS, no significant difference vs. sham infection.

Fig. 7.
Fig. 7.

MLN353 treatment attenuates CVB3-induced myocardial injury in mice. A/J mice were CVB3 infected in the presence of vehicle or MLN353. At 9 days after infection, heart tissue from both vehicle and MLN353 groups were collected and hematoxylin and eosin stained (top) and the extent of myocarditis was histologically graded (bottom) based on the intensity and character of injury and inflammatory infiltration as described in

materials and methods

. Results are means ± SE (vehicle group: n = 11; MLN353 group: n = 15). *P < 0.05 vs. vehicle-treated mice at 9 days after infection.

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