Ongoing coxsackievirus myocarditis is associated with increased formation and activity of myocardial immunoproteasomes

Abstract

A growing body of evidence indicates that viral infections of the heart contribute to ongoing myocarditis and dilated cardiomyopathy. Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the human disease and allow identification of susceptibility factors that modulate the course of viral myocarditis. Susceptible mouse strains develop chronic myocarditis on the basis of restricted viral replication, whereas resistant strains recover after successful virus elimination. In comparative whole-genome microarray analyses of infected hearts, several genes involved in the processing and presentation of viral epitopes were found to be uniformly up-regulated in acutely CVB3-infected susceptible mice compared with resistant animals. In particular, expression of the catalytic subunits LMP2, LMP7, and MECL-1, immunoproteasome proteins important in the generation of major histocom-patibility complex (MHC) class I-restricted peptides, was clearly enhanced in the susceptible host. Increased expression resulted in enhanced formation of immunoproteasomes and altered proteolytic activities of proteasomes in the heart. This was accompanied by a concerted up-regulation of the antigen-presenting machinery in susceptible mice. Thus, we propose that increased formation of immunoproteasomes in susceptible mice affects the generation of antigenic peptides and the subsequent T-cell-mediated immune responses.

Figure 1

Regulation of LMP7, LMP2, and MECL-1 expression in the course of CVB3 myocarditis. Comparison of changes in immunosubunit expression of CVB3-infected mice and uninfected controls at days 4, 8, and 28 p.i. obtained by microarray analysis (left) and quantitative PCR analysis (right). At day 8 p.i., the strongest up-regulation of LMP7, LMP2, and MECL-1 was detected in susceptible mice (A.BY/SnJ, SWR/J) compared with resistant mice (C57BL/6, DBA/1J). At days 4 and 28 p.i., up-regulation was less pronounced and nearly uniform in all four mouse strains. RNA was pooled from five animals per mouse strain, and experiments were repeated twice.

Figure 2

Time course of immunoproteasome expression in CVB3-infected susceptible and resistant mice. Quantitative RT-PCR analysis of expression of LMP7, LMP2, and MECL-1 at days 4, 8, 10, 12, and 28 p.i. revealed that the enhanced up-regulation in A.BY mice is prolonged up to day 10 p.i. compared with resistant C57BL/6 mice. At days 12 and 28 p.i., both mouse strains show expression levels of LMP7, LMP2, and MECL-1 that are similar to uninfected controls, as indicated by the dashed line. RNA was pooled from five animals per mouse strain, and experiments were repeated twice. Expression of immunoproteasomal subunits in infected animals was normalized to expression in noninfected controls by means of the comparative C_t method (2^−ΔΔCt).

Figure 3

Identification and quantification of LMP7 mRNA-expressing cells in the myocardium of CVB3-infected susceptible (A.BY/SnJ) and resistant (C57BL/6) mice by in situ hybridization. A: CVB3 infection is more pronounced in A.BY/SnJ mice (I) compared with C57BL/6 mice at day 8 p.i. (II). Increased LMP7 expression in cardiomyocytes of infected animals compared with uninfected controls (III and V and IV and VI) and in infiltrating cells (3A III and IV, inset) of infected animals. Bars = 20 μm. B: Quantification of LMP7-specific silver grains in single cardiomyocytes and infiltrating cells (n = 50 ± SD, pvalues were determined by Mann-Whitney U-test).

Figure 4

Enhanced incorporation of immunoproteasome subunits into 20S proteasomes of hearts of CVB3-infected resistant and susceptible mice. For purification of 20S proteasomes, 10 hearts of uninfected and CVB3-infected C57BL/6 and A.BY/SnJ mice were pooled, respectively, and Western blot analysis was performed. As controls, proteasomes of nontransfected T2 cells (c20S) and transfected T2 cells (i20S) were used. A: Purified 20S proteasomes from uninfected (control) or CVB3-infected mice were separated by SDS-PAGE and probed with specific antibodies to detect LMP2, MECL-1, LMP7, and α6 as indicated. Figure shows representative gel from two different 20S proteasome preparations. B: Densitometric analysis of the Western blot signals correspond to the indicated proteasome subunits in control and CVB3-infected mouse strains.

Figure 5

Two-dimensional IEF-SDS-PAGE of 20S proteasome purified from the pooled hearts of uninfected and CVB3-infected C57BL/6 and A.BY/SnJ mice. A: Purified 20S proteasomes from uninfected (left) or CVB3-infected (right) mice were separated by IEF-SDS-PAGE, and gels were silver stained. The top panel shows gels from A.BY/SnJ mice, the bottom panel from C57BL/6 mice. The constitutive subunits β1, β2, and β5 and the immunoproteasome subunits LMP2, LMP7, and MECL-1 are indicated. The 2-D gel patterns shown were reproducible in four gels of two independent 20S proteasome purifications. B: Densitometric evaluation of the indicated proteasome subunits (A). The intensity values of each corresponding pair of catalytic proteasome subunits are shown in single bars. In the fourth panel, bars represent percentage of combined constitutive β1, β2, and β5 subunits and of the immunosubunits LMP2, LMP7, and MECL-1.

Figure 6

Altered proteolytic activities of purified heart proteasomes of CVB3-infected resistant and susceptible mice. Proteolytic activities of 20S proteasome purified from pooled hearts of uninfected and CVB3-infected A.BY/SnJ and C57BL/6 mice are depicted as percent reduction of free AMC. A: Chymotrypsin-like activity. B: Caspase-like activity.