Identification, characterization, and gene expression profiling of endotoxin-induced myocarditis

Abstract

In septic shock, reversible cardiac dysfunction starts within 24 h. Myocardial depressant factors are thought to cause myocyte dysfunction, resulting in alterations of intrinsic cardiac function. Nitric oxide is a myocardial depressant factor candidate. Here we identify endotoxin-induced myocarditis (EIM) a previously uncharacterized pathophysiological entity. Features of EIM include differential patterns of inducible NO synthase (NOS2) mRNA induction in the left (LV) and right (RV) ventricles during the systemic response inflammatory syndrome (SIRS) and the presence of myocarditis with focal areas of aseptic necrosis in the RV 24 h after SIRS induction. Even though clinical data lead to the presumption of myocardial injury in sepsis, the underlying pathophysiological mechanisms have not been previously elucidated. Gene expression profiling was used to test the hypothesis of differential LV and RV responses in EIM, and revealed novel patterns of qualitative and quantitative expansion of transcription. Those genes are novel targets for drug development in SIRS and sepsis. Our results demonstrate spatial and temporal heterogeneity of myocardial responses in EIM. These findings justify the design of treatments to ameliorate tissue injury in the RV. Because the complexity of the inflammatory response increases substantially as time elapses, we suggest a stepwise and multitarget therapeutic approach for SIRS and sepsis. Our findings can help identify innate immune pathways that could become targets for immunotherapy in the treatment of disease caused by potential bioterrorism agents.

Fig. 1.

Induction of NOS2 mRNA in heart after LPS and identification of endotoxin-induced myocarditis in the RV. (A–D) A composite of film autoradiography showing the time course of NOS2 mRNA induction in the heart by in situ hybridization histochemistry. NOS2 mRNA induction starts at 2 h and progresses at 6 h after LPS administration. At 24 h, NOS2 levels return virtually to baseline in the LV, but are increased in the RV. (Scale bar = 2 mm.) (E–L)A composite of low-magnification (E, G, I, and K) and high-magnification (F, H, J, and L) images of NOS2 mRNA in heart ventricles. Shown are the LV at2h(E and F), 6 h (G and H), and 24 h (I and J) and the RV at 24 h (K and L). Arrows point to areas of increased NOS2 expression, which appear as white dots in dark-field (low magnification) and black dots in high-magnification images. Note high levels of NOS2 mRNA in the RV at 24 h (K and L). (Scale bar, 378 μm for E, G, I, and K and 30 μm for F, H, J, and L.) (M and N) Photomicrographs of hematoxylin/eosin-stained sections of heart ventricles 24 h after LPS administration. (M) In this section, the RV shows intense signs of inflammatory reaction. The myocardium is infiltrated by a moderate number of neutrophils that surround degenerate cardiomyocytes. Cardiomyocytes are fragmented, pale, and vacuolated, and in many areas the proteins are globular. Some globular proteins are surrounded by degenerate neutrophils characterized by fine nuclear debris and several neutrophils contain bright eosinophilic cytoplasmic material (phagocytosis). (N) Notice no signs of local inflammatory reaction in LV at 24 h after LPS administration. (Scale bar, 3.5 μm in M and 33.78 μm in N.)

Fig. 2.

Summary of microarray results. In each Venn diagram, circles represent the main effects of our ANOVA analysis: LPS treatment (red circle), LPS treatment by side (yellow circle), and side (blue circle). Numbers inside each compartment represents the number of transcripts that are significant for that effect. The intersections of the sets represent genes with P < 0.01 for each of the effects involved in the intersection. Note the expansion of transcripts that are significant for treatment effect from 6 to 24 h.

Fig. 3.

Transcriptional patterns during EIM. Cluster analysis of genes and EST that have fold change 5 or higher at 24 h after a single LPS injection. High transcriptional fold changes accompany the histological image of acute myocarditis with focal areas of aseptic necrosis. In each treatment group, genes colored in red are up-regulated, and genes colored in green are downregulated when compared to the average gene expression across all conditions. Note that expression appears to be higher in the RV for most transcripts. The number of transcripts that have fold change 5 or higher is significantly larger in the RV than in the LV (Inset). Accession number denotes EST. 24SALR, saline treated RV at 24 h; 24SALL, saline treated LV at 24 h; 24 LPSR, LPS treated RV at 24 h; 24 LPSL, LPS treated LV at 24 h.

Fig. 4.

Semiquantitative RT-PCR of genes induced at 24 h after LPS. Our results confirm large fold changes for 11 genes randomly selected out of genes and EST with fold change 5 or higher at 24 h (ANOVA, P < 0.05). GluT, protein-glutamine γ-glutamyltransferase; Gro, growth-related oncogene; ICBP, intracellular calcium-binding protein (MRP14); IL-1b, interleukin-1 β; Lcn, lipocalin 2; MIP1a, macrophage inflammatory protein-1 α; MIP2, macrophage inflammatory protein-2 precursor; MT2, metallothionein-2; Pap, pancreatitis-associated protein precursor; Reg, regenerating gene; TIMP, tissue inhibitor of metalloproteinase-1.

Fig. 5.

Diagram summarizing EIM data. LPS acts through toll-like receptor and initiates the heart's response to SIRS. Each spiral represents ≈50 genes or EST that are significantly changed in our microarray studies at P ≥ 0.01. Green spirals denote the number of genes that are common to the 6- and 24-h transcriptional response; they characterize a possible stereotypic response of the innate immune system in the heart. Red and black spirals designate genes that have a fold change of 2 or higher and 5 or higher, respectively. There is a small divergence in the response of the RV and LV at 6 h, which is intensified at 24 h. Note that the expansion of the transcriptional response in the heart at 24 h is more intense in the RV (it has more red and black spirals), the site for EIM. Hypothetically, the exacerbation of EIM would lead to death and the termination of the inflammatory process would result in recovery. R, right ventricle; L, left ventricle.