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Aims: Adiponectin (APN) is an immunomodulatory and cardioprotective adipocytokine. Toll-like receptor (TLR) 4 mediates autoimmune reactions that cause myocarditis resulting in inflammation-induced cardiac injury. Here, we investigated whether APN inhibits inflammation and injury in autoimmune myocarditis by interfering with TLR4 signalling., Methods and Results: APN overexpression in murine experimental autoimmune myocarditis (EAM) down-regulated cardiac expression of TLR4 and its downstream targets tumour necrosis factor (TNF)α, interleukin (IL)-6, IL-12, CC chemokine ligand (CCL)2, and intercellular adhesion molecule (ICAM)-1 resulting in reduced infiltration with cluster of differentiation (CD)3+, CD14+, and CD45+ immune cells as well as diminished myocardial apoptosis. Expression of TLR4 signalling pathway components was unchanged in hearts and spleens of APN-knockout (APN-KO) mice. In vitro APN had no effect on TLR4 expression in cardiac and immune cells but induced dissociation of APN receptors from the activated TLR4/CD14 signalling complex. APN inhibited the expression of a TLR4-mediated inflammatory phenotype induced by exogenous and endogenous TLR4 ligands as assessed by attenuated nuclear factor (NF)-κB activation and reduced expression of TNFα, IL-6, CCL2, and ICAM-1. Accordingly, following TLR4 ligation, splenocytes from APN-KO mice showed enhanced expression of TNFα, IL-6, IL-12, CCL2, and ICAM-1, whereas dendritic cells (DCs) from APN-KO mice demonstrated increased activation and T-cell priming capacity. Moreover, APN diminished TLR4-mediated splenocyte migration towards cardiac cells as well as cardiomyocyte apoptosis after co-cultivation with splenocytes. Mechanistically, APN inhibited TLR4 signalling through cyclooxygenase (COX)-2, protein kinase A (PKA), and meiosis-specific serine/threonine kinase (MEK)1., Conclusion: Our observations indicate that APN protects against inflammation and injury in autoimmune myocarditis by diminishing TLR4 signalling thereby attenuating inflammatory activation and interaction of cardiac and immune cells.

Aims: Coronary artery occlusion resulting in ischaemia/reperfusion (I/R) injury is a major cause of mortality in the western world. Circulating natural IgM has been shown to play a significant role in reperfusion injury, leading to the notion of a pathogenic response against self by the innate immune system. A specific self-antigen (non-muscle myosin heavy chain II) was recently identified as the major target of pathogenic natural IgM. Therefore, we hypothesized that a synthetic peptide mimetope (N2) or monoclonal antibodies directed against the self-antigen would prevent specific IgM binding to the self-antigen and reduce reperfusion injury in the heart., Methods and Results: We find that treatment with N2 peptide reduces infarct size by 47% and serum cardiac troponin-I levels by 69% following 1 h ischaemia and 24 h reperfusion. N2 peptide or an anti-N2 F(ab')(2) (21G6) is also effective at preventing IgM and complement deposition. Additionally, N2 peptide treatment significantly reduces monocyte and neutrophil infiltration at 24 h and collagen deposition at 5 days. Finally, we show that human IgM (hIgM) also includes specificity for the highly conserved self-antigen and that myocardial injury in antibody-deficient mice reconstituted with hIgM is blocked by treatment with N2 peptide or 21G6 F(ab')(2)., Conclusion: The findings in this study identify potential therapeutics [i.e. N2 peptide or 21G6 F(ab')(2)] that prevent specific IgM binding to ischaemic antigens in the heart, resulting in a significant reduction in cardiac I/R injury.

Objective: Myocardin is a recently discovered transcriptional regulator of cardiac and smooth muscle development. Its ability to transactivate smooth muscle-specific genes has been firmly established in animal cells but its effect on heart muscle genes has been investigated less extensively and the consequences of ectopic myocardin expression in human cells are unknown., Methods: In this study, primary human mesenchymal stem cells and foreskin fibroblasts were transduced with human adenovirus vectors expressing the longest splice variant of the human myocardin gene (hAd5/F50.CMV.myocL) or with control vectors. One week later, the expression of muscle-restricted genes in these cells was analyzed by reverse transcription-polymerase chain reactions and immunofluorescence microscopy., Results: Forced expression of myocardin induced transcription of cardiac and smooth muscle genes in both cell types but did not lead to activation of skeletal muscle-specific genes. Double labeling experiments using monoclonal antibodies directed against striated (i.e. sarcomeric alpha-actin and sarcomeric alpha-actinin) and cardiac (i.e. natriuretic peptide precursor A) muscle-specific proteins together with a polyclonal antiserum specific for smooth muscle myosin heavy chain revealed that hAd5/F50.CMV.myocL-transduced cells co-express heart and smooth muscle-specific genes., Conclusions: These data indicate that the myocardin protein is a strong inducer of both smooth and cardiac muscle genes, but that additional factors are necessary to fully commit cells to either cardiac or smooth muscle cell fates.

Objective: The differentiation of the coronary vasculature was studied to establish in particular the formation of the coronary venous system., Methods: Antibody markers were used to demonstrate endothelial, smooth muscle, and fibroblastic cells in serial sections of embryonic quail hearts. The anti-beta myosin heavy chain and the neuronal marker HNK-1 were added to our incubation protocol., Results: In HH32, the coronary vascular network has developed into a circulatory system with connections to the sinus venosus, the aorta and the right atrium. The connections between the aorta and the right atrium allow for direct arteriovenous shunting. Subsequently, differentiation into coronary arteries and veins occurs with an interposed capillary network. The smooth muscle cells of the coronary arterial media derive from the subepicardial layer, whereas the subepicardially located cardiac veins recrute atrial myocardium, as these cells express the beta-myosin heavy chain antigen. Ganglia are located in the subepicardium close to the vessels, while nerve fibres tend to colocalize with the formed vessel channels., Conclusions: A new finding is presented in which the subepicardial coronary veins have a media that consists of myocardial cells. The close positional relationship of neural tissue and coronary vessels that penetrate the heart wall is explained as inductive for vessel wall differentiation, but not for invasion into the heart.