0265 : Tumour necrosis factor- carried by microparticles from apoptotic RAW 264.7 macrophage cells triggers deleterious effects on cardiomyocytes from adult mice

Abstract

After ischaemic injury and in patients with atherosclerosis, the pool of inflammatory macrophages is enlarged in the heart and in atherosclerotic plaques. Monocyte/macrophage-derived microparticles (MPs) are part of the pathological process of unstable atherosclerotic plaques. In the present study, we have focused on MPs released by apoptotic murine RAW 264.7 macrophage cell line. The hypothesis that these MPs could induce deleterious effects in adult myocardial cells was evaluated. Flow cytometry and western blot analysis showed that these MPs contained the soluble form of tumour necrosis factor alpha (TNF-α). Furthermore, cardiomyocyte sarcomere shortening amplitudes and kinetics were reduced within 5 min of exposure to MPs. Conversely, Ca 2+ transient amplitudes and kinetics were not modified. The contractile effects of MPs were completely prevented after pre-treatment with nitric oxide synthase, guanylate cyclase or TNF-α inhibitors as well as blocking TNF-α receptor 1 with neutralizing antibody. Moreover, confocal microscopy showed that, after 1 h, MPs were clearly surrounding rod-shaped cardiomyocytes, and after 2 h they were internalized into cardiomyocytes undergoing apoptosis. Indeed, after 4 h of treatment with MPs, cardiomyocytes expressed increased apoptotic markers expression (caspase-3, caspase-8, Bax and cytochrome C) as demonstrated by Western Blot. The present study showed that these MPs carry functional TNF-α and activate TNFR1 in isolated adult murine ventricular cardiomyocytes, leading to cell shortening without affecting calcium signalling via a mechanism sensitive to NO synthase inhibitor and that the mitochondrial intrinsic pathway was implicated in their proapoptotic effects. Thus, the present study allows supporting the hypothesis that MPs from human carotid atherosclerotic plaques may contain active TNF-α, which could contribute to MP-induced inflammatory signals in human atherosclerotic lesions leading to myocardial infarction. The author hereby declares no conflict of interest