Human cardiomyocyte hypertrophy induced in vitro by gp130 stimulation.

OBJECTIVES: Recent in vivo and in vitro studies in animals have demonstrated that cytokines of the IL-6 family are involved in cardiac hypertrophy and in protection of cardiomyocytes against apoptosis. The present study aims to analyse the capacity of human atrial cardiac cells (i.e., cardiomyocytes and fibroblasts) to display the gp130 receptor subunit, and to evaluate its functionality. METHODS: Twenty human atrial biopsies were used for immunohistochemistry, in situ hybridisation, and western blot analysis or dissociated for isolation and primary culture of cardiac cells. RESULTS: Fibroblasts present in tissue or maintained in primary culture clearly express gp130 whereas the signal in cardiomyocytes is weaker. Culture of cardiac cells with a gp130 agonist antibody enhances atrial natriuretic peptide (ANP), beta myosin heavy chain (beta-MHC) expression in cardiomyocytes, and significantly increases the cell surface area microm(2)). This process could involve STAT3 (signal transducer and activator of transcription 3) phosphorylation. CONCLUSIONS: These results demonstrate that gp130 activation in human cardiac cells leads to cardiomyocyte hypertrophy. We discuss several hypotheses on the role of IL-6-type cytokines on cardiomyocyte functions.

A fusion protein of the gp130 and interleukin-6Ralpha ligand-binding domains acts as a potent interleukin-6 inhibitor.

Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an alpha-subunit (IL-6Ralpha) that binds IL-6 with low affinity and a beta-subunit (gp130) that binds the IL-6.IL-6Ralpha complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6.soluble IL-6Ralpha complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Ralpha (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies.

Secretion of IL-6, IL-11 and LIF by human cardiomyocytes in primary culture.

Interleukin (IL)-6-type cytokines are multifunctional proteins involved in cardiac hypertrophy and myocardial protection. Recent studies, performed on animal models, report the production of these cytokines by heart. The aim of this study was to analyse the capacity of myocytes and fibroblasts isolated from human atrium to secrete IL-6, leukaemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), IL-11, oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and the soluble receptor subunits sIL-6R and sgp130 during primary culture. We detected LIF, IL-11, sgp130 and a large amount of IL-6, but not OSM, CT-1, CNTF nor IL-6R in these culture supernatants. Both cardiomyocytes and fibroblasts are able to spontaneously produce IL-6. The increase of IL-6 production all along the culture period appears to be the consequence of fibroblast proliferation and gp130 stimulation. This is the first demonstration that human cardiac cells are able to secrete IL-6, but also LIF and IL-11 in vitro. These cytokines could be involved in an autocrine and/or a paracrine networks regulating myocardial cyto-protection, hypertrophy and fibrosis.

Reexpression of the nifedipine-resistant calcium channel during dedifferentiation of adult rat ventricular cardiomyocytes.

INTRODUCTION: Using an adult rat ventricular culture model and whole-cell patch-clamp technique, we investigated whether the nifedipine-resistant calcium current observed at the neonatal stage but not at the adult stage could be reobserved under dedifferentiating conditions. METHODS AND RESULTS: Application of 2 microM nifedipine totally inhibited the inward calcium current (carried by 5 mM Ba2+) in freshly isolated cells from adult rat heart, but it failed to block it completely when cells are cultured for 8 to 12 days. Dose-response curves of nifedipine in the range from 2 to 50 microM showed a residual current that represented, in the presence of 2 microM nifedipine, 16.4%+/-1.8% (n = 10) and 20.4%+/-1.5% (n = 10) of the total current in 8- and 12-day-old cultured cells, respectively. In these conditions, its density at 0 mV increased slightly during culture (-2.1+/-0.2 pA/pF, n = 7, and -3.2+/-0.4 pA/pF, n = 8, after 8 and 12 days in culture, respectively), without modification in the current-to-voltage curve, as well as in kinetics properties. CONCLUSION: These results show that nifedipine-resistant calcium current, which has been shown to be developmentally regulated in rat ventricular cardiomyocytes, can be reexpressed in cultured-dedifferentiated cells. Its possible expression and implication in physiopathologic function are suggested.