Cardiac fibroblasts play an important role in adverse cardiac remodelling. As in many cardiac diseases connexin43 (Cx43) is altered, we wanted to elucidate whether fibroblasts may influence cardiac Cx43 expression. We used four different cell culture systems of neonatal rat cardiomyocytes (CM) and fibroblasts (FB): type 1, pure CM culture; type 2, co-culture of CM/FB; type 3, pure FB culture; type 4, Transwell® system: CM/FB co-cultured but separated by a microporous membrane. Stimulation of types 1-3 cell culture models with isoprenaline significantly enhanced Cx43-protein and Cx43-mRNA expression as well as phosphorylation of ERK and translocation of AP1 and CREB only in the CM cultures; whereas, the CM/FB co-cultures and the FB cultures did not respond to isoprenaline. Similarly, if CM and FB were separated by a microporous membrane (Transwell® system) the isoprenaline-induced increase in CM Cx43 was completely suppressed, suggesting the existence of a soluble factor responsible for the suppressant effect of FB. Angiotensin II determination in types 1 and 2 cell culture supernatants revealed that the CM/FB co-cultures exhibited a significant higher angiotensin II release than the CM cultures. Furthermore, we aimed to inhibit angiotensin II signal transduction pathway: blockade of AT1 receptors or PKC inhibition restored the responsiveness of CM/FB co-cultures to isoprenaline. Moreover, external addition of angiotensin II to CM cultures also resulted in suppression of isoprenaline-stimulated Cx43 expression in an AT1-receptor- and PKC-dependent manner. Thus, our study indicates that cardiac fibroblasts inhibit β-adrenoceptor-dependent Cx43 signalling in CM involving angiotensin II.

Cyclic mechanical stretch (CMS) and angiotensin II (ATII) play an important role in cardiac remodelling. Thus, we aimed to examine how ATII affects CMS-induced changes in localisation and expression of the gap junction protein connexin43 (Cx43). Neonatal rat cardiomyocytes cultured on gelatin-coated Flexcell cell culture plates were kept static or were exposed to CMS (110 % of resting length, 1 Hz) for 24 h with or without additional ATII (0.1 μmol/L). Moreover, inhibitors of ATII receptors (AT-R) were used (for AT(1)-R: losartan 0.1 μmol/L, for AT(2)-R: PD123177 0.1 μmol/L). Thereafter, the cardiomyocytes were investigated by immunohistology, PCR and Western blot. After 24 h of CMS, cardiomyocytes were significantly elongated and orientated 75 ± 1.6° nearly perpendicular to the stretch axis. Furthermore, CMS significantly accentuated Cx43 at the cell poles (ratio Cx43 polar/lateral static: 2.32 ± 0.17; CMS: 10.08 ± 3.2). Additional ATII application significantly reduced Cx43 polarisation (ratio Cx43 polar/lateral ATII: 4.61 ± 0.42). The combined administration of ATII and losartan to CMS further reduced Cx43 polarisation to control levels, whilst the AT(2)-R blocker PD123177 restored polarisation. Moreover, CMS and ATII application resulted in a significant Cx43 protein and Cx43 mRNA up-regulation which could be blocked by losartan but not by PD123177. Thus, CMS results in a self-organisation of the cardiomyocytes leading to elongated cells orientated transversely towards the stretch axis with enhanced Cx43 expression and Cx43 accentuation at the cell poles. ATII enhances total Cx43 mRNA and protein expression probably via AT(1)-R (=inhibitory effect of losartan) and reduces Cx43 polarisation presumably via AT(2)-R, since PD123177 (but not losartan) inhibited the negative effects of ATII on polarisation.

Gut microbiota (GM) in the epigenetic mechanisms of diabetes mellitus and the reprogramming of the cells is a novel and emerging concept. The purpose of this chapter is to describe the modification of the GM and its relation with DM2. The increased risk of this disease is associated with changes in the amount of Bacteroides/Clostridium in the Firmicutes/Bacteroidetes ratio of people having DM. A dysbiosis state associated generates low-grade inflammation with similar characteristics that occur under metabolic syndrome, whose pattern is recognized by Toll-like receptor that recognizes important patterns of immunity. The synthesis of butyrate generated by intestinal microorganisms inhibits the metabolic pathway of histone deacetylase, promoting cellular differentiation, proliferation, and insulin resistance. On the other hand, the direct relationship between the neuroendocrine system and the GM has been demonstrated through the production of serotonin by enterochromaffin cells, whose action could influence the etiopathogenic factors of DM2.

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