A new role for AT1 and AT2-receptors in modulating cyclic stretch effects on organization of intercellular communication via gap junctions in cardiac cells

Objectives: Cyclic mechanical stretch is an important factor in self organization of cardiac cells leading to elongation of the cells and accentuation of the gap junction protein Cx43at the cell poles. Since in many cardiac diseases gap junctions (e.g. heart failure, atrial fibrillation) are no longer accentuated at the cell poles but are found at the lateral borders, and since in these diseases angiotensin-II often is elevated, we wanted to find out, which role the angiotensin-II receptors AT₁ and AT₂ play in the effects of cyclic mechanical stretch (CMS) on gap junctions.

Methods: Neonatal rat cardiomyocytes were cultured on flexible 6-well-plates coated with gelatine for 7 days. Thereafter, cells were kept static or stimulated with CMS (1Hz; 10% elongation) for 24h (±100 nM angiotensin-II) in absence or presence of the AT₁-antagonist losartan (100 nM) or the AT₂-antagonist PD123177 (100 nM). The specimen were investigated by immunohistology and Western blot for the gap junction protein connexin43.

Results: CMS lead to elongation of cardiomycytes within 24h which became rod shaped in 78±2%. These cells were orientated 75±1.5° perpendicular to the stretch axis. Elongation and orientation of the cells under CMS were not affected by angiotensin-II. However, while under static conditions connexin43 was equally distributed around the cells, within 24h CMS connexin43 was accentuated at the cell poles (pol: lateral 10±0.5: 1), which was significantly inhibited by additional angiotensin-II (4.8±0.4: 1). This effect of angiotensin-II was augmented by losartan but could be antagonized by PD123177. Since in presence of losartan angiotensin-II exerts effects no longer via AT₁ but via AT₂-receptors, this data indicates that AT₁-receptors favor polarization of Cx43 while AT₂ receptors inhibit polarization and favor lateralization. While under resting static conditions angiotensin-II increased Cx43 protein amount, the total protein expression of Cx43 was also increased by CMS, which was not further augmented by additional angiotensin-II.

Conclusion: Cyclic stretch induces elongation and orientation of cardiomyocytes together with accentuation of the gap junction protein Cx43at the cell poles. Angiotensin-II can inhibit Cx43-polarisation via AT₂-receptors, while AT₁ receptors mediate polarisation. Thus, angiotensin-II may contribute to the formation of an arrhythmogenic substrate by reduced polarisation of gap junctions via AT₂-receptor activation.