Re-Organization of Cardiac Band 3 (cAE1) in Neonatal Cardiomyocytes Submitted to Alkali Loading.

Band 3 was initially described as the first member of the anion exchanger family (AE1). It is an integral membrane protein, initially characterized in erythrocytes. The AE family is encoded by four genes, AE1, AE2, AE3 and AE4. Three AE1 isoforms have been described up to now: erythroid (eAE1), kidney (kAE1), and cardiac (cAE1). Increased expression and activity of AE1 and AE3, as well as of the Na⁺/H⁺ exchanger have been demonstrated in hypertrophic myocardium of rats. A physical association of band 3 with cardiac actin has been previously described by our group, using yeast two hybrid screening. It has also been suggested that this binding occurs in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane. In the present study, immunolocalization analysis by confocal laser scanning microscopy was used to investigate the band 3 re-organization in neonatal cardiomyocytes (Wistar rats 1 to 2-day-old) submitted to different stimuli, such as pH changing or mechanical stretch (a model leading to cardiac hypertrophy). In the untreated cells, band 3 was dispersedly distributed in the cytoplasm, preferentially in the perinuclear region. On the other hand, when these cells were submitted to cyclic stretch (2 hours), band 3 appears between actin filaments, in the sarcomeric units. Similar distribution was showed in cardiac cells submitted to alkali loading (pH 8.9 for 2 hours). However, in experiments where the culture medium was pH 6.8, band 3 was dispersed in the cytoplasm, but was not concentrated in the perinuclear region. Intracellular pH (pHi) is a major regulator of diverse cellular processes including metabolic pathways, Ca²⁺ homeostasis, cell contractility, cell excitability and cell death; a rise in pHi activates Cl⁻/HCO3⁻ exchanger. On the other hand, the mechanical stretch stimulates a rapid secretion of Angiotensin II, rise of pHi and activation of intracellular signaling molecules such as protein kinase C and tyrosine kinases. These processes induce cell proliferation, cardiac hypertrophy and reorganization of actin cytoskeleton. The demonstration of reorganization of band 3 and its localization in the sarcomeric units after these stimuli suggest that band 3 is involved in the cardiac hypertrophy physiopathology. Band 3 could function as an anchorage for the contractile apparatus, as well as in the transport of anions in the myocardium, which are well described processes in normal erythrocytes.