Characterization of cardiomyocyte membrane microdomains setup during postnatal development

Abstract

MAGUKs are one of the largest families of scaffolding proteins associated with intercellular junctions. Deletion of CASK is lethal, with full-term mice dying within hours of birth. The role of CASK was first established in neural development, but postnatal malformations have also been reported in the lungs and intestines. Previous results showed that CASK, in the adult cardiomyocyte, has an exclusive localization at the lateral membrane (LM), within the focal adhesion complexes. Preliminary data also suggest that CASK, in neonatal cardiomyocytes, plays a major role in the establishment and maintenance of intercellular junctions. To investigate the expression and organization of CASK and cardiomyocyte membrane microdomains during postnatal development of the Rat heart. Kinetics of CASK and polarity marker expression and localization during the developmental process from neonatal (P0) to adult (P60 days postnatal) were performed by western blot and immunofluorescence. CASK expression is maximal during early postnatal development (P0 to P5) and decreases from P20, to be strongly repressed at the adult stage. Structural polarity markers such as adherens (N-cadherin) and tight (ZO-1) junctions, as well as electrical polarity markers (NaV1.5, Kir2.1) follow the same kinetics. Only Cx43, a marker of electrical junctions, peaks at P20. CASK is very intense in the epicardial and subepicardial layers at P0 and P5 but weak in the midcardium. From P20 onward, as the fibers become oriented, CASK distributes throughout the myocardium and begins to become membranous at P20 to be fully localized to LM in a striated pattern at P60. Localization of N-cadherin to the intercalated disc (ID) is observed as early as P20, while Cx43 is still lateralized, as is ZO-1. At P60, Cx43 is restricted to the ID while ZO-1 retains a lateral localization but is also distributed to the ID. Like CASK, NaV1.5 is highly expressed in the epicardial and subepicardial layers at P0 and P5. From P20 onwards, both NaV1.5 and Kir2.1 adopt a cytosolic localization in the midcardium and concentrates at the ID, where CASK is excluded. This study shows that the expression level of polarity markers is inversely correlated with terminal cardiomyocyte maturation. In addition, the localization of CASK and NaV1.5 in the epicardial and subepicardial layers suggests that these proteins may have a role in early cardiac morphogenesis, before myocardial differentiation.