alpha 1 Connexin (connexin43) gap junctions and activities of cAMP-dependent protein kinase and protein kinase C in developing mouse heart

Abstract

alpha 1 Connexin (connexin43) is the dominant gap junction protein of the developing and mature heart where it forms channels that mediate intercellular electrical and metabolic coupling events that are critical for heart function. alpha1 connexin channels are rapidly and reversibly gated by actions of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), and disruption of consensus sites for these phosphorylations are associated with severe heart malformations. However, there have been no reports on the relative activities of PKA or PKC in early heart formation. Nor has the presence and phosphorylation state of alpha1 connexin been documented in these same developmental stages. To begin these studies, we used hearts from 8.5-18.5 dpc (days postcoitus) mouse embryos, postpartum pups, and adults. Membrane or supernatant fractions were used for immunoblots to assess the amounts and distribution of alpha1 connexin protein and each protein kinase. Phosphotransferase assays were done to document the endogenous activities of PKA and PKC. Three species of alpha1 connexin at 44, 46, and 49 kDa were evident in 8.5- and 9.5-dpc embryos and adult hearts, but the 49-kDa band was not consistently found in 10.5 dpc or embryos through 18.5 dpc, although it was robust in adult heart. The amount of PKA was minimal in 8.5-dpc hearts but rose thereafter and was maximal by 10.5 dpc and remained stable throughout development. Catalytic activity of this enzyme was minimal in 8.5-dpc hearts then rose thereafter and was maximal by 10.5 dpc of development. PKC delta was confined mainly to membrane fractions, whereas PKC epsilon had supernatant- and membrane-associated forms. Both enzyme isoforms showed large fluctuations throughout development. In 8.5- and 9.5-dpc hearts, PKC catalytic activity was maximal but, by 10.5 dpc, activity dramatically declined and remained low thereafter. The results demonstrate that alpha1 connexin is present at the heart tube stage (8.5 dpc) of development onward and provide evidence suggesting that channels formed by this protein are dynamically regulated by PKA and PKC, especially in 8.5- and 9.5-day embryonic hearts, which are crucial times for heart formation and left/right patterning in general.