Duncan John C, Fletcher William H
Department of Pathology and Human Anatomy, Division of Anatomy, Loma Linda University School of Medicine, Loma Linda, California, USA.
Dev Dyn. 2002 Jan;223(1):96-107. doi: 10.1002/dvdy.1232.
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.
α1连接蛋白（连接蛋白43）是发育中和成熟心脏中主要的间隙连接蛋白，它形成通道，介导对心脏功能至关重要的细胞间电和代谢偶联事件。α1连接蛋白通道可被环磷酸腺苷依赖性蛋白激酶（PKA）和蛋白激酶C（PKC）的作用快速且可逆地门控，这些磷酸化共有位点的破坏与严重的心脏畸形有关。然而，关于PKA或PKC在心脏早期形成中的相对活性尚无报道。在这些相同的发育阶段，α1连接蛋白的存在和磷酸化状态也未被记录。为了开展这些研究，我们使用了妊娠8.5 - 18.5天（受孕后天数）的小鼠胚胎、产后幼崽和成年小鼠的心脏。膜或上清液部分用于免疫印迹，以评估α1连接蛋白和每种蛋白激酶的含量及分布。进行磷酸转移酶测定以记录PKA和PKC的内源性活性。在妊娠8.5天和9.5天的胚胎以及成年心脏中，44 kDa、46 kDa和49 kDa的三种α1连接蛋白条带明显可见，但在妊娠10.5天的胚胎或直至18.5天的胚胎中，49 kDa条带并非始终存在，尽管在成年心脏中其条带较明显。PKA的量在妊娠8.5天的心脏中最少，但此后上升，在妊娠10.5天时达到最大值，并在整个发育过程中保持稳定。该酶的催化活性在妊娠8.5天的心脏中最小，此后上升，在发育至妊娠10.5天时达到最大值。PKCδ主要局限于膜部分，而PKCε有与上清液和膜相关的形式。两种酶同工型在整个发育过程中均表现出较大波动。在妊娠8.5天和9.5天的心脏中，PKC催化活性最大，但到妊娠10.5天时，活性急剧下降，此后保持在低水平。结果表明，α1连接蛋白在发育的心脏管阶段（妊娠8.5天）即已存在，并提供证据表明该蛋白形成的通道受PKA和PKC动态调节，尤其是在妊娠8.5天和9.5天的胚胎心脏中，这通常是心脏形成和左右模式化的关键时期。
