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Wnt/calcium signaling in cardiovascular development

Final Report Summary - WNT/CALCIUM IN HEART (Wnt/calcium signaling in cardiovascular development.)

Ca2+ homeostasis is central to signaling pathways that control cell fate, differentiation, and organogenesis. In cardiac biology Ca2+ ions play pivotal roles in multitude of cellular processes, ranging from excitation-contraction coupling to the regulation of hormone secretion and gene expression. Our ultimate goal is to understand the interplay between the morphogenetic pathways and physiologic cues, and the underlying molecular and cellular mechanisms that mediate the crosstalk between calcium signaling as well as other physiological stimuli, and major morphogenetic pathways such as Wnt signaling during cardiovascular development in zebrafish.
The main objective of the research project is aiming at understanding the role of Wnt/Ca2+ signaling in heart development as well as in cardiac function, and at elucidating the fundamental molecular mechanisms that lead to the attenuation of L-type Ca2+ channel (LTCC) by non-canonical Wnt signals. We have previously demonstrated that Wnt11 non-canonical signaling, a major developmental pathway regulating tissue morphogenesis and organ formation, patterns intercellular electrical coupling in the myocardial epithelium through effects on transmembrane Ca2+ conductance mediated via the LTCC. In order to define the molecular mechanisms, by which Wnt11 regulates LTCC at a subcellular resolution, we have moved from zebrafish embryonic hearts to cell-based assays. This allowed us to perform not only immunological experiments, but also more detailed biochemistry. We have used the immortalized rat cardiac myoblast H9c2 cell line, as it expresses the LTCC as well as the components of the Wnt11 signaling pathway. Our initial results suggested that Wnt11 does not alter the LTCC transcriptionally, as the loss of Wnt11 in H9c2 cells did not result in any significant changes in the LTCC mRNA levels. Furthermore, Wnt11 does not exert any effect on the localization or abundance of the main pore-forming subunit of the LTCC. Interestingly, we found that Wnt11 signaling regulates the generation of the C-terminal isoforms of the α1C channel subunit. Due to the fact that the distal C-terminal isoform is produced by β-adrenergic/PKA signaling pathway, we tested whether there is a crosstalk between Wnt11 and PKA signaling. We showed that blocking the PKA activity in the absence of Wnt11 prevented the formation of C-terminal isoforms, indicating that PKA signaling is required downstream of Wnt11. The spatial control of PKA activity is in part regulated by accurate localization of PKA to its targets through binding via A-kinase anchoring proteins (AKAPs). AKAPs play a key role in the regulation of the LTCC conductance, and in this respect the most studied AKAPs are AKAP-7 (AKAP-15/-18) and AKAP-5 (AKAP-79/-150). We tested whether Wnt11 signaling impinge upon the AKAP-PKA interaction, and observed that Wnt11 via its putative receptor Fzd7 affects the essential binding of PKA to LTCC via AKAP. Furthermore, we tested whether any of the two known AKAPs regulate LTCC downstream of Wnt11. Surprisingly, while loss of AKAP-5 alone or in combination with the loss of Wnt11 did not affect generation of the C-terminal isoforms, the absence of AKAP-7 alone or together with Wnt11 increased their formation, suggesting that AKAP-7 interaction with LTCC inhibits the C-terminal isoform generation. Our findings indicated that Wnt11 signaling prevents the LTCC phosphorylation by PKA mediated via another thus far uncharacterized AKAP. We have now identified the novel AKAP that binds the LTCC, and modulates its conductance in Wnt11-dependent manner. Markedly, we have discovered that intercellular electrical coupling gradients that are formed in the developing hearts depend on Wnt11 signaling preventing the binding of this novel AKAP to the LTCC. Thus, our data indicate that the Wnt11 signaling pathway may promote the binding of AKAP-7 to LTCC, and thus inhibit the AKAP-dependent formation of the C-terminal isoforms thereby regulating the LTCC conductance.
Next, we have studied the role of L-type Ca2+ channel and plasma membrane calcium fluxes in non-excitable cells. As a model we have chosen endothelial cell (EC) sprouting during angiogenesis. The presence of LTCC in EC is disputed as the depolarization of endothelial cell membrane abolishes the Ca2+ influx into the cell. First, we have established that the LTCC is indeed expressed in the EC. To determine whether the calcium fluxes through the LTCC play a role during angiogenic sprouting, we investigated the ISV outgrowth either in the absence of LTCC or upon its stimulation after treatment with the LTCC agonist Bay-K. The decrease of calcium fluxes through the LTCC significantly reduced the cell number of the ISVs. Conversely, the stimulation of calcium fluxes through the LTCC in the Bay-K treated embryos led to enhanced angiogenic behavior and overbranching of segmental artery sprouts. This phenotype was similar to the one observed in VEGF receptor mutant flt1, or in the loss of Notch signaling. To elucidate whether the increase in the EC number is due to the enhanced proliferation or migration, we performed time-lapse imaging of the growing ISVs, and quantified the number of proliferatory and migratory events. The stimulation of the LTCC conductance strongly increased the anterograde migration of the EC from the dorsal aorta. Markedly, in the absence of the LTCC there is the enhanced retrograde cell migration from the ISVs back to the dorsal aorta. In comparison, the cell proliferation is only slightly affected, as stalk but not tip cells proliferated less in the loss of LTCC. Thus, we found that Ca2+ fluxes through the LTCC are implicated in modulating the endothelial cell motility, and that the distinct inter and intracellular calcium concentrations are crucial for proper angiogenesis.
Last, we have investigated the mechanisms that govern the development of the chambered-heart from the linear heart tube. We first showed that epithelial remodeling drives the formation of the heart chambers through the cell neighbor exchange. We have demonstrated that Wnt11 non-canonical signaling branch: planar cell polarity (PCP) pathway regulates the cellular processes guiding the epithelial remodeling. We have shown that PCP affects actomyosin-based tension locally at the sites of the cell neighbor exchange, and on a tissue-scale where it planary polarizes actomyosin in the outflow tract. The generation of the tissue-scale tension is required for driving cardiac looping process and aids the expansion of the cardiac chambers.
All together, we have achieved most of the goals projected for the planned project with slight deviations. We have established that the same molecules that control the cellular events underlying the formation of the cardiac chambers, i.e. the signaling machinery of Wnt non-canonical pathway, also regulate the extent of intercellular electrical coupling, and thus actively contribute to the patterning of heart function. Our work expands the understanding of one of the central themes in biology - how function and form interact, by investigating not only the developmental processes that lead to the proper organ formation, but also their integrative physiology. Thus, our research contributes to our understanding of not only developmental processes required for organ form and function, but also pathophysiology of common disease states.