Mechanisms of signaling interaction between endocardium and myocardium

Communication between the myocardium and endocardium, separated by a layer of cardiac jelly, is essential for heart morphogenesis, especially trabecular formation. Trabeculae are sheet-like structures that increase surface area when the coronary system is not yet established. Lack of trabeculation causes embryonic demise, and

excess trabeculation causes left ventricular noncompaction cardiomyopathy (LVNC). The mortality of patients with LVNC ranges from 5% to 47%. Despite its clinical importance, the mechanisms of trabeculation are not fully understood, and how the communication between cardiomyocytes (CMs) in the myocardium and endocardial

cells (ECs) in the endocardium is achieved and regulates trabeculation is not fully known. Some ligands and receptors engaged in myocardium-endocardium communication are localized to membranes of CMs and ECs, yet how ligand/receptor-mediated interactions occur across the cardiac jelly has yet to be deciphered. Unlike direct cell-cell interactions, distant cell-cell communication can be achieved via several

mechanisms, including diffusible factors such as morphogens and recently discovered novel microstructures such as tunneling nanotubes (TNTs) in cultured mammalian cells or cytonemes in flies. Whether a microstructure that regulates signaling interaction among distant cells in vivo in mammals was not reported, and whether a

similar structure regulates the signaling interaction between the CMs and ECs during cardiovascular morphogenesis is unknown. Via genetic labeling, electron microscopy (EM), and cryogenic-EM (Cryo-EM), our preliminary data show a nanotube-like microstructure, which is named signaling bridges (SBs), extend from CMs

across cardiac jelly to reach ECs temporally; SB is sufficient to activate Notch signaling; disruption of SB by deleting Cdc42 or by chemicals hinders Notch activation and alters CM cellular behaviors, resulting in trabeculation defects. These novel preliminary data establish the essential roles of SBs in signal transduction

and trabeculation during cardiac morphogenesis; however, details regarding SB structure, function, and regulatory mechanisms impacting heart morphogenesis remain unknown. We hypothesize that SBs are required for the signaling interaction between the CMs and ECs to control cellular behaviors during trabeculation (Fig. 1).

Three aims are proposed: we will apply various electron microscopy to determine the ultrastructure and function of SBs in Aim I; interrogate how SBs transduce signaling between ECs and CMs in Aim II; elucidate how SB mediated interaction regulate trabecular morphogenesis in Aim III. Completing the proposed studies will determine how signal interaction between the CMs and ECs is achieved

and how SBs regulate cellular behavior and trabecular formation. The discovery of SBs and their functions in vivo opens new avenues for understanding intercellular interaction during cardiac morphogenesis. Successful completion of this study will expand our understanding of the etiology of trabeculation defects and, ultimately,

the etiology of LVNC, thereby providing a base for developing new therapeutic strategies for mitigating LVNC.