Myong-Ho Jeong^a,b,1, Hyun-Ji Kim^b,c,1, Jung-Hoon Pyun^a,b, Kyu-Sil Choi^b, Dong I. Lee^d, Soroosh Solhjoo^d, Brian O'Rourke^d, Gordon F. Tomaselli^d, Dong Seop Jeong^e, Hana Cho^b,c,2, and Jong-Sun Kang<sup>a,b,2

a</sup>Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Korea 16419;
^bSamsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 06351;
^cDepartment of Physiology, Sungkyunkwan University School of Medicine, Suwon, Korea 16419;
^dDivision of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
^eDepartment of Thoracic and Cardiovascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 06351

Abstract
On pathological stress, Wnt signaling is reactivated and induces genes associated with cardiac remodeling and fibrosis. We have previously shown that a cell surface receptor Cdon (cell-adhesion associated, oncogene regulated) suppresses Wnt signaling to promote neuronal differentiation however its role in heart is unknown. Here, we demonstrate a critical role of Cdon in cardiac function and remodeling. Cdon is expressed and predominantly localized at intercalated disk in both mouse and human hearts. Cdon-deficient mice develop cardiac dysfunction including reduced ejection fraction and ECG abnormalities. Cdon^-/- hearts exhibit increased fibrosis and up-regulation of genes associated with cardiac remodeling and fibrosis. Electrical remodeling was demonstrated by up-regulation and mislocalization of the gap junction protein, Connexin 43 (Cx43) in Cdon^-/- hearts. In agreement with altered Cx43 expression, functional analysis both using Cdon^-/- cardiomyocytes and shRNA-mediated knockdown in rat cardiomyocytes shows aberrant gap junction activities. Analysis of the underlying mechanism reveals that Cdon^-/- hearts exhibit hyperactive Wnt signaling as evident by β-catenin accumulation and Axin2 up-regulation. On the other hand, the treatment of rat cardiomyocytes with a Wnt activator TWS119 reduces Cdon levels and aberrant Cx43 activities, similarly to Cdon-deficient cardiomyocytes, suggesting a negative feedback between Cdon and Wnt signaling. Finally, inhibition of Wnt/β-catenin signaling by XAV939, IWP2 or dickkopf (DKK)1 prevented Cdon depletion-induced up-regulation of collagen 1a and Cx43. Taken together, these results demonstrate that Cdon deficiency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac fibrosis. Cdon exhibits great potential as a target for the treatment of cardiac fibrosis and cardiomyopathy.

Cdon, gap junction, connexin 43, cardiac fibrosis, Wnt

¹M.-H.J. and H.-J.K. contributed equally to this work.
²To whom correspondence may be addressed.

Author contributions: M.-H.J., H.-J.K., J.-H.P., K.-S.C., D.I.L., S.S., B.O., G.F.T., D.S.J., H.C., and J.-S.K. designed research; M.-H.J., H.-J.K., J.-H.P., K.-S.C., D.I.L., S.S., and D.S.J. performed research; M.-H.J., H.-J.K., J.-H.P., K.-S.C., D.I.L.., S.S., B.O., G.F.T., D.S.J., H.C., and J.-S.K. analyzed data; and M.-H.J., H.-J.K., H.C., and J.-S.K. wrote the paper.