Characterizing the function of the novel Xin genes in the mouse heart

Previously, our lab identified the novel striated muscle-specific Xin gene and showed that abnormal cardiac morphogenesis results from cXin antisense oligonucleotide treatment. In mouse, two Xin genes, mXinalpha and mXinbeta, exist, which are expressed beginning at embryonic day 8.0 in the developing heart tube. mXinalpha co-localizes with adherens junction proteins N-cadherin and beta-catenin to the intercalated disc of adult hearts. My thesis focuses on elucidating the function of these mXin genes.; An mXinalpha-null mouse line was generated to characterize the function of mXinalpha. mXinalpha-null mouse hearts are hypertrophied and exhibit fibrosis, indicative of cardiomyopathy. A significant upregulation of mXinbeta likely provides partial compensation and accounts for the viability of the mXinalpha-null mice. Ultrastructural analysis of mXinalpha-null mouse hearts reveals intercalated disc disruption and myofilament disarray. The observed decreased expression of intercalated disc proteins could compromise the integrity of the intercalated discs and functionally weaken adhesion, leading to cardiac defects and conduction abnormalities. Thoracic aortic banding was used to investigate mXinalpha's role in the hypertrophy response. Accelerated hypertrophy and aberrant gene expression was observed in banded mXinalpha-null mice. Cloning and subsequent characterization of mXinbeta was also carried out to further understand the function of this novel gene family.; To understand the molecular mechanisms leading to such cardiac defects, I investigated the functional domains and interacting partners of mXinalpha. I have shown that mXinalpha directly interacts with beta-catenin, and the beta-catenin binding site on mXinalpha was mapped to amino acids #535-636, which overlaps with the actin-binding domain composed of Xin repeats. A stronger interaction was found between mXinalpha C-terminal deletion and actin, as compared to full-length mXinalpha and actin. Expression of GFP-mXinalpha C-terminal deletion in cells showed increased stress fiber localization compared to GFP-mXinalpha, suggesting a novel regulatory mechanism for mXinalpha function.; In summary, this thesis demonstrates that mXinalpha functions in maintaining intercalated disc structural integrity in normal mice, likely through its association with adherens junction components and the actin cytoskeleton. The mXinalpha knockout mouse line provides a novel model of cardiac hypertrophy and cardiomyopathy with conduction defects.