The intercalated disk protein Xin was originally discovered in chicken striated muscle and implicated in cardiac morphogenesis. In the mouse, there are two homologous genes, mXinα and mXinβ. The human homolog of mXinα, Cmya1, maps to chromosomal region 3p21.2-21.3, near a dilated cardiomyopathy with conduction defect-2 locus. Here we report that mXinα-null mouse hearts are hypertrophied and exhibit fibrosis, indicative of cardiomyopathy. A significant upregulation of mXinβ likely provides partial compensation and accounts for the viability of the mXinα-null mice. Ultrastructural studies of mXinα-null mouse hearts reveal intercalated disk disruption and myofilament disarray. In mXinα-null mice, there is a significant decrease in the expression level of p120-catenin, β-catenin, N-cadherin, and desmoplakin, which could compromise the integrity of the intercalated disks and functionally weaken adhesion, leading to cardiac defects. Additionally, altered localization and decreased expression of connexin 43 are observed in the mXinα-null mouse heart, which, together with previously observed abnormal electrophysiological properties of mXinα-deficient mouse ventricular myocytes, could potentially lead to conduction defects. Indeed, ECG recordings on isolated, perfused hearts (Langendorff preparations) show a significantly prolonged QT interval in mXinα-deficient hearts. Thus mXinα functions in regulating the hypertrophic response and maintaining the structural integrity of the intercalated disk in normal mice, likely through its association with adherens junctional components and actin cytoskeleton. The mXinα-knockout mouse line provides a novel model of cardiac hypertrophy and cardiomyopathy with conduction defects. KeywordsXin repeat proteins; N-cadherin; β-catenin; p120-catenin; connexin 43The intercalated disk contains adherens junctions, desmosomes, and gap junctions that maintain the integrity of the association between cardiomyocytes and enable the myocardium Address for reprint requests and other correspondence: J. J.-C. Lin, Dept. of Biological Sciences, Univ. of Iowa, 340 Biology Bldg. East, 210 E. Iowa Ave., Iowa City, IA 52242 (e-mail: jim-lin@uiowa.edu).. NIH Public Access Author ManuscriptAm J Physiol Heart Circ Physiol. Author manuscript; available in PMC 2008 November 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript to function in synchrony. The expression and distribution of many of these junctional components are often altered in many types of heart disease (5,8,13,35). However, direct evidence to support a role for these proteins in contributing to cardiomyopathies remains incomplete. The best-characterized example involves the effects of deletion of a key adherens junction component, N-cadherin, on the intercalated disk. N-cadherin functions to mediate Ca 2+ -dependent homophilic cell-cell adhesion. Conditional deletion of N-cadherin in the adult mouse heart leads to a complete dissolution of the intercalated disk structure and a significant decrease in the express...

Rationale: The Xin repeat-containing proteins mXin␣ and mXin␤ localize to the intercalated disc of mouse heart and are implicated in cardiac development and function. The mXin␣ directly interacts with ␤-catenin, p120-catenin, and actin filaments. Ablation of mXin␣ results in adult late-onset cardiomyopathy with conduction defects. An upregulation of the mXin␤ in mXin␣-deficient hearts suggests a partial compensation. Objective: The essential roles of mXin␤ in cardiac development and intercalated disc maturation were investigated. Methods and Results: Ablation of mXin␤ led to abnormal heart shape, ventricular septal defects, severe growth retardation, and postnatal lethality with no upregulation of the mXin␣. Postnatal upregulation of mXin␤ in wild-type hearts, as well as altered apoptosis and proliferation in mXin␤-null hearts, suggests that mXin␤ is required for postnatal heart remodeling. The mXin␤-null hearts exhibited a misorganized myocardium as detected by histological and electron microscopic studies and an impaired diastolic function, as suggested by echocardiography and a delay in switching off the slow skeletal troponin I. Loss of mXin␤ resulted in the failure of forming mature intercalated discs and the mislocalization of mXin␣ and N-cadherin. The mXin␤-null hearts showed upregulation of active Stat3 (signal transducer and activator of transcription 3) and downregulation of the activities of Rac1, insulin-like growth factor 1 receptor, protein kinase B, and extracellular signal-regulated kinases 1 and 2. Conclusions: These findings identify not only an essential role of mXin␤ in the intercalated disc maturation but also mechanisms of mXin␤ modulating N-cadherin-mediated adhesion signaling and its crosstalk signaling for postnatal heart growth and animal survival. (Circ Res. 2010;106:1468-1478.)Key Words: N-cadherin-mediated adhesion signaling Ⅲ Xin repeat-containing protein Ⅲ intercalated disc maturation Ⅲ diastolic dysfunction Ⅲ postnatal heart growth A regulatory network of transcription factors is known to control cardiac morphogenesis. Although the core players in this network are highly conserved, from organisms with simple heart-like cells to those with complex four-chambered hearts, it has been theorized and proven that expansion of this regulatory network by adding new transcription factors is a major force for the heart to evolve new structures. 1,2 However, the addition of new transcription factors can only be a part of the mechanism underlying the formation of complex hearts. The transcription factors must act through their downstream targets, which are directly involved in cardiac morphogenesis, growth and function. However, our inventory of such downstream targets remains incomplete.The Xin repeat-containing proteins from chicken and mouse hearts (cXin and mXin␣, respectively) were first identified as a target of the Nkx2.5-Mef2C pathway. 3,4 Another mouse Xin protein, mXin␤ (or myomaxin), has been subsequently identified as a Mef2A downstream target. 5 Evolutionary studies suggest t...

. (2007) Am. J. Physiol. 293, H2680 -H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXin␣ and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXin␣ directly interacts with ␤-catenin. The ␤-catenin-binding site on mXin␣ was mapped to amino acids 535-636, which overlaps with the known actin-binding domains composed of the Xin repeats. The overlapping nature of these domains provides insight into the molecular mechanism for mXin␣ localization and function. Purified recombinant glutathione S-transferase-or His-tagged mXin␣ proteins are capable of binding and bundling actin filaments, as determined by co-sedimentation and electron microscopic studies. The binding to actin was saturated at an approximate stoichiometry of nine actin monomers to one mXin␣. A stronger interaction was observed between mXin␣ C-terminal deletion and actin as compared with the interaction between full-length mXin␣ and actin. Furthermore, force expression of green fluorescent protein fused to an mXin␣ C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXin␣. These results suggest a model whereby the C terminus of mXin␣ may prevent the fulllength molecule from binding to actin, until the ␤-catenin-binding domain is occupied by ␤-catenin. The binding of mXin␣ to ␤-catenin at the adherens junction would then facilitate actin binding. In support of this model, we found that the actin binding and bundling activity of mXin␣ was enhanced in the presence of ␤-catenin.The striated muscle-specific Xin genes encode proteins containing several proline-rich regions, a highly conserved sequence homologous to the Myb-A and Myb-B DNA-binding domain, and a region with 15-28 16-amino acid (aa) 4 repeating units (called the Xin repeats) (1-3). In the mouse, two Xin genes, mXin␣ and mXin␤, exist, whereas only one cXin gene is found in the chick. The expression of both cXin and mXin␣ is regulated by the muscle transcription factor, MEF2C, and the homeodomain transcription factor, Nkx2.5 (1, 2). Similarly, the expression of mXin␤ (also termed myomaxin) is also under the control of MEF2A (4). Treatment of chick embryos with cXin antisense oligonucleotides results in abnormal cardiac morphogenesis and a disruption in cardiac looping, suggesting that Xin plays an essential role in cardiac development (2). Embryonic lethality was expected based on this antisense oligonucleotide experiment in chicks; however, viable and fertile mXin␣ knock-out mice were observed. This viability probably results from functional compensation through the up-regulation of mXin␤ at both message and protein levels (5). Consistent with the compensatory role of mXin␤, we have previously shown that mXin␤, like mXin␣ (2, 6), localizes to the intercalated disc of the adult heart (5). Despite this compensation, the adult mXin␣-deficient mouse hearts are hypertrophied and exhibit c...