The cardiac intercalated disk is a complex structure found at the longitudinal ends of cardiomyocytes [1
]. The adherens junction region of the intercalated disks is where myofibrils terminate and link to complexes of membrane-associated proteins inside the cardiomyocyte, and where cardiomyocytes are mechanically coupled to each other through homophilic interaction of N-cadherin molecules embedded in the membranes of adjacent cells. These connections allow mechanical force generated by the myofibrils to be transmitted to the membrane and neighboring cells. Intercalated disks are also responsible for electrical coupling between neighboring cardiomyocytes via gap junctions formed by connexin-43.
NRAP is a striated muscle-specific protein that is concentrated at intercalated disks in cardiac muscle and at myotendinous junctions in skeletal muscle [2
]. Ultrastructural studies showed that NRAP is located in the terminal actin bundles that link the ends of the myofibrils to the membranes [3
]. NRAP is a multidomain scaffolding protein with many potential binding partners [5
]. Its N-terminal LIM domain can bind talin and α-actinin; its single nebulin repeats can bind α-actinin, actin, muscle LIM protein (MLP) and Krp1; and its nebulin-related super repeats can bind actin, vinculin, filamin and Krp1. On the basis of its domain organization, subcellular localization, and multiple binding partners, we hypothesized that NRAP may serve a mechanical role, linking the terminal actin filaments of myofibrils to the specific proteins concentrated in these junctional regions [7
]. This model was further supported by biochemical copurification of NRAP with cardiac intercalated disks, as well as with isolated, detergent washed myofibrils, demonstrating NRAP's tight association with both myofibril ends and the membrane-associated structures to which they attach [4
]. Subsequent experiments have implicated NRAP as an obligatory molecular scaffold in the first steps of myofibril assembly [8
Changes in the expression of NRAP and other components of cardiac intercalated disks have been observed in dilated cardiomyopathy (DCM) [6
]. DCM is a prevalent disease of the heart characterized by dilated and poorly functioning ventricles [13
]. Greater than 20% of cases appear to have a genetic basis, and a diverse group of genes have been identified as dominant loci for the disease [14
]. Several of these genes encode cytoskeletal proteins, leading to the suggestion that compromised force transmission is one primary cause of DCM [14
]. The DCM-causing genes at many other loci remain unidentified [14
Previous studies showed that NRAP protein levels are increased in two genetic mouse models of DCM [6
]. One of these is the muscle LIM protein (MLP) knockout mouse [16
], while the second is a tropomodulin-overexpressing transgenic (TOT) mouse [17
]. MLP protein is also decreased in the TOT mouse [6
]. In both mouse models, NRAP upregulation occurs soon after birth, preceding other molecular and morphologic changes associated with the cardiomyopathy. MLP directly binds NRAP in vitro [6
], and MLP mutations associated with human cardiomyopathy have been shown to lead to decreased MLP binding to NRAP and α-actinin [18
]. These findings implicate increased NRAP expression in the pathogenesis of cardiomyopathy.
Here we use the cardiac-specific tet-off transgenic mouse system [19
] to directly test the effects of NRAP overexpression on cardiac structure and function. We found that the NRAP-overexpressing transgenic animals exhibited right ventricular dilation and dysfunction, with little effect on the left ventricle. The results demonstrate that the excess NRAP expression observed in the MLP knockout mouse and the TOT mouse is unlikely to account for the development of left ventricular DCM in those systems.