Abstract 922: Mutations in Nebulette Have Different Effects on Protein Function

Nebulette, a 109kDa Z-disk protein, expressed with the nascent myofibrils during myofibril-logenesis, is believed that it helps align thin filaments in the sarcomere and determines the Z-band width in cardiomyocytes. We have previously identified K60N, Q128R, G202R, and A592E variants in nebulette in patients with dilated cardiomyopathy (DCM). The purpose of this study was to characterize the role of these variants in the development of DCM. We created transgenic mice with cardiac-restricted over-expression of human wild-type or mutant nebulette. Embryos were collected at E7.5, 9.5, and 12.5 stages, while adult mice were examined at 4 and 6 months of age. Cardiac function was measured by echocardiographic and MRI analyses and the hearts were assessed by histopathologic, immunohistochemical, western blot and co-immunoprecipitation analyses. Expression of G202R and A592E nebulette resulted in morphological, histological and ultrastuctural characteristics of DCM by 6 months of age. Echocardiographic and MRI analyses of these mice revealed impaired left ventricular contractility with ventricular dilation. The cytoarchitecture of cardiomyocytes was altered in length and width, with significant disruption and altered expression of α-actinin 2, F-actin, troponin T and myopalladin noted in the hearts of both mutant Tg mice lines compared with NTg and WT Tg. In contrast, expression of the N-terminal mutants, K60N and Q128R, appears to result in lethal cardiac structural defects in the embryonic hearts. In conclusion, these data suggest that variants found in nebulette are disease-causing mutations for DCM and have different effects on protein function during myofibrillogenesis and in mature cardiomyocytes. Thus, it appears that integrity of the N-terminal domain of nebulette, which binds F-actin, is crucial during cardiac development, while mutations in other domains may be crucial for protein function in the mature cardiomyocyte leading to impaired structural integrity and disorganization of the Z-disk, resulting in sarcomeric disruption and cardiac dysfunction.