Normal cardiomyocyte homeostasis and the increase in cardiac mass associated with cardiac hypertrophy require regulated synthesis, transport, and subcellular compartmentalization of proteins. Whereas increased protein synthesis is well documented in myocardial hypertrophy, the mechanisms regulating myocardial protein trafficking have not been examined and possible pathological consequences of aberrant cardiomyocyte protein transport are unknown. Our laboratory developed an interest in this question when a general survey of small G-protein expression in transgenic mouse models of cardiac hypertrophy/failure revealed increased expression of several Rab family members. Rab proteins are Ras-like G-proteins whose sole function is to regulate protein trafficking between intracellular organelles. To determine if increased Rab expression and altered cardiomyocyte vesicular transport could contribute to cardiac pathology, Rab1a was transgenically expressed in mouse hearts. Rab1a over-expressors exhibited ultrastructural vesicular abnormalities and developed myocardial hypertrophy that progressed to dilated cardiomyopathy. Having thus demonstrated that modified Rab function and resulting alterations in protein trafficking are sufficient to cause cardiac pathology, we propose to determine the effects of the four major myocardial Rab family members through in vivo and in vitro gain and loss of function studies.
In Specific Aim 1, TG mice expressing constitutively active (GTP-bound form) or dominant inhibitory (GDP-bound form) mutants of Rabs1, 4, 5, and 6 will be created and the effects of regulated in vivo cardiac Rab activity measured on physiologic and pathologic cardiac hypertrophy.
In Specific Aim 2, the effect of altered Rab activity on cardiac function will be assessed with an emphasis on AR internalization and recycling, which will be monitored in dual TG mice expressing mutant Rab proteins and epitope-tagged 2ARs. Parallel experiments in Specific Aim 3 will utilize adenoviral vectors to express the same mutant Rabs in cultured neonatal rat cardiomyocytes for studies of cellular hypertrophy, protein transport, and cellular ultrastructure that are not possible with the in vivo models. Together, these studies will comprise a comprehensive evaluation of Rab function in cardiac tissue, the first such studies ever performed, and will establish roles for these regulated small GTPases in normal and pathological cardiac states.
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