Adaptation is of utmost importance in overloaded or failing hearts because the total myofibrillar mass in the heart determines cardiac strength. My long-term goal is to understand how striated muscle adapts its molecular composition in response to changes in its physiological demands. To dissect mechanisms between stimulation and hypertrophy, I examine early changes occurring in the first hour. My hypotheses proposes that early growth is governed by two processes; phosphorylation of a major transcription factor and control of cytoplasmic mRNA. The study requires rapid pharmacological interventions to the beta-adrenergic pathway. Therefore, I use a primary cell culture from neonatal rat heart. This diverse, mixed cell population is best studied by cell biological techniques at the single cell level. I use immunochemistry to monitor activation by phosphorylation of the transcription factor, cAMP regulatory element binding protein (CREB). I use in situ hybridization to examine changes in subcellular message distribution.
Specific aim 1 characterizes the pathways of CREB phosphorylation, determines an effective paradigm of PKA activation for cell hypertrophy, and establishes conditions that increase alpha-myosin protein deposition in myofibrils.
Specific aim 2 quantifies the outward migration of mRNA from the nucleus and distinguishes whether the mRNA distribution pattern is due to diffusion, transport, or translational trapping.
Specific aim 3 tests if the untranslated region, 3'UTR, is required for transport by microtubules or control of translation. I use transfection with a chimeric construct of beta-galactosidase coding region and alpha-myosin 3'UTR. Study of cytoplasmic mRNA has not been sufficiently explored as a rapid regulatory event in cardiac hypertrophy.
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