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Effect of a dominant negative ras on myocardial hypertrophy by using adenoviral-mediated gene transfer
J B Pracyk1, D D Hegland, K Tanaka
1Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md., USA.
Background:
The small guanosine triphosphate-binding protein ras regulates a signal transduction cascade linking cell surface receptors to mitogen-activated protein kinase (MAPK). Because the molecular signaling mechanisms underlying cardiac hypertrophy remain unclear, the current study examined the regulatory role of ras in both the biochemical and morphologic aspects of hypertrophy.
Methods:
Adenoviral-mediated gene transfer was used to express a dominant negative mutant of ras (rasN17) at high efficiency in primary neonatal ventricular myocytes. Beta-galactosidase staining and Western blot analysis confirmed successful transfection and expression of the rasN17 gene product. MAPK activity was measured by an in vitro kinase assay resulting in radioactive phosphorus labeled product. Morphologic hypertrophy was assessed by fluorescein-conjugated phalloidin.
Results:
Compared with uninfected or control adenoviral-infected cells, myocytes infected with rasN17 demonstrated attenuated basal MAPK activity. In contrast, rasN17 expression did not affect endothelin 1-induced MAPK activation. Morphologic studies showed that although rasN17 produced a phenotypic difference in the basal state, the ability of cardiac myocytes to morphologically respond to endothelin 1 stimulation, as manifested by sarcomeric reorganization, remained unaltered by the expression of the rasN17 gene product.
Conclusions:
Endothelin 1-stimulated MAPK activation and endothelin 1-induced morphologic hypertrophy are ras-independent processes.
Insights
Ras protein does not regulate cardiac hypertrophy. Endothelin 1-stimulated MAPK activation and hypertrophy are independent of ras signaling pathways in cardiac myocytes.
Area of Science:
- Cardiology
- Molecular Biology
- Cell Signaling
Background:
- Ras, a small GTP-binding protein, controls signal transduction from cell surface receptors to mitogen-activated protein kinase (MAPK).
- Molecular mechanisms of cardiac hypertrophy are not fully understood.
- This study investigates the role of ras in cardiac hypertrophy's biochemical and morphological changes.
Purpose of the Study:
- To determine the regulatory role of ras in cardiac hypertrophy.
- To investigate ras-independent pathways in cardiac myocyte signaling.
Main Methods:
- Adenoviral gene transfer used to express dominant-negative ras (rasN17) in neonatal ventricular myocytes.
- MAPK activity measured via in vitro kinase assay.
- Morphological hypertrophy assessed using phalloidin staining.
Main Results:
- RasN17 expression reduced basal MAPK activity but did not affect endothelin 1-induced MAPK activation.
- RasN17 altered basal cell morphology but did not impede endothelin 1-induced hypertrophic response.
- Cardiac myocyte hypertrophy signaling is not dependent on ras.
Conclusions:
- Endothelin 1-induced MAPK activation is ras-independent.
- Endothelin 1-induced cardiac myocyte hypertrophy is a ras-independent process.