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CIP4 is required for the hypertrophic growth of neonatal cardiac myocytes.

Rusconi F, Thakur H, Li J, Kapiloff MS - J. Biomed. Sci. (2013)

Bottom Line: CIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking.Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.These results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Stem Cell Institute, Department of Pediatrics, Leonard M. Miller School of Medicine, University of Miami, Miami, FL 33101, USA.

ABSTRACT

Background: CIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking. Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.

Results: We now show using RNA interference that CIP4 expression in neonatal rat ventricular myocytes is required for the induction of non-mitotic, hypertrophic growth by the α-adrenergic agonist phenylephrine, the IL-6 cytokine leukemia inhibitor factor, and fetal bovine serum, as assayed using morphometry, immunocytochemistry for the hypertrophic marker atrial natriuretic factor and [3H]leucine incorporation for de novo protein synthesis. This requirement was consistent with the induction of CIP4 expression by hypertrophic stimulation. The inhibition of myocyte hypertrophy by CIP4 small interfering oligonucleotides (siRNA) was rescued by expression of a recombinant CIP4 protein, but not by a mutant lacking the N-terminal FCH domain responsible for CIP4 intracellular localization.

Conclusions: These results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.

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Related in: MedlinePlus

CIP4 localization in myocytes. Myocytes expressing myc-CIP4 WT were stained with myc antibodies (green) and α-actinin (red) antibodies and Hoechst nuclear stain (blue). Bar = 20 μm. Panel A shows the green channel alone. n > 3. Panel B is a composite image of myc and actinin antibody staining and the nuclear stain.
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Figure 2: CIP4 localization in myocytes. Myocytes expressing myc-CIP4 WT were stained with myc antibodies (green) and α-actinin (red) antibodies and Hoechst nuclear stain (blue). Bar = 20 μm. Panel A shows the green channel alone. n > 3. Panel B is a composite image of myc and actinin antibody staining and the nuclear stain.

Mentions: As found for adult heart tissue [4], there were two CIP4 bands detectable by western blot of neonatal rat ventricular myocyte extracts (Figure 1B). These bands represent CIP4h and CIP4a, that are identical except for a 56 amino acid residue insertion C-terminal to the F-Bar domain present in CIP4h due to alternative mRNA splicing (Figure 1A) [12]. The expression of both isoforms was consistently induced 2–3 fold by culture of the myocytes in the presence of hypertrophic agonists (Figure 1B), including by the α-adrenergic agonist phenylephrine (PE), the IL-6 cytokine leukemia inhibitory factor (LIF), and serum (FBS). We attempted to determine the localization of CIP4 in the cardiac myocyte by immunocytochemistry. However, the available CIP4 antibodies did not afford a significant signal for endogenous CIP4 protein in the myocytes, regardless of the culture condition. We were able to express myc-tagged CIP4h protein in the myocytes by adenoviral infection (Figure 2). myc-CIP4 was expressed throughout the cytosol in a reticular/punctate pattern that did not coincide with Z-line staining by an α-actinin antibody. The staining pattern was reminiscent of the CIP4 distribution in other cell types in which CIP4 is associated with microtubules, endosomes, membrane tubules and other membrane structures [8,13,14].


CIP4 is required for the hypertrophic growth of neonatal cardiac myocytes.

Rusconi F, Thakur H, Li J, Kapiloff MS - J. Biomed. Sci. (2013)

CIP4 localization in myocytes. Myocytes expressing myc-CIP4 WT were stained with myc antibodies (green) and α-actinin (red) antibodies and Hoechst nuclear stain (blue). Bar = 20 μm. Panel A shows the green channel alone. n > 3. Panel B is a composite image of myc and actinin antibody staining and the nuclear stain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750294&req=5

Figure 2: CIP4 localization in myocytes. Myocytes expressing myc-CIP4 WT were stained with myc antibodies (green) and α-actinin (red) antibodies and Hoechst nuclear stain (blue). Bar = 20 μm. Panel A shows the green channel alone. n > 3. Panel B is a composite image of myc and actinin antibody staining and the nuclear stain.
Mentions: As found for adult heart tissue [4], there were two CIP4 bands detectable by western blot of neonatal rat ventricular myocyte extracts (Figure 1B). These bands represent CIP4h and CIP4a, that are identical except for a 56 amino acid residue insertion C-terminal to the F-Bar domain present in CIP4h due to alternative mRNA splicing (Figure 1A) [12]. The expression of both isoforms was consistently induced 2–3 fold by culture of the myocytes in the presence of hypertrophic agonists (Figure 1B), including by the α-adrenergic agonist phenylephrine (PE), the IL-6 cytokine leukemia inhibitory factor (LIF), and serum (FBS). We attempted to determine the localization of CIP4 in the cardiac myocyte by immunocytochemistry. However, the available CIP4 antibodies did not afford a significant signal for endogenous CIP4 protein in the myocytes, regardless of the culture condition. We were able to express myc-tagged CIP4h protein in the myocytes by adenoviral infection (Figure 2). myc-CIP4 was expressed throughout the cytosol in a reticular/punctate pattern that did not coincide with Z-line staining by an α-actinin antibody. The staining pattern was reminiscent of the CIP4 distribution in other cell types in which CIP4 is associated with microtubules, endosomes, membrane tubules and other membrane structures [8,13,14].

Bottom Line: CIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking.Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.These results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Stem Cell Institute, Department of Pediatrics, Leonard M. Miller School of Medicine, University of Miami, Miami, FL 33101, USA.

ABSTRACT

Background: CIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking. Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.

Results: We now show using RNA interference that CIP4 expression in neonatal rat ventricular myocytes is required for the induction of non-mitotic, hypertrophic growth by the α-adrenergic agonist phenylephrine, the IL-6 cytokine leukemia inhibitor factor, and fetal bovine serum, as assayed using morphometry, immunocytochemistry for the hypertrophic marker atrial natriuretic factor and [3H]leucine incorporation for de novo protein synthesis. This requirement was consistent with the induction of CIP4 expression by hypertrophic stimulation. The inhibition of myocyte hypertrophy by CIP4 small interfering oligonucleotides (siRNA) was rescued by expression of a recombinant CIP4 protein, but not by a mutant lacking the N-terminal FCH domain responsible for CIP4 intracellular localization.

Conclusions: These results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.

Show MeSH
Related in: MedlinePlus