Limits...
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.

Show MeSH

Related in: MedlinePlus

CIP4 is important for neonatal rat ventricular myocyte hypertrophy. A. Myocytes were transfected with control or CIP4 siRNA oligonucleotides and cultured ± 10% fetal bovine serum (FBS), 10 μM PE, or 1000 U/mL LIF. After treatment for 2 days, the myocytes were stained for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. B. Cross-section area of myocytes in A. C. Fraction of myocytes expressing ANF in A. n = 5 for B and C. ANOVA (two-factor with replication): p-value (CIP4 siRNA vs. control siRNA) = 6 × 10-5(B) and = 0.046 (C); p-value (between culture conditions) < 10-7 for both B and C. Post-hoc testing: *p-values vs. control siRNA-transfected myocytes treated with the same agonist; †p-values vs. no drug control. D. [3H]leucine incorporation. n = 2. *p-value vs. PE-treated, control siRNA-transfected myocytes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750294&req=5

Figure 3: CIP4 is important for neonatal rat ventricular myocyte hypertrophy. A. Myocytes were transfected with control or CIP4 siRNA oligonucleotides and cultured ± 10% fetal bovine serum (FBS), 10 μM PE, or 1000 U/mL LIF. After treatment for 2 days, the myocytes were stained for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. B. Cross-section area of myocytes in A. C. Fraction of myocytes expressing ANF in A. n = 5 for B and C. ANOVA (two-factor with replication): p-value (CIP4 siRNA vs. control siRNA) = 6 × 10-5(B) and = 0.046 (C); p-value (between culture conditions) < 10-7 for both B and C. Post-hoc testing: *p-values vs. control siRNA-transfected myocytes treated with the same agonist; †p-values vs. no drug control. D. [3H]leucine incorporation. n = 2. *p-value vs. PE-treated, control siRNA-transfected myocytes.

Mentions: Hypertrophic myocytes are physically larger, have increased sarcomeric organization, express “fetal” genes such as that for atrial natriuretic factor (ANF), and have increased de novo protein synthesis [11,15]. Myocytes were transfected with the CIP4 or control siRNA and then cultured in the presence of PE, LIF, or FBS. After two days, cross-section area was measured using morphometric software and a qualitative assessment of sarcomeric organization was performed by α-actinin staining (Figure 3A,B). ANF expression was assayed by staining for prepro-ANF, and [3H]leucine incorporation was used to assay protein synthesis (Figure 3C,D). As expected, PE and FBS induced the growth in width and length of the control siRNA-transfected myocytes, while LIF, that activates the gp130-LIF cytokine receptor, induced an elongated myocyte phenotype. PE induced the most prominent sarcomeric organization as detected by α-actinin Z-line staining. All three stimuli induced ANF expression strongly. Regardless of the hypertrophic stimulus, myocytes transfected with the CIP4 siRNA were smaller in cross-section area, while not qualitatively different in overall proportion or sarcomeric organization (Figure 3A,B). Importantly, the induction of ANF expression by the hypertrophic agonists was attenuated by the CIP4 siRNA, albeit significantly only for the PE-treated cells (Figure 3C; 45%, 35%, and 15% less for PE, LIF, and FBS treated, CIP4 siRNA-transfected myocytes, respectively). To corroborate these data, we tested by [3H]leucine incorporation whether CIP4 siRNA could inhibit the de novo protein synthesis associated with PE-induced hypertrophy. Consistent with the requirement for CIP4 expression for PE-induced ANF expression, PE-stimulated [3H]leucine incorporation was significantly attenuated 48% by the CIP4 siRNA (Figure 3D). Taken together these data imply that the CIP4 scaffold contributes to signal transduction important for agonist-induced hypertrophy.


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

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

CIP4 is important for neonatal rat ventricular myocyte hypertrophy. A. Myocytes were transfected with control or CIP4 siRNA oligonucleotides and cultured ± 10% fetal bovine serum (FBS), 10 μM PE, or 1000 U/mL LIF. After treatment for 2 days, the myocytes were stained for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. B. Cross-section area of myocytes in A. C. Fraction of myocytes expressing ANF in A. n = 5 for B and C. ANOVA (two-factor with replication): p-value (CIP4 siRNA vs. control siRNA) = 6 × 10-5(B) and = 0.046 (C); p-value (between culture conditions) < 10-7 for both B and C. Post-hoc testing: *p-values vs. control siRNA-transfected myocytes treated with the same agonist; †p-values vs. no drug control. D. [3H]leucine incorporation. n = 2. *p-value vs. PE-treated, control siRNA-transfected myocytes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: CIP4 is important for neonatal rat ventricular myocyte hypertrophy. A. Myocytes were transfected with control or CIP4 siRNA oligonucleotides and cultured ± 10% fetal bovine serum (FBS), 10 μM PE, or 1000 U/mL LIF. After treatment for 2 days, the myocytes were stained for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. B. Cross-section area of myocytes in A. C. Fraction of myocytes expressing ANF in A. n = 5 for B and C. ANOVA (two-factor with replication): p-value (CIP4 siRNA vs. control siRNA) = 6 × 10-5(B) and = 0.046 (C); p-value (between culture conditions) < 10-7 for both B and C. Post-hoc testing: *p-values vs. control siRNA-transfected myocytes treated with the same agonist; †p-values vs. no drug control. D. [3H]leucine incorporation. n = 2. *p-value vs. PE-treated, control siRNA-transfected myocytes.
Mentions: Hypertrophic myocytes are physically larger, have increased sarcomeric organization, express “fetal” genes such as that for atrial natriuretic factor (ANF), and have increased de novo protein synthesis [11,15]. Myocytes were transfected with the CIP4 or control siRNA and then cultured in the presence of PE, LIF, or FBS. After two days, cross-section area was measured using morphometric software and a qualitative assessment of sarcomeric organization was performed by α-actinin staining (Figure 3A,B). ANF expression was assayed by staining for prepro-ANF, and [3H]leucine incorporation was used to assay protein synthesis (Figure 3C,D). As expected, PE and FBS induced the growth in width and length of the control siRNA-transfected myocytes, while LIF, that activates the gp130-LIF cytokine receptor, induced an elongated myocyte phenotype. PE induced the most prominent sarcomeric organization as detected by α-actinin Z-line staining. All three stimuli induced ANF expression strongly. Regardless of the hypertrophic stimulus, myocytes transfected with the CIP4 siRNA were smaller in cross-section area, while not qualitatively different in overall proportion or sarcomeric organization (Figure 3A,B). Importantly, the induction of ANF expression by the hypertrophic agonists was attenuated by the CIP4 siRNA, albeit significantly only for the PE-treated cells (Figure 3C; 45%, 35%, and 15% less for PE, LIF, and FBS treated, CIP4 siRNA-transfected myocytes, respectively). To corroborate these data, we tested by [3H]leucine incorporation whether CIP4 siRNA could inhibit the de novo protein synthesis associated with PE-induced hypertrophy. Consistent with the requirement for CIP4 expression for PE-induced ANF expression, PE-stimulated [3H]leucine incorporation was significantly attenuated 48% by the CIP4 siRNA (Figure 3D). Taken together these data imply that the CIP4 scaffold contributes to signal transduction important for agonist-induced hypertrophy.

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