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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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The CIP4 FCH domain is important for neonatal rat ventricular myocyte hypertrophy. Neonatal rat ventricular myocytes were transfected with control or CIP4 siRNA and then infected with adenovirus expressing myc-tagged CIP4 WT or ΔFCH protein. Myocytes were stimulated with 10 μM PE for two days as indicated. A. CIP4 proteins were detected using a mouse anti-CIP4 antibody against human CIP4 aa 411–501. (Rat and human CIP4 are 92% identical.) B. Immunocytochemistry for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. C. Cross-section area of myocytes. n = 7. D. Fraction of myocytes expressing ANF. n = 6. ANOVA (two-factor with replication): p-value (among the four CIP4 expression conditions) = 0.005 (C) and = 0.02 (D); p-value (± PE) < 10-6 for both B and C. Post-hoc testing: *p-values vs. CIP4 siRNA-transfected myocytes; †p-values comparing myocytes cultured ± PE; ‡p-values vs. myc-CIP4 WT expressing myocytes.
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Figure 4: The CIP4 FCH domain is important for neonatal rat ventricular myocyte hypertrophy. Neonatal rat ventricular myocytes were transfected with control or CIP4 siRNA and then infected with adenovirus expressing myc-tagged CIP4 WT or ΔFCH protein. Myocytes were stimulated with 10 μM PE for two days as indicated. A. CIP4 proteins were detected using a mouse anti-CIP4 antibody against human CIP4 aa 411–501. (Rat and human CIP4 are 92% identical.) B. Immunocytochemistry for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. C. Cross-section area of myocytes. n = 7. D. Fraction of myocytes expressing ANF. n = 6. ANOVA (two-factor with replication): p-value (among the four CIP4 expression conditions) = 0.005 (C) and = 0.02 (D); p-value (± PE) < 10-6 for both B and C. Post-hoc testing: *p-values vs. CIP4 siRNA-transfected myocytes; †p-values comparing myocytes cultured ± PE; ‡p-values vs. myc-CIP4 WT expressing myocytes.

Mentions: The requirement for CIP4 in hypertrophic growth was confirmed by rescue of CIP4 expression (Figure 4A). CIP4 siRNA or control siRNA-transfected myocytes were infected with adenovirus expressing myc-tagged wildtype (WT) CIP4 or the CIP4 ΔFCH N-terminal truncation mutant that cannot properly localize to membranes or bind microtubules [16,17]. Note that due to the PE-enhanced activity of the CMV promoter, the adenoviral-based expression of the wildtype and mutant CIP4 was higher in PE-treated cells, similar to endogenous CIP4. In this experiment (Figure 4C), the PE-induced increase in myocyte cross-section area was attenuated 42% by the CIP4 siRNA. In addition, PE-induced ANF expression was inhibited 63% by CIP4 siRNA (Figure 4D). Importantly, CIP4 siRNA-transfected myocytes infected with myc-CIP4 WT adenovirus were not different in size or ANF expression than control siRNA-transfected myocytes, both in the absence and presence of hypertrophic stimulus. Moreover, PE-stimulated, CIP4 siRNA-transfected myocytes expressing myc-CIP4 ΔFCH were significantly smaller and expressed less ANF than those rescued by myc-CIP4 WT expression, while similar in size and ANF expression to the non-rescued CIP4 siRNA-transfected myocytes (Figure 4B,C). Since the FCH domain confers CIP4 association with the plasma membrane and microtubules [16,17], this result suggests that the proper localization of the CIP4 scaffold is required to mediate hypertrophic signal transduction in cardiac myocytes.


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

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

The CIP4 FCH domain is important for neonatal rat ventricular myocyte hypertrophy. Neonatal rat ventricular myocytes were transfected with control or CIP4 siRNA and then infected with adenovirus expressing myc-tagged CIP4 WT or ΔFCH protein. Myocytes were stimulated with 10 μM PE for two days as indicated. A. CIP4 proteins were detected using a mouse anti-CIP4 antibody against human CIP4 aa 411–501. (Rat and human CIP4 are 92% identical.) B. Immunocytochemistry for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. C. Cross-section area of myocytes. n = 7. D. Fraction of myocytes expressing ANF. n = 6. ANOVA (two-factor with replication): p-value (among the four CIP4 expression conditions) = 0.005 (C) and = 0.02 (D); p-value (± PE) < 10-6 for both B and C. Post-hoc testing: *p-values vs. CIP4 siRNA-transfected myocytes; †p-values comparing myocytes cultured ± PE; ‡p-values vs. myc-CIP4 WT expressing myocytes.
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Figure 4: The CIP4 FCH domain is important for neonatal rat ventricular myocyte hypertrophy. Neonatal rat ventricular myocytes were transfected with control or CIP4 siRNA and then infected with adenovirus expressing myc-tagged CIP4 WT or ΔFCH protein. Myocytes were stimulated with 10 μM PE for two days as indicated. A. CIP4 proteins were detected using a mouse anti-CIP4 antibody against human CIP4 aa 411–501. (Rat and human CIP4 are 92% identical.) B. Immunocytochemistry for α-actinin (green), ANF (red) and Hoechst (blue); bar = 20 μm. C. Cross-section area of myocytes. n = 7. D. Fraction of myocytes expressing ANF. n = 6. ANOVA (two-factor with replication): p-value (among the four CIP4 expression conditions) = 0.005 (C) and = 0.02 (D); p-value (± PE) < 10-6 for both B and C. Post-hoc testing: *p-values vs. CIP4 siRNA-transfected myocytes; †p-values comparing myocytes cultured ± PE; ‡p-values vs. myc-CIP4 WT expressing myocytes.
Mentions: The requirement for CIP4 in hypertrophic growth was confirmed by rescue of CIP4 expression (Figure 4A). CIP4 siRNA or control siRNA-transfected myocytes were infected with adenovirus expressing myc-tagged wildtype (WT) CIP4 or the CIP4 ΔFCH N-terminal truncation mutant that cannot properly localize to membranes or bind microtubules [16,17]. Note that due to the PE-enhanced activity of the CMV promoter, the adenoviral-based expression of the wildtype and mutant CIP4 was higher in PE-treated cells, similar to endogenous CIP4. In this experiment (Figure 4C), the PE-induced increase in myocyte cross-section area was attenuated 42% by the CIP4 siRNA. In addition, PE-induced ANF expression was inhibited 63% by CIP4 siRNA (Figure 4D). Importantly, CIP4 siRNA-transfected myocytes infected with myc-CIP4 WT adenovirus were not different in size or ANF expression than control siRNA-transfected myocytes, both in the absence and presence of hypertrophic stimulus. Moreover, PE-stimulated, CIP4 siRNA-transfected myocytes expressing myc-CIP4 ΔFCH were significantly smaller and expressed less ANF than those rescued by myc-CIP4 WT expression, while similar in size and ANF expression to the non-rescued CIP4 siRNA-transfected myocytes (Figure 4B,C). Since the FCH domain confers CIP4 association with the plasma membrane and microtubules [16,17], this result suggests that the proper localization of the CIP4 scaffold is required to mediate hypertrophic signal transduction in cardiac myocytes.

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