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Down-regulation of replication factor C-40 (RFC40) causes chromosomal missegregation in neonatal and hypertrophic adult rat cardiac myocytes.

Ata H, Shrestha D, Oka M, Ochi R, Jong CJ, Gebb S, Benjamin J, Schaffer S, Hobart HH, Downey J, McMurtry I, Gupte R - PLoS ONE (2012)

Bottom Line: Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts.Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers.Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry & Molecular Biology, University of South Alabama, Mobile, Alabama, United States of America.

ABSTRACT

Background: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied.

Methods: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12.

Results: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers.

Conclusion: Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

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Related in: MedlinePlus

Chromosomal missegregation/Aneuploidy was observed in the CM nuclei from the hypertrophied RVs.Control (Con) and SuHxNx-5 wks (5 wks) RV sections were used to perform FISH analyses by co-hybridization of the tissues with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue) and slides were imaged with Spectral Imaging Software using an Olympus BX61 microscope with 1000X magnification. Two FISH signals for Cen12-ROX was observed in the CM nuclei from the control RV (A-i), however, one (B-i) and three (C-i) signals were observed in the CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (A-ii) and hypertrophied (B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). (D) Graph represents the number of FISH signals for Cen12-ROX observed per CM nuclei in the control and hypertrophied RVs. Fifty CM nuclei were measured from three individual animals in control and hypertrophied RVs. (E) Diving CM nuclei undergoing chromosomal missegregation in the hypertrophied RVs, with one nucleus receiving three sister chromatids while the other receiving only one chromatid.
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pone-0039009-g006: Chromosomal missegregation/Aneuploidy was observed in the CM nuclei from the hypertrophied RVs.Control (Con) and SuHxNx-5 wks (5 wks) RV sections were used to perform FISH analyses by co-hybridization of the tissues with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue) and slides were imaged with Spectral Imaging Software using an Olympus BX61 microscope with 1000X magnification. Two FISH signals for Cen12-ROX was observed in the CM nuclei from the control RV (A-i), however, one (B-i) and three (C-i) signals were observed in the CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (A-ii) and hypertrophied (B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). (D) Graph represents the number of FISH signals for Cen12-ROX observed per CM nuclei in the control and hypertrophied RVs. Fifty CM nuclei were measured from three individual animals in control and hypertrophied RVs. (E) Diving CM nuclei undergoing chromosomal missegregation in the hypertrophied RVs, with one nucleus receiving three sister chromatids while the other receiving only one chromatid.

Mentions: In addition, to observing two and four signals for Cen12-ROX, we also observed one (Figure 6B-i), and three (Figure 6C-i) signals in several CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (Figure 6A-ii) and hypertrophied (Figure 6B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). Furthermore, statistical analyses revealed that there was significant decrease (P<0.05) in the number of diploid CM nuclei and an increase in the number of aneuploid nuclei (Figure 6D). Additionally, we also observed some dividing CMs nuclei exhibiting chromosomal missegregation in the hypertrophied RVs with one nucleus receiving three sister chromatids of chromosome 12 while the other receiving only one sister chromatid (Figure 6E).


Down-regulation of replication factor C-40 (RFC40) causes chromosomal missegregation in neonatal and hypertrophic adult rat cardiac myocytes.

Ata H, Shrestha D, Oka M, Ochi R, Jong CJ, Gebb S, Benjamin J, Schaffer S, Hobart HH, Downey J, McMurtry I, Gupte R - PLoS ONE (2012)

Chromosomal missegregation/Aneuploidy was observed in the CM nuclei from the hypertrophied RVs.Control (Con) and SuHxNx-5 wks (5 wks) RV sections were used to perform FISH analyses by co-hybridization of the tissues with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue) and slides were imaged with Spectral Imaging Software using an Olympus BX61 microscope with 1000X magnification. Two FISH signals for Cen12-ROX was observed in the CM nuclei from the control RV (A-i), however, one (B-i) and three (C-i) signals were observed in the CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (A-ii) and hypertrophied (B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). (D) Graph represents the number of FISH signals for Cen12-ROX observed per CM nuclei in the control and hypertrophied RVs. Fifty CM nuclei were measured from three individual animals in control and hypertrophied RVs. (E) Diving CM nuclei undergoing chromosomal missegregation in the hypertrophied RVs, with one nucleus receiving three sister chromatids while the other receiving only one chromatid.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375256&req=5

pone-0039009-g006: Chromosomal missegregation/Aneuploidy was observed in the CM nuclei from the hypertrophied RVs.Control (Con) and SuHxNx-5 wks (5 wks) RV sections were used to perform FISH analyses by co-hybridization of the tissues with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue) and slides were imaged with Spectral Imaging Software using an Olympus BX61 microscope with 1000X magnification. Two FISH signals for Cen12-ROX was observed in the CM nuclei from the control RV (A-i), however, one (B-i) and three (C-i) signals were observed in the CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (A-ii) and hypertrophied (B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). (D) Graph represents the number of FISH signals for Cen12-ROX observed per CM nuclei in the control and hypertrophied RVs. Fifty CM nuclei were measured from three individual animals in control and hypertrophied RVs. (E) Diving CM nuclei undergoing chromosomal missegregation in the hypertrophied RVs, with one nucleus receiving three sister chromatids while the other receiving only one chromatid.
Mentions: In addition, to observing two and four signals for Cen12-ROX, we also observed one (Figure 6B-i), and three (Figure 6C-i) signals in several CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (Figure 6A-ii) and hypertrophied (Figure 6B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). Furthermore, statistical analyses revealed that there was significant decrease (P<0.05) in the number of diploid CM nuclei and an increase in the number of aneuploid nuclei (Figure 6D). Additionally, we also observed some dividing CMs nuclei exhibiting chromosomal missegregation in the hypertrophied RVs with one nucleus receiving three sister chromatids of chromosome 12 while the other receiving only one sister chromatid (Figure 6E).

Bottom Line: Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts.Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers.Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry & Molecular Biology, University of South Alabama, Mobile, Alabama, United States of America.

ABSTRACT

Background: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied.

Methods: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12.

Results: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers.

Conclusion: Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

Show MeSH
Related in: MedlinePlus