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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

Cardiac myocyte polyploidy was observed in 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), whereas two (B-i) and four (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) Graph represents the nuclear area of >100 CM nuclei measured from three individual animals in control and hypertrophied RVs. Values are mean ± SE. *indicates P<0.05 vs. control. (F) In vitro DNA synthesis was carried out using primed M13mp18 ss DNA (100 ng) and protein lysates (20 µg) obtained from fetal, control-RV (Con), SUHx-3 wks-RV and SUHxNx-5 wks-RV tissues, respectively, before (F-i) and after (F-ii) immunodepletion of RFC40. The primer extension products were separated on a 7 M urea-6% (v/v) polyacrylamide gel and visualized by the chemiluminescent nucleic acid detection kit. Lane 1: Biotinylated 2-log DNA ladder (0.5 µg; M) in kilobases (B-lane 1; exposed for only 5 secs to visualize all the bands, is separated by a line). Lane 2: unannealed biotinylated M13 80-mer primer (BLM13P; 1 fmole). Blot is a representative of three such individual experiments.
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pone-0039009-g005: Cardiac myocyte polyploidy was observed in 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), whereas two (B-i) and four (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) Graph represents the nuclear area of >100 CM nuclei measured from three individual animals in control and hypertrophied RVs. Values are mean ± SE. *indicates P<0.05 vs. control. (F) In vitro DNA synthesis was carried out using primed M13mp18 ss DNA (100 ng) and protein lysates (20 µg) obtained from fetal, control-RV (Con), SUHx-3 wks-RV and SUHxNx-5 wks-RV tissues, respectively, before (F-i) and after (F-ii) immunodepletion of RFC40. The primer extension products were separated on a 7 M urea-6% (v/v) polyacrylamide gel and visualized by the chemiluminescent nucleic acid detection kit. Lane 1: Biotinylated 2-log DNA ladder (0.5 µg; M) in kilobases (B-lane 1; exposed for only 5 secs to visualize all the bands, is separated by a line). Lane 2: unannealed biotinylated M13 80-mer primer (BLM13P; 1 fmole). Blot is a representative of three such individual experiments.

Mentions: To determine whether the increased expression of the DNA replication and S-phase proteins can support DNA synthesis during the pathogenesis of hypertrophy, we performed Fluorescent in-situ hybridization (FISH) on control and SUHxNx-5 wks-RVs, respectively. We used the BAC clone representing the bands 12p11–12q11 of rat chromosome 12 (Cen 12) labeled with 5-carboxyl-x-rhodamine (5-ROX; Cen12-ROX) as probe for FISH analyses (Figure S3 for the third supporting information figure). Interestingly, we noted two different observations in the CM nuclei of the hypertrophied RV- polyploidy and chromosomal missegregation/aneuploidy. Polyploidy: We observed two signals for Cen12-ROX (Red) in the CM nuclei from the control RV (Figure 5A-i). However, two (Figure 5B-i) and four (Figure 5C-i) signals in the CM nuclei from the hypertrophied RVs was observed. Localization of the CM nuclei in the control (Figure 5A-ii) and hypertrophied (Figure 5B–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 polyploid nuclei (Figure 5D) with concomitant doubling in the nuclear area of the CM nuclei (Figure 5E) in the hypertrophied RVs as compared to the control RVs.


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)

Cardiac myocyte polyploidy was observed in 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), whereas two (B-i) and four (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) Graph represents the nuclear area of >100 CM nuclei measured from three individual animals in control and hypertrophied RVs. Values are mean ± SE. *indicates P<0.05 vs. control. (F) In vitro DNA synthesis was carried out using primed M13mp18 ss DNA (100 ng) and protein lysates (20 µg) obtained from fetal, control-RV (Con), SUHx-3 wks-RV and SUHxNx-5 wks-RV tissues, respectively, before (F-i) and after (F-ii) immunodepletion of RFC40. The primer extension products were separated on a 7 M urea-6% (v/v) polyacrylamide gel and visualized by the chemiluminescent nucleic acid detection kit. Lane 1: Biotinylated 2-log DNA ladder (0.5 µg; M) in kilobases (B-lane 1; exposed for only 5 secs to visualize all the bands, is separated by a line). Lane 2: unannealed biotinylated M13 80-mer primer (BLM13P; 1 fmole). Blot is a representative of three such individual experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375256&req=5

pone-0039009-g005: Cardiac myocyte polyploidy was observed in 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), whereas two (B-i) and four (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) Graph represents the nuclear area of >100 CM nuclei measured from three individual animals in control and hypertrophied RVs. Values are mean ± SE. *indicates P<0.05 vs. control. (F) In vitro DNA synthesis was carried out using primed M13mp18 ss DNA (100 ng) and protein lysates (20 µg) obtained from fetal, control-RV (Con), SUHx-3 wks-RV and SUHxNx-5 wks-RV tissues, respectively, before (F-i) and after (F-ii) immunodepletion of RFC40. The primer extension products were separated on a 7 M urea-6% (v/v) polyacrylamide gel and visualized by the chemiluminescent nucleic acid detection kit. Lane 1: Biotinylated 2-log DNA ladder (0.5 µg; M) in kilobases (B-lane 1; exposed for only 5 secs to visualize all the bands, is separated by a line). Lane 2: unannealed biotinylated M13 80-mer primer (BLM13P; 1 fmole). Blot is a representative of three such individual experiments.
Mentions: To determine whether the increased expression of the DNA replication and S-phase proteins can support DNA synthesis during the pathogenesis of hypertrophy, we performed Fluorescent in-situ hybridization (FISH) on control and SUHxNx-5 wks-RVs, respectively. We used the BAC clone representing the bands 12p11–12q11 of rat chromosome 12 (Cen 12) labeled with 5-carboxyl-x-rhodamine (5-ROX; Cen12-ROX) as probe for FISH analyses (Figure S3 for the third supporting information figure). Interestingly, we noted two different observations in the CM nuclei of the hypertrophied RV- polyploidy and chromosomal missegregation/aneuploidy. Polyploidy: We observed two signals for Cen12-ROX (Red) in the CM nuclei from the control RV (Figure 5A-i). However, two (Figure 5B-i) and four (Figure 5C-i) signals in the CM nuclei from the hypertrophied RVs was observed. Localization of the CM nuclei in the control (Figure 5A-ii) and hypertrophied (Figure 5B–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 polyploid nuclei (Figure 5D) with concomitant doubling in the nuclear area of the CM nuclei (Figure 5E) in the hypertrophied RVs as compared to the control RVs.

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