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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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Up-regulation/re-expression of replication proteins in hypertrophied hearts occurs at the transcription level.Total RNA (50 ng) extracted from LV and RV tissues (n = 5) of the control (Con), SUHx-3 wks (3 wks) and SUHxNx-5 wks (5 wks) was subjected to real-time one-step-RT-PCR. The amplified products of RFC40, p125, and GAPDH mRNA/cDNA were visualized on 3% agarose gels at the end of each run. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). Graphs represent the changes in the mRNA levels for RFC40-mRNA (A) and p125-mRNA (B) calculated from the crossing point deviation of all the samples and normalized by GAPDH values. Values are mean ± SE. *indicates P<0.05 vs. control.
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pone-0039009-g003: Up-regulation/re-expression of replication proteins in hypertrophied hearts occurs at the transcription level.Total RNA (50 ng) extracted from LV and RV tissues (n = 5) of the control (Con), SUHx-3 wks (3 wks) and SUHxNx-5 wks (5 wks) was subjected to real-time one-step-RT-PCR. The amplified products of RFC40, p125, and GAPDH mRNA/cDNA were visualized on 3% agarose gels at the end of each run. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). Graphs represent the changes in the mRNA levels for RFC40-mRNA (A) and p125-mRNA (B) calculated from the crossing point deviation of all the samples and normalized by GAPDH values. Values are mean ± SE. *indicates P<0.05 vs. control.

Mentions: To determine whether the up-regulation/re-expression of the replication proteins in the hypertrophied myocardium occurred at the transcriptional level, we quantified mRNA for RFC40 and p125 in fetal, control and hypertrophied hearts by qRT-PCR and the amplified samples were ran on agarose gel to determine their molecular size and specificity at the end of each run (Figure S2 for the second supporting information figure). Quantitative analysis demonstrated that RFC40-mRNA and p125–mRNA in adult LV and RV decreased to 5–10% of those in fetal hearts (which may probably account for that present in the CFs). Interestingly, while RFC40-mRNA increased by 1.2 to 3.4-fold (P<0.05) in SUHx-3 wks and SUHxNx-5 wks hearts (Figure 3A), p125-mRNA increased by 7.6 to 40-fold in SUHx-3 wks and SUHxNx-5 wks hearts, respectively (Figure 3B) as compared to the control hearts. Nonetheless, this increase was marginal as compared to the fetal hearts.


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)

Up-regulation/re-expression of replication proteins in hypertrophied hearts occurs at the transcription level.Total RNA (50 ng) extracted from LV and RV tissues (n = 5) of the control (Con), SUHx-3 wks (3 wks) and SUHxNx-5 wks (5 wks) was subjected to real-time one-step-RT-PCR. The amplified products of RFC40, p125, and GAPDH mRNA/cDNA were visualized on 3% agarose gels at the end of each run. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). Graphs represent the changes in the mRNA levels for RFC40-mRNA (A) and p125-mRNA (B) calculated from the crossing point deviation of all the samples and normalized by GAPDH values. Values are mean ± SE. *indicates P<0.05 vs. control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375256&req=5

pone-0039009-g003: Up-regulation/re-expression of replication proteins in hypertrophied hearts occurs at the transcription level.Total RNA (50 ng) extracted from LV and RV tissues (n = 5) of the control (Con), SUHx-3 wks (3 wks) and SUHxNx-5 wks (5 wks) was subjected to real-time one-step-RT-PCR. The amplified products of RFC40, p125, and GAPDH mRNA/cDNA were visualized on 3% agarose gels at the end of each run. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). Graphs represent the changes in the mRNA levels for RFC40-mRNA (A) and p125-mRNA (B) calculated from the crossing point deviation of all the samples and normalized by GAPDH values. Values are mean ± SE. *indicates P<0.05 vs. control.
Mentions: To determine whether the up-regulation/re-expression of the replication proteins in the hypertrophied myocardium occurred at the transcriptional level, we quantified mRNA for RFC40 and p125 in fetal, control and hypertrophied hearts by qRT-PCR and the amplified samples were ran on agarose gel to determine their molecular size and specificity at the end of each run (Figure S2 for the second supporting information figure). Quantitative analysis demonstrated that RFC40-mRNA and p125–mRNA in adult LV and RV decreased to 5–10% of those in fetal hearts (which may probably account for that present in the CFs). Interestingly, while RFC40-mRNA increased by 1.2 to 3.4-fold (P<0.05) in SUHx-3 wks and SUHxNx-5 wks hearts (Figure 3A), p125-mRNA increased by 7.6 to 40-fold in SUHx-3 wks and SUHxNx-5 wks hearts, respectively (Figure 3B) as compared to the control hearts. Nonetheless, this increase was marginal as compared to the fetal hearts.

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