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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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Differential expression of the DNA replication proteins in CMs and CFs.(A) CMs (n = 5) and CFs (n = 5) were isolated from normal adult heart and lysates (50 µg) were then analyzed by 12% or 9% SDS-polyacrylamide gels. Purity of the two fractions were confirmed by Western blot analyses for Troponin I-C (CMs) and S100A4 (CFs) proteins respectively. (B) Expression of RFC37, RFC38, RFC40, RFC140, PCNA and p125 proteins in the CMs and CFs was determined by Western blot analyses. Whole hearts isolated from 15 day-old fetus (n = 5) were used as a positive control (Ft). GAPDH was used as the loading control. (C) Graphs represent protein expression of RFC40 and p125, normalized by GAPDH in the CMs and fibroblasts, respectively. (D) Total RNA (50 ng) extracted from CMs and fibroblasts pellets (n = 5) was subjected to real-time one-step-RT-PCR. At the end of each run the amplified products of RFC40, p125 and GAPDH mRNA/cDNA were visualized on 3% agarose gels. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). (E) Graphs represent the mRNA levels in the CMs and CFs calculated from the crossing point deviation and normalized by GAPDH values. Values are mean ± SE. BDL: Below detectable levels.
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pone-0039009-g001: Differential expression of the DNA replication proteins in CMs and CFs.(A) CMs (n = 5) and CFs (n = 5) were isolated from normal adult heart and lysates (50 µg) were then analyzed by 12% or 9% SDS-polyacrylamide gels. Purity of the two fractions were confirmed by Western blot analyses for Troponin I-C (CMs) and S100A4 (CFs) proteins respectively. (B) Expression of RFC37, RFC38, RFC40, RFC140, PCNA and p125 proteins in the CMs and CFs was determined by Western blot analyses. Whole hearts isolated from 15 day-old fetus (n = 5) were used as a positive control (Ft). GAPDH was used as the loading control. (C) Graphs represent protein expression of RFC40 and p125, normalized by GAPDH in the CMs and fibroblasts, respectively. (D) Total RNA (50 ng) extracted from CMs and fibroblasts pellets (n = 5) was subjected to real-time one-step-RT-PCR. At the end of each run the amplified products of RFC40, p125 and GAPDH mRNA/cDNA were visualized on 3% agarose gels. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). (E) Graphs represent the mRNA levels in the CMs and CFs calculated from the crossing point deviation and normalized by GAPDH values. Values are mean ± SE. BDL: Below detectable levels.

Mentions: We isolated CMs and CFs from normal adult heart and performed Western blot analyses for RFC, PCNA and p125 proteins to determine the cell type in which they are expressed. Purity of each fraction was first confirmed by cell specific markers, viz; Troponin I for CMs and S100A4 for CFs (Figure 1A). RFC40 (Figure 1B and C) protein was not detected in both CMs and CFs, while RFC140 and RFC38 proteins (Figure 1B) were expressed in both. Furthermore, PNCA (Figure 1B), RFC37 (Figure 1B) and p125 (Figure 1B and C) proteins were expressed only in the CFs. Real time qRT-PCR demonstrated that p125-mRNA was transcribed in both cell types (Figure 1D-middle panel), with significant higher expression in the CFs as compared to the CMs (Figure 1E). Strikingly, however, RFC40-mRNA was transcribed only in the CFs but not in the CMs (Figure 1D-top panel and 1 E).


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)

Differential expression of the DNA replication proteins in CMs and CFs.(A) CMs (n = 5) and CFs (n = 5) were isolated from normal adult heart and lysates (50 µg) were then analyzed by 12% or 9% SDS-polyacrylamide gels. Purity of the two fractions were confirmed by Western blot analyses for Troponin I-C (CMs) and S100A4 (CFs) proteins respectively. (B) Expression of RFC37, RFC38, RFC40, RFC140, PCNA and p125 proteins in the CMs and CFs was determined by Western blot analyses. Whole hearts isolated from 15 day-old fetus (n = 5) were used as a positive control (Ft). GAPDH was used as the loading control. (C) Graphs represent protein expression of RFC40 and p125, normalized by GAPDH in the CMs and fibroblasts, respectively. (D) Total RNA (50 ng) extracted from CMs and fibroblasts pellets (n = 5) was subjected to real-time one-step-RT-PCR. At the end of each run the amplified products of RFC40, p125 and GAPDH mRNA/cDNA were visualized on 3% agarose gels. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). (E) Graphs represent the mRNA levels in the CMs and CFs calculated from the crossing point deviation and normalized by GAPDH values. Values are mean ± SE. BDL: Below detectable levels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375256&req=5

pone-0039009-g001: Differential expression of the DNA replication proteins in CMs and CFs.(A) CMs (n = 5) and CFs (n = 5) were isolated from normal adult heart and lysates (50 µg) were then analyzed by 12% or 9% SDS-polyacrylamide gels. Purity of the two fractions were confirmed by Western blot analyses for Troponin I-C (CMs) and S100A4 (CFs) proteins respectively. (B) Expression of RFC37, RFC38, RFC40, RFC140, PCNA and p125 proteins in the CMs and CFs was determined by Western blot analyses. Whole hearts isolated from 15 day-old fetus (n = 5) were used as a positive control (Ft). GAPDH was used as the loading control. (C) Graphs represent protein expression of RFC40 and p125, normalized by GAPDH in the CMs and fibroblasts, respectively. (D) Total RNA (50 ng) extracted from CMs and fibroblasts pellets (n = 5) was subjected to real-time one-step-RT-PCR. At the end of each run the amplified products of RFC40, p125 and GAPDH mRNA/cDNA were visualized on 3% agarose gels. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). (E) Graphs represent the mRNA levels in the CMs and CFs calculated from the crossing point deviation and normalized by GAPDH values. Values are mean ± SE. BDL: Below detectable levels.
Mentions: We isolated CMs and CFs from normal adult heart and performed Western blot analyses for RFC, PCNA and p125 proteins to determine the cell type in which they are expressed. Purity of each fraction was first confirmed by cell specific markers, viz; Troponin I for CMs and S100A4 for CFs (Figure 1A). RFC40 (Figure 1B and C) protein was not detected in both CMs and CFs, while RFC140 and RFC38 proteins (Figure 1B) were expressed in both. Furthermore, PNCA (Figure 1B), RFC37 (Figure 1B) and p125 (Figure 1B and C) proteins were expressed only in the CFs. Real time qRT-PCR demonstrated that p125-mRNA was transcribed in both cell types (Figure 1D-middle panel), with significant higher expression in the CFs as compared to the CMs (Figure 1E). Strikingly, however, RFC40-mRNA was transcribed only in the CFs but not in the CMs (Figure 1D-top panel and 1 E).

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