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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

Endogenous knock-down of RFC40 in rat neonatal cardiac myocytes results in chromosomal missegregation/aneuploidy. Rat Neonatal cardiac myocytes (RNCMs) were isolated as described previously and grown in 12 well plates and 2-chambered slides for 48 hr. RNCMs were then treated with non-targeting-siRNA (NT) and On-Target plus smartpool RFC40-siRNA respectively, for 72 hr. (A–D and F–G) RNCMs grown in 2-chambered slides were subjected to FISH analysis following treatment with RFC40-siRNA. Untransfected (UT) and RFC40-siRNA-RNCM slides were co-hybridization with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue). RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (A–D-ii and F–G-ii; Green) in each sample. Merge images are shown in A–D-iii and F–G-iii. Panels A–D represents RNCM nuclei aneuploidy and panels F–G represent chromosomal missegregation. White arrows point to Cen12-ROX signals. Yellow arrow points to micronuclei in G-i & iii. (E) Graph represents the number of signals for Cen12-ROX observed per RNCM nuclei in the UT and RFC40-siRNA treated samples. Fifty RNCM nuclei in control and RFC40-siRNA treated samples from three individual experiments were measured. (H) Before lysing the cells grown in 12-well plates for western blot analyses (Fig. S4), the RNCMs (n = 3) were trypsinized, resuspended in 1X PBS and counted using a hemocytometer. Graph represents the number of RNCMs vs the different RNCM treated samples. Values are mean ± SE. * indicates P<0.05 vs. Untransfected (UT).
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pone-0039009-g007: Endogenous knock-down of RFC40 in rat neonatal cardiac myocytes results in chromosomal missegregation/aneuploidy. Rat Neonatal cardiac myocytes (RNCMs) were isolated as described previously and grown in 12 well plates and 2-chambered slides for 48 hr. RNCMs were then treated with non-targeting-siRNA (NT) and On-Target plus smartpool RFC40-siRNA respectively, for 72 hr. (A–D and F–G) RNCMs grown in 2-chambered slides were subjected to FISH analysis following treatment with RFC40-siRNA. Untransfected (UT) and RFC40-siRNA-RNCM slides were co-hybridization with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue). RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (A–D-ii and F–G-ii; Green) in each sample. Merge images are shown in A–D-iii and F–G-iii. Panels A–D represents RNCM nuclei aneuploidy and panels F–G represent chromosomal missegregation. White arrows point to Cen12-ROX signals. Yellow arrow points to micronuclei in G-i & iii. (E) Graph represents the number of signals for Cen12-ROX observed per RNCM nuclei in the UT and RFC40-siRNA treated samples. Fifty RNCM nuclei in control and RFC40-siRNA treated samples from three individual experiments were measured. (H) Before lysing the cells grown in 12-well plates for western blot analyses (Fig. S4), the RNCMs (n = 3) were trypsinized, resuspended in 1X PBS and counted using a hemocytometer. Graph represents the number of RNCMs vs the different RNCM treated samples. Values are mean ± SE. * indicates P<0.05 vs. Untransfected (UT).

Mentions: It has been previously demonstrated that RFC40 is required for accurate chromosomal segregation in Drosophila[31]. Since we observed significant aneuploidy and chromosomal missegregation in the CM nuclei, and since the re-expression of RFC40 in the hypertrophied RVs was less than 10% of that in the fetal hearts, we explored the possibility of whether insufficient re-expression of RFC40 must be responsible for aneuploidy and the chromosomal segregation defects observed in the hypertrophied hearts. To test this possibility, we knocked-down endogenous RFC40 protein in the neonatal (day 2) rat cardiac myocytes (RNCMs) using an On-Target plus smartpool RFC40-SiRNA for 72 hr. Western blot analysis demonstrated approximately 85% down-regulation of the RFC40 protein in the RFC40-siRNA treated samples as compared to untransfected (UT) and non-targeting-siRNA (NT) treated RNCMs (Figure S4 for the fourth supporting information figure). UT and RFC40-siRNA treated RNCMs were subjected to FISH analyses and RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (Green) in each sample (Figure 7A–D-ii and F–G-ii). As expected, we observed only two signals for Cen12-ROX (Red) in the UT RNCM nuclei (Figure 7A-i). Interestingly, we observed one (Figure 7B-i) and three (Figure 7D-i) signals in the RFC40-siRNA treated RNCM nuclei. Although, we observed two signals (Figure 7C-i) for few RNCM nuclei treated with RFC40-siRNA, however, the RFC40 staining for these RNCM nuclei was stronger (Figure 7 C-ii) than that observed for the RFC40-siRNA treated RNCM nuclei with one (Figure 7B-ii) and three (Figure 7 D-ii) signals, respectively, suggesting that perhaps RFC40 was not knocked-down in these RNCMs (thus behaving as an internal control). Statistical analyses revealed significant increase in the number of aneuploid RNCMs (monosomy and trisomy for chromosome 12) following RFC40-siRNA treatment (Figure 7E). Furthermore, we also observed few bi-nucleated RNCMs with unequal chromosomal distribution (Figure 7F), similar to those observed in the hypertrophied RVs, and nuclei that exhibited both nondisjunction for Cen12-ROX (three signals) and micronuclei formation (Figure 7G). Moreover, cell number analyses demonstrated that RFC40-siRNA treated RNCMs significantly (P<0.05) decreased in cell numbers, as compared to Untransfected (UT) RNCMs (Figure 7H). Taken together, these data suggests that RFC40 is required for accurate chromosomal segregation as well as cell survival/proliferation in RNCMs.


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)

Endogenous knock-down of RFC40 in rat neonatal cardiac myocytes results in chromosomal missegregation/aneuploidy. Rat Neonatal cardiac myocytes (RNCMs) were isolated as described previously and grown in 12 well plates and 2-chambered slides for 48 hr. RNCMs were then treated with non-targeting-siRNA (NT) and On-Target plus smartpool RFC40-siRNA respectively, for 72 hr. (A–D and F–G) RNCMs grown in 2-chambered slides were subjected to FISH analysis following treatment with RFC40-siRNA. Untransfected (UT) and RFC40-siRNA-RNCM slides were co-hybridization with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue). RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (A–D-ii and F–G-ii; Green) in each sample. Merge images are shown in A–D-iii and F–G-iii. Panels A–D represents RNCM nuclei aneuploidy and panels F–G represent chromosomal missegregation. White arrows point to Cen12-ROX signals. Yellow arrow points to micronuclei in G-i & iii. (E) Graph represents the number of signals for Cen12-ROX observed per RNCM nuclei in the UT and RFC40-siRNA treated samples. Fifty RNCM nuclei in control and RFC40-siRNA treated samples from three individual experiments were measured. (H) Before lysing the cells grown in 12-well plates for western blot analyses (Fig. S4), the RNCMs (n = 3) were trypsinized, resuspended in 1X PBS and counted using a hemocytometer. Graph represents the number of RNCMs vs the different RNCM treated samples. Values are mean ± SE. * indicates P<0.05 vs. Untransfected (UT).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375256&req=5

pone-0039009-g007: Endogenous knock-down of RFC40 in rat neonatal cardiac myocytes results in chromosomal missegregation/aneuploidy. Rat Neonatal cardiac myocytes (RNCMs) were isolated as described previously and grown in 12 well plates and 2-chambered slides for 48 hr. RNCMs were then treated with non-targeting-siRNA (NT) and On-Target plus smartpool RFC40-siRNA respectively, for 72 hr. (A–D and F–G) RNCMs grown in 2-chambered slides were subjected to FISH analysis following treatment with RFC40-siRNA. Untransfected (UT) and RFC40-siRNA-RNCM slides were co-hybridization with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue). RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (A–D-ii and F–G-ii; Green) in each sample. Merge images are shown in A–D-iii and F–G-iii. Panels A–D represents RNCM nuclei aneuploidy and panels F–G represent chromosomal missegregation. White arrows point to Cen12-ROX signals. Yellow arrow points to micronuclei in G-i & iii. (E) Graph represents the number of signals for Cen12-ROX observed per RNCM nuclei in the UT and RFC40-siRNA treated samples. Fifty RNCM nuclei in control and RFC40-siRNA treated samples from three individual experiments were measured. (H) Before lysing the cells grown in 12-well plates for western blot analyses (Fig. S4), the RNCMs (n = 3) were trypsinized, resuspended in 1X PBS and counted using a hemocytometer. Graph represents the number of RNCMs vs the different RNCM treated samples. Values are mean ± SE. * indicates P<0.05 vs. Untransfected (UT).
Mentions: It has been previously demonstrated that RFC40 is required for accurate chromosomal segregation in Drosophila[31]. Since we observed significant aneuploidy and chromosomal missegregation in the CM nuclei, and since the re-expression of RFC40 in the hypertrophied RVs was less than 10% of that in the fetal hearts, we explored the possibility of whether insufficient re-expression of RFC40 must be responsible for aneuploidy and the chromosomal segregation defects observed in the hypertrophied hearts. To test this possibility, we knocked-down endogenous RFC40 protein in the neonatal (day 2) rat cardiac myocytes (RNCMs) using an On-Target plus smartpool RFC40-SiRNA for 72 hr. Western blot analysis demonstrated approximately 85% down-regulation of the RFC40 protein in the RFC40-siRNA treated samples as compared to untransfected (UT) and non-targeting-siRNA (NT) treated RNCMs (Figure S4 for the fourth supporting information figure). UT and RFC40-siRNA treated RNCMs were subjected to FISH analyses and RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (Green) in each sample (Figure 7A–D-ii and F–G-ii). As expected, we observed only two signals for Cen12-ROX (Red) in the UT RNCM nuclei (Figure 7A-i). Interestingly, we observed one (Figure 7B-i) and three (Figure 7D-i) signals in the RFC40-siRNA treated RNCM nuclei. Although, we observed two signals (Figure 7C-i) for few RNCM nuclei treated with RFC40-siRNA, however, the RFC40 staining for these RNCM nuclei was stronger (Figure 7 C-ii) than that observed for the RFC40-siRNA treated RNCM nuclei with one (Figure 7B-ii) and three (Figure 7 D-ii) signals, respectively, suggesting that perhaps RFC40 was not knocked-down in these RNCMs (thus behaving as an internal control). Statistical analyses revealed significant increase in the number of aneuploid RNCMs (monosomy and trisomy for chromosome 12) following RFC40-siRNA treatment (Figure 7E). Furthermore, we also observed few bi-nucleated RNCMs with unequal chromosomal distribution (Figure 7F), similar to those observed in the hypertrophied RVs, and nuclei that exhibited both nondisjunction for Cen12-ROX (three signals) and micronuclei formation (Figure 7G). Moreover, cell number analyses demonstrated that RFC40-siRNA treated RNCMs significantly (P<0.05) decreased in cell numbers, as compared to Untransfected (UT) RNCMs (Figure 7H). Taken together, these data suggests that RFC40 is required for accurate chromosomal segregation as well as cell survival/proliferation in RNCMs.

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