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.
References
[1]
Porter KE, Turner NA (2009) Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther. 123: 255–278.
[2]
Koudssi F, López JE, Villegas S, Long CS (1998) Cardiac fibroblasts arrest at the G1/S restriction point in response to interleukin (IL)-1beta. Evidence for IL-1beta-induced hypophosphorylation of the retinoblastoma protein. J Biol Chem. 273: 25796–803.
[3]
Soonpaa MH, Field LJ (1997) Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol. 272: H220–6.
[4]
Laflamme MA, Murry CE (2005) Regenerating the heart. Nat Biotechnol. 23: 845–856.
[5]
Ahuja P, Sdek P, MacLellan WR (2007) Cardiac myocyte cell cycle control in development, disease, and regeneration. Physiol Rev. 87: 521–544.
[6]
Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, et al. (2001) Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 344: 1750–1757.
[7]
Engel FB (2005) Cardiomyocyte proliferation: a platform for mammalian cardiac repair. Cell Cycle. 4: 1360–1363.
[8]
Bersell K, Arab S, Haring B, Kühn B (2009) Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury. Cell 138: 257–70.
[9]
Granata R, Trovato L, Gallo MP, Destefanis S, Settanni F, et al. (2009) Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia-reperfusion injury in rat heart. Cardiovasc Res. 83: 303–12.
[10]
Gupte RS, Weng Y, Liu L, Lee MY (2005) The second subunit of the replication factor C complex (RFC40) and the regulatory subunit (RIalpha) of protein kinase A form a protein complex promoting cell survival. Cell Cycle. 4: 323–329.
[11]
Majka J, Burgers PM (2004) The PCNA-RFC families of DNA clamps and clamp loaders. Prog Nucleic Acid Res Mol Biol. 78: 227–260.
[12]
Marino TA, Cao W, Lee J, Courtney R (1996) Localization of proliferating cell nuclear antigen in the developing and mature rat heart cell. Anat Rec. 245: 677–684.
[13]
Zhang SJ, Lee MY (1987) Biochemical characterization and development of DNA polymerases alpha and delta in the neonatal rat heart. Arch Biochem Biophys. 252: 24–31.
[14]
Chim SS, Fung K, Waye MM, Lee C, Tsui SK (2000) Expression of replication factor C 40-kDa subunit is down-regulated during neonatal development in rat ventricular myocardium. J Cell Biochem. 78: 533–540.
[15]
Bogaard HJ, Abe K, Vonk Noordegraaf A, Voelkel NF (2009) The right ventricle under pressure: cellular and molecular mechanisms of right-heart failure in pulmonary hypertension. Chest. 135: 794–804.
[16]
Bogaard HJ, Natarajan R, Henderson SC, Long CS, Kraskauskas D, et al. (2009) Chronic pulmonary artery pressure elevation is insufficient to explain right heart failure. Circulation. 120: 1951–1960.
[17]
Quaini F, Cigola E, Lagrasta C, Saccani G, Quaini E, et al. (1994) End-stage cardiac failure in humans is coupled with the induction of proliferating cell nuclear antigen and nuclear mitotic division in ventricular myocytes. Circ Res. 75: 1050–1063.
[18]
Leeuwenburgh BP, Helbing WA, Wenink AC, Steendijk P, de Jong R, et al. (2008) Chronic right ventricular pressure overload results in a hyperplastic rather than a hypertrophic myocardial response. J Anat. 212: 286–294.
[19]
Meckert PC, Rivello HG, Vigliano C, González P, Favaloro R, et al. (2005) Endomitosis and polyploidization of myocardial cells in the periphery of human acute myocardial infarction. Cardiovasc Res. 67: 116–23.
[20]
Setoguchi M, Leri A, Wang S, Liu Y, De Luca A, et al. (1999) Activation of cyclins and cyclin-dependent kinases, DNA synthesis, and myocyte mitotic division in pacing-induced heart failure in dogs. Lab Invest. 79: 1545–1558.
[21]
Bogaard HJ, Natarajan R, Mizuno S, Abbate A, Chang PJ, et al. (2010) Adrenergic receptor blockade reverses right heart remodeling and dysfunction in pulmonary hypertensive rats. Am J Respir Crit Care Med(182): 652–660.
[22]
Abe K, Toba M, Alzoubi A, Ito M, Fagan KA, et al. (2010) Formation of plexiform lesions in experimental severe pulmonary arterial hypertension. Circulation(121): 2747–2754.
[23]
Tsujikawa H, Song Y, Watanabe M, Masumiya H, Gupte SA, et al. (2008) Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its -adrenergic enhancement in ventricular myocytes. Am J Physiol Heart Circ Physiol. 294: H285–292.
[24]
MacDougall CA, Byun TS, Van C, Yee MC, Cimprich KA (2007) The structural determinants of checkpoint activation. Genes Dev. 21: 898–903.
[25]
Tasara T, Angerer B, Damond M, Winter H, Dorhofer S, et al. (2003) Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA. Nucleic Acids Res. 31: 2636–2646.
[26]
Tsurimoto T, Stillman B (1989) Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 8: 3883–3889.
[27]
Kozlovskis PL, Smets MJ, Strauss WL, Myerburg RJ (1996) DNA synthesis in adult feline ventricular myocytes. Comparison of hypoxic and normoxic states. Circ Res. 78: 289–301.
[28]
Jong CJ, Azuma J, Schaffer S (2012) Mechanism underlying the antioxidant activity of taurine: prevention of mitochondrial oxidant production. Amino Acids. 42: 2223–32.
[29]
Matouskova E, Kudlackova I, Chaloupkova A, Brozova M, Netikova I, et al. (2005) Origin of cells cultured in vitro from human breast carcinomas traced by cyclin D1 and HER2/neu FISH signal numbers. Anticancer Res. 25: 1051–7.
Krause SA, Loupart ML, Vass S, Schoenfelder S, Harrison S, et al. (2001) Loss of cell cycle checkpoint control in Drosophila Rfc4 mutants. Mol Cell Biol. 21: 5156–68.
[32]
Walsh S, Ponten A, Fleischmann BK, Jovinge S (2010) Cardiomyocyte cell cycle control and growth estimation in vivo–an analysis based on cardiomyocyte nuclei. Cardiovasc Res. 86: 365–373.
[33]
Chen X, Wilson RM, Kubo H, Berretta RM, Harris DM, et al. (2007) Adolescent feline heart contains a population of small, proliferative ventricular myocytes with immature physiological properties. Circ Res. 100: 536–544.
[34]
Ahuja P, Perriard E, Pedrazzini T, Satoh S, Perriard JC, et al. (2007) Re-expression of proteins involved in cytokinesis during cardiac hypertrophy. Exp Cell Res. 313: 1270–83.
[35]
Sarkar S, Chawla-Sarkar M, Young D, Nishiyama K, Rayborn ME, et al. (2004) Myocardial cell death and regeneration during progression of cardiac hypertrophy to heart failure. J Biol Chem. 279: 52630–42.
[36]
Kajstura J, Leri A, Finato N, Di Loreto C, Beltrami CA, et al. (1998) Myocyte proliferation in end-stage cardiac failure in humans. Proc Natl Acad Sci U S A. 95: 8801–5.
[37]
Vliegen HW, Eulderink F, Bruschke AV, van der Laarse A, Cornelisse CJ (1995) Polyploidy of myocyte nuclei in pressure overloaded human hearts: a flow cytometric study in left and right ventricular myocardium. Am J Cardiovasc Pathol. 5: 27–31.
[38]
Kellerman S, Moore JA, Zierhut W, Zimmer HG, Campbell J, et al. (1992) Nuclear DNA content and nucleation patterns in rat cardiac myocytes from different models of cardiac hypertrophy. J Mol Cell Cardiol. 24: 497–505.
[39]
Gerdes AM, Liu Z, Zimmer HG (1994) Changes in nuclear size of cardiac myocytes during the development and progression of hypertrophy in rats. Cardioscience. 5: 203–8.
[40]
Lee HO, Davidson JM, Duronio RJ (2009) Endoreplication: polyploidy with purpose. Genes Dev. 23: 2461–77.
[41]
Wohlschlaeger J, Levkau B, Brockhoff G, Schmitz KJ, von Winterfeld M, et al. (2010) Hemodynamic support by left ventricular assist devices reduces cardiomyocyte DNA content in the failing human heart. Circulation. 121: 989–96.
[42]
King RW (2008) When 2+2 = 5: the origins and fates of aneuploid and tetraploid cells. Biochim Biophys Acta. 1786: 4–14.
[43]
Yang AH, Kaushal D, Rehen SK, Kriedt K, Kingsbury MA, et al. (2003) Chromosome segregation defects contribute to aneuploidy in normal neural progenitor cells. J Neurosci. 23: 10454–62.
[44]
Geller LN, Potter H (1999) Chromosome missegregation and trisomy 21 mosaicism in Alzheimer’s disease. Neurobiol Dis. 6: 167–79.
[45]
Mosch B, Morawski M, Mittag A, Lenz D, Tarnok A, et al. (2007) Aneuploidy and DNA replication in the normal human brain and Alzheimer’s disease. J Neurosci. 27: 6859–67.
[46]
Yang Y, Geldmacher DS, Herrup K (2001) DNA replication precedes neuronal cell death in Alzheimer’s disease. J Neurosci. 21: 2661–8.
[47]
Shi Q, King RW (2005) Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines. Nature. 437: 1038–42.
[48]
Kanellis P, Agyei R, Durocher D (2003) Elg1 forms an alternative PCNA-interacting RFC complex required to maintain genome stability. Curr Biol. 13: 1583–95.
[49]
Petronczki M, Chwalla B, Siomos MF, Yokobayashi S, Helmhart W, et al. (2004) Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-alpha-associated protein Ctf4 is essential for chromatid disjunction during meiosis II. J Cell Sci. 17: 3547–3559.
[50]
Mayer ML, Gygi SP, Aebersold R, Hieter P (2001) Identification of RFC (Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae. Mol Cell. 7: 959–70.
[51]
Minegishi S, Kitahori K, Murakami A, Ono M (2011) Mechanism of pressure-overload right ventricular hypertrophy in infant rabbits. Int Heart J. 52: 56–60.
[52]
Anversa P, Olivetti G, Leri A, Liu Y, Kajstura J (1997) Myocyte cell death and ventricular remodeling. Curr Opin Nephrol Hypertens. 6: 169–76.
[53]
Arendt T, Brückner MK, Mosch B, L？sche A (2010) Selective cell death of hyperploid neurons in Alzheimer’s disease. Am J Pathol. 177: 15–20.
[54]
Hernández-Ortega K, Quiroz-Baez R, Arias C (2011) Cell cycle reactivation in mature neurons: a link with brain plasticity, neuronal injury and neurodegenerative diseases? Neurosci Bull. 27: 185–96.
