Critical Role of Cyclin D1 Nuclear Import in Cardiomyocyte Proliferation

Abstract

Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.

Differential regulation of cyclin D1 and D2 in protecting against cardiomyocyte proliferation

“…Immunostaining of these cells showed that cyclin D1 was not expressed in the nuclei as previously reported. 12,13 By contrast, cyclin D2 expression was clearly localized in the nuclei of cells positive for a cardiac marker, suggesting that cyclin D2 is inducible in the nuclei upon serum treatment. We next examined the relative expression of cyclin D1 and D2.…”

“…13 Cancellation of this inhibitory mechanism, the nuclear expression of cyclin D1 (D1NLS)/CDK4, is sufficient to induce cell cycle progression leading to cell division of neonatal cardiomyocytes. In the current study, we showed that cyclin D2 is expressed in the nuclei of cardiomyocytes, consistent with a previous report.…”

“…Rat neonatal cardiomyocytes were isolated from three-day-old postnatal Sprague-Dawley rats, purified by Percoll gradient centrifugation, and cultured as previously described. 12,13,19 The cardiomyocytes were incubated in MEM supplemented with 5% calf serum for 24 hr at 37°C, after which the medium was replaced with serum-free medium. Following a 48-hr incubation, the cells were subjected to various analyses.…”

“…The DNA content of cells positive for tropomyosin was analyzed using a laser scanning cytometer (LSC 101, Olympus) as previously described. 13,14,19 Use of rats in this study was approved by the Institutional Animal Care and Use Committee of our university.…”

“…[12][13][14] Further recent data indicated that overexpression of cyclin D2 can enhance cardiac regeneration in transgenic mice. 15,16 In this study, to investigate whether cyclin D2 induces cardiomyocyte proliferation as well as D1 under a similar level of expression in the nuclei, we generated an adenovirus vector expressing NLS-tagged mouse cyclin D2 and compared their effects on the induction of cardiomyocyte proliferation.…”