Author + information
Cardiomyocytes proliferate rapidly during fetal life but exit the cell cycle soon after birth in mammals and become terminally differentiated. Recent studies indicated that postnatal cardiomyocytes are still capable of cell cycle reentry, which results in cardiac hypertrophy, Triptolide (TP) can regulate the expressions of various cell cycle regulators in tumor cells. However, the effects of TP on cell cycle regulators in myocardial hypertrophy and its mechanism are at present poorly understood.
Neonatal rat ventricular myocytes (NRVM) were isolated and coincubated with 1.0 μmol/L angiotensin II (Ang II) and 1.0 μg/L TP for scheduled times (0, 5, 10, 30 min, or 1, 2, 3, 6, 12, 24, 48 h). Mice were randomly divided into isoprenaline (ISO) and TP treated group, and administrated with ISO (10mg/kg, sc. bid) or ISO plus TP (10 μg/kg, ip. q.d.) for 1, 3, 7, 14, 21 days, respectively. NRVM myocytes were analyzed with flow cytometry to detect the cell cycle, and myocytes area was measured by immunofluorescence assay. The pathological morphology changes were observed after HE, lectin and Masson’s trichrome staining. The mRNA expression levels of β-myosin heavy chain (β-MHC) and cyclin A, B, D, E, CDK 1, 2, 4, 6, and p21, p27 were detected by real-time PCR, and the protein expression levels of β-MHC and cyclin D, CDK 4, 6, p21 were detected by WB.
The cell size and β-MHC expression level increased dramatically after stimulation with Ang II. Cell cycle analysis indicated that the myocytes number in phase of S+G2 increased and that in G1 phase decreased significantly after Ang II stimulation. The mRNA expressions of cyclin A, p21 and p27 increased soon after stimulation for 5 min, after mRNA expressions of all cell cycle factors showed a decrease trend, and reached lowest at 1-3h. Only the expression of CDK1, p21, p27 mRNA increased significantly after stimulation of Ang II for 24-48h in NRVM. After treated with ISO for 3 days, heart indexes and myocardial cross sectional area increased significantly increased significantly. Morphological analysis showed myocardium injury, inflammatory cell infiltration and collagen deposition. The CDKs and cyclins expression levels all reached the peak at day 3-7, followed by a decrease trend, while p21 and p27 mRNA were expressed at a high level at day 21. All protein expression levels were consistent with the results of mRNA in NRVM or mice. Interestingly, it has been shown that the effects of Ang II or ISO on the protein expression was more obvious compared to those of mRNA. TP treatment not only significantly decreased cell size and heart index, improve tissue injury and attenuate myocardial fibrosis but also effectively prevented the imbalance expressions of cell cycle regulators compared hypertrophy model group.
Cell cycle regulators express imbalancedly during the process of cardiac hypertrophy. Triptolide can effectively attenuate cardiac hypertrophy and ameliorate myocardial damage, and the effects of correcting abnormal expressions of various cell cycle regulators is involved in the underlying mechanisms.