Abstract
Adult mammalian cardiomyocytes may reenter the cell cycle and cause cardiac hypertrophy. Triptolide (TP) can regulate the expressions of various cell cycle regulators in cancer cells. However, its effects on cell cycle regulators during myocardial hypertrophy and mechanism are unclear. This study was designed to explore the profile of cell cycle of cardiomyocytes and the temporal expression of their regulators during cardiac hypertrophy, as well as the effects of TP. The hypertrophy models employed were neonatal rat ventricular myocytes (NRVMs) stimulated with angiotensin II (Ang II) for scheduled times (from 5 min to 48 h) in vitro and mice treated with isoprenaline (Iso) for from 1 to 21 days, respectively. TP was used in vitro at 1 μg/L and in vivo at 10 μg/kg. NRVMs were analyzed using flow cytometry to detect the cell cycle, and the expression levels of mRNA and protein of various cell cycle regulators were determined using real-time PCR and Western blot. It was found NRVM numbers in phases S and G2 increased, while that in the G1 phase decreased significantly after Ang II stimulation. The mRNA expression levels of p21 and p27 increased soon after stimulation, and thereafter, mRNA expression levels of all cell cycle factors showed a decreasing trend and reached their lowest levels in 1–3 h, except for cyclin-dependent kinase 1 (CDK1) and CDK4 mRNA. The mRNA expression levels of CDK1, p21, and p27 increased markedly after stimulation with Ang II for 24–48 h. In myocardium tissue, CDK and cyclin expression levels peaked in 3–7 days, followed by a decreasing trend, while those of p21 and p27 mRNA remained at a high level on day 21. Expression levels of all protein were consistent with the results of mRNA in NRVMs or mice. The influence of Ang II or Iso on protein expression was more obvious than that on mRNA. TP treatment effectively prevented the imbalance in the expression of cell cycle regulators in the hypertrophy model group. In Conclusion, an imbalance in the expression of cell cycle regulators occurs during cardiac hypertrophy, and triptolide corrects these abnormal expression levels and attenuates cardiac hypertrophy.
Highlights
Cardiac hypertrophy is considered to be a compensatory adaptation in response to intrinsic and extrinsic stimuli and is characterized by the reactivation of fetal genes such as b-myosin heavy chain and brain natriuretic peptide (BNP), enhanced protein synthesis, increased sarcomere organization, and an increase in heart mass with the enlargement of cardiomyocytes (Tan et al, 2011; Pillai et al, 2015; Martens et al, 2020)
After Ang angiotensin II (II) stimulation for 24 h, we observed that 69.63% of neonatal rat ventricular myocyte (NRVM) were found in the G1 phase, and 28.33% were found in the S+G2 phase
These results indicate that the NRVM cell cycle can be reinitiated by disease-related hypertrophic stimuli, which is represented by the increased cardiomyocyte number in the S+G2 phase while the number of cells in the G1 phase decreased significantly compared with that in the control group (Figures 1A, B)
Summary
Cardiac hypertrophy is considered to be a compensatory adaptation in response to intrinsic and extrinsic stimuli and is characterized by the reactivation of fetal genes such as b-myosin heavy chain (bMHC) and brain natriuretic peptide (BNP), enhanced protein synthesis, increased sarcomere organization, and an increase in heart mass with the enlargement of cardiomyocytes (Tan et al, 2011; Pillai et al, 2015; Martens et al, 2020). Considering that all the cell cycle regulators vary dynamically and play roles synergistically with the progression of cardiac hypertrophy, it is necessary to elucidate the expression pattern of the cell cycle regulator family and explore the possible mechanisms of the hypertrophic response
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