Overexpression Of PTEN(Phosphatase And Tensin Homologue Deleted On Chromosome 10) Negatively Regulates Cardiac Hypertrophy Induced By Angiotensin Ⅱ

Background:Myocardial hypertrophy is crucial for the transition by which cardiac function from adaptive to maladaptive and progression by which cardiac remodeling from reversible to irreversible. Diverse intracellular signal pathways, such as Ca2+/CaN/ NFAT3 and PI3K/Akt signal pathway, have been shown being implicated in myocardial hypertrophy. Meanwhile, it was proved that there were several negative regulators involved in the progression of cardiac hypertrophy, which interacting with the pro-hypertrophic signals to balance the organization, function and gene expression profiles of cardiomyocytes. PTEN(Phosphatase and tensin homologue deleted on chromosome 10)may participate in the negative regulation of myocardial hypertrophy. It was shown that Isoproterenol up-regulated PTEN expression in the myocardium and overexpression of PTEN in myocardiocytes resulted in dramatic suppression of hypertrophy induced by IGF-1 and significantly enhanced apoptosiss of myocardiocytes. PTEN may be involved in different physiopathologic course via its interacting with distinct isoforms of PI3K, PTEN/PI3Kαsignaling may suppress adaptive hypertrophy, while PTEN/PI3Kγpositively improve cardiac contractibility and was expected to negatively regulate maladaptive hypertrophy. The findings mentioned above suggested that while pro-hypertrophic stimula activated positive signals to induce hypertrophy, negative regulators such as PTEN were activated as well. This study was aimed to further elucidate the role of PTEN in maladaptive myocardial hypertrophy via observing the effects of PTEN overexpression on primary cultured cardiomyocytes hypertrophy induced by Ang Ⅱ, and the mechanism by which PTEN acted as an endogenously negative regulator of hypertrophy. Methods: 1. Directed cloning and homologous recombination were employed to generate adenovirus containing wild-type PTEN (Ad-PTEN) or G129E which is point-mutated to loss lipid phosphatase activity but retaining protein phosphatase activity(Ad-G129E). 2. Primary cultured neonatal rat cardiomyocytes were infected with the adenovirus containing PTEN or G129E. Following up expression of GFP, we detected the infective efficiency of virus response to different M.O.I., and the positive rate of GFP expression at 24h, 48h and 72h after adenovirus infection, then combining with viability of cardiomyocytes evaluated by MTT, we ascertained the suitable infection programme and M.O.I.. 3. RT-PCR and Western Blot were used to detect the level of PTEN mRNA and protein in the infected cell to ensure PTEN or G129E overexpression as a result of adenovirus infection. 4. Using Ang Ⅱas pro-hypertrophic stimuli, we observed the effects of PTEN or G129E overexpression on the hypertrophic indices of cardiomyocytes, such as ANF, β-MHC and diameter of cells, taking empty adenovirus containing GFP (Ad-GFP) as control. 5. Furo-2/AM ratio imaging analysis was applied to detect intracellular Ca2 + concentration;and the level of CaNAβmRNA , and CaNAβ, ERK1/2 and pERK1/2 protein were evaluated with RT-PCR and Western Blot, respectively. Furthermore, CaN phosphatase activity was determined as well. Results: 1. Via directed cloning and homologous recombination, infection-competent adenovirus was generated in the AD293 package cell line; following repeatedly infecting, high titer adenovirus particles yielded. 2. After Ad-PTEN,Ad-G129E or Ad-GFP infection, the positive rate of GFP expression in the host cardiomyocytes was growing up in M.O.I.-dependent and time-dependent manner; When M.O.I. increased up to 100 or above, the percent of cardiomyocytes expressing GFP exceeded 90%; For each strata of M.O.I., there was no more increase in the positive rate of GFP expression as the expression time exceeded 48h, suggesting that this timepoint seemed be suitable for consequent procedures. 3. MTT showed that at 48h after the cardiomyocytes were infected by Ad-GFP Ad-PTEN or Ad-G129E, along with the increase of M.O.I., viability of myocytes decreased, especially when M.O.I. exceeded 100(Ad-GFP:β=-0.217,p>0.05;Ad-PTEN:β=-0.470,p<0.05;Ad-G129E:β=-0.372,p<0.05). So we took M.O.I. 100 and 48h as appropriate standard to continue the subsequent study. 4. Findings with RT-PCR and Western Blot showed that infection of adenovirusresulted in PTEN or G129E overexpression in the cardiomyocytes. 5. Ang Ⅱsitimulation resulted in cardiomyocytes enlargement, and enhangcement in ANF ,β-MHC expression; Overexpression of PTEN suppressed cardiomyocytes hypertrophy and expression of embryonic gene, which was significantly blunted in G129E overexpressing cells. 6. Ang Ⅱsitimulation was followed by increase in[Ca2+]i,Overexpression of PTEN significantly inhibited the increase in[Ca2+]i, [Ca2+]i in cardiomyocytes overexpressing G129E was significantly higher than that of cardiomyocytes overexpressing PTEN。7. Ang Ⅱsitimulation was followed by increase in expression of CaNAβmRNA and protein, Overexpression of PTEN significantly inhibited the increase in expression of CaNAβmRNA and protein, those of cardiomyocytes overexpressing G129E were significantly higher than PTEN overexpressing cells。8. Ang Ⅱsitimulation was followed by increase in CaN Phosphatase activity. Overexpression of PTEN significantly suppressed the increase of CaN Phosphatase activity, that of cardiomyocytes overexpressing G129E was significantly higher than PTEN overexpressing cells。9. Ang Ⅱsitimulation was followed by increase in expression of ERK1/2 and pERK1/2 protein. Overexpression of PTEN significantly suppressed the increase of expression of ERK1/2 and pERK1/2 protein, those of cardiomyocytes overexpressing G129E were significantly higher than PTEN overexpressing cells。Conclusions: Overexpression of PTEN in the cardiomyocytes resulted in dramatically repression of hypertrophic response to Ang Ⅱ, while overexpression of G129E lacked of sufficient repressive ability as much as overexpression of PTEN, which suggested that lipid phosphatase activity was involved in the negative regulative procedure of PTEN for cardiac hypertrophy induced by Ang Ⅱ; Furthermore, Ca2+/CaN/NFAT3 and MEK1/ERK1/2 activated by AngⅡwere suppressed by PTEN as well, these signal pathway may be participated in the negative regulation mediated by PTEN. In addition, to some degree, overexpression of G129E reduced cellular hypertrophy induced by Ang Ⅱ, which implied that the negative regulation of myocardial hypertrophy by PTEN might be associated with the protein phosphatase activity of PTEN as well.