| BackgroundCardiac hypertrophy is an adaptive process due to the pressure overload or the volume overload, and is a chronic and progressive disease which involved a variety of factors. Usually, it is associated with other cardiovascular disease, such as hypertension, cardiac valve disease, inherited heart disease, cardiac ischemia and so on. When the heart is under the pressure overload or the volume overload, it will develop to cardiac hypertrophy to decrease the overload, which will meet the demand of the body. In addition, it can be defined as a thickening of left ventricular wall and a reduction of the ventricular chamber. Besides it is characterized by an increase in the size of cardiomyocytes and the alterations of the sarcoma. Moreover, prolonged cardiac hypertrophy will initiate the cardiac remodeling, lead to the apoptosis of cardiomyocytes and cardiac fibrosis, which finally develop to heart failure, even death. However, the effective treatment for the cardiac hypertrophy is still lacking and the mechanism underlying is not clear. Thus, it is very important to seek the effective treatment or drugs and illustrate the underlying mechanisms.Arachidonic acid (AA) is one of the most abundant polyunsaturated fatty acids in the organism. It has several metabolic pathways. One of them is the cytochrome P450 epoxygenase metabolic pathway, which metabolize the AA to epoxyeicosatrienoic acid (EETs). EETs are very important to the human bodies and have a plenty of protective effects on the cardiovascular system. It is reported that EETs could dilate the arteries and decrease the blood pressure. Besides, EETs could protect the vascular from the arteriosclerosis and aortic aneurysm. Moreover, it has been reported that EETs play a vital role in the cardiac hypertrophy. Interesting, publications suggested that EETs not only prevented the development of the cardiac hypertrophy, but also reversed the cardiac hypertrophy. However, the underlying mechanism is remained unknown. Fortunately, we found that peroxisome proliferator activated receptor a (PPARa) might be the receptor for EETs and we hypothesized that EETs might ameliorate the cardiac hypertrophy via PPARa. In order to verify our hypothesis and explore the underlying mechanisms, we used the PPARa null mice as the object and overexpressed CYP 2J2 in the hearts through the recombinant Adeno Associated Virus (rAAV) system. Angiotensin Ⅱ (Ang II) pumped by the subcutaneous mini-pump was used to induce the cardiac hypertrophy in these mice.Methods and results1. First of all, we constructed and purified the rAAV-2J2 and rAAV-GFP virus. And the 293T cells was used to detect the infective efficiency of the virus. Moreover, the virus was injected into the mice via the tail vein and 4 weeks later, the heart, as well as the blood and urine were collected. The expression of CYP 2J2 in protein level and mRNA level were detected by Western blot and real time RT-PCR, while the level of 11,12-EET,11,12-DHET, 14,15-EET and 14,15-DHET in the blood and urine were determined by ELISA. Results turned out that these virus had a very high efficiency of infection. Besides, compared to the control group, the hearts of the group received the rAAV-2J2 virus showed higher expression of CYP 2J2 both in the protein level and the mRNA level. In addition, the level of the EETs and DHETs in the blood and urine which received rAAV-2J2 virus were higher than that of the control group.2. The PPARa null mice and wild type (WT) mice in the same background were used in the present study. All the animals were male and about 8 weeks old. CYP 2J2 was overexpressed by the rAAV system and the Ang II was continuously pumped via the subcutaneous mini-pump (1.5 μgKg-1min-1). Both mice were divided into 4 groups as below:NS+rAAV-GFP, NS+rAAV-2J2, Ang II+rAAV-GFP, Ang II+rAAV-2J2, n=8 for each gorup.2 weeks after the Ang II stimulation, the animals were subjected into the echocardiographic examination and the Millar pressure volume systems to determine the cardiac morphology and the cardiac function. Result showed that, in both mice, Ang II induced the morphological changes (increase in the ventricular wall and reduction in the ventricular chamber) and the function changes (decrease in the Dp/Dt and increase in the LVEDP). As expected, overexpression of CYP 2J2 ameliorated these changes induced by Ang II in WT mice. However, in PPARa null mice, CYP 2J2 overexpressing had no effects on cardiac hypertrophy.3. In addition, the animals were sacrificed and the heart were separated. We recorded the heart weight and calculated the ratio of heart weight to the body weight. Moreover, we prepared the section of the heart, and used the hematoxylin-eosin staining and the WGA staining to observe the cross section area of the cardiomyocytes. Meanwhile, we extracted the mRNA of the heart and real time RT-PCR was employed to detect the expression of the PMHC and BNP. Western blot was used to determine the phosphorylation of the ERK1/2 and the MAPK-P38 and the expression of the IκBα At the same time, the nuclear fraction and the plasma fraction of heart tissue were extracted, and the expression of the NFκB was determined. It was turned out that Ang II induced the cardiac hypertrophy in both WT mice and PPARa null mice by increasing the heart weight, promoting the size of the cardiomyocytes, and inducing the expression of βMHC and BNP. However, overexpression of CYP 2J2 inhibited the effects induced by Ang II in WT mice but showed no effects in PPARa null mice. Moreover, results from the western blot showed that compared to that of the NS+rAAV-GFP group, the phosphorylation level of the ERK1/2 and MAPK-P38 and the expression of IκBα was significantly higher in Ang II+rAAV-GFP group from both mice. Even more, Ang II induced the NFκB-P65 moved from plasma to nucleus. Similarly, overexpression of CYP 2J2 inhibited the activation of MAPK and IκBα pathway, and the re-localization of NFκB in wild type mice, while there was no difference between Ang Ⅱ+ rAAV-GFP and Ang II+rAAV-2J2 in PPARa null mice.4. In vitro, we used the H9c2 cell line and the primary cultured cardiomyocytes from the neonatal rats as the object, EETs and PPARa antagonist GW6471 was added as the intervention while Ang II was used as the stimulus. After treatment, F-actin staining was employed to evaluate the size of the cells and real time RT-PCR was used to determine the expression level in mRNA of βMHC and BNP. Meanwhile, western blot was carried out to detect the expression of the cav-1, IκBa, and the activation of NFκB and MAPK (ERK1/2 and MAPK-P38). Immunoprecipitation using the Ras-GTP antibody was used to assess the activation of Ras. Results showed that compared to the control group, the Ang Ⅱ group showed larger cardiomyocytes and higher expression of PMHC and BNP. Besides, Ang II was responsible for the activation of the Ras/MAPK and NFκB pathway. And treatment with 11,12-EET showed a significant inhibition on these effects induced by Ang II, which could be blocked by further treatment with 14,15-EEZE or GW6471. Intriguingly, 11,12-EET could induce the expression of cav-1 while GW6471 could inhibit the effects of 11,12-EET. It meant that 11,12-EET might induce the expression of cav-1 via PPARa.5. In order to investigate the relationship of PPARa and cav-1, we did the on the website and we found that there may be a transcription binding site of PPARa in cav-1 promoter. Following, we constructed several PGL-3 plasmid which carried the cav-1 promoters in different length, including-8 to-262,-8 to-492 and-8 to-1278. These clones were co-transfected with the plasmid expressing human PPARa or eGFP to 293 cells and the reporter gene activity was measured. To further ascertain that PPARa was recruited to the cav-1 promoter, we performed ChIP in 293 cells after gene transfer of PPARa. Results turned out that PPARa overexpression significantly enhanced the reporter gene activity of the-8 to-492 and-8 to-1278 constructs. And the results from the ChIP showed that the region between-199 to-455 of cav-1 promoter was enriched in the PPARa overexpressing group. Taken together, it was suggested that PPARa was recruited to this region of cav-1 promoter and initiated the expression of cav-1.6. Finally, we used the siRNA of cav-1 to silence the expression of cav-1 in the neonatal rat ventricular myocytes and employed the F-actin staining, real time RT-PCR, as well as Western blot to investigate that whether cav-1 was necessary for EETs in inhibition of cardiac hypertrophy. It is turned out that Ang Ⅱ enlarged the size of the cardiomyocytes, induced the expression of PMHC and BNP and activated the MAPK and NFκB pathway, while 11,12-EET showed a significant inhibition on the effects of Ang II. And it was interested to observed that silence of cav-1 blocked the 11,12-EET and promoted the effects induced by Ang II, suggesting that cav-1 played a vital role in the inhibition of cardiac hypertrophy by 11,12-EET.Conclusions1. In in vivo study, treatment with Ang II induced the cardiac hypertrophy, including the change of morphology (thickness of the ventricular wall and enlargement of the heart), the change of hemodynamics (decrease of Dp/Dt and increase of LVEDP), the change of pathology (the cardiomyocyte enlargement) as well as the induction of the fetal gene expression (pMHC and BNP), while overexpression of CYP 2J2 significantly inhibited these effects induced by Ang Ⅱ in WT mice, which might mediated by PPARa.2. In in vivo study, treatment with Ang Ⅱ induced hypertrophy of the cardiomyocytes and increased the expression of pMHC and BNP, while 11,12-EET inhibited the hypertrophy of the cells, which was blocked by the treatment with GW6471, suggesting that PPARa was necessary in the inhibition of cardiac hypertrophy by EETs.3. Also, the underlying mechanism was investigated and concluded as below: overexpression of CYP 2J2 and its product EETs acted as ligands to PPARa and activated the receptor, which further up-regulated the expression of cav-1. Following, the up-regulation of cav-1 inhibited the Ras/MAPK (ERK1/2 and MAPK-P38) and the NFκB pathway, which finally inhibited the cardiac hypertrophy. |
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