p53-mediated miR-18 repression activates HSF2 for IGF-IIR-dependent myocyte hypertrophy in hypertension-induced heart failure

Figures

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  Figure 1. HSF2 was required for ANG II-mediated IGF-IIR expression via transcriptional upregulation. (a) NRVMs were simultaneously transfected with the pGL4-IGF-IIR promoter construct (pGF4.1–1.3 kb) and siRNA against HSF2 for 24 h and then treated with 100 nM ANG II for 24 h. The cell lysates were assayed for luciferase activity. Quantification of the results is shown (n=3). *P<0.05 and **P<0.01. NC: negative control. (b) The schematic diagram of deletion mutations of IGF-IIR promoter constructs. NRVMs were transfected with each IGF-IIR promoter construct for 24 h and then treated with 100 nM ANG II for 24 h. Promoter activity was assessed by luciferase activity. Quantification of the results is shown right (n=3). *P<0.05 and ***P<0.001. (c) Sequence analysis and transcription factor site prediction identified one putative HSF2 binding element at the IGF-IIR promoter (nt -493 to -463). After treatment with ANG II, the NRVMs were lysed and analyzed by chromatin immunoprecipitation (ChIP). HSF2 binding to the IGF-IIR promoter was quantified using PCR. (d) The schematic diagram of point mutations on putative HSEs located on the IGF-IIR promoter. Two HSEs located on the IGF-IIR promoter were…

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  Figure 2. HSF2 nuclear translocation and activation were indispensable for ANG II-mediated IGF-IIR hypertrophic signaling. (a) NRVMs were treated with ANG II for 24 h and then the localization of HSF2 was observed by confocal microscopy. A total of 100 cells were counted for the assessment of clear nuclear foci of HSF2. Quantification of the results is shown right (n=3). *P<0.05 and **P<0.01. (b) H9c2 cells were treated with ANG II for 24 h. The cells were fractionated into the cytosolic and nuclear fraction. (c) NRVMs were transfected with a siRNA against HSF2 for 24 h and then treated with ANG II for 24 h. The hypertrophic marker BNP was detected by immunoblotting. Quantification of the results is shown right (n=3). **P<0.01. (d) NRVMs were transfected with Flag-HSF2 for 24 h and then treated with ANG II for 24 h. The hypertrophic markers BNP, ANP and p-cTnI were detected by immunoblotting. Quantification of the results is shown right (n=3). *P<0.05 and **P<0.01. (e) NRVMs were transfected with a HSF2 siRNA or Flag-HSF2 for 24 h and then treated with ANG II for 24 h. The cardiomyocyte size was measured using rhodamine phalloidin staining. A total of 100 cells were counted for statistic…

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  Figure 3. miR-18 downregulation by ANG II resulted in an increase in HSF2 that contributed to IGF-IIR-mediated cardiac hypertrophy. (a) NRVMs were treated with ANG II for 24 h. The expression profiles of the miR17-92 cluster were detected by qRT-PCR. Quantification of the results is shown right (n=3). ***<0.001. (b) Sequence alignment of the miR-18 targeting site in the 3′UTR of human, mouse and rat HSF2. The hsf2-3′UTR (~300 bp) is inserted into the pEGFP-C1 vector downstream of GFP. The hsf2 mu-3′UTR was generated by point mutation in the seed region of the miR-18 targeting sequence. (c) NRVM cells were transfected with pcDNA3-miR17~92 and GFP-hsf2-3′UTR or GFP-hsf2-mu-3′UTR for 24 h. Then, the cells were treated with ANG II for 24 h and the fluorescent intensities were assessed. Quantification of the results is shown right (n=3). *P<0.05, **P<0.01 and ***<0.001. (d) NRVMs were transfected with 3 μg and 5 μg of pcDNA3-miR17-92 for 24 h and then the cell were treated with ANG II for 24 h. The HSF2 expression level was measured by immunoblotting. (e) NRVMs were transfected with pcDNA3-miR17-92 for 24 h and then the cells were treated with ANG II for 24 h. The relative expression levels of…

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  Figure 4. miR-18 is negatively regulated by p53 to manipulate HSF2 expression and thus IGF-IIR-induced cardiac hypertrophy. (a) H9c2 cells and NRVMs were treated with various dosages of ANG II for 24 h. The regulatory factors of the miR17-92 cluster were detected by immunoblotting. (b) NRVMs were treated with 100 nM ANG II and 10 μM of the p53 inhibitor pifithrin-α (PFT) for 24 h. The miRNA expression levels were analyzed by qRT-PCR. Quantification of the results is shown (n=3). *P<0.05, **P<0.01 and ***P<0.001. (c) NRVMs were treated with 100 nM ANG II and PFT for 24 h and then analyzed by immunoblotting. (d) NRVMs were transfected with GFP-p53 for 24 h and then treated with 100 nM ANG II for 24 h. The cell lysates were analyzed by immunoblotting. (e) NRVMs were transfected with GFP-p53 for 24 h and then treated with 100 nM ANG II for 24 h. The relative expression levels of ANP, BNP, HSF2 and IGF-IIR were detected by qRT-PCR. Quantification of the results is shown (n=3). *P<0.05 and **P<0.01. (f) NRVMs were transfected with p53-WT, p53-S240R (only 50% of the p53 transcriptional activity) and p53-R249S (no p53 transcriptional activity) for 24 h and then treated with 100 nM ANG II for 24 h.…

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  Figure 5. miR-18 was negatively correlated with HSF2 expression in a spontaneous hypertension-induced heart failure animal model. (a) The angiotensin II receptor blocker (ARB) irbesartan was consecutively administered to 16-week-old spontaneously hypertensive rats (SHRs) for 6 weeks. The animals were sacrificed and the heart tissues were isolated for further analysis. The miR-18 expression pattern was detected by fluorescence in situ hybridization (FISH). (b) The rat left ventricular heart tissues were homogenized and extracted for analysis. miR-18 expression was verified by qRT-PCR. Quantification of the results is shown (n=4 per group). *P<0.05 and **P<0.01. (c,d) The cardiomyopathy markers ANP and BNP were detected by qRT-PCR. Quantification of the results is shown (n=4 per group). **P<0.01. (e,f) HSF2 and IGF-IIR expression was detected by qRT-PCR. Quantification of the results is shown (n=4 per group). **P<0.01. (g,h) Rat left ventricular heart tissues were homogenized and extracted for analysis. The p53, p-p53, HSF2, IGF-IIR, ANP, BNP and cTnI expression levels were measured by western blotting analysis. Quantification of the results is shown (n=4 per group). **P<0.01 and ***P<0.001.…

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  Figure 6. Restoration of cardiac AAV2-MLC0.26-miR-18 transduction improves cardiac function. (a) The schematic diagram of cardiac-specific AAV2-MLC0.26-pre-miR-18. Gross morphology of heart samples 8 weeks post-injection. Scale bars: 0.5 mm. (b) miR-18 expression in the hearts was detected by qRT-PCR. Quantification of the results is shown (n=3 per group). ***P<0.001. (c) HE and masson’s trichrome staining of heart samples from SHRs injected with AAV2-miR-18 and WKY rats injected with AAV2-miR-18 or the AAV2-vector control. Scale bars: 50 μm. (d) Fibrosis of the heart samples by assessing masson’s trichrome staining. Quantification of the results is shown (n=4 per group). **P<0.01 and ***P<0.001. (e) M-mode echocardiography images and left ventricular FS and EF of SHRs injected with AAV2-miR-18 and WKY rats injected with AAV2-miR-18 or the AAV2-vector control. Quantification of the results is shown (n=5 per group). *P<0.05 and **P<0.01. (f) ANP and BNP mRNA levels in the hearts of SHRs injected with AAV2-miR-18 and WKY rats injected with AAV2-miR-18 or the AAV2-vector control. Quantification of the results is shown (n=3 per group). ***P<0.001. (g) HSF2 and IGF-IIR mRNA levels in the hearts…