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. 2012;7(12):e52522.
doi: 10.1371/journal.pone.0052522. Epub 2012 Dec 31.

Cardiomyocyte-specific overexpression of HEXIM1 prevents right ventricular hypertrophy in hypoxia-induced pulmonary hypertension in mice

Noritada Yoshikawa  1 , Noriaki Shimizu, Takako Maruyama, Motoaki Sano, Tomohiro Matsuhashi, Keiichi Fukuda, Masaharu Kataoka, Toru Satoh, Hidenori Ojima, Takashi Sawai, Chikao Morimoto, Akiko Kuribara, Osamu Hosono, Hirotoshi Tanaka
Affiliations

Cardiomyocyte-specific overexpression of HEXIM1 prevents right ventricular hypertrophy in hypoxia-induced pulmonary hypertension in mice

Noritada Yoshikawa et al. PLoS One. 2012.

Abstract

Right ventricular hypertrophy (RVH) and right ventricular (RV) contractile dysfunction are major determinants of prognosis in pulmonary arterial hypertension (PAH) and PAH remains a severe disease. Recently, direct interruption of left ventricular hypertrophy has been suggested to decrease the risk of left-sided heart failure. Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is a negative regulator of positive transcription elongation factor b (P-TEFb), which activates RNA polymerase II (RNAPII)-dependent transcription and whose activation is strongly associated with left ventricular hypertrophy. We hypothesized that during the progression of PAH, increased P-TEFb activity might also play a role in RVH, and that HEXIM1 might have a preventive role against such process. We revealed that, in the mouse heart, HEXIM1 is highly expressed in the early postnatal period and its expression is gradually decreased, and that prostaglandin I(2), a therapeutic drug for PAH, increases HEXIM1 levels in cardiomyocytes. These results suggest that HEXIM1 might possess negative effect on cardiomyocyte growth and take part in cardiomyocyte regulation in RV. Using adenovirus-mediated gene delivery to cultured rat cardiomyocytes, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced phosphorylation of RNAPII, cardiomyocyte hypertrophy, and mRNA expression of hypertrophic genes, whereas a HEXIM1 mutant lacking central basic region, which diminishes P-TEFb-suppressing activity, could not. Moreover, we created cardiomyocyte-specific HEXIM1 transgenic mice and revealed that HEXIM1 ameliorates RVH and prevents RV dilatation in hypoxia-induced PAH model. Taken together, these findings indicate that cardiomyocyte-specific overexpression of HEXIM1 inhibits progression to RVH under chronic hypoxia, most possibly via inhibition of P-TEFb-mediated enlargement of cardiomyocytes. We conclude that P-TEFb/HEXIM1-dependent transcriptional regulation may play a pathophysiological role in RVH and be a novel therapeutic target for mitigating RVH in PAH.

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Conflict of interest statement

Competing Interests: With respect to funding from Actelion Pharmaceuticals, the authors do not have any other relevant declarations such as employment, consultancy, patents, products in development or marketed products, and their funding does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Protein expression of HEXIM1 in the heart.
(A) HEXIM1 protein expression in human tissues and rodent hearts. Twenty micrograms of extracts from different human tissues were subjected to Western blotting (top left). Immunohistochemistry using anti-human HEXIM1 antibodies showed HEXIM1 expression in the nucleus of human cardiomyocytes (top right). The lysates from wild-type (WT) mouse hearts at each time point and neonatal rat cardiomyocytes (NRCM) were subjected to Western blotting for evaluation of postnatal changes of HEXIM1 protein expression in mouse hearts (bottom). (B) Effect of the drugs for treatment of PAH on HEXIM1 protein expression. NRCM were treated with vehicle (water), 1 µg/ml PGI2, BQ123, sildenafil, or 5 mmol/L hexamethylene bisacetamide (HMBA) for 24 hr, and were analyzed by Western blotting. (C) Effect of PGI2 on HEXIM1 protein expression. NRCM were treated with vehicle (water), indicated concentration of PGI2, or 5 mmol/L HMBA for 24 hr, and were analyzed by Western blotting. (D) Decreased expression of HEXIM1 in the heart of PGI synthetase (PGIS) knockout mice. Thirty micrograms of the tissue extracts obtained from the hearts of 24-week-old male WT or PGIS knockout mice (PGIS−/−) were subjected to Western blotting. Representative Western blotting of HEXIM1 and actin expression from 5 independent experiments are shown in panels A–D. In panels B–D, the band densities of HEXIM1 detected by Western blotting were quantified and normalized to those of actin. Relative band densities compared to the values obtained from vehicle-treated cells or WT mice are presented (means ± SD, n = 5). *P<0.05. (E) Effect of PGI2 on endothelin-1 (ET-1)-induced cardiac myocyte hypertrophy. NRCM were infected with control adenovirus Adsictrl or recombinant adenovirus AdsiHEXIM1, which expresses siRNA against HEXIM1, were treated with or without 100 nmol/L ET-1 in the presence or absence of 1 µg/ml PGI2, and were further cultured for 72 hr. The indirect immunofluorescence for alpha-actinin was performed, the cell area was quantified, and relative cell areas compared to the values obtained from vehicle-treated and Adsictrl-infected cells are presented (means ± SD, n = 400). *P<0.05.
Figure 2
Figure 2. Overexpression of HEXIM1 prevents ET-1-induced phosphorylation of RNA polymerase II and cellular hypertrophy in NRCM.
NRCM were infected with irrelevant AxCALNLZ (C) or recombinant adenoviruses, which express FLAG-tagged human HEXIM1 (HEX) or its mutant lacking P-TEFb-binding activity (mt) in the co-presence of Cre recombinase, at MOI of 100 along with Cre recombinase-expressing recombinant adenovirus and further cultured for 24 hr. (A) Effect of HEXIM1 on the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) after treatment of NRCM with ET-1. The cells were treated with or without 100 nmol/L ET-1 for 15 min. Expression and phosphorylation levels of RNAPII were analyzed by Western blotting. Left, representative images of Western blotting of hyper- (II0) and hypo- (IIa) phosphorylated RNAPII, phosphoserine 2 (P-Ser2), phosphoserine 5 (P-Ser5), cyclin T1 (CycT1), Cdk9, exogenous and endogenous HEXIM1 (FLAG and rat HEX, respectively), and actin expression from 5 independent experiments are shown. Right, band densities of II0, and P-Ser2 and P-Ser5 detected by Western blotting were quantified and normalized to those of IIa and actin, respectively, and relative band densities compared to the values obtained from control cells (AxCALNLZ-infected and vehicle-treated cells) are presented in the right panel (means ± SD, n  = 5). *P<0.05. (B) Effect of HEXIM1 on hypertrophic cell growth in response to ET-1 in NRCM. The cells were treated with or without 100 nmol/L ET-1 and further cultured for 72 hr. Left, indirect immunofluorescence was performed. Alpha-actinin and HEXIM1 are shown in red and green, respectively. Representative fluorescent microscopic images from 5 independent experiments are shown. Right, recombinant adenoviruses were infected at indicated amount. The indirect immunofluorescence for alpha-actinin was performed, the cell area was quantified, and relative cell areas compared to the values obtained from vehicle-treated cells are presented (means ± SD, n  = 400). *P<0.05. (C) Effect of HEXIM1 on the phosphorylation status of ERK1/2 and MAP kinases, and mTOR activity in response to ET-1 in NRCM. Left, the cells were treated with or without 100 nmol/L ET-1 and further cultured for 1 hr. Right, the medium was replaced to amino acid-deprived DMEM, and the cells were treated with or without 100 nmol/L ET-1 in the presence or absence of 10 mmol/L BCAA cocktail and further cultured for 1 hr. Expression and phosphorylation levels of ERK1/2, JNK (p54 and p46), p38MAPK, S6K1, exogenous and endogenous HEXIM1 (FLAG and rat HEX, respectively), and actin were analyzed by Western blotting. Representative images of Western blotting from 5 independent experiments are shown.
Figure 3
Figure 3. Overexpression of HEXIM1 prevents ET-1-induced mRNA expression of cardiac hypertrophic genes in NRCM.
NRCM or cardiac fibroblasts were infected with irrelevant AxCALNLZ (C) or recombinant adenoviruses, which express FLAG-tagged human HEXIM1 (HEX) or its mutant lacking P-TEFb-binding activity (mt) in the co-presence of Cre recombinase, along with Cre recombinase-expressing recombinant adenovirus. After 24 hr, the cells were treated with vehicle or 100 nmol/L ET-1 and further cultured for 24 hr. Total RNA was extracted from the cells and expression levels of indicated mRNA were assessed in qRT-PCR analysis. Results were normalized to GAPDH mRNA levels and are shown as relative mRNA expression to expression levels in the control cells (AxCALNLZ-infected and vehicle-treated cells). Error bars represent SD (n = 5). *P<0.05 vs. vehicle-treated cells.
Figure 4
Figure 4. Generation of the transgenic mice with cardiomyocyte-specific overexpression of HEXIM1.
(A) Characterization of cardiomyocyte-specific HEXIM1 transgenic (HEX-Tg) mice. Left, representative photographs of 19-week-old male WT and HEX-Tg (Tg) mice and their hearts. Right, body weight of 10- and 19-week-old WT and HEX-Tg mice. Error bars represent SD (n = 5). (B) Heart-specific expression of FLAG-tagged human HEXIM1 in HEX-Tg mice. Left, bacterially expressed purified recombinant FLAG-tagged human and mouse HEXIM1 proteins were analyzed by Western blotting using anti-FLAG antibody or mouse HEXIM1-specific antiserum. Right, tissue extracts obtained from heart, lung, liver, and skeletal muscle of WT or HEX-Tg mice were analyzed by Western blotting. (C) Semi-quantification of endogenous and exogenous HEXIM1 protein in WT and HEX-Tg mouse hearts. Left, one hundred micrograms of extracts of the hearts from adult WT or HEX-Tg mice and the indicated amounts of bacterially expressed recombinant FLAG-tagged human or mouse HEXIM1 proteins were analyzed by Western blotting. Right, endogenous and exogenous HEXIM1 in the heart of WT and HEX-Tg mice exposed to different oxygen conditions were analyzed by Western blotting. N, normoxia. H, hypoxia. In panels B and C, representative images of Western blotting from 5 mice in each condition (genotype and oxygen concentration) and 5 independent experiments are shown.
Figure 5
Figure 5. Pathophysiological changes of pulmonary artery, hemodynamic, plasma ET-1 levels, and ET-1 mRNA expression of the lung in a hypoxia-induced pulmonary arterial hypertension model.
WT and HEX-Tg (Tg) mice were placed in normoxic or hypoxic conditions for 10 weeks. (A) Pulmonary vascular remodeling in WT and HEX-Tg mice exposed to chronic hypoxia. Representative photographs of Elastica Van Gieson stains of the lung sections of WT and HEX-Tg mice under normoxic and hypoxic conditions are shown (from 10 mice in each condition and genotype). Br, bronchiole. Arrow, pulmonary artery. (B) Right ventricular systolic pressure (RVSP) in WT and HEX-Tg mice exposed to chronic hypoxia. (C) Plasma ET-1 levels in WT and HEX-Tg mice exposed to chronic hypoxia. (D) mRNA expression levels of ET-1 in the whole lung extracts. Total RNA was extracted from lung tissues in each condition (genotype and oxygen concentration), and expression levels of mRNA of ET-1 were assessed in qRT-PCR analysis. Results were normalized to GAPDH mRNA levels and are shown as relative mRNA expression levels in the WT mice placed in normoxic condition. Error bars represent SD (n = 10). *P<0.05.
Figure 6
Figure 6. Cardiomyocyte-specific overexpression of HEXIM1 attenuates right ventricular hypertrophy in a hypoxia-induced PAH model.
WT and HEX-Tg (Tg) mice were placed in normoxic or hypoxic conditions for 10 weeks. (A) Effect of HEXIM1 on the development of RVH in mice exposed to chronic hypoxia. Left, representative photographs of cross-sections of the hearts stained with Hematoxylin-Eosin solution from 10 mice in each condition (genotype and oxygen concentration) are shown. Right, assessment of the RV weight to LV+S weight (RV/(LV+S)), RV weight to body weight (RV/BW), and LV+S weight to BW ((LV+S)/BW) are shown. Arrows, RV wall. (B) Effect of HEXIM1 on cardiomyocyte hypertrophy in mice exposed to chronic hypoxia. Left, representative photographs of Hematoxylin and Eosin stains of RV and LV sections of WT and HEX-Tg mice under normoxic and hypoxic conditions are shown. Right, 200 myocytes in each condition (genotype and oxygen concentration) were counted in randomly selected fields and myocyte width was measured. (C) Right ventricular end-diastolic diameter (RVDd) and ejection fraction of left ventricle (LV%EF) measured by ultrasound cardiography. Error bars represent SD (n = 10). *P<0.05.
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Grants and funding

This work was supported by Grants-in-Aid for Scientific Research (B) to HT (24390236), for Young Scientists (B) to NY (22790693) and NS (23791050), and for Encouragement of Scientists to TM (24930026), grants from the Ministry of Health, Labour, and Welfare to HT, grants from Takeda Science Foundation and Suzuken Memorial Foundation to NS, and grant from Actelion Academia Prize (Actelion Pharmaceuticals) to NY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.