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.