Arf1 And Creg In Pressure Overload-induced Cardiac Hypertrophy

Objective: ADP-ribosylation factor 1 (ARF1), a member of ARF subfamily of small G proteins, is a component and regulator of COP1 (coat proteins 1) vesicles and clathrin-coated vesicles and is also a factor for regulation of many signal molecules. But function of ARF1 in cardiocytes has not been studied. We found expressions of ARF1 increased in pressure-overloaded hearts by mRNA microarray method. To investigate its effects on pressure-overloaded cardiac hypertrophy, rat ARF1 gene was isolated and cloned, and its function was primarily studied.Methods: (1) A rat model of cardiac hypertrophy in vivo was established by constriction of abdominal aorta. (2) A neonatal rat model of cardiomyocyte hypertrophy of in vitro was established by stretching. (3) ARF1 has two different structures, one is in active state, binding by GTP, and the other is in inactive state, binding by GDP. Switching of two states is regulated by AFF1-GAP (GTPase-activating protein) and ARF-GEF (guanine nucleotide exchange factor). The former keeps it in GDP state, and the latter keeps it in GTP state. In this study, ARF1 was researched in five different aspects, including adenovirus-mediated expression of sense-ARF1, antisense-ARF1, sense-GAP and antisense-GAP and BFA, an inhibitor of ARF-GEF. (4) Northern blot analysis was used to test the expression of ARF1 gene in cardiac hypertrophic rats. (5) Western blot anlysis was taken to test the expressions of PKC α , PKCβ₁, PKC β₂, PKC γ , PKC ε , PKC δ , ERK1/2, JNK1/2, P38, P53, Bax, Bcl2 and Fas in neonatal cardiac cells. (6) Numbers of cardiac cells were determined by MTT assay.Results: (1) The average values of carotid systolic pressure (mmHg) of operated and sham operated rats at 28 days and 56 days after operation were 108.9 ± 8.7 vs 86.9 ± 6.6 (p<0.01); 109.7 ± 8.1 vs 84.4 ± 5.8 (p<0.01) respectively, and the cardiomyocyte areas (μm²) were 301.2 ± 32.2 vs 240.7 ± 19.9 (p<0.01); 335.7 ± 23.0 vs 263.7 ±11.4 (p<0.01) respectively. The simultaneous significant increase of systolic pressures and cardiomyocyte areas demonstrated that a rat model of pressure-overloaded cardiac hypertrophy in vivo had been established successfully. (2) Total proteins of cardiomyocytes increased gradually with prolonged 20% stretching from 2 h to 72 h. At the same time, c-fos protein levels increased rapidly after 1 h stretching by Western blot hybridization. These indicated that a stretch-induced cardiomyocyte hypertrophy in vitro was established successfully. (3) Northern blot analysis displayed that ARF1 gene expressed in kidneys, livers, brains, hearts and skeletal muscles. ARF1 mRNA levels in the hearts, including all the rats, were two times higher than those in the livers and one time in other tissues. (4) ARF1 mRNA levels increased gradually in the hearts of operated rats with the development of pressure-overload. Compared with sham-operated group, its levels increased by 15% and by 30% at 28 and 56 days after operation, respectively (p<0.05). (5) Adenoviruses mediated expression vectors of sense-ARF1, antisense-ARF1, sense-GAP and antisense-GAP were constructed successfully. Compared with control which were neither infected with adenovirus nor treated with BFA, the percents (%) of increase (+) or decrease (—) were, in turn, +13, —28, —18, +20 and —28 (p<0.05) in protein contents and +9, —7, —19, +26 and —30 (p<0.05) in cell areas after rat neonatal cardiomyocytes were infected with adenovirus-sense-ARF1, adenovirus-antisense-ARF1, adenovirus-sense-GAP, adenovirus-sense-GAP or treated with BFA. While protein contents in cardiac fibroblasts had not changed too much after being infected with those except an 8% (p<0.05) decrease after being treated with BFA. (6) Compared with control which were neither infected with adenovirus nor treated...