Effects Of RhoA/BK_Ca-ROCK Pathway On Artery Remodeling Induced By Simulated Microgravity In Rats

The weightlessness environment during space flight could induce a series of postflight cardiovascular dysfunction(PFCD) phenomenon, including postflight orthostatic intolerance(POI) and reduced upright aerobic exercise capacity, producing adverse effects on spaceflight activities and the re-adaptive capacity of astronauts after returning back to 1 G gravity environment on earth. The mechanisms underlying POI induced by exposure to weightlessness environment are complicated and not fully understood, thus, no effective countermeasures have been established at the moment. Previous studies have demonstrated that real/simulated microgravity-induced regional specific remodeling of the artery system, in particular the enhanced cerebral vasoconstriction and reduced peripheral vasoconstriction leading to cerebral blood volume reduction and decreased total peripheral resistance(TPR), respectively, might be one of the main mechanisms accounting for the occurrence of POI. Therefore, it has significant importance to investigate the regional specific structural and functional remodeling of the artery system induced by real/simulated microgravity for revealing the mechanisms underlying POI and proposing pertinent countermeasures.Previous data have documented the participation of several mechanisms in the mediation of structural and functional artery remodeling induced by real/simulated microgravity in rats, including vascular smooth muscle cells(VSMCs) ion channel remodeling, local renin-angiotensin system(L-RAS), reactive oxygen spices(ROS), nitric oxide-nitric oxide synthase(NO-NOS) pathway and inflammation, etc.. Large amounts of vascular biological studies have demonstrated that RhoA-Rho kinase(Rho-associated coiled-coil forming kinase, ROCK) pathway play an important role in the regulation of all the mechanisms mentioned above. RhoA is one of the Rho protein family members, functioning as an important mediator for actin cytoskeleton and several cellular functions, such as cell contraction, migration, proliferation and apoptosis. ROCK, which belongs to serine/threonine protein kinase, is Rho-associated coiled-coil forming kinase and the most widely studied downstream effector of RhoA. Up to now, the important role of RhoA-ROCK pathway in the regulation of abnormal vasoreactivity in many cardiovascular diseases such as hypertension and atherosclerosis has been well established. And our previous studies also showed participation of ROCK in the altered vasoconstriction of big elastic arteries induced by simulated microgravity in rats. Thus, we speculate that RhoA-ROCK pathway may function as a key factor, coordinating other mechanisms to contribute to the occurrence of artery remodeling, especially structural and functional remodeling of resistant arteries, induced by real/simulated microgravity.The present study applied 28-day hindlimb unweighted(HU) rat as the animal model to simulate the deconditioning effects of microgravity on the cardiovascular system, and targeted at femoral artery(FA), basilar artery(BA) and third-order mesenteric artery(MA) to detect the change of receptor- and non-receptor-mediated vasoconstriction and the possible role of RhoA-ROCK pathway in it. Besides, we further investigated the structural and functional relationship between RhoA and BKCa and possible role of this relationship in microgravity-induced functional remodeling of the artery system. Finally, we preliminarily discussed the co-localization of Caveolae/RhoA/BKCa and the possibility of these three structures to form buffering system on the surface of VSMCs to compensate exogenous stress changes.The main findings of the present study are as follows:(1) Simulated microgravity reduces the protein expression of ROCK and depresses its activity in FA.(1) As compared with control(CON) rats, HU significantly decreased phenylephrine(PE)- and potassium chloride(KCl)-elicited vasoconstriction of FA.(2) The application of Y-27632, a specific ROCK inhibitor, restrained FA vasoconstriction to the same extent in CON and HU.(3) HU significantly decreased the expression of ROCK II and the phosphorylation of myosin light chain phosphatase(MLCP) and MLC, and significantly increased the nuclear translocation of p65 and the expression of inducible NOS(iNOS).(4) HU significantly increased NO production and Y-27632 significantly amplified this effect.(2) RhoA-ROCK pathway may compensatorily impose a protective effect on altered vasoconstriction of small resistant arteries including cerebral and mesenteric arteries by simulated microgravity.(1) As compared with CON, PE-, serotonin(5-HT)- and KCl-elicited vasoconstriction of HU rats was significantly increased in BA and decreased in MA, respectively.(2) Except 5-HT-elicited vasoconstriction in BA of CON rats, Y-27632 significantly restrained vasoconstriction of BA and MA in both CON and HU rats, and the restrained extent of HU rats was significantly decreased in BA but increased in MA, respectively.(3) Y-27632 significantly increased 5-HT-elicited BA vasoconstriction of CON rats.(4) Compared with CON, U-46619-elicited vasoconstriction of HU rats was significantly decreased in BA and increased in MA.(5) HU induced differential changes of RhoA-ROCK pathway in BA and MA, specifically, the protein expression of RhoA and ROCK was not changed but their activity was significantly decreased in BA, and both the expression and activity of the two proteins were significantly increased in MA.(3) RhoA/BKCa complex may compensate the functional remodeling of small resistant arteries induced by simulated microgravity as buffering system.(1) As compared with CON, the protein expression of α and β subunits of BKCa of HU rats was significantly increased in BA but not changed in MA.(2) As shown by co-immunoprecipitation(co-IP), when we pulled down RhoA, both the expression of α and β subunits of BKCa was detected in BA, but only the expression of β subunit was detected in MA. And HU significantly upregulated the aforementioned expression level of BKCa subunits.(3) When we pulled down β subunit of BKCa, the expression of RhoA was detected in both BA and MA, and HU also significantly increased RhoA expression in these two arteries.(4) Confocal microscopy further confirms the aforementioned results, as relative to CON, the merged fluorescence by colocolization of RhoA and BKCa α/β subunits was significantly strengthened in both BA and MA of HU rats. Moreover, this merged fluorescence was better organized and significantly closer to the membrane and the two poles of the cell.(5) In the in vitro BKCa subunits overexpression HEK293 T cell model, we also detected the expression of RhoA or BKCa β subunit when pulled down β subunit or RhoA, and the α subunit of BKCa was detected only when β subunit existed.(6) As shown by whole cell patch clamp study, the selective Rho A inhibitor Clostridium botulitum C3 exoenzyme(C3) significantly amplified BKCa current density, while RhoA agonist U-46619 significantly inhibited this current density.(7) HU significantly decreased Caveolae number in aortic VSMCs of rats, but had no effects on the expression of Caveolin-1(Cav-1), the structural subunit of Caveolae.(8) When we pulled down Cav-1, both RhoA and BKCa α/β subunits were detected in TA and AA, and HU significantly decreased the expression of RhoA in these two different parts of aorta. In TA, HU also significantly decreased the co-expression of α subunit, but increased co-expression of β subunit. On the contrary, HU significantly increased and decreased the expression of α and β subunit, respectively, in AA.These results indicate that 1) simulated microgravity for 28 days reduces the expression of ROCK and inhibits its effects on downstream pathways, including the MLCP/MLC pathway and NF-κB/iNOS/NO pathway directly and indirectly related with FA vasoconstrictor reactivity, respectively, both of which may contribute to depressed FA vasoconstriction by simulated microgravity in rats; 2) RhoA co-localizes with BKCa, and simulated microgravity may compensate the functional remodeling of small resistant arteries through modification of the co-localization between RhoA and BKCa; 3) Caveolae/RhoA/BKCa may be a supplement of the classic buffering system on the surface of VSMCs, and function together in a protective way to compensate the adverse stimuli by exogenous stress changes on VSMCs.