The Study On The Effects And Underlying Mechanism Of Simulated Microgravity On β-adrenoceptor Signaling Transduction In Rat Heart

Increasing evidence have demonstrated that real/simulated microgravity leads to the disfunctions of several organs and systems including cardiovascular system. Elucidation of the underlying mechanism is of importance and significance for understanding the reason of cardiovascular disfunction after astronaut flying and providing aid measures. Recent observations during spaceflight and ground-based simulation studies have both suggested that postflight orthostatic intolerance may be due to the changes of cardiac tissue, low cardiovascular response to low circulating blood volume, and impaired regulation of cardiac function. The sympathetic nervous-catecholamine (CA) system has important effect in maintaining cardiac physiological function and blood pressure homeostasis. It is well known that theβ-adrenoceptor is a predominant receptor in regulation of cardiac function. The sympathetic nervous system releases norepinephrine which stimulatesβ-adrenoceptor to regulate cardiac function. Recent studies suggested that the responsiveness of cardiac contractility toβ-adrenoceptor stimulation is reduced after weightlessness. Depressedβ-adrenoceptor responsiveness may be due to down-regulation of theβ-adrenoceptor itself or to the impaired postreceptor events. Which are still not well understood and has not been reported.In our present study, hindlimb unweighted ( HLU) rat model was used to simulate microgravity, andβ-adrenoceptor mediated signaling of Gs protein /AC/cAMP/PKA/Ca2+ after 4 weeks microgravity in HLU rat has been studied to identify the mechanism of microgravity leading to reduced cardiac contractility.Objectives1. To observe the effect of 4-weeks microgravity on cardiac function.2. To observe the change ofβ-adrenoceptor after 4-weeks microgravity.3. To observe the effect of 4-weeks microgravity on cAMP-dependent pathway mediated byβ-adrenoceptor.MethodsIn the present study we adopted the rat tail suspension for 4 weeks to simulate weightlessness. Mean arterial blood pressure (MABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (?dP/dtmax) were monitored during the in vivo experiment.β-adrenoceptor density was quantitated by radioactive ligand binding. Single rat ventricular myocyte was obtained by enzymatic dissociation method. Gs protein was also determined by Westernblot. Myocyte contraction and cAMP in response toβ-adrenoceptor stimulation with isoproterenol or adenylyl cyclase stimulation with forskolin were measured. Myocyte contraction, intracellular [Ca2+]i transient in response toβ-adrenoceptor stimulation with isoproterenol were measured by Sony moving edges detection system. And L-type calcium current in response toβ-adrenoceptor stimulation with isoproterenol were measured by Whole-cell patch clamp technique.Results1. Compared with the control group, no significant changes were found in heart weight, body weight and MABP, whereas LVP and±dP/dtmax were significantly reduced. It showed that depressed cardiac contractility may not be due to the changes of cardiac tissue. LVP and±dP/dtmax were significantly attenuated in the HLU group in response to isoproterenol administration. It showed that the responsiveness of cardiac contractility toβ-adrenoceptor stimulation is reduced after weightlessness.2. Effects of isoproterenol on electrically induced single myocytes contraction in HLU rats were significantly attenuated. It showed that the depressedβ-adrenoceptor responsiveness(β-adrenoceptor desensitization) happened in myocytes of ventricles, which may be an important reason for the depressed myocytes contraction.3. In the in vitro study, theβ-adrenoceptor density and affinity were unchanged compared with that of the control group. It showed that the depressedβ-adrenoceptor responsiveness may not be due to downregulation and function of theβ-adrenoceptor itself.4. The biologically active isoform, Gsa (45 kDa) in the HLU rat heart, was unchanged. It showed thatβ-adrenoceptor responsiveness may not be due to Gs protein.5. Both the increased electrically induced contraction and cAMP in response to forskolin were also significantly attenuated in simulated weightlessness rats. Results indicate that impaired function of adenylyl cyclase causesβ-adrenoceptor desensitization,which may be partly responsible for the depressed cardiac function.6. Effects of isoproterenol on [Ca2+]i transient in myocytes of ventricles in HLU rats were significantly attenuated. It showed thatβ-adrenoceptor postreceptor signaling may be impaired and calcium homeostasis is impaired during weightlessness.7. The enhanced L-type Ca2+ current elicited by isoproterenol in cardiomyocytes was significantly decreased in the HLU group. The result indicated that impaired function of L-type Ca2+ current caused the impaired calcium homeostasis and the depressed responsiveness of theβ-adrenoceptor stimulation.Conclusion1. The depressed responsiveness of theβ-adrenoceptor stimulation causes the depression of cardiac function during weightlessness.2. Depressedβ-adrenoceptor responsiveness may not be due to downregulation of theβ-adrenoceptor itself, but to the impaired postreceptor events such as Gs protein/adenylyl cyclase (AC)/cAMP/protein kinase A/Ca2+ cascades.3. The impaired function of AC and L-type Ca2+ current may be partially responsible for the depressed responsiveness of theβ-adrenoceptor stimulation, which may be partially responsible for the depression of cardiac function.