Study On Astragali And Its Active Components For Treatment Of Experimental Heart Failure And The Calcium Signaling-related Mechanisms

ObjectiveThe purposes of this study were to determine the therapeutic effect of astragali and its active components on experimental heart failure and investigate the calcium signaling-related mechanisms so as to provide the valuable evidence to support the treatment of heart failure with astragali and its active components in clinic and display a clue to create a novel approach to treat the energetic dysfunction-related heart diseases.Methods1. Preperation of experimental heart failure in ratsThe healthy male SD rats were randomized into Normal control (NC) group, Model group, Digoxin (DG) group, Astragalosides (AST) group, Astragalus polysaccharides (APS) group Astragalus extract mixture (AEM) group and Astragaloside IV (ASIV) group. The rats in Model group were given intra-peritoneal injection of adriamycin (ADR) once every other day in 8 consecutive days at a dose gradually increased. In the NC group the rats were given with same volume of saline in the same way as ADR was given to the rats in Model group. AEM, AST, APS, ASIV and DG were administered into the stomach once a day for 15 consecutive days in AEM, AST, APS, ASIV and DG groups respectively. In the NC and Model groups the rats were given with same volume of saline in the same way.2. The confirmation of the therapeutic effects of astragali and its active components on experimental heart failureAfter the HF model was prepared and the different drugs were administered the hearts were cut off and perfused with Tyrode's solution in a retrograde manner at the Langendorff perfusion system. Left ventricular systolic pressure (LVSP), heart rate (HR), and +dp/dtmax were measured to confirm the therapeutic effect of astragali and its active components on experimental heart failure.3. Study on the possible role of the Ca²⁺ signaling modulators in the mediation of the effects of astragali and its active componentsAfter administration of electro-acupuncture, the heart of the rat was isolated and ventricular myocytes were dissociated by collagenaseâ… for the following experiments.(1) The responsiveness of intracellular calcium [Ca²⁺]imarked with Fura-2/AM to ruthenium red were detected in a single ventricular myocyte in order to explore if RyR is involved in the mediation of the effects of astragali and its active components on experimental heart failure;(2) The content of SERCA2 protein was measured by Western Blotting technique and the responsiveness of intracellular calcium [Ca²⁺]i marked with Fura-2/AM to thapsigargin was detected in a myocardial cell to explore if SERCA-ATPase is involved in the mediation of the effects of astragali and its active components on experimental heart failure;(3) The content of PLB protein was measured by Western Blotting technique to explore if PLB is involved in the mediation of the protection effect of astragali and its active components;(4) The responsiveness of single ventricular myocyte to Sulmazole, a specific calcium sensitizer, in both contraction and [Ca²⁺]i was detected to determine if the sensitivity of the myocytes to [Ca²⁺]i protein is involved in the mediation of the effects of astragali and its active components.(5) Troponin C protein was also measured by Western Blotting technique to determine if troponin C protein is involved in the mediation of the inotropic effect of astragali and its active components, with special focus on the role of troponin C in the sensitization of the contraction-related components in the cells to intracellular calcium.Results1. Effects of astragali and its active components on the myocardial contractility。Indicated by LVSP, +dp/dtmax, and HR, the myocardial contractility in Model group was significantly decreased as compared with NC group (all P<0.01). However, the effect of astragali and its active components on LVSP, +dp/dtmax and HR in AST, APS, AEM and ASIV groups was stronger than Model group, but not significantly different from DG group(all P>0.05). 2. Determination of the possible role of the Ca²⁺ signaling modulators in the mediation of the effects of astragali and its active components (1) Effect of RyR inhibition on the intracellular Ca²⁺ in single ventricular myocyte isolated from the failed hearts in the rats which were pretreated with astragali and its active components The amplitude of electrically-induced intracellular calcium ([Ca2+]i) transient was decreased by ruthenium red, a specific antagnist of RyR, in all the groups. However, as compared with Model group, the ruthenium red-induced decrease of [Ca²⁺]i transient was more significant (all P<0.01 except P<0.05 in AST group) in NC, AST, AEM, ASIV and DG groups, with no significant differences between DG group and the groups pretreated by astragali and its active components mentioned above (all P>0.05). The results suggest that RyR may be involved in the mediation of the effect of astragali and its active components on experimental heart failure.(2) Involvement of SERCA protein in the myocardial protection of astragali and its active components in the rats with experimental heart failure The myocardial contents of SERCA2 protein in the groups pretreated by astragali and its active components was not significantly different from those in either Model group or DG group, (all P>0.05).(3) Involvement of SERCA responsiveness in the myocardial protection of astragali and its active components in the rats with experimental heart failure The amplitude of electrically-induced [Ca2+]i transient in DG group and the groups pretreated by astragali and its active components was significantly decreased by thapsigargin, a specific antagonist of SERCA; the amplitude of [Ca²⁺]i transient in all above groups was much lower than that in Model group (all P<0.01 except P<0.05 in APS and AEM group); there was no significant difference between DG group and each of the groups pretreated by astragali and its active components (all P>0.05). The results indicate that SERCA responsiveness may contribute to the mediation of the effect of astragali and its active components on experimental heart failure.(4) Role of PLB protein in the effects of astragali and its active components Similarly, no significant difference in the myocardial contents of PLB protein between each of the groups pretreated by astragali and its active components and either Model group or DG group, (all P>0.05) , showing that PLB protein is not the key factor to mediate the effects of astragali and its active components.(5) Involvement of Ca²⁺ sensitivity in the effects of astragali and its active componentsThe amplitude of electrically-induced [Ca²⁺]i transient and +dL/dt in DG group and the groups pretreated by astragali and its active components were significantly increased respectively by sulmazole, a calcium sensitizer; the increasing rate of [Ca²⁺]i transient and +dL/dt in each of all above groups were much higher than that in Model group (all P<0.01). No significant difference was observed between DG group and each of the groups pretreated by astragali and its active components (all P>0.05). The results suggest that sensitivity of the myocytes to the calcium may contribute to the mediation of the effects of astragali and its active components.(6) Possible role of troponin C protein in the mediation of the effects of astragali and its active components and the sensitivity of the myocytes to [Ca²⁺]i The optical density of the protein band of troponin C in NC group was obviously enhanced as compared with that in Model group (P<0.05). In AST, APS, AEM, ASIV and DG group, the optical densities of troponin C protein were also significantly enhanced as compared with that in Model group respectively (all P<0.05); No significant difference was observed between DG group and each of the groups pretreated by astragali and its active components (all P>0.05).Conclusion1. The present results showed that the attenuation of LVSP, reduction of HR and decrease in +dp/dtmax were produced by intra-peritoneal injection of ADR at a gradually-increased dose. The results showed that the model of experimental heart failure was successfully established and it can be used in the study related.2. It was exhibited in the present study that astragali and its active components can increase LVSP and +dp/dtmax in the heart isolated from the rats with experimental heart failure.3. The mechanisms underlying the enhancement of cardiac contractility in the failed heart produced by Astragali and its active components may involve several Ca²⁺ signaling modulators.(1) Astragali and its active components increase the release of Ca²⁺ from sarcoplasmic reticulum (SR, Ca²⁺ pool) via increasing the responsiveness of RyR on the sarcoplasmic membrane, and subsequently enhance cardiac contractility;(2)Astragali and its active components enhance the cardiac contractility by, at least partially, increasing the responsiveness of SERCA on the membrane of sarcoplasmic reticulum followed by elevating the uptake of Ca²⁺ into the SR;(3)Astragali and its active components enhance the cardiac contractility due partially to the increment of Troponin C content, so as to elevate the sensitivity of the contractile components to the intracellular Ca²⁺;(4)The fact that Astragali and its active components enhance the intracellular Ca²⁺ transient and cardiac contractility seems not related to phospholamban (PLB).