Energetic Mechanisms Underlying The Effects Of Astragalus Extract Mixture And The Effective Components Of Astragalus On Cardiac Contactility In The Rats With Experimental Heart Failure

BackgroundHeart failure (HF) refers to a serious phase of many cardiac diseases caused by various factors and is the final consequence of most cardiovascular diseases. HF is the leading cause of death and disability in the industrialized world. The mortality rate of HF is about50%, and although there has been a reduction in mortality from acute myocardial infarction over the last30years, there has been a concomitant rise in morbidity of HF. The syndrome of HF was described by Hippocrates over two millennia ago but therapeutic efficacy, even at present, is so poor that its death rate is always on high level. Thereby, finding out the new ways to enhance the therapeutic efficacy is very significant for the treatment of HF.The incidence of HF is determined by a variety of factors, while a growing number of clinical and experimental studies have shown that there is a depletion of myocardial energy in HF. Myocardial energy metabolism plays an important role in the development of heart failure. In fact, one of the hallmarks of HF is lack of energy and obstruction of energy metabolism is one of the main indicators of HF. Modern research showed that in the progression of HF there are several crucial causes including energy deficiency, over-activity of metabolic enzymes and abnormal gene expression, which jointly induce the remodeling of left ventricle. In the fetal and newborn heart, glucose is the primary energy substrate for energy production, while fatty acid oxidation as a way to produce energy (ATP) in myocardium is at a low level, providing only a small proportion of overall ATP production. The heart is very reliant on glycolysis as a source of energy during this period due to the higher activities of the enzymes in the glycolytic pathway. However, after birth, there is a dramatic10-fold increase in fatty acid oxidation, which is accompanied by a parallel decrease in glycolytic rates. This is opposite to the switch towards reduced fatty acid oxidation and increased glucose oxidation observed in some forms of severe HF. Interestingly, if the newborn heart is subjected to a volume-overload hypertrophy, the expression and activity of key enzymes controlling fatty acid oxidation remain in the "fetal state". A number of key enzymes involved in fatty acid oxidation are altered in the transitions from fetal to adult metabolism, illustrating that there is not a simple switch to fetal metabolic regulation in response to chronic cardiac stress. Presumably, impact of the metabolic remodelling in failing heart is associated with not only the substrate metabolism switch about FFA supply but also the key enzyme convert to "fetal state".Therefore, HF is actually an overloaded cardiomyopathy induced by the abnormal gene expression following the energy deficiency. It was confirmed by the study with measuring high energy phosphates in myocardium of the patient with HF that the ventricular remodeling accompanied with the disorder of energy metabolism is pathophysiologically the fundamental mechanism. Occurring in the whole course of HF development throughout from compensatory myocardial hypertrophy to terminal cardiac failure, disorder of energy metabolism plays an important role in the progression of HF. Based on the fact that both impairment of cardiac mechanical contractility and disorder of energy metabolism are the dominant manifestations of HF, it was proposed by the scientists that modulating myocardial energy metabolism may enhance the cardiac work efficiency so as to treat HF effectively. More and more studies showed that modulating myocardial substrate utilization can significantly improve LV function and mechanical efficiency of failed heart., suggesting that the metabolic modulation as a new therapeutic way is very significant for the treatment of HF and even other heart diseases like myocardial ischemia which is known to be also related to the disorder of energy metabolism.Reinforcing qi to treat heart failure differentiated as the type of deficiency of cardiac qi in Chinese medicine is conceptually similar with modulating the disorder of energy metabolism. Astragalus membranaceus is a Chinese herbal medicine with function of supplementing qi. It was reported that Astragalus membranaceus can improve cardiac function significantly and has been widely using in the treatment of various heart diseases. Astragalus membranaceus contains various active components with cardiac effect, including astragalosides, polysaccharides and flavones.As one of the primary active components of Astragulas, Astragaloside IV has been found to produce potent cardioprotective effects. Both in vivo and in vitro studies have provided evidences indicating that an antioxidant effect may be one of the underlying mechanisms by which astragaloside IV protects myocardium. Although there were previous studies involving the metabolic modulation in other organ or tissues by Astragalus membranaceus or its components, no any one of them was emphasized on exploring whether Astragalus membranaceus or its components can directly modulate the course of myocardial energetics such as the utilization of metabolic substrates in energy production so as to improve the function of failed heart.With aim at systemically exploring the energetic mechanisms underlying the improvement of cardiac mechanical function produced by the extract and effective components of AEM, the present study was conducted to compare the various mechanic and energetic alterations before and after administrating the extract and the effective components of AEM. We compared and analyzed the changes in the utilization of energetic substrates by myocardium, partial pressure of oxygen (PO2), myocardial energetic enzymes and transporters for transportation of energy or energetic substrates in myocardium. It was anticipated that the evidence shown in present study could provide a scientific explanation from a new angle of view on the mechanisms underlying the improvement of cardiac mechanical contractility produced by qi-invigorating herbal medicine. Modulating the disorder of myocardial energetic metabolism may potentially be an effective new way to screen Chinese herbal medicine effective in the treatment of various diseases characterized with the disorder of energetic metabolism such as heart failure and myocardial ischemia.ObjectiveAs aforementioned, the present study was aimed at systemically exploring the energetic mechanisms underlying the improvement of cardiac mechanical function produced by the extract and the effective components of AEM, the present study was conducted to compare the various mechanic and energetic alterations before and after administrating the extract and the effective components of AEM. The study contains two parts of experiments. The actions of the extract and the effective components of AEM on the cardiac mechanic contractility in the rats with experimental heart failure were confirmed firstly with evaluating system of cardiac contractile function; based on the above confirmation of the effects produced by AEM, the second part of the study was conducted to address the energetic mechanism underlying the cardiac mechanic effect of AEM. The evidence provided by present study is prospected to be helpful to create a new way (with focus on modulating energetic metabolism) for screening more Chinese herbs which are effective in treating the heart diseases characterized with disorder of energetic metabolism.MethodsNinety male SD rats were randomized into7groups with18rats in HF group and12rats in each of other groups, including Control group, Digoxigein (DG) group, Astragaloside (ASIV) group, Astragalus Polysaccharide (APS) group, Total Saponins of Astragalus (AST) group and Astragalus Extract Mixture (AEM) group. The animals in all groups except Control group were treated with intraperitoneal injection of adriamycin once two days, while the animals in all groups except Control and HF groups were treated by intragastric administration of astragalus extract mixture and active components in the therapeutic groups at the same time. At the last day of treatment, the hearts were then isolated and perfused with modified Krebs buffer. Thereafter, two parts of experiments including effect confirming and mechanism analyzing ones were conducted.1. In the first part of the experiments cardiovascular hemodynamic indices and the changes of substrate energy metabolism were measured to evaluate the actions of AEM and the effective components on the cardiac mechanic contractility in the rats with the experimental HF induced by ADR.Animal survival rate (SR) was evaluated and the indices of cardiac mechanic contractility including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), T-dp/dtmax and Rate-Pressure Product (RPP) were recorded by a transducer connecting with a cardiac catheter inserted into left ventricle.2. In the second part of the experiments, the energetic mechanisms underlying the effects of AEM and the effective components in improvement of cardiac mechanic contractility of failed heart were explored. The changes in the utilization of energetic substrates by myocardium, partial pressure of oxygen (PO2), myocardial energetic enzymes and transporters for transportation of energy or substrates were detected.2.1. Fundamental energetic mechanisms underlying the cardiac mechanic effects of AEM and the effective components of AS were addressed as follows:By using of the techniques of biochemistry and HPLC etc., FFA, glucose, pyruvic acid and PO2in the perfusion buffer before and after perfusion and the lactic acid in perfusion buffer after perfusion were measured so as to explore the mechanisms mentioned above.2.2. Further mechanisms underlying energetic alterations responsible for the production of cardiac mechanic effects of AEM and the effective components of AS were explored in the following experiments:2.2.1. Effects of AEM and the effective components of AS on the transporters of the myocardial energetic metabolism in the failed heart induced by ADRAlso, the contents of the transporters for the transportation of energy and energetic substrates, including FAT, FABPpm, CPT and UCP protein were measured by Western Blotting technique to determine which one(s) is (are) involved in the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.2.2.2. Effects of AEM and the effective components of AS on the key enzymes of the myocardial energetic metabolism in the failed heart induced by ADRIn this part of experiment, the contents or gene expression of the energetic metabolism-related key enzyme(s) in myocardium including PFK, PK, MACD, LCAD and CK were measured to determine which enzyme(s) is(are) involved in the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.2.2.3. Effects of AEM and the effective components of AS on the myocardial energy storage in the failed heart induced by ADRFurthermore, The content of ATP, ADP and PCr were also determined by HPLC to explore if the state of energy production and storage contribute to the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.Results1. Confirmation of the improvement of cardiac mechanic contractility produced by AEM and the effective components of AS in the rats with ADR-induced HF.The results showed that the general physical condition, the ratio of heart weight over body weight and the survival rate were declined in HF group as compared with normal control group (P<0.05or P<0.01). An amelioration of the general physical condition was observed in all treating groups (P<0.01or P<0.05). The ratio of heart of weight over body weight was elevated in all treating groups except AEM group (P<0.05or P<0.01). In the HF group all the hemodynamic indices were impaired significantly as compared with normal control group (P<0.05or P<0.01); As compared with HF group, LVSP and+dp/dtmax were significantly enhanced in all treating groups (P<0.05or P<0.01). In addition, Rate-Pressure Product (RPP) was also improved significantly by AEM and the components of AS in comparison with that in HF group (P<0.01). Comparatively, the strongest cardiac mechanic effect was observed in AEM group among all of treating groups including DG group. Further experiment showed that AEM can even enhance HR and shorten T-dp/dtmax in the rats with HF. The results also showed that with similar cardiac mechanic effect of AST, APS significantly enhanced LVSP and+dp/dt max.2. Exploration of the mechanisms underlying the effects of AEM and the effective components in the improvement of cardiac mechanic contractility of failed heart2.1. Fundamental energetic mechanisms underlying the cardiac mechanic effects of AEM and the effective components of ASThe results showed that there was a metabolic disorder exhibited as a sharp switch away from fatty acid towards carbohydrate oxidation in HF group in comparison with normal control group. The switch of substrate uptake in HF group was evidenced by a sharp rise in glucose uptake and a fall in fatty acid uptake that coincided with contractile dysfunction. PO2was greatly enhanced by AEM and the effective components of AS(P <0.01or P<0.05as compared with HF group); An Obvious increase in the uptake of free fatty acid and pyruvic acid was found in all of treating groups(P<0.01or P<0.05); Likewise, as compared with HF group, the glucose uptake and the ratio of cardiac work (indicated as RPP) over glucose uptake were significantly reduced by AEM and the effective components of AS(P<0.01); The ratio of cardiac work over pyruvic acid uptake was raised in all treating groups except APS group(P<0.01as compared with HF group); In all treating groups, both the concentration of Lactic acid and the ratio of the uptake of glucose over the uptake of free fatty acid were reduced significantly(P <0.01or P<0.05). The data above suggests that the disorder in energetic metabolism in rats with ADR-induced HFwas significantly attenuated by AEM and the effective components of AS. Comparatively, the effect of AEM on modulation of glucose uptake and that of ASIV on regulation of FFA uptake were comparatively stronger than other effective components of AS respectively.2.2. Further mechanisms underlying energetic alterations responsible for the production of cardiac mechanic effects of AEM and the effective components of AS2.2.1. Effects of AEM and the effective components of AS on the transporters of the myocardial energetic metabolism in the failed heart induced by ADRThe results showed that the expression of UCP in HF group was elevated to1.5folds of that in control group; while the HF-induced enhancement of UCP expression was significantly diminished by APS and AEM respectively (P<0.01as compared with HF group).In HF group CPT-I, a key transferase for energy production and transportation, was substantially reduced to a very low level as only32%of that in control group(P<0.01). As compared with HF group, the content of CPT-I was significantly enhanced by AEM and the effective components of AS (P<0.01). The CPT-I-enhancing effect in ASIV and AEM groups was even obviously stronger than that in DG group P<0.01), suggesting CPT-I is involved in the mediation of both cardiac mechanic and energetic effects produced by AEM and the components of AS. The content of FAT-CD36was reduced in HF group (P<0.01as compared with control group) and the HF-reduced FAT-CD36was significantly enhanced in ASIV and AEM group to the control level, while in DG and APS group the FAT-CD36content was not significantly different from that of HF group (P>0.05). The protein of FABP, measured also by Western Blotting technique, was diminished markedly in HF group (P<0.01as compared with control group). However, no statistical difference was shown in terms of FABE content between HF group and each of all treating groups including AEM, ASIV, APS and AST groups respectively (P>0.05).2.2.2. Effects of AEM and the effective components of AS on the key enzymes of the myocardial energetic metabolism in the failed heart induced by ADRThe content of PFK in HF group increased to about1.3folds of that in normal control group (P<0.01). However, PFK in all the treating groups was not significantly different from that in HF group. There is no significant difference in content of PK among all groups. The results above suggest that PFK and PK are not involved in the mediation of both the cardiac mechanic and energetic effects produced by AEM and the effective components of AS.As another important enzyme in the beta-oxidation of free fatty acid, MCAD was reduced in HF group, to about50%of that in control group. In ASIV and AEM groups both protein content of MCAD and the gene expression of LACD were enhanced respectively as compared with HF group (P<0.01). No significant effect on LACD was observed in APS group. Further results showed that CKMB content was enhanced obviously in HF group (P<0.01as compared with Control group). The HF-induced increment of CKMB was significantly attenuated by APS and AEM respectively (P<0.05).2.2.3. Effects of AEM and the effective components of AS on the myocardial energy storage in the failed heart induced by ADRThe results achieved in present study showed that there was no significant difference in terms of ATP content among all groups. However, PCr content in HF group was significantly lower than that in control group (P<0.01), indicating that although ATP content wasn’t influenced by HF, the capability of energy pool for energy storage which is manifested as a transition from ATP to PCr was impaired in the failed heart. PCr content in ASIV and AEM groups were recovered almost to the level of control group (P<0.01as compared with HF group). Myocardial ADP content in HF group was markedly reduced as compared with control group (P<0.01). The HF-reduced ADP content was significantly enhanced by AST (P<0.05), but was only mildly elevated by DG, APS and AEM without statistical significance as compared with HF group (P>0.05).Conclusion1. Under the condition of the ADR-induced experimental HF a bad general physical condition, reduced ratio of heart weight over body weight and impaired cardiac mechanic contractility are observed. AEM and the effective components of AS significantly improve the general physical condition, increase the ratio of heat/body weight and survival rate and enhance the cardiac mechanic contractility in the rats with ADR-induced HF; 2. A significant disorder of myocardial energetic metabolism is obvious in the ADR-induced heart failure. The disorder is actually an abnormal alteration in the uptake and use of energetic substrates including FFA, glucose, pyruvic acid, lactic acid and even oxygen. In the ADR-induced HF a so-called "fetal state" is shown as that myocardial uptake of FFA as the substrate for energy production is reduced while glucose uptake is concomitantly enhanced. The disorder of energetics may be responsible for the impairment cardiac mechanics as the energetic disorder is concomitant with mechanic impairment in myocardium.3. The energetic disorder in the uptake of substrates is significantly ameliorated by AEM and the effective components of AS so as to improve the HF-impaired cardiac mechanic contractility.4. AEM and the effective components of AS significantly attenuate the content of UCP, enhance protein expression of CPT-I and FAT-CD36, which may contribute to the improvement of the HF-induced energetic disorder.5. Another mechanism underlying the improvement of the HF-induced energetic disorder is that AEM and the effective components of AS significantly modulate the contents of the key enzymes of energetic metabolism, including increase in MCAD and LACD and decrease in the gene expression of CKMB.6. Elevating the PCr level by AEM and the components of AS means increase in the energy storage, which is also one of the mechanisms underlying the cardiac mechanic and energetic effects produced by AEM and the effective components of AS in the ADR-induced HF.In summary, AEM and the effective components of AS significantly enhance the cardiac mechanic contractility in ADR-induced heart failure via modulating the myocardial key enzymes of energetic metabolism, transporters of energy and substrates and the energy storage so as to correct the energetic disorder including in particular the abnormal uptake of energetic substrates by myocardium.