Salvia Myocardial Systolic And Diastolic Function Of Mast Intervention Mechanism Based On Sarcoplasmic Reticulum Calcium Transport Astragalus

Heart failure is the terminal stage of a variety of cardiovascular diseases, such as hypertension, coronary heart disease, valvular heart disease. Once heart failure occurs, the prognosis is extremely poor, and accounted for40%of the total mortality of cardiovascular disease mortality. The basic mechanism of heart failure is myocardial remodeling, and the pathphysiology is myocardial calcium homeostasis balance disorders, leading to impaired myocardial function.The early manifestation of heart failure is diastolic dysfunction and reduced myocardial compliance, thereby affecting systolic function, eventually leading to heart dysfunction. Excitation-contraction coupling (ECC) barriers are an important part of the heart systolic and diastolic dysfunction. In normal excitation-contraction coupling (ECC), firstly, small amounts of calcium ions enter the cell through L-type calcium channel during depolarization, secondly, a large of calcium release from sarcoendoplasmic reticulium (SR) by ryanodine receptors. This is called the calcium-induced calcium release. The result is a rapid rise of cytosolic calcium concentration that causes myocardium contraction. Then during repolarization, most of the cytosolic calcium is uptaken into sarcoendoplasmic reticulum by its Ca+-ATPase (SERCA2) under the regulation of phospholamban. So the concentration of calcium in cytosol decreases and the myocardium relaxes. This change of intracellular calcium ions during excitation-contraction coupling is called calcium transient, and it is the molecular basis of myocardial contraction. The above of calcium transport protein is regulated by calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ). Calcium transport has become to be considered is one of the most mechanisms during the development of heart failure, and may be the target for intervention in the future. Use of inhibitors or gene knockout method to inhibit excessive activation of CaMK Ⅱ can delay heart failure progress, but how to promote clinical applications, still need a lot of tests. Therefore no corresponding drugs can be used for clinical application.Traditional Chinese medicine has certain advantages in early intervention in heart failure. During the clinical studies, the researcher suggests that deficiency qi and blood stasis is the cause of heart failure occurred, and a key factor for heart failure. Previous experiments also suggested that traditional Chinese medicine has different regulation for sarcoplasmic reticulum calcium transport. Salvia and Astragali is effective to heart failure which is commonly used drugs, with the exact clinical efficacy. There can improve cardiac hypertrophy, but also can delay the progress of heart failure.Our research is based on sorting out and analyzing a large of reference data and previous research. Use cardiac sarcoplasmic reticulum calcium transport mechanism to explain Astragalus, Sal via treatment of cardiac hypertrophy and pharmacological basis for the prevention of heart development.Methods1. Cardiomyocytes characteristics observed experiment:The cultured of neonatal rat cardiomyocytes were divided into control group, Model group, Salvia group (DS), Astragali Group (HQ), Salvia and Astragali group (DQ), Losartan group. At24th hour,48th hour,72nd hour, use inverted phase contrast microscope to observe the characteristics of cardiomyocytes. HE staining and image analysis record the model of the nucleus/cytoplasm area and diameter of cardiomyocytes; Use phase contrast microscope to record beating frequency of cardiomyocytes; The cell area before and after cell contraction was measured by the living cells workstation.2. Effects of Salvia and Astragali on the Calcium Transients:The cardiomyocytes were loaded with Fluo-4/AM. The Salvia and Astragali on the contractility, diastolic [Ca2+] and calcium transients were assessed by the Fluorescence Measurement and Cell Dimensioning Systems. Observe the changes of Salvia and Astragali effect on calcium transients at24th hour,48th hour,72nd hour.3. Effects of Salvia and Astragali on the expression of calcium handing proteins:Using immunofluorescence and Western bolt method observed Salvia and Astragali on the fluorescence intensity and protein content of calcium handing protein:CaMK Ⅱ, SERCA2a and PLB at different time points (24h,48h,72h).4. Effects of Salvia and Astragali on the Calcium transients after KN-93blocked CaMK Ⅱ:The Salvia and Astragali on calcium transients were assessed by the Fluorescence Measurement and Cell Dimensioning Systems and Laser Scanning Confocal Microscopy (LSCM). Using KN-93to block CaMK Ⅱ, observe the change of Salvia and Astragali effect on calcium transients in24th hour,48th hour, and72nd hour. Results1. The nuclear/cytoplasm area and diameter of myocardial are significantly increased at24th hour,48th hour, and72nd hour by10-7mol/L angiotensin Ⅱ. The total protein content of cardiomyocytes increased significantly; the beating rate decreased significantly with time.2.24th hour, compare to the control group, the relaxation time to50%baseline (RT50) and τ of model group significantly increased (P<0.05). In herbs group, RT50and τ decreased.48th hour, the F0, τ and RT50of model group increased, the△F and F decreased (P<0.05). The herbs group not only reduced RT50, τ and Fo, but also recovered the△F and F to normal levels. The Losartan group reduce RT50, τ and Fo.72nd hour, the F0,△and RT50of model group still increased, the△E and F were significantly lower (P<0.01). HQ and DS can improve the change by Ang Ⅱ, and the two drugs used in combination are better than their individual use to change τThe Losartan group increased△F, F and-dp/dt max.3. Effects of Salvia and Astragali on the mean IOD of calcium handing proteins.(1)24th hour, the CaMK11IOD of model group compared with control group increased significantly(P<0.05), SERCA2a mean IOD and PLB mean IOD decreased slightly, but not statistically significant. Each dose group can be varying degrees of improvement.(2)48th hour, model group CaMK Ⅱ mean IOD increased, while SERCA2a mean IOD and PLB mean IOD was significantly decreased(P<0.05). Each dose group can significantly reduce the CaMK Ⅱ mean IOD, and SERCA2a mean IOD and PLB mean IOD increased.(3)72nd hour, the model group mean IOD of CaMK Ⅱ, SERCA2a and PLB is still continuing to change(F <0.05). Each dose group increased SERCA2a mean IOD. Only the combined group and Losartan group increased PLB mean IOD. Effects of Salvia and Astragali on the expression of calcium handing proteins.(1)24th hour, compared to the model group and the control group CaMK Ⅱ expression level was higher(P <0.05) and the SERCA2a expression level decreased(P<0.05). The HQ group reduced the expression level of CaMK Ⅱ, and increased SERCA2a protein content to normal levels, while significantly elevated SERCA2a/PLB ratio. The DS group compared with the model group can be reduced CaMK Ⅱ protein expression.(2)48th hour, the model group continued to increase CaMK Ⅱ expression level and the expression of SERCA2a, PLB contimued to drop; SERCA2a/PLB ratio dropped significantly. HQ group and DS group can significantly reduce the CaMK Ⅱ protein expression, recovered SERCA2a and PLB protein expression, and SERCA2a/PLB ratio. The therapeutic effect of the QD group was better than.single-agent group.4.48th hour, the F0and RT50of model group increased, the△F decreased (P<0.05). HQ/DS can reduce RT50and FO, and recovery the△F to normal levels. The Losartan group can reduce RT50and recovery the△F. We study the calcium transients of KN-93blocked CaMK Ⅱ at48h. Compared of the model group and the control group F0and△F were no significant difference, but RT50increased significantly(P<0.05). The result indicated that the systolic function of cardiomyocytes was improved and the diastolic function exist abnomal. Each dose group can be further reduced on RT50.ConclusionAng Ⅱ causes the myocardial cell dynamic changes of survival ratio during the process of hypertrophy to heart failure, and causing myocardial nuclear/cytoplasmic area and diameter increased significantly. The diastolic function changed at24th hour, and the systolic dysfunction at48th hour by using Ang Ⅱ. Ang Ⅱ cause CaMKⅡ, PLB and SERCA2a dynamic changes of protein expression levels. Those affect the intracellular calcium ion concentration, thus affecting the changer of normal calcium transient.Astragalus, Salvia regulated calcium ions through the intervention of calcium transports regulatory proteins CaMK Ⅱ, PLB, SERCA2a, which resulting in improved myocardial systolic and diastolic function of the cell. Astragali can improve abnormal calcium transient, and regulate protein expression in early cardiomyocyte hypertrophy. Note to Ang Ⅱ could induce the changes of transcription and expression of calcium handing protein resulting in sarcoplasmic reticulum calcium transport barriers. This progress can be inhibitited by Salvia and Astragali in failure of myocardial cell. They improve cardiacmyocyte excitation-contraction coupling and slow the progress of heart failure. The combination of two drugs used in the early was not superior to Astragli, but in the failure stage the combination therapy was better. The two drugs combination not only protect the heart’s overall function, but also has a protective effect in a single cardiac cell contraction, during the long course of treatment in heart failure. They also can antagonize the abnormal changes of calcium transient and calcium handing protein in heart failure. Losartan improve either systolic function or diastolic function, but the Chinese medicine is different from western medicine. The herbs improve not only the systolic function but also the diastolic function of myocardial cells, which reflects the integrity of regulation.The effect of Salvia and Astragali on regulating systolic dysfunction, partly by regulating the changes of CaMK Ⅱ δ pathway, but the improvement of diastolic dysfunction may be by another pathway. Further research is needed.