| BACKGROUNDIn ischemic myocardium, myocardial injury is mainly caused by intracellular calcium overload and excess oxygen free radicals. During the ischemia in myocardium, proteins relevant for calcium homeostasis in cardiac myocytes altered, sarcoplasmic reticulum (SR) Ca2+-ATPase activity and sarcolemmal calcium pump were inhibited, in addition to energy metabolism disorder, intracellular calcium overload occurred. Intracellular excess Ca2+ actived phospholipase A2 and degradated sarcolemmal phospholipid, cell membranes were impaired. Meanwhile, excess oxygen free radicals were produced by xanthine oxidase pathway, directly disturbed the synthesis of ATP in mitochondria, accelerated energy metabolism disorder and cell damage. It has been proved that many drugs could prevent myocardial injury by regulation calcium homeostasis, Chinese traditional medicine-Astragalus membranaceus is one of the drugs. Astragalosides was the main effective component of Astragalus membranaceus, it reduced the myocardial damage in cultured cardiomyocytes, attenuated the area of myocardial infarction and improved cardiac function in ischemic rats. However, its molecular mechanism of myocardial protection is still unclear, especially on calcium handling and energy metabolism. In the present study, we attempted to investigate the mechanism of Astragalosides on calcium homeostasis regulation, in order to provide theoretical foundation for investigation the new component of Chinese medicine. AIMTo extract Astragalosides from Astragalus membranaceus and investigate Astragalosides' effect on L-type calcium channel, [Ca2+]i, SRcalcium load, SR Ca2+-ATPase activity, calcium regulatory proteins, energy metabolism and lipid peroxidation in injured cultured cardiomyocytes and rats by ion image system, patch clamp and molecular biology techniques. To discuss the calcium regulation mechanism of Astragalosides in myocardial injury. METHODS1. Cultured neonatal rat cardiomyocytes were exposed to isoproterenol as a cell injury model. Following drugs were added for 72 h, groups included: Normal control group (C), Isoproterenol (ISO), Astragalosides] (AS], 10 mg/L), Astragalosides2 (AS2, 100 mg/L), ISO and AS, (ISO+AS,), ISO and AS2 (ISO+AS2), ISO and metoprolol (ISO+M), ISO and verapamil (ISO+V). The cardiac troponin I (cTnl) in cultured medium was determined.2. Cardiomyocytes were loaded with Fura-2/AM, Cytosolic free calcium ([Ca2+]j) measurement was performed by ion imaging system. Caffeine was added rapidly to the cell for observation of calcium transient, the amplitude of calcium transient was used as an index of SR calcium load. The mRNA expressions of calcium regulatory proteins (SERCA, phospholamban, ryanodine receptor, L-calcium channel) and SR calcium-ATPase (SERCA) activity were also detected.3. Superoxide dismutase (SOD) activity and intracellular maleic dialdehyde (MDA) content in each group were measured.4. Adult male Sprague Dawley rats weighing 230 to 270 g were injected with isoproterenol (5 mg/kg/day, s.c.) for 2 days to produce myocardial injury. It was designed with following groups: Normal group (N), Injured group (ISO), Astragalosides treatment group (As), trimetazidine treatment group (TMZ), propranolol treatment group (PRO). With administration of above drugs for 7 days, a cardiectomy was performed. Cardiac apexs were stained with hematoxylin and eosin for assessment of pathology; lactate and MDA contents in left ventricular tissues were measured; ATP, ADP, AMP in left ventriculartissues were also determined by high performance liquid chromatography.5. Heart of rat was enzymatically dissociated by langendorff-perfusion apparatus. [Ca2+]i and SR calcium release in isolated cells were determined.6. Currents of L-type calcium channels were recorded by whole cell patch clamp technique. The maximum current density, current density-voltage curve and time constant of current inactivation in eac...
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