The Protection Of Dantrolene On Myocardial Ischemia-reperfusion Injury In Rats

Objective:The purpose is to investigate the protective effect of dantrolene on myocardial ischemia-reperfusion injury in rats. Tetrazolium chloride (TTC) staining, LDH release and hemodynamics were used to evaluate the tissue injury. The effect of dantrolene on energy metabolism was evaluated by measuring the amounts of high-energy phosphates, purine metabolites, i.e. adenosine, inosine, hypoxanthine, xanthine, and uric acid, and the activity of enzymes in purine nucleotide metabolism: 5'-nucleotidase (5'-NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), xanthine oxidase (XO) and those in purine salvage synthesis: hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyl transferase (APRT)Methods:Seventy-two male Wistar rats were randomly divided into three groups:control group, ischemia-reperfusion group and dantrolene-treated ischemia-reperfusion group. The hearts were perfused in a Langendorff system. They were first perfused with Krebs buffer for 30min. Pre-ischemic control continued to reperfuse for 90min; For ischemia-reperfusion, the hearts were subjected to global ischemia for 30min and reperfusion for 60min. The dantrolene-treated group was perfused in the presence of 5μmol/L dantrolene before ischemia. Heart rate (HR), aortic flow (AF), coronary fiow (CF), and cardiac output (CO) were determined at different concentrations of dantrolene; Intracellular high-energy phosphates, purine metabolites and enzyme activities were assayed by high-performance liquid chromatography (HPLC); The activity of LDH in perfusate was detected by ultraviolet spectrophotometry; Myocardium infarct size was measured on the basis of Tetrazolium chloride (TTC) staining at the end of each experiment.Results:1. Hemodynamic variables and myocardial protection of dantrolene Pretreatment with dantrolene significantly reduced the myocardium infarct size and LDH activity as compared with the ischemia-reperfusion hearts without dantrolene treatment (P<0.01)Dantrolene did not affect hemodynamics, except for a slight increase in coronary flow (P<0.05)2. The effect of dantrolene on energy metabolismThe levels of high-energy phosphates in dantrolene-treated group were significantly increased as compared with the ischemia-reperfusion group without dantrolene treatment (P<0.05).Dantrolene significantly reduced the contents of purine metabolites (P<0.05).3. The effect of dantrolene on the enzyme activities in purine nucleotide metabolism and purine salvage synthesis.The activity of 5'-NT, ADA, and XO in dantrolene-treated group was significantly increased (P<0.05), and the activity of PNP was significantly reduced as compared with the ischemia-reperfusion group without dantrolene treatment (P<0.05).The hearts receiving dantrolene showed a significantly higher levels of high-energy phosphates and a lower activity of HGPRT and APRT than those hearts in the ischemia-reperfusion group without dantrolene treatment (P<0.05).Conclusions:1. Dantrolene treatment could delay the development of myocardial ischemia-reperfusion injury. Dantrolene did not produce any significant hemodynamic effect, except for a slight increase in coronary flow. It produced a slight negative inotropic effect, but whether this hemodynamic action is cardioprotective is controversial.2. Energy metabolism blockage is the initiation factor of ischemia-reperfusion injury. Cardiac contraction depends on Ca2+ influx from extracellular space, which triggers the opening of RyR channel, resulting in a large amount of Ca+ release into the cytoplasm and the activation of the contractile system. During myocardial ischemia, energy metabolism is blocked and ATP re-synthesis is inadequate. On the other hand, Ca2+ overload leads to cardiac hypercontracture, which further aggravates ATP depletion, and results in cell damage. As an inhibitor of Ca2+ release, dantrolene can inhibit Ca2+ release through RyR channel, prevent Ca2+ overload, and thus reduce the activity of the myosin ATPase and SR Ca2+ -ATPase, which are the major ATPase in myocytes. By decreasing ATP consumption, dantrolene can relieve myocardial hypercontracture and improve energy recovery.In the myocardium, a primary pathway for ATP degradation is ATP-ADP-AMP-adenosine-inosine-hypoxanthine-xanthine-uric acid. The substrates were accumulated after ischemia-reperfusion. An increase of purine metabolism results in an accelerated degradation of purine nucleotides and ATP re-synthesis was decreased. The accumulation of purine metabolites suggested that dantrolene improved energy recovery.3. Calcium overload is a feature of ischemia-reperfusion injury. The increased Ca+ activates calpain-1, which leads to the degradation of membrane skeleton proteins and structural proteins. Cellular integrity is destroyed and enzymes are released into the extracellular space. Dantrolene inhibits calcium overload, reduces Ca2+ dependent activation of calpain, inhibits the degradation of membrane skeleton proteins and structural proteins, increase the stability of membrane phospholipids to maintain cell integrity and to keep metabolic enzymes within the cells. Therefore, the hearts receiving dantrolene showed significantly higher enzyme activities than the ischemia-reperfusion group without dantrolene treatment. ATP degradation product hypoxanthine and adenine participate the re-synthesis ATP though the phosphotransferase pathway. But after dantrolene treatment, the activities of HGPRT and APRT were decreased, suggesting that the phosphotransferase pathway does not make a significant contribution to ATP re-synthesis in rats with myocardial ischemia-reperfusion injury.