Protective Effects And Mechanism Of Glycine Through The Pathway Of Glycine Receptors On Early Myocardial Damage

"Shock heart", occurring at the early stage following severe burns, is not only the pathophysiological basis of inducement and aggravation of burn shock, but also one of the main causes for ischemia/hypoxia and dysfunctions of other organs such as kidney and gut. Therefore, it is of importance to protect myocardium from damage in the early stage postburn. Evidence has been found to indicate the notable protective effect of free glycine. Glycine obviously relieves endotoxemia and lowers the mortality of rat treated with endotoxin, lessens injury of skeletal muscle from ischemic/reperfusion, decreases the release of mediators of inflammation, calcium overload and reactive oxygen species. But no reports concerning the protective effect of glycine on myocardium and its mechanism following severe burns have been found.In the present study, the protective function of glycine on schemia/hypoxia cardiomyocytes and postburn myocardial damage and its mechanism is identified according to the changes of myocardial morphology, enzymology, energy, glycine receptor, free calcium ions, membrane potential, mitochondrial membrane potential, mPTP, capase-3 and apoptosis.Materials and MethodsIn vitro study1. Cell model and groupsNeonatal murine cardiomyocytes were collected, the cells were cultured in a hypoxic mixed gas containing 1% oxygen and used as hypoxic model. Cells were divided into normal(N) group, glycine-treated group (GN), schemia/hypoxia group(H) , schemia/hypoxia combined with glycine grope(GH), schemia/hypoxia combined with glycine and anti- GlyRα1 polyclonal antibody grope(GHA), schemia/hypoxia treated with glycine and taurine group(GHT). 2. Observation of indexes and methods①The cells were cultured in a ischemia/hypoxia .The content of ATP, ADP and AMP were tested by high performance liquid chromatogram(HPLC)and ATP/ADP and energy charge(EC) was calculated. The expression of TNF-αmRNA was detected by RT-PCR from cardiomyocytes after ischemia/hypoxia 1, 3, 6, 12 and 24h. LDH in the supernate fluid and the survival rate of cardiomyocytes after 6h ischemia/hypoxia were determined with routine methods. The cell survival rate was detected after 6h ischemia/hypoxia by CCK-8 and PI dyeing.②The changes of free calcium ions, membrane potential, mitochondrial membrane potential, mPTP and apoptosis in the cardiomyocytes were determined using laser confocal microscopy following 6h ischemia/hypoxia. Capase-3 was determined with routine methods.③Using nerve cell as positive control, glycine receptor on membrane of cardiomyocytes were detected by immunohistochemistry, RT-PCR and Western-blot.In vivo animal study1. Establishment of burn model and groupsThe study was carried out using a model of 30% TBSA of full-thickness burns in SD rats. The burn rats were grouped as normal (N), glycine-treated(G) and untreated(B) groups.2. Observation indexes and methods①The expression of GlyRα1 in myocardium were detected by western-blot after burn 1, 3, 6, 12 and 24h.②The content of ATP, ADP, AMP and EC in myocardium tissue were tested by high performance liquid chromatogram(HPLC). The contents of LDH in serum were checked by normal methods.③The content of TnI in serum was determined by enzyme-linked immunospecific assay.Results:In vitro study:1. The changes of energy in cardiomyocytes: In the ischemia/hypoxia group, ATP, EC and ATP/ADP levels were decreased gradually obviously after1, 3, 6, 12 and 24h ischemia/hypoxia. At the same time,ADP and AMP levels were increased gradually and obviously. However, the changes of energy in the glycine treated group were markedly improved when1, 3, 5 and 10mmol/L of glycine were added.2. The cardiomyocytes were obviously damaged by ischemia/hypoxia, including the levels of LDH increased in supernate fluid; the quantity of positive cells of PI dyeing increased and the expression of TNF-αmRNA increased obviously. However, the levels of LDH decreased in supernate fluid, the quantity of positive cells of PI dyeing decreased and the expression of TNF-αmRNA decreased obviously in the glycine treated group. Meanwhile, the glycine protection was the best in 5mmol/L.3. Apoptosis of cardiomyocytes was detected by Annexin-V. The 5mmol/L glycine can obviously lighten apoptosis after ischemia/hypoxia 6h.4. Glycine receptors exist in cardiomyocytes. Nerve cells as positive control, the expression of GlyRα1, GlyRα2 and GlyRβmRNA were founded by RT-PCR in cardiomyocytes. The expression of GlyRα1 and GlyRβwere founded by western-blot in cardiomyocytes. GlyRα1 subunits on membrane of cardiomyocytes were stained by immunohistochemistry. The changes of expression of GlyRα1 subunits on membrane of cardiomyocytes were checked after 1, 3, 6, 12 and 24h ischemia/hypoxia .5. Fluorescence intensity of membrane potential of cardiomyocytes after 6h schemia/hypoxia became lower than that in normal cardiomyocytes; Fluorescence intensity of calcium of cardiomyocytes after 6h schemia/hypoxia became stronger than that in normal cardiomyocytes; Fluorescence intensity of mitochondrial membrane potential of cardiomyocytes after 6h schemia/hypoxia became lower than that in normal cardiomyocytes; The opening of mPTP of cardiomyocytes after 6h schemia/hypoxia became stronger than that in normal cardiomyocytes; The release of capase-3 of cardiomyocytes after 6h schemia/hypoxia became lower than that in normal cardiomyocytes. However, the changes of membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 when glycine was added were opposites to the groups of schemia/hypoxia 6h. The changes of membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 when Taurine(0.5mmol/L) was added were concoed with the groups of glycine treated. The changes of membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 when GlyRα1(1:100) was added were opposites to the groups of glycine treated.In vivo study:1. The GlyRα1 exist in myocardium, the expression of GlyRα1 became stronger after burn 1h, reached the maximum at 6h then depressed gradually.2. In the burn untreated rats, levels of LDH in serum increased obviously, reached the maximum at 6h then recovered gradually. However, the levels of LDH in serum decreased obviously in the glycine-treated group.3. The content of ATP and EC were decreased and TDP and TMP were increased obviously after 3h burns. However, the content of ATP were increased and TDP and TMP were decreased obviously after 3h burns when glycine were added.4. The content of TnI in serum was elevated dramatically at 1 h after burns and came to the summit in 6 h, slightly recovered at 12 and 24 h, which was consistent with the pathological changes of myocardial tissues. The content of TnI in serum was decreased obviously in burn rats treated with glycine.Discussion and conclusion1. The results of cardiomyocytes culture in vito on schemia/hypoxia studies indicate that glycine has an obvious protective effect, including ATP, EC and ATP/ADP levels increased gradually and obviously after ischemia/hypoxia, at the same time,ADP and AMP levels decreased gradually and obviously, the levels of LDH decreased in supernate fluid, the quantity of positive cells of PI dyeing decreased, apoptosis of cardiomyocytes decreased and the expression of TNF-αmRNA decreased obviously. Meanwhile, the glycine protection was the best in 5mmol/L.2. Glycine receptors exist in cardiomyocytes. Nerve cells as positive control, the expression of GlyRα1, GlyRα2 and GlyRβmRNA were founded by RT-PCR in cardiomyocytes, the expression of GlyRα1 and GlyRβwere founded by western-blot in cardiomyocytes, GlyRα1 subunits on membrane of cardiomyocytes were stained by immunohistochemistry. The expression of GlyRα1 subunits on membrane of cardiomyocytes were founded after 1, 3, 6, 12 and 24 h ischemia/hypoxia.3. Cardiomyocytes membrane depolarized after 6h ischemia/hypoxia. Opened L-type voltage-dependent calcium channels and inflow of calcium increased. Mitochondrial membrane potential of cardiomyocytes decreased. The opening of mPTP increased. Kinase of apoptosis activated. In the end, led to apoptosis in cardiomyocytes.4. The possible mechanism that glycine inhibited apoptosis of cardiomyocytes on schemia/hypoxia may be that glycine receptors existing in cardiomyocytes and myocardial tissues. Membrane of cardiomyocytes was depolarized after schemia/hypoxia. As a consequence, agonist-induced opening of L-type voltage-dependent calcium channels and inflow of calcium increased. Mitochondrial membrane potential of cardiomyocytes decreased. The opening of mPTP increased. The releasing of caspase-3 inreased. In the end, these responses led to apoptosis in cardiomyocytes. However, the membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 were changed markdely when glycine were added. Then, these results decreased apoptosis in cardiomyocytes after schemia/hypoxia.5. The GlyRα1 exist in myocardium. In a model of 30% TBSA of full-thickness burns in SD rats, the expression of GlyRα1 became stronger after burn 1h and reached the maximum at 6h then depressed gradually. Glycine has an obvious protective effect on myocardial tissues in burn rats, including increasing the content of ATP and EC, decreasing the content of TDP and TMP obviously after 3h burns, decreasing the content of LDH and TnI in serum dramatically after burns. In a word, the new strategy and methods were provided from the results for preventing and treatment"shock heart"after burn early, especially for providing exogenous protection to myocardium of postburn.