The Influence Of Myocardial Metabolic State On NPY Receptors Subtypes Expression In Pressure Overload Rats

BackgroundHypertension is one of the most common cardiovascular diseases. Themyocardium in pressure overload faces the ventricular remodeling and the metabolicremodeling. Cardiac energy state in pressure overload is relatively negativebalance,including the decrease of Phosphocreatine, and AdenosineTriphosphate,increased myocardial free ADP levels, the higher ratio of ADP/ATP.Ithas also been shown that the reduction in fatty acid oxidation in hypertrophied heartsis accompanied by an increase in glucose oxidation.Neuropeptide Y(NPY)is one of the most abundant peptides in heart and braintissues, which is present in, and released together with Norepinephrine (NE) fromperipheral sympathetic nerve terminals. NPY is critical in the modulation of energyhomeostasis. NPY release may affect all receptor subtypes simultaneously, however,the relative contribution of each receptor to a given physiological response may varysince receptor expression, at least at the mRNA level, is modulated by the metabolicstate. For example, in obesity, Y1and Y5mRNA levels are reduced while that for Y2mRNA is elevated; this would be consistent of a concerted and organized effort toreduce the orexigenic drive in obese states. Therefore, Y1, Y2and Y5receptors are thekey effect of NPY activation on the metabolic state. The Y1receptor stimulated foodintake in satiated rats, Y2reduces food intake in humans and rodents, and reducesbody weight in models of obesity, Y5receptor was able to potently increase foodintake in rats. Y1and Y5can promote the protein turnover and MHC expression inneonatal rat cardiomyocytes. However Y2is protective and can promote theangiogenesis in the heart. Although the similarity is existing between the enegy metabolic regulation ofNPY and the myocardial metabolism of pressure overload, but there is no relativereport wether myocardial metabolic state can regulate the NPY receptors subtypesexpression.We put forward the hypothesis: the myocardial metabolic state regulates NPYreceptors subtypes expression in pressure overload heart of rats, NPY takes part in thecardiac enegy regulation and NPY play differential action through each receptorexpression.Thus, our study focused on three aspects: the first is whether metabolic stateregulates myocardial NPY receptors subtypes expression in pressure overload rats;thesecond is whether the negative energy balance regulates the NPY receptors subtypesexpression in neonatal rat cardiomyocytes and the possible mechanism throughAMPK pathway;the third is wether the NPY receptors subtypes,agonist may regulatethe glucose uptake in neonatal rat cardiomyocytes. we hope this study can raise newidea between NPY receptors and myocardial energy regulation. Part1Metabolic State Regulates Myocardial NPY Receptors SubtypesExpression in Pressure Overload RatsObjectivesTo observe the changes of myocardial Metabolic state, the expression of NPYreceptors subtypes and myocardial remodeling in pressure overload rats on2,7,14day. To analyse the dynamic relationship between the myocardial metabolic state,the expression NPY receptors subtypes and myocardial remodeling pressureoverload rat.Methods1. The method of abdominal aorta constriction was used to build pressureoverload model. By their constricting or not, they were randomly divided into theoperated and sham operated groups and2days,7days,14days groups. Use ofnon-invasive tail artery blood pressure monitor to detect the systolic blood pressureand heart rate.Ultrasonic cardiogram examination determination of left ventricularmorphology and function of the heart. The parts of ventricles were fixed in formalinand microtome sections were stained with hematoxylin and eosin (H&E) andMasson-trichrome. Cross-sectional area of myocardial cells was measured by usingIPP6.0.Fibrosis was determined by measuring collagen staining using IPP6.0andexpressed as a percentage of staining (green) areas to the total areas examined.2. Using luninometer to detecte the ADP/ATP ratio in myocardial tissue;3. Using real-time PCR assay to detecte the mRNA expression of Y1, Y2andY5receptor in myocardium.4. Adopted Western blot to analyse Y1, Y2and Y5receptor protein;5. Immunohistochemical staining for Y1, Y2and Y5receptors distribution andexpression in the myocardium.Rezults1.Systolic blood pressure of AAC rats after2days,7days,14days significantlyincreased compared to sham group rats (P<0.05) at each time point. Heart rate ofAAC rats compared with sham group were significantly increased (P<0.05) at eachtime point. The ratio of HW/BW(mg g-1) was significantly increased at14days, compared with sham group (P<0.05). The ratio of LVW/BW(mg g-1) at7days,14days increased compared with the sham group (P<0.05), indicating the LVW/BWratio is more sensitive than the HW/BW ratio. The IVST(mm) changes is consistentwith the LVW/BW ratio. Changes of cardiac myocyte cross-sectional area isconsistent with the HW/BW ratio. However, myocardial fibrosis is not obvious. Thesedata indicate that pressure overload model was successfully established, the cardiachypertrophy of AAC after2days,7days did not happen, the myocardium was still inthe early stages of pressure overload, the myocardium of AAC after14days havehypertrophy characteristics,the left ventricle has been at the compensatoryhypertrophy stage in pressure overload in the rat.2.AAC rats after2days,7days,14days, the ADP/ATP ratio is significantlyhigher than the sham group rats (P<0.05), Gradually decreased with time extension, today14was significantly decreased, indicating that energy consumption and oxygenconsumption is higher in the early pressure load myocardial compared withdecompensated.3.AAC rats after2days,7days,14days Y1, Y2and Y5receptors subtypesmRNA expression was consistent with protein expression in the pressure overloadmyodium, distributed in the myocardial cell membrane and cytoplasm, the myocardialgene and protein expression was elevated in the early stage of pressure overload, todecompensated stage was decreased, compared with sham rats (P <0.05).Brief conclusion1.The myocardium in AAC rats after2days,7days was not hypertrophic,stayed in the early stages of pressure overload, to14days, the myocardium hasoccurred hypertrophy and been in the decompensated stage of pressure overload.2.AAC rats in the early stages of pressure overload, the myocardial energyconsumption increased and was in the apparent negative energy balance state, therehas been compensatory changes at14days.3.AAC rats in the early stages of pressure overload, the Y1, Y2, Y5receptormRNA expression and protein expression increased compared to the sham groups, butthey decreased in hypertrophic cardium at14days, the trend is consistent with the change of myocardial ADP/ATP ratio.Part2The Negative Energy Balance Regulates the NPY ReceptorsSubtypes Expression and the Mechanism in Neonatal Rat CardiomyocytesObjectiveTo observe the negative energy balance on the effect of Y1, Y2, Y5receptorsexpression in neonatal rat cardiomyocytes and the changes with AICAR (AMPKagonist)pretreatment. By detecting the level of phosphorylated AMPK andphosphorylated ACC,To explore the signal regulatory mechanism of these biologicalchanges.Methods1.Neonatal rats cardiomyocytes were isolated and cultured in DMED mediumwith10%fetal calf serum (FCS).2.Using AngII10-6M, AICAR0.5M, Compound C1μM, AICAR0.5M+AngII10-6M to stimulate the cultured neonatal rat cardiomyocytes in vitro.3.Using luninometer to detecte the ADP/ATP ratio in the treatedcardiomyocytes, To assess the metabolic state of neonatal rat cardiomyocytes.4.For assessing the Y1, Y2, Y5receptor expression and the level ofphosphorylated AMPK and phosphorylated ACC, the detection methods of mRNAexpression and protein expression were same as the methods mentioned in part1.Results1. AngII and Compound C treated groups can induce the increase of ADP/ATPratio in neonatal rat cardiomyocytes compared to the control(P<0.05),If given AICARpretreatment, the increased ADP/ATP ratio was decreased(P<0.05), indicating AngII,Compound C can induce the negative energy balance of cardiomyocytes in vitro.2. Myocardial cells under the state of negative energy balance, Y1, Y2, Y5receptors mRNA expression and protein expression increased(P<0.05).Given AICARpretreatment, they decreased with the improvement of ADP/ATP ratio(P<0.05).3. Cardiomyocytes in the basal state, the level of AMPK-PThr172and ACC-PSer79is low,using AngII stimulation of Cardiomyocytes two hour,the level ofAMPK-PThr172and ACC-PSer79slightly increased compared to the control group.AICAR can significant active AMPK-PThr172and ACC-PSer79. Given AICARpretreatment, their level was increase. Compound C can slightly inhibit the level ofAMPK-PThr172and ACC-PSer79.Given pretreatment, their level was increased again; Total AMPK and ACClevel has no significant change.Brief conclusion1.AngII, Compound C can induce the negative balance of myocardial energymetabolism, AICAR can improve this effect.2.Under the state of negative energy balance, Y1, Y2, Y5receptors mRNAexpression and protein expression increased, their expression decreased by improvingthe state of cellular energy metabolism,.3.Myocardial energy metabolism levels may regulate the expression of NPYreceptors subtypes through the AMPK pathway.Part3The NPY Receptors Subtypes’ Agonist Regulates the GlucoseUptake in Neonatal Rat CardiomyocytesObjectivesTo objective the effect of NPY receptors subtypes’ agonist on the glucoseuptake in neonatal rat cardiomyocytes.Methods1.Neonatal rat cardiomyocytes were isolated and cultured in DMED mediumwith10%fetal calf serum (FCS).2.Experimental groups:①the Control②The Y1agonist10-7M③The Y2agonist10-6M④The Y5agonist10-6M⑤The negative control.3.To assess cardiomyocytes take up2-NBDG, fluorescence microscopy andflow cytometry were used to detected fluorescence intensity between different groups.4.By immunocytochemistry to detect the expression and distribution ofGLUT4.Results1. Fluorescence imaging and flow cytometry analysis showed that: Comparedwith control group, Y1agonist can inhibit glucose uptake(P<0.05), Y2agonist in neonatal rat cardiomyocytes(P<0.05), Y5agonist can stimulate glucose uptake inneonatal rat cardiomyocytes(P<0.05).2.Y1agonist can inhibit cardiac GLUT4expression or translocation;while Y2,Y5agonists can promote cardiac myocyte expression of GLUT4translocation.Brief conclusionY1agonist by inhibiting GLUT4expression in cardiac myocytes inhibitsmyocardial glucose uptake; Y2, Y5agonist by promoting GLUT4expression topromote myocardial uptake of glucose.Conclusion1.AAC rats in the early stages of pressure overload, the myocardium was in theapparent negative energy balance state, while to14days there has beencompensatory and adaptive changes.2.AAC rats in the early stages of pressure overload, the Y1, Y2, Y5receptor geneand protein expression increased, while to14days decreased in hypertrophic cardium.3.Under the state of negative energy balance, Y1, Y2, Y5receptor gene andprotein expression increased; Myocardial energy metabolism levels may regulate theexpression of NPY receptors subtypes through the AMPK pathway.4.Y1agonist inhibited myocardial glucose uptake in cardiac myocytes; while Y2,Y5agonist promoted uptake of glucose. It was relative with the GLUT4expression.