Effects And Mechanism Of Action Of Interleukin-2 On Cardiovascular Functions In Diabetic Rats

Diabetes mellitus is an endocrine and metabolic disease caused by either lack of insulin secretion or hyposensitivity to insulin,and is characterized by hyperglycemia.Its incidence and prevalence have increased in recent decades,so that it has become the principal disease that endangers health and influences the quality of life.Diabetes mellitus has several sequelae, among which the cardiovascular complications are the most important and the principal cause of death in affected patients.So,it is of clinical importance to study the pathogenesis of diabetes and its cardiovascular complications and to investigate strategies for their prevention and treatment at an early stage.Inflammation plays a very important role in the pathology of diabetes.Many cytokines are involved in the inflammatory response,and interleukin-2(IL-2)is closely involved in the pathological process of diabetes and its related cardiovascular diseases.Serum IL-2 concentration changes in diabetic patients and animal models.Administration of IL-2 partly blocks the development of diabetes in non-obese diabetic mice.Accumulating evidence suggests a relationship between IL-2 and myocardial infarction,myocarditis,hypotension and cardiomyopathy.The mRNA level of IL-2 increases in the ischemic myocardium of rats subjected to left anterior descending artery occlusion.Our previous studies showed that pretreatment with IL-2 reduces the cardiac myocyte injury caused by hypoxia and reoxygenation,and IL-2 induces endothelium-dependent vasodilatation.Also,preliminary experiments showed that IL-2 prevents the inhibition of endothelium-dependent vasodilation caused by high glucose So we proposed that IL-2 has an important effect on cardiovascular function in diabetes or high glucose.However,the effects of IL-2 on cardiovascular function in diabetes remain unclear.Objectives1.To investigate the effects and mechanism of action of IL-2 on contractility in rat cardiomyocytes in vitro and heart in vivo,and on vascular responses in aortic rings.2.To assess the changes in cardiovascular functions in streptozotocin(STZ)-induced diabetic rats at early stages.3.To determine the effect of IL-2 on vascular responses in diabetes(or high glucose concentration)and explore the related underlying mechanisms.4.To explore the effect and mechanism of action of IL-2 on cardiac function in STZ-induced diabetic rats.Methods1.Isolated ventricular myocytes were prepared by enzymatic isolation.2.A video-tracking system was used to measure the peak velocity of cell shortening (+dL/dtmax),the peak velocity of cell relengthening(-dL/dtmax),the amplitude of contraction(dL)and the end-diastolic length of isolated myocytes.3.Calcium transients in ventricular myocytes were determined by a spectrofluorometric technique using the sensitive dye fura-2 as the Ca²⁺indicator.4.A diabetic rat model was established in male Sprague-Dawley rats by injection of a single dose of STZ(60 mg/kg,i.p.);rats with glucose levels＞16.7 mmol/L after about 72 h were considered diabetic.A model of vascular injury induced by diabetes was established by perfusing aortas with high glucose concentration.5.Aortic rings were mounted between two stainless steel hooks and suspended in an organ bath containing Krebs-Henseleit solution(K-H solution).The tension in the aortic rings was recorded with a data acquisition system(MedLab,Nanjing Medease Co.Ltd., China).6.Serum NO level,and NOS,SOD and GSH-PX activity in tissue were measured spectrophotometrically.7.Rat hearts were isolated and perfused at a constant flow rate.Immediately after stunning and cervical dislocation,normal male Sprague-Dawley rat hearts were rapidly excised, cannulated via the aorta and perfused retrogradely under constant flow(calibrated roller pump at 10 ml/min)with K-H solution containing a high(33.3 mmol/L)or normal(11 mmol/L)concentration of glucose.Coronary perfusion pressure(CPP)was monitored with a data acquisition system(MedLab,Nanjing Medease Co.Ltd.,China);8.Echocardiography was used to measure or calculate anteroposterior diameter(D)and short-axis area(A)of the left ventricle(LV)at the papillary muscle level at end diastole (ED)and end systole(ES),left ventricular eject fraction(EF),fractional shortening fraction(FS%),intraventricular septum thickness at the end of systole(IVSs),and left ventricular posterior wall(LVPW)thickness at the end of systole(LVPWs).9.Hemodynamic measurements were made throught an aortic catheter advanced into the left ventricle to record left ventricular systolic pressure(LVSP),left ventricular end diastolic pressure(LVEDP),left ventricular developed pressure(LVDP),maximal rate of left ventricle pressure development and decline(+/-dP/dtmax)and heart rate.10.Staining for pathology was carried out after embedding the heart in paraffin and cutting 5μm sections.Sections were stained with hematoxylin and eosin(HE)and fibrosis was quantitated using Masson's trichrome.11.The reverse transcription polymerase chain reaction(RT-PCR)was carried out as follows: Cardiac tissue was pulverized and total RNA was extracted using Trizol.The primers were designed by primer3,then synthesized by Sangon Co.The one-step RT reaction was amplified using TaqDNA polymerase and primers for Bcl IL-2 and TGF-μ1mRNA. GAPDH was the reference.The half-quantity of the mRNA expression of different genes was measured by image scanning and analysis after electrophoresis.12.A laser scanning confocal microscope was used to measure NO production in isolated cardiomyocytes.Results1.Effect and mechanism of action of IL-2 on cardiovascular function in vivo and in vitro(1)Echocardiography:LVEF,FS%,IVS%,and LVPW%were decreased by subcutaneous administration of IL-2 for 7 weeks,but LVIDd was not affected.(2)Hemodynamics:LVSP,LVDP and±dP/dt_maxwere decreased by subcutaneous administration of IL-2 for 7 weeks,but LVEDP was not affected.(3)Serum NO level:NO level in serum was increased by subcutaneous administration of IL-2 for 7 weeks.(4)Isolated myocyte contraction:Dose-dependent inhibition of±dL/dtmax, -dL/dtmax,dL and end-diastolic cell length were induced by IL-2 at 2-1000 U/ml.(5)Contraction of myocytes pretreated with L-NAME or ODQ:Pretreatment with the NO synthase inhibitor L-NAME and the soluble guanylyl cyclase inhibitor ODQ attenuated the IL-2-induced inhibition of contractility,while aminoguanidine,an inhibitor of inducible NO synthase,had no effect.(6)NO production in myocytes:IL-2 enhanced the fluorescence intensity of DAF-FM in cardiac myocytes time-and dose-dependently.Pretreatment with L-NAME attenuated the effect of IL-2.(7)[Ca²⁺]_i in myocytes:IL-2 markedly reduced the amplitude of the[Ca²⁺]_i transient in isolated myocytes. (8)[Ca²⁺]_i in myocytes in the presence of L-NAME or ODQ:The IL-2-induced decrease in the amplitude of the intracellular Ca²⁺transient was attenuated by L-NAME or ODQ.(9)Vascular function in aortic rings:ACh-induced endothelium-dependent vasodilatation was notably increased by treatment with IL-2(5000 or 50000 U/kg/d,s.c.),but SNP-induced endothelium-independent vasodilatation was not affected.Co-incubation with IL-2 increased ACh-induced endothelium-dependent vasodilatation,which was reversed by pretreatment with L-NAME,but had no effect on SNP-induced endothelium-independent vasodilatation.(10)tNOS and cNOS activity in aortic tissue:Incubation of isolated aortic segments with IL-2 resulted in an increase of total NOS,constitutive NOS(cNOS)and SOD activity.2.Changes in cardiovascular function in diabetic rats at an early stage(1)Body weight(BW):The mean BW of diabetic rats was reduced compared with the control group.(2)Ventricular weight/BW,liver weight/BW and kidney weight/BW:The ventricular weight/BW,liver weight/BW and kidney weight/BW values in diabetic rats were higher than controls.(3)Echocardiography:LVEF,FS%,IVS%,LVPW%and LVIDd values in diabetic rats were lower than controls.(4)Hemodynamics:LVSP,LVDP and±dP/dt_maxof diabetic rats were lower and LVEDP was higher than controls.(5)Vascular function in diabetes:ACh-induced endothelium-dependent vasodilatation(EDR)was notably lower in diabetic rats,but SNP-induced endothelium-independent vasodilatation was not affected.(6)Vascular function in high glucose:ACh-induced endothelium-dependent vasodilatation was time-dependently decreased in high glucose,but SNP-induced endothelium-independent vasodilatation in high glucose was not affected. 3.Effect and mechanism of action of IL-2 on vascular function in vivo and in vitro in diabetes(1)Blood glucose level and BW:IL-2 treatment(5000 or 50000 U/kg/d,s.c.)for 7 weeks had no effect on blood glucose level in diabetic rats,but lessened the decrease in BW induced by diabetes.(2)Vascular function in diabetes:ACh-induced endothelium-dependent vasodilatation was clearly impaired in diabetic rats,and this was attenuated by treatment with IL-2.SNP-induced endothelium-independent vasodilatation did not change.(3)Serum NO level:Treatment with IL-2 markedly attenuated the reduction of serum NO level induced by diabetes.(4)Vascular function in high glucose:Co-incubation with IL-2 prevented the inhibition of ACh-induced endothelium-dependent vasodilatation caused by high glucose in a concentration-dependent manner,and this was reversed by pretreatment with L-NAME. SNP-induced endothelium-independent vasodilatation was not affected.(5)NOS and SOD activity in aortic tissue in high glucose:Treatment with IL-2 attenuated the decrease of total NOS,cNOS and SOD activity caused by high glucose in aortic tissue.(6)CPP in high glucose;Pretreatment with IL-2(200 U/ml)for 10 rain attenuated the increase of CPP induced by high glucose,and this effect was not abolished by pretreatment with norbinaltorphimine,a selectiveκ-opioid receptor antagonist.4.Effect and mechanism of action of IL-2 on cardiac function in diabetes(1)Ventricular weight/BW:Treatment with large dosage of IL-2 reduced ventricular weight/BW in diabetes.(2)Echocardiography:The large dosage of IL-2 reduced the diabetes-induced decrease in LVEF,FS%,IVS%and LVPW%,but had no effect on the diabetes-induced decrease in LVIDd.(3)Hemodynamics:LVDP,LVSP,and±dP/dtmax in diabetic rats treated with the large dosage of IL-2 were elevated,whereas this had no effect on the diabetes-induced increase in LVEDP and decrease in HR.(4)Cardiac morphology:In the myocardium of diabetic rats,disorganized cardiac muscle,fibrosis in interstitial cells and perivascular fibrosis were markedly increased in HE and Masson staining.The large dosage of IL-2 reduced the number of collagen-positive interstitial cells and the perivascular fibrosis caused by diabetes.(5)SOD and GSH-Px activity in ventricular tissue:Treatment with the large dose of IL-2 increased the activity of SOD and GSH-Px in diabetic rats.(6)TGF-β₁mRNA and Bcl-2 mRNA expression in ventricular tissue:Increased expression of TGF-β₁mRNA and reduced expression of Bcl-2 mRNA in ventricular tissue were measured in untreated diabetic rats.The expression Bcl-2 mRNA was upregulated and that of TGF-β₁mRNA was downregulated after treatment with the large dose of IL-2.Conclusions1.IL-2 is negatively inotropic in the heart in vivo and in vitro.A direct action of IL-2 on cardiomyocyte contraction,possibly through increased NO production,activation of sGC and reduction of intracellular Ca²⁺level.IL-2 increases ACh-induced endothelium-dependent vasodilatation,possibly through increased NOS activity and NO production.2.Diabetes and hyperglycemia markedly inhibit endothelium-dependent vasodilatation. IL-2 attenuates the inhibition of endothelium-dependent vasodilatation induced by high glucose or diabetes,in which activation of the NO pathway and decrease of oxidative stress injury are involved.Pretreatment with IL-2 attenuates the increase of CPP induced by high glucose,and this effect is not abolished by pretreatment with norbinaltorphimine, a selectiveκ-opioid receptor antagonist.3.STZ-induced diabetic rats at the 7th week display systolic and diastolic dysfunction. Administration of IL-2 improves diabetic cardiac function,which is related to increased activity of antioxidant enzymes and inhibited cardiac remodeling.Upregulation of Bcl-2 mRNA and downregulation of TGF-β1mRNA may be involved.