The Roles Of TLR-4 And IGF-1 In Ischemia/reperfusion And Endotoxin-induced Cardiac Dysfunction In Murine Cardiomyocytes

Background and ObjectiveWith the widespread application of percutaneous coronary intervention(PCI) and coronary artery bypass grafting(CABG) in ischemia heart disease,myocardial ischemia and reperfusion(I/R) injury received intensive attention from heart disease experts.In the past few years,except that applying medicines and the gene treatment method,growing evidence from animal experiments and even clinical observations indicates that Toll-like receptor 4 contributes significantly to subsequent functional and cellular injury through a variety of pathological pathways such as in liver or kidney ischemia and reperfusion,and it has been one of the most intensely studied topics in biomedical research and clinical practice.However,the signaling mechanisms in the affection of Toll-like receptor 4 in cardiac ischemia and reperfusion(I/R) injury are incompletely understood.Myocardial ischemia resulted from blockade in the coronary arteries caused by atherosclerosis can impair oxygen delivery.Toll-like receptor 4(TLR-4),a proximal signaling receptor in innate immune responses to lipopolysaccharide of Gram-negative pathogens is expressed in the heart and vasculature.TLR-4 binds lipopolysaccharide(LPS) from the gram-negative bacteria and autogenous ligands such as heat shock proteins and fibronectin which are released during oxidative stress, following ischemic injury.Increased TLR-4 expression has been observed in human heart failure and ischemic hearts.Mitogen-activated protein kinases(MAPKs) such as p38,ERK,and JNK are located downstream of TLR-4.Ischemia activates p38,which demonstrated negative inotropic and restrictive diastolic effects.The AMP-activated protein kinase(AMPK) is an energy-sensing enzyme that can be activated by ischemia,and it has been found that AMPK plays an important role in cardioprotection against ischemic injury.In cardiac muscle,AMPK activity is increased by stimuli such as exercise,hypoxia,ischemia,and stress.It plays a central role in monitoring the cellular energy status and controlling energy production and consumption.Intriguingly,it has been reported that AMPK protects cardiomyocytes against hypoxic injury by attenuation of endoplasmic reticulum(ER) stress.In eukaryotic cells,the ER plays a vital role in the maturation,processing and transporting secretory and membrane associated proteins.The ER is exquisitely sensitive to alteration in homeostasis,with perturbations in the environment resulting in a condition known as ER stress.However,the molecular mechanisms behind TLR-4 deficiency-induced cardioprotection against ischemia/reperfusion damage remain unclear.Here we examined whether the cardioprotection of TLR-4 deficient hearts against ischemia/reperfusion injury mediated by AMPK signaling pathways and what's the role of TLR-4 downstream,the MAPK signaling pathway.We also try to address the relationship between ischemic stress and ER stress in the heart.Materials and methodsIn vivo regional ischemia was induced by occlusion of the left anterior descending(LAD) coronary artery in wild type(WT) C3H/HeN and TLR-4 deficient C3H/HeJ mice that were purchased from the Jackson Laboratory(Bar Harbor,ME). C3H/HeJ do not express functional TLR-4 because of naturally occurring mutations in the TLR-4 gene(Tlr4^Lps-d).C3H/HeN and C3H/HeJ Mice were anesthetized with ketamine(95 mg/kg) and xylazine(40 mg/kg) intraperitoneally,incubated,and ventilated with a respirator.After thoracotomy,a suture was placed to ligate the proximal left anterior descending(LAD) coronary artery for different time requirement.Control mice underwent sham thoracotomy.Mouse hearts were divided into 8 experimental groups:(1) C3H/HeN mice(WT) sham(n=7);(2) C3H/HeN(WT) I/R 60 rains(n=7);(3) C3H/HeN(WT) I/R 120 rains(n=7);(4) C3H/HeN(WT) ischemia 15 mins(n=7);(5) C3H/HeJ(TLR-4) sham(n=7);(6) C3H/HeJ(TLR-4) I/R 60 mins(n=7);(7) C3H/HeJ(TLR-4) I/R 120 mins(n=7) and(8) C3H/HeJ(TLR-4) ischemia 15 mins(n=7).After ischemia/reperfusion,the myocardial infarct size was determined by means of a double-staining technique.Evan's blue stained area,TTC stained area and TTC stained negative area were measured digitally using Image Pro Plus software.The degree of apoptotic activity was assessed by measuring caspase-3 activity in tissue homogenates using a commercially available fluorimetric assay.In addition,TUNEL assessment of myonuclei positive for DNA strand breaks was determined using a fluorescence detection kit.Heart tissue was analyzed for tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6 levels as determined by Enzyme-linked immunosorbent assay(ELISA).Immunoblotting analysis was performed to assess the activation of AMPK and its downstream protein ACC and eEF2 and expression of the stress signaling the mitogen-activated protein kinase (MAPK) molecules JNK,p38 and ERK as well as the ER stress markers GRP78, Gadd153 and IRE1α.Data were Means±SEM.Differences between groups were assessed by variance (ANOVA) followed by Newman-Keuls post hoe test using GraphPad Prism 4.A P value less than 0.05 was considered statistically significant.ResultsThe extent of myocardial infarction was measured with dual staining to define the degree of necrosis within the ischemic region at risk.C3H/HeJ hearts demonstrated significantly smaller infarct size than C3H/HeN hearts.Ischemia and reperfusion can initiate pathways leading to cardiac apoptosis.Our data showed that there was an increase in caspase-3 activity cardiac myocytes in C3H/HeN hearts compared with C3H/HeJ hearts,suggesting greater activation of apoptotic pathways in C3H/HeN hearts.In addition,confocal microscopy revealed a greater number of TUNEL-positive nuclei following ischemia/reperfusion in C3H/HeN hearts compared with C3H/HeJ hearts.We determined proinflammatory cytokines TNF-α,IL-1βand IL-6 levels in serum.For all cytokines,significantly higher serum levels were seen in WT and C3H/HeJ hearts following MI/R compared to their shams.As would be expected,for WT hearts,all cytokines were higher with I/R vs.C3H/HeJ hearts. These results indicate that TLR-4 deficiency leads to a cardioprotective effect to prevent ischemia and reperfusion injury.To assess whether AMPK signaling pathway is involved in the resistance to I/R injury in C3H/HeJ mice,we first examined the AMPK phosphorylation of C3H/HeJ and C3H/HeN hearts during in vivo regional of I/R.The phosphorylation at Thr172 and activation of AMPK was significantly augmented in C3H/HeJ hearts as compared to that in C3H/HeN hearts.Moreover,the phosphorylation of the AMPK downstream targets,acetyl-CoA-carboxylase(ACC) and eukaryotic elongation factor 2(eEF2), were also enhanced in C3H/HeJ hearts compared to C3H/HeN hearts.In order to determine the role of the TLR-4 downstream effectors,such as MAPKs in ischemic injury,we measured the activation of p38,JNK and ERK.The results demonstrated that ischemia time-dependently stimulated p38,JNK and ERK activation in both C3H/HeN and C3H/HeJ hearts as manifested by phosphorylation immunoblots.Intriguingly,p38 and JNK signaling were blunted in C3H/HeJ hearts compared to C3H/HeN hearts during ischemia.In contrast,ERK signaling was enhanced in C3H/HeJ hearts compared to C3H/HeN hearts during ischemia.It has been reported that ischemia leads to the accumulation of misfolded proteins in the endoplasmic reticulum(ER),causing ER stress.Under conditions of ER stress,inhibition of protein synthesis and up-regulation of ER chaperone expression to reduce the misfolded proteins in the ER.We therefore examined whether TLR-4-deficiency affected ischemia and reperfusion induced ER stress in the hearts.The results demonstrated that ischemic treatment promoted ER stress,as shown by up-regulation of the ER chaperone,Gadd153/CHOP andGrp78/BiP,and the integral protein of ER membrane,IRE1α,while up-regulation in all these ER stress makers was blunted in C3H/HeJ hearts during I/R.Conclusions1.TLR-4 deficiency reduces infarct size after Ischemia/Reperfusion;2.Reduced Apoptosis in TLR-4-deficient Hearts during Ischemia/Reperfusion;3.TLR-4 deficiency reduces proinflammatory cytokines TNF-α,IL-1βand IL-6 levels in serum;4.Augmented AMPK Activation in TLR-4-deficient Mice during Ischemia/Reperfusion;5.Impaired Activation of MAPK Signaling in C3H/HeN vs.C3H/HeJ during Ischemia/Reperfusion;6.Resistance to Cardiac ER Stress in TLR-4-deficient Heart during Ischemia/Reperfusion. Background and ObjectiveInsulin-Like Growth Factor I(IGF-1) is essential for the maintenance of bodily structure and function.IGF-1 participates in the regulation of tissue remodeling, glucose metabolism,insulin sensitivity,lipid profile,myocardial growth and myocardial function in both physiological and pathophysiological conditions.The deficiency of which is associated with altered body composition,cytokine and neuroendocrine activation,cardiac atrophy and impaired cardiac function.A fall in the serum IGF-1 level,which often accompanies the biological aging process,leads to abnormal body composition and metabolism.The severely impairment of lifespan in IGF-1-deficient Ames dwarf mice compromised cardiac excitation-contraction coupling in cardiomyocytes.Similarly,patients with IGF-1 deficiency exhibit cardiac dysfunctions reminiscent of aging,which is mainly manifested as reduced left ventricular mass,ejection fraction and diastolic filling.Lipopolysaccharide(LPS) is a major component of the outer membrane of Gram-negative bacteria,contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack.LPS also increases the negative charge of the cell membrane and helps stabilize the overall membrane structure.It is of crucial importance to gram negative bacterial cells;death results if it is mutated or removed.LPS is an endotoxin,and induces a strong response from normal animal immune systems.It acts as the prototypical endotoxin because it binds the CD14/TLR4/MD2 receptor complex,which promotes the secretion of pro-inflammatory cytokines in many cell types,but especially in macrophages.Severe sepsis remains a major cause of death in hospitalized patients in the Unites States,and these deaths are caused frequently by shock,which results in multiple organ failure. Myocardial depression is a common feature of endotoxemia in patients and in experimental model of lipopolysacharide(LPS)-induced sepsis,and has been recognized as a key component contributing to the development of septic shock.Low levels of LPS may mediate vascular inflammation in atherosclerosis.Low circulating levels of insulin-like growth factorâ… (IGF-1) are found in sepsis although the influence of IGF-1 on septic cardiac defect is still unknown.This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca²⁺ dysfunction,activation of stress signal and endoplasmic reticulum(ER) stress.Materials and methodsWeight-(20-25g) and age-(4-5 months of age) matched adult male FVB and IGF-1 transgenic mice were used in this study.All animals were kept in our institutional animal facility with free access to standard laboratory chow and tap water. On the day of experimentation,both FVB and IGF-1 transgenic mice were injected intraperitoneally with 4 mg/kg Escherichia Coli LPS dissolved in sterile saline or an equivalent volume of pathogen-free saline(for control groups).The dosage of LPS injection was chosen based on previous observation of overt myocardial dysfunction without significant mortality.Six hrs following LPS challenge,mice were sacrificed for experimentation.Serum IGF-1 was analyzed using an enzyme-linked immunosorbent assay(ELISA)in accordance with manufacturer's recommendations (R & D System Inc,Minneapolis,MN,USA).Cardiomyocytes were isolated by Liberase Blendzyme 4 for 20 mins using Langendorff system.Myocyte yield was～70%which was not overtly affected by either LPS or IGF-1.Only rod-shaped myocytes with clear edges were selected for mechanical and intracellular Ca²⁺ transient studies.To elucidate the cardiac-specificity, if any,of LPS-induced cardiomyocyte mechanical response,a sub-lethal dose of Escherichia coli LPS(1μg/ml) was administered to cardiomyocytes isolated from FVB mice.The cells were incubated with LPS for 4-6 hrs in the presence or absence of exogenous recombinant IGF-1(50 nM).The mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system(IonOptix Corporation,Milton,MA,USA).An IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening.Cell shortening and relengthening were assessed using the following indices:peak shortening(PS)-indicative of the amplitude a cell can shorten during contraction;maximal velocities of cell shortening and relengthening(±dL/dt)-indicative of peak ventricular contractility;time-to-PS(TPS)-indicative of systolic duration;time-to-90%relengthening(TR₉₀)-indicative of diastolic duration(90% rather 100%relengthening was used to avoid noisy signal at baseline concentration).Myocytes were loaded with fura-2/AM(0.5μM) for 10 mins and fluorescence measurements were recorded with a dual-excitation fluorescence photo multiplier system(Ionoptix).The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca²⁺ concentration were inferred from the ratio of fura-2 fluorescence intensity(FFI) at two wavelengths(360/380). Fluorescence decay time was measured as an indication of the intracellular Ca²⁺ clearing rate.Both single and bi-exponential curve fit programs were applied to calculate the intracellular Ca²⁺ decay constant.Production of cellular ROS was evaluated by analyzing changes in fluorescence intensity resulting from oxidation of the intracellular fluoroprobe 5-(and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate(CM-H₂DCF-DA, Molecular Probes,Eugene,OR,USA).The carbonyl content of protein was determined using protein carbonyl assay.The degree of apoptotic activity was assessed by measuring caspase-3 activity in tissue homogenates using a commercially available fluorimetric assay.Expression of the stress signaling molecules JNK,p38 and ERK,the apoptotic proteins Bax and Bcl-2 as well as the ER stress markers GRP78 and Gadd153 was assessed using Western blotting.GAPDH was used as the internal loading control.Data were Means±SEM.Differences between groups was assessed by variance (ANOVA) followed by Tukey post hoc test using GraphPad Prism 4 or student's t-test wherever appropriate.Ap value less than 0.05 was considered statistically significant.ResultsAcute LPS treatment failed to affect plasma IGF-1 levels in either IGF-1 or FVB mice.Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening as well as prolonged duration of relengthening in LPS-treated FVB cardiomyocytes associated with reduced intracellular Ca²⁺ decay. Accumulation of ROS,protein carbonyl and apoptosis were elevated following LPS treatment.Western blot analysis revealed activated p38 and JNK,upregulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2.Total protein expression of p38,JNK and ERK was unaffected by either LPS or IGF-1.Interestingly,these LPS-induced changes in mechanical and intracellular Ca²⁺ properties,ROS,protein carbonyl,apoptosis,stress signal activation and ER stress markers were effectively ablated by IGF-1.In vitro LPS exposure(1μg/ml) produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting,which was alleviated by exogenous IGF-1(50 nM).Conclusions:1.Acute LPS treatment failed to affect plasma IGF-1 levels in either IGF-1 or FVB mice;2.In vitro study shows that LPS-elicited cardiomyocyte defect and IGF-1-offered cardioprotection may be attributed,at least in part,to changes in intrinsic cardiomyocyte properties; 3.IGF-1 itself did not elicit any overt effect on mechanical and intracellular Ca²⁺ properties.Overexpression of the growth factor significantly attenuated LPS-induced cardiomyocyte mechanical and intracellular Ca²⁺ dysfunction;4.IGF-1 reduced LPS-elicited effects on ROS generation,protein damage and apoptosis:5.IGF-1 ablated LPS-induced Stress signaling activation,apoptotic proteins and ER stress.These data collectively suggested a beneficial of IGF-1 in the management of cardiac dysfunction under sepsis.