mRNA In Myocardium Research Articles

Abstract 11926: Selective Inhibition of Rho Kinase 2 Limits No Re-Flow in Rat Hearts Following Ischemia/Reperfusion

Introduction: Myocardial infarction causes several types of injury to the myocardium including lethal cell injury and ‘no-reflow’ (NRF) /microvascular obstruction (MVO). Nonselective Rho Kinase (ROCK1/2) inhibitors such as Fasudil, ameliorate myocardial ischemia/reperfusion (I/R) injury but cause unwanted hypotension. Selective ROCK2 inhibitors (e.g.: KD025) are safe in clinical trials without causing haemodynamic compromise, however they have not been investigated in myocardial I/R. ROCK inhibitors prevent vascular smooth muscle cell (VSMC) contraction; such VSMC contraction/coronary spasm being features of ‘no-reflow’ (NRF) and microvascular obstruction (MVO). At present, there are limited therapies to improve ischemic MVO outcomes, and prognosis is poor. Hypothesis: Using a rat model, we hypothesised that, i) ROCK2 mRNA is expressed in myocardium and coronary vasculature and ii) The selective ROCK2 inhibitor KD025, would reduce infarct size (IS%) and NRF% (MVO) following I/R. Methods: RNA scope in-situ hybridisation was performed with a fluorescent, multiplex assay for ROCK1/2 & VSMC mRNA in myocardium and coronary vasculature. Male SD rats underwent in-vivo myocardial infarction with 30min ischemia, 180min reperfusion. 15min prior to reperfusion, the ROCK inhibitors Fasudil and KD025 or vehicle (DMSO) were administered i.p. For IS%, myocardium was stained with TTC, and regions not perfused with 1.5% Thioflavin S (NRF%), were visualised under UV light. Results: RNAscope confirmed the presence of ROCK2 mRNA within myocardium and VSMC of coronary arteries. Fasudil ( 10mg/kg ) vs control significantly reduced regional IS% ( 30.3 ± 4.4 vs 52.9 ± 3.8 , p=0.02, n=15 ) and area of NRF% ( 12.4 ± 2.8 vs 28.6 ± 2.2 , p=0.001, n=15 ). However, there was significant hypotension;- Mean BP (mmHg) (72±3.9) vs control (84±2.3, p=0.007 ). KD025 ( 100mg/kg) did not reduce IS%, but significantly reduced the area of NRF% vs control ( 18.4 ± 2.8 vs 28.6 ± 2.2, p=0.02, n=14 ) without hypotensive effect. Conclusions: Our results suggest that ROCK2 may be a prospective target in the management of coronary circulation reperfusion injury and ischemic MVO.

Role of Phosphorylated AMP-Activated Protein Kinase (AMPK) in Myocardial Insulin Resistance After Myocardial Ischemia-Reperfusion During Cardiopulmonary Bypass Surgery in Dogs.

BackgroundThe aim of this study was to determine the role of AMP-activated protein kinase (AMPK) in myocardial insulin resistance after myocardial ischemia-reperfusion during cardiopulmonary bypass surgery in dogs.Material/MethodsTwenty-four mongrel dogs were randomly assigned to 4 groups. The control group did not undergo aortic cross-clamping; the model group underwent 60 mins of aortic cross-clamping with 150 ml cardioplegic solution. The treatment group, the inhibition group respectively with 0.11mg/kg AICAR (AMPK agonist) in 150 ml cardioplegic solution and 0.11 mg/kg Compound C (AMPK inhibitor) in 150 ml cardioplegic solution. The blood flow was determined and left ventricular myocardial tissue were taken at pre-bypass, 15, 60, and 90 min after aorta declamping, respectively. Expression of AMPK mRNA, p-AMPK and GLUT-4 proteins was determined by RT-PCR, IHC and WB.ResultsCompared with the control group, receiving 60 min ischemia at 15 min after reperfusion, Myocardial Glucose Extraction Ratio were significantly decreased in the other 3 groups, it was significantly decreased from 20.0% to 1.2% at 60 min of reperfusion, and recovered to 6.1% after 90 min reperfusion in model group, while recovered to 4.1%, 12.0% after 90 min reperfusion respectively exposed to Compound C and AICAR. The expressions of p-AMPK, GLUT-4 protein and AMPK mRNA in myocardium were decreased in different experiment groups, but these changes occurred to a lesser extent in the treatment group.ConclusionsThe inability of GLUT-4 expression induced by the decreases in p-AMPK protein expression that may be one of the reasons for myocardial insulin resistance.

Expression of ADAMTS-1 mRNA in myocardium of viral heart disease mice and its clinical significance.

The expression of ADAMTS-1 mRNA in myocardium of viral heart disease (VHD) mice was investigated to explore its role in myocardial fibrosis. A total of 150 purebred inbred Balb/c mice were used in this study. According to the principle of similar body weight, 50 mice were selected to make an acute viral myocarditis (VMC) animal model (acute VMC group), and 50 mice were selected to make a chronic VMC animal model (chronic VMC group), and the remaining 50 mice were selected as a control group. RT-qPCR was used to detect the relative expression of transforming growth factor-β1 (TGF-β1) mRNA and ADAMTS-1 mRNA in myocardial tissue of three groups of mice, and their relationship in myocardial fibrosis was analyzed. Compared with the control group, the collagen volume fraction (CVF) in the myocardial tissue of the acute VMC group was significantly increased, and the increase of CVF in the myocardial tissue of the chronic VMC group was the most significant (p<0.001). Compared with the control group, the relative expression of TGF-β1 mRNA and ADAMTS-1 mRNA in myocardial tissue of the mice in the acute and chronic VMC group were significantly increased (p<0.001). The relative expression of TGF-β1 mRNA and ADAMTS-1 mRNA in myocardial tissue of chronic VMC group was significantly higher than that of acute VMC group (p<0.001). Pearson's correlation test results showed that ADAMTS-1 mRNA was positively correlated with CVF and TGF-β1 mRNA, and the correlation coefficients were (r=0.351, p<0.01, r=0.401, p<0.01). ADAMTS-1 is involved in the occurrence and development of myocardial fibrosis, and it is positively correlated with CVF and TGF-β1. It may play a role in promoting myocardial fibrosis during the development of VHD. It can be used as a biological index for predicting myocardial fibrosis.

Differential expression of B-type natriuretic peptide between left and right ventricles, with particular regard to sudden cardiac death

The aim of the present study was to investigate the differential expression of B-type natriuretic peptide (BNP) between the left and right ventricle (RV) in sudden cardiac death (SCD). A total of 26 forensic autopsy cases of sudden death (survival time <30 min, postmortem interval <48 h or frozen within 6 h following death) in the present institute were examined. The cases consisted of acute ischemic heart disease (AIHD, n=15) with/without apparent myocardial necrosis as a sign of infarction (acute myocardial infarction, n=6; ischemic heart disease, IHD, n=9), and arrhythmogenic right ventricular cardiomyopathy (ARVC/D, n=5), in addition to traffic accidents and high falls without any pre existing heart disease as control (C, total n=6). BNP was investigated in all cases by the colloidal gold method, hematoxylin-eosin staining, immunohistochemistry (IHC) and the molecular pathological method. The IHC results demonstrated that a positive BNP immunostaining was detected in all groups; however, there was no difference between different causes of death. Pericardial N-terminal (NT)-proBNP concentration was significantly increased in deaths resulting from AIHD and ARVC/D compared with control group. The relative quantification of BNP mRNA demonstrated that relative expression levels of BNP mRNA were significantly increased in the left ventricle (LV) in the AIHD group, and in the RV of the ARVC/D group. The relative quantification difference and ratio of BNP mRNA between LV and RV demonstrated a significantly greater value in the AIHD group compared with control group. BNP mRNA in myocardium and NT-proBNP concentration in pericardial fluid were elevated in SCD patients, and left ventricular dysfunction predominated in AIHD patients, whereas right ventricular dysfunction predominated in ARVC/D patients. The results of the present study suggest the possible use of molecular pathology of BNP for the determination of terminal cardiac function in SCD and analysis of its fatal mechanism in forensic practice.

Expression of ryanodine receptor mRNA and sarcoplasmic Ca²⁺-ATPase mRNA in the myocardium and intracellular reorganization of cardiomyocytes in dyslipidemia and during verapamil treatment.

In experiments on rats, atherogenic diet led to hypercholesterolemia, while addition of verapamil to the diet led to the development of hypertriglyceridemia in these animals. Dyslipidemia induced significant changes in the cardiomyocyte ultrastructure (lytic changes in myofibrils and sarcoplasmic matrix and aggravation of autophagocytosis) that were most pronounced after addition of mercazolyl alone to the diet. After 30-day atherogenic diet, we observed a decrease in the relative content of RyR2 mRNA (by 67-73%, p<0.01) and SERCA2a mRNA (by 75-91%, p<0.01) in the myocardium. In 64 days these parameters remained reduced: by 64-72% (p<0.05) and 54-62% (p<0.05), respectively. Verapamil treatment reduced the severity and number of lytic lesions in cardiomyocytes, induced considerable glycogen accumulation in the sarcoplasm and its sequestration, promoted normalization, and prevented pronounced decrease of the relative RyR2 mRNA and SERCA2a mRNA in rat myocardium.

ASSA13-03-22 The Relation Between Mitofusin-2 and b3-Adrenergic Receptor and Its Possible Mechanism in Chronic Heart Failure

<h3>Objective</h3> To investigate the effects of β₃-adrenergic receptor (β₃-AR) on apoptosis and its possible mechanism of rats with isoproterenol (ISO)-induced chronic heart failure (HF). <h3>Methods</h3> (1) The rats were randomly divided into three groups: 7 weight– matched normal adult rats as controls, 18 rats with ISO induced heart failure and 21 rats with ISO induced heart failure but received SR59230A treatment for 6 weeks. (2) Echocardiography examinated left ventricular ejection fraction (EF), early peak diastolic velocity/late peak diastolic velocity (E/A), fractional shortening (FS), left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD). (3) Ratio of left ventricular weight and body weight (LVW/BW) was calculated. (4) The expression levels of β₃-AR, mitofusin-2 (Mfn2) and Caspase-3 mRNA in myocardium were detected by reverse transcriptio-polymerase chain reaction (RT-PCR). (5) The expression of β₃-AR, Mfn2, Caspase-, receptor serine/threonine kinases (Akt) and p-Akt protein were detected by western blot. <h3>Results</h3> (1) Compared with control group, the cardiac function of ISO group aggravated, LVW/BW and β₃-AR mRNA expression increased in heart failure rats; these changes significantly reduced in ISO+SR59230A group. (2) In ISO group, the expression of β₃-AR, Mfn2 and Caspase-3 protein rised as well as Akt decreased in relative to control group, myocardial apoptosis was significantly increased and that reverse significantly in ISO+SR59230A group. <h3>Conclusions</h3> In heart failure, the expression of β₃-AR increased, SR59230A inhibited β₃-AR and Mfn2, β₃-AR induced cardiomyocyte apoptosis through Mfn2. Mfn2 may become new therapeutic benefits with heart failure.

ASSA13-03-33 Effects of b3-Adrenergic Receptor Antagonist on SOCS3 Expression and Cell Apoptosis in Heart Failure Model Rats

<h3>Objective</h3> To observe the influence of β₃-AR antagonist on expression of Suppressor of cytokine signalling 3 (SOCS3), receptor serine/threonine kinases (Akt), pAkt and Caspase3 in heart failure (HF) model rats induced by isoprenaline (ISO), observe the influence of β₃-AR antagonist in HF, and provide a theoretical basis for prevention and control HF. <h3>Methods</h3> (1) The rats were randomly divided into three groups:Igroup (10 weight-matched normal adult rats as controls), IIgroup (25 rats with ISO induced heart failure) and IIIgroup (25 rats with ISO induced heart failure but received SR59230A treatment). (2) Echocardiography examinated left ventricular ejection fraction (EF), fractional shortenings (FS), left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD). (3) Ratio of left ventricular weight and body weight (LVW/BW) was calculated. (4) The expression levels of β₃-AR and SOCS3 mRNA in myocardium were detected by reverse transcription-polymerase chain reaction (RT-PCR). (5) The expression of SOCS3, Akt, pAkt and Caspase3 protein in myocardium were detected by western blot. <h3>Results</h3> (1) Compared withIgroup, FS, LVEF was decreased and LVEDD, LVESD was increased inIIgroup; compared withIIgroup, FS, LVEF was increased and LVEDD, LVESD was decreased in IIIgroup. (2) Compared withIgroup, LVW/BW was increased inIIgroup, compared withIIgroup, LVW/BW was decreased in IIIgroup. (3) Compared withIgroup, β₃-AR and SOCS3 mRNA expression was increased in IIgroup; compared withIIgroup, β₃-AR and SOCS3 mRNA expression was decreased inIIIgroup. (4) Compared withIgroup, SOCS3, Caspase3 protein expression was increased and pAkt/Akt was decreased in IIgroup; compared withIII group, SOCS3, Caspase3 protein expression was decreased and pAkt/Akt was increased in III group. <h3>Conclusions</h3> The expression of β₃-AR, SOCS3 and Caspase3 was increased, pAkt/Akt was decreased, cardiac performance was reduced and cell apoptosis was increased in heart failure rats; β₃-AR inhibitor may through SOCS3-pAkt/Akt-Caspase3 increase cardic function and decrease cell apoptosis, may provide a new signal path therapy for heart failure.

ASSA13-03-36 Effects of B3-Adrenergic Receptor Antagonist on Myocardial UCP2 Expression and Energy Metabolism in Chronic Heart Failure Rats

Objective To observe the effects of β 3 -adrenergic receptor (β 3 -AR) antagonist on myocardial Uncoupling protein2 (UCP 2 ) expression and energy metabolism in chronic heart failure rats. Methods 7 weight-matched normal adult rats (control group), 18 rats with ISO induced heart failure (ISO group) and 21 rats with ISO induced heart failure but received specific β 3 -AR inhibitor SR59230A (ISO+ SR59230A group) for 6 weeks were included in this research. At the end of the study, Echocardiography was performed; the ratio of left ventricular weight and body weight (LVW/BW) was calculated. The expression of β 3 -AR and UCP 2 mRNA in myocardium were detected by reverse transcription-polymerse chain reaction (RT-PCR), the UCP 2 protein in myocardium were detected by western blot. The myocardial contents of creatine phosphate (PCr) and adenosine triphosphate (ATP) were measured by high performance liquid chromatography (HPLC). Results Compared with control group, the cardic function was significantly reduced and myocardial β 3 -AR mRNA significantly increased, UCP 2 mRNA and protein was also significantly increased in ISO group, this change could be attenuated by the treatment with SR59230A, and the expression of myocardial UCP 2 protein negatively correlated with PCr contents. Conclusions In the chronic stage of heart failure, the expression of UCP 2 increases, which causes myocardial energy shortage, SR59230A improves myocardic energy efficiency and cardiac function by means of suppressing the the expression of UCP 2 .

CTLA4-Ig Relieves Inflammation in Murine Models of Coxsackievirus B3-Induced Myocarditis

BackgroundT-cell–mediated cellular immunity is one of the most important factors in viral myocarditis. As an important costimulatory molecule, cytotoxic T-lymphocyte–associated antigen 4 (CTLA4) alleviates autoimmunity by influencing the balance of helper T cell (TH) subtype 1 (TH1) to TH2 in autoimmune diseases. The effects and mechanisms of CTLA4 fusion protein (CTLA4-Ig) in mice with coxsackievirus B3 (CVB3)–induced myocarditis were investigated. MethodsBALB/c mice were randomly divided into a CVB3 group, an IgG group, a CTLA4-Ig group, and a group of healthy control mice. Mice were humanely killed on day 7 post CVB3 inoculation, then CVB3, IFN-γ, mouse IL-4 (mIL-4), and mouse IL-2 (mIL-2) expression in myocardium were examined by real-time quantitative polymerase chain reaction, and the serum concentrations of IFN-γ, mIL-4, and mIL-2 were measured by enzyme-linked immunosorbent assay. ResultsIFN-γ expression was significantly higher and mIL-4 levels in serum were lower in the CVB3 group when compared with those in the healthy control group (P < 0.01). In the CTLA4-Ig group, the mouse mortality and CVB3 mRNA in myocardium were reduced compared with those in the CVB3 group. Furthermore, IFN-γ expression was lower, and mIL-4 was significantly higher compared with those values in the CVB3 and the IgG groups. The levels of mIL-2 in all groups showed no statistical difference (P > 0.05). ConclusionsTH1 cytokines were predominant in the acute phase of viral myocarditis. CTLA4-Ig relieves myocardial inflammation, virus replication, and mouse mortality, probably by influencing the balance of TH1 to TH2.

Involvement of the STAT3-Th17 axis in viral myocarditis induced by Coxsackievirus B3

ObjectiveThis study was designed to explore whether the STAT3-Th17 axis is involved in the pathology of viral myocarditis (VMC) induced by Coxsackievirus B3 (CVB3).MethodsWe induced VMC with male BALB/c mice...

Effects of Chinese herbal medicines Shengmai injection and Xuesaitong injection on ventricular fibrillation threshold and connexin 43 expression in rats with myocardial infarction

To explore the effects of Shengmai injection and Xuesaitong injection, compound Chinese herbal medicines for replenishing qi and activating blood, on ventricular fibrillation threshold, heart structure and connexin 43 (Cx43) expression in rats with myocardial infarction (MI). One hundred male SD rats were randomly divided into sham operation group, model group, Yiqi Huoxue (YQHX) group (Shengmai injection plus Xuesaitong injection) and captopril group. MI model of rats was established by ligating left anterior descending coronary artery, and rats in sham operation group were prepared in the same way except for the ligation of coronary artery. Rats were treated with corresponding drugs for 1 month from next day after modeling. After treatment ventricular fibrillation threshold was detected, and heart weight index, left ventricular internal diameter and percentage of myocardial infarction were measured. Expression of Cx43 mRNA in myocardium was detected by real-time fluorescent quantitative polymerase chain reaction, and expression of Cx43 protein was observed by immunohistochemical method. Compared with the sham operation group, ventricular fibrillation threshold decreased significantly, heart weight index and left ventricular internal diameter increased, while expressions of Cx43 mRNA and protein decreased remarkably in the model group (P<0.01). Compared with the model group, ventricular fibrillation threshold was increased significantly, heart weight index, left ventricular internal diameter and percentage of myocardial infarction were decreased significantly in the YQHX group and captopril group (P<0.05 or P<0.01). When it comes to expression of Cx43, both Cx43 mRNA and protein expressions were increased remarkably in the YQHX group compared with the model group (P<0.05 or P<0.01), while only density mean and integral optical density of Cx43 protein expression were increased significantly in the captopril group (P<0.05). The enhancements on Cx43 mRNA and positive area sum of Cx43 protein induced by YQHX drugs were stronger than those induced by captopril (P<0.05). Shengmai injection and Xuesaitong injection have beneficial effects on ventricular fibrillation threshold in rats with MI. The mechanism is related with improving heart structure and reducing Cx43 expression after MI.

Effect of high volume hemofiltration on expression of Toll-like receptor 4 in myocardium in endotoxin induced shock in dogs

To investigate the effects of high volume hemofiltration (HVHF) on the expression of Toll-like receptor 4 (TLR4) mRNA in myocardium in endotoxin (lipopolysaccharide, LPS) induced shock in dogs. Sixteen healthy male dogs were injected with LPS 650 microg/kg via central vein to reproduce the model of endotoxin shock. All dogs were divided randomly into two groups: control group and therapy group, with 8 dogs in each group. Contents of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 in circulation were measured by radioimmunological method. The expression levels of TLR4 mRNA in all group were measured by reverse transcription-polymerase chain reaction (RT-PCR). Change in myocardial histopathology was observed and analyzed with the aid of electron microscope. The contents of TNF-alpha (microg/L: 0.59+/-0.15, 0.51+/-0.12, 0.41+/-0.10), IL-6 (ng/L: 11.08+/-2.83, 9.82+/-2.58, 8.25+/-2.05), IL-10 (microg/L: 57.28+/-5.93, 53.81+/-5.83, 50.67+/-6.33) in therapy group were found to have decreased significantly at 1, 2, and 4 hours after HVHF compared with those when the model was completed [(0.84+/-0.16) microg/L, (16.97+/-2.50) ng/L, (70.86+/-5.43) microg/L], showing a continuous trend of lowering (all P<0.01). The contents of TNF-alpha, IL-6, IL-10 in therapy group were lower than those in control group significantly at any time point [TNF-alpha (microg/L): 0.75+/-0.14, 0.74+/-0.11, 0.72+/-0.11, IL-6 (ng/L): 15.33+/-3.20, 14.66+/-3.24, 14.20+/-3.33, IL-10 (microg/L): 71.54+/-4.73, 70.71+/-4.34, 69.35+/-4.60, all P<0.01]. Compared with control group, HVHF treatment group could down-regulate mRNA expression of TLR4 in myocardium (t=3.58, P<0.01). Correlation analysis revealed significant positive-correlation between tissue TLR4 mRNA expression and contents of TNF-alpha, IL-6, IL-10 in circulation (r(1)=0.785, r(2)=0.569, r(3)=0.653, all P<0.05). Injury to the myocardium was significantly ameliorated in therapy group compared with control group as shown by electron microscopic observation. HVHF can down-regulate mRNA expression of TLR4 in myocardium in LPS induced shock in dogs, and myocardial inflammatory response was alleviated resulting in amelioration of myocardial injury.

Gene inflammatory expression profiling in right versus left ventricles in young urbanites: what is the long‐term impact of myocardial inflammation in the setting of air pollution?

Increased cardiac morbidity and mortality are associated with air pollution. Mexico City (MC) residents are exposed to high concentrations of fine particulate matter and endotoxin. South MC residents are exposed to higher endotoxin v North (Rosas 2007). SMC exposed mice had the highest CD14 and IL1beta mRNA in myocardium v N (Villarreal 2009). To test the hypothesis that residency within MC is a key factor for the degree of myocardial inflammation, we measured inflammatory mediator genes in autopsy samples from R and L ventricles of 6 South and 15 North age‐matched MC residents 18.5±5.2y. South residents had significant up‐regulation of CD14, IL1beta and TNFalpha v North (p&lt;0.001). An anti‐inflammatory biventricular response was also present: IL10 (RV p=0.001, LV p=0.02). Increases in myocardial IL1beta and TNFalpha have been implicated in the pathogenesis of myocardial dysfunction and cardiomyocyte death in ischemia‐reperfusion injury, sepsis, chronic heart failure, etc. Exposure to polluted urban air is associated with myocardial inflammation in young people, the RV responds differently from the LV and endotoxin is a key player in the inflammatory response. Occult cardiotoxicity (Golomb et al., 2009) may be a deleterious effect in young urbanites and the long‐term impact of sustained myocardial inflammation is uncertain.

Apoptosis and expression of uncoupling protein-2 in pressure overload-induced left ventricular hypertrophy

Objective — To explore the possible role of uncoupling protein-2 (UCP2) in cardiomyocyte apoptosis in pressure overload-induced left ventricular hypertrophy (LVH).Methods and results — Pressure overload was developed in 21 Sprague-Dawley rats by thoracic aortic constriction at 12 weeks of age.An equal number of sham-operated, age-matched rats served as controls.Aortic blood pressure (ABP), LVH, myocardial apoptosis index (MAI), and UCP2 mRNA expression were quantified in 7 subgroups of 3 treated and 3 control rats that were killed, at 1,2, 4, 7, 14, 21, or 30 days post-surgery, respectively., Compared to controls, ABP increased gradually throughout the study in the treated rats;LVH did not develop significantly until 4 days after surgery and increased progressively afterwards. The MAI increased immediately after surgery, reached a plateau from 4 to 7 days, and then declined rapidly;apoptosis was undetectable throughout the study in the cardiomyocytes of the control rats. In treated rats, the expression of UCP2 mRNA in myocardium was upregulated at 4 days, and developed progressively to the end of the experiment.Conclusions — (i) Apoptosis of cardiomyocytes is an important regulatory mechanism that is involved in the cardiac adaptive response to pressure overload, and (ii) the apoptosis of cardiomyocytes may be suppressed, in part, by UCP2.

Dramatic Accumulation of Triglycerides and Precipitation of Cardiac Hemodynamic Dysfunction during Brief Caloric Restriction in Transgenic Myocardium Expressing Human Calcium-independent Phospholipase A2γ

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Effects of glutamine on matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-3 expressions in myocardium of rats with sepsis

The underlying mechanisms for cardiac dysfunction in sepsis include the inhibitory effect of endotoxin and inflammatory factors on myocardium and the decrease in cardiac myocardial cells in number. However, whether there is ventricular remodeling resulted from the abnormalities of extracellular collagen metabolism and whether glutamine (Gln) can protect myocardium from LPS-induced damage as in reperfusion are unknown. The aim of the present study was to examine the effects of Gln on the expressions of matrix metalloproteinase-3 (MMP-3), tissue inhibitor of metalloproteinase-3 (TIMP-3) and their mRNA in myocardium of rats with sepsis. Classical rat model of sepsis was established by intraperitoneal injection of lipopolysaccharide (LPS) (4 mg/kg, from Escherichia coli O(55): B(5), Sigma). from 121 Wistar rats aged 18 days were divided into three groups randomly, 0 h control group (normal saline: 1 ml/kg, n = 11), LPS group (LPS: 4 mg/kg, n = 55) and Gln group (LPS: 4 mg/kg and immediately 13.64% glutamine 1 ml/kg, Fresenus, n = 55). Furthermore, LPS and Gln groups were examined at 2 h, 4 h, 6 h, 24 h and 72 h time points (n = 11). On each time point, rats of LPS and Gln groups as well as control group were anesthetized with 1% chloral hydrate injected intraperitoneally at a dosage of 1 ml/kg. Then, rats were sacrificed, and the hearts were isolated. Eight of them were frozen at minus 80 degrees C to measure the expression of TIMP-3 mRNA by using RT-PCR. The expressions of MMP-3 and TIMP-3 were observed with immunohistochemistry and the expression of MMP-3 mRNA was observed by using in situ hybridization. (1) Compared to 0 h, the mRNA expressions of MMP-3 and TIMP-3 in LPS group significantly increased (P < 0.01) with the peak at 6 - 24 h. While, in Gln group, they were significantly higher than those in controls but significantly lower than those in LPS group with the peak at 24 h (P < 0.01). Even at 72 h, they were still higher than those at 0 h (P < 0.05 and P < 0.01). (2) Compared to 0 h, the expressions of MMP-3 and TIMP-3 in LPS group were significantly lower at any other time point with the lowest at 6 h (P < 0.01). In Gln group, these expressions were also significantly lower than those in controls, but significantly higher than those in LPS group with the lowest being postponed to 24 h (P < 0.01). (3) The ultra structure changed obviously. Z line was unclear and the ridge of mitochondrion disappeared. While, in Gln group, the myocardial injury was slight compared to that in LPS group. MMP-3 mRNA expression was increased and TIMP-3 mRNA expression was depressed in LPS-induced sepsis. Myocardial extracellular matrix was damaged in sepsis. Glutamine might decrease the effects of LPS on MMP-3 and TIMP-3 expressions and postpone the time of myocardial matrix injury.

UPREGULATION OF 14‐3‐3 ISOFORMS IN ACUTE RAT MYOCARDIAL INJURIES INDUCED BY BURN AND LIPOPOLYSACCHARIDE

1. Burn-induced myocardial injuries can be acute due to loss of body fluid and blood redistribution, and subacute due to pathogenic toxins of infecting bacteria. The goal of this study was to examine expression of 14-3-3 in the injured myocardium. 2. Myocardial injury models were created in vivo by subjecting rats to severe burn and administration of lipopolysaccharide. RT-PCR and Western blotting were employed to assess the expression of 14-3-3 proteins and messenger ribonucleic acid (mRNA) for 14-3-3eta and gamma in the myocardium, respectively. 3. In the two models, we found that 14-3-3 proteins were induced in a time-dependent fashion. Such a change is at least in part attributed to increases in mRNAs for 14-3-3gamma and eta. In contrast to 14-3-3xi, whose mRNA was not detectable in the heart, mRNA for 14-3-3gamma was found significantly elevated between 24-48 h after burn. 14-3-3eta mRNA exhibited a marked increase at 3 h continuing to 12 h and then decreased nearly to a normal level after 48 h. In lipopolysaccharide-treated intact rats, 14-3-3gamma mRNA in myocardium showed a significant increase, reaching a peak at 4 h, followed by a decrease at 6 h. In contrast, 14-3-3eta mRNA had a slight increase without significance. 4. Our results suggest that 14-3-3 may play a role in both acute and subacute (postburn infectious) phases of severe burn.

The chemical inducibility of mouse cardiac antioxidants and phase 2 enzymes in vivo

The recognition of the critical involvement of oxidative and electrophilic stress in cardiac disorders has led to extensive investigation of the protective effects of exogenous antioxidants on cardiac injury. On the other hand, another strategy for protecting against oxidative/electrophilic cardiac injury may be through induction of the endogenous antioxidants and phase 2 enzymes in myocardium by chemical inducers. However, our understanding of the chemical inducibility of cardiac antioxidants/phase 2 enzymes in vivo is very limited. In addition, careful studies on the basal levels of a scope of endogenous antioxidants/phase 2 enzymes in myocardium as compared with other tissues, such as liver, are lacking. Accordingly, this study was undertaken to determine the basal levels of endogenous antioxidants/phase 2 enzymes, including superoxide dismutase (SOD), catalase, reduced glutathione (GSH), GSH peroxidase (GPx), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1), and investigate the inducibility of the above antioxidants/phase 2 enzymes by the chemoprotectant, 1,2-dithiole-3-thione (D3T), in cardiac as well as hepatic tissues in C57BL/6 mice. Our results demonstrated that in C57BL/6 mice, the levels of catalase, GSH, GPx, GR, and GST were significantly lower in cardiac tissue than in hepatic tissue. The level of total SOD did not differ significantly between mouse heart and liver. Notably, heart contained a much higher NQO1 activity than liver. Immunoblotting and RT-PCR analyses further demonstrated the high expression of NQO1 protein and mRNA in myocardium. Oral administration of D3T at 0.25 and 0.5 mmol/kg body weight for 3 consecutive days resulted in a significant induction of cardiac SOD, catalase, GR, GST, and NQO1. No significant induction of cardiac GSH and GPx was observed with the above D3T treatment. Only GR, GST, and NQO1 in mouse liver were induced by the D3T treatment. Unexpectedly, we observed a significant D3T dose-dependent decrease in hepatic GPx activity. Taken together, this study demonstrates for the first time that: (1) the expression of NQO1 is remarkably high in mouse myocardium though other cardiac antioxidants/phase 2 enzymes are relatively lower as compared with liver; (2) a number of endogenous antioxidants/phase 2 enzymes in mouse cardiac tissue can be significantly induced by D3T following oral administration; and (3) the inducibility of endogenous antioxidants/phase 2 enzymes by D3T differs between mouse cardiac and hepatic tissues. This study provides a basis for future investigation of the cardioprotection of chemically induced endogenous antioxidants and phase 2 enzymes in myocardium in animal models of oxidative/electrophilic cardiac disorders.

Intra-amniotic lipopolysaccharide leads to fetal cardiac dysfunction. A mouse model for fetal inflammatory response.

Intrauterine infection is associated with increased lipopolysaccharide (LPS) and proinflammatory cytokines in amniotic fluid. We hypothesized that intra-amniotic LPS launches a fetal inflammatory response leading to cardiac dysfunction. A mouse model was established. At 15-16 days of gestation, 52 fetuses of nine dams received LPS and 46 fetuses of nine dams vehicle intra-amniotically. Five dams underwent a sham operation. Echocardiography was performed before and 6 h after the injection to obtain inflow and outflow blood velocity waveforms. Outflow mean velocity (V(mean)) and the proportions of isovolumetric relaxation (IRT%) and contraction (ICT%) times of the cardiac cycle were calculated. Pulsatility indices (PI) were calculated from the umbilical and intracranial arteries and the descending aorta. Pulsatility indices for veins (PIV) were obtained from ductus venosus. Toll-like receptor-4 (TLR4) and several other inflammatory mediators were determined using ELISA, immunohistochemistry, or ribonuclease protection assay. In the LPS group, outflow V(mean) was significantly lower, and ICT% and IRT% longer than in the other groups. LPS increased PIs, except in the intracranial arteries, which showed a decrease in PIs. In ductus venosus, PIVs were increased after LPS. LPS increased interleukin (IL)-6 in amniotic fluid and induced the expression of proinflammatory cytokines in placenta and fetal membranes, but not in lung. In fetal myocardium, TLR4 was constitutional. LPS induced the expression of IL-1beta and tumor necrosis factor (TNF)-alpha mRNA in myocardium, whereas inducible nitric oxide synthase (NOS2) protein and nitrotyrosine remained undetectable. As a response to endotoxin in amniotic fluid, fetal myocardium acutely generates cytokines and severe fetal cardiovascular compromise develops. These two may be linked through a mechanism that does not include NO.

Changes of adrenomedullin and receptor activity modifying protein 2 (RAMP2) in myocardium and aorta in rats with isoproterenol-induced myocardial ischemia

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 2 (RAMP2). To explore the pathophysiological roles of adrenomedullin and its receptor component RAMP2 in ischemic cardiovascular diseases, we studied the changes of adrenomedullin and RAMP2 mRNA in myocardium and aorta in rats with isoproterenol (ISO)-induced myocardial impairment. In ISO-treated rats, heart became enlarged markedly, the ratio of heart to body weight was increased by 54% ( P<0.01), and myocardial malondialdehyde content and plasma lactate dehydrogenase activity was elevated by 43% ( P<0.01) and 138% ( P<0.01), respectively. Immunoreactive adrenomedullin (ADM) in plasma, myocardium and aorta was augmented by 116.7% ( P<0.01), 50.8% ( P<0.01) and 12.5% ( P>0.05), respectively. ADM mRNA in myocardium and aorta was increased by 96.8% ( P<0.01) and 38.5% ( P<0.01), respectively. RAMP2 mRNA in myocardium and aorta was increased by 19.6% ( P<0.05) and 15.8% ( P<0.01), respectively. These results suggest that the increase of ADM level and the up-regulation of ADM and RAMP2 gene in myocardium and aorta may be significant in the pathogenesis of ischemic myocardiopathy.