Establishment of a chronic insomnia rat model of sleep fragmentation using unstable platforms surrounded by water.

Chronic fragmented sleep is a very common type of insomnia that affects the daily lives of numerous people around the world. However, its pathogenesis is not very clear and a corresponding rat model has not been reported for this purpose at present. The present study aimed to establish a rat model of chronic insomnia with sleep fragmentation using self-made multiple strings of unstable platforms surrounded by shallow water. During the establishment of the models, changes in body weight and differences in food and water intake in the daytime and at night were acquired. The rat models were assessed using several tests, including the Morris water maze test, pentobarbital sodium-induced sleep, infrared monitoring and electroencephalogram/electromyography during sleep. The expression levels of certain inflammatory factors and orexin A were detected in the serum and brain tissues using ELISAs, immunohistochemistry and immunofluorescence. The expression levels of orexin 1 receptor (orexin 1r) were also detected in the brain. Polysomnography indicated that the model rats were successfully prepared with reduced non-rapid eye movement (non-REM) sleep in the daytime, which was increased at night, and considerably lower REM duration during the day and night. The number of instances of sleep arousals were also increased in the day and at night, and the average duration of each sleep bout was decreased in the daytime. The body weights of the model rats increased at a normal rate. However, the reduction of body weight in the daytime and increased in body weight at night were significantly less than those of the control rats. The daytime food and water consumption of the model rats increased significantly compared with that of the control rats, but was similar to that of the control group at night. The Morris water maze test indicated that the model rats were slow to learn to escape the platforms and performed a lower number of target crossings. The pentobarbital-induced sleep experiment confirmed that the model rats exhibited a longer sleep latency and shorter sleep time. The serum IL-1ß, IL-6, TNF-α and orexin A levels of the model rats were significantly increased, whereas their serum IL-10 levels were significantly decreased compared with those of the control rats. The expression levels of IL-1ß, IL-6, orexin A and orexin 1r in the brain tissues of the model rats were also significantly increased. In conclusion, these data indicate that learning and memory function, sleep time, arousal times, diurnal and nocturnal body weight changes, food and water intake, and expression levels of the specific inflammatory factors orexin A and orexin 1r were altered in the model rats. This suggests the chronic insomnia rat model with sleep fragmentation was successfully established using multiple strings of unstable platforms surrounded by water.

FGF21 Enhances Therapeutic Efficacy and Reduces Side Effects of Dexamethasone in Treatment of Rheumatoid Arthritis.

In order to investigate efficacy of FGF21 combine dexamethasone (Dex) on rheumatoid arthritis (RA) meanwhile reduce side effects of dexamethasone. We used combination therapy (Dex 15 mg/kg + FGF21 0.25 mg/kg, Dex 15 mg/kg + FGF21 0.5 mg/kg or Dex 15 mg/kg + FGF21 1 mg/kg) and monotherapy (Dex 15 mg/kg or FGF21 1 mg/kg) to treat CIA mice induced by chicken type II collagen, respectively. The effects of treatment were determined by arthritis severity score, histological damage, and cytokine production. The levels of oxidative stress parameters, liver functions, and other blood biochemical indexes were detected to determine FGF21 efficiency to side effects of dexamethasone. Oil red O was performed to detect the effects of FGF21 and dexamethasone on fat accumulation in HepG2 cells. The mechanism of FGF21 improves the side effects of dexamethasone which was analyzed by Western blotting. This combination proved to be therapeutically more effective than dexamethasone or FGF21 used singly. FGF21 regulates oxidative stress and lipid metabolism by upregulating dexamethasone-inhibited SIRT-1 and then activating downstream Nrf-2/HO-1and PGC-1. FGF21 and dexamethasone are highly effective in the treatment of arthritis; meanwhile, FGF21 may overcome the limited therapeutic response and Cushing syndrome associated with dexamethasone.

Establishment of a rat model with ageing insomnia induced by D-galactosef and para-chlorophenylalanine.

The current study aimed to establish a rat model of ageing insomnia induced by D-galactose and/or para-chlorophenylalanine. Following establishment of the model, body weights were measured, and Morris water maze and pentobarbital-induced sleep tests were performed. The serum levels of inflammatory mediators and the neural levels of neurotransmitters were detected. The results demonstrated that the body weights of PCPA+D-gal-induced ageing insomnia rats decreased significantly. Ageing insomnia rats exhibited longer latencies to the platform in the Morris water maze tests and fewer target crossings. The sleep latency of the model rats was longer and sleep time was shorter by contrast. The relative expression of hippocampal IL-6, TNF-α, NF-κB and mGluR2 mRNA of the PCPA+D-gal-induced ageing insomnia group was higher, while the relative expression of 5-HT1AR and GABAARa1 mRNA were lower. The serum levels of IL-1ß, IL-6, TNF-α and brain level of glutamate increased in the PCPA+D-gal-induced ageing insomnia group, while the levels of 5-HT and GABA decreased. In conclusion, memory function, sleep time, expression of inflammatory factors and neurotransmitters are altered in ageing insomnia rats induced by D-galactose and para-chlorophenylalanine, indicating the successful establishment of a murine model of ageing insomnia.

Sedative and Hypnotic Effects and Transcriptome Analysis of Polygala tenuifolia in Aged Insomnia Rats.

OBJECTIVE: To study the sedative and hypnotic effects and underlying mechanisms of Polygala tenuifolia (PT) on treating aged insomnia rats. METHODS: Sixty Sprague-Dawley male rats were divided into 6 groups by a random number table, including control group, model group, diazepam group (0.92 mg/kg), as well as PT low-, medium- and high-dose groups (0.0875, 0.175, 0.35 g/kg, respectively), 10 rats in each group. Aged insomnia rat model was established with subcutaneous injection of D-galactose for 42 days and then intraperitoneal injection of para-chlorophenylalanine for 3 days. PT and diazepam were respectively given to aged insomnia rats by intragastric administration for 7 days after model establishment. Then the rats were investigated by body weight, Morris water maze test, pentobarbital test, enzyme-linked immunosorbent assay, and transcriptome sequencing. RESULTS: Compared with the model group, PT increased the body weight, improved memory ability, and prolonged pentobarbital-induced sleep time of aged insomnia rats (P<0.01 or P<0.05). The medium dose of PT also increased the neurotransmitter levels of 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA), and decreased the level of Glu in the hippocampus of aged insomnia rats (P<0.05 or P<0.01). Twenty-four differentially expressed genes (DEGs) were overlapped among model group, medium-dose PT group, and diazepam group in transcriptome analysis. Fuom and Pcp2 were down-regulated by the treatment of medium-dose PT (P<0.01 or P<0.05). The metabolic pathways of PT were relatively less than diazepam (91 vs. 104). CONCLUSIONS: The sedative and hypnotic effects of PT in aged insomnia rats might be related to neuro, metabolism pathways, especially through GABAergic signaling pathway. It provided more effective herb choice for the treatment of senile insomnia.

A novel monoclonal antibody against the N-terminus of Aß1-42 reduces plaques and improves cognition in a mouse model of Alzheimer's disease.

Senile plaques consisting of Amyloid-beta (Aß) peptides, in particular Aß1-42, are the hallmark of Alzheimer's disease (AD) and have been the primary therapeutic targets. Passive immunotherapy with monoclonal antibodies (mAbs) has shown initial success in mouse models of AD. However, the existing Aß-directed mAbs mostly were tested on animal models or patients with advanced disease. The effects and mechanisms of mAbs on animals or human trial participants in the prodromal phase of AD are not fully clarified. In the current study, a novel mAb (3F5) directed against the 1-11 amino acids of Aß1-42 was generated by immunizing mice with an emulsion of full length human Aß1-42. The mAb (3F5) showed the ability to disrupt Aß1-42 aggregation and prevent Aß-mediated neurotoxicity in vitro. In a mouse model of AD, administration with 3F5 for 3 months in 6 months-old mice demonstrated that the mAb specifically bound with Aß1-42 to promote the depolymerization of Aß fibrils, facilitated endocytosis of Aß1-42 by microglia, and attenuated the death and apoptosis of neuronal cells, accompanied by neurite outgrowth. APP/PS1 double-transgenic mice treated with 3F5 mAb showed reduced memory loss, cognitive decline, and decreased levels of amyloid deposits in the brain. Aß1-42 levels in cerebral tissues were also significantly reduced, whereas serum Aß1-42 was markedly increased. Interestingly, the concentration of 3F5 in peripheral circulation is much higher than that in the brain. These results indicate that 3F5 is able to cross the blood-brain barrier (BBB) to bind Aß and initiates the phagocytosis of antibody/Aß complexes by microglia in the amyloid depositing mice. 3F5 also promotes Aß efflux from the brain. As a consequence, the antibody reduces plaques in the AD mouse brain, in association with reduction in the pathology of AD.

Association of serum uric acid and risk of hypertension in adults: a prospective study of Kailuan Corporation cohort.

Whether hyperuricemia is an independent risk factor for hypertension in adults is still under debate. To determine the association between serum uric acid and risk of hypertension in the Chinese population, we conducted a prospective study using the "Kailuan Corporation cohort." A total of 39,233 adult subjects with available data on serum uric acid were enrolled from 2006 to 2007. Subjects with established hypertension were excluded and were then grouped based on the gender and baseline quartile serum uric acid into F1-4 for women and M1-4 for men with F1 and M1 being the lowest quartiles. Incidence of newly described primary hypertension was reevaluated in 2010-2011. The median (interquantile range) baseline uric acid (UA) was 290 (243-344) µmol/L in men and 230 (194-274) µmol/L in women. During a 4-year follow-up period, 12,844 subjects (31.31 %) were newly diagnosed with hypertension. The incidence of hypertension was 14.36, 16.57, 19.06, and 22.35 % in F1 to F4 and 33.64, 33.97, 36.54, and 40.74 % in M1 to M4, respectively. Multiple logistic regression analysis showed that the odds ratios (ORs) of incident hypertension were 1.17 [95 % confidence interval (CI) 1.00-1.37, P = 0.055], 1.24 (95 % CI 1.06-1.45, P = 0.009), and 1.20 (95 % CI 1.02-1.41, P = 0.027) in F2 to F4 compared to the F1 and 0.98 (95 % CI 0.91-1.05, P = 0.534), 1.05 (95 % CI 0.98-1.13, P = 0.190), and 1.13 (95 % CI 1.05-1.22, P = 0.002) in M2 to M4 compared to the M1. Elevated level of serum uric acid is associated with an increased risk of hypertension in adults.

Changes in Synovial Fluid Biomarkers after Experimental Equine Osteoarthritis.

INTRODUCTION: The study aimed to clarify the changes in the concentration of inflammatory mediators, proteases, and cartilage degradation biomarkers in the synovial fluid of joints in an equine osteoarthritis model. MATERIAL AND METHODS: Osteoarthritis was induced in eight Mongolian horses by a sterile intra-articular injection of amphotericin B, which was injected into the left carpal joint in a dose of 2 mL (25 mg/mL). The control group comprised five horses which were injected with an equal dose of sterile physiological saline into the left carpal joint. Synovial fluid was obtained at baseline and every week after injection. Test methods were based on ELISA. RESULTS: In the course of the osteoarthritis, the concentration of biomarkers in joint synovial fluid showed an increasing trend. IL-1, IL-6, MMP-9, MMP-13, ADAMTS-5, CS846, GAG, HA, CTX-II, and COMP concentrations sharply increased before the onset of significant symptoms of lameness, whereas TNF-α, MMP-2, and MMP-3 concentrations rose sharply after the occurrence of such symptoms. CONCLUSION: The results obtained confirm that the concentrations of IL-1, IL-6, MMP-9, MMP-13, ADAMTS-5, CS846, GAG, HA, CTX-II and COMP increase substantially in equine osteoarthritis, which provides a theoretical basis for the rapid diagnosis of the disease.

The Cytoprotective Effect of Hyperoside against Oxidative Stress Is Mediated by the Nrf2-ARE Signaling Pathway through GSK-3ß Inactivation.

Glycogen synthase kinase-3ß (GSK-3ß) acts as a negative regulator of NF-E2 related factor 2 (Nrf2) by inducing Nrf2 degradation and nuclear export. Our previous study demonstrated that the flavonoid hyperoside elicits cytoprotection against oxidative stress by activating the Keap1-Nrf2-ARE signaling pathway, thus increasing the expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD) and catalase. However, the role of GSK-3ß in hyperoside-mediated Nrf2 activation is unclear. Here, we demonstrate that in a normal human hepatocyte cell line, (L02), hyperoside is capable of inducing the phosphorylation of GSK-3ß at Ser9 without affecting the protein levels of GSK-3ß and its phosphorylation at Thr390. Lithium chloride (LiCl) and short interfering RNA (siRNA)-mediated inhibition of GSK-3ß significantly enhanced the ability of hyperoside to protect L02 liver cells from H2O2-induced oxidative damage, leading to increased cell survival shown by the maintenance of cell membrane integrity and elevated levels of glutathione (GSH), one of the endogenous antioxidant biomarkers. Further study showed that LiCl and siRNA-mediated inhibition of GSK-3ß increased hyperoside-induced HO-1 expression, and the effect was dependent upon enhanced Nrf2 nuclear translocation and gene expression. These activities were followed by ARE-mediated transcriptional activation in the presence of hyperoside, which was abolished by the transfection of the cells with Nrf2 siRNA. Furthermore, the siRNA-mediated inhibition of Keap1 also enhanced hyperoside-induced Nrf2 nuclear accumulation and HO-1 expression, which was relatively smaller than the effects obtained from GSK-3ß siRNA administration. Moreover, Keap1 siRNA administration alone had no significant effect on the phosphorylation and protein expression of GSK-3ß. Collectively, our data provide evidence that hyperoside attenuates H2O2 -induced L02 cell damage by activating the Nrf2-ARE signaling pathway through both an increase in GSK-3ß inhibitory phosphorylation at Ser9 and an inhibition of Keap1 and that hyperoside-mediated GSK-3ß inhibition exhibits more significant effects.

Unplanned Reoperations in Neurosurgical Patients Due to Postoperative Bleeding: A Single-Center Experience and Literature Review.

The aim of this study is to investigate the incidence of unplanned reoperations from all causes due to bleeding in neurosurgical patients. The medical records of patients who received neurosurgical procedures at our hospital were retrospectively reviewed and data of patients who received reoperations were extracted and summarized. A literature review was conducted of the Medline, Cochrane, EMBASE, and Google Scholar databases up to November 2013. The main outcome measure was the rate of unplanned reoperations due to bleeding. At our hospital, 68 patients with a mean age of 41.5 ±â€Š21.5 years (range, 7 months to 76 years) received an unplanned reoperation. More than 70% of the patients were older than 18 years, 64.7% were males, and 94.1% had cranial surgery. Almost 60% of the patients received >1 blood transfusion (58.8%) after the first surgery. Of the 68 patients, 35 (51.5%) received a second operation due to bleeding. Univariate logistic regression analysis only showed that an increasing time interval between the first and second surgery was associated with a decreased chance of the reoperation being performed due to bleeding (odds ratio [OR] = 0.843, 95% confidence interval [CI]: 0.720-0.987; P = .033). Of 229 studies identified, 5 retrospective reports with a total of 1375 patients were included in the analysis. The rate of reoperations for bleeding in the 5 studies ranged from 4.2% to 31.5%. Employing measures to reduce postoperative bleeding may help reduce the rate of unplanned neurosurgical reoperations.

Remission of rheumatoid arthritis and potential determinants: a national multi-center cross-sectional survey.

The aim of this study is to investigate the remission rate of rheumatoid arthritis (RA) in China and identify its potential determinants. A multi-center cross-sectional study was conducted from July 2009 to January 2012. Data were collected by face-to-face interviews of the rheumatology outpatients in 28 tertiary hospitals in China. The remission rates were calculated in 486 RA patients according to different definitions of remission: the Disease Activity Score in 28 joints (DAS28), the Simplified Disease Activity Index (SDAI), the Clinical Disease Activity Index (CDAI), and the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) Boolean definition. Potential determinants of RA remission were assessed by univariate and multivariate analyses. The remission rates of RA from this multi-center cohort were 8.6% (DAS28), 8.4% (SDAI), 8.2% (CDAI), and 6.8% (Boolean), respectively. Favorable factors associated with remission were: low Health Assessment Questionnaire (HAQ) score, absence of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP), and treatment of methotrexate (MTX) and hydroxychloroquine (HCQ). Younger age was also predictive for the DAS28 and the Boolean remission. Multivariate analyses revealed a low HAQ score, the absence of anti-CCP, and the treatment with HCQ as independent determinants of remission. The clinical remission rate of RA patients was low in China. A low HAQ score, the absence of anti-CCP, and HCQ were significant independent determinants for RA remission.

S1P lyase: a novel therapeutic target for ischemia-reperfusion injury of the heart.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic preconditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible S1P catabolism. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, we evaluated SPL inhibition as a strategy for reducing cardiac ischemia-reperfusion (I/R) injury. We measured SPL expression and enzyme activity in murine hearts. Basal SPL activity was low in wild-type cardiac tissue but was activated in response to 50 min of ischemia (n = 5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size, and increased functional recovery after I/R compared with littermate controls (n = 5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is a Federal Drug Administration-approved food additive that inhibits SPL. When given overnight at 25 mg/l in drinking water, THI raised S1P levels and reduced SPL activity (n = 5, P < 0.01). THI reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and to 40 min of reperfusion in ex vivo hearts (n = 7, P < .01). These data correlated with an increase in MAP kinase-interacting serine/threonine kinase 1, eukaryotic translation initiation factor 4E, and ribosomal protein S6 phosphorylation levels after I/R, suggesting that SPL inhibition enhances protein translation. Pretreatment with an S1P1 and S1P3 receptor antagonist partially reversed the effects of THI. These results reveal, for the first time, that SPL is an ischemia-induced enzyme that can be targeted as a novel strategy for preventing cardiac I/R injury.

Heart failure switches the RV alpha1-adrenergic inotropic response from negative to positive.

Right ventricular (RV) failure is a serious common clinical problem that is poorly understood. Therefore, for failing and nonfailing hearts, we examined the distinctive inotropic responses induced in the RV myocardium after the stimulation of alpha(1)-adrenergic receptors (ARs). In RV trabeculae from nonfailing mouse hearts, alpha(1)-ARs induced a negative inotropic response, consistent with our previous study. In marked contrast, in RV trabeculae from failing hearts, 12 wk after coronary artery ligation, alpha(1)-ARs induced a positive inotropic response. Mechanistically, experiments with skinned trabeculae showed that alpha(1)-ARs decreased myofilament Ca(2+) sensitivity in the nonfailing RV myocardium, whereas alpha(1)-ARs increased Ca(2+) sensitivity in heart failure. This suggests that a switch in the Ca(2+) sensitivity response to alpha(1)-AR stimulation explained the switch in the RV alpha(1)-AR inotropic response in heart failure. Myosin light chain kinase (MLCK) can increase myofilament Ca(2+) sensitivity, and the smooth muscle isoform (smMLCK), which is also present in cardiomyocytes, was more abundant in the RV myocardium from failing versus nonfailing hearts. Moreover, the MLCK inhibitor ML-9 prevented the switch of the RV myocardium to a positive alpha(1)-AR inotropic response in heart failure. In the left ventricular myocardium, in contrast, alpha(1)-AR inotropic responses were not different in failing versus nonfailing hearts, and smMLCK abundance was not increased in heart failure. In relation to human disease, we found that smMLCK mRNA and protein levels were increased in RVs from failing human hearts. We conclude that the RV inotropic response to alpha(1)-ARs is switched from negative to positive in heart failure, through a pathway involving increased myofilament Ca(2+) sensitivity. Since alpha(1)-AR agonist catecholamines are elevated in heart failure, increased alpha(1)-AR inotropic responses in the RV myocardium may be adaptive in heart failure by helping the failing RV respond to increased pulmonary pressures.

A computationally efficient formal optimization of regional myocardial contractility in a sheep with left ventricular aneurysm.

A non-invasive method for estimating regional myocardial contractility in vivo would be of great value in the design and evaluation of new surgical and medical strategies to treat and/or prevent infarction-induced heart failure. As a first step towards developing such a method, an explicit finite element (FE) model-based formal optimization of regional myocardial contractility in a sheep with left ventricular (LV) aneurysm was performed using tagged magnetic resonance (MR) images and cardiac catheterization pressures. From the tagged MR images, 3-dimensional (3D) myocardial strains, LV volumes and geometry for the animal-specific 3D FE model of the LV were calculated, while the LV pressures provided physiological loading conditions. Active material parameters (T(max_B) and T(max_R)) in the non-infarcted myocardium adjacent to the aneurysm (borderzone) and in myocardium remote from the aneurysm were estimated by minimizing the errors between FE model-predicted and measured systolic strains and LV volumes using the successive response surface method for optimization. The significant depression in optimized T(max_B) relative to T(max_R) was confirmed by direct ex vivo force measurements from skinned fiber preparations. The optimized values of T(max_B) and T(max_R) were not overly sensitive to the passive material parameters specified. The computation time of less than 5 hours associated with our proposed method for estimating regional myocardial contractility in vivo makes it a potentially very useful clinical tool.

Expression of a Gi-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy.

Increased signaling by G(i)-coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted G(i)-coupled receptor (Ro1). Activation of G(i) signaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff-perfused hearts. However, acute termination of Ro1 signaling with the antagonist nor-binaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiation of Ro1 expression, there was a 40% reduction in the abundance of the sarcoplasmic reticulum Ca(2+)-ATPase (P < 0.05); thereafter, there was progressive impairment of both Ca(2+) handling and force development assessed with ventricular trabeculae. Six weeks after initiation of Ro1 expression, systolic Ca(2+) concentration was reduced to 0.61 +/- 0.08 vs. 0.91 +/- 0.07 microM for control (n = 6-8; P < 0.05), diastolic Ca(2+) concentration was elevated to 0.41 +/- 0.07 vs. 0.23 +/- 0.06 microM for control (n = 6-8; P < 0.01), and the decline phase of the Ca(2+) transient (time from peak to 50% decline) was slowed to 0.25 +/- 0.02 s vs. 0.13 +/- 0.02 s for control (n = 6-8; P < 0.01). Early after initiation of Ro1 expression, there was a ninefold elevation of matrix metalloproteinase-2 (P < 0.01), which is known to cause myofilament injury. Consistent with this, 6 wk after initiation of Ro1 expression, Ca(2+)-saturated myofilament force in skinned trabeculae was reduced to 21 +/- 2 vs. 38 +/- 0.1 mN/mm(2) for controls (n = 3; P < 0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure in which increased activity of G(i)-coupled receptors leads to impaired Ca(2+) handling and myofilament injury, contributing to impaired ventricular pump function and heart failure.

Contrasting inotropic responses to alpha1-adrenergic receptor stimulation in left versus right ventricular myocardium.

The left ventricle (LV) and right ventricle (RV) have differing hemodynamics and embryological origins, but it is unclear whether they are regulated differently. In particular, no previous studies have directly compared the LV versus RV myocardial inotropic responses to alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation. We compared alpha(1)-AR inotropy of cardiac trabeculae from the LV versus RV of adult mouse hearts. As previously reported, for mouse RV trabeculae, alpha(1)-AR stimulation with phenylephrine (PE) caused a triphasic contractile response with overall negative inotropy. In marked contrast, LV trabeculae had an overall positive inotropic response to PE. Stimulation of a single subtype (alpha(1A)-AR) with A-61603 also mediated contrasting LV/RV inotropy, suggesting differential activation of multiple alpha(1)-AR-subtypes was not involved. Contrasting LV/RV alpha(1)-AR inotropy was not abolished by inhibiting protein kinase C, suggesting differential activation of PKC isoforms was not involved. However, contrasting LV/RV alpha(1)-AR inotropic responses did involve different effects on myofilament Ca(2+) sensitivity: submaximal force of skinned trabeculae was increased by PE pretreatment for LV but was decreased by PE for RV. For LV myocardium, alpha(1)-AR-induced net positive inotropy was abolished by the myosin light chain kinase inhibitor ML-9. This study suggests that LV and RV myocardium have fundamentally different inotropic responses to alpha(1)-AR stimulation, involving different effects on myofilament function and myosin light chain phosphorylation.

Cardiac transgenic matrix metalloproteinase-2 expression directly induces impaired contractility.

OBJECTIVE: Matrix metalloproteinase-2 (MMP-2) plays a major role in dysfunctional ventricular remodeling following myocardial injury induced by ischemia/reperfusion and heart failure. To directly assess the role of MMP-2 in the absence of superimposed injury, we generated cardiac-specific, constitutively active MMP-2 transgenic mice. METHODS: Morphologic and functional studies were carried out using both intact and demembranated (skinned) right ventricular trabeculae dissected from hearts of 8-month-old MMP-2 transgenic mice and wild-type controls (WT). RESULTS: Electron micrographs showed that compared to WT, MMP-2 myocardium had no gross, ultrastructural changes (no myocyte dropout or gross fibrosis). However, MMP-2 myocardium contained fibroblasts with abundant rough endoplasmic reticulum, consistent with an activated synthetic phenotype, suggesting extracellular matrix remodeling in MMP-2 trabeculae. Consistent with remodeling, mechanical studies found increased stiffness of intact unstimulated trabeculae (increasing sarcomere lengths from 2 to 2.3 microm caused a greater rise of passive muscle force for MMP-2 trabeculae versus WT). With electrical stimulation, MMP-2 trabeculae generated substantially less active force at all sarcomere lengths. Moreover, inotropic responses to increases of bath [Ca2+], pacing frequency, and isoproterenol were all significantly reduced versus WT trabeculae. Skinned fiber assessment of myofilament function revealed that maximum Ca2+-activated force of skinned MMP-2 trabeculae was reduced to approximately 50% of WT, suggesting a myofilament contraction defect. CONCLUSION: Cardiac-specific, constitutively active MMP-2 expression leads to impaired contraction and diminished responses to inotropic stimulation. These findings indicate that MMP-2 can directly impair ventricular function in the absence of superimposed injury.

In vitro electrophysiologic effects of morphine in rabbit ventricular myocytes.

BACKGROUND: Morphine is widely used in patients undergoing surgical operations and is also reported to mediate cardioprotection of preconditioning. The current study determined effects of morphine at therapeutic to pharmacologic concentrations on cardiac action potential, L-type Ca2+ current (ICa.L), delayed rectifier K+ current (IK), and inward rectifier K+ current (IK1) in isolated rabbit ventricular myocytes. METHODS: Ventricular myocytes were enzymatically isolated from rabbit hearts. Action potential and membrane currents were recorded in current and voltage clamp modes. RESULTS: Morphine at concentrations from 0.01 to 1 microM significantly prolonged cardiac action potential, and at 0.1 and 1 microM slightly but significantly hyperpolarized the resting membrane potential. In addition, morphine at 0.1 microM significantly augmented ICa.L (at +10 mV) from 5.9 +/- 1.9 to 7.3 +/- 1.7 pA/pF (by 23%; P < 0.05 vs. control) and increased IK1 (at -60 mV) from 2.8 +/- 1.0 to 3.5 +/- 0.9 pA/pF (by 27%; P < 0.05 vs. control). Five microM naltrindole (a selective delta-opioid receptor antagonist) or 5 microM norbinaltorphimine (a selective kappa-opioid receptor antagonist) prevented the increase in ICa.L induced by morphine, but 5 microM CTOP (a selective mu-opioid receptor antagonist) did not. The three types of opioid antagonists did not affect the augmentation of IK1 by morphine. Morphine had no effect on IK. CONCLUSIONS: These results indicate that morphine prolongs action potential duration by increasing ICa.L, an effect mediated by delta- and kappa-opioid receptors. It also hyperpolarizes cardiac resting membrane potential by increasing IK1, which is not mediated by opioid receptors.