Isoproterenol Research Articles

Molecular mechanisms mediate roflumilast protective effect against isoprenaline-induced myocardial injury

BackgroundMyocardial necrosis is one of the most common cardiac and pathological diseases. Unfortunately, using the available medical treatment is not sufficient to rescue the myocardium. So that, we aimed in our model to study the possible cardioprotective effect of roflumilast (ROF) in an experimental model of induced myocardial injury using a toxic dose of isoprenaline (ISO) and detecting the role of vascular endothelial growth factor/endothelial nitric oxide synthase (VEGF/eNOS) and cyclic guanosine monophosphate/cyclic adenosine monophosphate/ sirtuin1 (cGMP/cAMP/SIRT1) signaling cascade.Materials and methodsAnimals were divided into five groups; control, ISO given group (150 mg/kg) i.p. on the 4th and 5th day, 3 ROF co-administered groups in different doses (0.25, 0.5, 1 mg/kg/day) for 5 days.ResultsOur data revealed that ISO could induce cardiac toxicity as manifested by significant increases in troponin I, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), malondialdehyde (MDA), tumor necrosis factor alpha (TNFα), and cleaved caspase-3 with toxic histopathological changes. Meanwhile, there were significant decreases in reduced glutathione (GSH), total antioxidant capacity (TAC), VEGF, eNOS, cGMP, cAMP and SIRT1. However, co-administration of ROF showed significant improvement and normalization of ISO induced cardiac damage.ConclusionWe concluded that ROF successfully reduced ISO induced myocardial injury and this could be attributed to modulation of PDE4, VEGF/eNOS and cGMP/cAMP/SIRT1 signaling pathways with antioxidant, anti-inflammatory, and anti-apoptotic properties.

The Effect of Astaxanthin on Mitochondrial Dynamics in Rat Heart Mitochondria under ISO-Induced Injury.

Mitochondria are dynamic organelles that produce ATP in the cell and are sensitive to oxidative damage that impairs mitochondrial function in pathological conditions. Mitochondria are involved not only in a healthy heart but also in the development of heart disease. Therefore, attempts should be made to enhance the body's defense response against oxidative stress with the help of various antioxidants in order to decrease mitochondrial damage and reduce mitochondrial dysfunction. Mitochondrial fission and fusion play an important role in the quality control and maintenance of mitochondria. The ketocarotenoid astaxanthin (AX) is an antioxidant able to maintain mitochondrial integrity and prevent oxidative stress. In the present study, we investigated the effect of the protective effect of AX on the functioning of rat heart mitochondria (RHM). Changes in the content of proteins responsible for mitochondrial dynamics, prohibitin 2 (PHB2) as a protein that performs the function of quality control of mitochondrial proteins and participates in the stabilization of mitophagy, and changes in the content of cardiolipin (CL) in rat heart mitochondria after isoproterenol (ISO)-induced damage were examined. AX improved the respiratory control index (RCI), enhanced mitochondrial fusion, and inhibited mitochondrial fission in RHM after ISO injury. Rat heart mitochondria (RHM) were more susceptible to Ca2+-induced mitochondrial permeability pore (mPTP) opening after ISO injection, while AX abolished the effect of ISO. AX is able to perform a protective function in mitochondria, improving their efficiency. Therefore, AX can be considered an important ingredient in the diet for the prevention of cardiovascular disease. Therefore, AX can be examined as an important component of the diet for the prevention of heart disease.

β‐Adrenergic receptor agonist alleviates memory interference control deficits in APPswe/PS1dE9 mice

AbstractBackgroundEarlier studies have demonstrated that memory dysfunction during early Alzheimer’s disease (AD) is associated with an increased susceptibility to memory interference (MI). In this study, we examined whether previously stored memory representations interfered with novel similar input patterns in 2‐month‐old APPswe/PS1dE9 (APP/PS1), mice. APP/PS1 over‐expresses the Swedish mutation of amyloid precursor protein together with presenilin1 deleted in exon 9. Amyloid beta (Aβ) plaque deposition is observed at 4‐6 months of age, not at 2 months providing us with a window to study the earliest behavioural deficits that are independent of Aβ plaque deposition.Method(1) We introduced a proactive interference step to the well‐established novel object recognition paradigm (NOR), by exposing the mice to multiple objects of different shape, size and colour. (2) Earlier studies have demonstrated that prolonged oral administration of β‐adrenoreceptor (β‐AR) agonist (−)‐Isoproterenol hydrochloride (IPE) to mice protected them from hippocampal impairment by Aβ oligomers, and enhanced hippocampal synaptic plasticity. We provided the IPE in drinking water for three weeks from P26 till P47.ResultOur results demonstrate that NOR is intact in APP/PS1 at 2 months of age. However, introducing a short‐term object based priming or interference prior to NOR leads to recognition memory deficits. This finding indicates that 2‐month‐old APP/PS1 is susceptible to proactive MI unlike their Wild Type counterpart who could clearly discriminate between a novel and a familiar object. Our findings indicate that, object‐based priming can activate its presence during target memory encoding, and thus negate learning in 2‐month‐old APP/PS1 mice before amyloidosis onset. β‐AR agonist treatment restored performance of APP/PS1 mice to normal levels protecting them from the effects of priming during the object recognition task.ConclusionThese results indicate that integration or discrimination component of object memory representations relies on and can be modulated by β‐AR activation.

The lncRNA ADAMTS9-AS1/miR-185-5p/KAT7 ceRNA network inhibits cardiomyocyte hypertrophy in hypertrophic obstructive cardiomyopathy.

Hypertrophic obstructive cardiomyopathy (HOCM) is a well-recognized inherited cardiac disease. This study was conducted to explore the role of lncRNA ADAMTS9 antisense RNA 1 (ADAMTS9-AS1) in HOCM-induced cardiomyocyte hypertrophy. The serum of HOCM patients was collected. AC16 cells were treated with isoproterenol (ISO) and transfected with oe-ADAMTS9-AS1 vector, miR-185-5p mimic, and lysine acetyltransferase 7 (KAT7) specific small interfering RNA. lncRNA ADAMTS9-AS1, miR-185-5p, KAT7, brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) in the serum or cells were determine by qRT-PCR or Western blot assay. Cell surface area was observed by Texas Red-Phalloidin staining. Subcellular localization of lncRNA ADAMTS9-AS1 was tested by nuclear/cytoplasmic fractionation assay, with RNA pull-down and dual-luciferase assay to validate gene interactions. lncRNA ADAMTS9-AS1 was downregulated in the serum of HOCM patients and ISO-treated AC16 cells. lncRNA ADAMTS9-AS1 overexpression inhibited ISO-induced cardiomyocyte hypertrophy and reduced levels of ANP and BNP. lncRNA ADAMTS9- AS1 was located in cytoplasm and inhibited miR-185-5p expression through targeted binding. miR-185-5p bound to KAT7 3'UTR and inhibited KAT7 expression. miR-185-5p overexpression and KAT7 knockdown both neutralized the inhibitory role of lncRNA ADAMTS9-AS1 in cardiomyocyte hypertrophy. Overall, lncRNA ADAMTS9-AS competitively bound to miR-185-5p to up-regulate KAT7 and thus inhibited cardiomyocyte hypertrophy.

Intravenous Semaphorin 3A Administration Maintains Cardiac Contractility and Improves Electrical Remodeling in a Mouse Model of Isoproterenol-Induced Heart Failure.

The effects of recombinant semaphorin 3A (Sema3A) on myocardial contractility and electrical remodeling in mice with isoproterenol (ISP) -induced heart failure were investigated.C57BL/6J mice intraperitoneally received ISP (480 mg/kg/day, ISP group; n = 24) or saline (control group; n = 31) for 14 days. Twenty-one ISP-treated mice received 0.5 mg/kg Sema3A intravenously on days 7 and 11 (ISP+Sema3A group). The sympathetic nervous system was activated upon ISP treatment, but was reduced upon Sema3A administration. Greater myocardial tissue fibrosis was observed in the ISP group than in the control group. However, fibrosis was not significantly different between the ISP+Sema3A and control groups. Fractional shortening of the left ventricle was lower in the ISP group than in the control group and was restored in the ISP+Sema3A group (control, 53 ± 8%; ISP, 37 ± 7%; ISP+Sema3A, 48 ± 3%; P < 0.05). Monophasic action potential duration at 20% repolarization (MAPD20) was prolonged in the ISP group (compared to control group), but this was reversed upon Sema3A administration (control, 29 ± 3 ms; ISP, 35 ± 6 ms; ISP+Sema3A, 29 ± 3 ms; P < 0.05). qPCR revealed Kv4.3, KChIP2, and SERCA2 downregulation in the ISP group and upregulation in the ISP+Sema3A group; however, Western blotting revealed similar changes only for Kv4.3 (P < 0.05).Intravenous Sema3A may maintain myocardial contractility by suppressing the sympathetic innervation of the myocardium and reducing myocardial tissue damage, in addition to restoring MAPD via Kv4.3 upregulation.

Exercise Training Attenuates Acute β-Adrenergic Receptor Activation-Induced Cardiac Inflammation via the Activation of AMP-Activated Protein Kinase.

Exercise has proven cardiac benefits, but the underlying mechanisms of exercise that protect the heart from acute sympathetic stress injuries remain unknown. In this study, adult C57BL/6J mice and their AMP-activated protein kinase α2 knockout (AMPKα2-/-) littermates were either subjected to 6 weeks of exercise training or housed under sedentary conditions and then treated with or without a single subcutaneous injection of the β-adrenergic receptor (β-AR) agonist isoprenaline (ISO). We investigated the differences in the protective effects of exercise training on ISO-induced cardiac inflammation in wild-type (WT) and AMPKα2-/- mice using histology, enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. The results indicated that exercise training alleviated ISO-induced cardiac macrophage infiltration, chemokines and the expression of proinflammatory cytokines in wild-type mice. A mechanism study showed that exercise training attenuated the ISO-induced production of reactive oxygen species (ROS) and the activation of NLR Family, pyrin domain-containing 3 (NLRP3) inflammasomes. In cardiomyocytes, the ISO-induced effects on these processes were inhibited by AMP-activated protein kinase (AMPK) activator (metformin) pretreatment and reversed by the AMPK inhibitor (compound C). AMPKα2-/- mice showed more extensive cardiac inflammation following ISO exposure than their wild-type littermates. These results indicated that exercise training could attenuate ISO-induced cardiac inflammation by inhibiting the ROS-NLRP3 inflammasome pathway in an AMPK-dependent manner. Our findings suggested the identification of a novel mechanism for the cardioprotective effects of exercise.

A Mouse Model of Dilated Cardiomyopathy Produced by Isoproterenol Acute Exposure Followed by 5-Fluorouracil Administration.

Appropriate dilated cardiomyopathy (DCM) animal models are highly desirable considering the pathophysiological and clinical heterogeneity of DCM. Genetically modified mice are the most widely and intensively utilized research animals for DCM. However, to translate discoveries from basic science into new and personalized medical applications, research in non-genetically based DCM models remains a key issue. Here, we characterized a mouse model of non-ischemic DCM induced by a stepwise pharmacologic regime of Isoproterenol (ISO) high dose bolus followed by a low dose systemic injection of the chemotherapy agent, 5-Fluorouracil (5-FU). C57BL/6J mice were injected with ISO and, 3 days after, were randomly assigned to saline or 5-FU. Echocardiography and a strain analysis show that ISO + 5FU in mice induces progressive left ventricular (LV) dilation and reduced systolic function, along with diastolic dysfunction and a persistent global cardiac contractility depression through 56 days. While mice treated with ISO alone recover anatomically and functionally, ISO + 5-FU causes persistent cardiomyocyte death, ensuing in cardiomyocyte hypertrophy through 56 days. ISO + 5-FU-dependent damage was accompanied by significant myocardial disarray and fibrosis along with exaggerated oxidative stress, tissue inflammation and premature cell senescence accumulation. In conclusions, a combination of ISO + 5FU produces anatomical, histological and functional cardiac alterations typical of DCM, representing a widely available, affordable, and reproducible mouse model of this cardiomyopathy.

Paclitaxel Protects against Isoproterenol-Induced Damage in Rat Myocardium: Its Heme-Oxygenase Mediated Role in Cardiovascular Research.

(1) Background: In cardiovascular applications, paclitaxel inhibits smooth muscle cell proliferation and migration and significantly reduces the occurrence of restenosis and target lesion revascularization. However, the cellular effects of paclitaxel in the myocardium are not well understood; (2) Methods: Wistar rats were divided into four groups: control (CTRL), isoproterenol (ISO) treated (1 mg/kg) and two groups treated with paclitaxel (PAC), which was administrated (10 mg/kg/day) for 5 days by gavage/per os alone or in combination (ISO + PAC) 3 weeks after ISO treatment. Ventricular tissue was harvested 24 h later for measurements of heme oxygenase (HO-1), reduced glutathione (GSH), oxidized glutathione (GSSG), superoxide dismutase (SOD), NF-κB, TNF-α and myeloperoxidase (MPO); (3) Results: HO-1 protein concentration, HO-1 activity, SOD protein concentration and total glutathione significantly decreased in response to ISO treatment. When PAC was administered in conjunction with ISO, HO-1, SOD concentration and total glutathione were not different from control levels. MPO activity, NF-κB concentration and TNF-α protein concentration were significantly increased in the ISO-only group, while the levels of these molecules were restored when PAC was co-administered; (4) Conclusions: Oral administration of PAC can maintain the expression of important antioxidants, anti-inflammatory molecules, HO-1, SOD and GSH, and suppress the production of TNF-α, MPO and NF-κB, which are involved in myocardial damage. The principal component of this cellular defense seems to be the expression of HO-1.

Effects of Taraxerol on Oxidative and Inflammatory Mediators in Isoproterenol-Induced Cardiotoxicity in an Animal Model.

Myocardial infarction (MI) continues to be an important issue in healthcare systems worldwide, leading to high rates of morbidity and mortality. Despite ongoing efforts towards the development of preventive measures and treatments, addressing the challenges posed by MI remains difficult both in developed and developing countries. However, researchers recently investigated the potential cardioprotective effects of taraxerol utilizing an isoproterenol (ISO)-induced cardiotoxicity model among Sprague Dawley rats. Specifically, subcutaneous tissue injections consisting of 5.25 mg/kg or 8.5 mg/kg ISO were administered over two consecutive days as stimuli to induce cardiac injury. To investigate the possibility of preventing damage caused by ISO-induced cardiotoxicity by taraxerol treatment, five groups were formed: a normal control group (1% Tween 80), an ISO control group, an amlodipine group administered 5 mg/kg/day, and various doses of taraxerol. The study results showed that treatment significantly reduced cardiac marker enzymes. Additionally, pretreatment with taraxerol increased myocardial activity in SOD and GPx, leading to significant reductions in serum CK-MB levels along with MDA, TNF-α, and IL-6. Further histopathological analysis supported these observations, as treated animals had less cellular infiltration compared to untreated ones. These multifaceted findings suggest that oral administration of taraxerol could potentially protect hearts from ISO-caused damage by increasing endogenous antioxidant concentrations while decreasing pro-inflammatory cytokines.

PO-01-217 FUNCTIONAL CHARACTERIZATION OF CALCIUM HANDLING KINETICS IN RE-ENGINEERED CARDIOMYOCYTES FROM A PATIENT WITH TYPE 2 LONG QT SYNDROME

Type 2 long QT syndrome (LQT2) is caused by pathogenic variants in the KCNH2-encoded Kv11.1 K+ channel resulting in prolongation of the action potential duration (APD) at the cellular level and QT prolongation in the patient, predisposing LQT2 patients to potentially lethal ventricular arrhythmias. Limited evidence, namely prolonged Ca2+ transient in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and enhanced Ca2+ leak in transgenic LQT2 rabbit model, suggests that KCNH2 pathogenic variants may be associated with perturbations in intracellular Ca2+ handling. To characterize the APD and Ca2+ handling properties of an iPSC-CM model of LQT2. iPSC-CMs were generated from a patient with a pathogenic KCNH2 frame-shift variant (p.Q901fs/71) and an unrelated healthy control. Confocal imaging of FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90) in iPSC-CMs. Ca2+ kinetics was characterized using Ca2+ sensing dye Fluo4-AM before and after treatment with 100 nM isoproterenol (ISO). Q901fs/71-iPSC-CMs had a significantly prolonged APD90 compared to wild-type (WT) iPSC-CMs (444 ±9 ms vs 631±8 ms, p<0.001). Q901fs/71-iPSC-CMs had a higher Ca2+ amplitude (DF/F0) before (0.423±0.043 vs 0.97±0.07, p=0.001) and after ISO treatment (0.602±0.051 vs 1.056 ±0.06, p<0.001) compared to WT. The Ca2+ transient duration (CTD90) was longer in Q901fs/71-iPSC-CMs than in WT before (1.062±0.041 s vs 1.306±0.038 s, p=0.01), but not after ISO (1.071±0.062 s vs 1.092±0.044 s, p=0.96). Peak to 90% Ca2+ decay time was increased in Q901fs/71-iPSC-CMs before (0.785±0.04 s vs 0.942±0.033 s, p=0.028), but not after ISO (0.748±0.054 s vs 0.767±0.038 s, p=0.95). Upstroke time was longer in Q901fs/71-iPSC-CMs compared to WT before (0.281±0.006 s vs 0.364±0.014 s; p=0.002) but not after ISO (0.327±0.014 s vs 0.344±0.025 s; p=0.88). Upstroke velocity (DF/F0/t) was increased in Q901fs/71-iPSC-CMs before (1.092±0.122 vs 2.278±0.278, p<0.001) and after ISO (1.310±0.117 vs 2.2±0.139, p=0.005). Ca2+ leak from sarcoplasmic reticulum was higher in Q901fs/71-iPSC-CMs before (16% vs 38%) and after ISO (16% vs 55%). We show that iPSC-CMs derived from the patient with a truncating LQT2-causative variant (yielding likely Kv11.1 channel haploinsufficiency) have prolonged APD90 and display abnormal Ca2+ handling properties such as higher amplitude, increase in Ca2+ leak, prolonged transient duration, Ca2+ decay time, and upstroke velocity compared to WT iPSC-CMs.

Control of the fight-or-flight response of heart rate by L-type Cav1.3 (α1D) calcium channels

The mechanisms underlying the increase in the frequency of heartbeat induced by catecholamines, so called fight-or-flight response (FFR) of heart rate (HR), are incompletely understood. cAMP-dependent regulation of f-(HCN) channels, underlying the “funny” current (If), and ryanodine receptor 2 (RyR2)-mediated Ca2+ release are considered as the predominant mechanisms of HR FFR. However, it is showed that If contributes to setting the basal HR, but is not required for the sympathetic regulation of pacemaking. In addition, impairment of RyR2-mediated [Ca2+]i release by ryanodine only reduces β-adrenergic regulation of pacemaker activity, suggesting that other mechanism(s) are involved in the generation of HR FFR. We have shown that L-type Cav1.3 calcium channels are required for β-adrenergic mediated initiation of firing in dormant sino-atrial node (SAN) pacemaker cells. To define the role of Cav1.3 channels in the FFR of HR and SAN automaticity. We crossed mice deficient in Cav1.3 channels (Cav1.3-/-) with mice (CNBD) lacking cAMP sensitivity of f-channels. In vivo and in isolated hearts ECG recordings were performed. Automaticity and diastolic current of SAN cells were evaluated using patch-clamp technique. Cav1.3-/- and CNBD mice displayed bradycardia at rest, but similar degree of increase in HR after β-adrenergic stimulation by injection of isoproterenol (ISO) in comparison to WT mice. However, Cav1.3-/-/CNBD mice did not show significant HR increase in vivo after injection of ISO or during physical exercise. ISO failed to increase the rate in isolated herats and in the SAN cells from Cav1.3-/-/CNBD mice. Consistent with these data, mutant mice in which L-type Cav1.2 channels have been rendered insensitive to dihydropyridines (Cav1.2DHP-/-) showed that concomitant selective pharmacologic inhibition of Cav1.3 and cAMP-dependent dependent regulation of f-HCN channels by the di-cyclic nucleotide di-cGMP prevented the positive chronotropic response to ISO of SAN cells automaticity. Under the same conditions, ISO did not significantly increase diastolic current, explaining loss of SAN FFR. Our study identifies L-type Cav1.3 channels as key effectors of β-adrenergic regulation of SAN automaticity with If.

The source of liberation of dipeptidyl peptidase 3 (DPP3), a newly emerged cardiac biomarker, in pathological situations

Dipeptidyl peptidase 3 (DPP3) is a zinc dependent aminopeptidase that cleaves dipeptides from the N- terminus of several substrates such as angiotensins. DPP3 is an intracellular protein. It has also been detected in the circulation of healthy and sick individuals being known as circulating DPP3 (cDPP3). cDPP3 has emerged as a biomarker and a therapeutic target in acute cardiac stress. Despite the well-known ubiquitous expression of DPP3 in physiological situations, its expression and source of release in pathological conditions remain to be explored. This study aimed to evaluate DPP3 expression in cardiac stress conditions. C57BL6/J mice were subjected to cardiac dysfunction using two different pharmacological models: 1- Isoproterenol (ISO) model: injection of 150 mg/kg of ISO every 12 h for two days; 2- Doxorubicin (DOX) model: injection of 4 mg/kg/day of DOX for 6 days. Blood samples, organs, and bone marrow (cells and supernatant) were collected at the end of each protocol. DPP3 protein expression was evaluated by western-blot. Enzymatic activity of DPP3 was measured by luminometric immunoassay (DPP3-LIA). Our results showed different patterns of DPP3 expression based on pathological condition. Mice in ISO group showed a significantly higher DPP3 protein expression in bone marrow, both cells and supernatant, compared to control group (1.8X, P < 0.05). In consistence with this, DPP3 enzymatic activity was significantly increased in both bone marrow supernatant and plasma in these mice (1.4X, P < 0.05) and (1.3X, P < 0.05) respectively, indicating that bone marrow might be responsible for releasing DPP3 into the surrounding microenvironment and into the circulation. However, DPP3 expression was decreased in the spleen of ISO mice (0.5X, P < 0.05). This decrease could be explained by spleen-contraction following ISO injection, a phenomenon already described in the literature, allowing greater release of red blood cells, highly expressing DPP3, into bloodstream. On the other hand, in Dox model, only an up-regulation of DPP3 in the spleen (1.7X, P < 0.05) was reported. An increase of DPP3 activity in plasma but not in bone marrow supernatant was remarked. Our results show that DPP3 expression varies based on the type of model used to induce cardiac dysfunction. It seems that main hematopoietic organs (spleen and bone marrow) might represent sources of cDPP3.

β-adrenergic receptor agonist promotes ductular expansion during 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced chronic liver injury

Intrahepatic nerves are involved in the regulation of metabolic reactions and hepatocyte-based regeneration after surgical resection, although their contribution to chronic liver injury remains unknown. Given that intrahepatic nerves are abundant in the periportal tissue, they may be correlated also with cholangiocyte-based regeneration. Here we demonstrate that isoproterenol (ISO), a β-adrenergic receptor agonist, promoted ductular expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in vivo. Immunofluorescence analysis shows that nerve fibers positive for tyrosine hydroxylase form synaptophysin-positive nerve endings on epithelial cell adhesion molecule-positive (EpCAM+) cholangiocytes as well as on Thy1+ periportal mesenchymal cells (PMCs) that surround bile ducts, suggesting that the intrahepatic biliary tissue are targeted by sympathetic nerves. In vitro analyses indicate that ISO directly increases cAMP levels in cholangiocytes and PMCs. Mechanistically, ISO expands the lumen of cholangiocyte organoids, resulting in promotion of cholangiocyte proliferation, whereas it increases expression of fibroblast growth factor 7, a growth factor for cholangiocytes, in PMCs. Taken together, the results indicate that intrahepatic sympathetic nerves regulate remodeling of bile ducts during DDC-injury by the activation of β-adrenergic receptors on cholangiocytes and PMCs.

Respiration in isolated cardiomyocytes and microviscosity of their membranes in rats of different ages with heart failure

Background. According to the latest epidemiological data, heart failure (HF) is diagnosed in 10% of adult population over 70 years old. However, currently this diagnosis is being increasingly made in young and middleaged people. The pathogenesis of HF may be based on a decrease in respiration in cardiomyocytes with age, which affects the function of energy-dependent processes in cells.Aim. To study respiration in cardiomyocytes and microviscosity of their membranes in rats aged 2 and 15 months with heart failure.Materials and methods. The study was carried out on male Wistar rats. The animals were divided into 4 groups: 2 groups of intact rats aged 2 and 15 months (n = 12) and 2 groups of animals of similar ages (n = 10) with a model of HF. In the latter, HF formed by day 28 after a double subcutaneous injection of isoproterenol hydrochloride at a dose of 170 mg / kg with an interval of 24 hours. Isolated cardiomyocytes were obtained from enzyme-washed rat hearts. Cell respiration was studied in a thermostated chamber in an incubation medium supplemented with phosphorylation (ADP) and oxidation (succinate) substrates. The respiratory control (RC) ratio was calculated by dividing V3 respiration state to V4. Membrane microviscosity characteristics were assessed by the eximerization coefficient of pyrene fluorescence in the areas of protein – lipid and lipid – lipid interactions.Results. RC in cardiomyocyte membranes of intact animals did not change with age. In 2-month-old rats with HF, respiratory control ratio (RCR) did not change compared with the intact age-matched controls. In 15-month-old rats with HF, there was a significant decrease in RC of cardiomyocytes (CM) compared with the intact animals of this age and 2-month-old rats with HF. An age-dependent decrease in the microviscosity of CM membranes in the areas of lipid – lipid interactions and no significant changes in the parameter at the sites of protein – lipid interactions were noted. In 2-month-old animals with HF, the microviscosity of CM membranes in the areas of protein – lipid interactions significantly decreased, and in 15-month-old rats it increased, compared with the intact controls. When carrying out an intergroup comparison, an age-dependent increase in the microviscosity of CM membranes in the areas of protein – lipid interactions and no differences in the parameter in the areas of lipid – lipid interactions were revealed.Conclusion. In the intact rats, the absence of significant changes in respiration with age was revealed. In the 15-month-old animals with HF, respiration in CM was significantly lower than in the intact controls and 2-monthold animals with HF. These changes may be due to the differences in the membrane microviscosity characteristics in different periods of ontogenesis.

Trim65 attenuates isoproterenol-induced cardiac hypertrophy by promoting autophagy and ameliorating mitochondrial dysfunction via the Jak1/Stat1 signaling pathway

Pathological cardiac hypertrophy is a major cause of heart failure, and there is no effective approach for its prevention or treatment. The Trim family is a recently identified family of E3 ubiquitin ligases that regulate cardiac hypertrophy. Trim65, which is a member of the Trim family, previous studies have not determined whether Trim65 affects cardiac hypertrophy. In this study, the effects of Trim65 on isoproterenol (ISO)-induced cardiac hypertrophy and the underlying mechanisms were investigated. In contrast to C57BL/6 mice, Trim65-knockout (Trim65-KO) mice developed more severe myocardial hypertrophy, fibrosis and cardiac dysfunction after being intraperitoneally injected with ISO for 2 weeks. Transmission electron microscopy (TEM) revealed that the autophagic flux was inhibited, mitochondria were swollen, and mitochondrial cristae were lost or decreased in the myocardium of Trim65-KO mice. In vitro studies demonstrated that overexpression of Trim65 inhibited ISO-induced cardiomyocyte hypertrophy by increasing mitochondrial density and membrane potential, and the Stat1 inhibitor fludarabine attenuated the effect of Trim65 knockdown on ISO-induced cardiomyocyte hypertrophy by reducing Reactive oxygen species (ROS) production and increasing the mitochondrial density and membrane potential. Our findings provide the first link between Trim65 and mitochondria, and we found for the first time that Trim65 inhibits mitochondria-dependent apoptosis and autophagy via the Jak1/Stat1 signalling pathway, ultimately attenuating ISO-induced cardiac hypertrophy; this effect of Trim65 might be mediated via the regulation of Jak1 ubiquitination. Taking these findings together, we suggest that genes that are related to mitochondria-dependent apoptosis and that are associated with Trim65 could be promising therapeutic targets for cardiac hypertrophy.

Effects of electroacupuncture on cardiac function and local field potential of sensory and motor cortices in mice with stress cardiomyopathy

To observe the effects of electroacupuncture (EA) on cardiac function and local field potential (LFP) in sensory and motor cortices in mice with stress cardiomyopathy (SC), and to explore the possible mechanism of EA in improving SC. Twenty-seven female C57BL/6 mice were randomized into a blank group, a model group and an EA group, 9 mice in each group. In the model group and the EA group, SC model was established by continuous intraperitoneal injection of isoproterenol (ISO) for 14 days. At the same time of modeling, EA was applied at "Neiguan" (PC 6) and "Shenmen" (HT 7) in the EA group, with disperse-dense wave, in frequency of 2 Hz/15 Hz, 15 min each time, once a day for 14 days. After intervention, the total movement distance, the number of crossing grid and the number of crossing central grid of open field test within 5 minutes were observed; the left ventricular function indexes (left ventricular diameter of end-diastole [LVIDd], left ventricular diameter of end-systole [LVIDs], left ventricular volume of end-diastole [LVEDV], left ventricular volume of end-systole [LVESV], ejection fraction [EF] and fraction shortening [FS]) were detected by echocardiography; the changes in ST-segment amplitude and PR interval of electrocardiogram were observed; the morphology of myocardial tissue was observed by HE staining; the serum levels of cortisol (CORT), cardiac troponin T (cTnT) and brain natriuretic peptide (BNP) were detected by ELISA; the changes of LFP in sensory and motor cortices were recorded by Plexon multi-channel acquisition system. Compared with the blank group, in the model group, the total movement distance, the number of crossing grid and the number of crossing central grid of open field test were decreased (P<0.05); LVIDd, LVIDs, LVEDV and LVESV were increased (P<0.05), EF and FS were decreased (P<0.05); ST-segment amplitude was increased (P<0.05) and PR interval was prolonged (P<0.05); irregular myocardial fiber arrangement, interstitial edema and inflammatory cell infiltration were observed; the serum levels of CORT, cTnT and BNP were increased (P<0.05); in the sensory cortex, the ratios of delta, theta, alpha and beta frequency bands were increased (P<0.05), the maximum energy spectrum of theta and beta frequency bands was increased (P<0.05), the power spectral density (PSD) of delta, theta, alpha, beta and gamma frequency bands was increased (P<0.05); in the motor cortex, the ratios of delta, theta, alpha and beta frequency bands were increased (P<0.05), the maximum energy spectrum as well as PSD of delta, theta, alpha, beta and gamma frequency bands were increased (P<0.05). Compared with model group, in the EA group, the total movement distance, the number of crossing grid and the number of crossing central grid of open field test were increased (P<0.05); LVIDd, LVIDs, LVEDV and LVESV were decreased (P<0.05), EF and FS were increased (P<0.05); ST-segment amplitude was decreased (P<0.05), and the PR interval was shortened (P<0.05); myocardial fiber injury and inflammatory cell infiltration were reduced; the serum levels of CORT, cTnT and BNP were decreased (P<0.05); in the sensory cortex, the ratios of theta, alpha and beta frequency bands were decreased (P<0.05), the ratio of gamma frequency band was increased (P<0.05), the maximum energy spectrum of theta frequency band as well as the PSD of theta, alpha, beta and gamma frequency bands were decreased (P<0.05); in the motor cortex, the ratios of theta, alpha and beta frequency bands were decreased (P<0.05) and the ratio of gamma frequency band was increased (P<0.05), the maximum energy spectrum of delta frequency band was increased (P<0.05), the maximum energy spectrum of theta frequency band as well as the PSD of theta and gamma frequency bands were decreased (P<0.05). EA can improve cardiac function in mice with stress cardiomyopathy, and its mechanism may be related to the regulation of local field potentials in sensory and motor cortices.

An Ethanol Extract of Perilla frutescens Leaves Suppresses Adrenergic Agonist-Induced Metastatic Ability of Cancer Cells by Inhibiting Src-Mediated EMT.

Previous studies have indicated that the adrenergic receptor signaling pathway plays a fundamental role in chronic stress-induced cancer metastasis. In this study, we investigated whether an ethanol extract of Perilla frutescens leaves (EPF) traditionally used to treat stress-related symptoms by moving Qi could regulate the adrenergic agonist-induced metastatic ability of cancer cells. Our results show that adrenergic agonists including norepinephrine (NE), epinephrine (E), and isoproterenol (ISO) increased migration and invasion of MDA-MB-231 human breast cancer cells and Hep3B human hepatocellular carcinoma cells. However, such increases were completely abrogated by EPF treatment. E/NE induced downregulation of E-cadherin and upregulation of N-cadherin, Snail, and Slug. Such effects were clearly reversed by pretreatment with EPF, suggesting that the antimetastatic activity of EPF could be related to epithelial-mesenchymal transition (EMT) regulation. EPF suppressed E/NE-stimulated Src phosphorylation. Inhibition of Src kinase activity with dasatinib completely suppressed the E/NE-induced EMT process. Transfecting MDA-MB-231 cells with constitutively activated Src (SrcY527F) diminished the antimigration effect of EPF. Taken together, our results demonstrate that EPF can suppress the adrenergic agonist-promoted metastatic ability of cancer cells by inhibiting Src-mediated EMT. This study provides basic evidence supporting the probable use of EPF to prevent metastasis in cancer patients, especially those under chronic stress.

Biochanin-A alleviates fibrosis and inflammation in cardiac injury in mice.

Biochanin-A (BCA),is an isoflavonoid, exhibits protective effects against various diseases. This study was conducted to observe the effect of BCA on isoprenaline (ISP)-inducedcardiac fibrosis and explore the underlying mechanism. The curative effect of BCA was investigated with oral administration for 14 days in ISP-inducedcardiac fibrosis in mice. The fibrotic biomarkers, like collagen I and III, were estimated by ELISA. Commercial kits were used to estimate cholesterol, triglycerides, and creatine kinase-myocardial band (CK-MB)levels. Themessenger ribonucleic acid(mRNA) expression studies were performed by quantitative real-time polymerase chain reaction. Gelatin zymography was used to study the expression of matrix metalloproteinases-2 (MMP-2).BCAco-administration significantly improved the morphometric parameters; including heart weight, heart weight to body weight, heart weight to tibial length, and lipid profile. BCA treatment showed a reduction in inflammatory cells and collagen deposition as depicted in the histopathology of heart tissues. The enhanced levels of collagen-I, III, and hydroxyproline were significantly decreased by BCA co-treatment, whereas CK-MBlevel was reduced slightly. BCA co-administrationincreased the activity of reduced glutathione enzyme, showing the antioxidativeeffects of BCA. BCAtreatment significantly reduced interleukin-6 (Il6) inflammatory cytokine along with partially decreased mRNA expression of fibrotic signaling markers such as natriuretic peptide type B (Nppb), α-smooth muscle actin (Acta2), connective tissue growth factor (Ctgf), transforming growth factor β (Tgfb), small mothers against decapentaplegic homolog-3 (Smad-3). However, BCA did not modify Mmp-2expression, which was significantly increased by ISP. In conclusion, BCA exerts an antifibrotic effect by modulating lipid profile, enhancing antioxidant enzyme, and reducing collagen content and inflammation.

GRK2 participation in cardiac hypertrophy induced by isoproterenol through the regulation of Nrf2 signaling and the promotion of NLRP3 inflammasome and oxidative stress.

In cases of heart failure, cardiac hypertrophy may be caused by the upregulation of G-protein-coupled receptor kinase 2 (GRK2). Both NLRP3 inflammasome and oxidative stress contribute to cardiovascular disease. In this study, we clarified the effect of GRK2 on cardiac hypertrophy in H9c2 cells induced by isoproterenol (ISO) and examined the underlying mechanisms. We randomly categorized H9c2 cells into five groups: an ISO group, a paroxetine plus ISO group, a GRK2 small-interfering RNA (siRNA) plus ISO group, a GRK2 siRNA combined with ML385 plus ISO group, and a control group. To determine the effect of GRK2 on cardiac hypertrophy induced by ISO, we carried out CCK8 assays, RT-PCR, TUNEL staining, ELISA assay, DCFH-DA staining, immunofluorescence staining, and western blotting. By using paroxetine or siRNA to inhibit GRK2, we significantly decreased cell viability; reduced the mRNA levels of ANP, BNP, and β-MHC; and limited the apoptosis rate and protein levels of cleaved caspase-3 and cytochrome c in H9c2 cells treated with ISO. We also found that oxidative stress induced by ISO could be mitigated with paroxetine or GRK2 siRNA. This result was validated by decreased activities of the antioxidant enzymes CAT, GPX, and SOD and increased MDA levels and ROS production. We observed that the protein expression of NLRP3, ASC, and caspase-1 and the intensity of NLRP3 could be inhibited by paroxetine or GRK2 siRNA. Both paroxetine and GRK2 siRNA were able to abolish the increase in GRK2 expression induced by ISO. They also could increase protein levels of HO-1, nuclear Nrf2, and Nrf2 immunofluorescence intensity; however, they could not change the protein level of cytoplasmic Nrf2. By combining treatment with ML385, we were able to reverse GRK2 inhibition on H9c2 cells treated with ISO. According to the results of this study, GRK2 participated in cardiac hypertrophy induced by ISO by mitigating NLRP3 inflammasome and oxidative stress through the signaling of Nrf2 in H9c2 cells.

Surface hydrolysis-designed AuNPs-zwitterionic-glucose as a novel tool for targeting macrophage visualization and delivery into infarcted hearts.

Macrophages, innate immune cells, are key players in the maintenance of myocardial homeostasis under normal conditions and tissue repair after injury. The infiltration of macrophages into the injured heart makes them a potentially appealing vehicle for noninvasive imaging and targeted drug delivery of myocardial infarction (MI). In this study, we demonstrated the use of surface hydrolysis-designed AuNPs-zwitterionic-glucose to label macrophages and track their infiltration into isoproterenol hydrochloride (ISO)-induced MI sites noninvasively using CT. The AuNPs-zwitterionic-glucose did not affect the viability or cytokine release of macrophages and were highly taken up by these cells. The in vivo CT images were obtained on Day 4, Day 6, Day 7, and Day 9, and the attenuation was seen to increase in the heart over time compared to the Day 4 scan. In vitro analysis also confirmed the presence of macrophages around injured cardiomyocytes. Additionally, we also addressed the concern of cell tracking or merely AuNP tracking, which is the inherent problem for any form of nanoparticle-labeled cell tracking by using zwitterionic and glucose-functionalized AuNPs. The glucose coated on the surface of AuNPs-zwit-glucose will be hydrolyzed in macrophages, forming only zwitterionic protected AuNPs that cannot be taken up again by endogenous cells in vivo. This will greatly improve the accuracy and precision of imaging and target delivery. We believe this is the first study to noninvasively visualize the infiltration of macrophages into MI hearts using CT, which could be used for imaging and evaluating the possibility of macrophage-mediated delivery in infarcted hearts.