BDNF mimetics recover palmitic acid-induced injury in cardiomyocytes by ameliorating Akt-dependent mitochondrial impairments.

Cardiac lipotoxicity is a prevalent consequence of lipid metabolism disorders occurring in cardiomyocytes, which in turn precipitates the onset of heart failure. Mimetics of brain-derived neurotrophic factor (BDNF), such as 7,8-dihydroxyflavone (DHF) and 7,8,3'-trihydroxyflavone (THF), have demonstrated significant cardioprotective effects. However, it remains unclear whether these mimetics can protect cardiomyocytes against lipotoxicity. The aim of this study was to examine the impact of DHF and THF on the lipotoxic effects induced by palmitic acid (PA), as well as the concurrent mitochondrial dysfunction. H9c2 cells were subjected to treatment with PA alone or in conjunction with DHF or THF. Various factors such as cell viability, lactate dehydrogenase (LDH) release, death ratio, and mitochondrial function including mitochondrial membrane potential (MMP), mitochondrial-derived reactive oxygen species (mito-SOX) production, and mitochondrial respiration were assessed. PA dose-dependently reduced cell viability, which was restored by DHF or THF. Additionally, both DHF and THF decreased LDH content, death ratio, and mito-SOX production, while increasing MMP and regulating mitochondrial oxidative phosphorylation in cardiomyocytes. Moreover, DHF and THF specifically activated Akt signaling. The protective effects of DHF and THF were abolished when an Akt inhibitor was used. In conclusion, BDNF mimetics attenuate PA-induced injury in cardiomyocytes by alleviating mitochondrial impairments through the activation of Akt signaling.

7,8,3'-Trihydroxyflavone prevents doxorubicin-induced cardiotoxicity and mitochondrial dysfunction via activating Akt signaling pathway in H9c2 cells.

Clinical application of the widely used chemotherapeutic agent, doxorubicin (DOX), is limited by its cardiotoxicity. Mitochondrial dysfunction has been revealed as a crucial factor in DOX-induced cardiotoxicity. 7,8,3'-Trihydroxyflavone (THF) is a mimetic brain-derived neurotrophic factor with neuroprotective effects. However, the potential effects of THF on DOX-induced cardiomyocyte damage and mitochondrial disorders remain unclear. H9c2 cardiomyoblasts were exposed to DOX and/or THF at different concentrations. Cardiomyocyte injury was evaluated using lactate dehydrogenase (LDH) assay and Live/Dead cytotoxicity kit. Meanwhile, mitochondrial membrane potential (MMP), morphology, mitochondrial reactive oxygen species (mito-ROS) production, and the oxygen consumption rate of cardiomyocytes were measured. The protein levels of key mitochondria-related factors such as adenosine monophosphate-activated protein kinase (AMPK), mitofusin 2 (Mfn2), dynamin-related protein 1 (Drp1), and optic atrophy protein 1 (OPA1) were examined. We found that THF reduced LDH content and death ratio of DOX-treated cardiomyocytes in a concentration-dependent manner, while increasing MMP without significantly affecting the routine and maximum capacity of mitochondrial respiration. Mechanistically, THF increased the activity of Akt and protein levels of Mfn2 and heme oxygenase 1 (HO-1). Moreover, inhibition of Akt reversed the protective role of THF, increased mito-ROS levels, and repressed Mfn2 and HO-1 expression. Therefore, we conclude, THF relieves DOX-induced cardiotoxicity and improves mitochondrial function by activating Akt-mediated Mfn2 and HO-1 pathways. This finding provides promising therapeutic insights for DOX-induced cardiac dysfunction.

BDNF mimetic 7,8-dihydroxyflavone rescues rotenone-induced cytotoxicity in cardiomyocytes by ameliorating mitochondrial dysfunction.

The relationship between mitochondrial dysfunction and cardiovascular disease pathogenesis is well recognized. 7,8-Dihydroxyflavone (7,8-DHF), a mimetic of brain-derived neurotrophic factor, inhibits mitochondrial impairments and improves cardiac function. However, the regulatory role of 7,8-DHF in the mitochondrial function of cardiomyocytes is not fully understood. To investigate the potential mito-protective effects of 7,8-DHF in cardiomyocytes, we treated H9c2 or HL-1 cells with the mitochondrial respiratory complex I inhibitor rotenone (Rot) as an in vitro model of mitochondrial dysfunction. We found that 7,8-DHF effectively eliminated various concentrations of Rot-induced cell death and reduced lactate dehydrogenase release. 7,8-DHF significantly improved mitochondrial membrane potential and inhibited mitochondrial reactive oxygen species. Moreover, 7,8-DHF decreased routine and leak respiration, restored protein levels of mitochondrial complex I-IV, and increased ATP production in Rot-treated H9c2 cells. The protective role of 7,8-DHF in Rot-induced damage was validated in HL-1 cells. Nuclear phosphorylation protein expression of signal transducer and activator of transcription 3 (STAT3) was significantly increased by 7,8-DHF. The present study suggests that 7,8-DHF rescues Rot-induced cytotoxicity by inhibiting mitochondrial dysfunction and promoting nuclear translocation of p-STAT3 in cardiomyocytes, thus nominating 7,8-DHF as a new pharmacological candidate agent against mitochondrial dysfunction in cardiac diseases.

7,8-Dihydroxyflavone alleviates cardiac fibrosis by restoring circadian signals via downregulating Bmal1/Akt pathway.

Brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) pathway is a therapeutic target in cardiac diseases. A BDNF mimetic, 7,8-dihydroxyflavone (7,8-DHF), is emerging as a protective agent in cardiomyocytes; however, its potential role in cardiac fibroblasts (CFs) and fibrosis remains unknown. Thus, we aimed to explore the effects of 7,8-DHF on cardiac fibrosis and the possible mechanisms. Myocardial ischemia (MI) and transforming growth factor-ß1 (TGF-ß1) were used to establish models of cardiac fibrosis. Hematoxylin & eosin and Masson's trichrome stains were used for histological analysis and determination of collagen content in mouse myocardium. Cell viability kit, EdU (5-ethynyl-2'-deoxyuridine) assay and immunofluorescent stain were employed to examine the effects of 7,8-DHF on the proliferation and collagen production of CFs. The levels of collagen I, α-smooth muscle actin (α-SMA), TGF-ß1, Smad2/3, and Akt as well as circadian rhythm-related signals including brain and muscle Arnt-like protein 1 (Bmal1), period 2 (Per2), and cryptochrome 2 (Cry2) were analyzed. Treatment with 7,8-DHF markedly alleviated cardiac fibrosis in MI mice. It inhibited the activity of CFs accompanied by decreasing number of EdU-positive cells and downregulation of collagen I, α-SMA, TGF-ß1, and phosphorylation of Smad2/3. 7,8-DHF significantly restored the dysregulation of Bmal1, Per2, and Cry2, but inhibited the overactive Akt. Further, inhibition of Bmal1 by SR9009 effectively attenuated CFs proliferation and collagen production of CFs. In summary, these findings indicate that 7,8-DHF attenuates cardiac fibrosis and regulates circadian rhythmic signals, at least partly, by inhibiting Bmal1/Akt pathway, which may provide new insights into therapeutic cardiac remodeling.

Small-molecule 7,8-dihydroxyflavone counteracts compensated and decompensated cardiac hypertrophy via AMPK activation.

BACKGROUND: Pathological cardiac hypertrophy is a compensated response to various stimuli and is considered a key risk factor for heart failure. 7,8-Dihydroxyflavone (7,8-DHF) is a flavonoid derivative that acts as a small-molecule brain-derived neurotrophic factor mimetic. The present study aimed to explore the potential role of 7,8-DHF in cardiac hypertrophy. METHODS: Kunming mice and H9c2 cells were exposed to transverse aortic constriction or isoproterenol (ISO) with or without 7,8-DHF, respectively. F-actin staining was performed to calculate the cell area. Transcriptional levels of hypertrophic markers, including ANP, BNP, and ß-MHC, were detected. Echocardiography, hematoxylin-eosin staining, and transmission electron microscopy were used to examine the cardiac function, histology, and ultrastructure of ventricles. Protein levels of mitochondria-related factors, such as adenosine monophosphate-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), were detected. RESULTS: 7,8-DHF inhibited compensated and decompensated cardiac hypertrophy, diminished the cross-sectional area, and alleviated the mitochondrial disorders of cardiomyocytes. Meanwhile, 7,8-DHF reduced the cell size and repressed the mRNA levels of the hypertrophic markers of ISO-treated cardiomyocytes. In addition, 7,8-DHF activated AMPK and PGC-1α signals without affecting the protein levels of mitochondrial dynamics-related molecules. The effects of 7,8-DHF were eliminanted by Compound C, an AMPK inhibitor. CONCLUSIONS: These findings suggest that 7,8-DHF inhibited cardiac hypertrophy and mitochondrial dysfunction by activating AMPK signaling, providing a potential agent for the treatment of pathological cardiac hypertrophy.

Honokiol Inhibits Atrial Metabolic Remodeling in Atrial Fibrillation Through Sirt3 Pathway.

Background and Purpose: Atrial metabolic remodeling plays a critical role in the pathogenesis of atrial fibrillation (AF). Sirtuin3 (Sirt3) plays an important role in energy homeostasis. However, the effect of Sirt3 agonist Honokiol (HL) on AF is unclear. Therefore, the aim of this study is to determine the effect of HL on atrial metabolic remodeling in AF and to explore possible mechanisms. Experimental Approach: irt3 and glycogen deposition in left atria of AF patients were examined. Twenty-one rabbits were divided into sham, P (pacing for 3 weeks), P + H treatment (honokiol injected with pacing for 3 weeks). The HL-1 cells were subjected to rapid pacing at 5 Hz for 24 h, in the presence or absence of HL and overexpression or siRNA of Sirt3 by transfection. Metabolic factors, circulating metabolites, atrial electrophysiology, ATP level, and glycogens deposition were detected. Acetylated protein and activity of its enzymes were detected. Key Results: Sirt3 was significantly down-regulated in AF patients and rabbit/HL-1cell model, resulting in the abnormal expression of its downstream metabolic key factors, which were significantly restored by HL. Meanwhile, AF induced an increase of the acetylation level in long-chain acyl-CoA dehydrogenase (LCAD), AceCS2 and GDH, following decreasing of activity of it enzymes, resulting in abnormal alterations of metabolites and reducing of ATP, which was inhibited by HL. The Sirt3 could regulate acetylated modification of key metabolic enzymes, and the increase of Sirt3 rescued AF induced atrial metabolic remodeling. Conclusion and Implications: HL inhibited atrial metabolic remodeling in AF via the Sirt3 pathway. The present study may provide a novel therapeutical strategy for AF.

The regulatory role of the BDNF/TrkB pathway in organ and tissue fibrosis.

Fibrosis across diverse organ systems is one of the leading causes of morbidity and mortality by inducing progressive architectural remodeling and organ dysfunction. Brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase receptor B (TrkB) play crucial roles in regulating neural survival, development, function and plasticity in the central and the peripheral nervous system. Previous studies demonstrated that the BDNF/TrkB pathway is widely distributed in different cell types such as neuron, epithelial cell, hepatocyte, and cardiomyocyte. Recently, there is increasing recognition that BDNF and TrkB are also expressed in fibroblasts in different organs. Moreover, growing evidence was obtained regarding the functional roles of BDNF/TrkB signaling in organ and tissue fibrosis. Thus, this review summarizes the basic molecular characteristics of the BDNF/TrkB cascade and the findings of the crucial roles and therapeutic value in organ and tissue fibrosis including pulmonary fibrosis, hepatic fibrosis, renal fibrosis, cardiac fibrosis, bladder fibrosis and skin fibrosis. Small molecule BDNF mimetic and BDNF-related non-coding RNAs are also discussed for developing new therapeutic approaches for fibrotic disorders.

MicroRNA-98 ameliorates doxorubicin-induced cardiotoxicity via regulating caspase-8 dependent Fas/RIP3 pathway.

Cardiotoxicity is one of the primary limitations in the clinical use of the anticancer drug doxorubicin (DOX). However, the role of microRNAs (miRNAs) in DOX-induced cardiomyocyte death has not yet been covered. To investigate this, we observed a significant increase in miR-98 expression in neonatal rat ventricular myocytes after DOX treatment, and MTT, LIVE/Dead and Viability/Cytotoxicity staining showed that miR-98 mimic inhibited DOX-induced cell death. This was also confirmed by Flow cytometry and Annexin V-FITC/PI staining. Interestingly, the protein expression of caspase-8 was upregulated by miR-98 mimics during this process, whereas Fas and RIP3 were downregulated. In addition, the effect of miR-98 against the expression of Fas and RIP3 were restored by the specific caspase-8 inhibitor Z-IETD-FMK. Thus, we demonstrate that miR-98 protects cardiomyocytes from DOX-induced injury by regulating the caspase-8-dependent Fas/RIP3 pathway. Our findings enhance understanding of the therapeutic role of miRNAs in the treatment of DOX-induced cardiotoxicity.

The Emerging Role of BDNF/TrkB Signaling in Cardiovascular Diseases.

Brain-derived neurotrophic factor (BDNF) is one of the most abundantneurotrophins in the central nervous system. Numerous studies suggestthat BDNF has extensive roles by binding to its specific receptor, tropomyosin-related kinase receptor B (TrkB), and thereby triggering downstream signaling pathways. Recently, growing evidence highlightsthat the BDNF/TrkB pathway is expressed in the cardiovascular system andclosely associated with the development and outcome of cardiovascular diseases (CVD), including coronary artery disease, heart failure, cardiomyopathy, hypertension, and metabolic diseases. Furthermore, circulating BDNF has also been revealed as a new potential biomarker for both diagnosis and prognosis of CVD. In this review, we discuss the current evidence of the emerging role of BDNF/TrkBsignalingand address the challenges that remain in translating these discoveries to novel therapeutic strategies for CVD.

Aloe-emodin exerts cholesterol-lowering effects by inhibiting proprotein convertase subtilisin/kexin type 9 in hyperlipidemic rats.

Hyperlipidemia (HPL) characterized by metabolic disorder of lipids and cholesterol is one of the important risk factors for cardiovascular diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a potent circulating regulator of LDL through its ability to induce degradation of the low-density lipoprotein cholesterol receptor (LDLR) in the lysosome of hepatocytes. Aloe-emodin (AE) is one of potentially bioactive components of Chinese traditional medicine Daming capsule. In this study we evaluated the HPL-lowering efficacy of AE in both in vivo and in vitro HPL models. High-fat diet-induced rats were treated with AE (100 mg/kg per day, ig) for 6 weeks. We found that AE administration significantly decreased the levels of total cholesterol (TC) and LDL in the serum and liver tissues. Moreover, AE administration ameliorated HPL-induced hepatic lipid aggregation. But AE administration did not significantly inhibit HMG-CoA reductase activity in the liver of HPL rats. A cellular model of HPL was established in human hepatoma (HepG2) cells treated with cholesterol (20 µg/mL) and 25-hydroxycholesterol (2 µg/mL), which exhibited markedly elevated cholesterol levels. The increased cholesterol levels could be reversed by subsequent treatment with AE (30 µM). In both the in vivo and in vitro HPL models, we revealed that AE selectively suppressed the sterol-regulatory element-binding protein-2 (SREBP-2) and hepatocyte nuclear factor (HNF)1α-mediated PCSK9 signaling, which in turn upregulated LDL receptor (LDLR) and promoted LDL uptake. This study demonstrates that AE reduces cholesterol content in HPL rats by inhibiting the hepatic PCSK9/LDLR pathway.

Brain-derived neurotrophic factor mimetic, 7,8-dihydroxyflavone, protects against myocardial ischemia by rebalancing optic atrophy 1 processing.

Brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) pathway is associated with ischemic heart diseases (IHD). 7,8-dihydroxyflavone (7,8-DHF), BDNF mimetic, is a potent agonist of TrkB. We aimed to investigate the effects and the underlying mechanisms of 7,8-DHF on cardiac ischemia. Myocardial ischemic mouse model was induced by ligation of left anterior descending coronary artery. 7,8-DHF (5 mg/kg) was administered intraperitoneally two days after ischemia for four weeks. Echocardiography, HE staining and transmission electron microscope were used to examine the function, histology and ultrastructure of the heart. H9c2 cells were treated with hydrogen peroxide (H2O2), 7,8-DHF or TrkB inhibitor ANA-12. The effects of 7,8-DHF on cell viability, mitochondrial membrane potential (MMP) and mitochondrial superoxide generation were examined. Furthermore, mitochondrial fission and protein expression of mitochondrial dynamics (Mfn2 [mitofusin 2], OPA1 [optic atrophy 1], Drp1 [dynamin-related protein 1] and Fis-1 [fission 1]) was detected by mitotracker green staining and western blot, respectively. 7,8-DHF attenuated cardiac dysfunction and cardiomyocyte abnormality of myocardial ischemic mice. Moreover, 7,8-DHF increased cell viability and reduced cell death accompanied by improving MMP, inhibiting mitochondrial superoxide and preventing excessive mitochondrial fission of H2O2-treated H9c2 cells. The cytoprotective effects of 7,8-DHF were antagonized by ANA-12. Mechanistically, 7,8-DHF repressed OMA1-dependent conversion of L-OPA1 into S-OPA1, which was abolished by Akt inhibitor. In conclusion, 7,8-DHF protects against cardiac ischemic injury by inhibiting the proteolytic cleavage of OPA1. These findings provide a novel pharmacological effect of 7,8-DHF on mitochondrial dynamics and a new potential target for IHD.

MicroRNA-132 regulates total protein of Nav1.1 and Nav1.2 in the hippocampus and cortex of rat with chronic cerebral hypoperfusion.

Nav1.1 and Nav1.2 are the voltage-gated sodium channel alpha subunit1 and 2, encoded by the genes of SCN1A and SCN2A. Previous studies have shown that chronic cerebral hypoperfusion (CCH) could induce neuropathological and cognitive impairment and increased total Nav1.1 and Nav1.2protein levels, yet the detailed mechanisms are not fully understood. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are involved in the regulation of dementia. miR-132 is known to play a key role in neurodegenerative disease. Here, we determined that miR-132 regulates Nav1.1 and Nav1.2 under CCH state. In this study, the expression of miR-132 was decreased in both the hippocampus and cortex of ratsfollowing CCH generated by bilateral common carotid artery occlusion (2VO). Lentiviral-mediated overexpression of miR-132 ameliorated dementia vulnerability induced by 2VO. At the molecular level, miR-132 repressed the increased protein expression of Nav1.1 and Nav1.2 in both the hippocampus and cortex induced by 2VO. MiR-132 suppressed, while AMO-miR-132 enhanced, the level of Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs) detected by both western blot analysis and immunofluorescence analysis. Results obtained by dual luciferase assay showed that overexpression of miR-132 inhibited the expression of Nav1.1 and Nav1.2 in human embryonic kidney 293 (HEK293T) cells. Additionally, binding-site mutation failed to influence Nav1.1 and Nav1.2, indicating that Nav1.1 and Nav1.2 are potential targets for miR-132. Taken together, our findings demonstrated that miR-132 protects against CCH-induced learning and memory impairments by down-regulating the expression of Nav1.1 and Nav1.2, and SCN1A and SCN2A are the target genes of miR-132.

Combination of microRNA-21 and microRNA-146a Attenuates Cardiac Dysfunction and Apoptosis During Acute Myocardial Infarction in Mice.

Recent studies have revealed the cytoprotective roles of microRNAs (miRNAs) miR-21 and miR-146a against ischemic cardiac injuries. While these studies investigated each of these miRNAs as an independent individual factor, our previous study has suggested the possible interaction between these two miRNAs. The present study was designed to investigate this possibility by evaluating the effects of miR-21 and miR-146a combination on cardiac ischemic injuries and the underlying mechanisms. MiR-21 and miR-146a synergistically decreased apoptosis under ischemia/hypoxic conditions in cardiomyocytes compared with either miR-21 or miR-146a alone. Mice coinjected with agomiR-21 and agomiR-146a had decreased infarct size, increased ejection fraction (EF), and fractional shortening (FS). These effects were greater than those induced by either of the two agomiRs. Furthermore, greater decreases in p38 mitogen-associated protein kinase phosphorylation (p-p38 MAPK) were observed with miR-21: miR-146a combination as compared to application of either of the miRNAs. These data suggest that combination of miR-21 and miR-146a has a greater protective effect against cardiac ischemia/hypoxia-induced apoptosis as compared to these miRNAs applied individually. This synergistic action is mediated by enhanced potency of inhibition of cardiomyocyte apoptosis by the miR-21-PTEN/AKT-p-p38-caspase-3 and miR-146a-TRAF6-p-p38-caspase-3 signal pathways.

Fenofibrate inhibits atrial metabolic remodelling in atrial fibrillation through PPAR-α/sirtuin 1/PGC-1α pathway.

BACKGROUND AND PURPOSE: Atrial metabolic remodelling is critical for the process of atrial fibrillation (AF). The PPAR-α/sirtuin 1 /PPAR co-activator α (PGC-1α) pathway plays an important role in maintaining energy metabolism. However, the effect of the PPAR-α agonist fenofibrate on AF is unclear. Therefore, the aim of this study was to determine the effect of fenofibrate on atrial metabolic remodelling in AF and explore its possible mechanisms of action. EXPERIMENTAL APPROACH: The expression of metabolic proteins was examined in the left atria of AF patients. Thirty-two rabbits were divided into sham, AF (pacing with 600 beats·min(-1) for 1 week), fenofibrate treated (pretreated with fenofibrate before pacing) and fenofibrate alone treated (for 2 weeks) groups. HL-1 cells were subjected to rapid pacing in the presence or absence of fenofibrate, the PPAR-α antagonist GW6471 or sirtuin 1-specific inhibitor EX527. Metabolic factors, circulating biochemical metabolites, atrial electrophysiology, adenine nucleotide levels and accumulation of glycogen and lipid droplets were assessed. KEY RESULTS: The PPAR-α/sirtuin 1/PGC-1α pathway was significantly inhibited in AF patients and in the rabbit/HL-1 cell models, resulting in a reduction of key downstream metabolic factors; this effect was significantly restored by fenofibrate. Fenofibrate prevented the alterations in circulating biochemical metabolites, reduced the level of adenine nucleotides and accumulation of glycogen and lipid droplets, reversed the shortened atrial effective refractory period and increased risk of AF. CONCLUSION AND IMPLICATIONS: Fenofibrate inhibited atrial metabolic remodelling in AF by regulating the PPAR-α/sirtuin 1/PGC-1α pathway. The present study may provide a novel therapeutic strategy for AF.

Verapamil reverses cardiac iron overload in streptozocin-induced diabetic rats.

Accumulating evidence shows that iron overload is a new risk factor for diabetes mellitus. L-type Ca(2+) channel (LTCC) has been identified as an important mediator for ferrous iron uptake into cardiomyocytes. In this study, we aimed to examine the effects of verapamil, the LTCC blocker, on myocardial iron metabolism in diabetic rats. Diabetes was induced by intraperitoneal injection of streptozocin after intragastric administration of fat emulsion, and then treated by verapamil (5 mg · kg(-1) · day(-1)) for 1 week. The results showed that verapamil did not alter the blood glucose level of diabetic rats. However, elevated levels of superoxide dismutase, malonaldehyde, and serum ferritin in diabetic rats were decreased significantly by verapamil treatment. Moreover, serum, myocardial, and urine iron were elevated remarkably along with a decrease of hepatic iron in diabetic rats. After verapamil administration, serum and myocardial iron in diabetic rats were reduced significantly but urine and hepatic iron were increased. Furthermore, confocal microscopy demonstrated that intracellular-free iron concentration was elevated dramatically in cardiomyocytes of diabetic rats, which was markedly attenuated after verapamil treatment. In summary, our data demonstrated that verapamil prevented myocardial iron overload by inhibiting intracellular iron accumulation in diabetic cardiomyocytes.

Vitexin protects against cardiac hypertrophy via inhibiting calcineurin and CaMKII signaling pathways.

Vitexin is a flavone glycoside isolated from the leaf of Crataeguspinnatifida Bunge, the utility of which has been demonstrated in several cardiovascular diseases. However, its role in cardiac hypertrophy remains unclear. In the present study, we aimed to determine whether vitexin prevents cardiac hypertrophy induced by isoproterenol (ISO) in cultured neonatal rat ventricular myocytes in vitro and pressure overload-induced cardiac hypertrophy in mice in vivo. The results revealed that vitexin (10, 30, and 100 µM) dose-dependently attenuated cardiac hypertrophy induced by ISO in vitro. Furthermore, vitexin (3, 10, and 30 mg kg(-1)) prevented cardiac hypertrophy induced by transverse aortic constriction as assessed by heart weight/body weight, left ventricular weight/body weight and lung weight/body weight ratios, cardiomyocyte cross-sectional area, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that vitexin inhibited the increment of the resting intracellular free calcium induced by ISO. Vitexin also inhibited the expression of calcium downstream effectors calcineurin-NFATc3 and phosphorylated calmodulin kinase II (CaMKII) both in vitro and in vivo. Taken together, our results indicate that vitexin has the potential to protect against cardiac hypertrophy through Ca2+-mediated calcineurin-NFATc3 and CaMKII signaling pathways.

Choline inhibits angiotensin II-induced cardiac hypertrophy by intracellular calcium signal and p38 MAPK pathway.

Choline, an agonist of M(3) muscarinic acetylcholine receptors, is a precursor and metabolite of acetylcholine and is also a functional modulator of cellular membrane. However, the effect of choline on cardiac hypertrophy is not fully understood. The present study was therefore designed to explore whether choline could prevent cardiac hypertrophy induced by angiotensin II (Ang II) and clarify its potential mechanisms. Cardiac hypertrophy was induced by 0.6 mg kg(-1) day(-1) Ang II for 2 weeks in the presence or absence of choline pretreatment, while cardiomyocyte hypertrophy was induced by Ang II 0.1 µM for 48 h. We found that choline pretreatment attenuated the increment cell size of cardiomyocytes induced by Ang II both in vivo and in vitro. The high ANP and ß-MHC levels induced by Ang II were also reversed by choline in cardiomyocytes. Meanwhile, choline pretreatment prevented the augment of reactive oxygen species (ROS) and intracellular calcium concentration in Ang II-treated cardiomyocytes. Furthermore, the upregulated phospho-p38 mitogen-activated protein kinase (MAPK) and calcineurin levels by Ang II in ventricular myocytes were attenuated by choline. In conclusion, choline prevents Ang II-induced cardiac hypertrophy through inhibition of ROS-mediated p38 MAPK activation as well as regulation of Ca(2+)-mediated calcineurin signal transduction pathway. Our results provide new insights into the pharmacological role of choline in cardiovascular diseases.

Reciprocal regulation between M3 muscarinic acetylcholine receptor and protein kinase C-epsilon in ventricular myocytes during myocardial ischemia in rats.

We have studied the association between M(3) muscarinic acetylcholine receptors (M(3)-mAChR) and protein kinase C-epsilon (PKC-epsilon) during ischemic myocardial injury using Western blot analysis and immunoprecipitation technique. Myocardial ischemia (MI) induced PKC-epsilon translocation from cytosolic to membrane fractions. This translocation participated in the phosphorylation of M(3)-mAChR in membrane fractions, which could be abolished by the inhibitor of PKC, chelerythrine chloride. On the other hand, M(3)-mAChR could also regulate the expression of PKC-epsilon in ischemic myocardium. Choline (choline chloride, an M(3) receptor agonist, administered at 15 min before occlusion) strengthened the association between PKC-epsilon and M(3)-mAChR. However, blockade of M(3)-mAChR by 4-diphenylacetoxy-N-methylpiperidine methiodide (an M(3) receptor antagonist, administered at 20 min before occlusion) completely inhibited the effect of choline on the expression of PKC-epsilon. We conclude that the translocation of PKC-epsilon is required for the phosphorylation of M(3)-mAChR; moreover, increased PKC-epsilon activity is associated with M(3)-mAChR during MI. This reciprocal regulation is likely to play a role in heart signal transduction during ischemia between ventricular myocytes.