Deficiency of Smad7 Enhances Cardiac Remodeling Induced by Angiotensin II Infusion in a Mouse Model of Hypertension

Smad7 has been shown to negatively regulate fibrosis and inflammation, but its role in angiotensin II (Ang II)-induced hypertensive cardiac remodeling remains unknown. Therefore, the present study investigated the role of Smad7 in hypertensive cardiopathy induced by angiotensin II infusion. Hypertensive cardiac disease was induced in Smad7 gene knockout (KO) and wild-type (WT) mice by subcutaneous infusion of Ang II (1.46 mg/kg/day) for 28 days. Although equal levels of high blood pressure were developed in both Smad7 KO and WT mice, Smad7 KO mice developed more severe cardiac injury as demonstrated by impairing cardiac function including a significant increase in left ventricular (LV) mass (P<0.01),reduction of LV ejection fraction(P<0.001) and fractional shortening(P<0.001). Real-time PCR, Western blot and immunohistochemistry detected that deletion of Smad7 significantly enhanced Ang II-induced cardiac fibrosis and inflammation, including upregulation of collagen I, α-SMA, interleukin-1β, TNF-α, and infiltration of CD3⁺ T cells and F4/80⁺ macrophages. Further studies revealed that enhanced activation of the Sp1-TGFβ/Smad3-NF-κB pathways and downregulation of miR-29 were mechanisms though which deletion of Smad7 promoted Ang II-mediated cardiac remodeling. In conclusions, Smad7 plays a protective role in AngII-mediated cardiac remodeling via mechanisms involving the Sp1-TGF-β/Smad3-NF.κB-miR-29 regulatory network.     

小鼠胚胎干细胞分化心肌细胞血管紧张素Ⅱ 1型和2型受体的表达特征

胚胎干细胞(ESC)具有无限增殖和分化为体内3个胚层来源的各种类型组织细胞的潜能,经过体外诱导能够分化为心肌细胞,亦称为胚胎干细胞分化心肌细胞(ESCM).本研究探讨了ESC诱导分化心肌细胞过程中血管紧张素Ⅱ受体(ATR)的亚型AT1R和AT2R的表达特征.10-4mol/L维生素C体外诱导小鼠R1胚胎干细胞分化为自发搏动的心肌细胞,用免疫荧光法检测分化后的细胞表达心肌细胞特异性标志物α辅肌动蛋白.小鼠胚胎干细胞在诱导分化为心肌细胞以后,逆转录聚合酶链反应(RT-PCR)和实时定量RT-PCR(Real-timeRT-PCR)方法检测到ESCM表达AT1R,并且呈时间依赖性逐渐增加的特点,在第14d达到高峰.Western印记法检测AT1R表达特征与RT-PCR结果相符.Western印记法的结果显示,血管紧张素Ⅱ(10-6mol/L)可作为AT1R激动剂激活AT1R,并使其下游的细胞外信号调节激酶(ERK1/2)磷酸化水平上调,预孵育AT1R抑制剂Losartan(10-6mol/L),此作用被抑制.RT-PCR方法显示,与新生小鼠心室肌细胞相比,ESCM的AT2R表达水平较低.     

人参皂甙Rb1改善转基因扩张型心肌病模型小鼠的心功能和心脏重构

目的利用cTnT^R141W转基因扩张型心肌病小鼠,研究人参皂甙Rb1对遗传性扩张型心肌病心功能及心脏重构的作用及其可能机制。方法将cTnT^R141W转基因小鼠随机分为模型组和人参皂甙Rb1治疗组（70 mg/kg/d）,连续给药7个月,取野生型小鼠作为对照组。心脏超声检测心脏功能及几何构型。HE染色观察心肌细胞变化。透射电镜分析心肌超微结构。RT-PCR检测心肌粘附蛋白的表达。免疫荧光激光共聚焦观察心肌粘附分子Itga8的表达与分布。结果Rb1长期给药能显著改善该模型的心脏功能及几何构型。光镜和透射电镜观察显示Rb1能减轻心肌细胞排列紊乱及超微结构的破坏。RT-PCR结果显示,在模型中Cx40表达降低,E-cad、itga8和itgb1bp3表达升高,但在Rb1组中接近正常水平。免疫荧光激光共聚焦结果显示Rb1可降低Itga8的表达量并调节其分布。结论Rb1可改善扩张型心肌病模型的心功能,抑制心脏重构,其作用可能部分通过调节粘附蛋白的表达而实现的。     

A Food-Derived Flavonoid Luteolin Protects against Angiotensin II-Induced Cardiac Remodeling

Oxidative stress has been implicated in cardiac remodeling (cardiac fibrosis and hypertrophy), which impairs cardiac function and metabolism; therefore, it is anticipated antioxidative compounds will have protective properties against cardiac remodeling. Luteolin (3’,4’,5,7-tetrahydroxyflavone), a widely distributed flavonoid found in many herbal extracts including celery, green pepper, perilla leaves and seeds, and chamomile, is a known to be a potent antioxidant and was previously demonstrated to exert an antifibrotic effect in the lungs and the liver. In this study, we clearly demonstrate that oral pretreatment with the higher-luteolin diet (0.035% (wt/wt)) protected against cardiac fibrosis and hypertrophy as well as a hyperoxidative state in Ang II-infused rats. In cardiac tissue, increased gene expression levels of TGFβ1, CTGF, Nox2, Nox4, ANP, and BNP induced by Ang II were restored by oral pretreatment of this high-luteolin diet. In cultured rat cardiac fibroblasts, H₂O₂-induced TGFβ1 expression and the phosphorylation of JNK were suppressed by luteolin pretreatment. In conclusion, food-derived luteolin has protective actions against Ang II-induced cardiac remodeling, which could be mediated through attenuation of oxidative stress.     

Perinatal Nicotine Exposure Increases Angiotensin II Receptor-Mediated Vascular Contractility in Adult Offspring

Previous studies have reported that perinatal nicotine exposure causes development of hypertensive phenotype in adult offspring.

Aims

The present study was to determine whether perinatal nicotine exposure causes an epigenetic programming of vascular Angiotensin II receptors (ATRs) and their-mediated signaling pathway leading to heightened vascular contraction in adult offspring.

Main methods

Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps from day 4 of gestation to day 10 after birth. The experiments were conducted at 5 months of age of male offspring.

Key Findings

Nicotine treatment enhanced Angitension II (Ang II)-induced vasoconstriction and 20-kDa myosin light chain phosphorylation (MLC₂₀-P) levels. In addition, the ratio of Ang II-induced tension/MLC-P was also significantly increased in nicotine-treated group compared with the saline group. Nicotine-mediated enhanced constrictions were not directly dependent on the changes of [Ca²⁺]_i concentrations but dependent on Ca²⁺ sensitivity. Perinatal nicotine treatment significantly enhanced vascular ATR type 1a (AT_1aR) but not AT_1bR mRNA levels in adult rat offspring, which was associated with selective decreases in DNA methylation at AT_1aR promoter. Contrast to the effect on AT_1aR, nicotine decreased the mRNA levels of vascular AT₂R gene, which was associated with selective increases in DNA methylation at AT₂R promoter.

Significance

Our results indicated that perinatal nicotine exposure caused an epigenetic programming of vascular ATRs and their-mediated signaling pathways, and suggested that differential regulation of AT₁R/AT₂R gene expression through DNA methylation mechanism may be involved in nicotine-induced heightened vasoconstriction and development of hypertensive phenotype in adulthood.     

Angiotensin II Type 2 Receptor-Mediated Stimulation of Protein Phosphatase 2A in Rat Hypothalamic/Brainstem Neuronal Cocultures

Abstract: Recent studies have suggested a role for an inhibitory guanine nucleotide binding (G_i) protein and protein (serine/threonine) phosphatase 2A (PP2A) in the angiotensin II type 2 (AT₂) receptor-mediated stimulation of neuronal K⁺ currents. In the present study we have directly analyzed the effects of angiotensin II on PP2A activity in neurons cultured from newborn rat hypothalamus and brainstem. Angiotensin II elicited time (30 min–24 h)- and concentration (10 n M -1 µ M )-dependent increases in PP2A activity in these cells, an effect mimicked by the AT₂ receptor ligand CGP-42112A. These effects of angiotensin II and CGP-42112A involve AT₂ receptors, because they were inhibited by the AT₂ receptor-selective ligand PD 123,319 (1 µ M ) but not by the angiotensin II type 1 receptor antagonist losartan (1 µ M ). Furthermore, the stimulatory effects of angiotensin II and CGP-42112A on PP2A activity were inhibited by pretreatment of cultures with pertussis toxin (200 ng/ml; 24 h), indicating the involvement of a G_i protein. These effects of angiotensin II and CGP-42112A appear to be via activation of PP2A, and western blot analyses revealed no effects of either peptide on the protein levels of the catalytic subunit of PP2A in cultured neurons. In summary, these data suggest that PP2A is a cellular target modified following neuronal AT₂ receptor activation.     

Ca Sparks Do Not Explain all Ryanodine Receptor-Mediated SR Ca Leak in Mouse Ventricular Myocytes

Diastolic Ca leak from the sarcoplasmic reticulum (SR) of ventricular myocytes reduces the SR Ca content, stabilizing the activity of the SR Ca release channel ryanodine receptor for the next beat. SR Ca leak has been visualized globally using whole-cell fluorescence, or locally using confocal microscopy, but never both ways. When using confocal microscopy, leak is imaged as “Ca sparks,” which are fluorescent objects generated by the local reaction-diffusion of released Ca and cytosolic indicator. Here, we used confocal microscopy and simultaneously measured the global ryanodine-receptor-mediated leak rate (J_leak) and Ca sparks in intact mouse ventricular myocytes. We found that spark frequency and J_leak are correlated, as expected if both are manifestations of a common phenomenon. However, we also found that sparks explain approximately half of J_leak. Our strategy unmasks the presence of a subresolution (i.e., nonspark) release of potential physiological relevance.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

Diminished Autophagy Limits Cardiac Injury in Mouse Models of Type 1 Diabetes

Cardiac autophagy is inhibited in type 1 diabetes. However, it remains unknown if the reduced autophagy contributes to the pathogenesis of diabetic cardiomyopathy. We addressed this question using mouse models with gain- and loss-of-autophagy. Autophagic flux was inhibited in diabetic hearts when measured at multiple time points after diabetes induction by streptozotocin as assessed by protein levels of microtubule-associated protein light chain 3 form 2 (LC3-II) or GFP-LC3 puncta in the absence and presence of the lysosome inhibitor bafilomycin A1. Autophagy in diabetic hearts was further reduced in beclin 1- or Atg16-deficient mice but was restored partially or completely by overexpression of beclin 1 to different levels. Surprisingly, diabetes-induced cardiac damage was substantially attenuated in beclin 1- and Atg16-deficient mice as shown by improved cardiac function as well as reduced levels of oxidative stress, interstitial fibrosis, and myocyte apoptosis. In contrast, diabetic cardiac damage was dose-dependently exacerbated by beclin 1 overexpression. The cardioprotective effects of autophagy deficiency were reproduced in OVE26 diabetic mice. These effects were associated with partially restored mitophagy and increased expression and mitochondrial localization of Rab9, an essential regulator of a non-canonical alternative autophagic pathway. Together, these findings demonstrate that the diminished autophagy is an adaptive response that limits cardiac dysfunction in type 1 diabetes, presumably through up-regulation of alternative autophagy and mitophagy.     

Angiotensin II Induced Cardiac Dysfunction on a Chip

In vitro disease models offer the ability to study specific systemic features in isolation to better understand underlying mechanisms that lead to dysfunction. Here, we present a cardiac dysfunction model using angiotensin II (ANG II) to elicit pathological responses in a heart-on-a-chip platform that recapitulates native laminar cardiac tissue structure. Our platform, composed of arrays of muscular thin films (MTF), allows for functional comparisons of healthy and diseased tissues by tracking film deflections resulting from contracting tissues. To test our model, we measured gene expression profiles, morphological remodeling, calcium transients, and contractile stress generation in response to ANG II exposure and compared against previous experimental and clinical results. We found that ANG II induced pathological gene expression profiles including over-expression of natriuretic peptide B, Rho GTPase 1, and T-type calcium channels. ANG II exposure also increased proarrhythmic early after depolarization events and significantly reduced peak systolic stresses. Although ANG II has been shown to induce structural remodeling, we control tissue architecture via microcontact printing, and show pathological genetic profiles and functional impairment precede significant morphological changes. We assert that our in vitro model is a useful tool for evaluating tissue health and can serve as a platform for studying disease mechanisms and identifying novel therapeutics.     

Angiotensin II Inactivation Process in Cultured Mouse Spinal Cord Cells

The pattern of hydrolysis of [3H]angiotensin II ( [3H]AII; 20 nM) by intact cells was studied on cultured mouse spinal cord cells. Degradation products were identified by HPLC analysis after incubation for 2 h at 37 degrees C. In the absence of peptidase inhibitors, 70% of [3H]AII was degraded, and the main labeled metabolite was [3H]tyrosine (40% of total radioactivity). Minor quantities of [3H]AII1-5 and [3H]AII4-8 were formed. Results obtained in the presence of various inhibitors indicate that several enzymes were involved in the AII-hydrolyzing process. Dipeptidyl aminopeptidase III (EC 3.4.14.4) could play a critical role, as suggested by the formation of [3H]Val3-Tyr4 and [3H]-Tyr4-Ile5 in the presence of bestatin (2 X 10(-5) M). This hypothesis was confirmed by the potency of dipeptidyl amino-peptidase III inhibitors to inhibit both [3H]AII hydrolysis and formation of these 3H-labeled dipeptides. An arylamidase-like activity could also be participating in [3H]AII hydrolysis, because higher concentrations of bestatin (10(-4) M) in association with dipeptidyl aminopeptidase III inhibitors totally inhibited [3H]tyrosine formation, increased protection of [3H]AII and [3H]AII1-7 formed, and provoked a slight accumulation of [3H]AII2-8. These results suggest that the formation of [3H]AII2-8 is due to the action of a bestatin-insensitive acidic aminopeptidase and that the Pro7-Phe8 cleavage is also a step of AII hydrolysis, resulting from the action of an unidentified peptidase different from prolyl endopeptidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

In cardiomyocytes, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca²⁺ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca²⁺-activated K⁺ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca²⁺ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca²⁺-activated K+ current (I_K,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the I_K,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on I_K,Ca and Ca²⁺ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in I_K,Ca (p<0.05) and [Ca²⁺]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca²⁺ release is responsible for the activation and modulation of SK channels in cardiac myocytes.     

Arrhythmogenic Transient Dynamics in Cardiac Myocytes

Cardiac action potential alternans and early afterdepolarizations (EADs) are linked to cardiac arrhythmias. Periodic action potentials (period 1) in healthy conditions bifurcate to other states such as period 2 or chaos when alternans or EADs occur in pathological conditions. The mechanisms of alternans and EADs have been extensively studied under steady-state conditions, but lethal arrhythmias often occur during the transition between steady states. Why arrhythmias tend to develop during the transition is unclear. We used low-dimensional mathematical models to analyze dynamical mechanisms of transient alternans and EADs. We show that depending on the route from one state to another, action potential alternans and EADs may occur during the transition between two periodic steady states. The route taken depends on the time course of external perturbations or intrinsic signaling, such as β-adrenergic stimulation, which regulate cardiac calcium and potassium currents with differential kinetics.     

低氧与心肌细胞凋亡

细胞凋亡是心肌细胞低氧损伤的主要死亡形式之一。低氧引起心肌细胞凋亡可以通过外部的死亡受体通路以及内部的线粒体通路,两条通路之间又存在复杂的交互作用,其中,线粒体通路在低氧诱导的心肌细胞凋亡中起重要作用。另外,心肌细胞本身也具有多种内源性的凋亡抑制因子。因此,低氧时心肌细胞凋亡的产生是多种因素综合作用的结果,Bcl-2家族蛋白、线粒体通透性改变、细胞色素c的释放以及caspases的活化等参与了低氧引起的心肌细胞凋亡的调控。对低氧时心肌细胞凋亡的认识和深入研究,为人类在缺血性心脏病的防治中提供了一个新的治疗措施。