CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro ‘patient-specific cell-based system'' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after β-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca²⁺/calmodulin-dependent serine–threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.     

Mapping of reentrant spontaneous polymorphic ventricular tachycardia in a Scn5a+/- mouse model

Two major mechanisms have been postulated for the arrhythmogenic tendency observed in Brugada Syndrome (BrS): delays in conduction or increased heterogeneities in repolarization. We use a contact mapping system to directly investigate the interacting roles of these two mechanisms in arrhythmogenesis using a genetic murine model for BrS for the first time. Electrograms were obtained from a multielectrode recording array placed against the left ventricle and right ventricle (RV) of spontaneously beating Langendorff-perfused wild type (WT) and Scn5a+/- mouse hearts. Scn5a+/- hearts showed activation waves arriving at the epicardial surface consistent with slowed conduction, which was exacerbated in the presence of flecainide. Lines of conduction block across the RV resulting from premature ventricular beats led to the formation of reentrant circuits and polymorphic ventricular tachycardia. WT hearts showed an inverse relationship between activation times and activation recovery intervals measured at the epicardial surface, which resulted in synchronicity of repolarization times. In contrast, Scn5a+/- hearts, despite having smaller mean activation recovery intervals, demonstrated a greater heterogeneity compared with WT. Isochronal maps showed that their normal activation recovery interval gradients at the epicardial surface were disrupted, leading to heterogeneity in repolarization times. We thus directly demonstrate the initiation of arrhythmia in the RV of Scn5a+/- hearts. This occurs as a result of the combination of repolarization heterogeneities leading to lines of conduction block and unidirectional conduction, with conduction slowing allowing the formation of reentrant circuits. The repolarization heterogeneities may also be responsible for the changing pattern of block, leading to the polymorphic character of the resulting ventricular tachycardia.     

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

Introduction  

Vasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.     

PiggyBac转座酶转基因小鼠模型的建立

目的 建立表达PiggyBac转座酶转基因小鼠模型,为研究PiggyBac转座子介导基因修饰在小鼠中的应用提供工具.方法 利用Cytomegalovirus( CMV)启动子驱动PiggyBac转座酶基因的表达,经显微注射法建立C57BL/6J表达PiggyBac转座酶的转基因小鼠.PCR鉴定转基因小鼠的基因型,RT-PCR检测PiggyBac转座酶在小鼠生殖系睾丸中的表达情况.PiggyBac转座酶转基因小鼠活性的检测,是通过与转座子供体转基因小鼠杂交检测供体位置变化来确定的.结果 显微注射产生7只转基因小鼠并能传代,经RT-PCR筛选出一株在睾丸中相对高表达PiggyBac转座酶的转基因小鼠.随后与转座子供体转基因小鼠杂交,子代双阳小鼠与野生型小鼠杂交基因型分离,产生的子代转座子供体单阳性小鼠中具有转座子供体片段的转座反应.结论 成功建立了表达PiggyBac转座酶转基因小鼠动物模型,该模型为PiggyBac转座子技术在小鼠中的应用提供了有价值的工具动物.     

Cardiac ischemia activates calcium-independent phospholipase A2beta,precipitating ventricular tachyarrhythmias in transgenic mice: rescue of the lethal electrophysiologic phenotype by mechanism-based inhibition

Murine myocardium contains diminutive amounts of calcium-independent phospholipase A2 (iPLA2) activity (<5% that of human heart), and malignant ventricular tachyarrhythmias are infrequent during acute murine myocardial ischemia. Accordingly we considered the possibility that the mouse was a species-specific knockdown of the human pathologic phenotype of ischemiainduced lethal ventricular tachyarrhythmias. Transgenic mice were generated expressing amounts of iPLA2beta activity comparable to that present in human myocardium. Coronary artery occlusion in Langendorff perfused hearts from transgenic mice resulted in a 22-fold increase in fatty acids released into the venous eluent (29.4 nmol/ml in transgenic versus 1.35 nmol/ml of eluent in wild-type mice), a 4-fold increase in lysophosphatidylcholine mass in ischemic zones (4.9 nmol/mg in transgenic versus 1.1 nmol/mg of protein in wild-type mice), and malignant ventricular tachyarrhythmias within minutes of ischemia. Neither normally perfused transgenic nor ischemic wild-type hearts demonstrated these alterations. Pretreatment of Langendorff perfused transgenic hearts with the iPLA2 mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) just minutes prior to induction of ischemia completely ablated fatty acid release and lysolipid accumulation and rescued transgenic hearts from malignant ventricular tachyarrhythmias. Collectively these results demonstrate that ischemia activates iPLA2beta in intact myocardium and that iPLA2beta-mediated hydrolysis of membrane phospholipids can induce lethal malignant ventricular tachyarrhythmias during acute cardiac ischemia.     

Analysis of ubiquitination in vivo using a transgenic mouse model

The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.     

人载脂蛋白A1基因转基因小鼠的建立

目的建立系统性表达人载脂蛋白A1(APOA1)基因的转基因小鼠。方法 将人APOA1基因插入系统性表达启动子下游,构建转基因表达载体,通过显微注射法建立人APOA1转基因C57BL/6J小鼠。并利用特异引物PCR法鉴定转基因小鼠的基因型,Western blot检测基因表达水平,血生化分析检测不同月龄转基因小鼠与同龄野生型小鼠的血脂指标。结果建立了2个不同表达水平的人APOA1基因的转基因小鼠品系;转入的人APOA1基因在血液、肝脏、心脏、肾脏、脾脏、血管组织中均有明显表达;血生化分析结果显示不同月龄转基因小鼠的血浆高密度脂蛋白胆固醇水平高于同龄的野生型小鼠,甘油三酯水平低于同龄野生型小鼠。结论成功建立了系统性表达人APOA1基因的转基因小鼠,为研究高血脂以及高血脂相关的心血管病提供了工具。     

Phenotypical features of long Q-T syndrome in transgenic mice expressing human Na-K-ATPase alpha(3)-isoform in hearts

To understand why the adult human heart expresses three isoforms of the sodium pump, we generated transgenic mice (TGM) with 2.3- to 5. 5-fold overexpression of the human alpha(3)-isoform of Na-K-ATPase in the heart. Hearts from the TGM had increased maximal Na-K-ATPase activity and ouabain affinity compared with control hearts, even though the density of Na-K-ATPase pump sites (of all isoforms) was similar to that of control mice. In perfused hearts, contractility both at baseline and in the presence of ouabain tended to be greater in TGM than in controls. Surface electrocardiograms in anesthetized TGM had a steeper dependence of Q-T on sinus cycle length, and Q-T intervals measured during atrial pacing were significantly longer in TGM. Q-T dispersion during sinus rhythm also tended to be longer in TGM. Thus TGM overexpressing human alpha(3)-isoform have several of the phenotypical features of human long Q-T syndrome, despite the absence of previously described mutations in Na(+) or K(+) channels.     

In vivo monitoring of pancreatic beta-cells in a transgenic mouse model

We generated a transgenic mouse model (RIP-luc) for the in vivo monitoring of pancreatic islet mass and function in response to metabolic disease. Using the rat insulin promoter fused to firefly luciferase, and noninvasive technology to detect luciferase activity, we tracked changes in reporter signal during metabolic disease states and correlated the changes in luciferase signal with metabolic status of the mouse. Transgene expression was found to be specific to the pancreatic islets in this transgenic model. Basal transgene expression was tracked in male and female mice fed either a chow or a high-fat diet and in response to treatment with streptozotocin. Pancreatic bioluminescent signal increased in mice fed a high-fat diet compared with chow-fed animals. In a model of chemically induced diabetes, the bioluminescent signal decreased in accordance with the onset of diabetes and reduction of islet beta-cell number. Preliminary studies using islets transplanted from this transgenic model suggest that in vivo image analysis can also be used to monitor transplanted islet viability and survival in the host. This transgenic model is a useful tool for in vivo studies of pancreatic beta-cells and as a donor for islet transplantation studies.     

Calcineurin-induced energy wasting in a transgenic mouse model of heart failure

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.     

Gender and aging in a transgenic mouse model of hypertrophic cardiomyopathy

Mutations in the cardiac myosin heavy chain (MHC) can cause familial hypertrophic cardiomyopathy (FHC). A transgenic mouse model has been developed in which a missense (R403Q) allele and an actin-binding deletion in the alpha-MHC are expressed in the heart. We used an isovolumic left heart preparation to study the contractile characteristics of hearts from transgenic (TG) mice and their wild-type (WT) littermates. Both male and female TG mice developed left ventricular (LV) hypertrophy at 4 mo of age. LV hypertrophy was accompanied by LV diastolic dysfunction, but LV systolic function was normal and supranormal in the young TG females and males, respectively. At 10 mo of age, the females continued to present with LV concentric hypertrophy, whereas the males began to display LV dilation. In female TG mice at 10 mo of age, impaired LV diastolic function persisted without evidence of systolic dysfunction. In contrast, in 10-mo-old male TG mice, LV diastolic function worsened and systolic performance was impaired. Diminished coronary flow was observed in both 10-mo-old TG groups. These types of changes may contribute to the functional decompensation typically seen in hypertrophic cardiomyopathy. Collectively, these results further underscore the potential utility of this transgenic mouse model in elucidating pathogenesis of FHC.     

Maintained contractile reserve in a transgenic mouse model of myocardial stunning

Cardiac excitation-contraction (E-C) coupling is impaired at the myofilament level in the reversible postischemic dysfunction known as "stunned" myocardium. We characterized tension development and calcium cycling in intact isolated trabeculae from transgenic (TG) mice expressing the major proteolytic degradation fragment of troponin I (TnI) found in stunned myocardium (TnI(1-193)) and determined the ATPase activity of myofibrils extracted from TG and non-TG mouse hearts. The phenotype of these mice at baseline recapitulates that of stunning. Here, we address the question of whether contractile reserve is preserved in these mice, as it is in genuine stunned myocardium. During twitch contractions, calcium cycling was normal, whereas tension was greatly reduced, compared with non-TG controls. A decrease in maximum Ca2+-activated tension and Ca2+ desensitization of the myofilaments accounted for this contractile dysfunction. The decrease in maximum tension was paralleled by an equivalent decrease in maximum Ca2+-activated myofibrillar ATPase activity. Exposure to high calcium or isoproterenol recruited a sizable contractile reserve in TG muscles, which was proportionately similar to that in control muscles but scaled downward in amplitude. These results suggest that calcium regulatory pathways and beta-adrenergic signal transduction remain intact in isolated trabeculae from stunned TG mice, further recapitulating key features of genuine stunned myocardium.     

Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ

The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues.     

Inducible gene expression in transgenic Xenopus embryos

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.     

人sCD40L转基因小鼠模型的构建

目的研究阻断CD40-CD40L共刺激信号通路对移植皮肤免疫排斥反应的影响。方法通过RT-PCR技术克隆了呈可溶性表达的CD40L分子胞外区(sCD40L),利用K14启动子构建了sCD40L皮肤特异性表达载体,并利用该载体制备了转基因小鼠。结果所克隆的CD40L胞外区片段其大小及序列符合预期;以哺乳动物表达载体PCI为骨架,通过DNA重组,获得了含K14启动子和sCD40L编码区的皮肤特异性表达载体K14-sCD40L;通过显微注射和胚胎移植,PCR筛选检测:49只G0代小鼠有1只小鼠扩增出特异性条带,阴性对照无条带。结论成功建立sCD40L转基因阳性小鼠。