Mitochondrial Dysfunction in Friedreich's Ataxia: From Pathogenesis to Treatment Perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Mitochondrial dysfunction in Friedreich's ataxia: from pathogenesis to treatment perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Heart hypertrophy and function are improved by idebenone in Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neuro-degenerative disease causing limb and gait ataxia and hypertrophic cardiomyopathy. It results from a triplet expansion in the first intron of the frataxin gene encoding a mitochondrial protein of yet unknown function. Cells with low frataxin content display generalized deficiency of mitochondrial iron-sulfur cluster-containing proteins, which presumably denotes overproduction of superoxide radicals in these organelles. Idebenone, a short-chain quinone, may act as a potent free radical scavenger protecting mitochondria against oxidative stress. We therefore carried out an open trial of idebenone (oral supplementation; 5mg/kg/day) in a large series of FRDA patients and followed their left ventricular mass and function. Consistent and definitive worsening being observed in the natural course of the disease and cardiac hypertrophy having no chance of spontaneous reversal and to be subject to a placebo effect, the patient's heart status before and after the treatment was used to unambiguously establish the effect of the drug. After six months, heart ultrasound revealed more than 20% reduction of left ventricular mass in about half of the patients (p < 0.001) and no significant change in the other half. Since any measurable reversion of this pathogenic trait is highly significant, this demonstrates the efficiency of idebenone in controlling heart hypertrophy in FRDA. Owing to the absence of side effects of the drug, idebenone (up to 15mg/kg/day) should be prescribed for FRDA patients continuously as early as possible.     

Proteomic analysis of hearts from frataxin knockout mice: marked rearrangement of energy metabolism, a response to cellular stress and altered expression of proteins involved in cell structure, motility and metabolism

A frequent cause of death in Friedreich's ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed 2-DE to characterize the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in protein expression profile were observed in 9 week-old KO mice with severe cardiomyopathy. In contrast, only several proteins showed altered expression in asymptomatic 4 week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilization and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.     

缬沙坦联合氨氯地平治疗高血压伴左心肥厚的疗效及对心功能的影响

目的:通过探讨缬沙坦联合氨氯地平治疗高血压伴左心肥厚患者的疗效及对心功能的影响,为临床治疗提供依据。方法:选择2010年1月~2014年12月我院收治的高血压伴左心室肥厚患者共120例,按照随机数字表法随机分为观察组和对照组。对照组患者给予氨氯地平,观察组患者缬沙坦联合氨氯地平治疗,治疗6个月后,观察两组患者舒张压(DBP)、收缩压(SBP)、心率(HR)、室间隔厚度(IVST),左室后壁厚度(LVPWT)、左室舒张末期内径(LVDd)和左室重量指数(LVMI)。结果:治疗后,两组患者SBP、DBP和HR均较治疗前显著降低,差异有统计学意义(P0.05);观察组患者SBP、DBP和HR均低于对照组,差异有统计学意义(P0.05)。治疗后,两组患者IVST、IVPWT、LVDd和LVMI均较治疗前显著降低,差异有统计学意义(P0.05);观察组患者IVST、IVPWT、LVDd和LVMI均低于对照组,差异有统计学意义(P0.05)。结论:缬沙坦联合氨氯地平治疗高血压伴左心肥厚患者,能够降低患者血压、逆转左心室肥厚,改善患者心功能,疗效优于氨氯地平单独治疗,值得临床推广应用。     

Phenotyping of Left and Right Ventricular Function in Mouse Models of Compensated Hypertrophy and Heart Failure with Cardiac MRI

Background

Left ventricular (LV) and right ventricular (RV) function have an important impact on symptom occurrence, disease progression and exercise tolerance in pressure overload-induced heart failure, but particularly RV functional changes are not well described in the relevant aortic banding mouse model. Therefore, we quantified time-dependent alterations in the ventricular morphology and function in two models of hypertrophy and heart failure and we studied the relationship between RV and LV function during the transition from hypertrophy to heart failure.

Methods

MRI was used to quantify RV and LV function and morphology in healthy (n = 4) and sham operated (n = 3) C57BL/6 mice, and animals with a mild (n = 5) and a severe aortic constriction (n = 10).

Results

Mice subjected to a mild constriction showed increased LV mass (P<0.01) and depressed LV ejection fraction (EF) (P<0.05) as compared to controls, but had similar RVEF (P>0.05). Animals with a severe constriction progressively developed LV hypertrophy (P<0.001), depressed LVEF (P<0.001), followed by a declining RVEF (P<0.001) and the development of pulmonary remodeling, as compared to controls during a 10-week follow-up. Myocardial strain, as a measure for local cardiac function, decreased in mice with a severe constriction compared to controls (P<0.05).

Conclusions

Relevant changes in mouse RV and LV function following an aortic constriction could be quantified using MRI. The well-controlled models described here open opportunities to assess the added value of new MRI techniques for the diagnosis of heart failure and to study the impact of new therapeutic strategies on disease progression and symptom occurrence.     

Disruption of the ATP-binding cassette B7 (ABTM-1/ABCB7) induces oxidative stress and premature cell death in Caenorhabditis elegans

X-linked sideroblastic anemia with ataxia (XLSA/A) is a rare inherited disorder characterized by mild anemia and ataxia. XLSA/A is caused by mutations in the ABCB7 gene, which encodes a member of the ATP-binding cassette transporter family. Studies in yeast, mammalian cells, and mice have shown that ABCB7 functions in the transport of iron-sulfur (Fe-S) clusters into the cytoplasm. To further investigate the mechanism of this disease, we have identified and characterized the Caenorhabditis elegans homologue of the ABCB7 gene, abtm-1. We have studied the function of abtm-1 using mutants and RNAi. abtm-1-depleted animals produce arrested embryos that have morphogenetic defects and unusual premature, putative apoptotic events. abtm-1(RNAi) animals also show accumulation of ferric iron and increased oxidative stress. Despite the increased level of oxidative stress in abtm-1(RNAi) animals, they have an increased life span. We observed accumulation of DAF-16/FOXO in the nuclei of affected animals and elevation of the expression of SOD-3, a well established target of DAF-16, which may explain the increased life span extension of these animals. abtm-1 is strongly expressed in tissues with a high energy demand, and abtm-1(RNAi) animals have phenotypes that reflect the need for abtm-1 in these tissues. Finally, we show that reducing the function of other genes involved in Fe-S cluster production produces similar phenotypic consequences to abtm-1 loss of function. Therefore, ablation of abtm-1 in C. elegans provides a model in which to investigate the mechanism underlying XLSA/A.     

超声心动图对高血压心室肥厚患者左心功能的评价及意义

目的:本研究利用超声心动图检测高血压心室肥厚患者左心房结构,探讨当左心结构发生变化时心脏功能所受到的影响,为高血压及其并发症的临床诊断提供检测及诊断参考。方法:选取2011年5月-2013年1月在我院接受检查的高血压心室肥厚患者76例作为观察组,另选取同期经体检的健康人群60例为健康对照组,利用超声心动图观察左心功能和结构,比较两组研究对象的左心房内径(LAD)、心肌质量(LVMM)、舒张末容积(LVEDV)、收缩末容积(LVESV)、左心室射血分数(LVEF)及二尖瓣口舒张末期流速比值(E/A)。结果:两组间心室收缩功能无显著性差异(P0.05);高血压组LAD高于对照组,LVEF及E/A低于对照组,差异具有统计学意义(P0.05);高血压Ⅰ期、Ⅱ期、Ⅲ期患者间比较,左房内径随血压的升高逐渐递增,而左心室射血分数和二尖瓣口舒张期流速比值则逐渐递减,差异具有统计学意义(P0.05)。结论:超声心动图可以直观的显示高血压心室肥厚患者左心功能及血流动力学的变化,对临床诊断具有积极的意义。     

白藜芦醇甙对糖尿病小鼠心肌损伤的保护作用及机制研究

目的:探讨白藜芦醇甙对小鼠小鼠心肌损伤的影响及其可能的调控机制。方法:采用腹腔注射链脲霉素的方法建立1型糖尿病小鼠模型模型,随机将雄性C57/BL6J的正常小鼠和糖尿病小鼠分为3组(每组18只):对照组、糖尿病组、糖尿病+白藜芦醇甙组。其中糖尿病+白藜芦醇甙组给予白藜芦醇甙7.5 mg/kg/d腹腔注射治疗,对照组和糖尿病组给予同体积盐水腹腔注射。药物治疗12周后,小动物心脏超声检测小鼠的心功能;天狼星红染色观察胶原变化并进一步计算胶原容积分数(collagen volume fraction,CVF);柠檬酸合酶检测试剂盒和ATP生物荧光试剂盒分别检测检测心肌柠檬酸合酶活性和ATP含量;Western blot检测Sirt3和p-AMPK的蛋白表达情况。结果:与对照组相比,糖尿病组LVEDD和LVESD均明显增加(4.823±0.103 mm和3.701±0.121 mm,P0.05),LVEF和LVFS值均明显降低(37.121±4.298%和21.023±2.187%,P0.05),CVF显著增高(20.102±1.155%,P0.05),心肌柠檬酸合酶活性和ATP含量显著降低(5.267±0.202 nmol/g和105±7.638 U/g,P0.05),Sirt3和p-AMPK蛋白表达显著降低(P0.05);与糖尿病组相比,糖尿病+白藜芦醇甙组LVEDD和LVESD均显著降低(4.354±0.113 mm和3.056±0.130 mm,P0.05),LVFS和LVEF值均显著增加(58.435±8.143%和29.071±2.232%,P0.05),CVF显著降低(10.343±0.882%,P0.05),心肌柠檬酸合酶活性和ATP含量显著增加(6.233±0.176 nmol/g和132.7±5.774 U/g,P0.05),Sirt3和p-AMPK蛋白表达显著增加(P0.05)。结论:白藜芦醇甙可改善糖尿病小鼠心肌损伤,其机制可能与白藜芦醇甙激活Sirt3/AMPK信号通路有关。     

Genome Instability in Ataxia Telangiectasia (A-T) Families: Camptothecin-Induced Damage to Replicating DNA Discriminates between Obligate A-T Heterozygotes,A-T Homozygotes and Controls

Previously we used the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells primarily by inducing double strand breaks (DSBs) in replication forks, to show that ataxia telangiectasia (A-T) fibroblasts are defective in the repair of this particular subclass of DSBs. CPT treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges, viewed as prematurely condensed G2 chromosomes (G2 PCC), compared with normal cells where aberrations are mostly chromatid breaks. Here we show that A-T lymphoblastoid cells established from individuals with different mutations in the ATM gene also exhibit increased levels of chromosomal exchanges in response to CPT, indicating that the replication-associated DSBs are misrepaired in all these cells. From family studies we show that the presence of a single mutated allele in obligate A-T heterozygotes leads to intermediate levels of chromosomal exchanges in CPT-treated lymphoblastoid cells, thus providing a functional and sensitive assay to identify these individuals.     

改良大鼠左心室插管术及心衰大鼠左心功能指标的测定

目的完善大鼠左心室插管技术并确定充血性心力衰竭（CHF）大鼠心功能指标参数。方法将80只成年雄性SD大鼠随机分为2组：假手术组（SH）,腹主动脉缩窄模型组（CAA）。采用腹主动脉部分缩窄法制作CHF大鼠模型,BL-420E＋生物信号采集系统测定心功能参数。观察比较2组大鼠第6周后测定的心功能的各项指标。结果（1）经进一步完善大鼠左心室插管技术,成功率明显提高。（2）CAA组大鼠心功能明显减低,左室重量指数（LVMI）、左室舒张末压（LVEDP）升高,左心室内压上升、下降的最大变化速率（±dp/dtmax）下降（P〈0.01）。结论改良大鼠左心室插管术提高成功率,心衰大鼠的心功能指标明显改变。     

小鼠异位心脏移植模型的改良

目的探讨小鼠心脏移植模型的建立与改良。方法供体采用BLAB／c小鼠,受体采用C57小鼠。用24GACuff管法套扎供心肺动脉与颈外静脉,用自制Cuff管套扎供心升主动脉与颈总动脉。结果供心冷缺血时间少于15min,手术时间大大缩短,成功率明显提高。结论改进后的模型显著降低了手术难度,可用于移植免疫的各种研究。     

p18~(INK4C)调控小鼠T细胞的发育及功能

p18INK4C属于细胞周期蛋白激酶抑制剂,其突变或缺失与某些肿瘤的发生密切相关,如T细胞白血病,但目前关于p18调控T细胞发育及功能的研究还鲜有报道,其调控机制仍不明确.本研究利用p18基因敲除(p18KO)小鼠,系统地研究了胸腺中T细胞的早期发育及成熟T细胞的增殖和活化功能,并利用逆转录病毒的方法在Lin?造血干祖细胞上过表达p18,移植4个月后检测其对T细胞的影响.结果表明,p18的缺失对胸腺T细胞的早期发育影响不明显,但随着p18KO小鼠周龄的增加会促进CD4+CD8+双阳性T细胞的数量,此外,p18还通过影响CD3+成熟T细胞的细胞周期进程及IFN-?,GATA3,Tbx21和Foxp3等的表达增强脾脏T细胞的增殖和活化;进一步在造血干祖细胞上过表达p18后会影响T细胞的发育和成熟,进而纠正T细胞在数量上的异常.本研究阐释了p18在T细胞早期发育及后期活化中的调控机制,并证实可通过在干祖细胞水平改变p18的表达进而影响T细胞的分化,这对p18调控T细胞功能异常及参与T细胞白血病的发生提供了新的理论依据和重要的研究价值.     

交感神经阻滞对糖尿病心腔扩张顽固心衰的疗效

目的:探讨交感神经阻滞对有糖尿病、心腔扩张的顽固心衰患者心脏收缩功能和心腔大小的影响。方法:经胸3～胸4棘突间隙穿刺至硬膜外腔,留置硬膜外导管,0.5%利多卡因3～5ml每2h推注一次,持续4w,适当辅以常规治疗。测定并比较TEB治疗前及后4w射血分数(EF)、左室内径(LVED)和左房内径(LAED)等指标的变化。结果:治疗后左室射血分数(EF)由治疗前的(31.53±9.29)%升为(44.75±8.32)%(P0.05);左室内径(LVED)由治疗前的(70.59±6.25)mm缩减到(63.59±7.05)mm(P0.05);左房内径(LAED)由治疗前的(46.16±7.19)mm缩减到(39.05±7.23)mm(P0.05)。结论:心区交感神经阻滞结合常规用药对有糖尿病、心腔扩张的顽固心衰患者的心脏功能有明显改善作用,并使其扩大的心脏明显缩小。     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K_ATP channels directly associate with ankyrin-B in heart via the K_ATP channel α-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K_ATP channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K_ATP channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.