Cardioprotection by Hepc1 in cTnTR141W transgenic mice

Hepcidin 1 (Hepc1) is a peptide hormone secreted by the liver in response to iron loading. It is expressed in the heart and is thought to play a role in the regulation of iron homeostasis in an autocrine and paracrine fashion. We have shown that expression of Hepc1 is strongly down-regulated in the heart of the cTnT(R141W) transgenic mouse model of dilated cardiomyopathy (DCM) at 3?months of age. Transgenic mice with heart tissue-specific Hepc1 expression alone or in combination with the cTnT(R141W) mutation were produced to study the effects of Hepc1 on DCM. Transgenic expression of Hepc1 was found to be nonlethal and resulted in decreased mortality in cTnT(R141W) transgenic mice, from 29.6 to 7.4%(n =?27; P?相似文献   

Increased production of apolipoprotein B-containing lipoproteins in the absence of hyperlipidemia in transgenic mice expressing cholesterol 7alpha-hydroxylase

The finding that expression of a cholesterol 7alpha-hydroxylase (CYP7A1) transgene in cultured rat hepatoma cells caused a coordinate increase in lipogenesis and secretion of apoB-containing lipoproteins led to the hypothesis that hepatic production of apoB-containing lipoproteins may be linked to the expression of CYP7A1 (Wang, S.-L., Du, E., Martin, T. D., and Davis, R. A. (1997) J. Biol. Chem. 272, 19351-19358). To examine this hypothesis in vivo, a transgene encoding CYP7A1 driven by the constitutive liver-specific enhancer of the human apoE gene was expressed in C56BL/6 mice. The expression of CYP7A1 mRNA (20-fold), protein ( approximately 10-fold), and enzyme activity (5-fold) was markedly increased in transgenic mice compared with non-transgenic littermates. The bile acid pool of CYP7A1 transgenic mice was doubled mainly due to increased hydrophobic dihydroxy bile acids. In CYP7A1 transgenic mice, livers contained approximately 3-fold more sterol response element-binding protein-2 mRNA. Hepatic expression of mRNAs encoding lipogenic enzymes (i.e. fatty-acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, squalene synthase, farnesyl-pyrophosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and low density lipoprotein receptor) as well as microsomal triglyceride transfer protein were elevated approximately 3-5-fold in transgenic mice. CYP7A1 transgenic mice also displayed a >2-fold increase in hepatic production and secretion of triglyceride-rich apoB-containing lipoproteins. Despite the increased hepatic secretion of apoB-containing lipoproteins in CYP7A1 mice, plasma levels of triglycerides and cholesterol were not significantly increased. These data suggest that the 5-fold increased expression of the low density lipoprotein receptor displayed by the livers of CYP7A1 transgenic mice was sufficient to compensate for the 2-fold increase production of apoB-containing lipoproteins. These findings emphasize the important homeostatic role that CYP7A1 plays in balancing the anabolic lipoprotein assembly/secretion pathway with the cholesterol catabolic bile acid synthetic pathway.     

Increase in tension-dependent ATP consumption induced by cardiac troponin T mutation

How different mutations in cardiac troponin T (cTnT) lead to distinct secondary downstream cellular remodeling in familial hypertrophic cardiomyopathy (FHC) remains elusive. To explore the molecular basis for the distinct impact of different mutations in cTnT on cardiac myocytes, we studied mechanical activity of detergent-skinned muscle fiber bundles from different lines of transgenic (TG) mouse hearts that express wild-type cTnT (WTTG), R92W cTnT, R92L cTnT, and Delta-160 cTnT (deletion of amino acid 160). The amount of mutant cTnT is approximately 50% of the total myocellular cTnT in both R92W and R92L TG mouse hearts and approximately 35% in Delta-160 TG mouse hearts. Myofilament Ca2+ sensitivity was enhanced in all mutant cTnT TG cardiac muscle fibers. Compared with the WTTG fibers, Ca2+ sensitivity increased significantly at short sarcomere length (SL) of 1.9 microm (P < 0.001) in R92W TG fibers by 2.2-fold, in R92L by 2.0-fold, and in Delta-160 by 1.3-fold. At long SL of 2.3 microm, Ca2+ sensitivity increased significantly (P < 0.01) in a similar manner (R92W, 2.5-fold; R92L, 1.9-fold; Delta-160, 1.3-fold). Ca2+-activated maximal tension remained unaltered in all TG muscle fibers. However, tension-dependent ATP consumption increased significantly in Delta-160 TG muscle fibers at both short SL (23%, P < 0.005) and long SL (37%, P < 0.0001), suggesting a mutation-induced change in cross-bridge detachment rate constant. Chronic stresses on relative cellular ATP level in cardiac myocytes may cause a strain on energy-dependent Ca2+ homeostatic mechanisms. This may result in pathological remodeling that we observed in Delta-160 TG cardiac myocytes where the ratio of sarco(endo)plasmic reticulum Ca2+-ATPase 2/phospholamban decreased significantly. Our results suggest that different types of stresses imposed on cardiac myocytes would trigger distinct cellular signaling, which leads to remodeling that may be unique to some mutants.     

LMNA E82K mutation activates FAS and mitochondrial pathways of apoptosis in heart tissue specific transgenic mice

The lamin A/C (LMNA), nuclear intermediate filament proteins, is a basic component of the nuclear lamina. Mutations in LMNA are associated with a broad range of laminopathies, congenital diseases affecting tissue regeneration and homeostasis. Heart tissue specific transgenic mice of human LMNA E82K, a mutation causing dilated cardiomyopathy, were generated. Lmna(E82K) transgenic mouse lines exhibited thin-walled, dilated left and right ventricles, a progressive decrease of contractile function assessed by echocardiography. Abnormalities of the conduction system, myocytes disarray, collagen accumulation and increased levels of B-type natriuretic peptide (BNP), procollagen type III α1 (Col3α1) and skeletal muscle actin α1 (Actα1) were detected in the hearts of Lmna(E82K) transgenic mice. The LMNA E82K mutation caused mislocation of LMNA in the nucleus and swollen mitochondria with loss of critae, together with the loss of nuclear envelope integrity. Most interestingly, we found that the level of apoptosis was 8.5-fold higher in the Lmna(E82K) transgenic mice than that of non-transgenic (NTG) mice. In the presence of the LMNA E82K, both of FAS and mitochondrial pathways of apoptosis were activated consistent with the increase of FAS expression, the release of cytochrome c from mitochondria to cytosol and activation of caspase-8, -9 and -3. Our results suggested that the apoptosis, at least for the LMNA E82K or the mutations in the rod region of Lamin A/C, might be an important mechanism causing continuous loss of myocytes and lead to myocardial dysfunction. It could be a potential therapeutic means to suppress and/or prevent inappropriate cardiac cell death in patients carrying LMNA mutation.     

人参皂甙Rb1抑制扩张型心肌病发生中的HB-EGF表达和纤维化

目的HB-EGF过表达可促进心肌纤维化及心肌细胞凋亡,本文研究人参皂甙Rb1对cTnT^R141W转基因扩张型心肌病小鼠发病过程中的HB-EGF表达和心肌纤维化的影响。方法将cTnT^R141W转基因小鼠随机分为模型组和人参皂甙Rb1组（70 mg/kg/d）,连续给药7个月,取野生型小鼠作为对照组。用Kaplan-Meier法进行生存分析。心脏超声检测心功能及心脏几何构型。计算心重指数。光镜观察心肌细胞及间质变化。Western blot检测心脏HB-EGF,pSTAT3表达水平。结果Rb1长期给药能显著改善该模型的心功能和心脏几何构型,将死亡率降低50%。Rb1治疗组心重指数降低11.3%（P〈0.05）,光镜观察显示Rb1能减轻心肌细胞排列紊乱以及间质纤维化。Western blot结果显示Rb1能够显著降低模型中的HB-EGF及pSTAT3的表达。结论Rb1抑制心肌病发生中的HB-EGF表达及抑制下游信号pSTAT的激活,并改善扩张型心肌病模型的心功能及心脏重构。     

磁共振成像对扩张型心肌病转基因模型小鼠左右心室对比分析

目的对cTnTR141W扩张型心肌病转基因模型小鼠左、右心室进行对比分析,研究cTnTR141W转基因小鼠作为右心室心肌病的动物模型的可行性。方法利用7.0 T高场强磁共振成像(MRI)技术,定量分析了2、4、6和8月龄对照组及cTnTR141W转基因模型小鼠左、右心室的舒张末容积(EDV)、收缩末容积(ESV)和射血分数(EF)的变化情况,同时对6月龄对照组cTnTR141W转基因模型小鼠心肌组织进行组织学分析。结果转基因阴性对照小鼠相比,cTnTR141W转基因小鼠左、右心室的容积在2月龄时已有增大趋势,而射血分数有减小趋势。右心室射血分数减小出现最早也最显著(P<0.05)。随年龄增加,cTnTR141W转基因小鼠与转基因阴性对照小鼠相比,右心室的结构和功能的病理生理变化与左心室同时趋于严重。该小鼠左、右心室在4月龄后表现典型的扩张型心肌病表型。结论 cTnTR141W转基因模型小鼠左心室和右心室的扩张性心肌病表型同时出现,该小鼠可作为右室性心肌病等右心室功能下降相关疾病研究的动物模型。     

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。     

心脏特异表达人源FAM55A转基因小鼠的建立

目的建立心脏特异表达的人源FAM55A转基因小鼠,为研究该基因在心肌病发病中的作用提供模型。方法 Western blot检测FAM55A在野生型小鼠与cTnTR141W转基因小鼠心脏组织中的表达变化及其在野生小鼠的组织表达谱。克隆人源FAM55A基因入α-MHC启动子下游构建a-MHC-FAM55A表达载体,显微注射法建立FAM55A转基因小鼠。PCR鉴定转基因首建鼠的基因型。Western blot鉴定人源FAM55A在转基因小鼠心脏中的表达,超声检测转基因小鼠心脏的几何构型和功能。HE染色检测转基因小鼠心脏的病理改变。结果 FAM55A在野生型小鼠心脏中有少量表达,在扩张型心肌病小鼠的心脏中表达增加。建立了1个心脏组织特异表达人源FAM55A转基因小鼠品系。与野生型小鼠相比,FAM55A转基因小鼠的心脏收缩期和舒张期左室前壁从1月龄到5月龄持续增厚,3月龄转基因小鼠心脏射血分数和短轴缩短率稍有增强,1月龄和5月龄转基因小鼠心脏功能则与同龄野生型小鼠相比无变化。组织学检测显示,转基因小鼠心脏左室心肌细胞不均匀肥大,但不发生紊乱。结论 FAM55A在扩张型心肌病小鼠的心脏中表达上调,建立了心脏特异表达的人源FAM55A转基因小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。     

cTnT^（R141W）转基因小鼠扩张型心肌病模型的建立

目的建立cTnT^R141W扩张型心肌病的转基因小鼠模型。方法把cTnT^R141W基因插入-αMHC启动子下游,构建转基因表达载体,通过显微注射法建立cTnT^R141W转基因C57BL/6J小鼠。PCR鉴定cTnT^R141W转基因小鼠的基因表型,实时PCR检测基因的拷贝数,Northern blotting检测基因表达,光学显微镜和超声检测cTnT^R141W转基因小鼠心脏的病理改变。结果建立了3个系的cTnT^R141W转基因小鼠。3个系的基因拷贝数分别是15、20和59拷贝。cTnT^R141W基因在心脏组织的表达水平高于内源性cTnT。病理分析显示cTnT^R141W转基因小鼠心房心室明显大于野生型,心室壁明显变薄,心肌细胞不均匀肥大,心肌间质纤维增多。超声检查显示心室腔明显扩大,收缩期容积和舒张期容积显著增大,射血分数、短轴缩短率、室壁运动度明显降低。结论cTnT^R141W转基因小鼠的全心扩大,室壁变薄,心肌细胞肥大,间质纤维化以及心肌收缩力下降,说明成功建立了cTnT^R141W转基因小鼠扩张型心肌病模型,为研究扩张型心肌病发病机制和药物研发提供了有价值的动物模型。     

应用CRISPR/Cas13b编辑技术治疗TNNT2R141W转基因小鼠的扩张型心肌病

本研究旨在应用CRISPR/Cas13b系统对TNNT2R141W转基因扩张型心肌病（dilated cardiomyopathy,DCM）小鼠（DCM小鼠）进行探索性治疗,尝试发现治疗扩张型心肌病的一种新方式,为CRISPR/Cas13b系统在体内应用提供实验基础。随机设计11种Cas13b-TNNT2 gRNA并成功构建表达质粒,把它和人源TNNT2过表达质粒共同转染到293T细胞中,通过实时定量PCR（quantitative real-time PCR,Q-PCR）检测人源TNNT2 mRNA的表达水平。结果显示,gRNA 2引导Cas13b敲低目标基因的效率最高,达到80%（P<0.0001）。把gRNA2表达质粒包装到慢病毒载体中转导出生后1天的DCM小鼠原代心肌细胞,Q-PCR检测结果表明CRISPR/Cas13b系统对人源TNNT2 mRNA的敲低效率达到55%（P<0.01）。把PspCas13b和gRNA2的表达载体分别包装到AAV9病毒载体中,然后将200 μL 约1×1012 AAV9病毒颗粒通过尾静脉注射到4月龄DCM小鼠体内,待注射小鼠发育至5月龄时,Q-PCR检测结果显示,AAV9+DCM组TNNT2R141W表达水平较未注射组对照明显下降至40%（P<0.01）。对5月龄野生型（WT）、DCM（未注射病毒组）和AAV9+DCM（基因组编辑工具注射组）三组小鼠的心脏形态、心功能、心肌纤维化和心力衰竭等表型的观察结合显示：DCM小鼠的心脏形态异常,而AAV9+DCM小鼠心脏形态趋于正常;对三组小鼠的心脏进行超声心动图并对心功能指标进行统计发现,DCM组较WT组小鼠的左心室射血分数（left ventricular percent ejection fraction,LV EF%）、左心室短轴缩短率（left ventricular percent fractional shortening,LV FS%）分别下降了50.4%（P<0.0001）,55.1%（P<0.0001）,而AAV9+DCM组较DCM组小鼠的LV EF%、LV FS%分别上升了66.5%（P<0.01）,77.0%（P<0.01）;通过Q-PCR和天狼星红染色检测三组小鼠的心脏纤维化程度,结果显示DCM组较WT组小鼠的Col3a1和Postn两种纤维化基因,分别高表达5.2倍（P<0.001）、4.5倍（P<0.01）,而AAV9+DCM组较DCM组小鼠两种基因表达分别下降了2.0倍（P<0.05）、1.4倍（NS）,天狼星红染色结果显示纤维化区域明显下降;通过Q-PCR和蛋白质免疫印迹分别检测三组小鼠的心脏心力衰竭基因Nppb mRNA和Nppa蛋白质的表达水平,结果表明DCM组较WT组小鼠Nppb mRNA表达上升14.2倍（P<0.01）,而AAV9+DCM组较DCM组小鼠Nppb mRNA表达明显下降下降2.8倍（P<0.05）,Nppa蛋白质表达趋势与Nppb相同。把gRNA 5和含有R141W突变（gRNA 5T）和正常的TNNT2 mRNA（gRNA 5V）序列分别组合转染到293T细胞中,通过Q-PCR检测两种序列mRNA的表达水平。结果显示,gRNA 5T序列表达效率为30%（P<0.0001）,而并未检测到gRNA 5V mRNA的敲低。本研究通过设计靶向TNNT2R141W mRNA的gRNA,特异性敲低TNNT2R141W转基因小鼠体内突变的mRNA,有效改善了转基因小鼠的心功能,为临床进一步探索扩张型心肌病的治疗奠定了实验室基础。     

Chronic alcohol intake-induced oxidative stress and apoptosis: role of CYP2E1 and calpain-1 in alcoholic cardiomyopathy

Cytochrome P-450 2E1 CYP2E1 induction has been linked to oxidative stress in a number of experimental models. The aim of this study was to investigate the relationship between CYP2E1 activity and markers of oxidative stress and cardiac cell apoptosis during the development of alcoholic cardiomyopathy (ACM). Changes in left ventricular morphology were evaluated in 4 groups of chronically instrumented dogs (control; alcohol-receiving; and alcohol-receiving plus treatment with either valsartan or carnitine) after 6 months of treatment. CYP2E1 and calpain-1 protein expression were determined by Western blotting, and apoptosis evaluated by TUNEL and immunohistochemistry. Malonyl dialdehyde levels were assessed as a marker of oxidative stress, while superoxide dismutase and glutathione peroxidase levels were evaluated as markers of antioxidant defense mechanisms. Expression of CYP2E1 was increased in the alcohol-receiving group compared with controls (P<0.05) and was associated with oxidative stress. Similarly, expression of Bad and calpain-1 protein was increased after chronic alcohol exposure, while Bcl-xL protein expression remained at a low level. Bad and calpain-1 protein expressions were significantly inhibited by treatment with valsartan or carnitine, while expression of Bcl-xL protein was increased (P<0.05). Collectively, our results indicate a possibly significant role for CYP2E1 in the oxidative stress associated with chronic alcoholism. The resulting increase in oxidative stress is accompanied by cellular apoptosis and may ultimately contribute to tissue remodeling and ACM. Importantly, these alcohol-induced effects may be abrogated by means such as angiotensin 1 receptor blockade or carnitine supplementation.     

心脏特异表达肾素（原）受体转基因小鼠的建立

目的建立心脏特异表达（p）RR的转基因小鼠,研究（p）RR在心肌病发病过程中的调节作用。方法将（p）RR基因插入到心脏特异表达启动子α-MHC下游,构建转基因表达载体,通过显微注射的方法建立（p）RR转基因小鼠,PCR法鉴定转基因小鼠的基因型;Western blot和免疫组化的方法检测（p）RR在心脏组织中的表达;利用小动物超声仪检测转基因小鼠的心脏结构和功能;双色免疫荧光共聚焦进行（p）RR蛋白在转基因小鼠中的亚细胞定位,Western blot方法检测了钙泵（ATP2A2）、钠-钙交换体1（NCX 1）以及肌钙蛋白T（cTnT）在转基因小鼠心脏组织中表达量的变化情况。结果建立了心脏高表达（p）RR的转基因小鼠品系;内源性和转基因高表达的（p）RR蛋白均定位在细胞内高尔基体和内质网上;心脏特异高表达（p）RR蛋白使小鼠心脏收缩功能增强,主要表现在与同窝阴性对照小鼠相比,转基因小鼠收缩期左室内径（LVID,systolic）降低9%（P〈0.05,n=18）,收缩期容积（LVESV,systolic）减少了23%（P〈0.05,n=18）,射血分数EF（ejection fraction）升高14%（P〈0.01,n=18）,短轴缩短率FS（fraction shortening）升高20%（P〈0.01,n=18）;和心脏收缩功能和钙离子相关的ATP2A2和NCX 1表达均下调,而cTnT表达量上调。结论在心脏中过表达（p）RR能够引起心脏收缩功能明显增强,同时直接或间接调节ATP2A2、NCX 1和cTnT表达。提示（p）RR可能是调节心脏中的钙离子而参与影响心肌功能。     

Acute ethanol pretreatment increases FAS-mediated liver injury in mice: role of oxidative stress and CYP2E1-dependent and -independent pathways

This study evaluated whether acute ethanol pretreatment potentiates Fas-mediated liver injury and if oxidative stress and CYP2E1 play a role in any enhanced hepatotoxicity. There were 3-fold increases of transaminases and more extensive apoptotic necrosis of hepatocytes and focal hemorrhages of the hepatic lobule in mice treated with Jo2 Fas agonistic antibody plus ethanol compared to saline control or to mice treated with Jo2 or ethanol alone. CYP2E1 catalytic activity and protein were increased 2-fold by the acute ethanol pretreatment. There were 2- and 2.5-fold increases of caspase-8 and caspase-3 activity and 1.6-fold increases of apoptotic-positive cells in the Jo2 plus acute ethanol group compared to the Jo2 alone group. Levels of TNF-alpha, malondialdehyde, 4-hydroxynonenal, protein carbonyl formation, 3-nitrotyrosine protein adducts, and inducible nitric oxide synthase were increased in the Jo2 plus ethanol group. The enhanced hepatotoxicity of Jo2 plus ethanol and the elevated oxidative stress and TNF levels were lower in CYP2E1 knockout mice compared to wild-type mice expressing CYP2E1 but higher than saline controls. Toxicity also declined in mice treated with gadolinium chloride, an inhibitor of the inducible nitric oxide synthase or the antioxidant, N-acetyl-L-cysteine. These data indicate that acute ethanol pretreatment is capable of elevating hepatic apoptosis and liver injury induced by Jo2 Fas agonistic antibody. The enhanced hepatotoxicity involves increased oxidative and nitrosative stress, and appears to be mediated by CYP2E1-dependent and also CYP2E1-independent mechanisms.     

Apolipoprotein B production reduces lipotoxic cardiomyopathy: studies in heart-specific lipoprotein lipase transgenic mouse

Lipid accumulation is associated with cardiac dysfunction in diabetes and obesity. Transgenic mice expressing non-transferable lipoprotein lipase (LpL) with a glycosylated phosphatidyl-inositol (GPI) anchor in cardiomyocytes have dilated cardiomyopathy. However, the mechanisms responsible for lipid accumulation and cardiomyopathy are not clear. Hearts from 3-month-old mice expressing GPI-anchored human LpL (hLpLGPI) mice had increased fatty acid oxidation and heart failure genes and decreased glucose transporter genes. 6-month-old mice had increased mRNA expression and activation of the apoptosis marker caspase-3. Moreover, hLpLGPI hearts had significant cytochrome c release from mitochondria to cytosol. Low density lipoprotein uptake was greater in hLpLGPI hearts, and this was associated with more intracellular apolipoprotein B (apoB). To test whether lipid accumulation in the hLpLGPI heart is reduced by cardiac expression of apoB, hLpLGPI mice were bred with transgenic human apoB (HuB)-expressing mice. Hearts of HuB/hLpLGPI mice had less triglyceride (38%) and free fatty acids (19%), secreted more apoB, and expressed less atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and more glucose transporter 4 (GLUT4). The increased mortality of the mice was abrogated by the transgenic expression of apoB. Therefore, we hypothesize that cardiac apoB expression improves cardiomyopathy by increasing lipid resecretion from the heart.     

Mitochondria-targeted Cytochrome P450 2E1 Induces Oxidative Damage and Augments Alcohol-mediated Oxidative Stress

The ethanol-inducible cytochrome P450 2E1 (CYP2E1) is also induced under different pathological and physiological conditions. Studies including ours have shown that CYP2E1 is bimodally targeted to both the endoplasmic reticulum (microsomes) (mc CYP2E1) and mitochondria (mt CYP2E1). In this study we investigated the role of mtCYP2E1 in ethanol-mediated oxidative stress in stable cell lines expressing predominantly mt CYP2E1 or mc CYP2E1. The ER+ mutation (A2L, A9L), which increases the affinity of the nascent protein for binding to the signal recognition particle, preferentially targets CYP2E1 to the endoplasmic reticulum. The Mt+ (L17G) and Mt++ (I8R, L11R, L17R) mutant proteins, showing progressively lower affinity for signal recognition particle binding, were targeted to mitochondria at correspondingly higher levels. The rate of GSH depletion, used as a measure of oxidative stress, was higher in cells expressing Mt++ and Mt+ proteins as compared with cells expressing ER+ protein. In addition, the cellular level of F₂-isoprostanes, a direct indicator of oxidative stress, was increased markedly in Mt++ cells after ethanol treatment. Notably, expression of Mt++ CYP2E1 protein in yeast cells caused more severe mitochondrial DNA damage and respiratory deficiency than the wild type or ER+ proteins as tested by the inability of cells to grow on glycerol or ethanol. Additionally, liver mitochondria from ethanol-fed rats containing high mt CYP2E1 showed higher levels of F₂-isoprostane production. These results strongly suggest that mt CYP2E1 induces oxidative stress and augments alcohol-mediated cell/tissue injury.     

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes

Enhanced hepatic levels of cytochrome P450 2E1 (CYP2E1) may play a key role in the pathogenesis of some liver diseases because CYP2E1 represents a significant source of reactive oxygen species. Although a large fraction of CYP2E1 is located in the endoplasmic reticulum, CYP2E1 is also present in mitochondria. In this study, we asked whether ethanol, a known inducer of microsomal CYP2E1, could also increase CYP2E1 within mitochondria. Our findings indicated that ethanol increased microsomal and mitochondrial CYP2E1 in cultured rat hepatocytes and in the liver of lean mice. This was associated with decreased levels of glutathione, possibly reflecting increased oxidative stress. In contrast, in leptin-deficient obese mice, ethanol administration did not increase mitochondrial CYP2E1, nor it depleted mitochondrial glutathione, suggesting that leptin deficiency hampers mitochondrial targeting of CYP2E1. Thus, ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, thus favouring oxidative stress in these compartments.     

Abnormal heart rate regulation in murine hearts with familial hypertrophic cardiomyopathy-related cardiac troponin T mutations

Mutations in cardiac troponin T (cTnT), Δ160E and R92Q, have been linked to familial hypertrophic cardiomyopathy (FHC), and some studies have indicated that these mutations can lead to a high incidence of sudden cardiac death in the relative absence of significant ventricular hypertrophy. Alterations in autonomic function have been documented in patients with hypertrophic cardiomyopathy. We hypothesize that alterations in autonomic function may contribute to mutation-specific clinical phenotypes in cTnT-related FHC. Heart rate (HR) variability (HRV) has been used to assess autonomic function from an electrocardiograph. Nontransgenic, Δ160E, or R92Q mice were implanted with radiofrequency transmitters to obtain continuous electrocardiograph recordings during 24-h baseline and 30-min recordings after β-adrenergic receptor drug injections. Although Δ160E mice did not differ from nontransgenic mice for any 24-h HRV measurements, R92Q mice had impaired HR regulation, as measured by a decrease in the SD of the R-R interval, a decrease in the low frequency-to-high frequency ratio, a decrease in normalized low frequency, and an increase in normalized high frequency. β-Adrenergic receptor density measurements and HRV analysis after drug injections did not reveal any significant differences for Δ160E or R92Q mice versus nontransgenic mice. Arrhythmia analysis revealed both an increased incidence of heart block in R92Q mice at baseline and frequency of premature ventricular contractions after isoproterenol injections in Δ160E and R92Q mice. In addition, Δ160E and R92Q mice exhibited a prolonged P duration after drug injections. Therefore, between two independent and clinically severe cTnT mutations within the same functional domain, only R92Q mice exhibited altered autonomic function, whereas both mutations demonstrated abnormalities in conduction and ventricular ectopy.